Synthesis of phosphinic acids on the basis of hypophosphites: IV. Synthesis of pseudo-γ-glutamylglycine and its enantiomers

Article abstract of DOI:10.1007/s11176-005-0133-1

A new method for construction of pseudopeptide molecules is proposed, exemplified by the synthesis of [3-amino-3-(hydroxycarbonyl)propyl][2- (hydroxycabonyl)ethyl]phosphinic acid (I) (pseudo-γ-glutamylglycine), starting from ammonium hypophosphite. Enzyma

Full text of DOI:10.1007/s11176-005-0133-1

Russian Journal of General Chemistry, Vol. 74, No. 8, 2004, pp. 1177 1181. Translated from Zhurnal Obshchei Khimii, Vol. 74, No. 8, 2004,
pp. 1273 1278.
Original Russian Text Copyright
2004 by Ragulin.
Synthesis of Phosphinic Acids on the Basis of Hypophosphites:
IV.1 Synthesis of Pseudo- -glutamylglycine and Its Enantiomers
V. V. Ragulin
Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolovka, Russia
Received December 3, 2002
Abstract A new method for construction of pseudopeptide molecules is proposed, exemplified by the
synthesis of [3-amino-3-(hydroxycarbonyl)propyl][2-(hydroxycabonyl)ethyl]phosphinic acid (I) (pseudo- -
glutamylglycine), starting from ammonium hypophosphite. Enzymatic synthesis using immobilized penicill-
amidase allowed preparation of the enantiomers of acid I.
Phosphinic acids are useful as models of transition
states of biochemical reactions and can serve as
effective inhibitors of natural enzymes [2]. In this
connection elaboration of convenient synthetic ap-
proaches to phosphine analogs of peptides is a pros-
pective route to potentially physiologically active
compounds [3 5]. The key step in the construction of
new phosphoryl analogs of natural peptides is substi-
tution of the amide fragment C C(O) N of a natural
peptide by a phosphinate fragment C P(OH)(O) C.
The known methods of pseudopeptide synthesis [3 8]
involve two steps: synthesis of an aminoalkylphos-
phonous component B (pseudo-N peptide component),
a phosphonous analog of a natural amino acid and its
atoms and consists of three or four steps [9, 10]. The
synthesis of the pseudopeptide fragment, that forms
the second phosphorus carbon bond, most commonly
involves the addition of silyl esters of aminoalkyl-
phosphonous acids to acrylates by the procedure in
[11].
In this work we propose a new synthetic approach
to pseudopeptides with reversed construction steps:
The first phosphorus carbon bond is formed via
addition of a hypophosphite to an acrylate [12] and
the second, by construction and addition of the amino-
alkyl fragment [13]. This synthetic approach may
prove more facile and convenient in one-pot formation
of both phosphorus carbon bonds, and we develop it
aiming at preparing various functionally substituted
phosphinic acids [14 16].
subsequent addition to
-substituted acrylates
(pseudo-C peptide component) to form a pseudo-
peptide fragment (C) of the desired structure [6 8].
The proposed pseudo- -glutamylpeptide synthesis
is simplified by a convenient procedure [13, 17, 18]
for preparing vinylphosphoryl compounds. The latter
are used as starting materials for adding the amino
acid function in the form of diethyl acetamidomalo-
nate and forming a phosphoryl analog of glutaminic
acid, viz. fragment B of the pseudopeptide molecule.
B
R
O
O
O
A
P
N
H
OH
Here we propose a new method for synthesis of
[3-amino-3-(hydroxycarbonyl)propyl][2-(hydroxy-
cabonyl)ethyl]phosphinic acid (I) (pseudo- -glutamyl-
glycine; -Glu (PO2CH2)Gly) [5, 8] from ammonium
hypophosphite (II) (Scheme 1) and an enzymatic
synthesis of the enantiomers of acid I (Scheme 2).
O
C
The synthesis of the aminoalkylphosphonous com-
ponent, that forms the first phosphorus carbon bond,
has been satisfactorily developed [9, 10]. In this case,
the synthesis of component B requires introduction of
protective groups both to the nitrogen and phosphorus
The proposed synthesis of pseudo- -glutamylgly-
cine (I) is two-step. The first step involves one-pot
formation of both phosphorus carbon bonds and
construction of pseudopeptide fragment C via the
formation of a key intermediate of the synthesis,
vinylphosphinate III, through a sequence of reactions,
1
For communication III, see [1].
