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Exploration of a new synthetic route to 2,4-diamino glutaric acid

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Title:
Exploration of a new synthetic route to 2,4-diamino glutaric acid
Creator:
Miller, Nathaniel John
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Language:
English
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45 leaves : ; 28 cm

Thesis/Dissertation Information

Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry

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Subjects / Keywords:
2,4 diamino glutaric acid -- Synthesis ( lcsh )
Methionine ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 44-45).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Nathaniel John Miller.

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University of Colorado Denver
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Auraria Library
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ocm53993717
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LD1190.L46 2003m M54 ( lcc )

Full Text
EXPLORATION OF A NEW SYNTHETIC ROUTE TO 2,4-DIAMINO
GLUTARIC ACID
by
Nathaniel John Miller
B.S., University of Denver 1998
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2003
(A
i,.,-
*
*
t


This thesis for the Master of Science
degree by
Nathaniel John Miller
has been approved
by
Robert Damraurer
U;2aoZ
Date


Miller, Nathaniel John (M.S., Chemistry)
Exploration of a New Synthetic Route to 2,4-Diamino Glutaric Acid
Thesis directed by Professor Douglas F. Dyckes
ABSTRACT
A new route to the synthesis of 2,4-diamino glutarate was explored. Homoserine
lactone bromide salt (Compound IA) was produced by the reaction of methionine
and cyanogen bromide. It was converted to homoserine lactone nitrate salt
(Compound IB) which then was acylated to produce acetylhomoserine lactone
(Compound II). Ammonolysis was carried out with Compound II to yield
acetylhomoserine amide (Compound El). The attempted oxidation of Compound
m was conducted using three different oxidants. Pyridinium chlorochromate
(PCC) was first used but was found to contribute to the breakdown of Compound
III. Swem oxidation was also examined, but was found to be unsuccessful due to
the lack of solubility of Compound in. Pyridinium dichromate (PDC) was then
used, but the polar nature of the solvent required may have prevented or caused
over oxidation of Compound III.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Signed
m
Douglas F. Dyckes


CONTENTS
Figures..............................................................vi
Schemes....................;.........................................vii
Chapter
1. Introduction..........................................................1
1.1 Goal.................................................................1
1.2 Peptides.............................................................5
1.2.1 Peptide Synthesis..................................................6
1.2.2 Peptide Bond Formation.............................................6
1.3 Protection of the Functional Groups..................................7
1.3.1 N Protection.......................................................8
1.4 Racemization........................................................9
1.5 Diaminodicarboxylic Acids......................................... 9
1.6 Lactonization......................................................10
1.7 Acetylation........................................................12
1.8 Ammonolysis........................................................14
1.9 Oxidation..........................................................16
2. Results and Discussion...............................................19
IV


2.1 Synthesis of Homoserine Lactone Nitrate Salt
(Compound IB)..................................................19
2.2 Synthesis of Acetylhomoserine Lactone
(Compound II)..................................................23
2.3 Synthesis of Acetylhomoserine Amide
(Compound III).................................................28
2.4 Attempted Oxidation of Acetylhomoserine Amide..................33
2.5 Conclusion.....................................................34
3. Experimental...................................................36
3.1 Reagents.......................................................36
3.2 General Methods.............................................. 36
3.2.1 Thin Layer Chromatography....................................36
3.2.2 Nuclear Magnetic Resonance.................................. 37
3.3 Synthetic Procedures...........................................38
3.3.1 Synthesis of Homoserine Lactone Nitrate Salt
from Methionine (Compound IB).................................38
3.3.2 Synthesis of Acetylhomoserine Lactone
(Compound II).................................................39
3.3.3 Synthesis of Acetylhomoserine Amide
(Compound HI).................................................40
3.3.4 Attempted Oxidation of Acetylhomoserine Amide................41
References.........................................................44


FIGURES
1.1.1 Methionine and 2,4-Diamino Glutaric Acid........................1
1.1.2 The S,S and the S,R Configurations of 2,4-Diamino Glutaric Acid.2
1.1.3 Internally Cyclized Products of 2,4-Diamino Glutaric Acid........3
1.1.4 A Cyclic Oligomer of R,R or S,S 2,4-Diamino Glutaric Acid........4
1.1.5 Two Views of the R,S or S,R Polymer of 2,4 Diamino Glutaric Acid.5
2.1.1 ]H NMR Spectra of Compound LA..................................20
2.1.2 13C NMR Spectra of Compound IA.................................22
2.2.1 H NMR Spectra of Compound II..................................25
2.2.2 2D NMR Spectra of Compound II..................................26
2.3.1 !H NMR Spectra of Compound M...................................29
2.3.2 Representation of the trans and cis States
of Acyl-Homoserine Amide..........................................31
VI


