Stereochemistry of the reductive amination of 4-oxoproline derivatives with glycine esters

Article abstract of DOI:10.1023/A:1021237314299

An amination of 4-oxoproline derivatives with glycine methyl or benzyl ester and sodium cyanoborohydride led to the mixtures of corresponding diastereomeric 4-cis- and 4-trans-glycinoproline derivatives. We found that the ratio of diastereomers mainly depends on the structure of 4-oxoproline ester groups and, to a lesser extent, on the structure of N-acyl substituents. The best results were achieved with tert-butyl ester group; it ensured good yields of the amination products and the greatest prevalence of 4-cis-isomers. The structure of ester group in glycine molecule only scarcely affected the resulting ratio of N-(N-benzyloxycarbonylglycyl)-4-glycinoprolines.

Full text of DOI:10.1023/A:1021237314299

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 6, 2002, pp. 444–449. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 6, 2002, pp. 491–496.
Original Russian Text Copyright © 2002 by Yelin, Onoprienko, Miroshnikov.
Stereochemistry of the Reductive Amination of 4-Oxoproline
Derivatives with Glycine Esters
1
E. A. Yelin, V. V. Onoprienko, and A. I. Miroshnikov
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia
Received November 21, 2001; in final form, December 21, 2001
Abstract—An amination of 4-oxoproline derivatives with glycine methyl or benzyl ester and sodium
cyanoborohydride led to the mixtures of corresponding diastereomeric 4-cis- and 4-trans-glycinoproline deriv-
atives. We found that the ratio of diastereomers mainly depends on the structure of 4-oxoproline ester groups
and, to a lesser extent, on the structure of N-acyl substituents. The best results were achieved with tert-butyl
ester group; it ensured good yields of the amination products and the greatest prevalence of 4-cis-isomers. The
structure of ester group in glycine molecule only scarcely affected the resulting ratio of N-(N-benzyloxycarbo-
nylglycyl)-4-glycinoprolines.
Key words: N-glycyl-4-glycinoproline derivatives, 4-oxoproline derivatives, reductive amination, separation of
diastereomers
1
INTRODUCTION
RESULTS AND DISCUSSION
The mixture of 4-cis- and 4-trans-glycinoprolines
(VIa) and (VIIa), we obtained previously [1], was
chromatographically separated after its conversion into
the mixture of corresponding isomeric trifluoroacet-
amides (VIb) and (VIIb); their ratio was found to be
6.5 : 1.
The direct exchange of the protective benzyloxy-
carbonyl group in the molecule of (II) by benzyloxy-
carbonylglycyl residue could not be performed,
because its selective removal by an acidic treatment
(AcOH/HBr) was impossible due to the presence of
acid-labile tert-butyloxycarbonyl grouping in (II) and
its hydrogenolysis was impractical, since it is known to
result in the 4-oxoproline polymerization products [2].
Therefore, 4-trans-hydroxyproline was transformed to
We have previously synthesized conformationally
restricted proline derivative (I) [1]. During this synthe-
sis, we subjected N-benzyloxycarbonyl-4-oxoproline
tert-butyl ester (II) to a reductive amination with gly-
cine methyl ester and received a mixture of tert-butyl
esters of 4-cis- (VIa) and 4-trans-N-benzyloxycarbo-
nyl-4-methoxycarbonylmethylamino-L-prolines (VIIa).
The conversion of this mixture into γ-lactam (I) in yield
exceeding 60% demonstrated that 4-cis-diastereomer
(VIa) prevailed in this mixture [1].
In this case, stereoselectivity of the reductive amina-
tion is determined by a facilitated approach of hydride
ion from the side opposite to the tert-butyloxycarbonyl
grouping of pyrrolidine ring to the C=N bond of the N-benzyloxycarbonylglycyl-4-trans-hydroxyproline
(VIII) by the coupling with N-succinimide ester of
benzyloxycarbonylglycine, and the secondary hydroxy
group in (VIII) was oxidized to yield 4-oxoderivative
(III), which was then converted into the tert-butyl ester
(IV). The reductive amination of (IV) with glycine
methyl ester led to the diastereomeric mixture (VIc) +
(VIIc).
