Enzymatic synthesis of the enantiomers of 2-amino-2-methyl-4- phosphonobutyric acid [5]

Full text of DOI:10.1007/s11176-005-0158-5

Russian Journal of General Chemistry, Vol. 74, No. 8, 2004, pp. 1297 1299. Translated from Zhurnal Obshchei Khimii, Vol. 74, No. 8, 2004,
pp. 1400 1402.
Original Russian Text Copyright
2004 by Ragulin.
LETTERS
TO THE EDITOR
Enzymatic Synthesis of the Enantiomers
of 2-Amino-2-methyl-4-phosphonobutyric Acid
V. V. Ragulin
Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolovka, Russia
Received December 10, 2003
(S)-2-Amino-2-methyl-4-phosphonobutyric acid is
known as an antagonist of metabotropic glutamate
receptors III [1 3]. Stereoselectivity of these receptors
is often unexpected [2, 3], and, therefore, both enan-
tiomers are usually needed for complete biological
investigation. To this end, the enzymatic method of
resolution of racemic phosphonic aminocarboxylic
acids [4 8] may prove more suitable than the asym-
metric synthesis [7, 8]. Moreover, the stereoselective
introduction of the -phosphonoethyl radical [8] in
the chiral trans-2-phenyloxazolidinone, aimed at
preparing the S enantiomer of 2-amino-2-methyl-4-
phosphonobutyric acid (I) may be accompanied by
inversion of the chiral carbon center [7]. Ma et al. [8]
have reported the following optical rotation data for
25
S-I: [ ]
12.3 (c 0.31, MeOH). However, the
589
25
opposite value, [ ] +12.4 (c 1.0, 6 N HCl), is also
589
available in the literature [9].
In the present work we propose an enzymatic
synthesis of the R and S enantiomers of compound I
according to the following scheme. Penicillinamidase
(PcAm) is an enzyme that exhibits an extremely high
S/R stereoselectivity in hydrolysis of N-acyl deriva-
tives of -aminocarboxylic acids and peptides [4 6],
which makes this synthetic procedure feasible for re-
solving the above-mentioned controversy.
O
1) PhCH2C(O)Cl;
2) EtOH (H2O)
O
(Me3Si)2NH
R,S-I
Me3SiO P
OSiMe3
HO P
Me
NHSiMe3
Me
O
OH
O
OH
NH
O
OSiMe3
Ph
III
II
O
PcAm
HO P
OH
Me
_
---
Dowex (H+)
..
.
.
.
.
NH2
HO
S-I
O
O
O
Me
HO P
HO P
OH
Me
_
..
O
HCl, reflux
.
.
.
.
...
|
.
NH2
O
OH
OH
HO
NH
Ph
O
R-I
R-II
In the enantioselective hydrolysis of N-phenyl-
acetylated amino acid II according to our previously
proposed procedurefor resolution of racemic -amino-
-phosphonocarboxylic acids we made use of immobi-
lized PcAm. Attempted acylation of amino acid I by
the classical Schotten Baumann procedure [10] failed.
1070-3632/04/7408-1297 2004 MAIK Nauka/Interperiodica