SCHEMES
Scheme I. Peptide Bond Formation.....................................6
Scheme II. The Synthesis of Homoserine
Lactone Bromide Salt (Compound 1A) From Methionine.......11
Scheme III. The Synthesis of Acetylhomoserine
Lactone (Compond IT).....................................13
Scheme IV. The Synthesis of Acetylhomoserine
Amide....................................................15
Scheme V. Proposed Oxidation of Acetylhomoserine Amide
Using PDC................................................17
Scheme VI. Schematic Representation of Swem
Oxidation...............................................18
vii


1. Introduction
1.1 Goal
The goal of this research was to explore a new synthetic route to 2,4-
diamino glutaric acid (Fig. 1.1.1) from methionine.
CH3
S
ch2
ch2
CH
/ \
h3n co2

o2c

nh3
\/
CH
CH,
CH
/\ e
h3n co2
Figure 1.1.1 Methionine and 2, 4-Diamino Glutaric Acid.
The main challenge of this work was to figure out a synthetic pathway that was
efficient and produced 2,4-diamino glutaric acid in sufficient yield. What is
unique about this process is that one of the stereocenters of the product is
predetermined by using L-methionine. There are other ways to produce this 2,4-
diamino glutaric acid [1], but they result in three possible isomers, the R,R, S,S
and R,S (which is identical to the S,R). Naturally occurring amino acids are used
widely as a source for the synthesis of chiral compounds. They present an
1


attractive source of starting materials that can be used in asymmetric synthesis
[2]. Because L-methionine was used as the starting material, only two possible
isomers of the product will be formed. They are the S,S and S,R molecules (Fig.
1.1.2).
H
H
=
h3n*^^co2
,
H3N'
ch2
H3N^^ i^C02
H
H
Figure 1.1.2 The S,S and the S,R Configurations of 2,4-Diamino Glutaric Acid.
The two molecules shown in Fig 1.1.2 are diastereomers and can be separated.
Part of the long term goal of this study was to use the information obtained in the
separation of the two diastereomers to help identify the structures of isomers
from other synthetic pathways.
Another long term goal in this study was to look at how 2,4-diamino
glutaric acids can be internally cyclized and then polymerized. These isomers can
be internally cyclized to produce a five membered ring with a free amine and a
2


free carboxy terminus. The four possible stereoisomers of this cyclic molecule
are seen below (Fig 1.1.3).
o 0 0
A
NH or [ NH 0R L NH
V COOH XOOH COOH
R.R s,s S,R
Figure 1.1.3 Internally Cyclized Products of 2,4-Diamino Glutaric Acid.
From the four possible isomers of 2,4-diamino glutaric acid, one can create two
types of polymers, cyclic or linear. When the R,R or S,S isomers are
polymerized, they can form cyclic oligomers such as the one shown in (Fig
1.1.4). Such structures resemble naturally occurring ionophores.
3


o
o
Figure 1.1.4 A Cyclic Oligomer of R,R or S,S 2,4-Diamino Glutaric Acid.
When either the R,S or S,R isomer is polymerized, it is expected to form a linear
polymer that structurally resembles a stair case. The amide bond is seen as the
vertical component of the stair case, while the ring is seen as the horizontal
component of the stair case. The top view can be seen to the left and a side
profile can be seen to the right in Figure 1.1.5.
4


Figure 1.1.5 Two Views of the R,S or S,R Polymer of 2,4-Diamino Glutaric
Acid.
1.2 Peptides
a-Amino acids are chemically varied but all contain a carboxylic acid
group and ah amino group connected to the a-carbon. They serve as the building
blocks for proteins. Peptides are relatively small polymers of amino acids. Their
polymeric backbone is connected by an amide bond between the carboxy
terminus of one amino acid to the amino terminus of the next amino acid.
Peptides may be either linear or cyclic. Cyclic peptides have variable ring sizes.
Twenty common amino acids are found in proteins, and they all have different
side chains attached to the a-carbon. The chemical diversity proteins show arises
from the inherent properties of their amino acids. Fundamentally, the properties
5


of the amino acid side chains and their sequence determine the properties of the
protein and create a diverse set of functions for proteins [3].
1.2.1 Peptide Synthesis
In the past, peptide synthesis was an elaborate, time-consuming process.
Experiments that took years to complete are done in days now [4], thanks to solid
phase synthetic methods. Biologically, peptides and proteins can be considered
as encoded molecules, which serve a very specific function. Peptide synthesis
was used to determine the two possible configurations of 2,4-glutaric acid
through a series of chemical reactions from methionine.
1.2.2 Peptide Bond Formation
The formation of a peptide bond results in a dipeptide. This occurs
formally with the elimination of water (Scheme I).
O
Scheme I. Peptide Bond Formation
6