Unlike trifluoroacetamides (VIb) and (VIIb), 4-cis-
and 4-trans-trifluoroacetamide derivatives of all the
N-(N-benzyloxycarbonylglycyl)-4-glycinoproline
derivatives we obtained exhibited almost no differences
in their chromatographic behavior. In all the cases, we
achieved the separation of diastereomers after the con-
version of mixtures of the N-(N-benzyloxycarbonylgly-
intermediate Schiff base that arises in the reaction mix-
ture through the interaction of 4-oxoproline derivative
with the glycine ester.
In this work, we carried out the reductive amination
of a series of 4-oxo-L-proline derivatives and separated
the formed diastereomeric pairs of glycinoprolines. As
a result, the changes in the ratio of the 4-cis- and 4-
trans-diastereomers were traced in dependence on the
structures of starting 4-oxo-L-proline and glycine deri-
vatives.
1
The author may be contacted by phone, +7 (095) 336-1300 or e-
mail, onovv@mail.ibch.ru.
1068-1620/02/2806-0444$27.00 © 2002 MAIK “Nauka /Interperiodica”

STEREOCHEMISTRY OF THE REDUCTIVE AMINATION
445
(II) R1 = Cbz, R2 = But
COOR2
R1
Cbz
R1
(III) R1 = CbzGly, R2 = H
(IV) R1 = CbzGly, R2 = But
(V) R1 = CbzGly, R2 = Me
O
N
N
N
COOMe
(I)
O
COOR2
COOR2 R1
COOR
CbzGly
N
N
N
OH
COOR4
N
COOR4
N
R3
R3
(VIII) R = H
(IX) R = Me
(VI)
(VII)
a: R1 = Cbz, R2 = But, R3 = H, R4 = Me
b: R1 = Cbz, R2 = But, R3 = COCF3, R4 = Me
c: R1 = CbzGly, R2 = But, R3 = H, R4 = Me
d: R1 = CbzGly, R2 = But, R3 = Ac, R4 = Me
e: R1 = CbzGly, R2 = But, R3 = H, R4 = Bzl
f: R1 = CbzGly, R2 = But, R3 = Ac, R4 = Bzl
g: R1 = CbzGly, R2 = But, R3 = Ac, R4 = H
h: R1 = CbzGly, R2 = Me, R3 = H, R4 = Bzl
i: R1 = CbzGly, R2 = Me, R3 = Ac, R4 = Bzl
cyl)-4-glycinoproline free bases into the mixtures of the N-(N-benzyloxycarbonylglycyl)-4-oxoproline (IV) did
corresponding acetamides. For example, the (VIc) + not lead to substantial changes in the reaction stereose-
(VIIc) mixture was converted into the (VId) + (VIId) lectivity. The resulting diastereomeric mixture of free
mixture, which can be easily separated by chromatog- bases (VIe) + (VIIe) was transformed to a mixture of
raphy.
diastereomeric acetamides (VIf) + (VIIf), whose chro-
matographic separation showed that they are formed at
the ratio of 4.95 : 1. Acetamide (VIf) was identified
with the substance obtained from the 4-cis-acetami-
doglycinoproline (VId) by means of its saponification
to acid (VIg) followed by benzylation. This proved that
acetamides (VIf) and (VIIf) have 4-cis- and 4-trans-
configurations, respectively.
