Biomimetic synthesis of esters of natural amino acids

Article abstract of DOI:10.1002/hc.20427

A method for the synthesis of Gly, Ala, Phe, and Thr esters is proposed and considered as being a stage of possible biomimetic synthesis of peptides. The methyl esters of the said amino acids are obtained via intervention of 2-hydroxypropyl phosphonate. The resulting aminoacyl phosphonates reacts with methanol to produce the amino acid methyl esters, with the release of phosphoric acid. The reaction is carried out at room temperature in water.

Full text of DOI:10.1002/hc.20427

Heteroatom Chemistry
Volume 19, Number 3, 2008
Biomimetic Synthesis of Esters of Natural
Amino Acids
Ivan T. Devedjiev,1 Stanislav G. Bairyamov,2
and Vladimira S. Videva1
1Institute of Polymers, Bulgarian Academy of Sciences, “Acad. G. Bonchev” av., build. 103-A,
1113-Sofia, Bulgaria
2Institute of Organic Chemistry, Bulgarian Academy of Sciences, “Acad. G. Bonchev” av., build. 9,
1113-Sofia, Bulgaria
Received 20 April 2006; revised 28 March 2007, 17 July 2007
mation of peptide bonds even at high dilution and
ABSTRACT: A method for the synthesis of Gly,
physiological pH and at room temperature [2–5].
Ala, Phe, and Thr esters is proposed and consid-
The synthesis of peptides and proteins is one of the
ered as being a stage of possible biomimetic synthe-
most important biosynthetic processes in living sys-
sis of peptides. The methyl esters of the said amino
tems. Most of the amino acids are chemically ac-
acids are obtained via intervention of 2-hydroxypropyl
tivated via the formation of the respective aminoa-
phosphonate. The resulting aminoacyl phosphonates
cyl adenylate, which contains a macroenergetic acyl
reacts with methanol to produce the amino acid
phosphate bond. The carboxyl group is transferred
methyl esters, with the release of phosphoric acid.
to the hydroxyl one at the 3ꢁ-carbon atom in the ri-
The reaction is carried out at room temperature in
bose cycle of tRNA, resulting in the formation of an
ꢀ
C
water. 2008 Wiley Periodicals, Inc. Heteroatom Chem
acyl ester and the release of the phosphate residue.
In the end, peptization with a second amino acid at
the active 2-hydroxy ester and release of the alco-
19:252–255, 2008; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10.1002/hc.20427
hol (ribose in the tRNA) occurs. In this paper, we
propose a method for reproducing the biosynthesis
of peptides. In the biochemical reactions with the
INTRODUCTION
participation of phosphates, the reaction center is
the phosphate group. We have selected the starting
phosphorus-containing compound 2-hydroxypropyl
phosphonate, in which the substituents at the phos-
phoryl moiety and especially the presence of a vici-
nal hydroxyl group are the same as in 3ꢁ-position of
ribose. It has long been known that 2-hydroxy esters
of oxophosphorus acids can be used as phosphory-
lating reagents [6,7]. They are easy to obtain, simple
compounds and have been successfully used in the
phosphorylation of D-glucose [8], desoxynucleosides
[9], and glycine [10].
The first biopolymers have been formed by conden-
sation and dehydration in the primary ocean [1].
By the 60 years of the past century, the possibility
of the polymerization of free and substituted amino
acid phosphoanhydrides has been studied. The lat-
ter are energy-rich monomers, permitting the for-
Correspondence to: Ivan T. Devedjiev; idev@polymer.bas.bg,
itdevedjiev@yahoo.com.
Contract grant sponsor: National Science Foundation of the
Ministry of Education and Science, Bulgaria.
Contract grant number: X-1309/2003.
2008 Wiley Periodicals, Inc.
c
ꢀ
252

