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Synthesis of 1,4-diamino-1,4-cyclohexanedicarboxylic acid, a new polymerization monomer, and its derivatives

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Title:
Synthesis of 1,4-diamino-1,4-cyclohexanedicarboxylic acid, a new polymerization monomer, and its derivatives
Creator:
Wilson, Carolina Recuerda
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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English
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xv, 110 leaves : ; 28 cm

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Subjects / Keywords:
Cyclohexane ( lcsh )
Polymerization ( lcsh )
Cyclic compounds ( lcsh )
Monomers -- Synthesis ( lcsh )
Cyclic compounds ( fast )
Cyclohexane ( fast )
Monomers -- Synthesis ( fast )
Polymerization ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 108-110).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Carolina Recuerda Wilson.

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|University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
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SYNTHESIS OF 1,4-DIAMINO-1,4-CYCLOHEXANEDICARBOXYLIC
ACID, A NEW POLYMERIZATION MONOMER,
Carolina Recuerda Wilson
B.S., Universidad de Granada, 1994
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
1999
AND ITS DERIVATIVES
by


This thesis for the Master of Science
degree by
Carolina Recuerda Wilson
has been approved
by
Donald C. Zapien
Susan M. Schelble

Date


Wilson, Carolina Recuerda (M.S., Chemistry)
Synthesis of 1,4-Diamino-1,4-cyclohexanedicarboxylic Acid, a New Polymerization
Monomer, and its Derivatives
Thesis directed by Professor Douglas F. Dyckes
ABSTRACT
The first part of the research is directed toward the synthesis and characterization
of 1,4-diamino-1,4-cyclohexane dicarboxylic acid. This cyclic diamino acid was
prepared by Strecker synthesis using 1,4-cyclohexanedione, ammonium chloride,
and sodium cyanide as reagents. The hydrolysis of the dinitrile intermediate yielded
the desired amino acid. The molecule could be potentially used as a polymerization
monomer.
To be able to polymerize it, the molecule secondly needs to be derivatized protecting
its amine and/or carboxylic groups. Derivatization of the cyclic diamino acid proved
to be a more difficult task than was expected, due to the poor solubility of this
compound in organic solvents and the steric hindrance of the ring.
Synthesis of the diacetylated amino acid and the diformylated amino acid was
successfully accomplished. The synthesis of the diacetylated derivative was carried
m


out to determine better conditions for the N-acylation reaction with the more labile
trifluoroacetyl group. Unfortunately when trifluoroacetyl groups were used, the
reaction failed. The diformylated derivative was obtained in good yield. The yield
of this reaction was better when more concentrated formic acid was used.
The synthesis of methyl esters by standard Fischer esterification was not
successful.
The present studies have shown the possibility of derivatization of our monomer.
The finding of new organic solvent systems where the polymerization reactions
can be carried out is a priority for future studies.
This abstract accurately represents the content of the candidates thesis.
I recommend its publication.
Signed_
Douglas F. Dyckes
IV


DEDICATION
To Roger
for being the energy that keeps me going


ACKNOWLEDGMENTS
My special thanks to Professor Douglas Dyckes for his support, guidance and
patience throughout this work, which is the result of two years of gratifying learning
under his guidance. I also wish to express my gratitude to Professor Donald Zapien
and Professor Susan Schelble for their advice and assistance.
Thanks to mi familia for their support and love on the other side of the Atlantic
Ocean (with occasional visits to Denver). Special thanks to Mario, Margarita, and
Paco for their patience during their vacation.
Also, thanks to the Ramirez, the Wilson, the Carabellos, the Kuta and the
Cordova families, as well as, Mina Sumita and Mei-Chen Kuo for their help and
encouragement.


CONTENTS
Figures.............................................................xii
Schemes.............................................................xiv
Tables..............................................................xv
Chapter
1. Introduction................................................. 1
1.1 Research Goal............................................... 1
1.2 Polymerization Strategies.................................. 6
1.2.1 Activation of the Carboxyl Group.......................... 7
1.2.1.1 Formation of Active Esters.............................. 7
1.2.1.2 Formation of Mixed Anhydrides........................... 8
1.2.1.3 Coupling Reagents....................................... 9
1.2.2 Protection of theAmineGroup.............................. 10
1.2.2.1 The Benzyloxycarbonyl Group (Z Group)................... 10
1.2.2.2 The t-Butoxycarbonyl Group (Boc Group).................. 11
1.2.2.3 The 9-Flourenylmethoxycarbonyl Group (Fmoc Group)....... 12
1.2.2.4 The Formyl Group........................................ 14
vii


1.2.2.5 The Acetyl Group............................................... 14
1.3 Fragment Condensation versus Stepwise Condensation................. 15
1.3.1 Fragment Condensation............................................ 15
1.3.2 Stepwise Condensation............................................ 18
2. Results and Discussion.............................................. 21
2.1 Synthesis of 1-Amino-cyanocyclohexane (Compound I) and
1-Aminocyclohexanecarboxylic Acid (Compoundll)..................... 25
2.2 Synthesis of 1,4-Diamino- 1,4-dicyanocylcohexane (Compound III) and
1.4- Diamino-1 ,4-cyclohexanedicarboxylic Acid
. (Compound IV)...................................................... 29
2.3 Activation of Carboxylic Function by Formation of Methyl Ester: Synthesis
of Methyl 1-Aminocyclohexane carboxylate (Compound V)................ 43
2.4 Activation of Carboxylic Function by Formation of Methyl Ester:
Attempted Synthesis ofDimethyl l,4-Diaminocyclohexane-l,4-dicarboxylate
(CompoundVI)................................................... 45
2.5 Protection of Amino Function with a Boc Group: Synthesis of
1 -tert-Butoxycarbonylaminocyclohexanecarboxylic Acid
(Compound VII)..................................................... 47
2.6 Protection of Amino Function with an Acetyl Group: Synthesis of
1 -Acetylaminocyclohexanecarboxylic Acid (Compound VIII) and
1.4- Diacetylamino-l,4-cyclohexanedicarboxylic Acid (Compound IX). 46
viii


2.7 Protection of Amino Function with a Fmoc Group: Synthesis of
l-(9-Fluorenyl)aminocyclohexanecarboxylic Acid (Compound X) and
Attempted Synthesis of l,4-[Di-(9-Fluorenyl)amino]-1,4-cyclohexane
dicarboxylic Acid (Compound XI).......................... 51
2.8 Protection of Amino Function with a Formyl Group: Synthesis of
1-Formylaminocyclohexanecarboxylic Acid (Compound XII) and Synthesis
of l,4-Diformylamino-l,4-cyclohexanedicarboxylic Acid
(Compound XIII)..................................................... 57
2.8.1 Analysis of the NMR spectra of 1-Formylaminocyclohexanecarboxylic
Acid.............................................................. 58
2.8.2 Analysis of the NMR spectra of 1,4-Diformylamino-1,4-cyclohexane
Dicarboxylic Acid................................................. 63
2.9 Protection of Amino Function with a Trifluoroacetyl Group: Attempted
Synthesis of 1-Trifluoroacetylaminocyclohexanecarboxylic Acid
(Compound XIV).................................................. 68
2.10 Protection of Amino Group with a Trifluoroacetyl Group: Attempted
Synthesis 1,4-Ditrifluoroacetylamino-1,4-cyclohexanedicarboxylic
Acid (Compound XV)................................................ 70
2.11 A Search for Diastereoisomers of 1,4-Diamino-1,4-cyclohexane
dicarboxylic Acid (Compound IV)................................... 71
IX


3. Experimental.................................................... 75
3.1 Reagents........................................................ 75
3.2 General Methods................................................. 75
3.2.1 Thin Layer Chromatography...................................... 75
3.2.2 Nuclear Magnetic Resonance..................................... 76
3.2.3 Melting Points................................................. 77
3.3 Synthetic Procedures........................................... 78
3.3.1 Synthesis of 1-Amino-1-cyanocyclohexane (Compound I)........... 78
3.3.2 Synthesis of 1 -Aminocyclohexanecarboxylic Acid (Compound II).. 79
3.3.3 Synthesis of l,4-Diamino-l,4-dicyanocyclohexane (Compound III). 80
3.3.4 Synthesis of 1,4-Diamino-1,4-cyclohexanedicarboxylic Acid
(Compound IV).................................................. 82
3.3.5 Synthesis of Methyl 1-Aminocyclohexanecarboxylate (Compound V)... 84
3.3.6 Attempted Synthesis of
1,4-Dimethyl-1,4-diaminocyclohexanedicarboxylate (Compound VI).... 85
3.3.7 Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic Acid
(Compound VII)................................................. 88
3.3.8 Synthesis of 1-Acetylaminocyclohexanecarboxylic Acid
(Compound VIII)................................................ 90
x


CO
.9 Synthesis of l,4-Diacetylamino-l,4-cyclohexanedicarboxylic Acid
(Compound IX)..................................................... 92
3.3.10 Synthesis of l-(9-fluorenylmethoxycarbonyl)aminocyclohexane
carboxylic Acid (Compound X)................................... 93
3.3.11 Attempted Synthesis of l,4-Di[(9-fluorenyl)amino]- 1,4-cyclohexane
dicarboxylic Acid (Compound XI)................................ 95
3.3.12 Synthesis of 1-Formylaminocyclohexanecarboxylic Acid
(Compound XII)................................................. 97
3.3.13 Synthesis of l,4-Diformylamino-l,4-cyclohexanedicarboxylic Acid
(Compound XIII)................................................ 98
3.3.14 Attempted Synthesis of 1-Trifluoroacetylaminocyclohexanecarboxylic
Acid (Compound XIV)............................................ 99
3.3.15 Attempted Synthesis of 1,4-Ditrifluoroacetylamino-1,4-cyclohexane
dicarboxylic Acid (Compound XV)................................ 102
3.3.16 Characterization of Diastereoisomers of Compound IV........... 103
4. Conclusion for Future Studies...................................... 105
References............................................................ 108
xi


FIGURES
Figure
1.11,4-Diamino-1,4-cyclohexanedicarboxylic Acid.......................... 1
1.2 Peptide Bonds........................................................ 2
1.3 Diketopiperazines as Linkage Bonds of a New Polymer.................. 4
1.4 Proposed Hydrogen Bond Formation between Strands of Polymer.......... 5
1.2.1.1 p-Nitrophenol, Pentachlorophenol, and Pentafluorophenol Esters... 8
1.2.1.2 Mixed Pivalic Anhydride.......................................... 8
1.2.1.3 Dicyclohexylcarbodiimide (DCC)................................... 9
1.2.2.1 The Benzyloxycarbonyl Group...................................... 11
1.2.2.2 Boc Anhydride.................................................... 11
1.2.2.3 Fmoc Group....................................................... 12
2.1.1 13C-NMR at 50 MHz of Compound I in D20............................. 26
2.1.2 13C-NMR at 50 MHz of Compound II in D20/NaOD....................... 28
2.2.1 Configurations of Compounds III and IV............................. 31
2.2.2 Conformations of cis-isomers of Compounds III and IV............... 32
2.2.3 Conformations of trans-isomers of Compounds III and IV............. 32
xii


2.2.5 More Stable Conformation of trans-isomer of Compound IV........
2.2.4 H-NMR at 200 MHz of Compound III in D20.......................
2.2.6 I3C-NMR at 50 MHz of Compound III in D20.......................
2.2.7 H-NMR at 200 MHz of Compound IV in D20/DC1....................
2.2.8 H-NMR at 200 MHz of Compound IV in D,0/NaOD...................
2.7.1 H-NMR at 200 MHz of Compound X in CDC13.......................
2.8.2 13C-NMR at 50 MHz of Compound X in CDC13.......................
2.8.1.1 H-NMR at 200 MHz of Compound XII in DzO/NaOD................
2.8.1.2 Configurations of Compound XII...............................
2.8.1.3 13C-NMR at 50 MHz of Compound XII in D,0/NaOD................
2.8.2.1 More Stable Conformer of the trans-isomer of Compound XIII...
2.8.2.2 The Three Possible Configurations of Compound XIII...........
2.8.2.4 13C-NMR at 50 MHz of Compound XIII in D20/NaOD...............
2.11.1 Polymeric Salt between the trans-Compound IV and Ca+2 Cations.
2.11.2 A Possible Structure between the cis-Compound IV and Ca+2 Cations
36
37
39
41
42
53
55
59
60
62
64
64
66
72
73


SCHEMES
Scheme
Scheme I. The Formation of a Peptide Bond..................................6
Scheme II. Activation of a Carboxylic Group by DCC......................... 10
Scheme III. Cleavage of Boc Group.......................................... 12
Scheme IV. Cleavage of Fmoc Group.......................................... 13
Scheme V. Synthesis of N-formyl Derivatives................................ 14
Scheme VI. Synthesis of NCAs by Treatment of a-Amino Acids with Phosgene ... 15
Scheme VII. Synthesis of NCAs by Treatment of Benzyloxyamino Derivatives with
SOCl2......................................................... 16
Scheme VIII. Polymerization via NCAs Formation.............................. 17
Scheme IX. Stepwise Condensations........................................... 20
Scheme X. Synthesis of Model Amino Acid......................................21
Scheme XI. Different Approaches for Derivatization of the Model Molecule... 22
Scheme XII. Synthesis of 1,4-Diamino- 1,4-cyclohexanedicarboxylic Acid.......23
Scheme XIII. Different Approaches for Derivatization of
1,4-Diamino-1,4-cyclohexanedicarboxylic Acid.................. 24
xiv


TABLES
Table
2.2.1 Conformational Energies of Some Substituents
xv


1. Introduction
1.1 Research Goal
The main goal of our research is the synthesis of 1,4-diamino-1,4-cyclohexane
dicarboxylic acid, a cyclic diamino acid (Fig. 1.1), and derivatives which can be
suitable for peptide synthesis. The monomer could be used both to bridge two
peptide chains and as a potential unit of a new polymer.
The synthesis of peptides has been challenging chemists since the beginning of the
century. Peptide molecules consist of chains of amino acids that are linked together
by amide (or peptide) bonds as shown in Fig. 1.2
Fig. 1.1 1,4-diamino-1,4-cyclohexanedicarboxylic acid.
1


II II II II
Fig. 1.2 Peptide bonds
The formation of a peptide bond between two amino acids occurs when the
amino group of one amino acid is acylated by the carboxyl group of the second
amino acid. This reaction is not spontaneous under mild conditions so one of these
groups must be activated. Every amino acid contains at least one amino group and
one carboxyl group, meaning that every one of them has the capacity of acting as an
acylating reactant (carboxyl group) or as the compound to be acylated (amino
group). To form a specific peptide bond, protection of the amino group of one
amino acid and the carboxyl group of the other amino acid is necessary, so these
two groups will not interfere with the coupling reaction.
Protecting groups should be chemically stable under the conditions of peptide bond
synthesis. They also need to reduce the activity of the amino and carboxyl groups to
which they are attached and, finally, they must be easily removed under mild
conditions when necessary.
2


The introduction of 1,4-diamino-1,4-cyclohexanedicarboxylic acid in the main chain
of a protein can introduce structural restrictions to the chain. These restrictions
may direct the possible folded conformations of the protein. Various studies
(13,14) have proven the capacity of our model compound (1-aminocyclohexane
carboxylic acid) to restrict the conformations of peptides in which it has been
included. These restrictions can be explained on basis of the semi-rigid chair
conformation adopted by the cyclohexane ring. The synthetic cycloaliphatic amino
acid 1,4-diamino-1,4-cyclohexanedicarboxylic acid could also substitute for a
disulfide bridge formed between two cystine residues of the protein chain. Two
major advantages could arise with the introduction of 1,4-diamino-1,4-
cyclohexanedicarboxylic acid into a peptide: the peptide could resist the hydrolysis
of a large number of peptidases, and the stability of this peptide could increase since
its tertiary structure is stabilized by the inert cyclohexane ring and not by the
disulfide bridges that could be reduced.
In addition to its incorporation in peptide analogues, there is an attractive potential
use for this cycloaliphatic amino acid. The compound 1,4-diamino-1,4-
cyclohexanedicarboxylic acid could be used as the monomer for a new polymer.
The units of this polymer are envisioned as linked together by formation of
diketopiperazines.


Diketopiperazines are six-membered heterocyclic rings containing two peptide
bonds, as shown in Fig. 1.3
Fig. 1.3 Diketopiperazines as linkage bonds of the new polymer
This polymeric diketopiperazine can potentially present interesting characteristics
due to its conformational restrictions. The polymer would consist of cyclohexane
rings alternating with diketopiperazines. The polymer chain would thus be an
alternating sequence of two conformationally constrained units, each of which
would contribute rigidness to the polymer. There is also the possibility of formation
of hydrogen bonds between the diketopiperazines of different polymer strands that
can also contribute rigidness to this polymer (Fig. 1.4)
4


o
o
o
II
II
II
II
C----NH
II
O
\ II
C---NH
" l
9 o
li
NC,
C----NH
II
O
ii
II
O
Fig. 1.4 Proposed hydrogen bond formation between strands of polymer
During this research, we successfully synthesized 1,4-diamino-1,4-cyclohexane
dicarboxylic acid. We were also able to protect the amino groups of our monomer
by both acetyl and formyl groups. The diacetylated monomer is not useful per se
since it will be very difficult to remove using the mild conditions demanded
following the initial polymerization process. However, this reaction gave us some
important information for similar reactions with more labile amino protecting
groups (e.g. trifluoroacetyl groups). The diformylated monomer is a good
derivative for the polymerization process since the formyl group is easily hydrolyzed
using mild acidic conditions. We failed to find a procedure to protect the carboxyl
groups of our diamino diacid. Further studies need to be done in this particular
matter.
5


1.2 Polymerization Strategies
Peptide synthesis has been studied since the beginning of the century. Synthetic
peptides are interesting molecules because they can act as biologically active
substances and also as models with which to study the chemical and physical
properties of proteins. Peptide synthesis began with the isolation and determination
of amino acids constituting proteins. The studies of Franz Hofmeister (1850-1922)
led to the present concept of peptide structure: Long chains of a-amino acids linked
to each other through amide bonds between carboxyl and amino groups (15). The
principal reaction in the synthesis of peptide chains is the acylation of the amino
group of an amino acid by the carboxyl group of a second amino acid with the
formation of an amide bond as shown in scheme I.
O
RfNhk + R2~C'X
0
ii
R2-C-N-R-! + HX
I
H
Scheme I. The formation of a peptide bond
6


1.2.1 Activation of the Carboxyl Group
To allow the nucleophilic attack of the amino group to form a peptide bond,
activation of the carboxyl group is necessary, because carboxylic acids simply form
salts with the amines at room temperature. Transformation of these salts into the
corresponding amides requires severe heating that can destroy amino acids. The
most common methods of activation for the carboxyl groups are the formation of
active esters, the formation of mixed anhydrides, or the use of coupling reagents.
These methods are described in the following sections.
1.2.1.1 Formation of Active Esters
Simple alkyl esters of carboxylic acids methyl, ethyl and benzyl can be reactive
enough to acylate amines for peptide bond formation. Also, these alkyl esters are
sufficiently stable toward the amine nucleophilic attack to be used as carboxyl
protecting groups. The method for alkyl ester formation was first developed by
Emil Fischer (16) as a procedure for amino acid separation. An increase on the
reactivity of the esters is achieved with the use of electron-withdrawing substituents.
Examples of this are p-nitrophenyl (17), pentachlorophenyl (29), and
pentafluorophenyl esters (30) shown in Fig. 1.2.1.1.
7


02N
O-C-Ri Cl
0
11
Cl Cl F F
-C-R3
0
u
(a)
(b)
(c)
Fig. 1.2.1.1 p-nitrophenyl (a), pentachlorophenyl (b), and pentafluorophenyl (c)
1.2.1.2 Formation of Mixed Anhydrides
Treatment of mixed anhydrides of carboxylic acids with amines is the simplest
method for peptide bond formation. However, mixed anhydrides have two similar
electrophilic sites and this can introduce a certain ambiguity to the peptide bond
formation. The selectivity of the nucleophilic attack can be improved by
introducing steric hindrance and inductive depression of electrophilicity in one side
of the mixed anhydride. Mixed anhydrides between the carboxylic acid and pivalic
acid are normally used for this purpose (Fig. 1.2.1.2).
esters.
Fig. 1.2.1.2 Mixed pivalic anhydride
8


1.2.1.3 Coupling Reagents
The introduction of carbodiimides (18), particularly dicyclohexyl carbodiimide
(Fig. 1.2.1.3.1), as reagents for the peptide bond formation was a major step for the
peptide synthesis.
Fig. 1.2.1.3 Dicyclohexylcarbodiimide (DCC)
The major advantage of DCC is that can be added to the mixture of the carboxyl
and amino component for peptide bond formation. Amine groups react very slowly
with DCC while carboxyl groups react rapidly with one of the double bonds of the
carbodiimide. The first step of the reaction mechanism is the addition of the
carboxyl group of one amino acid to the carbodiimide to give an O-acylisourea
intermediate. This intermediate is an active acylating agent and reacts readily with
nucleophiles. The second step of the mechanism is the nucleophilic attack on the
carbonyl carbon of the O-acylisourea by an amino group of a second amino acid to
form the tetrahedral intermediate. Finally, the decomposition of this intermediate
gives a linear dipeptide and N-N-dicyclohexylurea (Scheme II).
9


r.-C-OjH
R-N=C=N-R"
Rrc--
R-£QC=N-R"
.. 4
R H Ni"
*PJ..
'0-Cn-r2
l H
Ri
o
n
H
H
Scheme II. Activation of the carboxyl group of an amino acid by DCC
1.2.2 Protection of the Amine Group
For directed peptide coupling, the a-amino group of the carboxyl component
must be prevented from reacting with the activated carboxyl group. This is
accomplished by introduction of substituents that reduce the normal nucleophilicity
of the amine. The most common N-protecting groups present different sensitivities
toward different acidic environments during their cleavage process. Some of these
groups are also sensitive to treatment with alkalis and catalytic hydrogenation. A
brief description of the N-protecting groups used in the research is given in this
section.
1.2.2.1 The Benzyloxycarbonyl Group (Z group)
Bergmann and Zervas (19) synthesized the Z group (Fig. 1.2.2.1) as an amino
protecting component for peptide synthesis.
10


o
coc
H2
II
Fig. 1.2.2.1 Benzyloxycarbonyl Group
The Z-group is normally introduced in its acid chloride form. It reacts rapidly and in
high yield with an amino acid to form its N-benzyloxycarbonyl derivative. The
Z-group is resistant to mildly basic conditions so peptide esters can be hydrolyzed
without affecting the Z-group protecting the amino component. The classic
cleavage conditions for this N-protecting group are HBr/AcOH or catalytic
hydrogenolysis. Under these conditions, complete cleavage requires normally a few
minutes.
1.2.2.2 The t-Butoxycarbonyl Group (Boc-group)
This protecting amino group was synthesized simultaneously by Carpino (20) and
McKay and Albertson (21). The Boc group (Fig. 1.2.2.2) is normally introduced in
its active form as a symmetric anhydride.
CH3 o o ch3
ch3- c o- c- o c- o c ch3
II
ch3
ch3
Fig. 1.2.2.2 Boc anhydride
11


This group can be also removed by acid catalyzed hydrolysis. Boc-groups are
more sensitive toward acidic conditions than Z-groups and conditions for the
cleavage of Boc-groups do not affect the Z-groups. Boc removal is often carried
out in TFA/CH2C12 at room temperature. The Boc-group is removed as a
carbocation and in the process, carbon dioxide is also produced. The carbocation
is spontaneously converted to isobutene (Scheme III). Basic hydrolysis and
catalytic hydrogenation do not affect the Boc-group.
1.2.2.3 The 9-Fluorenylmethoxycarbonyl Group
(Fmoc-Group)
The Fmoc-group was introduced by Carpino and Han (22) as an amino
protecting group. The Fmoc-group (Fig. 1.2.2.3) is normally introduced in its
chloroformate form.
(CH3)2CFCH2 +C02 + +H3N C-OH
Rl R2
o
II
Scheme HI. Cleavage of Boc group
H2c'o-c-
II
o
Fig. 1.2.2.3 Fmoc-group
12


The Fmoc-group is very stable to acidic reagents but is cleaved rapidly under basic
conditions (20% piperidine in DMF). Cleavage of Fmoc with piperidine takes only
a few seconds at room temperature. The mechanism of cleavage is one type of 1,2-
elimination reaction abbreviated as Elcb (elimination unimolecular conjugate base).
The removal of the proton from the (3-carbon forms a carbanion
(dibenzocyclopentadienyl carbanion) that is stabilized by aromatic effects. This
carbanion is the conjugate base of the original Fmoc-derivative giving the name to
the elimination reaction. The carbanion decomposes to give the free amino acid and
carbon dioxide and dibenzofulvene as coproducts as shown in scheme IV.
002 + H2N cooh
Ri R2
Scheme IV. Cleavage of Fmoc group
13