The 4-cis- and 4-trans-configuratios were ascribed
to (VId) and (VIId), respectively, after their counter
synthesis from the diastereomeric mixture of (VIa) and
(VIIa). The conversion of this mixture into the mixture
of chromatographically inseparable acetamides and the
successive removal of the benzyloxycarbonyl protec-
tive group and the acylation of the resulting free amino
group with N-benzyloxycarbonylglycine led to a mix-
A substantial decrease in stereoselectivity of the
ture of diastereomeric 4-cis- and 4-trans-acetamides, reductive amination was only observed at the amination
which were chromatographically separated and, of
N-(N-benzyloxycarbonylglycyl)-4-oxoproline
according to TLC and mass spectrometry, turned out to methyl ester (V), which was obtained by the treatment
be identical to (VId) and (VIId), respectively. The ratio of hydroxyacid (VIII) with diazomethane with the sub-
of diastereomers (VId) and (VIId) in the mixture sequent oxidation of the intermediate (IX) by chromic
obtained from the (VIa) + (VIIa) mixture was 6.0 : 1, acid. The amination of (V) with glycine benzyl ester
which was practically the same as the value 6.5 : 1 pre- resulted in a mixture of diastereomeric free bases N-(N-
viously obtained after the direct separation of the benzyloxycarbonylglycyl)-4-glycinoprolines (VIh) +
(VIb) + (VIIb) mixture. At the same time, the ratio of (VIIh), which was separated by chromatography; the
(VId) and (VIId) that were obtained from N-(N-benzyl- ratio of the bases was 2.7 : 1. In addition, the same mix-
oxycarbonylglycyl)-4-oxoproline (IV) (4.7 : 1) demon- ture of diastereomers (VIh) + (VIIh) was subjected to
strates that, in the 4-oxoproline(II) molecule, the acetylation and chromatographic separation; in this
replacement of N-benzyloxycarbonyl protective group case, the ratio of the resulting acetamides (VIi) and
by N-benzyloxycarbonylglycyl residue exerts a com- (VIIi) was found to be 2.3 : 1.
paratively little effect on the ratio of diastereomers that
are formed upon the reductive amination.
Acetamides (VIi) and (VIIi) were also obtained by
acetylation of individual (VIh) and (VIIh). Acetamide
Likewise, the substitution of glycine benzyl ester for (VIi) turned out to be identical to the compound
its methyl ester in the reaction of reductive amination of obtained from 4-cis-acetamide (VIf) by the removal of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

446
YELIN et al.
its tert-butyl ester group followed by the treatment of dride (96 µl) was added to a solution of a diastereo-
the intermediate acid with diazomethane. These trans- meric mixture of (VIa) + (VIIa) [1] (143.7 mg,
formations proved that (VIi) and its precursor (VIh) are 0.367 mmol) in dry pyridine (720 µl) cooled on an ice
4-cis-diastereomers, whereas (VIIi) and (VIIh) have bath. The reaction mixture was kept for 3.5 h under
trans-configuration. Note that the reductive amination cooling; diluted with ether (40 ml); washed with 1 N
of
N-(N-benzyloxycarbonylglycyl)-4-oxoproline HCl, water, saturated solution of sodium bicarbonate,
methyl ester (V) was less efficient than those of (II) and and water; dried with Na2SO4; and evaporated. The res-
(IV) and the total yield of (VIh) and (VIIh) was signif-
icantly lower, which is probably due to a partial sapon-
ification of the methyl ester (V) during the reaction.
idue (170 mg) was chromatographed on a silica gel col-
umn (17 g) eluted with a step gradient of ether (0–80%)
in hexane. The chromatographically pure oily 4-cis-tri-
fluoroacetamide (VIb) was eluted with a 3 : 7 hexane–
ether mixture, yield 124.8 mg (69.8%); it was crystal-
lized under storage in a refrigerator. After recrystalliza-
tion from an ether–hexane mixture, (VIb) had mp
50.5–53°ë; Rf 0.55 (3 : 1 ether–hexane); PD MS, m/z:
Thus, we established that the stereoselectivity of the
reaction of reductive amination of the 4-oxoproline
derivatives with glycine methyl or benzyl ester in the
presence of sodium cyanoborohydride is mainly deter-
mined by the nature of the ester group of proline deriv-
ative and, to a lesser extent, by the N-acyl substituent in
4-oxoproline molecule. The best results were obtained
when using tert-butyl ester, which provides for a rea-
sonable yield of the amination products and a suffi-
ciently high ratio of 4-cis- and 4-trans-diastereomers.
The nature of ester group in the glycine molecule only
scarcely affects the ratio of the resulting diastereomeric
N-glycyl-4-glycinoprolines.