Biomimetic Synthesis of Esters of Natural Amino Acids 253
MATERIALS AND METHODS
The following compounds and materials, commer-
cially supplied by Merck, Darmstadt, Germany were
used: Gly, Ala, Phe, and Thr; phosphoric acid, in
crystals; 1,2-propylene oxide; TLC aluminum sheets,
and silica gel 60 F254. NMR spectrometer Brucker
Advance operating at 250 MHz was used.
FIGURE 2 Phosphoaminoalanil acylphosphonate.
formed acylphosphate bond is stable even in the wa-
ter solution. The presence of acylphosphate bonding
offers the possibility of reaction with alcohols. The
formation of the methyl esters of the amino acids is
illustrated in Scheme 1.
The first stage is the breaking of the zwitterion
of the amino acid with the formation of a salt bond
with the phosphonate. This is the reason for the bet-
ter solubility of the products in the water/methanol
solution.
The second stage of the reaction involves a
transition state, characterized by the possibility of
the nucleophilic reaction at the phosphorus atom
(compound II).
The third stage consists in the release of glycol
and the formation of an acylphosphate bond, which
is relatively stable in water because of the six atomic
circles and the aminophosphoryl salt bonds.
In stage IV, methanol, which has nucleophile
higher than water, attacks the acylphosphate bond
with the formation of the methyl ester of the corre-
sponding amino acid.
In practice, the amino acid and phosphorous
acid were mixed in equimolar quantities, then dis-
solved in a methanol/water mixture and treated by
the addition of 2–4 mol of propylene oxide. (Accord-
ing to weight analysis, 2 mol equivalents of propy-
lene oxide reacted in the first stage.) The reaction was
carried out for 1 h at room temperature and 15 min
at 40◦C and was monitored by the use of TLC. The
final product was isolated from the reaction mixture
in the following order:
RESULTS AND DISCUSSION
In our previous paper [10], we have reported that
the reaction between 2-hydroxypropyl esters of ox-
ophosphorus acids and glycine in methanol solution
produces phosphoamidoacyl phosphates as shown
in Fig. 1.
It could be expected that the available acyl–
phosphorus bonds will be attacked by the alcohol to
form acyl esters. To overcome the low solubility of
the amino acids in methanol and to provide relevant
conditions for comparison with the biochemical re-
action, the esterification has been carried out in wa-
ter in the presence of small amounts of methanol,
just sufficient for the solubilization of the amino
acids. As the starting phosphorus-containing com-
pound, we have selected phosphorous acid, which
ensures the possibility of tracking the changes in the
reaction products by the changes in P H in 1H-NMR.
On the other hand, the use of phosphorous acid
avoids complications that would arise with the use
of Mg-containing phosphoric acid derivatives, which
are the corresponding reactants in live cells. The re-
action between 2-hydroxypropyl phosphonate and
the amino acid in water solution does not produce a
phosphamide bond (P N), but rather a salt-forming
bond, (P O− +NH3). The product, isolated by the
reaction of alanine with 2-hydroxypropyl phospho-
nate, represents, after precipitation in ethanol, crys-
tals with m.t. 208–210◦C and NMR (DMSO): 7.901–
5.417 d, JP,H = 621 Hz (1H, PH); 3.77 m, J = 7.2 Hz
(1H, CH); 1.47 d, 7.2 Hz (3H, CH3).
JP,H = 621 Hz is characteristic of salt-forming
bonds. The compound is phosphoaminoalanil
acylphosphonate (Fig. 2).
Owing to the presence of a salt bond between
the acidic P-OH group and the amino group, the
1. acidification with 0.5 N HCl for breaking of
the phosphamide bond. After evaporation, the
residue is a white oil;
2. extraction with methylene chloride (chloroform,
ethylacetate) for the removal of the remaining
phosphorous acid and glycols;
3. removal of solvents by vacuum distillation;
4. addition of triethylamine, dissolved in methylene
chloride, to liberate the amino acid ester from the
hydrochloride;
5. extraction of the organic phase with water;
6. isolation of the final product.
FIGURE 1 Phosphoamidoacyl phosphates.
Heteroatom Chemistry DOI 10.1002/hc