For this reaction to occur the carboxy terminus of one amino acid must be
activated, allowing the nucleophilic attack of the amino component to form the
peptide bond. The key to controlled peptide bond formation is the use of
protection groups, which eliminates the formation of other possible peptide
bonds [5]. The first step of peptide synthesis is the protection of the amino group
on the a-carbon of the amino acid. This also serves to eliminate the zwitterions
present as seen in (Fig 1.1.1). In the second step the N-protected amino acid must
be activated at the carboxy functional group. This allows the third step, formation
of the peptide bond. The fourth step is the removal of one or more protective
groups, in order to add another amino acid, or to end synthesis when the chain is
fully assembled.
1.3 Protection of the Functional Groups
There are two types o f protecting groups used in peptide synthesis. They
are temporary and semiperminant. The temporary protecting groups are most
often used for short-term protection of the amino or carboxy termini. They are
removed for the extension of the chain [6]. These groups must not interfere with
the reaction process, and must be able to be selectively cleaved at any point.
Semiperminant protecting groups are selectively cleaved at the end of peptide
synthesis. Protection of functional groups follows five simple rules.
7


Addition of the protecting group should lead to an amino acid that is no
longer in its zwitterionic form [6].
Cleavage must be done such that it does not alter the essential structure of
the amino acid.
The amino acid must not be racemized.
The inteimediate(s) should be stable and easily characterized.
The solubility of the protected amino acid should be favorable.
1.3.1 N Protection
The amino group can be blocked reversibly by acylation, alkylation, and
alkyl-acylation. There have been several hundred different types of N- protecting
groups developed over the years, which suggests that they can be tailored to
different reaction conditions [6]. They can be categorized two different ways, one
through their structure and the other through the cleavage condition. There are
various cleavage conditions: acidolysis, base cleavage, reduction/oxidation,
nucleophilic substitution, and photolysis [6].
Two frequently used examples of protecting groups are the tert-
butoxycarbonyl (Boc) group and the 9-fluorenylmethoxycarbonyl (Fmoc) group.
The Boc protecting group can be cleaved relatively easily in mild acidic
conditions, while the 9-fluorenylmethoxycarbonyl group (Fmoc) can be cleaved
8


under mild basic conditions [8]. Fmoc is the preferred method of amino
protection when a synthetic route requires acidic conditions. Protection groups
that are chosen must be easily cleaved under conditions that do not allow the
degradation of the peptide.
1.4 Racemization
The risk of racemization is an important factor to consider when dealing
with functional groups connected to the stereogenic center (in this study the a-
carbon in L-methionine). This is an important factor when peptide synthesis
occurs in several steps [10]. Each step could potentially alter one or more of the
stereogenic centers. The preservation of all stereogenic centers is of great
importance. The biological activity of peptides and amino acids normally
depends on the configuration of the stereogenic centers.
1.5 Diaminodicarboxylic Acids
Diaminodicarboxylic acids play an important part in biochemistry. They
can be complexed with transition metals to inhibit tumor activity and also be
used to inhibit the growth of bacteria [9]. Diaminodicarboxylic acids can be
found in a wide variety of cell types, especially in the cell wall. They serve as
cross-linking agents in all gram-positive and some gram-negative
peptidoglycans. They commonly serve as anchors of membrane-associated
macromolecules, such as lipoproteins in the cell wall [5,6], Methods of
9


synthesizing these compounds can be lengthy and time-consuming especially
because of the need for precise stereochemistry. The main goal of this research
project was to synthesize 2,4-diamino glutaric acid using a relatively abundant
amino acid (L-methionine) as a precursor, and to carry out the conversion in a
series of steps that would be both cost effective and efficient.
1.6 Lactonization
Lactones are cyclic, intramolecular esters derived from a hydroxy acid
[10]. Lactones are defined as a, |3, y, or 8 depending on the position of the
esterfied hydroxyl group. The most common lactones consist of five or six
membered rings. Small lactones are often very soluble in aqueous solutions and
pose a challenging problem when organic solvents are needed to carry out
modification.
The first step in this research was to chemically cleave the thioether bond
found in methionine using cyanogen bromide[l 1]. The nature of this cleavage is
very selective. It makes use of the intramolecular nucleophilic attack by the
amino acid carbonyl on the side chain after it has been activated by reaction with
an electrophilic reagent [12, 13]. It has been shown that cyanogen bromide is
specific for the cleavage of the methyl-thio group in methionine. The mechanism
of this reaction is shown below (Scheme II).
10


H O

h3n-

h3n-
H O
OH
ch2-ch2 Br_
' SC=N
ch3
- I Cyanogen Bromide
CH3
Methionine in Formic acid
Cyanosulfonium Bromide