579 [M + CH2C6H5]+, 523 [M – C4H8 + CH2C6H5]+, 489
[M + H]+, 433 [M + H – C4H8]+, 387 [M – CO2C4H9]+.
The chromatographically pure oily 4-trans-trifluoroac-
etamide (VIIb) was eluted with a 2 : 8 hexane–ether
mixture; yield 19.2 mg (10.7%); Rf 0.42 (3 : 1 ether–
hexane); mass spectrum was practically identical to that
of (VIb). The ratio of cis- and trans-diastereomers
(VIb) and (VIIb) was 6.5 : 1.
N-Benzyloxycarbonylglycine
N-succinimide
EXPERIMENTAL
ester, Cbz-Gly-ONSuc, was obtained from N-benzyl-
oxycarbonylglycine by the procedure in [4], mp 113.5–
114°ë (lit. mp 113–114°ë [4]).
We used in this work the following reagents: trans-
4-hydroxy-L-proline, tert-butyl acetate, sodium
cyanoborohydride, and trifluoroacetic acid from Sigma
(United States); 10% PdO/C, N,N'-dicyclohexylcarbo-
diimide, and 70% perchloric acid from Merck-Schu-
hardt (Germany); and 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) from Fluka (Switzerland). Other reagents,
including glycine methyl ester hydrochloride, were of
domestic manufacture. For analytical and semiprepara-
tive TLC, precoated plates (glass), TLC aluminium
sheets, and HPTLC precoated plates (glass, for Nano-
TLC) from Merck (Germany) were used. Glass PLC
precoated plates with the layer thickness of 2 mm were
used for preparative TLC. All the plates were prepared
on the basis of Silica gel 60 F254. Spots of substances
were detected by the plate visualization under UV light
with the wavelength of 254 nm or by spraying with
0.8% potassium permanganate or sulfuric acid with the
subsequent heating. Silica gel 60 (40–63 µm, Merck,
Germany) was used for column chromatography.
N-(N-Benzyloxycarbonylglycyl)-4-oxo-L-proline
(III). A solution of Cbz-Gly-ONSuc (721 mg,
2.36 mmol) in THF (16 ml) was added to a solution of
trans-4-hydroxyproline in 0.36 M KOH (13.1 ml,
4.72 mmol). The reaction mixture was stirred for 2.5 h
at 20°ë and evaporated in a vacuum, and the residue
was dissolved in water (20 ml) and thrice extracted with
ethyl acetate. The aqueous solution was cooled on an
ice bath, acidified with 5.5 N HCl to pH 2–3, saturated
with NaCl, and thoroughly extracted with ethyl acetate.
The extract was washed with a saturated solution of
NaCl, dried with Na2SO4; and evaporated to dryness.
The resulting oily residue (655.1 mg) of crude N-(N-
benzyloxycarbonylglycyl)-4-hydroxy-L-proline (VIII)
was oxidized by chromic acid mixture (1.935 ml) as
described in [2] to give a crude crystalline product
(579.6 mg), which was first washed with ether and then
recrystallized from an acetone–ether mixture. Keto-
dipeptide (III) was obtained; yield 361.6 mg (48%
from Cbz-Gly-ONSuc), mp 168–169.5°ë. After an
additional recrystallization, it had mp 169–172°ë,
Rf 0.31 (40 : 10 : 1 CHCl3–90% MeOH–AcOH); and
Mass spectra were determined under ionization by
electron impact (EI MS, 70 eV, Varian MAT 44) or by
plasma of the 252Cf scission products (PD MS, MSBKh
Elektron, Sumy, Ukraine). Melting points were deter-
mined on a micro heating plate Boetius and were not
corrected.
PD MS, m/z: 365 [M – H + 2 Na]+, 343 [M + Na]+, 321
[M + H]+.
N-Benzyloxycarbonylglycine and glycine benzyl
ester p-toluenesulfonate were obtained using the
known procedures [3].