1070-3632/04/7408-1177 2004 MAIK Nauka/Interperiodica

1178
RAGULIN
starting from ammonium hypophosphite (II), in a total
yield of 38 43% (depending on the isolation proce-
dure) without separation of intermediates IV VI)
(Scheme 1). The second step involves introduction of
the amino acid function and isolation of pseudo- -
glutamylglycine (I) (Scheme 1).
Scheme 1.
O
OSiMe3
OSiMe3
OSiMe3
EtO(O)CCH=CH2
(Me3Si)2NH
H2POONH4
H P
P
EtO
OSiMe3
IV
II
V
BrCH2CH2Br
EtOH
Me3SiBr
O
O
O
P
OEt
CH(OEt)3
Br
EtO
P
EtO
OH
O
III
VI
AcNHCH(COOEt)2
O
C(O)OEt
NHAc
C(O)OEt
O
O
O
P
OH
HCl
O
P
EtO
Dowex 50W (H+)
OH
HO
NH2
OEt
VII
I
Bis(triaminosilyl) hypophosphite (IV) is readily
formed from ammonium hypophosphite (II) and si-
lazane [19, 20] and added in situ to ethyl acrylate to
form bis(trimethylsilyl) 3-(ethoxycarbonyl)phopyl-
phosphonite (V) [12]. Treatment of the latter in situ
with excess 1,2-dibromoethane followed by alcoho-
lysis gave (2-bromoethyl)-[2-(ethoxycarbonyl)ethyl]-
phosphinic acid (VI) which was treated in situ with
excess triethyl orthoformate. Dehydrobromination of
the -bromoethyl fragment of acid VI is accompanied
by esterification of the C(O)OH and P(O)OH groups
and affords ethyl [2-(ethoxycarbonyl)ethyl]vinylphos-
phinate (III) [13, 17]. Phosphinate III as the target
product of the first step of the synthesis was isolated,
characterized, and used for further transformations in
the second step of the synthesis of aminophosphinic
acid I.
form [3-amino-3-(hydroxycarbonyl)propyl][2-(hydr-
oxycarbonyl)ethyl]phosphinic acid (I) in 71% yield
(per diethyl acetamidomalonate) after chromatography
on Dowex 50W(H+) as the final step of the synthesis
of racemic amino acid I [21, 22].
Comprehensive biological investigations often
require both enantiomers of a physiologically active
molecule. Amino acids are convenient to resolve
enzymatically. We propose an enzymatic synthesis of
the L and D enantiomers of amino acid I with use of
immobilized penicillamidase for the stereoselective
hydrolysis of N-phenylacetylated acid VIII by
Scheme 2 [23].
Earlier we took advantage of the high enantioselec-
tivity of penicillamidase in the enzymatic hydrolysis
of N-phenylacetylated amino acids in the synthesis of
the enantiomers of phosphoruscontaining -amino-
carboxylic acids [24] by analogy with the synthesis
of the enantiomers of -aminophosphonic acids [25].
Amino acid I was N-acylated with phenylacetyl
chloride by a classical Schotten Baumann procedure
The Michael addition of diethyl acetamidomalonate
to vinylphosphinate III leads to formation of ethyl
[3-(acetylamino)-3,3-bis(ethoxycarbonyl)propyl][2-
(ethoxycarbonyl)ethyl]phosphinate (VII) which was
subjected to acid hydrolysis in situ (Scheme 1) to
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 74 No. 8 2004

SYNTHESIS OF PHOSPHINIC ACIDS ON THE BASIS OF HYPOPHOSPHITES: IV.
1179
Scheme 2.
O
O
PhCH2C(O)Cl
OH
O
O
P
O
NH2
OH
O
HO
OH
P
N
H
HO
Ph
O
OH
L,D-I
L,D-VIII
PcAm
L-I
D-I
D-VIII
[26]. The hydrolyzed L form of acid I was separated
from the nonhydrolyzed D form of N-phenylacetyla-
ted amino acid VIII by ion-exchange chromatography.
Acid hydrolysis of D-VIII gave the D enantiomer of
acid I without noticeable racemization [23].
50WX8-200 (H+) (Lancaster) and Diasorb-Sulfo (H+)
(BioKhimMak).