1.2.2.4 The Formyl Group
Amines and amino acids can be converted to N-formyl derivatives by using a
mixture of acetic anhydride and formic acid. The reaction mechanism proceeds
through the mixed acetic-formic anhydride (Scheme V).
o
CH3-C-0-C-CH3
o
II
H-COH
0 o
II M
CH3-C-0-C-H + H3C-COOH
9 o
11 11
CH3-C-0-C-H +
K2-R1
o
II
CH3-C-OH
0
II ..
H-C-N-R1
H
Scheme V. Synthesis of N-formyl derivatives
Formyl groups are readily removed by treatment with 0.5 N hydrochloric acid in
methanol at room temperature according to Sheenan & Yang (8). Formyl groups
are sufficiently stable to alkali cleavage so that they are not affected by the cleavage
of peptide esters.
1.2.2.5 The Acetyl Group
The introduction of an acetyl group as an amino protecting component is not a
useful strategy. These groups cannot be removed using mild conditions. Important
information was collected from the acylation of our amino acids,- however.
14


1.3 Fragment Condensation versus
Stepwise Condensation
Peptide synthesis begins with amino acids. These units may be converted to
polymers in an undirected fashion (fragment condensation) or condensed by
addition of one residue at the time (stepwise condensation). A description of various
methods used for both types of condensations are given in this section.
1.3.1 Fragment Condensation.
Mixtures of peptides containing hundreds of amino acid units can be prepared by
polymerization in the laboratory. These mixtures may be called amino acid
polymers to distinguish them from the peptides produced by stepwise methods.
Free a-amino acids cannot be polymerized by heating to form the corresponding
polyamides because at that temperature, a-amino acids suffer degradative reactions.
The most common method for amino acid polymerization is the polymerization of
their corresponding N-carboxyanhydrides or NCAs (31). The NCAs of a-amino
acids can be synthesized by treatment of the amino acid with phosgene as shown in
scheme VI.
Scheme VI. Synthesis of NCAs by treatment of a-amino acids with phosgene
COOH
+
COCh
II
. o
15


NCAs can also be synthesized by treatment of benzyloxycarbonyl derivatives of the
corresponding amino acid with inorganic acid halides like SOCU as shown in
scheme VII, or by reaction with P205.
o
C6H5-CHrO-C-NHv^cOOH+ SOCfe
'R R"
O
II ..
CsHs-CHjO-C-N
O
it
C-CI + SO2 + HCI
'R RM
H2C C6H5
+ C6H5CH2CI
Scheme VII Synthesis of NCAs be treatment of benzyl oxyamino derivatives with
SOCl2
Polymerization of NCAs is initiated by nucleophilic attack of primary amines. The
nucleophilic attack to the carbonyl carbon corresponding to the original amino acid
originates the opening of the ring. The intermediate carbamic acid spontaneously
decarboxylates to generate a new amino group, and so on, as shown in scheme VIII
16


HI

C-
II
O
o=c
Scheme VIII. Polymerization via NCAs formation.
All the naturally occurring amino acids have been polymerized via their NCAs.
Yields are normally high and the reactions proceed at room temperature. Berlinguet
and Tailleur (23) synthesized the free dipeptide of 1-aminocyclopentanecarboxylic
acid by using the NCA of one amino acid condensed with the benzyl ester of the
other amino acid in the presence of DCC.
During manipulation of the free dipeptide, they discovered the strong tendency of
the free dipeptide to cyclize into the corresponding diketopiperazine when heated in
anhydrous conditions. These results show the possibility of our model molecule
(1-aminocyclohexanecarboxylic acid) to polymerize with the formation of
diketopiperazines.
A second method of polymerization is done via the unreactive esters of the
corresponding amino acids.
17


Pacsu and Wilson (32) reported the formation of various polyglycine methyl esters
that were obtained by condensation of their respective methyl and ethyl esters. As
mentioned in section 1.2, simple alkyl esters are reactive enough to acylate amine
groups for peptide bond formation.
1.3.2 Stepwise Condensation
I
Amino acids can be incorporated to an existing peptide in a directed fashion, one
amino acid at the time. In the early days of peptide synthesis, segment condensation
was the strategy used for their synthesis. This technique introduced racemization as
a frequent problem. A newer and now commonly used strategy for peptide
synthesis is the chain elongation from the C-terminal residue. This strategy was first
introduced by Bodanszky in 1960. This method is the best application for the active
esters of amino acids. Acylating reagents are derivatives from protected amino
acids. N-protected amino groups are cleaved to yield the free amino group for the
acetylating reaction toward the active ester of a second amino acid. Formation of
peptide bond between two chosen amino acids is then formed. This method permits
the directed formation of peptide bonds between determinate amino acids carrying
the minimal protection. Racemization problems also diminish with this method.
18


In this research, the monomer 1,4-diamino-1,4-cyclohexanedicarboxylic acid was
synthesized. Formyl and acetyl derivatives were also obtained.
The ultimate goal of this research is to polymerize this diamino diacid.
Diketopiperazines can be formed using two different approaches: (1) once the .
N-protecting groups are cleaved leaving the free amino groups, both amino groups
could be acylated forming diketopiperazines between monomers, or (2) only one of
the amino groups is acylated, forming a single peptide bond between monomers
(Scheme IX). Peptides, whose units are linked by single peptide bonds, may thus
have their unreacted esters and free amino groups alternating in the chain.
This type of polymer might be very interesting to obtain. It might be more flexible
than the polymer in which all the monomers are linked together by formation of
DKPs, with the possibility of being converted to the latter by heat treatment.
19


o
o
II
HN-----C
O
C----NH >-------' C-------NH
Jl II
0 O
DIKETOPIPERAZINE LINKS
HN-
\
ii
O
SINGLE PEPTIDE BOND LINKS
(2)
Scheme IX. Stepwise Condensations
20


2. Results and Discussions
The model amino acid, 1-aminocyclohexanecarboxylic acid, was synthesized from
cyclohexanone following the Strecker synthesis described by Cremlyn (2) as shown
in scheme X.
Scheme X. Synthesis of model amino acid
Derivatization of the model amino acid was successfully accomplished. Protection
of both the amino group and carboxylic group of the model amino acid were
achieved through different approaches. Scheme XI shows the different approaches
studied for derivatization of the model molecule.
21


o
1!
Scheme XL Different approaches for derivatization of the model molecule
22


The syntheses of 1,4-diamino-1,4-cyclohexanedicarboxylic acid and its derivatives
were attempted after the synthesis of the model amino acid and its derivatives were
mastered. The cycloaliphatic diamino diacid was synthesized from 1,4-
cyclohexadione following similar procedures used for Strecker synthesis described
by Cremlyn (2) as shown in Scheme XII.
Cvclohexanedione 1.4-Diaimno-l,4-dicyanocyclohexane Cycloaliphatic Diamino Diacid
Scheme XII. Synthesis of l,4-diamino-l,4-cyclohexanedicarboxylic acid
Several approaches were studied for the derivatization of 1,4-diamino-1,4-
cyclohexanedicarboxylic acid (Scheme XIII). Protection of the amino groups was
achieved by formylation and acetylation reactions. Several methods to protect the
carboxylic functions of the molecule were tried without success.
23


Compound VI
Scheme XIII. Different approaches for derivatization of 1,4-diamino-1,4-
cyclohexanedicarboxylic acid
24


2.1 Synthesis of 1-Amino-l-cyanocycIohexane (Compound I)
and 1-Amino cy do hexanecarboxvlic Acid (Compound II)
The model amino acid (Compound II) was synthesized using the Strecker method
described by Cremlyn (2). The first step entitled combining cyclohexanone with
ammonium chloride and sodium cyanide in a 50% water/methanol system yielding
1-aminocyanocyclohexane (Compound I). In the second step, the hydrolysis of
Compound I with concentrated HC1 solution converted the cyano group to a
carboxylic acid, yielding the model amino acid. This model experiment easily
produced a high yield product.
^-NMR spectra of Compounds I and II are not very useful in determining their
structure due to the complex set of peaks between 0.9-2.1 ppm given by the
cyclohexane protons. The assignment of the 13C-NMR spectrum of the cyano
intermediate (Fig. 2.1.1) is as follows: the resonance at 115.85 ppm corresponds to
the carbon of the cyano group, at 51.03 ppm corresponds to the alpha carbon of the
cyclohexane ring. The resonances at 31.88 ppm, 21.47 ppm and 20.37ppm
correspond to the beta, delta and gamma carbons of the cyclohexane ring
respectively.
25


.21.478


Compound II was synthesized by hydrolyzing of the cyano group to a carboxyl
group. 13C-NMR analysis of Compound II, using D20/NaOD as the solvent system,
confirmed the hydrolysis of the cyano group, as the peak at 115.85 ppm disappeared
and a new signal, further down field (183.88 ppm), was observed. This resonance
was assigned to the carbon of the more electronegative carboxylate function
(Fig. 2.1.2). Also, the resonance correponding to the alpha carbon of the
cyclohexane ring was shifted upfield to 56.54 ppm. This carbon is the most affected
by the electronegativity of the new carboxylate group. The beta carbon gives a
peak at 33.67 ppm, the delta carbon at 23.48 ppm and the gamma carbon at 20.13
ppm, respectively.
27


183.580
H2N\ ^COCT
Ca^
Cb^ ^Cb
to
oo
-iT-T-ir*r-rTr_|'T h-*"
160 140 120
Fig 2 1 2 ,3C-NMR spectrum at 50 MHz of Compound II in D20/NaOD.
i i i nTriTrncm-rr'rnTi
-23.67*


2.2 Synthesis of l,4-Diamino-l,4-dicyanocydohexane
(Compound HI) and l,4-Diamino-l,4-cyclohexanedicarboxyIic
Acid (Compound IV)
Synthesis of Compounds HI and IV was accomplished by the Strecker method.
The procedures were a modification of Cremlyns method, used to synthesize
1-aminocyclohexanecarboxylic acid, the model compound.
The synthesis of Compound III was easier than that of its analog, Compound I,
because the product conveniently precipitated from the aqueous methanol mixture
used for the reaction. Thus, the crude product was collected by filtration and
hydrogen chloride gas was not needed to obtain it. The crude product was easily
recrystallized from hot water. The procedures for the synthesis of Compound IV
were essentially identical to those used for the synthesis of the model amino acid
(Compound II). The hydrolysis of the dicyano intermediate was carried out in a two
step process. In the first step, Compound III was treated with concentrated HC1
solution to form the corresponding intermediate amide. In the second step, the
solution containing the amide was diluted and refluxed for 48 hours yielding
Compound IV.
To obtain Compound IV, the hydrolysis of the intermediate compound was carried
out in two steps. Any modification on the hydrolysis of Compound III gave
undesired products.
29


Compounds III and IV are polysubstituted cyclohexane rings. Sometimes,
cyclohexane rings can be found in two different conformations, the rigid chair and
the flexible boat. It was assumed that Compounds III and IV and all their
derivatives adopted only the less energetic chair conformation. There are two types
of bonds in the chair conformation, the axial bonds and the equatorial bonds.
During the chair inversion process, all the equatorial bonds become axial and and all
the axial bonds become equatorial. Chair inversion occurs rapidly at room
temperature. In Compounds III and IV, even though the geminal protons on each
carbon can occupy both the axial and the equatorial positions, they still experience
different chemical environments because the substituents on carbons one and four of
the rings are different. These pairs of protons are diastereotopic. Diastereotopic
hydrogens are generally distinct both spectroscopically and chemically. A distinct
NMR signal for each group of diastereotopic protons was thus expected.
The disubstituted Compounds III and IV also have two different configurations, the
trans and the cis as shown in Fig. 2.2.1
30


Trans-Compound III
Cis-Compound III
COOH
COOH
Trans-Compound IV Cis-Compound IV
Fig. 2.2.1 Configurations of Compounds III and IV
The more stable configuration is normally the one with the larger substituents
occupying equatorial positions. It was assumed that Compounds III and IV were
only present in their trans configurations with their larger substituent in the
equatorial positions. Due to the inversion of the ring process, the cis and the trans
isomers of Compounds III and IV present two different conformations. The two
possible conformations of the cis-isomers of III and IV are energetically equivalent
as shown in Fig. 2.2.2. Both of them have an amino group and a cyano or
carboxylic group occupying the equatorial positions.
31


Fig. 2.2.2 Conformations of cis-isomers of Compounds III and IV
However, the two conformations of the trans-isomers of Compounds III and IV are
not equivalent as shown in Fig. 2.2.3. One of them has two carboxylic or cyano
groups equatorial and the other has two amino groups equatorial. Therefore, the
two conformations of the trans-isomers are not present in equimolar quantities and
can be distinguished by NMR analysis.
Fig. 2.2.3 Conformations of trans-isomers of Compounds III and IV.
32


To determine what is likely to be the more stable conformation of the trans-
conformer of Compounds III and IV, the conformational energies (33) of each
substituent were studied. Table 2.2.1 presents some of the conformational energies
for the most common substituents of our molecules. These values of
conformational energies represent the difference on free energy between the
substituent occupying the more energetic axial position and the substituent
occupying the equatorial position.
Group
-CN
-nh2
-nh3+
-COOH
-COO'
-NHCOC6H5
Conformational Energy fKcal/molI
0.2
1.7
1.7-2.0
1.4
2.0
1.6
Table 2.2.1 Conformational energies of some substituents.
Based on the conformational energies, not only the preferred conformation, but also
the abundance of each conformer can be determined.


Making the reasonable assumption that the conformational energies of substituents
A and B are additive, the free energy change (AG) in a molecule will then be
(GA axial "t" Gg equatorial ^Ta equatorial Qb axial) (^' 1)
assuming that GAequatonal= GBcquatorial = 0, the free energy change of the process is
giving by
AG = (GAaxial Gb^ (2.2)
The formation of the two conformers of the trans-isomer occurs rapidly at room
temperature. It is an equilibrium process. The distribution of conformers is
calculated using
AG = RT InK (2.3)
the equation relating the equilibrium constant with the free energy change of the
process.
For the trans-isomer of Compound III, the predominant conformer is the one in
which the cyano groups are occupying axial positions. The much less abundant
conformer is the one in which the cyano groups are occupying equatorial positions.
The difference of energy between the less abundant and the more abundant
conformers is 3.0 Kcal/mol (1.7x2-0.2x2 Kcal/mol). The distribution then
corresponds to 99.4% axial-cyano conformer and 0.6% equatorial-cyano
conformer.
34


Calculations were performed for Compound IV in two different forms: the
deprotonated one with carboxylate and free amino groups, and the protonated one
with carboxylic acid and ammonium groups. When Compound IV is deprotonated,
the predominant conformer is the one with both carboxylate groups occupying
equatorial positions. The differences in energy between the less abundant
conformer and the more abundant conformer is only 0.6 Kcal/mol (1.7x2-1.4x2
Kcal/mol). The distribution corresponds to 64% carboxylate-equatorial conformer
and 36% carboxylate-axial conformer. When Compound IV is protonated, the
predominant conformer is the one in which both carboxylic groups are axial. The
energy difference between the less stable and the more stable conformers is also 0.6
Kcal/mol (2.0x2- 1.7x2 Kcal/mol). Therefore, the distribution also corresponds to
a 64:36 mixture, but in this case the carboxylate-axial conformer is predominant.
Analyzing the values obtained for the distribution of the different conformers, it was
expected that Compound III will only exist in its cyano-axial form (99.4%) and
Compound IV, protonated and deprotonated, will be a mixture of both conformers
in roughly 2:1 ratios. The distribution values obtained for the conformers of trans-
isomers of Compounds III and IV may help to explain the NMR spectra obtained
for these molecules. We have already seen, at the beginning of this section, that the
hydrogens attached to the cyclohexane ring in Compounds III and IV are
diastereotopic.
35


Diastereotopic hydrogens are spectroscopically distinguishable. Every proton of the
cyclohexane ring is expected to be coupled with two non-equivalent protons: one
geminal and one vicinal. The coupling constant (J) between two geminal protons is
larger (10-30 Hz) than the coupling constant between two vicinal protons, one axial
and the other equatorial (3-5 Hz). Therefore, the 'H-NMR spectra of Compounds
III and IV were expected to give two sets of doublet of doublets (eight peaks in
all).
The JH-NMR spectrum of Compound III showed only two doublets (Fig. 2.2.4).
One doublet at 2.26 and 2.21 ppm (J= 10 Hz) and the second doublet at 1.81 and
1.76 ppm (J= 10 Hz) respectively. Previously we have assumed that only the
trans-configuration of Compound III (the more stable one) is present. Calculations
indicate that only the conformer with both cyano groups occupying the axial
positions is significant (Fig.2.2.5)
Fig. 2.2.5 More stable conformation of trans-isomer of Compound III
36


-4
= Vy '
S-j >
Fig 2 2 4 spectrum at 200 MHz of Compound III in D20.


This conformer has equivalent axial protons antiperiplanar to cyano groups. Cyano
groups are electronegative. It is known (28) that the coupling constant between
vicinal protons diminishes by 1-2 Hz if one of the two coupled protons is
antiperiplanar to an electronegative group.
Since the normal value of the vicinal coupling constant between one axial proton
and one equatorial proton is 3-5 Hz, it is possible that the coupling between vicinal
protons is not seen in the spectrum, especially because the peaks are fairly broad.
Only the coupling between geminal protons seems to be present. The value of the
coupling constants of the two doublets is consistent with the average values
normally seen for geminal coupling.
The 13C-NMR spectrum of Compound III (Fig. 2.2.6) is interpreted as follows:
the resonance at 117.18 ppm corresponds to the cyano groups as seen in the 13C-
NMR spectrum of the model molecule (Compound I). The resonance at 48.59 ppm
corresponds to the Ca of cyclohexane ring. The resonance at 29.86 ppm
corresponds to the Cb of cyclohexane ring.
Compound IV was studied in its protonated and deprotonated form. The
distribution calculations, for the two forms of Compound IV described above,
assigned significant populations of the two possible conformers (64% vs. 36%). It
was assumed, based on the same explanation given for Compound III, that the
vicinal coupling between non-equivalent hydrogens are not seen in the ^-NMR
38


*
n
C=N
Cb^ ^Ca
-crH3N.c/Uc/ NH*CI
NSC
wt
'O


spectra of Compound IV. Only the coupling between non-equivalent geminal
hydrogens was recorded on the spectra. Both conformers of protonated Compound
IV exist in the mixture.
Hydrogens of carboxylic-axial conformer may experience slightly different
magnetic field than the hydrogens of carboxylic-equatorial conformer causing the
multiplet signal between 1.9 and 2.1 ppm in the ^-NMR spectrum. The coupling
constants for this multiplet are consistent (J= 9.6 Hz) with the average value for
geminal coupling constants (Fig. 2.2.7).
The 'H-NMR spectrum of deprotonated Compound IV (Fig. 2.2.8) was expected
to show a pattern of resonances similar to the one seen for the protonated form
because calculations determined a 36:64 ratio vs. 64:36 ratio between the two
conformers. Surprisingly, only two signals, resonating as doublets, were seen in the
spectrum. One of the doublets appearing at 1.31 and 1.26 ppm (J= 9.4 Hz) and the
second doublet appearing at 1.84 and 1.79 ppm (J= 10 Hz). Even though the
calculations predict the presence of both conformers, only one conformer seems to
be present in the basic solution.
The 13C-NMR spectrum of the protonated and deprotonated forms of Compound
IV are very similar showing only small differences in the peak positions. They cast
no light on the relative distribution of conformers.
40




Ha
'OOC
to
Fig. 2.2.8 H-NMR spectrum at 200 MHz of Compound IV in D20/NaOD


2.3 Activation of Carboxylic Function by Formation
of Methyl Ester: Synthesis of Methyl 1-Aminocyclohexanecarboxylate
(Compound V)
Amino acid esterification, first described by Fischer (8), is a standard method for
the synthesis of esters. This is a nucleophilic acyl substitution reaction where one
molecule of the Compound II reacts with an excess of methanol to form the methyl
ester of Compound II and one molecule of water in the presence of an acidic
catalyst. Kenner et al. (3) reported the synthesis of Compound V in 1965 following
Fischers method. In their procedures, the hydrochloride salt of the amino acid
methyl este'r was collected by filtration and recrystallized from methanol before the
methyl ester was obtained in a 52% yield. Here, neither the hydrochloride salt nor
the methyl ester were purified because Compound V was obtained as an oil. A
comparable yield of 53% for the crude product was obtained.
The :H-NMR (D20/DC1) spectrum obtained for Compound V was very similar to
the spectrum published by Paul et al. (13). The cyclohexane protons resonate
between 1.94 and 1.37 ppm and a new singlet for the methyl protons appears at 3.8
ppm.
The 13C-NMR for Compound V is similar to the 13C-NMR spectrum obtained for
1-aminocyclohexanecarboxylic acid (Compound II) with an extra peak at 50.40 ppm
corresponding to the methoxy carbon.
43


The resonances for the carbonyl carbon and Ca carbon of the cyclohexane ring are
shifted downfield in the spectrum of Compound V because these two groups are the
most affected ones by the electron density introduced by the methyl group.
44


2.4 Activation of Carboxylic Function by Formation of
Methyl Ester: Attempted Synthesis of Dimethyl
l,4-Diaminocyclohexane-l,4-dicarboxylate (Compound VI)
Synthesis of Compound VT was attempted following three modified Fischer
esterification procedures, without success. The major difficulty encountered during
these esterification reactions was the unpredictable behavior of the starting material
(Compound IV) in acidic conditions.
During the first reaction, the starting material was suspended in an excess of
anhydrous methanol. Bubbling hydrogen chloride through the suspension for a few
seconds dissolved Compound IV completely. As more hydrogen chloride was
passed into the clear solution, the starting material became insoluble again. After 10
minutes, at least 90% of the original amount used had precipitated from the
solution. The suspension was stirred at room temperature overnight but the starting
material never redissolved in the acidic solution.
The recovered starting material, from the first attempt, was collected by filtration
and used in a second esterification attempt. This time the hydrogen chloride was
passed into the suspension of Compound IV in methanol just long enough (a few
seconds) to obtain the clear solution. The solution was then stirred at room
temperature for five days. It was then taken to dryness and the residue suspended in
water.
45


The mixture was made basic with and extracted with an organic solvent. With this
extraction, the deprotonated amino acid ester was expected to be transferred to the
organic phase. No components were detected by any of the three visualization
methods when the organic fraction was analyzed by TLC. The aqueous fraction
was analyzed using the same system. Two concentrated, ninhydrin positive spots
with a Rf values of 0.00 and 0.36 were observed. Even thought the ninhydrin
positive spot with a Rf value of 0.36 was located where the methyl ester was
expected, the fact that this component was in the aqueous phase led us to discard it.
The second reaction was stopped at this point.
The third reaction was a variation of the Fischer esterification described by Kenner
et al. (3). This reaction was very similar to the first attempt. It was a more
aggressive procedure where hydrogen chloride was passed into the boiling mixture
of Compound IV in methanol for 1 hour and the suspension was then refluxed for
12 hours. After the heating period, the mixture was taken to dryness and the
residue partially dissolved in water. Basification of the mixture with 6 N NaOH
yielded a small amount of precipitate. 13C-NMR (CDC13) analysis of the precipitate
showed no signal for a methoxy carbon at 50 ppm.
46


2.5 Protection of Amino Function with a Boc Group:
Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic
Acid (Compound VII)
Synthesis of Compound VII was achieved following general procedures
described by Bodanszky et al. (34). The synthesis of Compound VII is achieved by
acylating the amino group of the starting material with a symmetric anhydride. The
major difficulty encountered during the synthesis arose from the solubility
differences between the two species involved in this reaction. On one hand, the
starting material, which is in its zwitterion form, needs to be converted to a
nucleophile by deprotonating the amine function. To do so, Compound II was
dissolved in an aqueous solution containing one equivalent of NaOH per molecule
of starting material. On the other hand, di-tert-butyldicarbonate is poorly soluble in
aqueous solutions but freely soluble in organic solvents like toluene or dioxane.
Therefore, the reaction was run in a two-phase solvent system. The heterogeneous
reaction mixture was vigorously stirred to allow the reaction to proceed.
When the toluene/water system was used, the synthesis of Compound VII was
obtained in a 22% yield. When dioxane/water system was used, the reaction failed.
47


The ^-NMR spectrum of Compound VII in CDC13 gave a multiplet resonance
between 1.98 and 1.61 assigned to the cyclohexane protons. The ^-NMR spectrum
of di-tert-butyldicarbonate (Aldrich) shows the tert-butyl group resonating as a
singlet at 1.40 ppm. In this spectrum, a similar resonance at 1.43 ppm was obtained.
However, the integration of this signal indicated the presence of hydrolyzed Boc
anhydride in the sample. The success of this acylation was proven when a broad
peak at 4.8 ppm integrating to 1 hydrogen appeared in the spectrum. This peak
corresponds to the amide hydrogen.
48


2.6 Protection of Amino Function with an Acetyl Group:
Synthesis of 1-Acetylaminocyclohexanecarboxylic Acid
(Compound Vill) and 1,4-Diacetylamino-l,4-cyclohexane
dicarboxylic Acid (Compound IX)
Synthesis of Compound VIII and Compound IX was accomplished following the
procedures described by Shotten and Bauman (11) with small yields of 33% and
14% respectively. Acetyl groups are not good amino protective functions. The
major disadvantage of using the acetyl groups as N-protecting groups is the
difficulty associated with removing them under mild conditions to avoid the
hydrolysis of peptide bonds. Therefore, the syntheses of Compounds VIII and IX
were done as a model experiments to gain information about the reaction conditions
for later attempts of N-acylation reactions with similar but more labile protecting
groups (e.g. trifluoroacetyl groups).
The acetylation was carried out using acetic anhydride which is only slowly
hydrolyzed by water, so it could be carried out in an aqueous medium.
The 'H-NMR spectrum of Compound VIII showed the expected acetyl methyl
protons as a singlet at 1.78 ppm, and the cyclohexane protons as a multiplet
between 1.10 and 1.86 ppm.
49