N-(N-Benzyloxycarbonylglycyl)-4-oxo-L-proline
tert-butyl ester (IV). A stirred cooled to 10°ë suspen-
sion of (III) (310 mg, 0.969 mmol) in tert-butyl acetate
(12 ml) was treated with 70% HClO4 (121 µl) and then
tert-Butyl esters of N-benzyloxycarbonyl-4-cis-
(VIb) and -4-trans-(methoxycarbonylmethyl)triflu- stirred until complete dissolution (2 h) and kept at the
oroacetamido-L-proline (VIIb). Trifluoroacetic anhy- same temperature for 46 h. A crude product obtained
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STEREOCHEMISTRY OF THE REDUCTIVE AMINATION
447
after a usual treatment was purified by a column chro-
B. A diastereomeric mixture (VIa) + (VIIa) [1]
matography on silica gel (10 g). Elution with a 4 : 1 (200 mg, 510 µmol) was acetylated in a usual manner,
ether–hexane mixture afforded partially crystallized and the reaction mixture was diluted with water and
chromatographically homogeneous (IV); yield extracted with ethyl acetate. The extract was succes-
182.1 mg (50%); after washing with ether, crystals had sively washed with 1 N HCl, 10% NaHCO3, and water;
mp 57–59°ë; Rf 0.44 (ether) and PD MS, m/z: 377
[M + H]+, 321 [M + H – C4H8]+, 277 [M + H –
CO2C4H8]+.
dried with sodium sulfate; and evaporated to give the
chromatographically homogeneous mixture of diaste-
reomeric acetamides as a viscous oil; Rf 0.10 (ether); EI
MS, m/z: 378 [M – C4H8]+, 334 [M – CO2C4H8]+, 303
N-(N-Benzyloxycarbonylglycyl)-4-cis/trans-metho-
xycarbonylmethylamino-L-proline tert-butyl ester
(VIc) + (VIIc). A solution of (IV) (74.7 mg,
0.199 mmol) and glycine methyl ester hydrochloride
(125 mg, 0.996 mmol) in absolute methanol (1.2 ml)
was treated at room temperature with portions of 250,
125, 63, and 32 µl of 1 N NaOH in absolute methanol
followed by delay for 10, 15, 20, and 30 min, respec-
tively. Sodium cyanoborohydride (14 mg, 0.22 mmol)
was then added; and the reaction mixture was kept for
1.5 h, diluted with a saturated solution of NaCl (4 ml),
and extracted with ethyl acetate. The extract was thor-
oughly washed with a saturated solution of NaCl, dried
over Na2SO4, and evaporated. The residue was chro-
matographed on a silica gel column (10 g) eluted with
a 99 : 1 chloroform–methanol mixture to give 48.6 mg
of the chromatographically pure oily (VIc) + (VIIc).
The fractions that contained the product with some
admixtures were combined and subjected to a prepara-
tive TLC in ethyl acetate (double development and elu-
tion of the substance from the UV-light-absorbing zone
with Rf 0.47 by 2 : 1 chloroform–methanol mixture).
Total yield of the product was 54.4 mg (61%); oil; Rf
0.31 (24 : 1 chloroform–methanol); PD MS, m/z: 450
[M + H]+, 392 [M – C4H9]+, 348 [M – CO2C4H9]+, 157
[378
–
CH3CO
–
ëH3OH]+, 247 [378
–
CH3CONHCH2CO2CH3]+, 202 [247 – CO2H]+, 158
[202 – CO2]+, 132 [CH3CONHCH2CO2CH3 + H]+.
