EXPLORING A NEW ROUTE TO a,a-DIAMINO-1,4-BENZENEDIACETIC
ACID DIMETHYL ESTER
B.A., University of Colorado at Boulder, 1995
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
This thesis for the Master of Science
has been approved
Sumita, Minako (M. S., Chemistry)
Exploring a New Route to oc,ac-Diamino-1,4-Benzenediacetic Acid Dimethyl Ester
Thesis directed by Professor Douglas F. Dyckes
The purpose of this research is to explore a new synthetic route to a molecule
that could serve as a subunit that can form a new polymer. The polymer is built with
diketopiperazine and benzene ring. The monomer which will be used for the new
polymer is a,a-Diamino-1,4-Benzenediacetic Acid Dimethyl Ester. This research is
an attempt to synthesize the monomer by the Schollkopf bis-lactim technique. In
this synthesis, amino acids are formed via diastereoselective alkylation of a bis-
lactim ether which is derived from a cyclic dipeptide (a diketopiperazine). The
electrophiles used for alkylation can be alkyl halides, aldehydes, ketones, and so on.
After the alkylation, acidic cleavage of the bis-lactim ether produces the substituted
amino acid ester. In this particular research, bis-lactim ether is synthesized from
glycine anhydride and isopropyl diketopiperazine. Also 1,4-cyclohexanedione is
used as the electrophile. Cyclohexanone is used for the model study. Dehydration
of the bis-adduct was attempted using the same conditions with the model study, but
double bond formation was not indicated.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
I dedicate this thesis to my high school founder, Dr. Daisaku Ikeda. He gave an
important guidance for my life. Without his guidance, it is impossible to get a
degrees in foreign institutions.
My thanks to my advisor, Prof. Douglas F. Dyckes, for his understanding and
patience with me for past two years. Special thanks to my parents for financial and
emotional supports. Without your support, I cannot complete my degree. I also with
thank all my friends for their support and understanding.
1.1 Research Objective......................................................1
1.3 Bis-lactim Ether Method........n*......................................3
1.4 Proposed Synthetic Route...............................................5
1.4.2 Preparation of the Diketopiperazines..................................7
1.4.3 Derivatization of Diketopiperazine.................................. 12
1.4.4 Conversion to the New Amino Acid.....................................12
2 Experimental Procedure..................................................13
2.2 General Methods.......................................................13
2.2.1 This Layer Chromatography (TLC)......................................13
2.2.2 Column Chromatography................................................14
2.2.3 Melting Points.......................................................14
2.2.4 Nuclear Magnetic Resonance (NMR).....................................14
2.3 Synthetic Procedure....................................................15
2.3.1 Synthesis of Bis-lactim Ether...........................................15
2.3.2 Model Study............................................................22
2.3.3 Synthesis of Amino Acid Derivatives....................................24
3 Result and Discussion......................................................28
3.1 Synthesis of Bis-lactim Ether............................................28
3.1.1 fert-Butoxycarbonyl-L-Valylglycine Methyl Ester (Compound I)............28
3.1.2 Glycine Anhydride (2,5-Piperazinedione) (Compound II-A)..............32
3.1.3 Isopropyl Diketopiperazine (3-S-Isopropyl-Piperazine-2,5-Dione) (Compound
3.1.4 2,5-Dihydro-3,6-Dimethoxypirazine (Compound III)..;.................. 35
3.1.5 2,5-Dihydro-3,6-Diethoxypirazine (Compound IV)........................ 36
3.1.6 3,6-Dihydro-275-Dimethoxy-3-S-Isopropyl-Pyrazine (Compound V)..........39
3.2 Model Study..............................................................43
3.2.1 Addition of Cyclohexanone to Compound IV................................43
3.2.2 Dehydration Reaction of the Model Compound.............................45
3.3 Synthesis of Amino Acid Derivatives....................................49
3.3.1 Addition of 1,4-CycIohexanedione to Compound IV (Preparation of Compound
3.3.2 Addition of 1,4-Cyciohexanedione to 3
Isopropyl-Pirazine (Preparation of Compound VII)...
5 Suggestions for Future Studies...............
1. The Preparation of Diketopiperazine.......................................6
2. The Proposed Synthetic Pathway to a,a1-Diamino-1,4-Benzenediacetic Acid
3. ]H NMR of rerr-Butoxycarbonyl-L-Valylglycine Methyl Ester (Compound I)...29
4. I3C NMR of terr-Butoxycarbonyl-L-Valylglycine Methyl Ester (Compound I)....31
5. ]H NMR of Isopropyl Diketopiperazine (3-S-Isopropyl-Piperazine-2,5-Dione)
6. NMR of 2,5-Dihydro-3,6-Diethoxypirazine (Compound IV)...........;.....37
7. 13C NMR of 2,5-Dihydro-3,6-Diethoxypirazine (Compound IV)...............38
8 NMR of 3,6-Dihydro-2,5-Dimethoxy-3-S-Isopropyl-Pyrazine (Compound V).41
9. 13C NMR of 3,6-Dihydro-2,5-Dimethoxy-3-S-Isopropyl-Pyrazine (Compound V)
10 NMR of Product by Dehydration of the Model Compound............47
11 HJH COSY of Product by Dehydration of the Model Compound.........48
12 1HNMR of Product by Cleavage of the Model Compound...............50
13 ]H NMR of Compound VI............................................52
14 13C NMR of Compound VI..................................53
15 ]H NMR of Compound VH...................................55
16 H,H-COSY of Compound VII................................56
17 13C NMR of Compound VII.................................57
1.1 Research Objective
In this research, the ultimate goal is to synthesize a,a -diamino-1,4-
benzenediacetic acid dimethyl ester by using Schollkopf s bis-lactim ether method
Schollkopf s method could shortcut many reactions compared to the published
strategy (Falck-Pedersen et al., 1996). The molecule itself may be useful for
directing peptide structures because the benzene ring bridges the two amino acids.
The benzene ring bridge can replace the disulfide linkage in the amino acid cystine.
The disulfide linkage is one of the main ways that the tertiary structure of a peptide
or protein is stabilized. For example, Nutt et al. (1980) used (S,S)-2,7-diamino
suberic acid to replace the disulfide linkage for bioactive peptides. Such peptides
will be more stable than the original peptides because of the absence of the reducible
disulfide bond (Falck-Pedersen et al., 1996). The benzene ring is not only resistant
to cleavage, it is also more rigid than the disulfide bond it replaces, so the peptide
will have higher conformational stability than the original. The conformation may
also change because of the rigidness of the benzene ring.
The polymerized form of a,a -di amino-1,4-benzenediacetic acid dimethyl
ester would be alternating diketopiperazine and benzene rings. The polymer may
have interesting characteristics structurally and chemically. The benzene ring will
make the polymer fairly rigid. Also, hydrogen bonding between the
diketopiperazines may create interaction between polymer strands.
Diketopiperazines are a condensed cyclic form of two amino acids bound
together by two peptide bonds. Ten diketopiperazines, including cyclo-alanine-
valine, that many of us encounter in daily life are in any cocoa product, like
chocolate. It is the bitter component of cocoa, and is formed during roasting of the
cocoa beans (Pickenhagen et al., 1975). Diketopiperazines have a stronger bitterness
than the corresponding dipeptides or the amino acids (Pickenhagen et al., 1975).