254 Devedjiev, Bairyamov, and Videva
SCHEME 1 The formation of the methyl esters of the amino acids.
The esters were purified and isolated by column
Amino acids are commonly esterified with al-
chromatography, using chloroform/methanol = 9:1.
The products were characterized by 1H- and 13C-
NMR (Table 1).
cohols in the presence of strong acids, ensuring
acid catalysis; for example, alanine was mixed with
methanol, saturated with hydrogen chloride [11]. A
TABLE 1 The Methyl Esters of Amino Acids and Its Characterization by 1H- and 13C-NMR
1H-NMR (CDCl3): 3.70 s, 3H, (OCH3); 3.56 s, 2H, (CH2)
13C-NMR (CDCl3): 175.2, (C O); 44.3 (CH2); 52.0, (OCH3)
1H-NMR (CDCl3): 3.78 m, J = 7.2 Hz, 1H, (CH); 3,70 s, 3H, (OCH3); 1.47 d, J = 7.2 Hz, 3H, (CH3)
13C-NMR (CDCl3): 175.7, (C O); 19.0 (CH3); 50.3 (CH); 51.8, (OCH3)
1H-NMR (CDCl3): 7.2 m, 5H, (C6H5); 3.75 dd, J = 5.2 Hz, 1H, (CHa); 3.72s, 3H, (OCH3);
3.07–3.14 dd, J = 5.2 Hz, 2.83–2.91 dd, J = 8.8 Hz, 2H, (CH2); 1.72 s, 2H, (NH2)
13C-NMR (CDCl3): 175, (C O); 128, (C6H5); 55.5, (CHa); 51.6, (OCH3); 40.7, (CH2)
1H-NMR (CDCl3): 3.86 m, J = 6.2 Hz, 1H, (Hb); 3.70 s, 3H, (OCH3); 3.23–3.25 d, J = 5.1 Hz, 1H,
(CHa); 2.38 s, 3H, (NH2, OH); 1.19–1.17 d, J = 6.3 Hz, 3H, (CH3)
13C-NMR (CDCl3): 174.5, (C O); 68.1, (CHb); 59.8, (CHa); 52.1, (OCH3); 19.7, (CH3)
Heteroatom Chemistry DOI 10.1002/hc

Biomimetic Synthesis of Esters of Natural Amino Acids 255
more convenient preparative method is the one us-
ing thionyl chloride [12], where the mechanism of
esterification is the same. We have carried out a
blank experiment to establish whether the presence
of phosphorous acid induced esterification without
the introduction of propylene oxide in the system.
Thus, equimolar quantities of phosphorous acid and
phenylalanine were dissolved in the methanol/water
mixture and was stirred for 1 h at room temperature
and 15 min at 40◦C. The mixture was titrated with
0.1 N NaOH to pH 5.88 (isoelectrical point of Phe)
and precipitated in ethanol. The nonreacted pheny-
lalanine was quantitatively isolated. No methyl ester
was detected by TLC in the system.
dry residue was dissolved in a mixture of chloro-
form/methanol = 9:1 and was isolated by column
chromatography. From the solution, 0.222 g methyl
ester of phenylalanine crystallized (62% of the theo-
retical yield).
The methyl esters of glycine, alanine, and threo-
nine were obtained by the same procedure. The es-
terification of threonine was performed with 3 mL
water and 7 mL methanol.
Yields were Gly.OMe (67%), Ala.OMe (73%), and
Thr.OMe (68%).
CONCLUSION
It could be expected that the esterification in
methanol solution (without water) would produce
methyl esters of amino acids with higher yields. The
control experiment with phenylalanine in methanol
indicated that the yield of methyl ester of pheny-
lalanine (45%) was less than in methanol/water. The
reason of this result has not been studied in detail.
After the successful esterification of alanine,
phenylalanine, and glycine, it was of interest to try
the preparation of the methyl ester of an amino acid
that has a hydroxyl group in the side chain. A possi-
ble attack of the side-chain OH group of the amino
acid onto the acylphosphate bond could produce oli-
goesters of the amino acid. We have chosen threo-
nine as the amino acid model for this purpose.
This method reproduces the biosynthesis of acti-
vated esters of amino acids. The authors consider
it as the first stage of a biomimetic synthesis of
peptides.
ACKNOWLEDGMENTS
The authors thank Eng. Martin F. Gaidusek, Aus-
trian Science and Research Liaison Office, Sofia, for
his efficient help.
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A flask equipped with a magnetic stirrer and a re-
flux condenser was loaded with 330 mg (2 mmol)
L-phenylalanine and 164 mg (2 mmol) phospho-
rous acid. The solvent mixture of 2 mL water and
10 mL methanol was added and, after dissolution,
0.46 mL (6 mmol) propylene oxide was introduced.
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ature, then a second portion (2 mmol) of propy-
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stirred for 15 min at 40◦C. Methanol was evapo-
rated, and 1 mL 0.5 N HCl was added. The water
was vacuum-evaporated, and the dry residue was
washed with ether. The residue was then mixed with
5 mL methylene chloride and treated with 0.417 mL
triethylamine, dissolved in 5 mL methylene chlo-
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Heteroatom Chemistry DOI 10.1002/hc