Convenient synthesis of deuterated glutamic acid, proline and leucine via catalytic deuteration of unsaturated pyroglutamate derivatives

Article abstract of DOI:10.1002/jlcr.1038

Novel 3,4-didehydropyroglutamate derivatives, the key intermediates in this synthesis, were obtained from a protected pyroglutamate by the well-known selenenylation-oxidative deselenenylation method and were catalytically deuterated using a slow-addition technique. The obtained deuterated pyroglutamates were converted to [3,4-²H₂]glutamic acid, [3,4,5-²H₃]proline, and [3,4,5,5,5-²H ₅]leucine via an appropriate functional group interconversions followed by the standard deprotection procedure. Copyright

Full text of DOI:10.1002/jlcr.1038

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 229–235.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1038
Research Article
Convenient synthesis of deuterated glutamic acid,
proline and leucine via catalytic deuteration of
unsaturated pyroglutamate derivatives
Makoto Oba*, Kosuke Ohkuma, Hiroshi Hitokawa, Ayumi Shirai and
Kozaburo Nishiyama
Department of Materials Chemistry, Tokai University, 317 Nishino, Numazu, Shizuoka
410-0395, Japan
Summary
Novel 3,4-didehydropyroglutamate derivatives, the key intermediates in this synthesis,
were obtained from a protected pyroglutamate by the well-known selenenylation-
oxidative deselenenylation method and were catalytically deuterated using a slow-
addition technique. The obtained deuterated pyroglutamates were converted to
[3,4-2H2]glutamic acid, [3,4,5-2H3]proline, and [3,4,5,5,5-2H5]leucine via an appro-
priate functional group interconversions followed by the standard deprotection
procedure. Copyright # 2006 John Wiley & Sons, Ltd.
Key Words: deuterated amino acids; catalytic deuteration; unsaturated pyrogluta-
mate
Introduction
Recently, we have been engaged in the asymmetric synthesis of regio- and
stereoselectively deuterium-labeled amino acids for structure analysis of
proteins by means of NMR spectroscopy,1–6 in which l-pyroglutamic acid ð1Þ
has been often used as a chiral starting material. We previously reported the
synthesis of deuterated proline and leucine using deuterated l-pyroglutamate
derivative 3 as a common intermediate, where a catalytic deuteration of
unsaturated l-pyroglutaminol 2 derived from l-pyroglutamic acid ð1Þ was
used as a key reaction to introduce deuterium atoms into the b- and g-
positions of the amino acid side chains (Scheme 1).3,5 However, the procedure
necessitates reduction of the a-carboxyl group of the l-pyroglutamic acid and
*Correspondence to: Makoto Oba, Department of Materials Chemistry, Tokai University, 317 Nishino,
Numazu, Shizuoka 410-0395, Japan. E-mail: makoto@wing.ncc.u-tokai.ac.jp.
Contract/grant sponsor: Japan Science and Technology Corporation
Copyright # 2006 John Wiley & Sons, Ltd.
Received 25 October 2005
Revised 22 November 2005
Accepted 22 November 2005