Another diketopiperazine, cyclo-histidine-proline, can be found in many places in
our body like the central nervous system. It has important relations in
neurophysiological functioning (Goolcharran et al., 1997). Cyclo-histidine-proline is
produced from thyrotropin-releasing hormone (TRH) by pyroglutamyl
aminopeptidase action (Miyashita et al., 1993).
There are potentially many kinds of diketopiperazine. The type is dependent
on its parent amino acids. When two of the same parent amino acids are used to
form a diketopiperazine, the diketopiperazine is symmetrical, or homogeneous. On
The other hand, if two different amino acids form a diketopiperazine, the
diketopiperazine is unsymmetrical or mixed. Also, many diketopiperazines have
optically active cis forms (R,R- and S,S-) and one optically inactive trans form (R,S-
). Glycine anhydride is an exception. It cannot form optically active cis or trans
isomers because both substituents of the 3 and 6 carbons are protons. An
unsymmetrical or mixed diketopiperazine generally has two optically active cis
forms and two optically active trans forms (Greenstein et al., 1961).
1.3 Bis-lactim Ether Method
There are many ways to synthesize amino acids. Traditionally, the Hell-
Volhard-Zelinsky reaction and Strecker synthesis have been the commonly used.
In the Hell-Volhard-Zelinsky reaction (Scheme I), the starting material is a
RCH2COOH ^/P RCHCOOH excess NH^ rcHCOOH
carboxylic acid. The carboxylic acid reacts with a halogen in the presence of small
amounts of phosphorous as a catalyst to form an a-halogenated carboxylic acid.
Then, the a-halogenated carboxylic acid is reacted with aqueous ammonia to
synthesize an a-amino acid.
RCH2CO ftCN/N*%- RCH2CHCN
In the Strecker synthesis (Scheme II), introduced by Adolf Strecker, an
aldehyde is converted to an a-amino nitrile by ammonia and cyanide ion. Then
hydrolysis of the nitrile group to a carboxylic group produces a desired amino acid.
Compared to these syntheses, the bis-lactim ether method is relatively new.
It was introduced by Ulrich Schollkopf in 1978 (Schdllkopf et al., 1978). In this
synthesis, amino acids are formed via diastereoselective alkylation of a bis-lactim
ether, which is derived from a diketopiperazine (Scheme HI).
The ring proton of the bis-lactim ether is more acidic than the original
diketopiperazine. Butyl lithium deprotonates the ring to form an anion. The
electrophiles used for alkylation can be alkyl halides, aldehydes, ketones, and so on.
After the alkylation, acidic cleavage of the bis-lactim ether produces the substituted
amino acid ester.
There are two major advantages to the synthesis. The first advantage is that
it has relatively few experimental steps. It means that complex amino acids often
can be produced in a more economical way. Another advantage is that the final
product of the synthesis is already derivatized, as a methyl ester. The product is
immediately useful for peptide synthesis.
1.4 Proposed Synthetic Route
Two amino acids (glycine methyl esters for II-A and te/Y-butoxycarbonyl
valine and glycine methyl ester for II-B) are the starting materials. After they are
coupled to form a linear dipeptide, it is cyclized to form a diketopiperazine (Figure
1). The major intermediate, a bis-lactim ether, is prepared from the diketopiperazine
with trimethyloxonium tetrafluoroborate or triethyloxonium tetrafluoroborate
(Figure 2). The three intermediate compounds shown in Figure 2 (HI, IV, and V) are
all known, well characterized compounds. The bis-lactim ether is deprotonated by
butyl lithium to form a homoaromatic anion, and the electrophile, 1,4-
cyclohexanedione, is used for alkylation. After the dehydration reaction is used to
form a cyclohexanediene bridge, a dehydrogenation reaction is carried out to
transform the bridge to the more stable benzene ring form. Finally, acidic cleavage
of the bis-lactim ether produces the a-a-diamino-1,4-benzenediacetic acid dimethyl
ester (Figure 1).
, ? f
t f f
Boc-Val-Gly-OMe (Compound I)
f ? ?
Boc-Val-Gly-OMe (Compound I)
Compound 0-A R=H
Compound H-B R=CH(CH3)2
2 NH2CH2COCH3 HCl
Figure 1 The Preparation of Diketopiperazine
Diketopiperazine Compound III R=H, R'=CH3
Compound IV R=H, R'=CH2CH3
Compound V R=CH(CH3>2, R^CHs
Figure 2 The Proposed Synthetic Pathway to a-ac-Diamino-1,4-benzenediacetic
Acid Dimethyl Ester
1.4.2 Preparation of the Diketopiperazines
18.104.22.168 Protecting Group Strategy
The required elements for success in the coupling reaction of amino acids are
protecting groups for amine and carboxyl groups, and activating reagents to raise the
reactivity of the carboxyl group. When two different amino acids are used for a
synthesis, a protecting group for the amino of one and the carboxyl group of the
other are required to avoid undesired products. There are several well-known
protecting groups like the benzyloxycarbonyl group, the ferr-butoxycarbonyl group,
and the 9-fluorenylmethoxycarbonyl group as amine protecting groups, methyl and
ethyl esters as carboxyl protecting groups (Bodanszky et al., 1976).
The tertbutoxycarbonyl group ((CH3)3COOC-), abbreviated Boc, is a well-
known amino protecting group. It was introduced by Carpino (Carpino, 1957).
Also, Mckay (Mckay et al., 1957) and Anderson (Anderson et al., 1957) proposed its
use for the protection of amino acids in peptide synthesis. Unlike the
benzyloxycarbonyl group, the Boc group is stable under the condition of catalytic
hydrogenation. The Boc group is smoothly cleaved as a carbocation by mild acidic
conditions, and produces carbon dioxide (Bodanszky et al., 1976). The carbocation
is converted to isobutene (2-methylpropene) (Scheme IV).
Trifluoroacetic acid (CF3COOH) is often used to cleave the tert-butoxycarbonyl
group. However, excess trifluoroacetic acid interacts with amino acids and forms
milky emulsions. When trifluoroacetic acid is used, triturating with dry ether is
(CH3>2C=CH2 + C02 + H3NCHCOOH
needed to get a solid product. Also, the excess trifluoroacetic acid sometimes makes
peptides more soluble in ether and results in low yields (Bodanszky et al., 1984). To
prevent a low yield and to make trituration easier, the important thing is to remove
the trifluoroacetic acid as much as possible. A water pump is good to remove most
of the trifluoroacetic acid, but will not completely remove it. The boiling point of
trifuoroacetic acid is 75C (Aldrich. 1997). An oil pump is the best way.
22.214.171.124 Coupling Amino Acids
An important consideration in coupling amino acids is the reactivity of the
carbonyl group and the amine group. There are several known coupling agents, such
as N,N-dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC).
N,N-dicyclohexylcarbodiimide is the most common reagent (Pennington et
al., 1994). J.C. Sheehan and J.P. Hess introduced this coupling reagent (Sheehan et
al., 1955). In the mechanism of the reaction (Scheme V), the first step is that the
carboxylic acid of an amino acid protonates a nitrogen atom of the DCC. Then, the
carboxylate attacks the carbon atom of DCC, and forms an O-acylisourea. The
structure of the O-acylisourea resembles a carboxylic acid anhydride. Like an
anhydride, the O-acylisourea, is an active acylating reagent. The next step is that a
free amino group of another amino acid attacks the carbonyl carbon of the O-
acylisourea to from a tetrahedral intermediate. The final step is that the tetrahedral
intermediate forms a linear dipeptide and N,N-dicyclohexylurea.