Analytical HPLC was performed on a Gilson
chromatograph with a US-Vis 118 detector at 254 nm,
column Diasorb 130 C16/T (4 250 mm), sorbent
particle size 5 m. Separation was performed under
conditions of ion-pair chromatography with a specific
counter ion, eluent acetonitrile distilled water
(gradient). For control of enzymatic hydrolysis and
quantitative analysis the column was calibrated with
phenylacetic acid solutions. The enzymatic reaction
was performed with immobilized penicillamidase with
Thus, we propose a new method for construction
of pseudo- -glutamylglycine [21], which may turn a
more convenient synthetic approach to various phos-
phinic analogs of glutamylpeptides compared with the
syntheses proposed in [3, 4]. We also propose an
enzymatic synthesis of the enantiomers of pseudo- -
glutamylglycine [23]. Valyaeva et al. [27] recently
applied our methodology [13, 21] for the synthesis of
a phosphinic analog of -glutamylglutamate.
1
an activity of 103 U g dry biocatalyst [24, 25].
Synthesis of ethyl [2-(ethoxycarbonyl)ethyl]-
vinylphosphinate (III). A mixture of 4.0 g of
ammonium hypophosphite (II) and 15 ml of hexa-
methyldisilazane was stirred for 2 h at 120 130 C.
After cooling to room temperature, 5.3 ml of ethyl
acrylate was slowly added dropwise to the mixture,
and it was stirred for 2 h at 40 C, and cooled. 1,2-Di-
bromoethane, 21 ml, was then added in one portion,
and stirring was continued for 5 h at 120 C. The tri-
methylbromosilane formed and excess 1,2-dibromo
ethane were removed in vacuo, and 50 ml of aqueous
ethanol (1:1) was added to the residue. The mixture
was refluxed for 0.5 h and then evaporated in vacuo.
The residue was partitioned between 100 ml of ethyl
acetate and 50 ml of water, and the aqueous layer was
extracted with ethyl acetate (2 50 ml). The combined
organic layer was evaporated in vacuo. The residue,
EXPERIMENTAL
1
The H, 31P and 13C NMR spectra were registered
on Bruker DPX-200 and Bruker CXP-300 spectro-
meter against internal TMS and external H3PO4. The
optical rotation was measured on Perkin Elmer 241
and Polamat A polarimeters. The mass spectra were
registered on a Finigan 4021 instrument. The melting
points were measured on a Boetius hot stage or in an
open capillary. All reactions with silyl esters of
hypophosphorus and phospinic acids in situ were
performed under argon. All solvents used were
carefully dried. Ammonium hypophosphite was
prepared by a slightly modified published method [11].
Triethyl orthoformate, diethyl acetamidomalonate,
propylene oxide, and phenylacetyl chloride were
purchased from Khimeks (St. Petersburg).
1
according to H and 31P data (CDCl3), was a 9:1
mixture of phosphinic acid VI ( 54.6 ppm) and
[2-(ethoxycarbonyl)ethyl]vinylphospPhinic acid
(
Thin-layer chromatography of individual com-
pounds and reaction mixtures was performed on
Silufol plates (eluent chloroform acetone, 5:1),
Merck glass plates with a 0.2-mm layer of silica gel
UV-254, and Kavalier Alufol plates (neutral alumina
on an aluminum foil, eluent 1-butanol acetic acid
water, 5:1:1), developer ninhydrin (110 C). Ion-
exchange chromatography was performed on Dowex
42.6 ppm). The latter was probably formed by partiaPl
dehydrobromination of acid VI in the presence of
hexamethyldisilazane. To the residue, 40 ml of tri-
ethyl orthoformate was added, and the reaction mix-
ture was refluxed in a Dean Stark trap for 2 3 h with
distillation of the ethyl formate and ethanol formed.