The resulting mixture of acetamides (220 mg,
507 µmol) was hydrogenated over 10% PdO/C as
described in [1]. The hydrogenation product was dis-
solved in anhydrous methylene chloride (2.5 ml), and
the solution was treated with N-benzyloxycarbonylgly-
cine (113 mg, 540.6 µmol) and DCC (113 mg,
548.5 µmol) upon cooling on an ice bath. The reaction
mixture was kept for 30 min upon cooling and 18 h at
room temperature. After the separation of dicyclohexy-
lurea, the mixture of acetamides (VId) and (VIId) was
chromatographically separated as described above in
the experiment Ä. 4-cis-Acetamide (VId) [191.8 mg,
79.0% from the (VIa) + (VIIa) mixture] and 4-trans-
acetamide (VIId) (32.0 mg, 13.2%) resulted; they were
identical (according to TLC and mass spectra) to the
corresponding acetamides obtained in the experiment
A. The ratio of their quantities was 6.0 : 1, which obvi-
ously reflects the diastereomer ratio in the starting
(VIa) + (VIIa) mixture.
tert-Butyl esters of N-(N-benzyloxycarbonylgly-
cyl)-4-cis- (VIe) and -4-trans-benzyloxycarbonylme-
thylamino-L-proline (VIIe). A solution of (IV)
(106.6 mg, 283 µmol) and glycine benzyl ester p-tolu-
enesulfonate (477 mg, 1.417 mmol) in absolute metha-
nol (2.6 ml) was treated with 1 N NaOH in absolute
methanol (360 µl and, after 10 min, 180 µl) at 26°ë.
The reaction mixture was kept for 20 min, treated with
sodium cyanoborohydride (17 mg, 270 µmol), after 2 h
diluted with a saturated NaCl solution (5 ml), and
extracted with ethyl acetate. The extract was washed
with water, dried with Na2SO4, and evaporated. The
product was isolated by column chromatography on sil-
ica gel (23 g), eluting with chloroform and 99.5 : 0.5
chloroform–methanol mixture. The resulting crude
product (88.7 mg) was additionally purified by prepar-
ative TLC upon the development with ethyl acetate.
The elution of the main UV-light-absorbing zone (Rf
0.39) with ethyl acetate gave 76.6 mg (51.5%) of the
chromatographically pure (VIe) + (VIIe) product; oil;
Rf 0.64 (ethyl acetate); PD MS, m/z: 526 [M + H]+, 470
[348 – PhCH2CONHCHCO]+.
tert-Butyl esters of N-(N-Benzyloxycarbonylgly-
cyl)-4-cis- (VId) and -4-trans-(methoxycarbonylme-
thyl)acetamido-L-proline (VIId). A. A solution of
(VIc) + (VIIc) mixture (54.0 mg, 120.3 µmol) in a 1 : 1
pyridine–acetic anhydride mixture (600 µl) was kept
for 3.5 h at room temperature, excess acetic anhydride
was decomposed with methanol, and the reaction mix-
ture was evaporated to dryness. Acetamides (VId) and
(VIId) were isolated by chromatography on a silica gel
column (6.3 g) with ethyl acetate elution. The interme-
diate fraction containing both substances was addition-
ally separated by TLC in ethyl acetate (thrice develop-
ment), while isolating them from the UV-light-absorb-
ing zones with Rf 0.61 and 0.71 for (VId) and (VIId),
respectively. There were totally obtained oily 4-cis- and
4-trans-acetamides (VId) and (VIId). (VId): yield
42.9 mg (72.7%); Rf 0.47 (ethyl acetate, double devel-
opment); PD MS, m/z: 492 [M + H]+, 436 [M + H –
C4H8]+, 392 [M + H – CO2C4H8]+, 302 [436 –
C6H5CHCO]+. (VIId): yield 9.1 mg (15.4%); Rf 0.57
(ethyl acetate, double development); PD MS was prac-
tically identical to that of (VId). The ratio of the diaste- cyl)-4-cis- (VIf) and -4-trans-(benzyloxycarbonyl-
reomers was 4.7 : 1.