The problem with using DCC is that one of the final products, N,N-
dicyclohexylurea, partially dissolves in the solvents used for the coupling reaction.
That is why it is possible that the linear dipeptide will be contaminated. To avoid
the urea contamination, N-ethyl-N,-(y-dimethylaminopropyl) carbodiimide is useful.
The corresponding urea and the intermediate are soluble in water, so they are easy to
separate from dipeptides (Bodanszky et al., 1976).
126.96.36.199 Cyclization of Diketopiperazine
The next step is to cleave the protecting group (Boc) of the linear dipeptide,
and cyclize by heat The cyclization reaction mixture should be of low concentration
to avoid undesired products such as dimers.
After the Boc group is cleaved by an acid, a nucleophilic amine group is free
to attack the carbon of a carbonyl group of another end. The carbonyl carbon
becomes tetrahedral and its oxygen atom will gain an extra lone pair of electrons and
become negative, because the double bond of the carbonyl group will change to a
single bond. After deprotonation of the intermediate, the extra electron pair of the
oxygen atom will reform the double bond with the carbonyl carbon as the -OCH3
group leaves (Scheme VI).
1.4.3 Derivatization of Diketopiperazine
In a diketopiperazine, the C-H hydrogens are less acidic than the N-H
hydrogens. The diketopiperazine is O-methylated by trimethyloxonium
tetrafluoroborate to raise the C-H acidity. The product is called a bis-lactim ether.
The reaction is a simple Lewis acid and Lewis base reaction. An acid is a proton
donor and a base is a proton acceptor in the Bronsted definition. The
trimethyloxonium tetrafluoroborate is a strong acid. It donates a methyl group
instead of a proton.
The bis-lactim ether is ionized by butyl lithium. The cation of the bis-lactim
ether is stable because of the homoaromaticity (67c electron system). A second
deprotonation of the ring will not happen. The electrophile, 1,4-cyclohexanedione,
can react with two molecules of the cation.
1.4.4 Conversion to the New Amino Acid
The intermediate alcohol can undergo elimination to form a diene by treatment with
thionyl chloride and 2,6-lutidine. The diene may be dehydrogenated by quinone
derivatives to form an aromatic ring. Finally, the diketopiperazine ring can be
cleaved by acid to synthesize the desired product, a,a-diamino-1,4-benzenediacetic
acid dimethyl ester.
2. Experimental Procedure
All chemicals used in this experiment were purchased from Aldrich
Chemical Company, Inc. and were of the highest purity available.
2.2 General Methods
2.2.1 Thin Layer Chromatography (TLC)
Plates used for thin layer chromatography were aluminum pre-coated with
250|tim layer of silica gel bound with a fluorescent dye (UV254) (Whatman Ltd.).
Samples were spotted on the plate from a lpL micropipet and the chromatograms
were generally developed in chloroform/methanol (9/1) system. The chromatograms
were visualized by the following methods:
1. The plate was observed under ultraviolet light (254nm). A dark spot
indicates a UV absorbing molecule, for example an aromatic compound.
2. The plate was put into ajar of silica gel saturated with I2 for 5 minutes. A
spot indicates a halogen absorbing organic molecule such as an amide.
3. The plate was sprayed with a solution of 2% ninhydrin in acetone, and
then heated for 3-5 minutes. A blue, purple or brown spot indicates a primary or
secondary amine group.
2.2.2 Column Chromatography
lOOmL of Merck 230-400 mesh silica gel was used for chromatography as the
stationary phase. Fractions were collected. Each fraction contained 5mL of sample.
TLC was used for detecting products in certain fractions. Appropriate fractions were
combined and the solvent evaporated by rotary evaporation.
2.2.3 Melting Points
Melting points were determined using a Mel-Temp Laboratory Devices
melting point apparatus. All melting points are reported without correction.
2.2.4 Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance were performed using a Gemini 200 Broad Band
spectrometer. lH spectra and 13C spectra were obtained. Two-dimensional
homonuclear (H,H)-correlated NMR spectroscopy, (HJ3-COSY), used to identify
intramolecular couplings was carried out with standard instrument software.
2.3 Synthetic Procedure
2.3.1 Synthesis of Bis-Iactim Ether
188.8.131.52 te/f-Butoxycarbonyl-L-Valylgiycine Methyl Ester (Compound I)
fcrt-Butoxycarbonyl-L-valylglycine methyl ester was synthesized according
to procedures described by Eugen Schaich et al. (1974), with some modifications.
N-(/err-butoxycarbonyl)-L-valine (1 l.Og, 0.05mol) and glycine methylester
hydrochloride (6.32g, O.OSmol) were dissolved into lOOmL of chlorofoim. The
reaction mixture was put in an ice bath and 7.0mL of triethylamine (0.05mol) was
added. N,N5~dicyclohexylcarbodiimide (DCC) was added to the cold reaction
mixture slowly with stirring. At this point, the pH of the vapor was checked by hold
a moist piece of indicator paper above the solution. Because the pH was still acidic,
triethylamine was added until the pH became basic. After the reaction mixture was
stirred at room temperature for about five minutes, a precipitate formed in the
reaction mixture. The mixture was stirred overnight. On the following day, it was
full of white precipitate. The white precipitate, assumed to be N,N-
dicyclohexylurea, was removed by filtration. The filtrate was washed sequentially
with 75mL of 1% acetic acid solution, 75mL of 10% sodium bicarbonate solution,
and 75mL of water, in this order. The organic solution was dried with magnesium
sulfate. The dried mixture was filtered to remove magnesium sulfate, and the
solvent was evaporated. The mass of crude product was 22. lg (>100%). The crude
product was dissolved into about 40mL of nearly boiling ethyl acetate, and filtered
hot. Petroleum ether was added to the filtered solution until the solution became a
little cloudy. The mixture was allowed to crystallize in a freezer. The white
powdery reciystallized substance was collected by vacuum filtration and dried in a
desiccator overnight. Yield: 11.7g (80.4%); Rf: 0.54; m.p.: 107C; JH NMR (CDC13)
S(ppm) 0.87-0.98 (dd, 6H, val R-CH3); 1.43 (s, 9H, Boc -CH3); 2/12-2.22 (m, 1H, val
R-CH); 3.74 (s, 3H, -OCH3); 3.95-3.98 (m, 1H, val -CH); 4.03-4.06 (d, 2H, gly -
CH2); 5.04 (d, 1H, val -NH); 6.56 (s, 1H, gly -NH) (Figure 3).
I3C NMR (CDCI3) 5(ppm) 16.0-17.6 (val R-CH3); 26.7 (Boc -CH3); 29.3 (val -CH);
39.5 (gly -CH2); 50.7 (-OCH3); 58.2 (val -CH); 78.4 (Boc -C); 154.4 (Boc carboxyl);
168.7-170.6 (carbonyl) (Figure 4).
184.108.40.206 Glycine Anhydride (2,5-Piperazinedione) (Compound II-A)
Glycine anhydride (2,5-piperazinedione) was synthesized according to
procedures described by Fisher (Greenstein et al., 1961) with some modifications.