The 13C-NMR spectrum showed resonances at 171.39 ppm (amide carbon) and
20.63 ppm (methyl carbon) in addition to the ring and carboxyl carbon signals seen
in the spectrum of the parent compound.
The 'H-NMR spectrum of Compound IX indicated two resonances, singlets at
1.81 ppm and 1.80 ppm respectively. The integration value for the pair of signals
was six hydrogens. It appears that the protons of the two acetyl methyl groups
experience slightly different environments within the molecule. The cyclohexane
protons appear as a multiplet, possibly two doublets, starting at 1.26 and partially
overlapping the methyl signals. The 13C-NMR showed the expected five signals with
no apparent differences between the two acetyl groups.
50


2. 7 Protection of Amino Function with a Fmoc Group: Synthesis
of l-(9-Fluorenyl)aminocyclohexanecarboxylic Acid (Compound X)
and Attempted Synthesis of l,4-[Di-(9Fluorenyl)amino]-l,4-
cyclohexanedicarboxylic Acid (Compound XI)
Based on the results obtained for the N-acylation carried out using Boc anhydride
on our model amino acid (see section 2.5), a new approach was tested for the
synthesis of Compounds X and XI. The two-phase solvent system used for the
reaction was replaced by a solution containing the phase transfer agent 18-crown-6
ether.
Crown ethers are cyclic macromolecules containing the repeating unit (-0-CH2-
CH2)n with the ability to crown cations by complexation thus rendering them
soluble in organic solvents. The 18-crown-6 ether molecule has a high selectivity
for potassium cations because of the size of its cavity.
A dioxane solution was made containing two moles of KOH and 18-crown-6 per
mole of starting material. A.C.S. reagent grade potassium hydroxide contains 15%
of H20. On a molar basis, this is almost a two-to-one mixture of KOH and water.
The presence of these water molecules normally facilitates the dissolution of KOH
in the dioxane/18-crown-6 ether solution because the salt dissolves better in the
mixture if it is first dissolved in water.
51


Here, an extra 10 to 15% (v/v) volume of water was needed to dissolve the
potassium hydroxide. Half of the 18-crown-6/KOH mixture was used to initiate the
N-acylation of 1-aminocyclohexanecarboxylic acid by Fmoc-Cl. The second half of
the 18-crown-6/KOH mixture was then added dropwise to the reaction mixture to
scavenge protons released in the reaction. The basicity of the solution was
regulated to minimize the hydrolysis of unreacted Fmoc-Cl by the OH" anions
present in this second fraction. The order of addition of reactants is therefore
slightly different from the one used for the N-acylation of the model molecule by
Boc anhydride. In the ^-NMR spectrum of Compound X (Fig.2.7.1), the
resonances appearing as a multiplet from 7.18 to 7.69 ppm correspond to the
aromatic protons of the Fmoc group. It is a very clean signal. This multiplet was
taken as the reference signal, integrating as 8 hydrogens, to assign integration values
to the remaining spectrum. The multiplet between 1.78 and 1.18 ppm corresponds
to the cyclohexane protons This signal integrates as 13 hydrogens (instead of 10)
mostly because it contains ethyl acetate as an impurity (the singlet at 1.98 ppm and
the triplet at 1.25 ppm). The resonance at 3.7 ppm is also an impurity that is
believed to be water. There is also a broad resonance integrating to less than one
proton at 9.25 ppm corresponding to the carboxylic proton. The second broad
signal at 5.1 ppm corresponds to the amide proton. The integration value for this
resonance is also smaller than expected. Both of these are exchangeable protons.
52


I'ig. 2.7.1 'I I-NMR. spectrum at 200 MHz ofConipound X in CDCIj.
-i


The doublet at 4.33 ppm integrating for 2 hydrogens corresponds to the -CH2-
group. The signal at 4.14 ppm integrating to one hydrogen corresponds to the -
CH- proton of the Fmoc group, partially obscured by the ethyl acetate impurity.
The 13C-NMR (CDC13) spectrum of Compound X (Fig. 2.7.2) is interpreted as
follows: the resonance at 177.87 ppm corresponds to the carbonyl carbon of
carboxylic group. The signal at 154.17 ppm corresponds to the carbonyl carbon of
the amide. The resonances at 142.4, 139.85, 126.18, 125.6, 123.58, 118.44 ppm
correspond to the aromatic carbons of the Fmoc group. The resonance at 65.45
ppm corresponds to the -CH- carbon of the Fmoc group. The resonance at 45.66
ppm corresponds to the -CH2- carbon of the Fmoc group. The resonance at 57.45
ppm corresponds to the Ca of the cyclohexane ring. The resonances at 30.73, 23.49
and 19.53 ppm correspond to the Cb, Cd and Cc carbons of the cyclohexane ring
respectively. The signals at 68.86, 58.94 and 12.57 ppm are believed to be
impurities.
54


Fig 2.7.2 I:,C-NMR spectrum at 50 MHz of Compound X in CDC13.


Synthesis of l,4-[Di-(9-Fluorenyl)amino]-l,4-cyclohexanedicarboxylic acid
(Compound XI) was attempted following the procedures used for the synthesis of
Compound X. Unfortunately, the reaction time allowed for the synthesis of
Compound XI was only 10 hours. This shorter period may have been decisive for
the failure to obtain Compound X3. The ^-NMR (d6-DMSO) spectrum of the
product obtained shows the characteristic multiplet resonance between 7.86 and
7.28 ppm corresponding to the aromatic hydrogens of the Fmoc group. If the
N-acylation of the two amino groups of the starting material was completed, this
multiplet should integrate to 16 hydrogens. Instead, the signal integrates to 9
hydrogens. This integration value could indicate that only one amino group had
been protected during the process. As seen with the model compound (X), the
Fmoc group also gives two sets of coupled signals for the methylene and -CH-
protons. However, these resonances, normally found between 4.30 and 4.10 ppm,
are missing in the spectrum of the product. The characteristic resonance pattern of
the disubstituted cyclohexane protons is also absent in this !H-NMR spectrum. The
spectrum indicates that the product was not the N-acylated starting material but
possibly a by-product arising from chemical transformation of the Fmoc-Cl.
56


2.8 Protection of Amino Function with a Formyl Group:
Synthesis of l-Formylaminocyclohexanecarboxylic Acid
(Compound XII) and 1,4-Diformylamino-l,4-cyclohexane
dicarboxylic Acid (Compound XIII)
The N-formyl groups were introduced readily in Compounds II and IV by
formylation in the presence of acetic anhydride. Compound XII was synthesized
following the procedures described by Kenner, Sheppard and Preston (3). This
method is a modification of the procedures described by du Vigneaud (9) for
formylation reactions. Kenners procedure involves the use of lower temperature
during the reaction to minimize the danger of racemization when optically active
amino acids are involved. Neither of our starting materials, Compound II or
Compound IV, are optically active, so racemization was not a problem in our case.
Compound XIII was synthesized following the procedures described by du
Vigneaud (9) because the starting material, Compound IV was not soluble in
formic acid at room temperature. The reaction needed to be done using higher
temperatures than in du Vigneauds method.
The first step of the formylation reaction is presumably the formation of the
mixed anhydride between the formic acid and acetic anhydride. Later, the
nucleophilic attack of the amino group of the starting material on the formyl group
of the mixed anhydride yields the desired formyl derivative.
57


Both methods have a reported yield generally greater than 80% of theory. Yields of
56% and 47% were obtained for Compounds XII and XIII respectively. The
procedures called for a 98% formic acid. In our case, a 96% formic acid was used
to carry out the reactions. Perhaps, an improvement of the yield can be obtained if
a more concentrated formic acid is used for these formylation reactions.
2.8.1 Analysis of the NMR Spectra of 1-Formylaminocyclohexanecarboxylic
acid (Compound XII)
The assignment of the ^-NMR (D20/NaOD) spectrum of Compound XII
(Fig. 2.8.1.1) is as follows: the singlets at 7.76 and 7.87 ppm which together
integrate to one proton, correspond to the formyl proton of the trans and cis
conformations of Compound XII respectively. The assignment of these peaks is
explained further in this section. The multiplet between 1.09 and 1.81 ppm
integrating to 10 protons corresponds to the protons of the cyclohexane ring. The
signal at 4.64 ppm corresponds to the solvent (D20).
The amide bond, formed between the formyl and the amino group in Compound
XII, is planar and has a high rotational energy barrier. The amide bond does not
rotate freely in the molecule.
58


787


Therefore, Compound XII presents two diastereoisomers with respect to the
peptide bond*. In the cis configuration, the formyl proton and the cyclohexane ring
are on opposite sides of the amide bond. In the trans configuration, the formyl
proton and the cyclohexane ring are on the same side of the amide bond. Figure
2.8.1.2 shows the two possible diastereoisomers of Compound XII with respect to
the rigid amide bond.
Trans-configuration Cis-configuration
Fig. 2.8.1.2 The two possible diastereoisomers of Compound XII
It is necessary to clarify at this point that, based on the conformational energies, it is
assumed that the more stable conformer of Compound XII is the one where the
carboxylate group is occupying the equatorial position and the carboxamide group
occupies the axial position.
* Although the cis and trans configurations do exchange, they do not do so on the NMR time scale. For the
purpose of clarity in this discussion, we will refer to the cis and trans amide forms as if they were true
configurational isomers. We will use the term conformer to mean only rotations around normal sigma bonds.
60


The enthalpy differences between conformers is small (0.4 Kcal/mol) so the
Boltzmann distribution equation predicts a nearly equimolar mixture of both
conformers.
The formyl protons experience different chemical environments depending upon the
amide configuration of the individual molecules, and their resonances are then
different. We believe that the trans diastereoisomer is more stable than its cis
diastereoisomer. In the trans configuration, there are 1,3-diaxial interactions
between the formyl proton and the axial hydrogens of the cyclohexane ring. In the
cis configuration, the 1,3-diaxial interactions increase when the carbonyl group of
the resulting amide interacts with the axial protons of the cyclohexane ring. It is
possible that these interactions are of such magnitude that they will force the
resulting amide out of the cyclohexane ring. Presumably, the amide group will rest
between the carboxylate anion and the second carbon of the cyclohexane ring (that
is, gauche to the carboxylate group).
The !H-NMR spectrum also reveals that the formyl proton for the trans
diastereoisomer resonates at higher field than the formyl proton for the cis
diastereoisomer. This difference in resonances cannot be explained as a simple
inductive effect.
The assignment of 13C-NMR (D20/NaOD) spectrum of Compound XII (Fig.
2.8.1.3) is as follows: the resonance at 180.01 ppm corresponds to the carboxylate
carbon.
61


ISO.oil
Fig. 2.8.1.3 l3C-NM.R spectrum at 50 MHz of Compound XII in D20/Na0D.


Assigned on the same basis as the XH-NMR spectrum, the resonance at 166.05 ppm
corresponds to the formyl carbon of the cis diastereoisomer and the resonance at
161.55 ppm corresponds to the formyl carbon of the trans diastereoisomer. The
resonances at 59.94 and 59.20 ppm correspond to the Ca carbons of the cyclohexane
ring in the cis and trans stereoisomers respectively. The resonances at 32.03 and
30.60 ppm correspond to the Cb carbons of the cyclohexane ring in the cis and trans
conformations of Compound XII respectively. The resonances at 23.17 and 19.40
ppm correspond to the Cd and Cc carbons of the cyclohexane ring respectively. The
two latter carbons may also generate two slightly different peaks.
Based on the height of the signal for the formyl carbon in the 13C-NMR spectrum, a
80:20 distribution occurs between the trans and the cis diastereoisomers of
Compound XII.
2.8.2 Analysis of the NMR Spectra of
l,4-DiformyIamino-l,4-cyclohexanedicarboxylic
Acid (Compound XIH)
Based upon the discussion for the starting material (Compound IV) in section
2.2, the more stable conformation of the trans-isomer of Compound XU3 is believed
to be the one where the carboxylate groups occupy the equatorial position as shown
in Fig. 2.8.2.1
63


o
II
HNC-H
'OOC
COCT
H-C-NH
O
Fig. 2.8.2.1 More stable conformer of the trans-isomer of Compound XHI.
An enthalpy difference of 0.8 Kcal/mol was calculated between the two possible
conformers of trans-Compound XIII. The distribution is then 74% for the
conformer with the carboxylates equatorial and 26% for the conformer with the
carboxylates axial. It is not surprising that in the ^-NMR spectrum of Compound
XIII, the cyclohexane hydrogens present the same multiplet signal pattern seen for
the protonated molecules of Compound IV (Fig. 2.2.7).
Compound XIII has two formyl groups attached to its two amino groups, therefore,
it presents three different configurations (Fig. 2.8.2.2)

H
H
Cis-trans-XIll
H
O
All-trans-XII!
H
All -cis-XI 11
Fig. 2.8.2.2 The three possible configurations of Compound XIII
64


In the most stable configuration, the carbonyl carbon of the formyl group and the
cyclohexane ring are on opposite sides of the amide bonds. This is the all-trans
configuration. The second more stable configuration corresponds to the cis-trans
isomer, with one cis-amide bond and one trans-amide bond respectively. The least
stable configuration corresponds to the all-cis stereoisomer.
The ^H-NMR (D20/NaOD) spectrum of Compound XIII shows both cis and
trans amide protons but cannot distinguish among the three configurations.
The 13C-NMR (D20/NaOD) spectrum of Compound XIII (Fig. 2.8.2A) does
clearly show all three diastereoisomers. The resonances at 166.45 ppm and 162.07
ppm, for the amide carbonyl, and the Ca signals at 58.75 ppm and 58.12 ppm show
the expected cis and trans pairs. The Cb signal, however, is now more complex,
consisting of four peaks. The resonance at 28.56 ppm corresponds to the four Cb
carbons of the cyclohexane ring when Compound X3II presents the all-cis
configuration. The resonances at 26.91 and 25.25 ppm correspond to the Cb
carbons of the cyclohexane ring in the cis-trans configuration. The resonance at
25.63 ppm corresponds to the Cb carbons of the cyclohexane ring when Compound
XIII presents the all-trans configuration. A distribution of 80:20 was calculated for
the two conformers of the model, Compound XII, based on the height of the peaks
of its 13C-NMR spectrum.
65


o
II H
H-C-Nn /COO-
Ca
Cb^ ^Cb
ON
On
Cb\r /Cb
Ob
r*
'U OOC N-C-H
(4 H ||
O
U]
in
Fig. 2.8.2.3 ,3C-NMR spectmm at 50 MHz of Compound XIII in D20/NaOD.


If the substituents of the cyclohexane ring in Compound XIII are assumed to
behave as independent groups, an estimation of the distribution of possible
conformers can be calculated. A 64:32:4 distribution is obtained for the all-trans,
cis-trans, and all-cis isomers, respectively. When the height of the Cb peaks in
spectrum (Fig. 2.8.2.3) were measured, then base on the assignments made above,
a experimental distribution of 69:26:7 is obtained. The agreement between these
two distributions is very good.
67


2.9 Protection of Amino Function with a Trifluoroacetyl Group:
Attempted Synthesis of 1-Trifluoroacetylaminocyclohexane
carboxylic Acid (Compound XIV)
The synthesis of Compound XIV was attempted modifying the procedures
described by Weygand and Geiger (10). This method called for trifluoroacetic
anhydride as reagent. This compound was not available in our laboratory so it was
synthesized, as described in section 2.3.10, from trifluoroacetic acid and
dicyclohexylcarbodiimide (DCC).
Although DCC is better known as a coupling reagent for the formation of peptide
bonds, it has also been used for the preparation of esters and anhydrides. For the
synthesis of Compound XfV, the starting material (Compound II) was dissolved in
trifluoroacetic acid. To this clear solution, the freshly prepared trifluoroacetic
anhydride solution was added dropwise. The oily yellow residue, obtained after the
reaction mixture was taken to dryness, was divided into two equal fractions. The
first fraction was dissolved in ether and extracted with a basic aqueous solution.
The aqueous fraction was then acidified and extracted again with ether. The acyl
derivative (Compound XIV) was expected in the organic fraction. The analysis of
the organic fraction by TLC analysis in chloroform/methanol (9:1) system revealed
no spots. The procedure was stopped at this point.
68


The absence of a product corresponding to trifluoroacetylation of the amino group
led us to investigate the possible formation of the amino acid-trifluoroacetyl
anhydride. To test this idea, the second fraction was treated as if the mixed
anhydride had been formed. The yellow oil was dissolved in ethanol and an excess
of sodium ethoxide was then added with stirring. If the mixed anhydride was
present in solution, the nucleophilic ethoxide anion would attack the reactive
anhydride forming the corresponding the ethyl ester of the formic acid. The
reaction mixture was analyzed by TLC in chloroform/methanol (9:1) system. Only
a ninhydrin positive spot at the origin was found. The experiment was stopped at
this point.
69


2.10 Protection of Amino Function with a Trifluoroacetyl Group:
Attempted Synthesis of l,4-Ditrifluoroacetylamino-l,4-
cyclohexanedicarboxylic Acid (Compound XV)
The synthesis of the trifluoroacetylated model amino acid (Compound XIV) was
attempted following the procedures described by Weygand and Geiger (10). A
major obstacle did not allow us to use this procedure to synthesize Compound XV:
the starting material (Compound IV) was not soluble in trifluoroacetic acid.
Therefore, the synthesis of Compound XV was attempted following a modification
of the du Vigneauds procedure, specifically described for formylation reactions (9).
Compound IV was dissolved in formic acid. To this clear mixture, a solution of
trifluoroacetic anhydride in formic acid was added dropwise. The formation of the
mixed anhydride between the trifluoroacetic group and the formic group was
expected. The procedure was carried out and worked up in the same way as the
formylation reactions, but the ^-NMR of the resulting product only showed
resonances corresponding to the starting material. A possible cause for the failure of
this synthesis may have been the high concentration of water in the reaction mixture.
Water molecules are good nucleophiles and may have hydrolyzed a portion of the
trifluoroacetic anhydride molecules. The hydrolysis of the symmetric anhydride
would have prevented the synthesis of Compound XV.
70


2.11 A Search for Diastereoisomers of 1,4-Diamino-l,4-cyclohexane
carboxylic Acid (Compound IV)
Compound IV can exist as two diastereoisomers: the cis and the trans. As
discussed in section 2.2, the more stable configuration is the one where the more
bulky substituents are occupying the equatorial positions to diminish the 1,3-diaxial
interactions on the molecule. Both conformations of cis-1,4-diamino-1,4-
cyclohexanedicarboxylic acid have one amino and one carboxylic group axial. The
two cis-conformers are then energetically equivalent, present as an equimolar
mixture, and indistinguishable. The two trans-conformers of 1,4-diamino-1,4-
cyclohexanedicarboxylic acid are not equivalent. One conformer has its two
carboxylic groups occupying axial positions and the other conformer has the two
carboxylic groups equatorial. When Compound IV is deprotonated (free amine),
the more stable conformer of the trans-isomer is the one with the carboxylate
groups equatorial. A distribution of 64:36 was calculated for the carboxylate-
equatorial versus the carboxylate-axial conformers respectively based on the
standard conformational energies (section 2.2). When Compound IV is protonated
(hydrochloric salt), the more stable conformer of the trans-isomer is the one where
the two carboxylic groups are axial. A distribution of 64:36 was calculated for the
carboxylic-axial and carboxylic equatorial conformers respectively.
71


Therefore, the predominant conformer for the trans-isomer of Compound IV
depends upon the molecules protonation state, however both forms are well
represented in either case.
To study the diastereoisomerism of Compound IV, a chemical analysis was carried
out. This analysis tried to differentiate between the two possible diastereoisomers,
based on their solubility difference in the presence of the divalent cation Ca+2. To
deprotonate the two carboxylic groups of Compound IV, the diamino acid was
dissolved in a NaOH solution containing just two moles of NaOH per mole of
Compound IV. To this solution, one equivalent of CaCl, was added. Every
molecule of the cycloaliphatic diamino diacid is attracted by electrostatic
interactions to one ion of Ca"2 If the molecule is the trans diastereoisomer, the
following polymeric salt is likely to be formed (Fig.2.11.1):
Fig. 2.11.1 Polymeric salt between the trans-Compound IV and Ca+2 cations.
The polymeric salt between the Ca*2 and the trans-Compound IV is probably not
soluble in aqueous solution.
72


This type of rearrangement produces extensive precipitate in aqueous solution.
If the molecule is the cis-diastereoisomer, the polymeric salt may not be formed and,
instead, the following arrangement could be found (Fig.2.11.2):
Fig. 2.11.1 A possible structure formed between the cis-Compound IV and the
Ca+2 cations.
The structure formed between the cis-Compound IV and the Ca+2 cations could
have a different solubility from the polymeric salt formed between the trans-
Compound TV and the Ca+2 cations.
When the CaCl2 was added to the basic solution containing Compound IV, 90% of
the original amount of starting material came out of solution. The remaining 10%
of Compound IV in solution was then collected and treated again with CaCl2 to see
if the solubility of this fraction of starting material in the presence of calcium cations
was truly different.
A different behavior in solubility could indicate a different diastereoisomer in this
fraction of starting material. Unfortunately, treatment of the 10% fraction of
Compound IV with calcium cations also produced extensive precipitation.
73


This is not a good method to differentiate between the two possible
diastereoisomers of Compound IV.
74


3. Experimental
3.1 Reagents
Benzyl chloroformate (95%), 18-crown-6 ether (99.5%), dioxane (95%),
trifluoroacetic acid (99%), acetic anhydride (99%), trifluoroacetic anhydride (99%),
hydrogen chloride anhydrous (99+%), 1,4-cyclohexanedione (98%), cyclohexanone
(98%), formic acid (96%), di-tert-butyldicarbonate (98%) were all purchased from
Aldrich Chemical Company.
Ammonium chloride (95%), calcium hydroxide (95%), potassium hydroxide (85%,
pellets) were purchased from J.T. Baker.
Sodium cyanide (95%) was purchased from E.M. Science.
3.2 General Methods
3.2.1 Thin Layer Chromatography
The plates used for thin layer chromatography (TLC) were aluminum sheets pre-
coated with 0.2 mm layer of silica gel (UV254). Micropipetes with capacity of lpL
were used to spot the samples on the silica plates. Chromatograms were developed
either in chloroform/ methanol (9:1) or butanol/ acetic acid/ water (4:1:1) systems.
The solvent system used in each case is described specifically in the synthetic
procedures section of this paper. Once the chromatograms were developed, the
components of each sample were visualized following three methods in the
75


following order:
Method A: The plate was observed under ultraviolet light at 254 nm. The TLC
plates contain a flourescent material whose emission is quenched by
\
most solutes. After the solvent evaporates and the plate is viewed with
the UV lamp, the solute spots appear dark while the rest of the plate is
bright.
Method B: The plate was immersed in silica gel saturated with iodine for a brief
period. The I2 vapor is reversibly adsorbed on many substances,
particulary amides, and creates a dark spot wherever an adsorbing
compound is located.
Method C: The plate was sprayed with a 2% solution of ninhydrin in acetone and
then heated at about 80-90 C also for a brief period of time. Ninhydrin
is the reagent most commonly used for the detection of free amino
groups, yielding a blue color under the proper conditions.
Spots observed on the plate using methods A,B,C are described as UV positive,
iodine positive and ninhydrin positive, respectively.
3.2.2 Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) analyses were performed using a Varian
200 MHz spectrometer. Samples were dissolved in either D20, D20/DC1, D20/
NaOD, CDC13 or d6-DMSO for the analysis.
76


All solvents contained 0.03% of TMS as an internal reference. Proton and carbon
spectra were obtained for each sample. Chemical shifts are reported in ppm.
3.2.3 Melting Points
A mel-temp Laboratory device was used to determine the melting points of
individual samples. An open, thin-walled capillary tube (1mm 100 mm) was used
to contain the solid sample. All melting points are reported without corrections.
77


3.3 Synthetic Procedures
3.3.1 Synthesis of 1-Amino-l-cyanocyclohexane
(Compound 1)
Cyclohexanone (20 g, 0.2 mole) was dissolved in 250 mL of aqueous methanol.
Ammonium chloride (15 g, 0.4 mole) and sodium cyanide (15 g, 0.31 mole) were
added to the solution, which changed from colorless to intense burgundy after a
stirring period of 24 hours at room temperature. The reaction mixture was then
diluted with water (30 mL) and extracted with ether (500 mL). The extract was
washed with water, dried over MgS04 and concentrated to 300 mL. Dry hydrogen
chloride gas was bubbled through the concentrated organic mixture to obtain a
yellowish precipitate (17.0 g). Crystallization of the precipitate from absolute
ethanol gave 13.8 g of glistening white plates of Compound I. Yield: 54%. A
ninhydrin positive spot at a Rf value of 0.66 was obtained with TLC analysis of
Compound I in chloroform/methanol (9:1) system. Melting point: 173-180C (lit:
182C). 'H-NMR (D20): 6 (ppm) 2.1 (2 H, cyclohexane ring); 1.1-1.9 (6 H,
cyclohexane ring); 0.9-1.1 (2 H, cyclohexane ring). 13C-NMR (D20): 6 (ppm)
115.8 (CN); 51.03 (Ca cyclohexane ring); 31.88 (Cb cyclohexane ring); 21.48 (Cd
cyclohexane ring); 20.37 (Cc cyclohexane ring).
78