[M + H – C4H8]+, 426 [M + H – CO2C4H8]+, 334 [M –
CO2C4H8 – PhCH]+.
tert-Butyl esters of N-(N-benzyloxycarbonylgly-
methyl)acetamido-L-proline (VIIf). The diastereo-
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448
YELIN et al.
meric mixture (VIe) + (VIIe) (75 mg, 142.8 µmol) was graphically pure (V); oil; Rf 0.35 (24 : 1 chloroform–
methanol); PD MS, m/z: 335 [M + H]+.
acetylated under usual conditions and then separated by
preparative TLC in ethyl acetate. Elution of the UV-
absorbing zones with Rf 0.41 and 0.24 resulted, respec-
tively, in (VIIf) and (VIf). (VIIf): yield 12.9 mg (16%);
oil; Rf 0.65 (ethyl acetate); PD MS, m/z: 568 [M + H]+,
Methyl esters of N-(N-benzyloxycarbonylglycyl)-
4-cis (VIh) and 4-trans-benzyloxycarbonylmethyl-
amino-L-proline (VIIh). A 1 N solution of NaOH in
absolute methanol (2 ml) was added to a solution of (V)
(297.4 mg, 0.890 mmol) and glycine benzyl ester p-tol-
uenesulfonate (1.5 g, 4.45 mmol) in absolute methanol
(8.1 ml) at room temperature. The reaction mixture was
kept for 30 min, treated with sodium cyanoborohydride
(54 mg, 0.857 mmol), and, after 70 min, diluted with
ether. The precipitated solid was thoroughly extracted
with water, and the product was additionally extracted
from the ether solution with 1 N HCl. The acidic layer
was washed with ether and then treated with sodium
bicarbonate to alkaline reaction and extracted with
ether. The extract was washed with a saturated solution
of NaCl, dried with Na2SO4, and evaporated. The resi-
due was purified by preparative TLC in a 24 : 1 chloro-
form–methanol system at double development. The
UV-light-absorbing zone (Rf 0.31) was eluted with a
2 : 1 chloroform–methanol mixture to give (VIh) +
(VIIh) mixture (157.7 mg, 36.7%), which was sepa-
rated by preparative TLC; the plates were developed
five times with a 49 : 1 chloroform–methanol system. 4-
cis-Isomer (VIh) was eluted from the zone with Rf 0.52;
yield 67.2 mg (15.6%); oil; PD MS, m/z: 506 [M +
Na]+, 484 [M + H]+. 4-trans-Isomer (VIIh) was eluted
from the zone with Rf 0.36; yield 24.7 mg (5.7%); oil;
512 [M + H – C4H8]+, 468 [M + H – CO2C4H8]+. (VIf):
yield 63.9 mg (79%) that was identical (TLC and PD
MS) to the substance obtained from (VId) (see the next
experiment). The ratio of (VIf) and (VIIf) was 4.95 : 1.
N-(N-Benzyloxycarbonylglycyl)-4-cis-benzyloxy-
carbonylmethyl)acetamido-L-proline
tert-butyl
ester (VIf). A solution of (VId) (95 mg, 193 µmol) in
methanol (4.2 ml) was mixed with 1 N NaOH (575 µl),
and the mixture was stirred for 3.5 h at 20°ë, acidified
with 5.5 N HCl to pH 2, and methanol was removed in
a vacuum. The remaining aqueous solution was
extracted with ethyl acetate, the extract was washed
with water, dried over Na2SO4, and evaporated. The
residue (91.8 mg, 192.5 µmol), which was the chro-
matographically pure acid (VIg), Rf 0.28 (40 : 5 : 1
ëHCl3–MeOH–AcOH), was dissolved in dry benzene
(2.3 ml), benzyl bromide (42.5 µl, 297 µmol) and DBU
(43.8 µl, 293 µmol) were added, and the reaction mix-
ture was stirred under reflux for 5.5 h. After cooling and
dilution with ether, the precipitated DBU hydrobro-
mide was filtered off and washed with ether and ethyl
acetate. The combined filtrates were washed with
water, 1 N HCl, 10% NaHCO3, and water, dried with
Na2SO4, and evaporated. The residue was dissolved in
ether and applied onto a silica gel column (5 g). Low-
polar admixtures were eluted with ether, and the prod-
uct, with ethyl acetate. The yield of (VIf) was 92 mg
[84% from (VId)]; oil; Rf 0.48 (ethyl acetate); PD MS,
PD MS, m/z: 506 [M + Na]+, 484 [M + H]+. The ratio of
the isomers was 2.7 : 1.
