A practical synthesis of fully protected L-γ-carboxyglutamic acid

Article abstract of DOI:10.1016/j.tetlet.2005.10.121

We have developed a new synthetic route for the preparation of Fmoc protected L-γ-carboxyglutamic acid in 60% overall yield (>99% ee) via a six-step synthesis from D-Garner's aldehyde. An aldol condensation and the selective cleavage of the acetonide prot

Full text of DOI:10.1016/j.tetlet.2005.10.121

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 23–25
A practical synthesis of fully protected L-c-carboxyglutamic acid
Sheng Jiang, Christopher C. Lai, James A. Kelley and Peter P. Roller*
Laboratory of Medicinal Chemistry, CCR, National Cancer Institute–Frederick, NIH, Frederick, MD 21702, USA
Received 28 September 2005; revised 19 October 2005; accepted 25 October 2005
Available online 10 November 2005
Abstract—We have developed a new synthetic route for the preparation of Fmoc protected L-c-carboxyglutamic acid in 60% overall
yield (>99% ee) via a six-step synthesis from D-GarnerÕs aldehyde. An aldol condensation and the selective cleavage of the acetonide
protective group are key steps.
Published by Elsevier Ltd.
1. Introduction
Fmoc protected L-c-carboxyglutamic acid was synthe-
sized using the procedure described in Scheme 1. The
starting material, D-GarnerÕs aldehyde, can be easily
prepared from D-serine on a large scale.20 The latter
intermediate 2 was reacted with di-tert-butyl malonate
by using aldol condensation conditions, and then the
b-hydroxy group was eliminated by treatment with
(CF3CO)2O and Et3N.21,22 Using YaoÕs procedure23 to
selectively deprotect the acetonide group with bismuth
bromide provided compound 4 in 93% yield. The purity
of the resulting product was very high and without the
need for further purification. We also found that chang-
ing the reaction conditions by addition of 1 equiv water
in anhydrous acetonitrile enhanced selectivity with no
deprotection of tert-butyl ester being observed. Subse-
quent transformation of aminoalcohol 4 to amino acid
1 involved a three-step reaction sequence: (i) selective
oxidation using PDC as an oxidant,24 (ii) a-amino group
deprotection by catalytic reduction using H2/10% Pd–C,
yielding the enantiomerically pure L-Gla 6, and (iii) the
L-Gla amino acid was reacted with Fmoc–OSu without
purification to yield the final product. The structures of
final product 1 and intermediates 3 and 4 were fully
L-c-Carboxyglutamic acid (L-Gla) is formed in proteins
via the post-translational modification of L-glutamic
acid by vitamin K carboxylase.1 It has been found in
several vertebrate calcium-binding proteins such as osteo-
calcin.2,3 L-Gla has also been identified unexpectedly in
some of the neuroactive peptides such as conantoxin GV
and conantoxin T.4,5 We have found that replacement
of L-Glu with L-Gla in cyclic peptides, such as G1TE,
substantially improves the latterÕs binding affinity to
the Grb2–SH2 domain protein, thereby improving its
cellular signaling inhibitory properties.6–8 Numerous
syntheses of L-Gla or its derivatives have appeared in
the literature, including both racemic syntheses9–11 and
chiral syntheses.12–17 In these approaches, the overall
yields of resolved L-Gla9–16 have been on the order of
6–13%, except for HiskeyÕs method17 whereby they ob-
tained a 60% yield of the L-Gla product. In this method,
the preparation of chiral L-Gla was achieved by using
expensive starting materials. Considering that the fully
Fmoc protected L-Gla has been used in a number of
peptide synthesis,4,6–8,18 and this substance is consider-
ably more expensive,19 we developed, and present here
a practical, concise and economical route to the prepara-
tion of Fmoc protected L-c-carboxyglutamic acid for
easier availability.
1
characterized and confirmed by H NMR, 13C NMR,
optical rotation, IR, and MS. The enantiomeric purity
was determined by measurement of optical rotation.
In summary, we developed a new synthetic route to effi-
ciently prepare the Fmoc protected L-c-carboxyglutamic
acid in 60% overall yield (>99% ee) via a six-step proce-
dure. In this synthesis route, only the intermediate 3 and
the final product 1, need a flash column chromatography
purification. The key synthetic steps involving aldol con-
densation and selective cleavage of the acetonide protect-
ing group have been optimized for yield and selectivity.
Keywords: L-c-Carboxyglutamic acid; Aldol condensation; D-GarnerÕs
aldehyde; Stereoselective synthesis.
*
Corresponding author. Tel.: +1 301 846 5904; fax: +1 301 846
6033; e-mail: proll@helix.nih.gov
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2005.10.121