3.3.2 Synthesis of 1-Aminocyclohexanecarboxylic Acid
(Compound II)
Compound I (12.5 g, 0.1 mole) was suspended at 0C in concentrated
hydrochloric acid (12 M, 70 mL) and stirred at room temperature for 12 hours. The
acidic suspension was then diluted with 40 mL of water and boiled under reflux
overnight. After the refluxing period, rotary evaporation of the reaction mixture
yielded a yellowish residue. The crude product was suspended with stirring in 40
mL of water and a saturated solution of NajCOj was used to neutralize the
suspension. Compound II was then collected by filtration and dried over P2Os
overnight. Yield: 73%. Melting point >300C (lit.:350C in sealed tubes).
Compound II was analyzed by TLC in the chloroform/methanol (9:1) system. A
single, ninhydrin positive spot at the origin was obtained. 'H-NMR (D20/DC1):6
(ppm) 1.7-1.82 (2 H, cyclohexane protons); 1.4-1.7 (6 H, cyclohexane protons);
1.1-1.4 (2 H, cyclohexane protons). 13C-NMR (D20/Na0H):5 (ppm) 183.88
(carboxylate group); 56.54 (Ca cyclohexane ring); 33.67 (Cb cyclohexane ring);
23.48 (Cd cyclohexane ring); 20.13 (Cc cyclohexane ring).
79


3.3.3 Synthesis of l,4-Diamino-l,4-dicyanocyclohexane
(Compound IK)
The starting material 1,4-cyclohexanedione (7.46 g, 0.066 mole) was
dissolved in 90 mL of a 50% solution of aqueous methanol. Ammonium chloride
(7.08 g, 0.13 mole) was then added to this solution with stirring. Once the
ammonium chloride was totally dissolved, sodium cyanide (6.48 g, 0.13 mole) was
added and the reaction mixture turned a deep yellow color almost immediately. The
mixture was stirred at room temperature for 48 hours. A precipitate formed. It was
collected by filtration, washed with fresh aqueous methanol and partially dissolved
in hot water for its recrystallization. A large fraction of the original crude product
did not dissolve in hot water and was collected by filtration, dried, and weighed :
5.83 g. The filtrate was cooled at 0C overnight and the crystals that formed were
collected by filtration, dried, and weighed: 1.29 g Total yield: 7.12 g, 65%. Both
fractions, the insoluble fraction of the crude product and the fraction that
recrystallized from hot water, were analyzed by NMR. The 'H-NMR spectra of the
two fraction were identical indicating that both fractions corresponded to the same
compound. The melting point of Compound III in open capillary tubes was 190-
192C. Compound III was dissolved in acidic solution (pH 2) for TLC analysis in
chloroform/methanol (9:1) system. An iodine, ninhydrin positive spot with a Rf
80


value of 0.33 was obtained.
^-NMR (D20/DC1):6 (ppm) 2.28-2.24 (d, hydrogens of cyclohexane ring); 1.84-
1.79 (d, hydrogens of cyclohexane ring). The two doublets integrated to 1:1 ratio.
13C-NMR (D20/DC1):8 (ppm) 117.18 (CN); 48.59 (Cc carbons of cyclohexane
ring); 29.87 (Cp carbons of cyclohexane ring).
81


3.3.4 Synthesis of l,4-Diamino-l,4-CyclohexanedicarboxyIic
Acid (Compound IV)
Compound III (1.99 g, 0.012 mole) was suspended in 40 mL of 12 M
hydrochloric acid solution and cooled at 0C. The reaction mixture was then stirred
at room temperature for 48 hours. After the stirring period, the white suspension
was diluted with 30 mL of water and heated under reflux for another 48 hours. The
reaction mixture remained as a suspension changing its color to slightly yellow
during the heating process. The mixture was then taken to dryness with an oil
pump and the residue resuspended in 25 mL of fresh water. The acidic suspension
was neutralized with a saturated solution of sodium carbonate. The desired cyclic
diamino acid (Compound V) was collected by filtration, washed and dried over P205
in a desiccator. Yield: 62%. Melting point: >260C. Compound IV was dissolved
in acidic (pH 2) and basic (pH 10) solutions for TLC analysis in
chloroform/methanol (9:1) system. A single, UV, iodine, and ninhydrin positive
spot at the origin of the plate was obtained. H-NMR (D,0/NaOD):6 (ppm) 1.84-
1.79 (d, hydrogens of cyclohexane ring); 1.31-1.26 (d, hydrogens of cyclohexane
ring). The two doublets integrated to 1:1 ration.
13C-NMR (D20/NaOD): 5 (ppm) 183.27 (carboxylate carbon); 55.72 (Ca carbon of
cyclohexane ring); 29.31 (Cp carbon of cyclohexane ring).
82


'H-NMR (D20/DC1): 5 (ppm) 2.10-1.93 (m, hydrogens of cyclohexane ring).
13C-NMR (D20/DC1):8 (ppm) 170.82 (COOH); 55.19 (Cc carbons of cyclohexane
ring); 25.41 (Cp carbons of cyclohexane ring).
83


3.3.5 Synthesis of Methyl 1-Aminocyclohexanecarboxylate
(Compound V)
Dry hydrogen chloride was passed through a suspension of Compound II (1.51 g,
0.01 mole) in anhydrous methanol (150 mL) during 30 minutes. During this period
of time, the reaction mixture was placed in an ice bath to avoid evaporation of
methanol. The cloudy yellow solution was then sealed and stirred at room
temperature overnight. After the stirring process, the reaction mixture was filtered
and the clear filtrate taken to dryness using an oil pump. The residual product was
partially dissolved in 10 mL of water and the pH value of the mixture was raised to
10 with a saturated solution of sodium carbonate. The aqueous mixture was then
extracted with ethyl acetate (3x30 mL). The combined organic fractions were
dried with MgS04 and evaporated with an oil pump to obtain 1 mL of the oily
Compound V. Yield: 53%. TLC analysis of Compound V in chloroform/methanol
(9:1) system gave an elongated, UV, iodine and ninhydrin positive spot with a Rf
value of 0.55. H-NMR (D20/DC1): 6 (ppm) 3.672 (3H, CH30-); 1.94-1.92 (2H,
cyclohexane ring); 1.66-1.37 (8H, the rest of the hydrogens in the cyclohexane
ring). l3C-NMR ( d6-DMSO): 6 (ppm) 176.26 (carbonyl group); 55.51 (Ca
cyclohexane ring); 50.39 (CHsO-); 33.83 (Cp cyclohexane ring); 23.99 (C5
cyclohexane ring); 20.38 (Cy cyclohexane ring).
84


3.3.6 Attempted Synthesis of l,4-DimethyI-l,4-diaminocyclohexane
dicarboxylate (Compound VI)
Three attempts were used to synthesize Compound XI by Fischer esterification
(8). First attempt: Compound IV (0.4 g, 1.98 10'3 mole) was suspended in
anhydrous methanol (50 mL). The suspension was placed in an ice bath and dry
hydrogen chloride was passed into it for a few seconds to obtain a clear solution.
Further addition of hydrogen chloride gas (10 minutes) transformed the clear
solution back to a suspension that was then stirred at room temperature for 24
hours. After the stirring period, a white precipitate was collected by filtration,
dried, and weighed. Yield: 0.36 g (90%). 'H-NMR analysis of the white precipitate
(0.04 g) dissolved in 0.75 mL of D20/NaOD solution showed no singlet at 3.8 ppm
(CH3-CO-). Resonances at 1.26, 1.31, 1.79, and 1.84 ppm, corresponding to
Compound IV, were obtained respectively.
The filtrate was taken to dryness with an oil pump to obtain a yellow residue that
was redissolved in 10 mL of water. A 6 N solution of sodium hydroxide was used
to raise the pH value of this sample to 12. Neither precipitate or separation of
layers were seen at this pH value.
The basic solution was analyzed by TLC in the chloroform/methanol (9:1) system.
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SYNTHESIS OF 1,4-DIAMIN0-1,4-CYCLOHEXANEDICARBOXYLIC ACID, A NEW POLYMERIZATION MONOMER, AND ITS DERIVATIVES by Carolina Recuerda Wilson B.S., Universidad de Granada, 1994 A thesis submitted to the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Science Chemistry 1999

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This thesis for the Master of Science degree by Carolina Recuerda Wilson has been approved by Donald C. Zapien Susan M. Scheible Date

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Wilson, Carolina Recuerda (M.S., Chemistry) Synthesis of 1 4Diamino-1 ,4-cyclohexanedicarboxylic Acid, a New Polymerization Monomer, and its Derivatives Thesis directed by Professor Douglas F. Dyckes ABSTRACT The first part of the research is directed toward the synthesis and characterization of 1,4-diamino-1,4-cyclohexane dicarboxylic acid. This cyclic diamino acid was prepared by Strecker synthesis using 1,4-cyclohexanedione, ammonium chloride, and sodium cyanide as reagents. The hydrolysis of the dinitrile intermediate yielded the desired amino acid. The molecule could be potentially used as a polymerization monomer To be able to polymerize it, the molecule secondly needs to be derivatized protecting its amine and/or carboxylic groups. Derivatization of the cyclic diamino acid proved to be a more difficult task than was expected, due to the poor solubility of this compound in organic solvents and the steric hindrance of the ring. Synthesis of the diacetylated amino acid and the diformylated amino acid was successfully accomplished. The synthesis ofthe diacetylated derivative was carried ll1

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out to determine better conditions for the N-acylation reaction with the more labile trifluoroacetyl group. Unfortunately when tri.fluoroacetyl groups were used, the reaction failed. The diformylated derivative was obtained in good yield The yield of this reaction was better when more concentrated formic acid was used The synthesis of methyl esters by standard Fischer esterification was not successful. The present studies have shown the possibility of derivatization of our monomer. The finding of new organic solvent systems where the polymerization reactions can be carried out is a priority for future studies This abstract accurately represents the content of the candidate's thesis I recommend its publication. Douglas F. Dyckes IV

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DEDICATION To Roger for being the energy that keeps me going

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ACKNOWLEDGMENTS My special thanks to Professor Douglas Dyckes for his support, guidance and patience throughout this work which is the result of two years of gratifYing learning under his guidance I also wish to express my gratitude to Professor Donald Zapien and Professor Susan Scheible for their advice and assistance Thanks to mi familia for their support and love on the other side of the Atlantic Ocean (with occasional visits to Denver) Special thanks to Mario, Margarita, and Paco for their patience during their vacation Also thanks to the Ramirez the Wilson, the Carabellos the Kuta and the Cordova families, as well as, Mina Sumita and Mei-Chen Kuo for their help and encouragement.

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CONTENTS Figures .............. ....... .......... ... . .... ....... . ........ ........... . ........ ............ ........ xii Schemes ...... ........................................ ........ .... ...... ......... ..................... .... xiv Tables .. ..... ............ ............ ......... . ....... .......... ........... ......... ..... . .......... . .... xv Chapter 1. Introduction........ .......... ............... ....... .... .... ............ ...... ................. 1 1. 1 Research Goal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Polymerization Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.1 Activation ofthe Carboxyl Group ... ............. ....... .... ........... .... .... 7 1.2.1.1 Formation of Active Esters. ........... . .................................. .... 7 1.2.1.2 Formation of Mixed Anhydrides .. .. ... .... ..... ..... .............. .......... 8 1.2 .1.3 Coupling Reagents. .... ... .................... . ... ..... ...... ...... .... ..... ... 9 1.2.2 Protection ofthe.AJ:nineGroup........ ....... .... .... ..... . ..... .... ........ ..... 10 1.2.2 1 The Benzyloxycarbonyl Group (Z Group) . . . . . . . . .. .. . . . . . 10 1.2.2 2 The t-Butoxycarbonyl Group (Boc Group).. ...... ...................... 11 1.2.2.3 The 9-Flourenylmethoxycarbonyl Group (Fmoc Group)...... ...... 12 1.2 2.4 The Formyl Group.. .................. .... ........... .... ..... .... ......... ......... 14 Vll

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1.2.2.5 The Acetyl Group .... ......... .... ......... .... . . . .... ........ .... . .... ... .... .... 14 1.3 Fragment Condensation versus Stepwise Condensation.. ... .... ...... ......... 15 1 3. 1 Fragment Condensation......... . ............................. . ..... .... ............. . ... 15 1.3.2 Stepwise Condensation .... .... . 18 2. Results and Discussion .... ..... ... .... ..... . ............ . . ... .... .... ... .... ... . ...... ... ... 21 2 1 Synthesis of 1-Amino-cyanocyclohexane (Compound I) and 1-Aminocyclohexanecarboxylic Acid (Compoundll) . . . . . ...... .. . . ... ... 25 2.2 Synthesis of 1,4-Diamino-1,4-dicyanocylcohexane (Compound III) and 1, 4Diamino-1, 4-cyclohexanedicarboxylic Acid (Compound IV) . ..... .... ............ ....... . . ............. ..... .... .... . .............. ... 29 2 3 Activation of Carboxylic Function by Formation ofMethyl Ester : Synthesis ofMethyl1-Aminocyclohexane carboxylate (Compound V) . ... ......... . 43 2.4 Activation of Carboxylic Function by Formation ofMethyl Ester : Attempted Synthesis of Dimethyl 1, 4-Diaminocyclohexane-1, 4-dicarboxylate (Compound VI)............. ... . ..... . ...... ............ ... ... ........... . ...... ........ ... 45 2.5 Protection of Amino Function with a Boc Group: Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic Acid (Compound VII) ... .... ..... ....... ... . ... . ............... ... ....... ....... ..... .... ...... 47 2 6 Protection of Amino Function with an Acetyl Group: Synthesis of 1-Acetylaminocyclohexanecarboxylic Acid (Compound VIII) and 1 4-Diacetylamino-1,4-cyclohexanedicarboxylic Acid (Compound IX) ..... 46 Vlll

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2. 7 Protection of Amino Function with a Fmoc Group: Synthesis of 1-(9-Fluorenyl)aminocyclohexanecarboxylic Acid (Compound X) and Attempted Synthesis of 1, 4-[Di-(9-Fluorenyl)amino ]-1, 4-cyclohexane dicarboxylic Acid (Compound XI)........ ............. ... ... ........................ 51 2.8 Protection of Amino Function with a Formyl Group: Synthesis of 1-Formylaminocyclohexanecarboxylic Acid (Compound XII) and Synthesis of 1, 4-Diformylami no-1, 4-cyclohexanedicarboxylic Acid (Compound XIII). ........................ .......................... . ..... ........... ...... 57 2.8.1 Analysis of the NMR spectra of 1-Formylaminocyclohexanecarboxylic Acid.................. ...................................... ... ............ ...... ............... 58 2 8 2 Analysis of the NMR spectra of 1,4-Diformylamino-1,4-cyclohexane Dicarboxylic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 2.9 Protection of Amino Function with a Trifluoroacetyl Group: Attempted Synthesis of 1-Trifluoroacetylaminocyclohexanecarboxylic Acid (Compound XIV) ... ...... ....................... .............. .... ......... ......... . 68 2.10 Protection of Amino Group with a Trifluoroacetyl Group : Attempted Synthesis l, 4Ditrifluoroacetylamino-1, 4-cyclohexanedicarboxylic Acid (Compound XV) ....... ........................ . :.... ....... . . .... . ... ...... 70 2.11 A Search for Diastereoisomers of 1, 4Diarnino-1, 4-cyclohexane dicarboxylic Acid (Compound IV) ... ...... .... ......... ....... ........ ......... 71 lX

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3 Experimental. .... ....... ....... . . . .... .... ...... ..... ......... . ... .. ..................... . 7 5 3.1 Reagents . ...... .... ........... . ..... . ...... ... ........... ... .. ... . .......... . .... ..... .... . . 75 3.2 General Methods....... .... ... .... .... ........... ............. ......... ....... ........ ..... 75 3 2 1 Thin Layer Chromatography ....... .......... ..... ....... . . ........................... 75 3 2.2 Nuclear Magnetic Resonance ............................................. .......... . . . 76 3.2.3 Melting Points........ ... ............ .............. ... ..... ....... . ........ ..... ..... ....... ... 77 3.3 Synthetic Procedures........ ................. ... ... ..... . ........ . .............. ............ 78 3. 3 1 Synthesis of 1Amino-1-cyanocyclohe x ane (Compound I)...... ... ... ..... 78 3.3.2 Synthesis of 1-Aminocyclohexanecarbo xy lic Acid (Compound II). ...... 79 3 3 3 Synthesis of 1,4-Diamino-1 4-dicyanocyclohex ane (Compound III) ...... 80 3 3 4 Synthesis of 1 4Diamino-1 4-cyclohe x aned i carboxylic Acid (Compound IV) . ........ ...... ........ .... ............. .... ... .. ... ..... . . ....... ......... . 82 3.3 5 Synthesis ofMethyl 1 Aminocyclohexanecarboxylate ( Compound V) ... 84 3 3 6 Attempted Synthesis of (Compound VI) . . 85 3.3. 7 Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic Acid ( Compound VII) .... ........ ............... ........ .......... . ............. . ..... .... ...... 88 3. 3. 8 Synthesis of 1-Acetylaminocyclohe x anecarboxylic Acid (Compound VIII) . ................. ............. ...... ....... ......... ................ ........ 90 X

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3. 3. 9 Synthesis of I, 4Diacetylamino-1, 4-cyclohexanedicarboxylic Acid (Compound IX) ................................................................... ................ 92 3.3 .I 0 Synthesis of I-(9-fluorenylmethoxycarbonyl)aminocyclohexane carboxylic Acid (Compound X)..... .. ... ..... ............ ... .... . ................. 93 3 3 I1 Attempted Synthesis of 1,4-Di[(9-fluorenyl)amino]-1,4-cyclohexane dicarboxylic Acid (Compound XI). . ......... ........................................ 95 3 3 .12 Synthesis of 1-F ormylaminocyclohexanecarboxylic Acid (Compound XII). .... .............. ........................................ ..... ........ ..... 97 3.3.13 Synthesis of 1,4-Diformylamino-I,4-cyclohexanedicarboxylic Acid (Compound XIII) .... .... ................. .................................................... 98 3.3 .14 Attempted Synthesis of 1-Trifluoroacetylaminocyclohexanecarboxylic Acid (Compound XIV) ...... ....... ... ..................................... ...... .... .... 99 3. 3 .15 Attempted Synthesis of 1, 4-Ditrifluoroacetylamino-1, 4-cyclohexane dicarboxylic Acid (Compound XV) .... ........ . .... . ........................... I02 3 3.16 Characterization ofDiastereoisomers of Compound IV........... ... ...... 103 4 Conclusion for Future Studies. ..... ..... ........ ............. ... ........ ........ .... ..... . 105 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 08 XI

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FIGURES Figure 1.1 1,4-Diamino-1,4-cyclohexanedicarboxylic Acid...... .... ........... ......... ... .. ........ 1 1.2 Peptide Bonds .. . . . . .. . . . . .. . . . .. . . . . ..... .. ... .. . . ... . .. . . . . . . . . . . . . .. . .. 2 1.3 Diketopiperazines as Linkage Bonds of a New Polymer. .. .. . . . .. . . . .. . .. . . 4 1.4 Proposed Hydrogen Bond Formation between Strands ofPolymer ...... ........... 5 1.2 .1.1 p-Nitrophenol, Pentachlorophenol, and Pentafluorophenol Esters . . . . . . 8 1 2.1.2 Mixed Pivalic Anhydride .. . . . . . . .. . . . . . . . . . . . . . . . . .. . . . .. . .. .. .. . . . 8 1.2.1.3 Dicyclohexylcarbodiimide (DCC) .. .. .. .. .. .. .. .. .. .. ... .. .. ...... ............................ 9 1.2 2.1 The Benzyloxycarbonyl Group .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 11 1.2 2.2 Boc Anhydride . . . . . . . . .. .. . . .. .. . . . .. . .. . .. .. ... . ... .. . ... .. .... . . . . .. .. 11 1.2 2 3 Fmoc Group........ ................... ..... ... ....... . ............ ........ ........ ... ...... .... .... 12 2 1 1 13C-N1vlR at 50 :tv1Hz of Compound I in D20 .... ... .. . ...... . .. ... . .. .. . . ........ 26 2.1.2 13C-N1vlR at 50 :MH.z of Compound II in D20/NaOD ... ............. ......... ....... 28 2.2.1 Configurations of Compounds III and IV .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 31 2.2.2 Conformations of cis-isomers of Compounds III and IV............................. 32 2.2.3 Conformations of trans-isomers of Compounds III and IV........... ....... ....... 32 Xll

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2.2 5 More Stable Confonnation of trans-isomer of Compound IV. .... . ... . ......... 36 2.2.4 1H-NMR at 200 l\1Hz of Compound III in D20 ..... ........ .... .... ... ...... .... .... . 37 2 2 6 13C-NMR at 50 MHz of Compound III in D20 ... .... ....... ... . ................ . .... 39 2 2 7 1H-NMR at 200 MHz of Compound IV in D20/DC1 . ....... ... . .... ... ...... ... : ... 41 2 2.8 1H-NMR at 200 MHz of Compound IV in D20/Na0D .. .. ... . . ... ...... . ...... ... 42 2 .7.11H-NMR at 200 MHz of Compound X in CDCI3 ............................ .......... 53 2 8 2 13C-NMR at 50 MHz ofCompound X in CDC13 ......................... ......... ....... 55 2.8.1.11H-NMR at 200 MHz of Compound XII in D20/NaOD ......... .... ....... . . 59 2.8 1.2 Configurations ofCompound XII.... ... .... ...... ... ........... ....... .................. ... 60 2.8.1.3 13C-NMR at 50 MHz of Compound XII in D20/Na0D ... ......... . ... .......... 62 2.8 .2.1 More Stable Confonner ofthe trans-isomer ofCompound XIII ..... ..... . 64 2 8 2.2 The Three Possible Configurations of Compound XIII........ .... ........... . ... 64 2.8 2.4 13C-NMR at 50 MHz of Compound XIII in D20/NaOD ................. ......... 66 2.11.1 Polymeric Salt between the trans-Compound IV and Ca+2 Cations .. ... ... 72 2 11.2 A Possible Structure between the cis-Compound IV and Ca+2 Cations .... ... 73 Xlll

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SCHEMES Scheme Scheme I. The Formation of a Peptide Bond .... ... ..... ............ .... ... :. . . . . . . . . . . . 6 Scheme II. Activation of a Carboxylic Group by DCC . . . . . . . . . . . . . . . . . . . . . 10 Scheme III. Cleavage ofBoc Group ...... ...... . . ....... ...... ... ..... ........... .... ..... .... ..... 12 Scheme IV. Cleavage ofFmoc Group ...... .. ................. . . ........ ........ ..... ...... . . ..... 13 Scheme V. Synthesis ofN-formyl Derivatives .... . ..... .. .... ....... ... ................ .. . .. . . 14 Scheme VI. Synthesis ofNCAs by Treatment of a:Amino Acids with Phosgene ... 15 Scheme VII. Synthesis ofNCAs by Treatment ofBenzyloxyamino Derivatives with SOC12 . ........ ........... . . ....... .... ...... .......... ................... ... ........ ..... 16 Scheme VIII. Polymerization via NCAs Formation ........ . . ......... .. ....... ... .... ...... 17 Scheme IX. Stepwise Condensations . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. 20 Scheme X. Synthesis ofModel Amino Acid .... . ... .......... .. . ..... ........ ..... ..... ... ... 21 Scheme XI. Different Approaches for Derivatization of the Model Molecule....... . 22 Scheme XII. Synthesis of 1,4-Diamino-1,4-cyclohexanedicarboxylic Acid ..... . ..... 23 Scheme XIII. Different Approaches for Derivatization of 1, 4-Diamino-1, 4-cyclohexanedicarboxylic Acid . . . . . . . . . . . . . . . 24 XIV

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TABLES Table 2 2 1 Conformational Energies of Some Substituents . . . .. . . . . . . . . . . . . . .. ... . 33 XV

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1. Introduction 1.1 Research Goal The main goal of our research is the synthesis of I, 4-diamino-I 4-cyclohexane dicarboxylic acid, a cyclic diamino acid (Fig. I l ), and derivatives which can be suitable for peptide synthesis. The monomer could be used both to bridge two peptide chains and as a potential unit of a new polymer Fig. I.l I 4-diamino-I, 4-cyclohexanedicarboxylic acid. The synthesis of peptides has been challenging chemists since the beginning of the century Peptide molecules consist of chains of amino acids that are linked together by amide (or peptide) bonds as shown in Fig. 1.2