Br
h3

Homoserine Lactone Bromide Salt
(Compound 1A)
H
Protonated
Homoserine Lactone

: S---CEEEN
I
CH3
Methyl thiocyanate
Scheme II. The Synthesis of Homoserine Lactone Bromide Salt (Compound 1 A)
From Methionine.
11


1.7 Acetylation
The next step in synthesis was to add a protecting group on the free amine
of the homoserine lactone. This was done to increase the solubility of this
compound in an aprotic solvent. The route chosen was to acylate the free amine
of the lactone. There are a variety of ways to acylate the lactone[14,15,13,16,17].
Five separate procedures were used to produce the acetylhomoserine lactone. All
but one seemed to produce the desired product in sufficient yield. The route that
was chosen as best in terms of ease and yield was first used by Huffman and
Ingersoll [17]. In this reaction the lactone amino group, after deprotonation, is
reacted with acetic anhydride. This procedure is done under moderate heating.
The scheme is shown below (Scheme HI).
12


o
o
H2
-HzC\ 7 +
N(? CHc^s.
V %
HiC
c c
/ v/ \
Homoserine Lactone Nitrate Salt
(Coiripound IB)
CH3
Glacial acetic acid
h2
JC^
100 C / 5 minutes
HiC( /

CHc-

HN
V
J Acetylhomoserine Lactone
Hs (Compound U)
H
Scheme III. The Synthesis of Acetylhomoserine Lactone (Compound IE).
13


1.8 Ammonolysis
The next step involved adding a protecting group to the carboxyl end
while opening up the lactone. For this purpose a mild base was needed. In the
early stages of synthesis, benzyl amine was chosen due to its qualities as a base
and its solubility in organic solvents. Several attempts were made under a variety
of conditions, but the reaction did not seem to work for reasons that remain
unexplained. The second method was the reaction with ammonia gas in
methanol. This method effectively opened the lactone ring. The mechanism can
be seen below (Scheme IV).
14


H2
H
HZC\ /
CH"C^N
HN
(NH3 / methanol) 0 0 C
-----------------
S'
/
h3c
OH
I
ch2
I
CH,
CH n
O NH V*
V \
/ nh2
h3c
Acetylhomoserine Amide
(Compound 111)

O NH4
CHz
I
ch2
h3c
X
CH r
/ \
LJ 'q-''
NH
\
nh2
H,C.
H3C
h3c
H,Cv
Ox NH C
\X \
/c nh2
Scheme IV. The Synthesis of Acetylhomoserine Amide.
15


The reaction occurs by a nucleophilic acyl substitution. The lactone ring is
opened by the addition of ammonia to create an amide bond. This reaction also
creates the primary alcohol functional group.
1.9 Oxidation
The need to oxidize alcohols to aldehydes with greater efficiency and
ease has led to the extensive study of Cr(VI) reagents [18]. Corey et. al. found
pyridinium chlorochromate (PCC) a superior agent in such oxidation
reactions[ 19,20]. The conversion of primary alcohols to aldehydes by PCC is
very selective. It did not further oxidize the aldehyde to a carboxylic acid. The
only drawback to the use of PCC is its mild acidic nature. Primary alcohols that
are acid sensitive could pose a problem. Pyridinium dichromate (PDC) was soon
invented by Coates and Corrigan but was exploited by Corey and Schmidt [19].
The reagent was found to be non-acidic and was used to oxidized compounds
that were acid labile. The reaction scheme for PDC oxidation can be seen below
(Scheme V).
16


Scheme V. Proposed Oxidation of Acetylhomoserine Amide Using PDC.
Daniel Swem found that DMSO could be activated by the addition of
oxalyl chloride [21, 22]. The reaction is conducted at very low temperature to
control its exothermic nature. It was found that primary and secondary alcohols
can be oxidized to aldehydes and ketones by this reagent.The addition of triethyl
amine greatly improved the yields of aldehydes and ketones. This is shown
schematically below (Scheme VI).
17


,CI
b
h3c
S----Cl + co2 + CO + Cl
HaC^
H3C
HaC
---O-
5
>
Cl
Cl
SchemeVI. Schematic Representation of Swem Oxidation.