Unreacted triethyl orthoformate was removed in vacuo,
and the residue was distilled. Vinylphosphinate III
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 74 No. 8 2004

1180
RAGULIN
was isolated as a yellowish oily substance. Yield 3.8 g
(38% per ammonium hypophosphite, after 5 steps). bp
[3-(phenylacetyl)amino-3-(hydroxycarbonyl)pro-
pyl]phosphinic acid (VIII). A solution of 4.6 ml of
phenylacetyl chloride in 5 ml of dioxane was added
at 0 2 C to a solution of 6.9 g of racemic amino acid
I in 60 ml of aqueous dioxane (2:1), maintaining the
solution pH at about 9.5 with 50% KOH. The reaction
mixture was then stirred at room temperature for 10
15 h, acidified to pH 5, and extracted with ether (2
30 ml), after which it was acidified to pH 1 2 and
extracted with ethyl acetate (3 50 ml). The combined
extracts were dried over MgSO4 and evaporated. The
residue was recrystallized from a mixture of hexane
(or pentane) and acetone. The product was pure
enough to be brought in further transformations. An
analytically pure sample of acid VIII was obtained by
recrystallization from a mixture of hexane (or pentane)
125 129 C (3 mm Hg), n2D0 1.4600. H NMR spec-
1
trum (CDCl3), , ppm: 1.25 t (3H, CH3), 1.34 t (3H,
CH3), 2.07 m (2H, PCH2), 2.60 m [2H, C(O)CH2],
4.03 m (2H, CH2OC), 4.15 m (2H, CH2OP), 6.00
6.50 m (3H, CH=CH2). 31P NMR spectrum (CDCl3),
P, ppm: 41.3. Found, %: C 49.14; 49.33; H 7.97,
8.11. C9H17O4P. Calculated, %: C 49.09; H 7.78.
Mass spectrum: found 221, calculated 221. Column
chromatography (eluent chloroform hexane 2:1) gave
43% of vinylphosphinate III; TLC: Rf 0.4 (chloro-
form acetone, 8:1).
Synthesis of [3-amino-3-(hydroxycarbonyl)-
propyl][2-(hydroxycarbonyl)ethyl]phosphinic acid
(I). A mixture of 6.8 g of ethyl acetamidomalonate,
7.5 g of vinylphosphinate II, 8.6 g of potassium car-
bonate, and 0.5 g of tetrabutylammonium bromide in
20 ml of THF was refluxed for 11 13 h with stirring
until complete consumption of ethyl acetamidomalo-
nate (control by TLC on Silufol, eluent chloroform
acetone 4 5:1, Rf 0.5 0.6). The reaction mixture was
partitioned between 50 ml of chloroform and 25 ml of
water, and the aqueous layer was neutralized and ex-
tracted with chloroform (2 25 ml). The combined
organic extract was evaporated in vacuo. The residue
1
and acetone. Yield 8.2 g (79%), mp 127 129 C. H
NMR spectrum (CD3OD), , ppm: 1.80 m (2H),
2.00 m (4H), 2.55 m (2H), 3.65 s (2H), 4.46 t (1H),
7.30 m (5H). 31P NMR spectrum (CD3OD), P, ppm:
53.4. Found, %: C 49.34, 49.33; H 5.64, 5.61; P 8.31,
8.29. C15H20NO7P 0.5H2O. Calculated, %: C 49.18;
H 5.78; P 8.46. Mass spectrum: calculated for
(C15H20NO7P 0.5H2O + H
322.5.
CO2)+ 322.4, found
Enzymatic synthesis of the enantiomers of
amino acid I. Immobilized penicillinamidase, 3 5 g,
was added to a solution of 7.1 g of D,L-VIII at pH
7.5, maintaining the solution pH at 7.5. The enzy-
matic hydrolysis was monitored by HPLC by the
formation of phenylacetic acid. After 50% hydrolysis,
the enzyme was filtered off, and the reaction mixture
was acidified to pH 5 and washed with ether (2
40 ml). The aqueous layer was acidified to pH 2 with
6N HCl, concentrated to 20 ml, and passed through
a column (3 25 30 cm) of a cation exchanger, eluent
water. A strongly acidic eluate with a negative nin-
hydrin test was evaporated in vacuo. To the oily
residue whose 1H and 31P spectra were similar to those
of VIII, 70 ml of 8 N HCl was added, and the mix-
ture was refluxed for 10 h, cooled, washed with ether
(2 40 ml), and evaporated in vacuo. The residue was
dissolved in aqueous ethanol and treated with excess
1,2-propylene oxide. Recrystallization from aqueous
1
(ca. 14 g), according H and 31P NMR data (CDCl3),
was a 10:1 mixture of phosphinate VII ( P 55.8 ppm)
and unreacted vinylphosphinate III ( 41.3).