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0 0 0 0 II 11 II II -----HN x--HN yc-HN ')(C-HN :x:--R1 R2 'R4 \6 R7 a Fig 1.2 Peptide bonds The formation of a peptide bond between two amino acids occurs when the amino group of one amino acid is acylated by the carboxyl group of the second amino acid. This reaction is not spontaneous under mild conditions so one of these groups must be activated. Every amino acid contains at least one amino group and one carboxyl group, meaning that every one of them has the capacity of acting as an acylating reactant (carboxyl group) or as the compound to be acylated (amino group). To form a specific peptide bond, protection of the amino group of one amino acid and the carboxyl group of the other amino acid is necessary, so these two groups will not interfere with the coupling reaction. Protecting groups should be chemically stable under the conditions of peptide bond synthesis. They also need to reduce the activity of the amino and carboxyl groups to which they are attached and, finally, they must be easily removed under mild conditions when necessary 2

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The introduction of 1, 4-diamino-1, 4-cyclohexanedicarboxylic acid in the main chain of a protein can introduce structural restrictions to the chain. These restrictions may direct the possible folded conformations of the protein. Various studies ( 13, 14) have proven the capacity of our model compound ( 1-aminocyclohexane carboxylic acid) to restrict the conformations of peptides in which it has been included These restrictions can be explained on basis of the semi-rigid chair conformation adopted by the cyclohexane ring. The synthetic cycloaliphatic amino acid 1, 4-diamino-1, 4-cyclohexanedicarboxylic acid could also substitute for a disulfide bridge formed between two cystine residues of the protein chain. Two major advantages could arise with the introduction of 1,4-diamino-1,4cyclohexanedicarboxylic acid into a peptide: the peptide could resist the hydrolysis of a large number of peptidases, and the stability of this peptide could increase since its tertiary structure is stabilized by the inert cyclohexane ring and not by the disulfide bridges that could be reduced. In addition to its incorporation in peptide analogues, there is an attractive potential use for this cycloaliphatic amino acid. The compound 1,4-diamino-1,4cyclohexanedicarboxylic acid could be used as the monomer for a new polymer. The units of this polymer are envisioned as linked together by formation of diketopiperazines. 3

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Diketopiperazines are six-membered heterocyclic rings containing two peptide bonds, as shown in Fig.l.3 0 0 0 II II 11 II II 0 0 0 Fig.l.3 Diketopiperazines as linkage bonds of the new polymer This polymeric diketopiperazine can potentially present interesting characteristics due to its conformational restrictions The polymer would consist of cyclohexane rings alternating with diketopiperazines The polymer chain would thus be an alternating sequence of two confonnationally constrained units each of which would contribute rigidness to the polymer There is also the possibility of formation of hydrogen bonds between the diketopiperazines of different polymer strands that can also contribute rigidness to this polymer (Fig 1 4) 4

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0 0 0 " -II 0 I 11 I 0 Q I 0 0 0 " 0 0 0 Fig. I 4 Proposed hydrogen bond fonnation between strands of polymer During this research, we successfully synthesized 1, 4-diamino-1, 4-cyclohexane dicarboxylic acid. We were also able to protect the amino groups of our monomer by both acetyl and fonnyl groups. The diacetylated monomer is not useful per se since it will be very difficult to remove using the mild conditions demanded following the initial polymerization process. However, this reaction gave us some important infonnation for similar reactions with more labile amino protecting groups (e.g. trifluoroacetyl groups). The difonnylated monomer is a good derivative for the polymerization process since the fonnyl group is easily hydrolyzed using mild acidic conditions. We failed to find a procedure to protect the carboxyl groups of our diamino diacid. Further studies need to be done in this particular matter. 5

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1.2 Polymerization Strategies Peptide synthesis has been studied since the beginning of the century. Synthetic peptides are interesting molecules because they can act as biologically active substances and also as models with which to study the chemical and physical properties of proteins Peptide synthesis began with the isolation and determination of amino acids constituting proteins. The studies ofF ranz Hofineister ( 1850-1922) led to the present concept of peptide structure: "Long chains of a-amino acids linked to each other through amide bonds between carboxyl and amino groups" (15). The principal reaction in the synthesis of peptide chains is the acylation of the amino group of an amino acid by the carboxyl group of a second amino acid with the formation of an amide bond as shown in scheme I. 0 II R2-C-N-R + HX I 1 H Scheme I. The formation of a peptide bond 6

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1.2.1 Activation of the Carboxyl Group To allow the nucleophilic attack of the amino group to form a peptide bond, activation of the carboxyl group is necessary, because carboxylic acids simply form salts with the amines at room temperature Transformation of these salts into the corresponding amides requires severe heating that can destroy amino acids The most common methods of activation for the carboxyl groups are the formation of active esters, the formation of mixed anhydrides-, or the use of coupling reagents. These methods are described in the following sections. 1.2.1.1 Formation of Active Esters Simple alkyl esters of carboxylic acids methyl ethyl and benzyl can be reactive enough to acylate amines for peptide bond formation Also, these alkyl esters are sufficiently stable toward the amine nucleophilic attack to be used as carboxyl protecting groups. The method for alkyl ester formation was first developed by Emil Fischer (16) as a procedure for amino acid separation. An increase on the reactivity of the esters is achieved with the use of electron-withdrawing substituents Examples ofthis are p-nitrophenyl (17), pentachlorophenyl (29), and pentafluorophenyl esters (30) shown in Fig 1.2.1.1. 7

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Cl Cl Cl (a) (b) F F (c) 0 II -C-Rs Fig 1.2.1.1 p-nitrophenyl (a) pentachlorophenyl (b), and pentafluorophenyl (c) esters. 1.2.1.2 Formation of Mixed Anhydrides Treatment of mixed anhydrides of carboxylic acids with amines is the simplest method for peptide bond formation However, mixed anhydrides have two similar electrophilic sites and this can introduce a certain ambiguity to the peptide bond formation. The selectivity of the nucleophilic attack can be improved by introducing steric hindrance and inductive depression of electrophilicity in one side of the mixed anhydride Mixed anhydrides between the carboxylic acid and pivalic acid are normally used for this purpose (Fig 1.2 1 2). o o II II I I Fig. 1.2 1 2 Mixed pivalic anhydride 8

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1.2.1.3 Coupling Reagents The introduction of carbodiimides (18), particularly dicyclohexyl carbodiimide (Fig 1 2 1 3 .1 ), as reagents for the peptide bond formation was a major step for the peptide synthesis. Fig. 1.2.1.3 Dicyclohexylcarbodiimide (DCC) The major advantage ofDCC is that can be added to the mixture of the carboxyl and amino component for peptide bond formation Amine groups react very slowly with DCC while carboxyl groups react rapidly with one of the double bonds of the carbodiimide. The first step of the reaction mechanism is the addition of the carboxyl group of one amino acid to the carbodiimide to give an 0-acylisourea intermediate. This intermediate is an active acylating agent and reacts readily with nucleophiles The second step of the mechanism is the nucleophilic attack on the carbonyl carbon of the 0-acylisourea by an amino group of a second amino acid to form the tetrahedral intermediate. Finally, the decomposition ofthis intermediate gives a linear dipeptide and N-N'-dicyclohexylurea (Scheme II) 9

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0 .. II f R -c-O-H + R'-N=C=N-R" 1 :.,; 0 II/ '?' .. R 1-c-o + .. R'-N-C=tf-R" H I .. -o-c-N-R2 I H R1 0 II II R1-C-N-R2 + H R'-N-C-N-R" H H Scheme II. Activation of the carboxyl group of an amino acid by DCC 1.2.2 Protection of the Amine Group For directed peptide coupling the a-amino group of the carboxyl component must be prevented from reacting with the activated carboxyl group. This is accomplished by introduction of substituents that reduce the normal nucleophilicity of the amine. The most common N-protecting groups present different sensitivities toward different acidic environments during their cleavage process. Some of these groups are also sensitive to treatment with alkalis and catalytic hydrogenation. A brief description of theN-protecting groups used in the research is given in this section 1.2.2.1 The Benzyloxycarbonyl Group (Z group) Bergmann and Zervas ( 19) synthesized the Z group (Fig 1.2.21) as an amino protecting component for peptide synthesis. 10

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0 H2 Fig. 1.2.2 1 Benzyloxycarbonyl Group The Z-group is normally introduced in its acid chloride form. It reacts rapidly and in high yield with an amino acid to form its N-benzyloxycarbonyl derivative. The Z-group is resistant to mildly basic conditions so peptide esters can be hydrolyzed without affecting the Z-group protecting the amino component. The classic cleavage conditions for this N-protecting group are HBr/AcOH or catalytic hydrogenolysis. Under these conditions, complete cleavage requires normally a few minutes. 1.2.2.2 The t-Butoxycarbonyl Group (Boc-group) This protecting amino group was synthesized simultaneously by Carpino (20) and McKay and Albertson (21 ). The Boc group (Fig. 1.2.2 2) is normally introduced in its active form as a symmetric anhydride Fig. 1.2.2 2 Boc anhydride 11

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This group can be also removed by acid catalyzed hydrolysis. Bee-groups are more sensitive toward acidic conditions than Z-groups and conditions for the cleavage ofBoc-groups do not affect the Z-groups. Boc removal is often carried out in TF A/CH2Cl2 at room temperature. The Boc-group is removed as a carbocation and in the process, carbon dioxide is also produced. The carbocation is spontaneously converted to isobutene (Scheme III). Basic hydrolysis and catalytic hydrogenation do not affect the Boc-group. 0 II (CH3)2c:;::CH2 + + +H3N)(C-OH R1 R2 Scheme III. Cleavage ofBoc group 1.2.2.3 The 9-Fluorenylmethoxycarbonyl Group (Fmoc-Group) The Fmoc-group was introduced by Carpino and Han (22) as an amino protecting group The Fmoc-group (Fig. 1.2.2.3) is normally introduced in its chloroformate form Fig. 1.2 2.3 Fmoc-group 12

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The Fmoc-group is very stable to acidic reagents but is cleaved rapidly under basic conditions (20% piperidine in DMF). Cleavage ofFmoc with piperidine takes only a few seconds at room temperature The mechanism of cleavage is one type of 1 ,2elimination reaction abbreviated as E 1 cb (elimination unimolecular conjugate base) The removal of the proton from the forms a carbanion (dibenzocyclopentadienyl carbanion) that is stabilized by aromatic effects. This carbanion is the conjugate base of the original Fmoc-derivative giving the name to the elimination reaction. The carbanion decomposes to give the free amino acid and carbon dioxide and dibenzofulvene as coproducts as shown in scheme IV. + H2NXCOOH R1 R2 /COOH "L II A 0 R1 R2 Dibenzocyclopentadienyl carbanion + Dibenzofulvene Scheme IV Cleavage ofFmoc group 13

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1.2.2.4 The Formyl Group Amines and amino acids can be converted to Nformyl derivatives by using a mixture of acetic anhydride and formic acid The reaction mechanism proceeds through the mixed acetic-formic anhydride (Scheme V). 0 II H-C-OH t> CH3C-OC-CH3 + 0 0 II II + ri3C-COOH Scheme V. Synthesis ofN-formyl derivatives Formyl groups are readily removed by treatment with 0 5 N hydrochloric acid in methanol at room temperature according to Sheenan & Yang (8). Formyl groups are sufficiently stable to alkali cleavage so that they are not affected by the cleavage of peptide esters 1.2.2.5 The Acetyl Group The introduction of an acetyl group as an amino protecting component is not a useful strategy. These groups cannot be removed using mild conditions. Important information was collected from the acylation of our amino acids,however. 14

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1.3 Fragment Condensation versus Stepwise Condensation Peptide synthesis begins with amino acids These units may be converted to polymers in an undirected fashion (fragment condensation) or condensed by addition of one residue at the time (stepwise condensation) A description of various methods used for types of condensations are given in this section. 1.3.1 Fragment Condensation. Mixtures of peptides containing hundreds of amino acid units can be prepared by polymerization in the laboratory These mixtures may be called amino acid polymers to distinguish them from the peptides produced by stepwise methods Free a-amino acids cannot be polymerized by heating to form the corresponding polyamides because at that temperature a-amino acids suffer degradative reactions The most common method for amino acid polymerization is the polymerization of their corresponding N-carboxyanhydrides or NCAs (31 ) The NCAs of a-amino acids can be synthesized by treatment of the amino acid with phosgene as shown in scheme VI 'R R" + 'R 0 "R+-< HN, /0 c II .0 Scheme VI Synthesis ofNCAs by treatment of a-amino acids with phosgene 15

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NCAs can also be synthesized by treatment ofbenzyloxycarbonyl derivatives of the corresponding amino acid with inorganic acid halides like SOCI., as shown in scheme VII, or by reaction with P 205 0 II .. CeHsCH1-OC+ SOCi2 R 0 .I "R-c-c I I' <.' HN \ Cl \ -o c{b I 1-+.!C-CsHs 'R R" .., 0 0 II 11 + S0:2 + HCI 'R 0 +-II "R HN, ......-0 c II 0 'R R" Scheme VII Synthesis ofNCAs be treatment ofbenzyloxyamino derivatives with Polymerization ofNCAs is initiated by nucleophilic attack of primary amines. The nucleophilic attack to the carbonyl carbon corresponding to the original amino acid originates the opening of the ring The intermediate carbamic acid spontaneously decarboxylates to generate a new amino group, and so on, as shown in scheme \t1II. 16

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'R 0 I II H R"-c-c-N-R1 + I 'R 0 I 11 H R"-c-c-N-R1 I o=c I Scheme VIII Polymerization via NCAs formation. All the naturally occurring amino acids have been polymerized via their NCAs. Yields are normally high and the reactions proceed at room temperature Berlinguet and Tailleur (23) synthesized the free dipeptide of 1-aminocyclopentanecarboxylic acid by using the NCA of one amino acid condensed with the benzyl ester of the other amino acid in the presence of DCC. During manipulation of the free dipeptide they discovered the strong tendency of the free dipeptide to cyclize into the corresponding diketopiperazine when heated in anhydrous conditions These results show the possibility of our model molecule ( 1-aminocyclohexanecarboxylic acid) to polymerize with the formation of diketopiperazines A second method of polymerization is done via the unreactive esters of the corresponding amino acids 17

PAGE 33

Pacsu and Wilson (32) reported the formation of various polyglycine methyl esters that were obtained by condensation of their respective methyl and ethyl esters As mentioned in section 1.2, simple alkyl esters are reactive enough to acylate amine groups for peptide bond formation 1.3.2 Stepwise Condensation Amino acids can be incorporated to an existing peptide in a directed fashion one amino acid at the time. In the early days of peptide synthesis, segment condensation was the strategy used for their synthesis This technique introduced racemization as a frequent problem. A newer and now commonly used strategy for peptide synthesis is the chain elongation from the C-terminal residue This strategy was first introduced by Bodanszky in 1960. This method is the best application for the active esters of amino acids. Acylating reagents are derivatives from protected amino acids N-protected amino groups are cleaved to yield the free amino group for the acetylating reaction toward the active ester of a second amino acid Formation of peptide bond between two chosen amino acids is then formed. This method permits the directed formation of peptide bonds between determinate amino acids carrying the minimal protection. Racemization problems also diminish with this method 18

PAGE 34

In this research, the monomer 1, 4-diamino-1, 4-cyclohexanedicarboxylic acid was synthesized Formyl and acetyl derivatives were also obtained. The ultimate goal ofthis research is to polymerize this diamino diacid Diketopiperazines can be formed using two different approaches: (1) once the N-protecting groups are cleaved leaving the free amino groups, both amino groups could be acylated forming diketopiperazines between monomers, or (2) only one of the amino groups is acylated, forming a single peptide bond between monomers (Scheme IX). Peptides, whose units are linked by single peptide bonds, may thus have their unreacted esters and free amino groups alternating in the chain This type of polymer might be very interesting to obtain. It might be more flexible than the polymer in which all the monomers are linked together by formation of DKPs, with the possibility of being converted to the latter by heat treatment 19

PAGE 35

0 II --x-OCH3 H2N ---HN--DIKETOPIPERAZINE LINKS (1) .----NH SINGLE PEPTIDE BOND LINKS (2) Scheme IX. Stepwise Condensations 20 c----11 0

PAGE 36

2. Results and Discussions The model amino acid 1-aminocyclohexanecarbox:ylic acid, was synthesized from cyclohexanone following the Strecker synthesis described by Cremlyn (2) as shown in scheme X. 0 NaCN Cyclohexanone HOOC HCI12M 1-Amino-1-cyanocyclobexane Model Amino Acid Scheme X. Synthesis of model amino acid Derivatization of the model amino acid was successfully accomplished. Protection ofboth the amino group and carboxylic group of the model amino acid were achieved through different approaches Scheme XI shows the different approaches studied for derivatization of the model molecule. 21

PAGE 37

Compound X Compound VIII Compound V l CH30H, anhydrous HCI gas .....___ HOOC NH2 18-crown-S, KOH in NaOH Compound VII 0 ________., dioX2ne, Fmoc-CI (BOC):zO in toluene J CF3COOH Formic Ac i d 96% Compound XIV Acetic anhydride Formic Ac i d 96% Compound XII Scheme XI Different approaches for derivatization ofthe model molecule 22

PAGE 38

The syntheses of 1, 4-diamino-1, 4-cyclohexanedicarboxylic acid and its derivatives were attempted after the synthesis of the model amino acid and its derivatives were mastered. The cycloaliphatic diamino diacid was synthesized from 1,4cyclohexadione following similar procedures used for Strecker synthesis described by Cremlyn (2) as shown in Scheme XII. 0 0 2NH/CI 2 NaCN Cyclohexanedione 1.4-Diamino-1.4-dicyanocyclohexane HOOC Cycloaliphatic Diamino Diacid Scheme XII. Synthesis of 1, 4-diam.ino-1, 4-cyclohexanedicarboxylic acid Several approaches were studied for the derivatization of 1, 4-diam.ino-1, 4cyclohexanedicarboxylic acid (Scheme XIII) Protection of the amino groups was achieved by formylation and acetylation reactions Several methods to protect the carboxylic functions of the molecule were tried without success. 23

PAGE 39

0 0 II Ho-OCH, H3co-c NH II 2 0 Compound VI CH30H. anhydrous ''\. HCI gas HOCQC NaOH Acetic Anhydride H c-c-NH COOH 0 H II HOOQC N--c-CF3 CF3COOH Formic Acid 96% ...... 3 II 0 Compound IX d Fmoc-CI y\ 18-crown-6, KOH in dioxane 0 H II "Qc-o-2, COOH II 0 Compound XI F3C-c-N COOH II H 0 Acetic anhydride Formic Acid 96"..(, H-C-N II H 0 Compound XV Compound XIII Scheme XIII. Different approaches for derivatization of 1, 4-diamino-1, 4cyclohexanedicarboxylic acid 24

PAGE 40

2.1 Synthesis of 1-Amino-1-cyanocyclohexane (Compound I) and 1-Aminocyclohexanecarboxylic Acid (Compound IT) The model amino acid (Compound II) was synthesized using the Strecker method described by Cremlyn (2). The first step entitled combining cyclohexanone with ammonium chloride and sodium cyanide in a 50% water/methanol system yielding 1-aminocyanocyclohexane (Compound I). In the second step, the hydrolysis of Compound I with concentrated HCl solution converted the cyano group to a carboxylic acid, yielding the model amino acid. This model experiment easily produced a high yield product. 1H-NMR spectra ofC0mpounds I and II are not very useful in determining their structure due to the complex set of peaks between 0.9-2 1 ppm given by the cyclohexane protons. The assignment of the 13CNMR spectrum of the cyano intermediate (Fig. 2.1.1) is as follows: the resonance at 115.85 ppm corresponds to the carbon of the cyano group, at 51.03 ppm corresponds to the alpha carbon of the cyclohexane ring. The resonances at 31.88 ppm, 21.47 ppm and 20.37ppm correspond to the beta, delta and gamma carbons of the cyclohexane ring respectively. 25

PAGE 41

N 0\ zoo 160 160 1<10 120 -ci+H3N-___. C::: N Ca cb/ ""'-cb I I Cc""' /Cc Cd 100 60 60 40 Fig. 2.1.1 13C-NMR spectrum at 50 MHz ofCompound I in 020 .. .. .. . l .. ... .. I ... .. l .zo

PAGE 42

Compound II was synthesized by hydrolyzing of the cyano group to a carboxyl group 13C-NMR analysis of Compound II, using D20/NaOD as the solvent system, confirmed the hydrolysis ofthe cyano group, as the peak at 115.85 ppm disappeared and a new signal, further down field (183.88 ppm) was observed This resonance was assigned to the carbon of the more electronegative carboxylate function (Fig. 2.1.2) Also, the resonance correponding to the alpha carbon of the cyclohexane ring was shifted upfield to 56.54 ppm. This carbon is the most affected by the electronegativity of the new carboxylate group. The beta carbon gives a peak at 33. 67 ppm, the delta carbon at 23.48 ppm and the gamma carbon at 20 .13 ppm, respectively. 27

PAGE 43

N 00 H2N......._ ,......cooca, Cb/ Cb I I cc ............... _..........cc Cd .. .. "! .. .. .. ,.. .. .. :.: .. .. ' 1 -r . .. . ,." ?.00 11\0 160 140 120 100 80 60 40 ZO 0 11pm Fig. 2. J .2 13C-NMR spectmm at 50 MHz of Compound II in D20/NaOD.

PAGE 44

2.2 Synthesis of 1,4-Diamino-1,4-dicyanocyclohexane (Compound III) and 1,4-Diamino-1,4-cyclohexanedicarboxylic Acid (Compound IV) Synthesis of Compounds III and IV was accomplished by the Strecker method. The procedures were a modification ofCremlyn's method, used to synthesize 1-aminocyclohexanecarboxylic acid, the model compound. The synthesis of Compound III was easier than that of its analog, Compound I, because the product conveniently precipitated from the aqueous methanol mixture used for the reaction. Thus, the crude product was collected by filtration and hydrogen chloride gas was not needed to obtain it. The crude product was easily recrystallized from hot water. The procedures for the synthesis of Compound IV were essentially identical to those used for the synthesis of the model amino acid (Compound II). The hydrolysis of the dicyano intermediate was carried out in a two step process. In the first step, Compound III was treated with concentrated HCl solution to form the corresponding intermediate amide. In the second step, the solution containing the amide was diluted and refluxed for 48 hours yielding Compound IV. To obtain Compound IV, the hydrolysis ofthe intermediate compound was carried out in two steps. Any modification on the hydrolysis of Compound III gave undesired products. 29

PAGE 45

Compounds III and IV are polysubstituted cyclohexane rings. Sometimes, cyclohexane rings can be found in two different conformations, the rigid chair and the flexible boat. It was assumed that Compounds III and IV and all their derivatives adopted only the less energetic chair conformation. There are two types of bonds in the chair conformation, the axial bonds and the equatorial bonds. During the chair inversion process, all the equatorial bonds become axial and and all the axial bonds become equatorial. Chair inversion occurs rapidly at room temperature. In Compounds III and IV, even though the geminal protons on each carbon can occupy both the axial and the equatorial positions, they still experience different chemical environments because the substituents on carbons one and four of the rings are different. These pairs of protons are diastereotopic. Diastereotopic hydrogens are generally distinct both spectroscopically and chemically. A distinct NMR signal for each group of diastereotopic protons was thus expected. The disubstituted Compounds III and IV also have two different configurations, the trans and the cis as shown in Fig. 2.2 1 30

PAGE 46

Trans-Compound III CN NH3+cr Cis Compound III COOH HOOC Trans-Compound IV Cis-Compound IV Fig. 2 .2.1 Configurations of Compounds III and IV The more stable configuration is normally the one with the larger substituents occupying equatorial positions It was assumed that Compounds III and IV were only present in their trans configurations with their larger substituent in the equatorial positions. Due to the inversion of the ring process, the cis and the trans isomers of Compounds III and IV present two different conformations. The two possible conformations of the cis-isomers of ill and IV are energetically equivalent as shown in Fig. 2.2.2 Both of them have an amino group and a cyano or carboxylic group occupying the equatorial positions. 31

PAGE 47

CN NCPNHs cr ci'HsN NH3 +cr Cis-Compound III COOH HOOC COOH NH3+cr Cis-Compound IV NH 3+cr Fig 2.2.2 Conformations of cis-isomers of Compounds III and IV However, the two conformations of the trans-isomers of Compounds III and IV are not equivalent as shown in Fig. 2.2.3 One of them has two carboxylic or cyano groups equatorial and the other has two amino groups equatorial. Therefore, the two conformations of the trans-isomers are not present in quantities and can be distinguished by NN1R analysis. CN NC CN NH3 +cr Trans-Compund III COOH HOOC COOH NH +cr 3 Trans-Compound IV COOH Fig. 2.2.3 Conformations of trans-isomers of Compounds III and IV. 32

PAGE 48

To determine what is likely to be the more stable conformation of the transconformer of Compounds III and IV, the conformational energies (33) of each substituent were studied. Table 2.2.1 presents some of the conformational energies for the most common substituents of our molecules. These values of conformational energies represent the difference on free energy between the substituent occupying the more energetic axial position and the substituent occupying the equatorial position. Group -CN -NH2 -NH3+ -COOH -coo--NHcoc6Hs Conformational Energy (KcaVmol) 0.2 1.7 1.7-2 0 1.4 2.0 1.6 Table 2 2 1 Conformational energies of some substituents. Based on the conformational energies, not only the preferred conformation, but also the abundance of each conformer can be determined. 33