24
S. Jiang et al. / Tetrahedron Letters 47 (2006) 23–25
O
Ref.20
NCbz
NCbz
a
b
D-Serine
O
OtBu
OtBu
O
c
H
O
O
2
3
O
O
NH2
O
O
O
NHCbz
NHCbz
OtBu
OtBu
O
d
O
OtBu
OtBu
HO
OtBu
OtBu
HO
HO
O
6
5
4
O
O
NHFmoc
OtBu
OtBu
O
e
HO
1
Scheme 1. The synthesis of Fmoc protected L-c-carboxyglutamic acid. Reagents and conditions: (a) (i) LDA, THF–HMPA, di-tert-butyl malonate,
ꢀ78 ꢀC; (ii) (CF3CO)2O, Et3N, CH2Cl2, 80% for two steps; (b) BiBr3 (10 mol %), 1 equiv H2O, anhydrous acetonitrile, rt, 12 h, 93%; (c) PDC, DMF,
83%; (d) H2, 10% Pd–C, methanol, 100%; (e) Fmoc–Su, NaHCO3, CH3CN/H2O (1/1), 96%.
Advantages of this route include the simplicity of the pro-
cedure, the use of inexpensive, nontoxic reagents and use
of an inexpensive D-serine as a starting material, which
may strengthen industrial interest in this route.
2360, 2341, 1708, 1403, 1367, 1348, 1161, 733 cmꢀ1
.
25
½aꢁD ꢀ50.8 (c 17.45, CHCl3). FAB-MS m/z (relative
intensity) 462 (M+H+, 5), 406 (M+H+ꢀt-Bu, 12), 350
(M+H+ꢀ2t-Bu, 49). HRMS (FAB): calcd for
C25H36NO7 [M+H]+ 462.249, found 462.248.
2. Experimental
2.2. Synthesis of 2S-(2-benzyloxycarbonylamino-3-hy-
droxy-propylidene)-malonic acid di-tert-butyl ester (4)
2.1. Synthesis of 2S-(3-benzyloxycarbonyl-2,2-dimethyl-
oxazolidin-4-ylmethylene)-malonic acid di-tert-butyl ester
(3)
To a solution of cyclic N,O-aminal 3 (4.62 g, 10 mmol)
in anhydrous MeCN (100 mL) was added bismuth(III)
bromide (450 mg, 1.0 mmol) at room temperature. After
30 min, water (0.18 mL, 10 mmol) was added. The reac-
tion mixture was stirred at room temperature for 12 h.
When all starting material had disappeared (about
12 h), the reaction mixture was quenched by adding sat-
urated aqueous NaHCO3 (5 mL). The layers were sepa-
rated, and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated to afford the
pure product 4 (3.92 g, 93%). 1H NMR (400 MHz,
CDCl3): 7.33 (5H, br s), 6.70 (1H, d, J = 8.4 Hz), 5.44
(1H, d, J = 8.0 Hz), 5.09 (2H, d, J = 4.4 Hz), 4.65
(1H, br s), 3.75 (2H, m), 2.70 (1H, t, J = 6.0 Hz),
1.49–1.51 (18H, m) ppm. 13C NMR (100 MHz, CDCl3):
164.7, 155.9, 142.3, 136.1, 132.8, 128.5, 128.1, 82.9, 82.3,
To a solution of diisopropylamine (14.1 mL, 0.12 mol)
in anhydrous THF (150 mL) was added n-BuLi
(42.2 mL, 1.6 M in hexane, 0.068 mol) at 0 ꢀC under
N2, and the mixture were stirred for 30 min. After stir-
ring for an additional 30 min at ꢀ78 ꢀC, anhydrous
HMPA (22.6 mL, 0.13 mol) was added, and the mixture
was stirred for 30 min. A solution of di-tert-butyl mal-
onate (14.7 g, 0.068 mol) in THF (50 mL) was injected
into the above mixture. After 30 min, a solution of
D-GarnerÕs aldehyde (14.92 g, 0.057 mol) in THF
(100 mL) was introduced, and the reaction mixture
was stirred for 2 h at ꢀ78 ꢀC. The mixture was quenched
with saturated aqueous NH4Cl and extracted with ether.
The organic layer was washed with brine and dried
(Na2SO4). Removal of the solvents afforded a crude
oil. To the mixture of the above oil and Et3N (36 mL,
0.256 mol) in CH2Cl2 (80 mL) at 0 ꢀC was added
(CF3CO)2O (18 mL, 0.128 mmol). The reaction was stir-
red at 0 ꢀC for 12 h and at rt for 6 h, quenched with sat-
urated NH4Cl, and extracted with CH2Cl2. After drying
(Na2SO4), the extracts were filtered and concentrated
under reduced pressure. The crude product was purified
by flash column chromatography on silica gel to afford
pure 3 (20.9 g, 80%). 1H NMR (400 MHz, CDCl3):
7.26–7.36 (5H, m), 6.73 (1H, d, J = 8.8 Hz), 5.13 (2H,
m), 4.80 (1H, m), 4.22 (1H, m), 3.86 (1H, dd, J =
3.2, 9.2 Hz), 1.43–1.56 (24H, m) ppm. 13C NMR (100
MHz, CDCl3): 163.9, 163.1, 152.1, 146.7, 136.3, 131.3,
128.3, 128.1, 127.7, 127.6, 95.1, 82.2, 81.9, 68.4, 66.6,
55.7, 28.0, 26.1, 23.9 ppm. IR (neat): 2980, 2938, 2878,
67.0, 64.3, 52.0, 27.9 ppm. IR (neat): 3371, 2979, 2883,
25
2359, 1707, 1368, 1254, 1160, 754 cmꢀ1. ½aꢁD ꢀ 19:35
(c 5.85, CHCl3). FAB-MS m/z (relative intensity) 422
(M+H+, 3), 366 (M+H+ꢀt-Bu, 11), 310 (M+H+ꢀ2t-
Bu, 53). HRMS (FAB): calcd for C22H32NO7 [M+H]+
422.218, found 422.220.
2.3. Synthesis of N-a-Fmoc-L-c-carboxy-glutamic acid-
c,c-tert-butyl ester (1)
To a stirred mixture of alcohol 4 (1.79 g, 4.24 mmol), in
dry DMF (18 mL) at room temperature under argon
was added pyridinium dichromate (6.38 g, 16.96 mmol).
After stirring overnight, another portion of pyridinium
dichromate (1.595 g, 4.24 mmol) was added and the
resulting mixture was stirred for additional 4 h. The