Glycine methylester hydrochloride (5.05g, 0.04mol) was dissolved in 4.0mL of
water. The solution was put in an ice bath with stirring. Then, 5.0mLofN-
methylmorpholine was slowly added to the solution. After methylmorpholine was
added completely, the solution was stirred at room temperature overnight. On the
following day, the solution was put in a refrigerator for 30 minutes. A white
precipitate appeared. The white precipitate was collected by vacuum filtration. The
collected solid was washed with a small amount of cold water and cold ethanol,
twice each, then dried in a desiccator overnight. The dried product (1.22g, 53.7%)
was dissolved into about 25mL of nearly boiling water, and filtered hot. The filtered
solution was allowed to reciystallize at room temperature. The reciystallized
substance was collected by vacuum filtration and dried in a desiccator overnight.
Yield: 0.46g (20.3%); Rf: 0.22; m.p.; >300C (lit. m.p. 289->300C) (SchOllkopf et
al., 1981. Aldrich 1995).
220.127.116.11 Isopropyl Diketopiperazine (3-S-IsopropyI-Piperazine-2,5-Dione)
The procedure was from the description by Schollkopf et al. (1981) with
some modifications. Boc-L-valylglycine methyl ester (2.5g, 0.009mol) was partially
dissolved and suspended in 20.0mL of tetrahydrofuran. The mixture was put in an
ice bath, and dry hydrogen chloride gas was bubbled through the mixture for 5
minutes. During the addition of hydrogen chloride gas, the solid dissolved
completely. The completeness of the reaction was tested by thin layer
chromatography. When the reaction was completed, a single spot appeared at origin,
detected with ninhydrin. The starting material was not ninhydrin positive and its Rf
value was 0.68. The solvent was evaporated, and a yellow oil remained. The oil
was redissolved into 20.0mL of tetrahydrofuran, and the solution was put in an ice
bath. Triethylamine was added to the cold solution slowly until the pH of the vapor
became basic. When triethylamine was added, a white precipitate immediately
appeared. The mixture was filtered to remove the white precipitate. Again, the
solvent was evaporated, and a yellow oil remained. The yellow oil was dissolved
into 50.0mL of toluene, and refluxed for 12 hours. After 4 hours of reflux, a white
precipitate appeared. After 12 hours of reflux, the reaction mixture was put in a
freezer overnight. The mixture was full of white precipitate. The precipitate was
collected by filtration. The crude product was 1.92g (>100%). The crude product
was dissolved into about lOmL of nearly boiling water, then filtered hot. The
product was allowed to reciystalize at room temperature. The mass of the product
was 1.12g(83%); Rf: 0.52; m.p.; 253C (lit. m.p. 254C Schollkopfet al., 1981);
'HNMR (DMSO); 5(ppm) 0.80-0.91 (dd, 6H, isopropyl R-CHj); 2.03-2.12 (m, 1H,
isopropyl R-CH); 3.50-3.52 (m, 1H, ring H with isopropyl group); 3.64 (s, 1H, ring
H); 3.75-3.84 (dd, 1H, ring H); 8.02-8.20 (d, 2H, -NH) (lit. NMR. Bull et al., 1998)
13C NMR (DMSO); 5(ppm) 15.88,17.37 (isopropyl CH3); 31.37 (isopropyl CH);
42.98 (ring CH2) 58.67 (ring with isopropyl group); 165.1, 166.3 (carbonyl C) (lit
NMR. Bull et al., 1998).
18.104.22.168 Preparation of 2,5-Dihydro-3,6-Dimethoxypirazine (Compound HI)
O-methylated glycine anhydride (2,5-dihydro-3,6-dimethoxypyrazine) was
synthesized according to procedures described by Schollkopf (Schollkopf et al.,
1981) and Blake (Blake et al., 1972) With some modifications. Glycine anhydride
(0.5g, 0.004mol) was suspended in lO.OmL of dried methylene chloride.
Trimethyloxonium tetrafluoroborate (1.60g, O.Olmol) was added to the reaction
mixture. The reaction mixture was stirred under dried nitrogen at room temperature
for two days. After that, the reaction mixture was extracted with 20.0mL of
saturated sodium bicarbonate. The aqueous layer was extracted with 5.0mL of
methylene chloride three times. All of the organic layers were combined, and dried
with magnesium sulfate. The dried mixture was filtered to remove magnesium
sulfate, and the solvent was evaporated. The mass of crude product was 0.09g
(14.5%). The mass was too small for recrystallization. Rf: 0.79; NMR (CDC13);
8(ppm) 3.70 (s, 4H, 3 and 6-CH2), 4.04 (s, 6H, 2 and 5-CH3) (lit. NMR Groth et al.,
22.214.171.124 Preparation of 2,5-Dihydro-3,6-Diethoxypirazine (Compound IV)
Ethylated glycine anhydride (2,5-dihydro-3,6-diethoxypirazine) was
synthesized according to procedures described by Groth (Groth et al., 1993) with
some modifications. Glycine anhydride (2.00g, 0.02mol) was suspended in 38.0mL
of 1.0M triethyloxonium tetrafluoroborate. The reaction mixture was stirred under
dried nitrogen at room temperature for 5 days. Additional 1.0M triethyloxonium
tetrafluoroborate (4.0mL) was added on the 2nd and 4th days. During the reaction
time, a sticky solid was formed. After day 5, the reaction mixture was cooled down
to 0C, and 2.5N sodium hydroxide was added until its pH became nine. Two layers
formed. The aqueous layer was extracted with lOmL of methylene chloride twice.
The combined organic layers were again extracted with 15mL of water three times,
then dried over magnesium sulfate. The solvent was removed, leaving the crude
product, a brownish solid: 1.01 g (30% yield). The crude solid was recrystallized
from petroleum ether to give white, needle shaped crystals. Yield: 0.84g (25%);
Rf: 0.81; m.p.: 82C (lit ffi.p. 83-84C, Groth et al., 1993). ]H NMR (CDCIj);
8(ppm) 1.27 (t, 6H, CHj), 4.01 (s, 4H, ring CH2), 4.09 (q, 4H, -OCH2) (lit HNMR,
Blake et al., 1972) (Figure 6).
l3C NMR (CDClj); 8(ppm) 12.6' (CH3), 44.92 (ring C), 59.36 (-OCH2), 162.22 (ring
C with -OCH2CH3) (Figure 7).
126.96.36.199 Prepartation of 3,64)ihydro-2,5-Dimethoxy-3-S-IsopropyI-Pyrazine
The bis-lactim ether of isopropyl diketopiperazine was synthesized according
to procedures described by Schollkopf (Schollkopf et al., 1981). CompoundII-B
(l.OOg, 0.0064mol) was suspended in 21.0mL of methylene chloride (anhydrous).
Trimethyloxonium tetrafluoroborate (2.25g, 0.0152mol) was added to the mixture.
The mixture was stirred under nitrogen at room temperature. After 8 hours of
stirring, the mixture became a pale yellow solution. More trimethyloxonium
tetrafluoroborate (1,03g, 0.007mol) was added to the solution and stirring was
continued for two additional days. After a total of 3 days of stirring, the solution
turned to a brownish color. The solution was washed with 75mL of NaH2P04
2H20/Na2HP04 2H20 buffer solution. Then the aqueous layer was washed with
methylene chloride three times. The combined organic layer was dried with
magnesium sulfate. After the magnesium sulfate was removed, a crude product
(0.91g, 77%) was collected by evaporation. The crude product was tested by thin
layer chromatography, and two ninhydrin positive spots (Rf values are 0.69 and 0.55)
were clearly indicated. The crude oil was purified by column chromatography (the
solvent was ethylacetate). The product from early fractions (Rf= 0.69) was 0.64g
(54%). The product of Rf= 0.55 (0.12g) was recovered form die later fractions. The
desired product was from the early fractions. 1HNMR (CDC13); 8(ppm) 0.60,0.89
(dd, 6H, isopropyl CH3), 2.01-2.11 (m, 1H, isopropyl CH), 3.55 and 3.58 (s, 3H, -
OCH3), 3.87(m, 3H, ring H) (lit. NMR. Bull et al., 1998) (Figure 8).