PAGE 49

Making the reasonable assumption that the conformational energies of substituents A and Bare additive the free energy change (8G0 ) in a molecule will then be 8G0= (GAaxial + GBequatoria!-G Aequatorial + (2 .1) assuming that GAequatoria!= GBequatoria! = 0 the free energy change ofthe process is giving by (2 2) The formation of the two conformers of the trans-isomer occurs rapidly at room temperature It is an equilibrium process The distribution of conformers is calculated using 8G0 =-RT lnK (2 3) the equation relating the equilibrium constant with the free energy change of the process For the trans-isomer of Compound III the predominant conformer is the one in which the cyano groups are occupying axial positions The much less abundant conformer is the one in which the cyano groups are occupying equatorial positions. The difference of energy between the less abundant and the more abundant conformers is 3.0 Kcallmol (I.7x2-0. 2 x 2 Kcal/mol) The distribution then corresponds to 99.4% axial-cyano conformer and 0 6% equatorial-cyano conformer. 34

PAGE 50

Calculations were performed for Compound IV in two different forms: the deprotonated one with carboxylate and free amino groups, and the protonated one with carboxylic acid and ammonium groups. When Compound IV is deprotonated, the predominant conformer is the one with both carboxylate groups occupying equatorial positions. The differences in energy between the less abundant conformer and the more abundant conformer is only 0.6 Kcal/mol (1.7x2-1.4x2 Kcalfmol). The distribution corresponds to 64% carboxylate-equatorial conformer and 36% carboxylate-axial conformer. When Compound IV is protonated, the predominant conformer is the one in which both carboxylic groups are axial. The .energy difference between the less stable and the more stable conformers is also 0 6 Kcallmol (2.0x2-1. 7x2 Kcalfmol). Therefore, the distribution also corresponds to a 64:36 mixture, but in this case the carboxylate-axial conformer is predominant. Analyzing the values obtained for the distribution of the different conformers, it was expected that Compound III will only exist in its cyano-axial form (99.4%) and Compound IV, protonated and deprotonated, will be a mixture ofboth conformers in roughly 2: 1 ratios. The distribution values obtained for the conformers of transisomers of Compounds III and IV may help to explain the NMR spectra obtained for these molecules. We have already seen, at the beginning of this section, that the hydrogens attached to the cyclohexane ring in Compounds III and IV are diastereotopic. 35

PAGE 51

Diastereotopic hydrogens are spectroscopically distinguishable. Every proton of the cyclohexane ring is expected to be coupled with two non-equivalent protons : one geminal and one vicinal. The coupling constant (J) between two geminal protons is larger (10-30Hz) than the coupling constant between two vicinal protons, one axial and the other equatorial (3-5Hz). Therefore, the 1H-NMR spectra of Compounds III and IV were expected to give two sets of doublet of doublets (eight peaks in all). The 1H-NMR spectrum of Compound III showed only two doublets (Fig. 2.2.4) One doublet at 2.26 and 2.21 ppm (J= 10Hz) and the second doublet at 1.81 and 1 76 ppm (J= 10 Hz) respectively Previously we have assumed that only the trans-configuration of Compound III (the more stable one) is present. Calculations indicate that only the conformer with both cyano groups occupying the axial positions is significant (Fig.2 2.5) Fig 2.2.5 More stable conformation of trans-isomer of Compound III 36

PAGE 52

1.;) -..) Ha C:N ## Hb ci+H3N Hb NH3 +cr Ha N:::c Ha .......... .. .. ... ...... ___ -...... --------Iii -----------------------, . ... t' r .,. -,-,-r-r--r . ,.--..--,, -,--,---r-,-r--,--.-,--.-y""?-r-"t--,-1-r-r-r rr-r ,. "T ..... 1 1.0 6.5 G.o 5.5 5 n 4.5 3.5 3.0 2.5 z.o 1.5 1'1"" r. 2 2 4 1H-NMR spectrum at 200 MHz ofCompound III in 020. alg. .

PAGE 53

This conformer has equivalent axial protons antiperiplanar to cyano groups Cyano groups are electronegative. It is known (28) that the coupling constant between vicinal protons diminishes by 1-2 Hz if one of the two coupled protons is antiperiplanar to an electronegative group. Since the normal value of the vicinal coupling constant between one axial proton and one equatorial proton is 3-5Hz, it is possible that the coupling between vicinal protons is not seen in the spectrum, especially because the peaks are fairly broad. Only the coupling between gerninal protons seems to be present. The value of the coupling constants of the two doublets is consistent with the average values normally seen for gerninal coupling The 13C-NMR spectrum of Compound III (Fig. 2 2.6) is interpreted as follows: the resonance at 117. 18 ppm corresponds to the cyano groups as seen in the 13CNMR spectrum of the model molecule (Compound I). The resonance at 48.59 ppm corresponds to the Ca of cyclohexane ring. The resonance at 29.86 ppm corresponds to the Cb of cyclohexane ring. Compound IV was studied in its protonated and deprotonated form. The distribution calculations, for the two forms of Compound IV described above, assigned significant populations ofthe two possible conformers (64% vs. 36%). It was assumed, based on the same explanation given for Compound III, that the vicinal coupling between non-equivalent hydrogens are not seen in the 1H-NMR 38

PAGE 54

w \{) C::N I Cb--Ca -cl+ / Cb......-/ "'-+ H3N-c --Cb NH3 cr N=[ --cb .. =: ::. .. .. tun tr.u 1110 uu too au Gil
PAGE 55

spectra of Compound IV Only the coupling between non-equivalent geminal hydrogens was recorded on the spectra Both conformers of protonated Compound IV exist in the mixture. Hydrogens of carboxylic-axial conformer may experience slightly different magnetic field than the hydrogens of carboxylic-equatorial conformer causing the multiplet signal between 1.9 and 2 1 ppm in the 1H-NMR. spectrum. The coupling constants for this multiplet are consistent (J= 9.6 Hz) with the average value for geminal coupling constants (Fig 2 2 7) The 1H-NMR spectrum of deprotonated Compound IV (Fig 2.2 8) was expected to show a pattern of resonances similar to the one seen for the protonated form because calculations determined a 36:64 ratio vs. 64:36 ratio between the two conformers. Surprisingly, only two signals resonating as doublets, were seen in the spectrum. One ofthe doublets appearing at 1.31 and 1.26 ppm (J= 9.4 Hz) and the second doublet appearing at 1.84 and 1.79 ppm (J= 10Hz). Even though the calculations predict the presence of both conformers, only one conformer seems to be present in the basic solution. The 13C-NMR. spectrum of the protonated and deprotonated forms of Compound IV are very similar showing only small differences in the peak positions. They cast no light on the relative distribution of conformers. 40

PAGE 56

,_. Ha Ha COOH NH3+cr HOOC ( / .,., ;: lD ---------------/ . 0: .I"! s '1111 "' ::1 .. ) ...... . .. ... ... , . . .. . -,---,---, . ... ,.. rr-, ., .. ---, ,--1' .,r -,....--,-,.--,1-,.-,---,----,-r --.-r --, ,---, r """T--, --1---, ... 1 . r -... --,---,-G O !i. O 4 5 q o 3.5 3.0 Z-5 Z-0 LS 1-0 fljliR Fig. 2.2 7 11 1-NMR spectrum at 200 MHz ofCompound IV in 020/DCI.

PAGE 57

+:>. N ---------. ----I I Ha I Ha coo-I ----------NH2 .. _______ ___..____ ---iS, )I a "' 1--, . -rr -1---,---1 ...... . .... -,--,-----,--,--,-----.r-r---,--,---r --,----,----r. .... -r ,. I -. 2 6 5 u 4 ;, Fig. 2 2.8 1H-NMR spectrum at 200 MHz of Compound IV in 020/NaOD ppm

PAGE 58

2.3 Activation of Carboxylic Function by Formation of Methyl Ester: Synthesis of Methyl 1-Aminocyclohexanecarboxylate (Compound V) Amino acid esterification first described by Fischer (8), is a standard method for the synthesis of esters This is a nucleophilic acyl substitution reaction where one molecule of the Compound II reacts with an excess of methanol to form the methyl ester of Compound II and one molecule of water in the presence of an acidic catalyst. Kenner et al. (3) reported the synthesis of Compound V in 1965 following Fischer's method In their procedures the hydrochloride salt of the amino acid methyl was collected by filtration and recrystallized from methanol before the methyl ester was obtained in a 52% yield. Here, neither the hydrochloride salt nor the methyl ester were purified because Compound V was obtained as an oil. A comparable yield of 53% for the crude product was obtained The 1H-NMR (D20/DC1) spectrum obtained for Compound V was very similar to the spectrum published by Paul et al. (13). The cyclohexane protons resonate between 1.94 and 1.37 ppm and a new singlet for the methyl protons appears at 3.8 ppm The 13C-NMR for Compound Vis similar to the 13C-NMR spectrum obtained for 1-aminocyclohexanecarboxylic acid (Compound II) with an extra peak at 50.40 ppm corresponding to the methoxy carbon. 43

PAGE 59

The resonances for the carbonyl carbon and Ca carbon ofthe cyclohexane ring are shifted downfield in the spectrum of Compound V because these two groups are the most affected ones by the electron density introduced by the methyl group 44

PAGE 60

2.4 Activation of Carboxylic Function by Formation of Methyl Ester: Attempted Synthesis of Dimethyl 1,4-Diaminocyclohexane-1,4-dicarboxylate (Compound VI) Synthesis of Compound VI was attempted following three modified Fischer esterification procedures, without success. The major difficulty encountered during these esterification reactions was the unpredictable behavior of the starting material (Compound IV) in acidic conditions. During the first reaction, the starting material was suspended in an excess of anhydrous methanol. Bubbling hydrogen chloride through the suspension for a few seconds dissolved Compound IV completely. As more hydrogen chloride was passed into the clear solution, the starting material became insoluble again After 10 minutes, at least 90% of the original amount used had precipitated from the solution The suspension was stirred at room temperature overnight but the starting material never redissolved in the acidic solution. The recovered starting material, from the first attempt, was collected by filtration and used in a second esterification attempt. This time the hydrogen chloride was passed into the suspension of Compound IV in methanol just long enough (a few seconds) to obtain the clear solution The solution was then stirred at room temperature for five days. It was then taken to dryness and the residue suspended in water. 45

PAGE 61

The mixture was made basic with and extracted with an organic solvent. With this extraction, the deprotonated amino acid ester was expected to be transferred to the organic phase. No components were detected by any of the three visualization methods when the organic fraction was analyzed by TLC The aqueous fraction was analyzed using the same system. Two concentrated, ninhydrin positive spots with a Rfvalues ofO .OO and 0.36 were observed. Even thought the ninhydrin positive spot with a Rfvalue of0.36 was located where the methyl ester was expected, the fact that this component was in the aqueous phase led us to discard it. The second reaction was stopped at this point. The third reaction was a variation of the Fischer esterification described by Kenner et al. (3). This reaction was very similar to the first attempt. It was a more aggressive procedure where hydrogen chloride was passed into the boiling mixture of Compound IV in methanol for 1 hour and the suspension was then refluxed for 12 hours After the heating period, the mixture was taken to dryness and the residue partially dissolved in water Basification of the mixture with 6 N NaOH yielded a small amount of precipitate. 13C-NMR (CDCl3 ) analysis of the precipitate showed no signal for a methoxy carbon at 50 ppm. 46

PAGE 62

2.5 Protection of Amino Function with a Boc Group: Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic Acid (Compound Vll) Synthesis of Compound VII was achieved following general procedures described by Bodanszky et al. (34) The synthesis of Compound VII is achieved by acylating the amino group of the starting material with a symmetric anhydride. The major difficulty encountered during the synthesis arose from the solubility differences between the two species involved in this reaction On one hand, the starting material, which is in its zwitterion form, needs to be converted to a nucleophile by deprotonating the amine function. To do so, Compound II was dissolved in an aqueous solution containing one equivalent ofNaOH per molecule of starting material. On the other hand, di-tert-butyldicarbonate is poorly soluble in aqueous solutions but freely soluble in organic solvents like toluene or dioxane. Therefore, the reaction was run in a two-phase solvent system. The heterogeneous reaction mixture was vigorously stirred to allow the reaction to proceed. When the toluene/water system was used, the synthesis of Compound VII was obtained in a 22% yield. When dioxane/water system was used, the reaction failed 47

PAGE 63

The 1H-NMR. spectrum of Compound VII in CDC13 gave a multiplet resonance between 1.98 and 1.61 assigned to the cyclohexane protons. The 1H-NMR. spectrum of di-tert-butyldicarbonate (Aldrich) shows the tert-butyl group resonating as a singlet at 1.40 ppm In this spectrum, a similar resonance at 1.43 ppm was obtained. However, the integration of this signal indicated the presence ofhydrolyzed Boc anhydride in the sample. The success of this acylation was proven when a broad peak at 4 8 ppm integrating to 1 hydrogen appeared in the spectrum. This peak corresponds to the amide hydrogen. 48

PAGE 64

2.6 Protection of Amino Function with an Acetyl Group: Synthesis of 1-Acetylaminocyclohexanecarbo:xylic Acid (Compound Vlli) and 1,4-Diacetylamino-1,4-cyclohexane dicarboxylic Acid (Compound IX) Synthesis of Compound VIII and Compound IX was accomplished following the procedures described by Shotten and Bauman (11) with small yields of33% and 14% respectively Acetyl groups are not good amino protective functions. The major disadvantage of using the acetyl groups as N-protecting groups is the difficulty associated with removing them under mild conditions to avoid the hydrolysis of peptide bonds. Therefore, the syntheses of Compounds VIII and IX were done as a model experiments to gain information about the reaction conditions for later attempts ofN-acylation reactions with similar but more labile protecting groups (e.g. trifluoroacetyl groups). The acetylation was carried out using acetic anhydride which is only slowly hydrolyzed by water, so it could be carried out in an aqueous medium. The 1H-NN.IR spectrum of Compound VIII showed the expected acetyl methyl protons as a singlet at 1. 78 ppm, and the cyclohexane protons as a multiplet between 1.10 and 1.86 ppm. 49

PAGE 65

The 13C-NMR spectrum showed resonances at 171.39 ppm (amide carbon) and 20 .63 ppm (methyl carbon) in addition to the ring and carboxyl carbon signals seen in the spectrum of the parent compound. The 1H-NMR spectrum of Compound IX indicated two resonances, singlets at 1. 81 ppm and 1 80 ppm respectively The integration value for the pair of signals was six hydrogens. It appears that the protons of the two acetyl methyl groups experience slightly different environments within the molecule. The cyclohexane protons appear as a multiplet possibly two doublets, starting at 1.26 and partially overlapping the methyl signals The 13C-NMR showed the expected five signals with no apparent differences between the two acetyl groups 50

PAGE 66

2. 7 Protection of Amino Function with a Fmoc Group: Synthesis of 1-(9-Fluorenyl)aminocyclohexanecarboxylic Acid (Compound X) and Attempted Synthesis of 1,4-[Di-(9Fluorenyl)amino]-1,4cyclohexanedicarboxylic Acid (Compound XI) Based on the results obtained for theN-acylation carried out using Boc anhydride on our model amino acid (see section 2.5), a new approach was tested for the synthesis of Compounds X and XI The two-phase solvent system used for the reaction was replaced by a solution containing the phase transfer agent 18-crown-6 ether. Crown ethers are cyclic macromolecules containing the repeating unit ( -O-CH2 -CH2 )0 with the ability to "crown" cations by complexation thus rendering them soluble in organic solvents. The 18-crown-6 ether molecule has a high selectivity for potassium cations because of the size of its cavity. A dioxane solution was made containing two moles ofKOH and 18-crown-6 per mole of starting material. A.C S reagent grade potassium hydroxide contains 15% ofH20 On a molar basis, this is almost a two-to-one mixture ofKOH and water. The presence of these water molecules normally facilitates the dissolution ofKOH in the dioxane/18-crown-6 ether solution because the salt dissolves better in the mixture if it is first dissolved in water. 51

PAGE 67

Here, an extra 10 to 15% (v/v) volume of water was needed to dissolve the potassium hydroxide Half of the 18-crown-6/K.OH mixture was used to initiate the N-acylation of 1-aminocyclohexanecarboxylic acid by Fmoc-Cl. The second half of the 18-crown-6/K.OH mixture was then added dropwise to the reaction mixture to scavenge protons released in the reaction. The basicity of the solution was regulated to minimize the hydrolysis ofunreacted Fmoc-Cl by the OHanions present in this second fraction. The order of addition of reactants is therefore slightly different from the one used for theN-acylation of the model molecule by Boc anhydride. In the 1H-NMR spectrum of Compound X (Fig.2.7.1), the resonances appearing as a multiplet from 7.18 to 7.69 ppm correspond to the aromatic protons of the Fmoc group. It is a very clean signal. This multiplet was taken as the reference signal, integrating as 8 hydrogens, to assign integration values to the remaining spectrum The multiplet between 1 78 and 1.18 ppm corresponds to the cyclohexane protons. This signal integrates as 13 hydrogens (instead of 10) mostly because it contains ethyl acetate as an impurity (the singlet at 1.98 ppm and the triplet at 1.25 ppm). The resonance at 3. 7 ppm is also an impurity that is believed to be water. There is also a broad resonance integrating to less than one proton at 9.25 ppm corresponding to the carboxylic proton. The second broad signal at 5 .1 ppm corresponds to the amide proton. The integration value for this resonance is also smaller than expected. Both of these are exchangeable protons. 52

PAGE 68

Vl vJ C Q H COOH H2 'o-cQ-N Ha Ha Hb l-Ib He He-Hd Hd He He -/1 _____ ../'.....__-.../ jl_ ___ ___ .. -,- r' I 0 9 II 1 6 5 4 3 2 1 0 1 I' JIDI Fig. 2 7 1 '11-NMR spectrum at 200 MHz ofCompound X in CDCIJ.

PAGE 69

The doublet at 4 33 ppm integrating for 2 hydrogens corresponds to the -CH2 group. The signal at 4.14 ppm integrating to one hydrogen corresponds to the CHproton of the Fmoc group, partially obscured by the ethyl acetate impurity The 13C-NMR (CDC13 ) spectrum of Compound X (Fig 2.7.2) is interpreted as follows: the resonance at 177.87 ppm corresponds to the carbonyl carbon of carboxylic group. The signal at 154 17 ppm corresponds to the carbonyl carbon of the amide. The resonances at 142.4 139.85, 126.18, 125.6, 123. 58, 118.44 ppm correspond to the aromatic carbons of the Fmoc group The resonance at 65.45 ppm corresponds to the -CHcarbon of the Fmoc group. The resonance at 45.66 ppm corresponds to the -CH2 carbon of the Fmoc group. The resonance at 57.45 ppm corresponds to theCa of the cyclohexane ring. The resonances at 30 73, 23.49 and 19.53 ppm correspond to the Cb, Cd and Cc carbons of the cyclohexane ring respectively The signals at 68.86, 58 94 and 12.57 ppm are believed to be impurities 54

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VI VI ... : ... j ... .. "' .. .. .. I .. : i .. 111 ... .. .. .. ..... ....... T ... "' .. "' Fig. 2.7 2 uC NMR spectrum at 50 MHz ofCompound X in CDCI3 H C 9 H 2 '0-C-N COOH .. 0 ... -'ca .. .. ... Cb,....... 'cb I I Cc........_ .,..Cc Cd :;: ... ...

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Synthesis of 1, 4-[Di-(9Fluorenyl)amino ]-1, 4-cyclohexanedicarboxylic acid (Compound XI) was attempted following the procedures used for the synthesis of Compound X. Unfortunately, the reaction time allowed for the synthesis of Compound XI was only 1 0 hours. This shorter period may have been decisive for the failure to obtain Compound XI The 1H-NMR (d6-DMSO) spectrum of the product obtained shows the characteristic multiplet resonance between 7. 86 and 7.28 ppm corresponding to the aromatic hydrogens of the Fmoc group. If the N -acylation of the two amino groups of the starting material was completed, this multiplet should integrate to 16 hydrogens. Instead, the signal integrates to 9 hydrogens. This integration value could indicate that only one amino group had been protected during the process. As seen with the model compound (X), the Fmoc group also gives two sets of coupled signals for the methylene and -CH protons. However, these resonances, normally found between 4.3 0 and 4.1 0 ppm, are missing in the spectrum of the product. The characteristic resonance pattern of the disubstituted cyclohexane protons is also absent in this 1H-NMR spectrum. The spectrum indicates that the product was not the N-acylated starting material but possibly a by-product arising from chemical transformation of the Fmoc-CL 56

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2.8 Protection of Amino Function with a Formyl Group: Synthesis of 1-Formylaminocyclohexanecarboxylic Acid (Compound Xll) and 1,4-Diformylamino-1,4-cyclohexane dicarboxylic Acid (Compound Xlll) TheN-formyl groups were introduced readily in Compounds II and IV by formylation in the presence of acetic anhydride. Compound XII was synthesized following the procedures described by Kenner, Sheppard and Preston (3). This method is a modification of the procedures described by du Vigneaud (9) for formylation reactions. Kenner's procedure involves the use of lower temperature during the reaction to minimize the danger of racemization when optically active amino acids are involved. Neither of our starting materials, Compound II or Compound IV, are optically active, so racemization was not a problem in our case. Compound XIII was synthesized following the procedures described by du Vigneaud (9) because the starting material, Compound IV was not soluble in formic acid at room temperature. The reaction needed to be done using higher temperatures than in du Vigneaud's method. The first step of the formylation reaction is presumably the formation of the mixed anhydride between the formic acid and acetic anhydride. Later, the nucleophilic attack of the amino group of the starting material on the formyl group of the mixed anhydride yields the desired formyl derivative. 57

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Both methods have a reported yield generally greater than 80% of theory. Yields of 56% and 4 7% were obtained for Compounds XII and XIII respectively. The procedures called for a 98% formic acid. In our case, a 96% formic acid was used to carry out the reactions. Perhaps an improvement of the yield can be obtained if a more concentrated formic acid is used for these formylation reactions. 2.8.1 Analysis of the NMR Spectra of 1-Formylaminocyclohexanecarboxylic add (Compound Xll) The assignment of the 1H-NMR (D20/NaOD) spectrum of Compound XII (Fig. 2.8 1.1) is as follows: the singlets at 7.76 and 7.87 ppm which together integrate to one proton, correspond to the formyl proton of the trans and cis conformations of Compound XII respectively. The assignment of these peaks is explained further in this section. The multiplet between 1. 09 and 1 81 ppm integrating to 10 protons corresponds to the protons of the cyclohexane ring. The signal at 4 64 ppm corresponds to the solvent (D20). The amide bond, formed between the formyl and the amino group in Compound XII, is planar and has a high rotational energy barrier. The amide bond does not rotate freely in the molecule. 58

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I L r---1.111 J ' i J I ...... I I J I N J Oo i i J I z s:: ::::= :1> '"0 ('I) ("l .., 1: 3 :1) tv 0 0 ..,.. N 0 ("l 0 .::: OJ 0 1: Q. ::... :o 0 ......... z :1) 0 :0 i .... J 1 1 1 I I j wJ J I 1 j ... J J I J I l 1 -...: i I 7. 7S7 :r :r C) I n=o I z:c :r:r C'"GI

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Therefore, Compound XII presents two diastereoisomers with respect to the peptide bond* In the cis configuration, the formyl proton and the cyclohexane ring are on opposite sides of the amide bond. In the trans configuration, the formyl proton and the cyclohexane ring are on the same side of the amide bond. Figure 2.8 1.2 shows the two possible diastereoisomers of Compound XII with respect to the rigid amide bond. Trans-configuration H H I N-C 0 R1 Cis-configuration Fig. 2 8.1.2 The two possible diastereoisomers of Compound XII It is necessary to clarify at this point that, based on the conformational energies, it is assumed that the more stable conformer of Compound XII is the one where the carboxylate group is occupying the equatorial position and the carboxamide group occupies the axial position. Although the cis and trans configurations do exchange they do not do so on the NMR time scale For the purpose of clarity in this discussion we will refer to the cis and trans amide forms as if they were true configurational isomers We will use the term conformer to mean only rotations around normal sigma bonds. 60

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The enthalpy differences between conformers is small (0.4 KcaVmol) so the Boltzmann distribution equation predicts a nearly equimolar mixture ofboth conformers The formyl protons experience different chemical environments depending upon the amide configuration of the individual molecules, and their resonances are then different We believe that the trans diastereoisomer is more stable than its cis diastereoisomer. In the trans configuration, there are 1,3-diaxial interactions between the formyl proton and the axial hydrogens of the cyclohexane ring. In the cis configuration, the 1 3-diaxial interactions increase when the carbonyl group of the resulting amide interacts with the axial protons of the cyclohexane ring. It is possible that these interactions are of such magnitude that they will force the resulting amide out of the cyclohexane ring Presumably, the amide group will rest between the carboxylate anion and the second carbon of the cyclohexane ring (that is, gauche to the carboxylate group) The 1H-NMR spectrum also reveals that the formyl proton for the trans diastereoisomer resonates at higher field than the formyl proton for the cis diastereoisomer. This difference in resonances cannot be explained as a simple inductive effect. The assignment of13C-NMR (D20/NaOD) spectrum ofCompound XII (Fig. 2.8 1.3) is as follows : the resonance at 180 .01 ppm corresponds to the carboxylate carbon. 61

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0\ N ... .. 1 0 II H H-e-N coo / Ca cb/ ""-cb I I Cc""" /Cc Cd .. :: ... .. .. I .. N I ..... .... r M ., N .. J Fig. 2.8.1.3 13C-NMR spectrum at 50 MHz of Compound XII in 020/NaOD.