18


2. Results and Discussion
2.1 Synthesis of Homoserine Lactone Nitrate Salt
(Compound IB)
Homoserine lactone bromide salt, Compound IA, was synthesized by
combining methionine with cyanogen bromide in a 70% (v/v) solution of formic
acid and refluxing at 50 C for 24 hours (Scheme II). Several reaction conditions
were tested. They involved different reaction times along with different
temperatures. The method chosen as best was the one that provided the highest
yield (65%). This method is described in the experimental section. The cleavage
of methionine by cyanogen bromide yields the lactone bromide salt (Compound
IA). It was found that Compound IA would not dissolve in glacial acetic acid for
acetylation. The counter anion had to be switched. In the second step of the
synthesis silver nitrate was added to switch anions with the lactone. Silver
bromide precipitates out of the solution and the lactone nitrate, (Compound IB) is
left behind in solution.
The ^-NMR spectrum of Compound IA (Fig. 2.1.1) is very useful in
confirming its structure. The doublet of doublets found around 3.75 ppm can be
attributed to the alpha proton. The gamma protons, being split by the beta protons
and each other show up as a complex set of peaks between 4.1 and
19


1.5
| 1 : , >- I > ' < : ---1--'-1
4.0 3.5 3.0 2.5 2.0
Figure 2.1.1 'll NMR Spectra of Compound IA


4.24 ppm. The two sets of peaks centered around 2.0 and 2.5 ppm can be
attributed to the beta protons. They are being split by the protons found on the
alpha and gamma carbons, and showed geminal coupling as well. The
assignment of the 13C-NMR spectrum of Compound IA ( Fig. 2.1.2) is as
follows: The beta carbon corresponds to the resonance found at 25ppm. The
alpha carbon corresponds to the resonances found at 47. The gamma carbon
corresponds to the resonance found at 66 ppm. The resonance found at 173 ppm
corresponds to the carbonyl carbon.
21


Figure 2.1.2 13C NMR Spectra of Compound IA


2.2 Synthesis of Acetylhomoserine Lactone
(Compound II)
The solubility of the intermediates(Compound IA and IB) proved to be
the most difficult aspect of this study and the most challenging to overcome.
The homoserine lactone nitrate salt (Compound IB) would only dissolve in
protic media. The problem of solubility needed to be overcome in future steps
of the synthetic route. To encourage the solubility, two protecting groups were
evaluated. One was the use of the Fmoc protecting group, and the other was the
acetyl group. Acetylmethionine was first studied. It was synthesized by
acetylating methionine. Acetyl-methionine was reacted with cyanogen bromide
in formic acid while being refluxed at 50?C. The results of this experiment were
discouraging. No acetylhomoserine lactone resulted. A model study was
conducted using acetyl-alanine under the same conditions stated as above [21].
It was found that the acetyl group was being hydrolyzed under the conditions
used for the cyanogen bromide cleavage experiment. Fmoc-methionine was also
used in the cyanogen bromide reaction under the same conditions stated above,
and the same conclusion can be drawn. The conditions used for these cyanogen
bromide cleavage experiment are much more vigorous than those normally used
for the selective cleavage of peptides. They were found to hydrolyze both the
acetyl and the Fmoc group. These results indicate that the best way to add
23


protecting groups to the N-terminus was after the lactonization by cyanogen
bromide.
The synthesis of Compound II involved heating the lactone nitrate salt
(Compound IB) in a warm solution of glacial acetic acid. To precipitate out any
excess salts in the solution, the solution was allowed to cool to room
temperature. After filtering the solution, acetic anhydride was added and the
solution was set to boil. Acetylating the free amine yielded Compound II
(Scheme III). TLC analysis showed that Compound II did not stain with
ninhydrin, suggesting that there was no free amine. The ^-NMR spectrum of
Compound II (Fig.2.2.1) confirms the structure. A singlet found at 2.1 ppm
corresponds to the protons found on the N-acetyl group connected to the
lactone. The complex set of peaks found at 2.2 and 2.9 ppm correspond to the
beta protons found on the lactone ring. The amine peak can be seen as a broad
band at 6.3 ppm. The sets of peaks found at 4.3 and 4.5 ppm correspond to the
gamma protons on the lactone ring. The alpha proton can be seen as a complex
set of peaks at 4.6 ppm. A 2D NMR spectrum (Fig. 2.2.2) was also taken for
Compound II. This spectrum was primarily used to determine which signal in
the 4.2-4.7 ppm region arose from the alpha proton. When looking at the
topography of the spectrum, the alpha proton found at 4.6 ppm interacts
strongly with the beta protons found at 2.2 and 2.9 ppm, as evident by the off-
24


to
6
3
ppm
Figure 2.2.1 'H NMR Spectra of Compound II
5
i


FI (ppm]
c*> :
n1
w :
. N
1

,PN
N
' K>
.
Figure 2.2.2 2D NMR Spectra of Compound II
26


diagonal peaks. The spectrum also indicates no interaction between the alpha
and the gamma protons (4.3 and 4.5). The interactions between the beta protons
and the gamma protons, as well as the geminal proton couplings, can also be
seen as off-diagonal peaks, as expected.
27