13C NMR (CDCI3); 8(ppm) 15.27,17.34 (isopropyl CH3), 30.78 (isopropyl CH),
44.92 (ring CH2), 50.82,50.88 (-OCH3), 59.39 (ring C with isopropyl group), 160.8,
163.4 (ring C with -OCH3) (lit. NMR. Bull et al., 1998) (Figure 9).
2.3.2 Model Study
188.8.131.52 Preparation of Addition of Cyclohexanone to Compound IV
Compound IV (0.26g, l,7*10'3mol) was dissolved into 5.0mL of THF. The
mixture was stirred under nitrogen gas in a dry ice bath. Butyl lithium (0.8mL of a
2.0M solution in cyclohexane, 1.7* 10'3mol) was added to the mixture dropwise. The
mixture immediately changed its color from yellow to dark red. The mixture was
stirred for 15 minutes. Then, cyclohexanone (0.2mL, 1.9*10'3mol) was added to the
mixture slowly. The color changed back to yellow immediately. The reaction
mixture was stirred under the same conditions for 4 hours. Acetic acid solution
(O.lmL in 2.0mL of THF) was then added tot quench the reaction, and the reaction
mixture allowed to come to room temperature. The solvent was removed by rotary
evaporation, and a yellow oil remained. The oil was redissolved into lOmL of ethyl
ether and washed with 15mL of water twice. The organic layer was dried over
magnesium sulfate. Upon evaporation, 0.82g of yellow oil (>100%) was recovered.
The crude oil was purified by column chromatography. The eluent was 2%
methanol in chloroform. The yield was 0.60g (>100%). Rfi 0.43; lH NMR (CDC13),
5(ppm); 1.25-1.33 (m, CH3), 1.57 (m, cyclohexane ringH), 4.01 (m, bis-lactim ether
ring H), 4.13-4.17 (m, -OCH2). This intermediate was used without further
184.108.40.206 Dehydration Reaction
The alcohol from the previous step (0.6g, 2.13*10'3mol) was dissolved in
lOmL of toluene. 2,6-lutidine (0.50mL, 4.26*1 O3 mol) was added to the mixture.
Then, thionyl chloride (0.16mL, 2.13*10'3mol) was added to the mixture.
Immediately, some white precipitate appeared, and the reaction mixture became
more vivid yellow. The reaction mixture was stirred at room temperature overnight.
The precipitate was dissolved into water. The organic layer was collected and dried
over magnesium sulfate. The yellow oil was recovered after rotary evaporation. The
yield was 0.82g (>100%). The crude oil was purified by column chromatography.
The solvent was 2% methanol in chloroform. Yield: 0.66g (>100%); Rf: 0.92;
NMR (CDC13), Â§(ppm), 1.25 (t, CH3), 1.5 (m, cyclohexene ring H, 4.10 (q,
-OCH2), 4.27 (m, bis-lactim ether ring H), 5.4 (s, cyclohexene double bond H)
(Figure 10). This product was used in the following step without further
The alkene from the previous step (0.66g, 3.78*10'4mol) in 0.25N HC1
(3.0mL, 7.57*10~4mol) was stirred for 30 hours. The mixture was extracted with
5mL of ether. The aqueous phase was concentrated by water pump. Then, the
remaining l-2mL of product mixture was redissolved into about 5mL of ether. The
pH of the mixture was raised to a 8 -10 by saturated sodium carbonate solution. The
ether phase separated. The aqueous phase was extracted with ether three more
times. The organic layers were combined and dried over magnesium sulfate. The
yield was 0.43g (99%). The Rf was 0.5. 1HNMR (CDC13), S(ppm), 1.25 (CEB), 1.5
(cyclohexene ring H), 4.10 (-OCH2), 5.4 (cyclohexene double bond El) (Figure 11).
The product was not purified.
2.3.3 Synthesis of Amino Acid Derivatives
220.127.116.11 Addition of 1,4-Cyclohexanedione to Compound IV(Preparation of
Compound IV (0.22g, 1.5*10'3mol) was dissolved into 5.0mL of THF. The
mixture was stirred under nitrogen gas in a dry ice bath. Butyl lithium (0.8mL of a
2.0M solution in cyclohexane, 1.5*10'3mol) was added to the mixture dropwise. The
mixture immediately changed its color from yellow to dark red. The mixture was
stirred for 30 minutes. Then, 1,4-cyclohexanedione solution (0.09g in 2.0mL of
THF, 7.5*10'4mol) was added to the mixture slowly. The color changed back to
yellow immediately. The reaction mixture was stirred under the same conditions for
10 hours. Acetic acid solution (0.09mL in 2.0mL of THF) was then added to quench
the reaction, and the reaction mixture allowed to come to room temperature. The
solvent was removed by rotaiy evaporation, and a yellow oil remained. The oil was
redissolved into ethyl ether and washed with water twice. The organic layer was
dried over magnesium sulfate. Upon evaporation, 0.20g of yellow oil (67%) was
recovered. The crude oil was purified by column chromatography. The eluent was
3% methanol in chloroform. The yield (oil) was 0. lg (33%). The Rf was 0.69.
lHNMR (CDC13), 5(ppm), 1.26 (t, 12H, CH3), 1.55-1.75 (m, 5H, cyclohexane ring
H), 1.95-2.00 (m, 2H, cyclohexane ringH), 2.12-2.25 (m, 1H, cyclohexane ring H),
2.49 (s, 2H, -OH), 4.00 (t, 3H, bis-lactim ether ring H), 4.11 (q, 8H, -OCH2) (Figure
13C NMR (CDCI3), 8(ppm), 12.66 (CH3), 29.67 (cyclohexane ring Cp), 45.95
(-OCH2), 59.20 (cyclohexane ring Ca), 59.71 (bis-lactim ether C), 73.52 (bis-lactim
ether C with cyclohexane ring), 160.71 and 162.66 (bis-lactim ether C with
-OCH2CH3) (Figure 13).
18.104.22.168 Addition of 1,4-Cyclohexanedione to 3,6-Dihydro-2,5-Dimethoxy-3-S-
Isopropyl-Pirazine (Preparation of Compound VII)
Compound V(0.94g, 0.005mol) was dissolved into 15.0mL of dry THF. The
mixture was stirred under nitrogen gas and in a diy ice bath. Butyl lithium (5mL of a
2.0M solution in THF, O.Olmol) was added to the mixture dropwise. The mixture
changed color from yellow to dark red. The mixture was stirred for 10 minutes.