S. Jiang et al. / Tetrahedron Letters 47 (2006) 23–25
25
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reaction mixture was diluted with ethyl ether (100 mL)
and washed with 1 N HCl (5 · 100 mL). The organic
layer was washed with brine, dried over Na2SO4, and
evaporated to give a crude oil 5 (1.54 g, 83%). To the
mixture of the above oil, CH3OH (20 mL), and 10%
Pd–C (0.3 g) was stirred vigorously under a H2 atmo-
sphere until hydrogen was absorbed to saturation. The
mixture was filtrated and concentrated to give a crude
oil 6 (1.02 g, 100%). A mixture of the above oil, N-(9-
fluorenylmethoxycarbonyloxy)succinimide (1.43 g, 4.24
mmol) and NaHCO3 (1.43 g, 16.96 mmol) in water
and acetonitrile (1:1, 20 mL) was stirred at room tem-
perature overnight. Acetonitrile was evaporated under
reduced pressure, the resulting solution was adjusted
to pH = 4 using 10% citric acid. The mixture was
extracted with EtOAc. The combined organic layers
were washed with brine, dried over Na2SO4, and
concentrated and purified by flash chromatography
1
to afford 1 as a solid (1.79 g, 96% from 6). H NMR
(400 MHz, CDCl3): 7.76 (2H, d, J = 7.6 Hz), 7.59 (2H,
dd, J = 3.2, 7.2 Hz), 7.40 (2H, dd, J = 7.2 Hz), 7.40
(2H, ddd, J = 1.2, 7.6 Hz), 5.58 (1H, d, J = 8.0 Hz),
4.45 (2H, m), 4.35 (1H, m), 4.22 (1H, t, J = 6.8 Hz),
3.41 (1H, t, J = 7.2 Hz), 2.46 (1H, m), 2.21 (1H, m),
1.46–1.48 (18H, m) ppm. 13C NMR (100 MHz, CDCl3):
175.3, 168.4, 168.2, 156.2, 143.8, 143.6, 141.2, 127.7,
127.1, 125.1, 119.9, 82.4, 82.3, 67.9, 67.3, 52.4, 50.8,
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47.0, 30.7, 27.8, 27.7 ppm. IR (film): 3397, 3182, 2978,
25
1723, 1670, 1531, 1150, 1050 cmꢀ1. ½aꢁD ꢀ 8:7 (c 1.0,
MeOH) [lit.25 ꢀ8.6 (c 0.173, MeOH)]. FAB-MS m/z (rel-
ative intensity) 526 (M+H+, 1), 470 (M+H+ꢀt-Bu, 4),
414 (M+H+ꢀ2t-Bu, 31). HRMS (FAB): calcd for
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Acknowledgements
This research was supported by the Intramural Research
Program of the NIH, National Cancer Institute, Center
for Cancer Research. We are grateful to Dr. Krzysztof
Krajewski, for advice, helpful discussions, and a critical
reading of the manuscript.
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