2.3 Synthesis of Acetylhomoserine Amide
(Compound III)
Two approaches were studied for opening acetylhomoserine lactone. In
the beginning stages benzyl amine was evaluated as the aminolyzing base. The
incorporation of a benzyl group in Compound II would have increased its
solubility in an aprotic solvent. Several attempts were made under various
conditions but the results were unsatisfactory. Presence of the benzylated
compound could not be confirmed in the reaction mixture. Ammonia/methanol
was then employed for the possible ring opening of acetylhomoserine lactone.
Ammonolysis occurred, opening up the acetylhomoserine lactone and converting
the cyclic ester into acetylhomoserine amide (Compound III) (Scheme IV).
The 'H-NMR spectrum supports the identity of Compound ID. When
comparing the spectra between Compound II (Fig. 2.2.1) and Compound HI (Fig.
2.3.1), none of protons exchangeable with D2O can be seen in the latter. This
only leaves the protons on the acyl group and the protons on the alpha, beta, and
gamma carbon to be compared. For protons on the acyl group and alpha carbon
relatively little shifting occurs between spectra. Both can be seen at 2.1 and 4.4
ppm, respectively. The real focus is on the protons on the beta and gamma
carbons. The protons on the beta carbon of the lactone ring can be found as
complex signals centered at 2.2.and 2.9 ppm for Compound 13 (Fig. 2.2.1),but
28


NJ
VO
OH
:n---' 1* ------r
1.5 ppm
Figure 2.3.1 ]H NMR Spectra of Compound III


when looking at the spectrum for Compound III (Fig. 2.3.1) the corresponding
peaks merge into one complex signal at 1.75-2.25 ppm. The downfield proton
signal has shifted upfield by at least 0.7 ppm. The gamma protons, which were
seen as separate signals at 4.25 and 4.5 ppm on the lactone ring, have also moved
considerably upfield in Compound in. They are merged as a set of peaks
centered at 3.7ppm. This evidence strongly coincides with the model spectra of
homoserine found in Aldrich NMR library.
The influence of amide bond on the NMR spectrum of the compound
studied is of interest due to its electronic and steric properties. The bond length is
shorter than a regular C-N single bond, due to its partial double bond nature,
which arises from resonance delocalization of the non-bonded electrons on
nitrogen [11]. The same ir bonding results in the cis and the trans conformations
of acetylhomoserine amide shown below (Fig. 2.3.2).
30


OH
OH
CH2
ch2
ch2
ch2
o
HN'
CH
r
nh2
.o
h3c
0
ch3
Figure 2.3.2 Representation of the trans and cis States of Acetylhomoserine
Amide.
The rotation around the C-N bond is drastically reduced because of this partial
double bond character. The cis and trans conformations are slow to exchange and
give rise to separate NMR signals. This is evident in Fig. 2.3.1. When looking at
the alpha carbon signal at 4.2-4A ppm, the cis and trans conformations are
represented by separate signals. The doublet of doublets found centered at 4.35
has an integral of 0.7, while the doublet of doublets centered at 4.25 has an
integral of 0.3. Further testing needs to be done to determine which
conformations (cis or trans) is represented by which signal. Another interesting
aspect of this spectrum is the signal found for the acyl group. It, too, shows up as
two distinct signals centered around 2.0 ppm, a result of presumably the same
31


cis/trans isomerism. Due to the interference of the beta protons which are also
found in the same area, integrals could not be obtained.
32


2.4 Attempted Oxidation of Acetylhomoserine Amide
Several attempts to oxidize acetylhomoserine amide were conducted
using the reagent PCC in methylene chloride. Once the reaction was complete,
the solution was washed with water and both the aqueous and the organic layer
were analyzed. The NMR spectruins of both layers contained none of the original
peaks for the acetylhomoserine amide, and no aldehyde peak in the region of 9-
10 ppm. Acetone was used as an alternate solvent to increase the solubility of
Compound in. The NMR spectrum taken afterwards indicated the same results,
that none of the original peaks for Compound HI can be seen, and there is an
absence of the aldehyde peak at 9-10 ppm.
A model study was carried out using benzyl alcohol. Two types of
oxidation conditions were used. In the first study PDC in methylene chloride was
used as the reagent. Benzyl alcohol was successfully oxidized to benzaldehyde.
This was confirmed by NMR spectroscopy. There was a singlet peak at 10 ppm,
along with the characteristic aromatic peaks around 7.5 ppm. The oxidation of
acetylhomoserine amide was also attempted using PDC in dimethyl formamide.
The solution was then resuspended in methylene chloride and decanted. The
residual oil was then analyzed using NMR spectroscopy. Analysis of the NMR
spectrum showed there was no aldehyde peak present in either layer.
33