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Assigned on the same basis as the 1H-NMR spectrum, the resonance at 166.05 ppm corresponds to the formyl carbon of the cis diastereoisomer and the resonance at 161.55 ppm corresponds to the formyl carbon of the trans diastereoisomer. The resonances at 59 94 and 59.20 ppm correspond to theCa carbons of the cyclohexane ring in the cis and trans stereoisomers respectively The resonances at 32.03 and 30.60 ppm correspond to the Cb carbons of the cyclohexane ring in the cis and trans conformations of Compound XII respectively The resonances at 23.17 and 19.40 ppm correspond to the Cd and Cc carbons of the cyclohexane ring respectively. The two latter carbons may also generate two slightly different peaks . Based on the height of the signal for the formyl carbon in the 13C-NMR spectrum, a 80 : 20 distribution occurs between the trans and the cis diastereoisomers of Compound XII. 2.8.2 Analysis of the NMR Spectra of 1,4-Diformylamino-1,4-cyclohexanedicarboxylic Acid (Compound XTII) Based upon the discussion for the starting material (Compound IV) in section 2.2, the more stable conformation ofthe trans-isomer of Compound XIII is believed to be the one where the carboxylate groups occupy the equatorial position as shown in Fig. 2.8.2.1 63

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-ooc H-C-NH II 0 0 II HN-C-H coo-Fig. 2 8 2.1 More stable conformer of the trans-isomer of Compound XIII. An enthalpy difference of0.8 Kcal/mol was calculated between the two possible conformers of trans-Compound XIII. The distribution is then 74% for the conformer with the carboxylates equatorial and 26% for the conformer with the carboxylates axial. It is not surprising that in the 1H-NMR spectrum of Compound XIII, the cyclohexane hydrogens present the same multiplet signal pattern seen for the protonated molecules of Compound IV (Fig. 2.2. 7) Compound XIII has two formyl groups attached to its two amino groups, therefore, it presents three different configurations (Fig. 2.8.2.2) 'R H e-N /j \ 0 H H 0 ,, -c 'H R' All-trans-XIII H 0 ,, 0, N-C 'H 'R-rd-R' 'e-N \ H H cis-trans-XIII 'R H H H I N-C 0 R' All-cis-XIII Fig. 2.8.2.2 The three possible configurations of Compound XIII 64

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In the most stable configuration, the carbonyl carbon of the formyl group and the cyclohexane ring are on opposite sides of the amide bonds. This is the all-trans configuration The second more stable configuration corresponds to the cis-trans isomer, with one cis amide bond and one trans-amide bond respectively The least stable configuration corresponds to the all-cis stereoisomer. The1H-NMR (D20/NaOD) spectrum of Compound XIII shows both cis and trans amide protons but cannot distinguish among the three configurations. The 13C-NMR (D20/NaOD) spectrum of Compound XIII (Fig. 2.8 2.4) does clearly show all three diastereoisomers. The resonances at 166.45 ppm and 162 07 ppm for the amide carbonyl, and the ca signals at 58 7 5 ppm and 58. 12 ppm show the expected cis and trans pairs. The Cb signal, however, is now more complex, consisting of four peaks The resonance at 28.56 ppm corresponds to the four Cb carbons of the cyclohexane ring when Compound XIII presents the configuration The resonances at 26.91 and 25.25 ppm correspond to the Cb carbons of the cyclohexane ring in the cis -trans configuration. The resonance at 25.63 ppm corresponds to the Cb carbons of the cyclohexane ring when Compound XIII presents the all-trans configuration A distribution of80:20 was calculated for the two conformers of the model, Compound XII based on the height of the peaks of its 13C-NMR spectrum 65

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0\ 0\ ... tutJ ... .. 0 II H H-e-N, /coo ca cb/ "-cb I I Cb"-/Cb Ca / .. ooc N-C-H H II 0 "' .. .. .. i .. "' Fig 2 8.2.3 13C-NMR spectn1m at 50 MHz of Compound XIII in 020/NaOD. 0 .. .. I. "'

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If the substituents of the cyclohexane ring in Compound XIll are assumed to behave as independent groups, an estimation of the distribution of possible conformers can be calculated A 64 : 32:4 distribution is obtained for the all-trans, cis trans, and all-cis isomers respectively When the height of the Cb peaks in spectrum (Fig. 2.8 2.3) were measured then base on the assignments made above a experimental distribution of69:26:7 is obtained The agreement between these two distributions is very good 67

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2.9 Protection of Amino Function with a Trifluoroacetyl Group: Attempted Synthesis of 1Trifluoroacetylaminocyclohexane carboxylic Acid (Compound XIV) The synthesis of Compound XIV was attempted modifying the procedures described by Weygand and Geiger (10) This method called for trifluoroacetic anhydride as reagent. This compound was not available in our laboratory so it was synthesized, as described in section 2 3.10, from trifluoroacetic acid and dicyclohexylcarbodiimide (DCC). Although DCC is better known as a coupling reagent for the formation of peptide bonds, it has also been used for the preparation of esters and anhydrides. For the synthesis of Compound XIV, the starting material (Compound II) was dissolved in trifluoroacetic acid. To this clear solution, the freshly prepared trifluoroacetic anhydride solution was added dropwise The oily yellow residue, obtained after the reaction mixture was taken to dryness, was divided into two equal fractions The first fraction was dissolved in ether and extracted with a basic aqueous solution. The aqueous fraction was then acidified and extracted again with ether. The acyl derivative (Compound XIV) was expected in the organic fraction. The analysis of the organic fraction by TLC analysis in chloroform/methanol (9 : 1) system revealed no spots. The procedure was stopped at this point. 68

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The absence of a product corresponding to trifluoroacetylation of the amino group led us to investigate the possible formation of the amino acid-trifluoroacetyl anhydride To test this idea the second fraction was treated as if the mixed anhydride had been formed The yellow oil was dissolved in ethanol and an excess of sodium ethoxide was then added with stirring If the mixed anhydride was present in solution, the nucleophilic ethoxide anion would attack the reactive anhydride forming the corresponding the ethyl ester of the formic acid The reaction mixture was analyzed by TLC in chlorofonnlmethanol (9 : 1) system Only a ninhydrin positive spot at the origin was found The experiment was stopped at this point. 69

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2.10 Protection of Amino Function with a Trifluoroacetyl Group: Attempted Synthesis of 1,4Ditrifluoroacetylamino-1,4cyclohexanedicarboxylic Acid (Compound XV) The synthesis of the trifluoroacetylated model amino acid (Compound XIV) was attempted following the procedures described by Weygand and Geiger (10) A major obstacle did not allow us to use this procedure to synthesize Compound XV: the starting material (Compound IV) was not soluble in trifluoroacetic acid. Therefore, the synthesis of Compound XV was attempted following a modification of the du Vigneaud s procedure, specifically described for formylation reactions (9) compound IV was dissolved in formic acid To this clear mixture, a solution of trifluoroacetic anhydride in formic acid was added dropwise The formation of the mixed anhydride between the trifluoroacetic group and the formic group was expected. The procedure was carried out and worked up in the same way as the formylation reactions, but the 1H-NMR of the resulting product only showed resonances corresponding to the starting material. A possible cause for the failure of this synthesis may have been the high concentration of water in the reaction mixture Water molecules are good nucleophiles and may have hydrolyzed a portion of the trifluoroacetic anhydride molecules. The hydrolysis of the symmetric anhydride would have prevented the synthesis of Compound XV. 70

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2.11 A Search for Diastereoisomers of 1,4-Diamino-1,4-cyclohexane carboxylic Acid (Compound IV) Compound IV can exist as two diastereoisomers : the cis and the trans. As discussed in section 2 2, the more stable configuration is the one where the more bulky substituents are occupying the equatorial positions to diminish the 1,3-diaxial interactions on the Both conformations of cis-1 4-diamino-1, 4cyclohexanedicarboxylic acid have one amino and one carboxylic group axial The two cis-conformers are then energetically equivalent, present as an equimolar mixture, and indistinguishable. The two trans-conformers of 1, 4-diamino-1, 4cyclohexanedicarboxylic acid are not equivalent. One conformer has its two carboxylic groups occupying axial positions and the other conformer has the two carboxylic groups equatorial When Compound IV is deprotonated (free amine), the more stable conformer of the trans-isomer is the one with the carboxylate groups equatorial. A distribution 64 : 3 6 was calculated for the carboxylateequatorial versus the carboxylate-axial conformers respectively based on the standard conformational energies (section 2.2). When Compound IV is protonated (hydrochloric salt), the more stable conformer of the trans-isomer is the one where the two carboxylic groups are axial. A distribution of 64:36 was calculated for the carboxylic-axial and carboxylic equatorial conformers respectively. 71

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Therefore, the predominant conformer for the trans-isomer of Compound IV depends upon the molecule's protonation state, however both forms are well represented in either case To study the diastereoisomerism of Compound IV, a chemical analysis was carried out. This analysis tried to differentiate between the two possible diastereoisomers, based on their solubility difference in the presence of the divalent cation Ca +2 To deprotonate the two carboxylic groups of Compound IV, the diamino acid was dissolved in a NaOH solution containing just two moles ofNaOH per mole of Compound IV. To this solution, one equivalent ofCaC12 was added. Every molecule of the cycloaliphatic diamino diacid is attracted by electrostatic interactions to one ion ofCa*2 If the molecule is the trans diastereoisomer, the following polymeric salt is likely to be formed (Fig.2.11 1 ) : NH2 _ +2ooc Ca NH2 coo+200C Ca Fig. 2.11 1 Polymeric salt between the trans-Compound IV and Ca +2 cations. The polymeric salt between the Ca ... 2 and the trans-Compound IV is probably not soluble in aqueous solution. 72

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This type of rearrangement produces extensive precipitate in aqueous solution. If the molecule is the cis-diastereoisomer, the polymeric salt may not be formed and, instead the following arrangement could be found (Fig.2.11.2): 0 0 II II c-o-+20-c Ca Fig 2 11. 1 A possible structure formed between the cis-Compound IV and the ca+2 cations. The structure formed between the cis-Compound IV and the Ca +2 cations could have a different solubility from the polymeric salt formed between the trans-Compound IV and the Ca +2 cations When the CaCl2 was added to the basic solution containing Compound IV, 90% of the original amount of starting material came out of solution. The remaining 1 0% of Compound IV in solution was then collected and treated again with CaC12 to see if the solubility of this fraction of starting material in the presence of calcium cations was truly different. A different behavior in solubility could indicate a different diastereoisomer in this fraction of starting material. Unfortunately, treatment ofthe 10% fraction of Compound IV with calcium cations also produced extensive precipitation 73

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This is not a good method to differentiate between the two possible diastereoisomers of Compound IV. 74

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3. Experimental 3.1 Reagents Benzyl chloroformate (95%), 18-crown-6 ether (99.5%), dioxane (95%), trifluoroacetic acid (99% ), acetic anhydride (99% ), trifluoroacetic anhydride (99% ), hydrogen chloride anhydrous (99+%), 1,4-cyclohexanedione (98%), cyclohexanone (98% ), formic acid (96% ) di-tert-butyldicarbonate (98%) were all purchased from Aldrich Chemical Company Ammonium chloride (95% ), calcium hydroxide (95% ), potassium hydroxide (85%, pellets) were purchased from J. T Baker. Sodium cyanide (95%) was purchased from E.M. Science 3.2 General Methods 3.2.1 Thin Layer Chromatography The plates used for thin layer chromatography (TLC) were aluminum sheets pre coated with 0.2 mm layer of silica gel (UV254). Micropipetes with capacity of 1 !J.L were used to spot the samples on the silica plates. Chromatograms were developed either in chloroform/ methanol (9: 1) or butanoV acetic acid/ water ( 4: 1 : 1) systems The solvent system used in each case is described specifically in the synthetic procedures section of this paper. Once the chromatograms were developed, the components of each sample were visualized following three methods in the 75

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following order : Method A: The plate was observed under ultraviolet light at 254 nm The TLC plates contain a flourescent material whose emission is quenched by most solutes. After the solvent evaporates and the plate is viewed with the UV lamp, the solute spots appear dark while the rest of the plate is bright. Method B: The plate was immersed in silica gel saturated with iodine for a brief period. The 12 vapor is reversibly adsorbed on many substances, particulary amides, and creates a dark spot wherever an adsorbing compound is located Method C : The plate was sprayed with a 2% solution of ninhydrin in acetone and then heated at about 80-90C also for a brief period of time. Ninhydrin is the reagent most commonly used for the detection of free amino groups, yielding a blue color under the proper conditions. Spots observed on the plate using methods A, B C are described as UV positive, iodine positive and ninhydrin positive, respectively 3.2.2 Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) analyses were performed using a Varian 200 MHz spectrometer. Samples were dissolved in either D20, D20/DC1, D20/ NaOD, CDCI3 or d6-DMSO for the analysis 76

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All solvents contained 0.03% ofTMS as an internal reference Proton and carbon spectra were obtained for each sample. Chemical shifts are reported in ppm. 3.2.3 Melting Points A mel-temp Laboratory device was used to determine the melting points of individual samples. An open, thin-walled capillary tube (lmm x 100 rnm) was used to contain the solid sample. All melting points are reported without corrections 77

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3.3 Synthetic Procedures 3.3.1 Synthesis of 1-Amino-1-cyanocyclohexane (Compound I) Cyclohexanone (20 g, 0 2 mole) was dissolved in 250 mL of aqueous methanol. Ammonium chloride (15 g, 0 4 mole) and sodium cyanide (15 g, 0 .31 mole) were added to the solution, which changed from colorless to intense burgundy after a stirring period of 24 hours at room temperature. The reaction mixture was then diluted with water (30 mL) and extracted with ether (500 mL). The extract was washed with water, dried over MgS04 and concentrated to 300 mL. Dry hydrogen chloride gas was bubbled through the concentrated organic mixture to obtain a yellowish precipitate ( 17 0 g). Crystallization of the precipitate from absolute ethanol gave 13. 8 g of glistening white plates of Compound I. Yield: 54%. A ninhydrin positive spot at a Rf value of 0. 66 was obtained with TLC analysis of Compound I in chloroform/methanol (9:1) system Melting point: 173-180C (lit: 182C) 1H-N1v1R (D20): o (ppm) 2 1 (2 H, cyclohexane ring); 1.1-1.9 (6 H, cyclohexane ring); 0.9-1.1 (2 H cyclohexane ring). 13C-NMR (D20): o (ppm) 115.8 (CN); 51.03 (Ca cyclohexane ring) ; 31.88 (Cb cyclohexane ring); 21.48 (Cd cyclohexane ring); 20. 37 (Cc cyclohexane ring). 78

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3.3.2 Synthesis of 1-Aminocyclohexanecarboxylic Acid (Compound ll) Compound I (12.5 g, 0 1 mole) was suspended at ooc in concentrated hydrochloric acid (12M, 70 mL) and stirred at room temperature for 12 hours. The acidic suspension was then diluted with 40 mL of water and boiled under reflux overnight. After the refluxing period, rotary evaporation of the reaction mixture yielded a yellowish residue. The crude product was suspended with stirring in 40 mL of water and a saturated solution was used to neutralize the suspension. Compound II was then collected by filtration and dried over P 205 overnight. Yield: 73%. Melting point : >-300C (lit.:350C in sealed tubes). Compound II was analyzed by TLC in the chloroform/methanol (9 : 1) system A single, ninhydrin positive spot at the origin was obtained 1H-Nl\1R (D20/DCl):o (ppm) 1.7-1.82 (2 H, cyclohexane protons); 1.4-1.7 (6 H, cyclohexane protons); 1.1-1.4 (2 H, cyclohexane protons). 13C-Nl\1R (D20/NaOH):o (ppm) 183.88 (carboxylate group); 56.54 (Ca cyclohexane ring); 33.67 (Cb cyclohexane ring); 23.48 (Cd cyclohexane ring); 20.13 (Cc cyclohexane ring). 79

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3.3.3 Synthesis of 1,4-Diamino-1,4-dicyanocyclohexane (Compound Ill) The starting material1,4-cyclohexanedione (7.46 g, 0.066 mole)-was dissolved in 90 mL of a 50% solution of aqueous methanol. Ammonium chloride (7 08 g, 0. 13 mole) was then added to this solution with stirring. Once the ammonium chloride was totally dissolved, sodium cyanide (6.48 g, 0.13 mole) was added and the reaction mixture turned a deep yellow color almost immediately. The mixture was stirred at room temperature for 48 hours A precipitate formed It was collected by filtration washed with fresh aqueous methanol and partially dissolved in hot water for its recrystallization. A large fraction of the original crude product did not dissolve in hot water and was collected by filtration, dried, and weighed : 5 83 g. The filtrate was cooled at 0C overnight and the crystals that formed were collected by filtration dried, and weighed: 1.29 g Total yield: 7.12 g, 65%. Both fractions the insoluble fraction of the crude product and the fraction that recrystallized from hot water, were analyzed by NMR. The 1H-NMR spectra of the two fraction were identical indicating that both fractions corresponded to the same compound. The melting point of Compound III in open capillary tubes was 190192C. Compound III was dissolved in acidic solution (pH 2) for TLC analysis in chloroform/methanol (9 : 1) system An iodine ninhydrin positive spot with a Rf 80

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value of0.33 was obtained 1H-NMR (D20/DCl):o (ppm) 2 28-2 24 ( d, hydrogens of cyclohexane ring); 1.841.79 ( d, hydrogens of cyclohexane ring). The two doublets integrated to I: 1 ratio. 13C-NMR (D20/DCI):o (ppm) 117.18 (CN); 48.59 (Ca carbons of cyclohexane ring); 29.87 carbons of cyclohexane ring). 81

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3.3.4 Synthesis of 1,4-Diamino-1,4-Cyclohexanedicarboxylic Acid (Compound IV) Compound III (1.99 g, 0.012 mole) was suspended in 40 rnL of 12M hydrochloric acid solution and cooled at 0 o C. The reaction mixture was then stirred at room temperature for 48 hours. After the stirring period, the white suspension was diluted with 30 mL of water and heated under reflux for another 48 hours. The reaction mixture remained as a suspension changing its color to slightly yellow during the heating process. The mixture was then taken to dryness with an oil pump and the residue resuspended in 25 mL of fresh water. The acidic suspension was neutralized with a saturated solution of sodium carbonate. The desired cyclic diamino acid (Compound V) was collected by filtration, washed and dried over P205 in a desiccator. Yield: 62%. Melting point: >-260C. Compound IV was dissolved in acidic (pH 2) and basic (pH 1 0) solutions for TLC analysis in chloroforrn/methanol (9:1) system. A single, UV, iodine, and ninhydrin positive spot at the origin of the plate was obtained 1H-Mv.1R (D20/NaOD): o (ppm) 1.84-1. 79 ( d, hydrogens of cyclohexane ring); 1.31-1.26 ( d, hydrogens of cyclohexane ring). The two doublets integrated to 1 : 1 ration. 13C-NNIR (D20/NaOD): o (ppm) 183 .27 (carboxylate carbon); 55. 72 (Ca carbon of cyclohexane ring); 29 31 carbon of cyclohexane ring). 82

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1H-NMR (D20/DCI) : o (ppm) 2.10-1 .93 (m, hydrogens of cyclohexane ring) 13C-NMR (D20/DCI):o (ppm) 170.82 (COO H); 55.19 (Ca carbons of cyclohexane ring); 25.41 ( Cp carbons of cyclohexane ring) 83

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3.3.5 Synthesis of Methyl 1-Aminocyclohexanecarboxylate (Compound V) Dry hydrogen chloride was passed through a suspension of Compound II (1.51 g, 0.01 mole) in anhydrous methanol (150 mL) during 30 minutes. During this period of time, the reaction mixture was placed in an ice bath to avoid evaporation of methanol. The cloudy yellow solution was then sealed and stirred at room temperature overnight. After the stirring process, the reaction mixture was filtered and the clear filtrate taken to dryness using an oil pump. The residual product was partially dissolved in 10 mL of water and the pH value of the mixture was raised to 10 with a saturated solution of sodium carbonate The aqueous mixture was then extracted with ethyl acetate (3 x 30 mL). The combined organic fractions were dried with MgSO 4 and evaporated with an oil pump to obtain 1 mL of the oily Compound V. Yield: 53%. TLC analysis of Compound V in chloroform/methanol (9 : 1) system gave an elongated, UV, iodine and ninhydrin positive spot with a Rf value of0.55. 1H-NMR (D20/DC1) : o (ppm) 3 672 (3H, CH30-); 1.94-1.92 (2H, cyclohexane ring); 1. 66-1.3 7 (8H, the rest of the hydrogens in the cyclohexane ring) 13C-NI\1R. ( d6-DMSO) : o (ppm) 176 .26 (carbonyl group); 55.51 (Cu. cyclohexane ring); 50.39 (CH30-); 33.83 (C11 cyclohexane ring); 23.99 (C6 cyclohexane ring); 20 38 (Cy cyclohexane ring) 84

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3.3.6 Attempted Synthesis of 1,4-Dimethyl-1,4-diaminocyclohexane dicarboxylate (Compound VI) Three attempts were used to synthesize Compound XI by Fischer esterification (8). First attempt : Compound IV (0.4 g, 1.98 10-3 mole) was suspended in anhydrous methanol (50 mL). The suspension was placed in an ice bath and dry hydrogen chloride was passed into it for a few seconds to obtain a clear solution Further addition of hydrogen chloride gas (10 minutes) transformed the clear solution back to a suspension that was then stirred at room temperature .for 24 hours After the stirring period, a white precipitate was collected by filtration, dried, and weighed Yield: 0.36 g (90%). 1H-NMR analysis of the white precipitate (0.04 g) dissolved in 0.75 mL ofD20/NaOD solution showed no singlet at 3 8 ppm (CH3-CO). Resonances at 1.26, 1.31, 1. 79, and 1.84 ppm, corresponding to Compound IV, were obtained respectively The filtrate was taken to dryness with an oil pump to obtain a yellow residue that was redissolved in 10 mL of water. A 6 N solution of sodium hydroxide was used to raise the pH value of this sample to 12. Neither precipitate or separation of layers were seen at this pH value. The basic solution was analyzed by TLC in the chloroform/methanol (9 : 1) system 85

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A single, UV, iodine and ninhydrin positive spot at the origin of the plate was obtained. The reaction was stopped at this point. Second attempt: The white precipitate obtained in the first reaction (0.36 g, 90% of starting material) was partially dissolved in anhydrous methanol ( 40 mL). The mixture was again placed in an ice bath while dry hydrogen chloride was passed into it for a few seconds until a clear reaction mixture was obtained. The dry hydrogen chloride was then shut down, the clear mixture sealed and stirred at room temperature for 5 days. After the stirring period, the reaction mixture was taken to dryness using a rotary evaporator with an oil pump to obtain a white residue that was then mixed with 25 mL of water. The resulting suspension was brought to pH 10 with a solution of 6 N NaOH, resulting in a cloudy, pale yellow solution. The sample was then extracted with 10 mL of ethyl acetate ( x 2). Aqueous and organic fractions were analyzed by TLC in chloroform/methanol (9:1) system. Two concentrated, ninhydrin positive spots with a Rfvalues ofO.OO and 0.36 respectively were obtained for the aqueous fraction No components were detected by any of the three visualization methods for the organic fraction where the methyl ester was expected. The synthesis was stopped at this point 86

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Third attempt: A modified Fischer esterification reaction described by Sheppard et al. (3) was followed for this third reaction Dry hydrogen chloride was passed into a boiling mixture of Compound IV (0 22 g, 1 1 1 o-3 mole) and anhydrous methanol ( 10 mL) for an hour. The mixture was then refluxed for 12 hours and, after the heating period, the suspension was taken to dryness with a rotary evaporator attached to an oil pump. The brown residue was then partially dissolved in 20 mL of water and its pH was raised to a value of 12 with a solution of 6 N sodium hydroxide The precipitate was collected by filtration, washed with water, dried and weighed Yield: 0 .083 g. 1H-NMR analysis of the precipitate dissolved in CDCl3 showed no signal at 3.8 ppm (CH30CO-). The resonances seen in the 1H-NMR spectrum also did not correspond to those obtained for the starting material The filtrate was set aside overnight and some precipitate formed. The precipitate was collected by filtration and dried. 1H-NMR analysis of this precipitate dissolved in a D20/NaOD solution showed no singlet at 3.8 ppm but signals at 2 13, 2.16, 2.21 ppm respectively were obtained, matching the signals for the starting material in the same solvent system. The reaction was stopped at this point. 87