Then, 1,4-cyclohexanedione solution (0.38g in 5.0mL of THF, 0.003mol) was added
to the mixture slowly. The mixture was stirred under the same conditions for 10
hours. After a while, the color changed back to yellow. Acetic acid solution (0.6mL
acetic acid in 5.0mL of THF) was then added to the mixture, and the temperature
was allowed to come to room temperature. The solvent was removed by rotary
evaporation, and a black oily solid remained. The solid was redissolved into ethyl
ether and washed with water twice. The organic layer was dried over magnesium
sulfate. Upon evaporation, 1.1 lg of yellow oil (92.5%) was recovered. The crude
product was purified by a column chromatography. Yield: 0.1 lg (9.2%); Rf-: 0.50;
'HNMR(CDC13), 5(ppm), 0.63 and 1.03 (dd, 12H, isopropyl CH3), 1.51-1.71 (m,
4H, cyclohexane ring H), 1.85-1.92 (m, 2H, cyclohexane ring H), 2.01-2.16 (m, 2H,
cyclohexane ringH), 2.26-2.35 (m, 2H, isopropyl CH), 2.50 (s, 2H, -OH), 3.68 (d,
12H, -OCH3), 3.94 (t, 2H, bis-lactim ether ring H with isopropyl group), 4.05 (d, 2H,
bis-lactim ether ring H) (Figure 14).
l3C NMR (CDC13), 5(ppm), 14.82,17.61 (isopropyl CH3), 29.3 (isopropyl CH),
29.35,29.53 (cyclohexane ring Cp), 50.81, 51.16 (-OCH3), 59.04 (cyclohexane ring
Ca), 59.17 (bis-lactim ether C with isopropyl group), 73.05 (bis-lactim ether C),
160.68,163.91 (bis-lactim ether C with -OCH3) (Figure 16).
3. Result and Discussion
3.1 Synthesis of Bis-Lactim Ether
3.1.1 tert-Butoxycarbonyl-L-Valylglycine Methyl Ester (Compound I)
The reaction to produce tert-butoxycarbonyl-L-valylglycine methyl ester
went smoothly. The recrystallization was the only difficult part. The product was
partially soluble in ethyl acetate, so the usual hot and cold recrystallization method
may have caused the low yield. To increase the yield, the solvent pair
recrystallization method was used (Fessenden et al., 1993). Choosing the two
solvents is most important in this procedure. One solvent must dissolve the
compound easily and another solvent must not dissolve it. Usually, the solvents are
a polar/nonpolar pair. The crude product is dissolved into a minimum amount of hot
solvent that can dissolve the compound readily. Then, the non-solvent is added drop
by drop until the solution becomes cloudy. The result is a solution saturated by the
crude product, an ideal condition for recrystallization. The solution is allowed to
crystallize. In this experiment, ethyl acetate and petroleum ether was used for the
recrystallization, and 80.4% was yielded.
In the !H NMR spectrum (Figure 3) of the linear dipeptide, die methyl group
protons of valine appeared as doublet of doublets at 0.89-0.98ppm instead of as a
Figure 3. lINMR of /err-butoxycarbonyl valylglycine methyl ester (Compound I)
doublet. It is because the methyl groups are diastereotopic. When an isopropyl
group is attached to a chiral atom, the methyl groups are diastereotopic and may
generate separate signals because they have different magnetic shieldings (Friebolin.
The amide proton of glycine (6.56ppm) is more downfield than the amide
proton of valine (5.04ppm). The oxygen atom of an amide is an electron
withdrawing atom. Because the amide group of valine is attached to the oxygen
atom of a Boc group, the carbonyl group of the amide can pull electron density from
not only the nitrogen of the amide group but also the oxygen atom of the Boc group.
However, the carbonyl group of the glycine amide group can pull electron density
only from the nitrogen of the amide group, so the oxygen atom pulls electron density
more strongly from the nitrogen. That is why the amide hydrogen of glycine is more
down field than the amide hydrogen of valine.
In addition, some small signals in the !H NMR spectrum between 1 and
2ppm show the presence of some impurity. The impurity may be the contamination
of N,N-dicyclohexylurea N,N dicyclohexylurea is hard to separate from dipeptide
because there is weak hydrogen bonding between them (Thayer et al., 1993).
In the 13C NMR spectrum (Figure 4), there are two peaks for the methyl
groups of valine because they are diastereotopic. Also, there are two carbonyl peaks
Figure 4. 13CNMRof te/Y-butoxycarbonyl valylglycine methyl ester (Compound I)
at 168.7 and 170.6ppm. The peak at 168.7 may be the peptide carbonyl group, and
170.6ppm maybe the methoxy carbonyl group. The methoxy carbonyl group may be
less shielded because oxygen is more electronegative than nitrogen. To identify
these two peaks for sure, a two dimensional C-C spectrum is required.
3.1.2 Glycine Anhydride (2,5-Piperazinedione) (Compound II-A)
The key to success in this synthesis is a base which deprotonates the starting
material, glycine methylester hydrochloride. A nucleophilic amine group is then free
to attack the carbon of a carbonyl group of another glycine methylester. Several
bases were tested for this experiment. Pyridine found to be too weak as a base for
this reaction. N-Methylmorpholine, which is a stronger base than pyridine, worked
The yield (20.3%) of glycine anhydride using N-methylmorpholine was low
compared to the literature which is 49-54% (Greenstein et al., 1961). The reaction
time in this experiment was overnight; however, it is 36-48 hours in the literature.
The reaction is a slow process, and it may need more time to get a higher yield. A
longer reaction time is suggested. Also, some catalyst like dimethylaminopyridine
(DMAP) may be useful to get a better yield.
3.1.3 Isopropyl Diketopiperazine (3-S-IsopropyI-Piperazrae-2,5-d*one)
The reaction mechanism is similar to that for the cyclization of Compound II-
A. The reaction progress could be monitored by thin layer chromatography. The
starting material, terf-butoxycarbonyl-L-valylglycine methyl ester is ninhydrin
negative and its Rf value is high (RfD.68). After the Boc group is cleaved, the
amino group becomes free and the molecule becomes more polar. It appears at the
origin in TLC and is ninhydrin positive. After the cyclization reaction is done, the
product has no free amine group and it is ninhydrin negative. Also, the cyclic
dipeptide is less polar and migrates on TLC to a place which is close to the starting
It is hard to get a high yield in this reaction. Glycine nitrophenyl ester will be
a better starting material for preparation of the dipeptide because the nitrophenyl
ester is better leaving group than the methyl ester.
In the NMR spectrum (Figure 5), the diastereotopic methyl groups of
isopropyl group appear as a doublet of doublets, at 0.80-0.91 ppm, just as they did in
the linear dipeptide. Compared to the NMR of the starting material (Figure 3),
the Boc group peak at 1.46ppm and methyl ester peak at 3.74ppm are gone. All
other peaks are at about the same place as in the linear dipeptide except amide
Figure 5 1HNMR of isopropyl diketopiperazine (Compound II-B)
peaks. In the spectrum of the linear dipeptide, the amide peaks are farther apart (the
difference is 1.53ppm). These peaks are located close to each other in the cyclic
dipeptide (the difference is 0. lppm). The spectra used different solvents (CDCI3 and
DMSO), so the peaks appear in different positions. If CDC13 is used for the cyclic
compound, the amide peaks would likely be located close to the position of the
amide proton of glycine in the linear peptide. It is because the less deshielding Boc
group has been cleaved from valine and the amide protons of both amino acid units
now indicate more normal peptide bonds.