1298
RAGULIN
Contrary to -unsubstituted -amino acids, amino
(3 40 cm) of Dowex 50WX8-200 (H+) using water
as eluent.
acid I had to be preliminarily silylated [11, 12]. Silyl
derivative III was then reacted in situ with phenyl-
acetyl chloride to obtain target N-(phenylacetyl)amino
acid II after alcoholysis and removal of the silyl
group.
Strongly acidic fractions with a negative ninhydrin
test were evaporated in a vacuum. The oily residue
1
(about 7 g), R-II [the H and 31P NMR spectra are
identical to those of R,S-II], was dissolved in 30 ml
of 8 N HCl, and the resulting solution was boiled for
10 h and evaporated in a vacuum. The residue was
partitioned between water (50 ml) and ether (30 ml).
The aqueous layer was additionally washed with ether
(2 30 ml) and evaporated to dryness. The residue
was treated with excess propylene oxide in aqueous
ethanol. Additional crystallization from aqueous
ethanol gave 2.4 g of R-I, yield 57% per R,S-II,
mp 203 205 C (decomp.). Rf 0.2 (pyridine acetic
The enzymatic hydrolysis of compound II was
much slower compared with -unsubstituted amino
acids [5]. Hydrolyzed form S-I and N-phenylacetyla-
ted form R-II were resolved by ion-exchange chro-
matography. Acid hydrolysis of R-II gave the target
R enantiomer of I [4, 5].
Synthesis of 2-methyl-2-(phenylacetyl)amino]-
4-phosphonobutyric acid (R,S-II). A mixture of
6.7 g of (R,S)-I and 21.5 ml of hexamethyldisilazane
was stirred for 0.5 h at room temperature and then
gradually brought to the boiling point, stirred with
boiling for 2 h, cooled under argon, and evaporated
in a vacuum. The residue was dissolved in 10 ml of
absolute toluene, and a solution of 5.2 ml of phenyl-
acetyl chloride in 5 ml of toluene was slowly added
dropwise. The resulting mixture was boiled for 3 h,
cooled, and 10 ml of aqueous ethanol was slowly
added dropwise with vigorous stirring. The solvent
was removed in a vacuum, and the residue was passed
through a column (3 30 cm) of Dowex 50WX8-200
(H+) using water as eluent. A fraction with a negative
ninhydrin test was collected and evaporated in a
vacuum. Crystallization of the residue from ethereal
ethanol gave 8.9 g (82.4%) of product II, mp 181
acid water 2-methylpropan-1-ol
1:3:5:15).
1H
NMR spectrum (D2O + DCl), , ppm: 1.42 s (3H,
Me), 1.58 m (2H, CH2), 1.97 m (2H, CH2). 13C NMR
spectrum (D2O + DCl), C, ppm: 21.7 s (Me), 21.9 d
(C4, JPC 136.1 Hz), 30.8 d (C3, JPC 3.3 Hz), 60.3 d
(C2, JPC 18.7 Hz), 173.7 (C1). 31P NMR spectrum
(D2O + DCl), P, ppm: 25.0. Found, %: C 30.13,
30.17; H 6.21, 6.19; N 7.07, 7.03. C5H122N5 O5P. Cal-
culated, %: C 30.47; H 6.14, N 7.11. [ ]546 8.5 (c
25
546
25
589
1.2, H2O); [ ]
13.4 (c 1.1, 6 N HCl); [ ]
12.1 (c 1.1, 6 N HCl).
Fractions with a positive ninhydrin test were col-
lected and evaporated in a vacuum. The residue was
passed through a column (3 20 cm) of Dowex
50WX8-200 (H+), and fractions with a positive
ninhydrin test were evaporated. The residue was
crystallized from 2 N HCl acetone, 9:1, to obtain
3.1 g of S-I HCl, yield 62.0% per R,S-II, mp 198
199 C (decomp.). Rf 0.3 (pyridine acetic acid water
1
183 C. H NMR spectrum (D2O, pH 2), , ppm:
1.33 s (2H, Me), 1.57 m (2H, CH2), 1.92 m (1H,
CH2), 2.12 m (1H, CH2), 3.47 s (2H, CH2Ph), 7.22 m
1
(5H, Ph). H NMR spectrum (C3OD), , ppm: 1.47 s
(3H, Me), 1.62 m (2H, CH2), 2.18 m (2H, CH2),
3.52 br.s (2H, CH2Ph), 7.27 m (5H. Ph). 31P NMR
spectrum, P, ppm: (D2O, pH 1 2) 30.6, (CD3OD)
30.2. Found, %: C 49.27, 49.37; H 5.97, 5.83; N 4.46,
4.50. C13H18NO6P. Calculated, %: C 49.53; H 5.75,
N 4.44.
2-methylpropan-1-ol 1:3:5:15). The 1H, 13C, and 31
P
NMR spectra are identical to those of R-I. Found,
%: C 25.50, 2553; H 5.77, 5.70; N 6.07, 6.03.
C5H12NO5P HCl. Calculated, %: C 25.71; H 5.61; N
25
25
6.00. [ ]
+7.9 (c 1.0, H2O); [ ]
+9.1 (c 1.0,
589
25
546
25
H2O); [ ]
+12.2 C (c 0.1, MeOH); [ ]
+12.3
589
589
Enzymatic synthesis of the enantiomers of
2-methyl-2-amino-4-phosphonobutyric acid (I).
R,S-II, 13.5 g, was dissolved in 70 ml of water at
pH 7.5 which was maintained with 1 N KOH. Pre-
liminary prepared immobilized PcAm, 5 7 g, was
added to the solution, and the mixture was stirred at
25 C. Reaction progress was controlled by HPLC by
the formation of phenylacetic acid [5]. After the reac-
tion was complete, the enzyme was filtered off, and
the filtrate was washed with ether (2 20 ml). The
aqueous layer was concentrated in a vacuum to 30 ml
and treated with 1 N HCl to pH 5, was washed with
ether (2 20 ml), and passed through a column
(c1 1.0, 6 N HCl).
The optical rotations of (S)-I in various solvents
agree with those reported in [9]. The present work
offers the first example of the application of PcAm
for resultion of enantiomers of -substituted -amino
acids.
1
The H, 13C, and 31P NMR spectra were recorded
on a Bruker DPX-200 Fourier spectrometer. The
optical rotations were measured on a Perkin Elmer
1
On an account of the free amino acid.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 74 No. 8 2004

ENZYMATIC SYNTHESIS OF THE ENANTIOMERS
1299
241 and Polamat A polarimeters. The enzymatic stage
was carried using immobilized PcAm with a specific
activity of 103 U/g dry biocatalyst [4 6]. Analytical
HPLC was carried out on a Gilson chromatograph
with a UV-Vis-118 detector at 254 nm. Quantitative
analysis was carried out on a Diasorb C16/T column
preliminary calibrated by phenylacetic acid.
Tsvetkov, E.N., Zh. Obshch. Khim., 1999, vol. 69,
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Tetrahedron: Asymmetry, 1997, vol. 8, no. 6,
pp. 889 893.
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