In another set of studies, Swem oxidation was carried out with benzyl
alcohol. There was strong evidence that the oxidation of benzyl alcohol was a
success. The NMR spectrum once again indicated conversion to benzaldehyde.
Swem oxidation was attempted using acetylhomoserine amide (Compound III).
There was no evidence of oxidation, and all the original peaks of the starting
material were accounted for in the NMR spectrum of the product.
2.5 Conclusion
This study was conducted to find alternate pathways to synthesize 2,4-
diamino glutaric acid. Although we came up short of synthesizing 2,4-diamino
glutaric acid, many valuable lessons can be drawn from it. Solubility played an
important role in this study; many of the setbacks can be attributed to poor
solubility of the intermediates. In the case of PDC oxidation, the polarity of the
solvent can lead to over-oxidation [18]. To increase the solubility of the
intermediates, protecting groups were employed. Given the nature of the
synthetic route some protecting groups could not be employed the synthetic
process (Fmoc and benzyl amine). In order to oxidize homoserine lactone
successfully other protecting groups should be examined. Protecting groups that
are added must increase the solubility of homoserine in non-polar aprotic media.
Given enough time this feat can be accomplished. Once oxidation is complete,
34


the removal of the protecting groups followed by a Strecker synthesis will yield
the intended product, 2,4-diamino glutaric acid.
35


3. Experimental
3.1 Reagents
Methionine (98%), formic acid (88%), CNBr (97%), PDC (98%), and
(COCl)2 (98%) were all purchased from Acros Chemicals. AgNC>3 (99+%),
anhydrous NHa(g), PCC (98%) and DMF (99%) were purchased from Aldrich
Chemicals. Acetic anhydride was purchased from Fischer Products.
3.2 General Methods
3.2.1 Thin Layer Chromatography
The plates used for thin layer chromatography were aluminum based,
coated with 0.2mm silica gel(UV254). Samples for analysis were spotted from
lpL pipettes. Chromatograms were developed using two solvent systems:
chloroform/ methanol (CM) in a ratio of 9:1, and butanol/acetic acid/water
(BAW) in a ratio of 4:1:1. The solvent system used, CM or B AW, respectively,
is indicated in the synthetic procedures section. The chromatograms were
visualized using two methods in the following order:
Method A: The plate was immersed in a container filled with silica gel saturated
with iodine for a short period of time. The I2 vapor is absorbed onto
any substance containing polarizable bonds. A brown spot is seen
whenever a compound absorbs the I2 vapor.
36


Method B. The plate was sprayed with a 1% solution of ninhydrin in acetone and
heated briefly. Ninhydrin primarily reacts with primary amines to
give a dark purple color.
3.2.2 Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) analysis was performed using a
Varian 200 MHz spectrometer. A 50 pg sample of the substance to be analyzed
was dissolved in 0.7mL of one of the following solvents: D2O, CDCI3, d$-
DMSO, or CD3OD. Calibration of spectra was done either with 0.01% TMS, or
by referencing the minor proton peak(s) of the actual solvent itself. Chemical
shifts are reported in ppm, from TMS.
37


3.3 Synthetic Procedures
3.3.1 Synthesis of Homoserine Lactone Nitrate Salt
from Methionine (Compound IB)
Methionine (8 g, 60 mmol) was dissolved in 100 mL of 70% (v/v) formic
acid. Cyanogen bromide (16g, 160mmol) was added to the solution. The solution
was then boiled under reflux for 24 hours. After the refluxing period, rotory
evaporation of the reaction mixture yielded a light yellow precipitate. This
precipitate was then dissolved in methanol (100ml). Silver nitrate (27g,
160mmol) was added to the solution, and a yellow precipitate formed. The
mixture was filtered. Rotory evaporation of the filtrate yielded 6g of white
precipitate, Compound IB. Yield: 65%.TLC analysis of Compound IB revealed a
single ninhydrin positive spot at the origin(CM), or a single ninhydrin positive
spot with an Rf value of 0.60(BAW). H-NMR (D20): 5 (ppm) 1.8-2.1 (m, 1 H
); 2.4-2.6 (m, 1 H ); 3.7r3.9 ( dd,lH); 4.1-4.25 (m,lH); 4.25-4.4 (m, 1H).I3C-
NMR (D20): 5 (ppm) 25 (Ca); 47 (Cb); 66 (Cc); 173 (Cd).
38