3.1.4 2,5-Dihydro-*3,6-Dimethoxypirazine (Compound ITT)
Initially, trimethyloxonium tetrafluoroborate was used for the O-alkylation of
the diketopiperazine. The trimethyloxonium tetrafluoroborate is a strong acid,, so the
important thing is this experiment is that water should be avoided as much as
The yield (14.5%) is low compared to the literature, which is 25% (Groth et
al., 1993). For the same reactions, the reaction time in this experiment was two days
at room temperature: however, it is one day at room temperature and two days reflux
in the literature. The reaction may require high energy, and it may require higher
temperature to get a higher yield. Also, both trimethyloxonium tetrafluoroborate and
glycine anhydride are insoluble in methylene chloride so the insolubility may cause
the low yield (Groth et al., 1993).
The *H NMR (See the experimental procedure) spectrum of the product
clearly indicates the methoxy group peak at 4.04ppm. Compared to the spectrum of
the starting material, glycine anhydride, the ring proton peak is shifted downfield a
little. The shift may be caused by changing the NMR solvent. In this experiment,
the crude product was not purified because of the small amount.
3.1.5 2,5-Dihydro-3,6-Diethoxypyrazmc (Compound IV)
The reaction is basically the same as the synthesis of 2,5-dihydro-3,6-
dimethoxypirazine (Compound HI). Instead of trimethyloxonium tetrafluoroborate,
triethyloxonium tetrafluoroborate was used. Triethyloxonium tetrafluoroborate can
dissolve in the solvent, methylene chloride. That may be why the yield improved
(25%) compared to the Compound III (Groth et al., 1993). Also, a longer reaction
time and more triethyloxonium tetrafluoroborate may be required to get a higher
The H NMR spectrum (Figure 6) and 13C NMR spectrum (Figure 7) are a
little more complicated There are three major peaks in the ]H NMR spectrum. Two
peaks for ethoxy group which are triplet at 1.27ppm and quartet at 4. lOppm. Ring
protons are almost same place at 4.0 lppm as in Compound DL
Figure 6 HNMR of 2,5-dihydro-3,6-diethoxypyrazine (Compound IV)
In the C NMR spectrum, there are four major peaks. The methyl carbon is
at 12.6lppm, and the ethoxy carbon is at 59.36ppm. The ring carbon with ethoxy
groups is at 162.22ppm, and other ring carbon is at 44.92ppm.
3.1.6 3,6-Dihydro-2,5-Dimethoxy-3-S-Isopropyl-Pyrazine (Compound V)
A methylation of diketopiperazines takes time even though the
trimethyloxonium tetrafluoroborate acts as a strong acid because both starting
materials dont dissolve into methylene chloride. Triethyloxonium tetrafluoroborate
dissolves into the solvent easily, and the yields are better. The disadvantage for the
ethylated reagent is a more complicated NMR spectrum and less stereoselectivity
(Huniget al., 1994).
The methylation of isopropyl diketopiperazine produced one major product,
the bis-O-methylated molecule, and two minor products, the mono-O-methylated
molecules (Scheme VII). In the *11NMR of the mono-O-methylated products, every
signal is doubled. One set is taller than the other set. The ratio between the major
and minor monomethylated product was about 3:1 (data are not shown). The carbon
2 is further from the isopropyl group than the carbon 5, so the carbon 2 is less
hindered. 2-methoxydiketopiperazine may be more favorable than 5-
methoxydiketopiperazine (Bull et al., 1998).
These monomethylated isomers arise from the intermediates to the
dimethylated diketopiperazine. The second methylation is a slower process
compared to the first. After the first methylation, the nitrogen that is close to the
methoxy group is already positively charged Another methylation means another
positive charge. The second charged reaction is harder than the first one (Bull et al.,
1998). Longer time and more trimethyloxonium tetrafluoroborate should improve
the products yield (Rose et al., 1995).
Also, the procedure is good only for small amounts. For larger amounts, the
commercial product of trimethyloxonium tetrafluorborate gives poor yields (Bull et
al., 1998). The possible reason is trimethyloxonium tetrafluoroborate is highly
reactive with water. The reagent required more careful handling in humid places.
In the NMR spectrum (Figure 8), the diastereotopic methyl groups (0.60
and 0.89ppm) separate more than in the starting material, Compound II-B (Figure 5).
Hr-* 1--1--1 -r-J
o.ia 0.06 o.is
Figure 8 1HNMR of 3,6-dihydro-2,5-dimethoxy-3-S-isopropyl-pyrazine (Compound V)
-| l I 1- I T IT I I I I !| I I I I T- I I I...........Ill T~l I I I I I I I l-T-T-'T I I I I I- I-
Figure 9 13CNMR of 3,6-dihydro-2,5-dimethoxy-3-S-isopropyl-pyrazine (Compound V)
Also, the methoxy groups at about 3.6ppm are not equivalent In the 13C NMR
(Figure 9), there are new methoxy group peaks (50.82 and 50.88ppm).
3.2 Model Study
3.2.1 Addition of Cyclohexane to Compound IV
Before using a disubstituted compound, the reaction was tested by using a
monosubstited compound which is cyclohexane. The reaction progress was
indicated by a color change (Scheme VIE).
The initial color of the reaction solution was yellow. The ring protons of a bis-
lactim ether are quite acidic because of the allylic double bonds. The ionized bis-
lactim ether is a 6% electron homoaromatic system. Because of the homo
aromaticity, further ionization is suppressed. Because of the electronic change, the
color changed to dark red upon ionization. The electronic configuration of the
product obtained by adding cyclohexanone will be close to that of the starting
material, and the color returns to yellow.
The method used to purity the product was not efficient. After column
chromatography, the product yield was still more than 100%. The major
contaminant is probably cyclohexanone. Cyclohexanone has a high boiling point
(155C), and was not removed completely, even using an oil pump. Another
possibility is that there is hydrogen bonding between the product and cyclohexanone.
The product is likely to have high boiling point, so vacuum distillation may be better
In TLC, the Rf value of the product was 0.43. The spot is lower down than
the starting material, Compound in-B. The Rf value of Compound IH-B was 0:81.
The product became more polar because of the alcohol groups formed
In the 1H NMR spectrum (See the experimental procedure), there are
complex ethoxy peaks. The methyl signal looks like two triplets at 1.3ppm, and the -
CH2 group signal is multiplets at about 4.15ppm. One ethoxy group is closer to the
cyclohexane ring than another, so they are no longer equivalent Also, the bis-lactim
ether ring protons appear as mutiplets. It is because there are two different types of
protons. Two protons are the usual bis-lactim ether ring protons, but one proton is
near the alcohol group of the cyclohexane ring. The spectrum also indicates the
contamination. The peaks at 2.33 and 1.86 and some overlapping at 1.57ppm are
3.2.2 Dehydration of the Model Compound
Usually dehydration reactions of alcohol use acid catalysis. However, in this
research, strong acid may cleave the bis-lactim ether, so thionyi chloride and 2,6-
lutidine were used. A lone pair of electrons of the alcohol oxygen attack a sulfur of
the thionyi chloride, and form a better leaving group. A base (either chlorine ion or
2,6-lutidine) then removes a hydrogen from the (3 carbon, and a double bond forms
2,6-Lutidine is a kind of pyridine. Pyridine can absorb hydrochlorides produced in
this reaction (Vogel, 1972). The reaction will stay relatively neutral.
Again, the purification by column chromatography was not adequate.
Because the starting material was the product from the previous experiment, the
major contamination is still cyclohexanone. Vacuum distillation may again be a
better procedure for the purification.