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3.3. 7 Synthesis of 1-tert-Butoxycarbonylaminocyclohexanecarboxylic Acid (Compound Vll) Compound II (2.86 g, 0.021 mole) was slowly dissolved in a solution of 4 N NaOH (5.75 rnL, 0.021 mole). This brownish clear solution was placed in a water bath at room temperature (approximately 20C). With vigorous stirring, 6 rnL of 4 N NaOH solution (0 .021 mole) and 6 rnL of a solution of di-tert-butyldicarbonate (0.021 mole) in toluene were simultaneously added, drop by drop, during a 25 minute period The reaction mixture was then stirred overnight. By the end of the stirring process, extensive precipitate was collected by filtration dried over P 205 and weighed Yield: 1.36 g, 47% of original starting material. A small portion of this precipitate was dissolved in water for TLC analysis in chloroform/methanol (9 : 1) system A single, iodine and ninhydrin positive spot at the origin was obtained The filtrate was then extracted with 30 rnL of ethyl ether The aqueous fraction was cooled in an ice-water bath and 3.5 rnL of concentrated HCl was added to the mixture A first crop of precipitate appeared in the slightly acidic reaction mixture (pH 5) and was collected by filtration. The acidic filtrate was then taken to pH 3 by the addition of more concentrated HCl solution A second crop of precipitate was collected by filtration and combined with the first crop. The combined crude product was dried in a desiccator. Yield : 1.32g 88

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The crude product was analyzed by TLC in the chloroform/methanol (9 : I) system A single, ninhydrin positive spot with a Rfvalue of0.2 was observed. Recrystallization of the crude product from ethyl acetate/petroleum ether gave 1.06 g ofCompound VII. Yield: 22%. Melting point : 176-178C. 1H-NMR (CDC13 ) :o (ppm) 1.43 (s, 23H, tert-butyl group); 1.98-1.61 (m, IOH, cyclohexane ring hydrogens); 4 8 (s, broad peak, -NH-). 89

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3.3.8 Synthesis of 1-Acetylaminocyclohexanecarboxylic Acid (Compound VITI) The synthesis of 1-acetylaminocyclohexanecarboxylic acid was achieved following the Schotten-Bauman method (12) using acetic anhydride Compound II (0.1 g, 7.10-4 mole) was suspended in 3 mL of water. A 10% NaOH solution (0 .28 mL, 7.1 0-4 mole) was added to the suspension and a clear brownish solution was obtained with gentle warming Acetic anhydride (0.076 mL, 8.12 10-4 mole) and 10% NaOH solution (0 28 mL, 7.10-4 mole) were divided in five equal fractions and alternatively added to the solution during a 15 minute period The reaction mixture was vigorously shaken after the addition of every fraction of acetic anhydride A time gap of a few seconds was allowed between the addition of the NaOH and acetic anhydride solutions to assure that all the anhydride was consumed in the reaction and therefore, was not hydrolysed by the base. After the addition, the reaction mixture was acidified to pH 3 using a 1 N solution ofHCl. The precipitate which formed was collected by filtration dried over P 205 and weighed. Yield : 0.043 g, 33%. Melting point: 106-108C. 1H-NMR (D20/Na0D): o (ppm) I. 784 (3H, CH30); t865-l. 738 (2H, H cyclohexane ring); 1.518-1.104 (8H, the rest of hydrogens from the cyclohexane ring). 90

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13C-NMR (D20/Na0D): o (ppm) 180 68 (carbonyl of carboxylate group); 171.39 (carbonyl of acetyl group); 59.49 (Ca cyclohexane ring); 30.61 (Cp cyclohexane ring); 23.54 (C5 cyclohexane ring); 20.63 (methyl group) ; 19. 56 (Cy cyclohexane ring) 91

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3.3.9 Synthesis of 1,4Diacetylamino-1 ,4-cyclohexanedicarboxylic Acid (Compound JX) Synthesis of Compound IX was achieved following the procedures described by Shotten and Bauman (11) Compound IV (0 .05 g, 2.47 104 moles) was mixed with 5 mL ofwater, and 0.2 mL of a 10% NaOH solution (5 104 moles) was added to the suspension. A clear brownish solution was obtained after gentle heating (3 040C). With vigorous stirring, acetic anhydride (0.056 mL, 5 104moles) and 10% NaOH solution (0.2 mL, 5 104 moles) were added alternatively to the mixture during a 10 minute period. The reaction mixture was then stirred overnight at room temperature. After the stirring period, the cloudy solution was filtered. The clear filtrate was acidified to pH 2 with a 6 N HCl solution and set aside for 10 minutes Some precipitate formed from the clear solution. It was collected by filtration, dried over P205 and weighed. Yield : 0.01 g (14%). Melting point: >-260C. Compound IX was dissolved in basic solution for TLC analysis A single, ninhydrin positive spot at the origin of the plate was obtained. 1H-NMR (D20/NaOD): o (ppm) 1.81 (s, CH3CO-); 1.73-1.63 (m, H cyclohexane ring). 13C-NMR (D20/NaOD):o (ppm) 179.93 (Coo-); 171.89 (CH3CO-); 58 (Ca cyclohexane ring); 25. 62 (Cb cyclohexane ring); 20 .63 (CH3-). 92

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3.3.10 Synthesis of 1-(9-fluorenylmethyloxycarboxyl)amino cyclohexane carboxylic Acid (Compound X) Synthesis of Compound X was carried out following a different approach from that used for the amino protection reaction with the Boc group due to the low yields obtained in that case. A solution of0.3 M potassium hydroxide in 18-crown-6 ether was prepared by first dissolving 0. 3 6 g ( 1. 4 1 o-4 mole) of 18-crown-6 ether in 4 mL of anhydrous methanol, and then adding potassium hydroxide (86% pellets, 0 092 g, 1.4 104 mole) to the mixture to obtain a clear solution. The solution was diluted to 12 rnL with the addition of fresh dioxane, concentrated to 4 mL by rotary evaporation, diluted again to 12 mL with fresh dioxane and finally concentrated to 3 rnL, to obtain a greenish cloudy solution. The mixture was then divided into two equal fractions. One of the 18-crown-6/KOH fractions was diluted to 3 mL with fresh dioxane and Compound II ( 0.104 g, 7.1 04 mole) was added to it with vigorous stirring. The resulting suspension was diluted with 0.32 mL ofwater (11% ofthe total volume) and gently heated ( 30-40C) to obtain an almost clear solution To this mixture, a solution of9-fluorenylmethyl chloroformate (Fmoc-Cl, 0 188 g, 7.104 mole) in 0.5 mL of dioxane was added dropwise to obtain a cloudy solution with a pH value of7. 93

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The second fraction of 18-crown-6/K.OH was then added dropwise. Most of the cloudiness of the reaction mixture faded and the pH rose to 10. The reaction was stirred for three days at room temperature. After the stirring period, some precipitate was removed by filtration and the filtrate was mixed with 25 mL of a 1 : 1 ethylacetate/water mixture. The organic layer was separated and then reextracted with water. The combined aqueous fractions were acidified to a pH value 2 with a 6 N HCl solution and then extracted with 50 mL of ethyl acetate. The organic fraction was dried over MgS04 and evaporated by a rotary evaporatoration to obtain Compound X in its crude form as an oil. Compound X was not further purified and all the available data was collected from its crude form. Yield: 44%. 1H-NMR (CDC13):o (ppm) 9.25 (s, broad peak, -COOH); 7.69-7.18 (m, 8H, aromatic hydrogens); 5.1 (1H, broad peak, -NH-);4.35-4.33 (d, 2H, -CH2-); 4.17-4.14 (t, 1H, -CH-); 1.77-1.22 (m, IOH, cyclohexane ring); 1.98 (s, acetone peak). 13C-NMR (CDC13):o (ppm) 177.87 (-COOH); 154.17 (-CO-NH); 142.40,139.85, 126.18, 125.60, 123.58 and 118.44 (aromatic carbons ofFmoc group); 65.44 (sp3 carbon ofFmoc group); 57.45 (Ca carbon ofcyclohexane ring); 45.66 (-CH2 ofFmoc group); 30.73, 23.49 and 19.53 (Cb, Cd and Cc ofcyclohexane ring); 68.56, 58.94 and 12.57 are impurities. 94

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3.3.11 Attempted Synthesis of 1,4-Di [(9-fluorenyl)amino]-1,4cyclohexanedicarboxylic Acid (Compound XI) The low solubility of Compound IV in organic solvents made neccesary the introduction of a phase transfer catalyst in the solvent system used for the amino protection reaction_ The 18-crown-6 ether/KOH catalyst solution was prepared as described in page 98, and then divided into two equivalent fractions with a total volume of 1.5 mL_ Each individual solution contained two equivalents ofKOH and 18-crown-6 per mole of Compound IV Compound IV (0 05 g, 2.47 104 mole) was mixed with the first fraction ofthe 18-crown-6/K.OH solution previously diluted to 5 5 mL with fresh dioxane_ The suspension was stirred and gently heated (30-40C) while l mL of water was introduced to obtain an almost clear solution To this mixture, a solution ofFmoc-Cl (0.13 g, 5 104 mole) in 1 mL of dioxane was added dropwise during a 20 minute period Extensive precipitate formed during this addition and the resulting suspension was then stirred at room temperature for 40 minutes After the stirring period the second 18-crown-6/K.OH solution was added dropwise to the mixture during another 20 minute period and the heterogeneous suspension was then stirred overnight at room temperature. The next morning, the mixture was filtered and the yellow filtrate extracted with 25 mL of a 50% water/ethyl acetate mixture ( x2)_ 95

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The cloudy aqueous fraction was acidified to a pH value of 2 with a 6 N HCl solution and then extracted with 10 mL of ethyl acetate ( x 3). The organic phase was dried over MgSO 4 filtered and taken to dryness to obtain a brown residue The brown residue weighed 0 02 g Yield : 12%. The residue was dissolved in 0.75 mL of d6-DMSO for 1H-NMR. analysis. Signals at 7 86-7 25 ppm (aromatic protons) were obtained The expected characteristic chemical shifts 4 3 4 2 2.10 and 1.93 ppm were not observed 96

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3.3.12 Synthesis of 1-Formylaminocyclohexanecarboxylic Acid (Compound XII) Compound XII was synthesized according to the procedures described by Kenner, Preston, and Sheppard (3). 1-Aminocyclohexanecarboxylic acid (0.1 g, 7 .10-4 mole) was dissolved in 2 mL of 96% formic acid. The acidic solution was placed in an ice water bath to maintain its temperature around 5-10C while an excess of acetic anhydride (0 6 mL, 6.35 10-3 mole) was added in six equal fractions of 0.1 mL each during a 15 minute period. After all the acetic anhydride has been added, the reaction mixture was stirred at l2C for 30 minutes The clear mixture was then stirred at room temperature for 2 hours before being diluted with 0.6 mL of ice cold water. The diluted solution was evaporated giving a white precipitate as the crude product. Recrystallization of the crude product from water yielded 0.07 g of Compound XII. Yield: 58%. Melting point: 190C (lit: C). 1H-NMR (D20/NaOD): o (ppm) 7.87 and 7.76 (s, : 1H, HCO-); 1.81-1.09 (m, 10H, cyclohexane protons). 13C-NMR (D20/NaOD): o (ppm) 180.01 (-coo-); 166.05 and 161.55 (HCO-); 59.39 and 59.19 (Ca cyclohexane ring); 32.6 and 30. 6 (Cbcyclohexane ring); 23 17 and 22.99 (Cd cyclohexane ring); 19.4 and 19.23 (Cc cyclohexane ring). 97

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3.3.13 Synthesis of 1,4-Diformylamino-1,4-cyclohexanedicarboxylic Acid (Compound Xlll) Synthesis of Compound XIIT was achieved following the procedures described by du Vigneaud (9) Compound IV (0 .093 g, 4.6 104 mole) was dissolved with stirring and gentle heating in 5 mL of 96% formic acid. The solution was then placed in a water bath at 50 55C. An excess of acetic anhydride (1 mL, 0 .01 mole) was added dropwise to the mixture during a 15 minute period. Extensive precipitate formed in the reaction mixture after 2/3 of the total acetic anhydride was added After all acetic anhydride had been added, the resulting suspension was stirred at room temperature for 2 hours, at the end ofwhich, 1 mL of ice-water was introduced in the reaction mixture The precipitate was then collected by filtration, washed with hot water and dried Yield : 0.057 g, 47%. Melting point :>-260C. Compound XIII was dissolved in D20/NaOD for TLC analysis in the chloroform/methanol (9: 1) system A single, ninhydrin positive spot at the origin was obtained 1H-NMR (D20/Na0D): o (ppm) 7 87 (s, formyl group); 7 .83 (s, formyl proton); 1.87-1.71 (m, 8H cyclohexane ring). 13C-NMR (D20/NaOD): o (ppm) 179.13 (Coo-); 166.45 and 162.02 (HCO-); 58.75 and 58.12 (Ca cyclohexane ring); 28.56, 26.91, 25.63 and 25.25 (Cb cyclohexane ring). 98

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3.3.14 Attempted Synthesis of 1-Trifluoroacetylamino cyclohexanecarboxylic Acid (Compound XIV) Synthesis of Compound XIV was attempted following the procedures described by G .W. Kenner et al. (3) The synthesis of Compound XIV calls for trifluoroacetic anhydride, which was synthesized using trifluoroacetic acid and 1 ,3-dicyclohexylcarbodiimide (DCC) Triflouroacetic anhydride solution: DCC (0.1 g, 2 104 mole) was mixed with a minimum amount of ether until a clear solution was obtained Trifluoroacetic acid ( 0 1 mL, 1 23 1 o-3 mole) was added to the DCC solution with formation of precipitate and evolution of heat. The suspension was stirred for less than a minute before the precipitate dissolved completly in the reaction mixture giving a clear solution that was then stirred at room temperature for 1 0 minutes. After the stirring period the sample was analyzed by TLC in chlorofonn/methanol (9 : 1) system. Four spots developed on the plate Two concentrated spots with Rfvalues of0. 92 (UV positive) and 0.66 (iodine positive) were assigned to DCC and DHU respectively Also a concentrated spot staining negatively with ninhydrin with a Rf value of0.46 and an elongated, UV positive spot with a Rfvalue of0.16 were observed 99

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The trifluoroacetic anhydride mixture was added dropwise to a solution of Compound II (0.05 g, 1.4 104 mole) in trifluoroacetic acid (1.5 mL). The reaction mixture was then stirred at room temperature overnight at the end of which, it was taken to dryness using the rotary evaporator to obtain 0.4 g of a yellow oil. The residue was then separated into two approximately equal fractions to carry out two separate experiments. First experiment: The first fraction of yellow oil was dissolved in ethyl ether (1 0 mL) and extracted with 20 mL of0.05 M NaOH solution. During the extraction, intensive precipitate formed in the aqueous fraction Precipitate was then collected by filtration and a small portion of it dissolved in water for TLC analysis in the chloroform/methanol (9: 1) system A single, iodine positive spot with the characteristic Rfvalue for DHU was observed. The filtrate's volume was reduced by one-half using the rotary evaporator and the concentrated solution was acidified to pH 3 with 1 N HCl solution. Neither formation of precipitate nor separation of layers was seen The acidic solution was analyzed by TLC in the chloroform/methanol (9: 1) system. No components were detected on the TLC plate by any of the three visualization methods. 100

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Second experiment : A mixture of sodium ethoxide (0.35 g, 5.15 10-3 mole) in 8 rnL of anhydrous ethanol (6 28 g, 0 136 mole) was incorporated with stirring to the second fraction of the oily residue The suspension was then analyzed by TLC in the chlorofonnlmethanol (9 : 1) system. A single ninhydrin positive spot at the origin was observed. 4 101

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3.3.15 Attempted Synthesis of 1,4-Ditrifluoroacetylamino-1,4cyclohexanedicarboxylic Acid (Compound XV) Compound IV (0 .21 g, 1.04 10-3 mole) was dissolved with stirring and gentle heating in 8 rnL of 96% formic acid. The mixture was then placed in a water bath at a temperature of50-55C. A solution oftrifluoroacetic anhydride (4.37 rnL, 0.031 mole) in 5 mL of 96% formic acid was then added dropwise to obtain a slightly cloudy solution that was stirred for one hour at room temperature After the stirring period, the solution was filtered The filtrate was twice diluted with ice water and the solutions twice evaporated to eliminate any traces of formic acid in the sample. The residue was finally dissolved in ice water and extracted with 10 rnL of chloroform ( x 2) The aqueous fraction was taken to dryness and the white residue suspended in hot water for its recrystallization The hot suspension was filtered and the collected precipitate was then dried and weighed. Yield: 0.07 g (33%) A fraction ofthe white precipitate (0 .03 g) was dissolved in a D20/NaOD system for 1H-NMR analysis. No signals at 8 ppm (formyl group) were observed. Resonances at 1.84, 1.79, 1.31, and 1.26 ppm, corresponding to the starting material, were seen 102

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3.3.16 Characterization ofDiastereoisomers of Compound IV To test for the possible presence of stereoisomerism in Compound N a simple chemical procedure was tried. Compound IV (0.2 g, 9.9 10-4 mole) was suspended in 20 mL ofwater. To this mixture, 0.79 mL of a 10% NaOH solution (1.98 10-3 mole) was added with stirring to obtain a clear mixture. A solution ofCaC12 (0.22 g, 1 98 10-4 mole) in 3 mL of water was added dropwise to the clear solution and the mixture was then stirred at room temperature for a few minutes before some precipitate formed in solution. The resulting suspension was left stirring at room temperature for one hour. The precipitate was then collected by filtration, dried and weighed. Yield: 0 22 g, 91% The filtrate was neutralized with a 6 N HCl solution and then stirred overnight at room temperature. The next morning, some precipitate was collected by filtration, dried and weighed. Yield: 0.03 g, 12%: To determine if there were solubility differences between the first (91%) and the second ( 12%) crop of collected precipitates, the latter one was treated again with CaCl2 The total amount ofthe second crop of precipitate (0.03 g) was mixed with 3 mL of water. To the resulting mixture, 0 12 mL of a 10% NaOH solution was added obtaining a clear solution that was then allowed to stir at room temperature while a solution ofCaCl2 (0 018 g) in 0.64 mL ofwater was incorporated dropwise. 103

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Five minutes after the addition, precipitate formed in solution indicating that the formerly soluble molecules could be precipitated under these conditions. 104

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4. Conclusion for Future Studies During the first part of the research, the compound 1, 4-diamino-1, 4-cyclohexane dicarboxylic acid was synthesized To use it as a monomer for a possible polymer, suitable derivatives needed to be synthesize. Numerous amino protection reactions were studied for this monomer. The majority of them failed to protect the amino functions. Two major obstacles were encountered during these reactions : the poor solubility of the monomer in organic solvents and its apparent hindrance. The monomer is only soluble in acidic and basic aqueous systems. Meanwhile, the acylating agents most commonly used to protect amino groups (Fmoc-Cl, Z-Cl and (Boc)20) are primarily soluble in organic solvents and poorly soluble in water. To facilitate the reaction between the monomer and the protecting groups, three approaches were followed during these studies : a first approach was to carry out the amino protecting reactions in a two-phase solvent system (e.g. water/toluene) as with the acylation of the amino function by Boc anhydride. A second one was to carry out amino protecting reactions were done using the phase transfer catalyst, 18-crown-6 ether, as in the acylation ofthe amino function by Fmoc-CL Neither of the two systems gave good results for these reactions. A third approach was to 105

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carry out the amino protection reactions in an acidic medium such as formic acid (formylation reaction) or basic medium such as sodium hydroxide solution (Shotten and Baumann conditions) for acetylation reactions. These systems were very successful giving the formyl and acetyl derivatives of the monomer in good yields The formylation reaction was carried out in acidic aqueous medium Both the monomer and the acetic anhydride, used as reagent, were soluble in this solvent system. Also, the fortnyl group is less bulky than some of the common amino protecting groups (Z Fmoc and Boc groups) This group could have introduced less repulsion at the already crowded cyclohexane ring during the reaction. A 47% yield was obtained for this amino protection reaction. Formyl groups have been reportedly cleaved using mild acidic conditions. The cleavage of the formyl group from the monomer needs to be studied to find out the optimum conditions for this reaction. Also the formylated monomer needs further derivatization before it can be polymerized. The acetylation reaction was carried out to obtain information about the necessary conditions to synthesize the trifluoroacetyl derivative. The synthesis of this derivative was also tried using the Shotten and Baumann conditions but the reaction was a failure. This reaction needs further investigation. 106

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Based on our experiments, the amino protecting reactions that gave good results were those in which the acylating agents and the monomer were soluble in the same solvent system. Therefore, further work needs to be done to find a solvent system where the acylating agents and our monomer could co-exist during the reaction Ester formation is the most common form of carboxyl protection of amino acids. In this research, the synthesis of the methyl ester of our polymerization monomer was repeatedly tried without success. This reaction was carried out following the standard Fischer procedures. The protection of the carboxyl function is a necessary priority to be able to polymerize the molecule 1, 4-diamino-1, 4-cyclohexane dicarboxylic acid in a stepwise fashion Formation of the methyl ester by the use of methyl iodide may be a possible alternative to Fischer esterification. Also, an organic solvent system needs to be found to carry out the polymerization reactions. Compound 1, 4-diamino-1, 4-cyclohexanedicarboxylic acid can present two diastereoisomers. The trans diastereoisomer seems to be the more stable one. The polymerization monomer was, therefore, assumed to be found only as the trans isomer. Through the research, there was no evidence of the monomer present in both forms. Even though some studies were done to search for the diastereoisomerism ofthe monomer, the assumption remains to be proven 107

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5. References 1. Sheppard, R.C.; Atherton, E (1990) Solid Phase Peptide Synthesis: A Practical Approach, VHS New York, p 75-85 2 Cremlyn, R.J.W., J. Am Chem. Soc., 1962, 3977-3980 3 Kenner, G.W.; Preston, J.; Sheppard, R.C., ]. Am. Chern. Soc., 1965, 18, 6239-6244 4 Carpino, L.A.; Han, G Y., ] Org. Chern., 1972, 22, 3404-3409 5. Tsukube, H. (1992) Crown Ethers and Analogous Compounds, Hiraoka,M. (Ed ), Elevier Science Publishers, p. 100-110 6 Gokel G W.; Durst, H. D., Synthesis 1976, 168-184 7 Fieser, M.; Fieser, L. (1969) Reagents for Organic Synthesis, Vol. 2, Wiley Interscience, p.1 0-13 8. Sheehan, J.C. ; Yang, D H., J. Am. Chern. Soc., 1958, 80, 1154-1158 9 du Vigneaud, V.; Dorfinann R.; Loring, H.S., J. Bioi. Chern., 1932, 98, 577 10 Weygand F.; Geiger, R., Chern. Ber., 1955, 647-654 11. Greenstein, J P.; Winitz, M (1961) Chemistry of The Amino Acids, John Wiley and Sons (Ed ) p.582-584, 920-917 108

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12. Bodanszky M. ; Wieland, T (1991) The World of Peptides: A Brief History of Peptide Synthesis, Springer-Verlag (Ed ) 13. Paul, P K.C.; J Amer. Chern. Soc., 1986, 108 6363 14. Bardi R.; Int. J. Pept. Protein Res., 1985, 25 628-639 15. Hofmeister, F.; Naturwiss. Rundschau., 1902, 17, 529-545. 16. Fischer, E ; Hoppe-Say1er's Zetschr. Physiol. Chern. 1901, 33 151-171. 17 Bodanszky, M.; Nature 1955 175, 685. 18. Sheenan J.C. Hess, G.P. ; J Amer.Chem. Soc., 1955 77 1067-1068 19. Bergmann M. Zervas, L.; Chern. Ber., 1932 65, 1192-1201. 20. Carpino L.A.; J. Amer. Chern Soc 1957, 79, 98-.101. 21. McKay F.C. Albertson, N.F ; J. Amer. Chern Soc. 1957, 79, 4686-4690. 22. Carpino L.A. Han G Y.; J Amer. Chern. Soc. 1970, 92 5748-5749 23. Berlinguet L., Tailleur P.; Can. J. Chern., 1961, 39 1309-1320. 24 Jones, J. in Amino Acid and Peptide Synthesis; Oxford Science Publications; New York, 1992. 25 Kopple, K.D in Peptides and Amino Acids; W .A. Benjamin ; New York l966. 26 Eliel, E L. in Conformational Analysis; John Wiley & Son ; New York, 1965 27 Bodanszky, M. in Peptide Chemistry : A Practical Te:A1:book; Springer-Verlag; Berlin, 1988 109

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28. Eliel E.L. Wilen, S.H. in Stereochemistry of Organic Compounds; John Wiley & Son; New York, 1994. 29. Kupriszewski, G.; Rocz. Chern., 1961, 35, 595. 30 Kovacs, J.; Kisfaludi L. ; Ceprini, M. Q.; J. Amer. Chern. Soc., 1967, 89, 183-184 31. Leuchs, H ; Chern. Ber., 1906 69, 857 32 Pacsu E.; Wilson, E.J.; J. Amer. Chern. Soc., 1942, 64, 2268-2271. 33 Winstein S ; Holness, N J.; J. Amer. Chern Soc., 1955, 77 5562. 34. Bodanszky, M ; Bodanszky, A. in The Practice of Peptide Synthesis; Springer Verlag Heidelberg; New York, 1984 110