Methyl esters of N-(N-benzyloxycarbonylglycyl)-
4-cis- (VIi) and 4-trans-(benzyloxycarbonylme-
thyl)acetamido-L-proline (VIIi). A. A unseparated
(VIh) + (VIIh) mixture (117.2 mg, 243 µmol),
described in the preceding experiment, was acetylated
under usual conditions, and the resulting acetamides
were separated with preparative TLC in a 24 : 1 chloro-
form–methanol system at double development. The
substances were eluted from the UV-absorbing zones
with Rf 0.36 and 0.25 for (VIIi) and (VIi), respectively.
The resulting acetamides had admixtures of the corre-
sponding 4-epimers; and, therefore, they were addition-
ally separated by TLC at double development with
ethyl acetate; (VIi) and (VIIi) exhibited Rf values 0.43
and 0.50, respectively. The corresponding fractions
were combined; yields were 69.1 mg (54.2%) for (VIi)
and 29.6 mg (23.2%) for (VIIi). Both isomers exhibited
identical PD MS spectra with m/z 526 [M + H]+. The
isomer ratio was 2.3 : 1.
m/z: 658 [M + PhCH2]+, 590 [M + Na]+, 568 [M + H]+,
534 [M + Na – C4H8]+, 512 [M + H – C4H8]+, 468 [M + H
– CO2C4H8]+, 376 [M – CO2C4H8 – PhCH2]+.
N-(N-Benzyloxycarbonylglycyl)-4-trans-hydroxy-
L-proline methyl ester (IX). A solution of N-(N-ben-
zyloxycarbonylglycyl)-4-trans-hydroxy-L-proline
(VIII) (524.3 mg) in methanol (3 ml) was kept with
excess diazomethane ether solution for 40 min at 0°ë;
the diazomethane excess was decomposed with acetic
acid. The solution was evaporated, and the residue was
purified by column chromatography on silica gel (25 g)
using a step gradient of methanol (0–10%) in chloro-
form. Yield of (IX) was 441 mg (65% from Cbz-Gly-
ONSuc); viscous oil; Rf 0.20 (24 : 1 chloroform–meth-
anol); PD MS, m/z: 527 [M + PhCH2]+, 437 [M + H]+.
N-(N-Benzyloxycarbonylglycyl)-4-oxo-L-proline
methyl ester (V). N-Benzyloxycarbonylglycylhydrox-
yproline methyl ester (IX) (422.1 mg, 1.256 mmol) was
oxidized by chromic acid mixture by the procedure in
[2], and the reaction product was purified by column
chromatography on silica gel (25 g). A successive elu-
tion with chloroform and 99 : 1 and 99 :2 chloroform–
B. The acetylation under usual conditions of the pre-
liminarily separated (VIh) and (VIIh) led to quantita-
tive yields of the acetamides whose chromatographic
behavior and PD MS coincided with the corresponding
characteristics of the above described (VIi) and (VIIi).
N-(N-Benzyloxycarbonylglycyl)-4-cis-(benzyloxy-
methanol mixtures gave 316.8 mg (75%) of chromato- carbonylmethyl)acetamido-L-proline methyl ester
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

STEREOCHEMISTRY OF THE REDUCTIVE AMINATION
449
(VIi). A solution of (VIf) (3.1 mg, 5.47 µmol) in TFA
REFERENCES
(50 µl) was kept for 2.5 h at room temperature and
evaporated to dryness. The residue was dissolved in
methanol (100 µl), treated with an excess of ethereal
solution of diazomethane (40 min, 0°ë), and evapo-
rated. The reaction product was purified by TLC in
ethyl acetate at double development. The substance
from the main UV-absorbing zone gave (VIi); yield
1.9 mg [66% from (VIf)]; oil; Rf 0.43 (AcOEt, double
development); PD MS, m/z: 526 [M + H]+; the sub-
stance was identical to the sample obtained as
described in the preceding experiment.
1. Yelin, E.A. and Onoprienko, V.V., Bioorg. Khim., 1998,
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2. Patchett, A.A. and Witkop, B., J. Am. Chem. Soc., 1957,
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002