3.3.2 Synthesis of Acetylhomoserine Lactone
(Compound II)
Compound IB (5g, 50 mmol) was suspended at in glacial acetic acid
(lOOmL) and stirred at 50C for 10 minutes. The suspension was then cooled to
room temperature and allowed to sit for 12 hours. The suspension was then
filtered to remove brown crystals. Rotory evaporation was then conducted on the
filtrate and a white precipitate was collected. The purified nitrate salt was then
dissolved in glacial acetic acid (lOOmL) and heated to a boil. Acetic anhydride
(10 mL, 100 mmol) was added and the solution was allowed to boil for 5 more
minutes. The solution was allowed to cool to room temperature while being
stirred for 12 hours. The solution was filtered and a yellow filtrate was obtained.
Rotary evaporation of the filtrate yielded a yellowish precipitate Compound II.
Yield: 75%. An I2 positive stain at an Rf value of 0.25 was obtained with TLC
analysis(BAW). 'H-NMR (CDC13): 8 (ppm) 1.9-2.3 (m, 4H); 2.75-2.9 (m, 1H);
4.2-4.4 (m, 1H); 4.4-4.55 (m, 1H); 4.55-4.7 (m, 1H); 6.3 (s, 1H).
39


3.3.3 Synthesis of Acetylhomoserine Amide
(Compound III)
Compound II (5 g, 38 mmol) was dissolved at 0 C in 100 mL of dry
methanol. Ammonia gas was then bubbled through the solution, with stirring for
20 minutes. The flask was then sealed and the solution was stirred for 24 hours at
room temperature. After the period of stirring, rotary evaporation of the mixture
yielded brownish oil, Compound HI. Yield : 74%. Compound HI was analyzed
by TLC (BAW). A single, ninhydrin and I2 positive, broad spot at an Rf value of
0.6 was observed. 'H-NMR (D20): 5 (ppm) 1.8-2.2 (m, 4H); 3.6-3.8 (m, 2H),
4.2-4.4 (m, 1H).
40


3.3.4 Attempted Oxidation of Acetylhomoserine Amide
The first attempt to oxidize acetylhomoserine amide was conducted using
PCC. In a 100 mL round bottom flask 0.16 g (1.08 mmol) of acetylhomoserine
amide and 0.32g (1.5 mmol) of PCC were dissolved in 20 mL of dry methylene
chloride. The flask was purged with nitrogen and quickly sealed. The solution
was stirred at room temperature for 24 hours, during which time it turned black.
The methylene chloride was then evaporated using reduced pressure. The black
solid was resuspeded in 5mL of acetone, added to a 16 cm silica column and
eluted using a (chloroform: acetone)( 1:1) solvent system. The eluent was then
evaporated under reduced pressure and the residue analyzed using NMR
spectroscopy. None of the peaks found in the spectrum corresponded to those of
acetylhomoserine amide. The same procedure was also conducted using acetone
as the solvent. The results were very similar to those noted above.
A second attempt to oxidize acetylhomoserine amide was conducted
using PDC. In a 100 mL round bottom flask acetylhomoserine amide, (0.16 g,
1.08 mmol), and PDC (0.25g,l .5 mmol) were dissolved in 10 mL of dry DMF.
The flask was purged with nitrogen and quickly sealed. The solution was stirred
at 0 C for 4 hours, during which time it turned black. To the solution 100 mL of
methylene chloride was added. A black oil formed in the bottom of the flask and
the flask was decanted The black oil was resuspended in lOmL of water and
41


passed through a 2.5 cm silica column using a chloroform: acetone (1:1) solvent
system for elution. The eluent was evaporated using reduced pressure. Analysis
of the residue by NMR spectroscopy showed only peaks corresponding to
pyridinium ion.
Lastly, Swem oxidation was attempted on acyl-homoserine-amide.
Methylene chloride (50 mL) was placed in a 250 mL round bottom flask and
chilled to -60 0 C using a dry ice/acetone bath. The system was purged with
nitrogen to prevent the build up of moisture. To the methylene chloride 2.5 mL
(27.5 mmol) of oxalyl chloride was added while stirring. Dimethyl sufoxide 3.0
mL (40 mmol) was added dropwise to the mixture and stirred for 5 minutes. To
the solution 1.48 g (lOmmol) of Compound HI was added dropwise. The
solution was stirred for an additional 15 minutes. Then 7.0 mL (50 mmol) of
triethylamine (TEA) was then added. The solution turned cloudy white. The flask
was removed from the dry ice acetone bath and was allowed to stir until it
reached room temperature. Next 50 mL of H2O was added to the flask. The two
layers that formed were separated with a separatory funnel. The organic layer
was washed twice more with water and then twice with 50 mL of brine. The
organic layer was the evaporated using reduced pressure. No residue was
obtained from the organic layer. Rotory evaporation of the aqueous layer yielded
42


a yellow oil, which was analyzed. NMR spectroscopy showed peaks
corresponding to Compound III, and TEA.
Model studies were conducted using benzyl alcohol. Under the conditions
stated above, benzyl alcohol was successfully oxidized to benzaldehyde using
PCC, PDC and Swem oxidation.
43


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