P
To the oily residue, 70 ml of 8N HCl was added,
and the mixture was refluxed for 13 15 h, cooled,
washed with diethyl ether (3 20 ml), and evaporated
in vacuo. The residue was chromatographed on a
column (200 mm) of Dowex 50W(H+), eluent 0.5
0.7 N HCl. Fractions with a positive ninhydrin test
were concentrated, dissolved in 20 ml of aqueous
ethanol (1:4), and treated with excess propylene oxide
to isolate the target amino acid. Additional recrystal-
lization from aqueous ethanol gave 5.3 g (71% per
ethyl acetamidomalonate) of compound I. Total yield
28% (per ammonium hypophosphite), mp 205 207 C
1
(decomp.). H NMR spectrum (D2O + DCl), , ppm :
1.70 m (2H), 1.88 m (4H), 2.36 d.t (2H, JPH 12 Hz),
3.92 t (1H). 13C NMR spectrum (D2O), C, ppm.:
26.4 d (PCH2C, J 91.4 Hz), 27.3 d (PCH2C, J
90.2 Hz), 28.9 d (CCH2CH, J 2.4 Hz), 31.4 d
[CCH2C(O), J 3.1 Hz], 57.5 d (CH, J 15.8 Hz),
183.3 d [C(O)CH2, J 17.1 Hz], 183.6 s [C(O)CH].
31P NMR spectrum, P, ppm.: 50.0 (D2O), 44.0
(D2O + NaOD, pH 10). Found, %: C 34.06, 33.93; H
6.07, 6.08; N 5.89, 5.86. C7H14NO6P 0.5H2O.
Calculated, %: C 33.88; H 6.09; N 5.64.
ethanol gave 1.7 g [71.0 % per D,L-VIII] of D-I. mp
25
194 195 C (decomp.), [ ]
23.7 (c 1.2, 6 N HCl),
546
25
[ ]
13.4 (c 1.1, H2O), [ ]2D5 20.9 (c 1.2, 6 N
546
HCl). The H, 13C and 31P NMR spectra were the
same as those of the racemate. Found, %: C 34.33,
34.09; H 6.12, 6.08; N 5.86, 5.82. C7H14NO6P
0.5H2O. Calculated, %: C 33.88; H 6.09; N 5.64.
1
A less acidic eluate with a positive ninhydrin reac-
tion was evaporated in vacuo, and the residue was re-
crystallized from ethanol. Recrystallization from
Synthesis of D,L-[2-(Hydroxycarbonyl)ethyl]-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 74 No. 8 2004

SYNTHESIS OF PHOSPHINIC ACIDS ON THE BASIS OF HYPOPHOSPHITES: IV.
1181
aqueous ethanol gave 1.3 g [54.4% per D,L-VIII] of
10. McCleery, P.P. and Tuck, B., J. Chem. Soc., Perkin
Trans. 1, 1989, p. 1319.
1
L-I. mp 186 187 C (decomp.). The H, 13C and 31P
NMR spectra were the same as those of the racemate.
Found, %: C 33.80, 33.91; H 6.04, 5.99, N 5.89, 5.86.
C7H14NO6P 0.5H2O. Calculated, %: C 33.88; H 6.09;
N 5.64.
11. Boyd, E.A., Regan, A.C., and James, K., Tetrahedron
Lett., 1992, vol. 33, no. 3, p. 813.
12. Kurdyumova, N.R., Rozhko, L.F., Ragulin, V.V., and
Tsvetkov, E.N., Zh. Obshch. Khim., 1997, vol. 67,
no. 12, p. 1965.
ACKNOWLEDGMENTS
13. Kurdyumova, N.R., Ragulin, V.V., and Tsvetkov, E.N.,
Mendeleev Commun., 1997, no. 2, p. 69.
The work was supported by the Research and
Development Divisions of Nippon Chemical Industrial
and Iskra Industry (agreem. 98-99/add. no. 1) [20, 22].
The authors are grateful to S.G. Klochkov for HLPC
analysis of enzymatic steps and to G.D. Sishko for
elemental analysis of new compounds.
14. Ragulin, V.V. and Tsvetkov, E.N., Izv. Akad. Nauk
SSSR, Ser. Khim., 1988, no. 11, p. 2652.
15. Ragulin, V.V., Kurdyumova, N.R., and Tsvetkov, E.N.,
Phosphorus, Sulfur Silicon, 1994, vol. 88, nos. 3, 4,
p. 271.
16. Kurdyumova, N.R., Ragulin, V.V., and Tsvetkov, E.N.,
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