In the TLC analysis, the Rf of the product increases from 0.43 to 0,92. It is
because the alcohol group is gone. The product becomes less polar.
In the NMR (Figure 10) spectrum, there is a new significant peak at
5.4ppm. This is the range for vynilic protons, so the new peak may indicate a C-C
double bond. From the results of HJH-COSY spectrum (Figure 11), the vinylic peak
has some correlation with cyclohexane ring protons. It means that the double bond
is inside of the cyclohexane ring, not outside of the ring. According to the
Schollkopf (1981), there is an 80:20 ratio between forming a double bond inside of a
substituent vs. between the substituent and the ring. In this experiment, the
substituent is cyclic. An intracyclic double bond should be much more favorable
than an intercyclic double bond.
Acidic conditions will readily cleave a bis-lactim ether and form two amino
acid ethyl esters. The intermediate from the model study was cleaved using 0.25N
TLC indicates two ninhydiin positive spots in the product. The Rf values are
0.5 and 0.38. These spots were compared with glycine methylester, which Rf was
0.35. The lower spot therefore may be glycine ethylester, and the higher spot may be
1-cyclohexenyl glycine ethyl ester.
Because the product was not purified, The 1HNMR spectum (Figure 12)
indicates mixture. The mixture supposed to be glycine methylester and the desired
product. The important thing in the specturm is the indication of the double bond at
5.4ppm that is the same position before cleavage.
3.3 Synthesis of Amino Acid Derivatives
3.3.1 Addition of 1,4-CycIohexanedione to Compound IV (Preparation of
The reaction progress was indicated by color change as the model study. The
initial color of the reaction mixture was yellow. When the bis-lactim ether was
ionized, the color changed to dark red. After 1,4-cyclohexanedione was reacted with
the bis-lactim ether ion, the color returned to yellow.
6 5 4 3 2
Figure 12 lHNMR of the product of cleavage reaction
In the ]H NMR of Compound VI (Figure 13), the three major peaks (two for
ethoxy group and one for ring H) are still in the same positions. There are three
peaks for the cyclohexane protons (between 1.55 to 2.3ppm) and one peak (at
2.50ppm) for the alcohol group. The cyclohexane protons are not equivalent to each
other because of the equatorial and axial positions, so they show a complicated
pattern in the spectrum.
In the 13C NMR (Figure 14), there are many new peaks. Two peaks are from
the cyclohexane ring. Ca is at 59.2ppm, and Cp is at 29.7ppm. The ring carbon of
the bis-lactim ether with cyclohexane ring is shifted downfield (73.5ppm) from the
dihydrocarbon position (59.7ppm). The carbons with ethoxy groups show as two
peaks because one ethoxy carbon is closer to the cyclohexane ring than other ethoxy
3.3.2 Addition of 1,4-Cyclohexanedione to 3,6-Dihydro-2,5-Dimethoxy-3-S-
Isopropyl-Pyrazine (Preparation of Compound VII)
The reaction progress was indicated by color change as with Compound IV-
A. Schollkopf has shown that the proton without the isopropyl group is deprotonated
by butyl lithium. The ring proton with the isopropyl group is harder to ionize
because it is hindered. Anions of the bis-lactim ether attack the carbonyl carbons of
a v ia ) s
o e *> p
I IJ I 1
Figure 13 1HNMR of the compound VI
Figure 14 I3CNMR of the compound VI
1,4- cyclohexanedione to form a neutral molecule, so which is a 1,4-disubstituted
In the NMR spectrum (Figure 15), three new multiplet peaks appear
between 1.51 and 2.13ppm. They are the peaks for protons of cyclohexane ring.
Also, the broad peak (2.50ppm) indicates the alcohol proton. The significant peak
shift is for protons of bis-lactim ether ring. Before addition of 1,4-
cyclohexanedione, these protons are overlapped and show a single peak at 3.87ppm.
Now, these proton peaks are separated to a triplet (3.93ppm) and a doublet
(4.05ppm). From the results of RH-COSY spectrum (Figure 16), the triplet is the
proton adjacent to the isopropyl group. However, the peak for the triplet should be
doublet, and the doublet peak should be singlet theoretically. Because it is a big
molecule, the conformation is complicated. The triplet peak may be a doublet of
doublets, indicating two conformations.
In the 13C NMR (Figure 17), the carbon of the bis-lactim ether that
cyclohexane ring is attached to is shifted from 44.92 to 73.05ppm. The cyclohexane
ring carbons appear at 59.04ppm for the a carbon and 29.53 for the p carbon.
Figure 17 13CNMR of the compound YU
The Schollkopf s bis-lactim ether method is well established since 1981.
This research investigated the synthesis of a,a-diamino-1,4-benzenediacetic acid
dimethyl ester. To synthesize an amino acid by using Schollkopf s method, there are
two key reactions. One is the synthesis of the bis-lactim ether (Compound III, IV,
and V), and another is adding an electrophile to the bis-lactim ether anion
(Compound VI and VII). Synthesis procedures for addition of 1,4-cyclohexanedione
to two molecules of bis-lactim ether were successfully developed based on the model
study. Also, the model study of the dehydration reaction indicated that endocyclic
double bond formation is much more favorable than exocyclic double bond
formation. Confirmation of the structure was performed by HNMR, CNMR, and
RH-COSY NMR spectra.
Dehydration of the bis-adduct (Compound VIII) was attempted using the
same conditions with the model study, but double bond formation was not indicated.
5 Suggestions for Future Studies
Obviously, dehydration, aromatization, and cleavage reaction procedures for
the bis-adduct should be developed The dehydration and cleavage reactions may be
done by a procedure similar to that the model study. However, preliminary attempts
were not successful. This may indicate that the conjugated diene, if formed, is
unstable under the reaction conditions. Decomposition of related conjugated
systems under eliminating conditions has been reported (Kuo. 1999).
Another problem will be dehydrogenation to form an aromatic ring. The
common dehydrogenating agents are sulfur and palladium catalyst. However,
hydrogen gas will be produced by using such reagents. In this case, using a stream of
nitrogen gas is suggested to remove hydrogen gas. Also, chloranil and dichloro-
dicyanoquinone may be better reagents. They can transfer hydrogens to themselves
to form hydroquinones (House. 1972). In addition, the reaction progress will be
indicated as color change. As the reaction goes, the hydroquinone, which is not
soluble in benzene, separates as a pale yellow solid (House. 1972).
Schollkopf s method is not an economical way to synthesize the amino acids
sought in this study. A large amount of trimethyloxonium tetrafluoroborate or
triethyloxonium tetrafluoroborate are required to synthesize bis-lactim ether. Bull et
al. (1989) reported the commercial methoxy or ethoxy reagents give lower yield, and
they suggest the use of the fresh methoxy or ethoxy reagents.
Schollkopf s method is stereoselective when a bulky amino acid enantiomer
such as L-valine is used to form the diketopiperazine. The final amino acid should
be mostly one stereoisomer. But if glycine anhydride is used to synthesize the bis-
lactim ether, there are no bulky groups, so the final amino acid will be an equal
mixture of two stereoisomers. Also, use of ethoxy groups for the formation of the
bis-lactim ether gives less stereoselectivity than when methoxy groups are used
because of hindrance (Hunig 1994). After the final amino acid can be successfully
produced, a study of the stereoselectivity will be interesting.
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