230
M. OBA ET AL.
[3,4,5-2H3]Pro
D
R
D
R
D2
OTBDMS
CO2But
O
CO2H
O
O
N
H
N
Boc
N
Boc
[3,4,5,5,5-2H5]Leu
1
2
3
R = H, CH3, CD3
Scheme 1. Synthesis of deuterated proline and leucine via deuteration of
unsaturated pyroglutaminol derivatives 2
PhSe
1.NaN(SiMe3)2
R
2. PhSeCl
3. MeI (R = Me)
D
CO2But
1
CO2But
O
O
N
N
HO2C
CO2H
NH2
Boc
Boc
a: R = H
b: R = Me
4
5
1. 1 M HCl reflux
2. Dowex 50W-X8
D
8
D
D
D
D
R
R
ref. 2-4
D2, PdO
30% H2O2
D
CO2H
CO2H
CO2But
N
H
9
O
O
CO2But
N
N
Boc
Boc
6
7
ref. 5,6
R = Me
D
D3C
D
Me
NH2
10
Scheme 2. Synthesis of deuterated glutamic acid, proline, and leucine via
deuteration of unsaturated pyroglutamate derivatives 6
its regeneration after the incorporation of deuterium atoms has been
achieved. In fact, the overall yield of compound 3 ðR ¼ HÞ from 1 was 23%
for 10 steps.
If the catalytic deuteration of an unsaturated l-pyroglutamate derivative
such as compound 6 in Scheme 2 can be realized, the protocol would
become more straightforward. However, only a few reports regarding the
reactivity of the 3,4-didehydropyroglutamates can be seen in the literature.7–9
Furthermore, it is well known that 3,4-didehydropyroglutamates are
prone to racemize and isomerize via a double-bond shift. In 1995, Ezquerra
and co-workers reported the first trapping reaction of the olefin with
cyclopentadiene where the Diels-Alder adduct was obtained in 50% ee.8 To
our knowledge, there are no examples where a catalytic reduction is performed
with the olefin. In this paper, we describe a convenient synthesis of
[3,4-2H2]glutamic acid ð8Þ, [3,4,5-2H3]proline ð9Þ, and [3,4,5,5,5-2H5]leucine
ð10Þ via catalytic deuteration of novel unsaturated pyroglutamate derivatives 6
(Scheme 2).
Copyright # 2006 John Wiley & Sons, Ltd.
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CONVENIENT SYNTHESIS OF DEUTERATED GLUTAMIC ACIDS
231
Results and discussion
3,4-Didehydropyroglutamate 6a ðR ¼ HÞ is a new compound and can be
obtained from tert-butyl N-(tert-butoxycarbonyl)pyroglutamate ð4Þ by the
well-established procedure involving phenylselenenylation and oxidative
deselenenylation by hydrogen peroxide. In our case, olefin 6a was obtained
as the sole product in almost pure form; and the predicted side reactions
associated with the use of organoselenium compounds as reported by
Ezquerra et al.8 could not be observed. Since the unsaturated pyroglutamate
6a was chromatographically unstable, olefin 6a was prepared from the purified
phenyl selenide 5a and used in the next step without further purification.
First of all, olefin 6a was subjected to the conventional hydrogenation
conditions. When a mixture of 6a and 10% palladium on carbon in MeOD
was stirred at room temperature under an atmosphere of deuterium gas, the
deuterated pyroglutamate 7a was obtained with considerable hydrogen–
deuterium scrambling along with partial methanolysis to give an open-chain
by-product. It was found that a slow addition of a dilute solution of olefin 6a
to the suspension where the deuterium gas was pre-adsorbed on the catalyst
improved the extent of the hydrogen–deuterium scrambling. To explore the
optimization of this deuteration reaction, we surveyed other transition metal
catalysts. Finally, the use of palladium(II) oxide as a catalyst proved
advantageous, resulting in quantitative formation of [3,4-2H2]pyroglutamate
7a. Consequently, it has become feasible to obtain the deuterated pyroglu-
tamate 7a from 1 in 61% yield for 7 steps.
The pyroglutamate 7a was then deprotected in refluxing 1 M HC1 and
subsequent ion exchange purification afforded (2S,3S,4R)-[3,4-2H2]glutamic
acid ð8Þ in 84% yield. Although the relative stereochemistry of the three
contiguous stereocenters within the deuterated glutamic acid 8 was estab-
lished, the HPLC analysis using a chiral column showed that partial
racemization occurred at the a-position (63% ee), probably due to the high
acidity of the a-proton in compound 6a.
According to the procedure described in our previous papers,2–4 the
deuterated pyroglutamate 7a was also converted to [3,4,5-2H3]proline ð9Þ in
46% yield for 3 steps. The cis selective deuteration of the d-position was
achieved by a stepwise reduction of the lactam carbonyl group with LiEt3BH
followed by Bu3SnD in the presence of BF3.OEt2.10
In order to obtain [3,4,5,5,5-2H5]leucine ð10Þ, unsaturated 4-methylpyr-
oglutamate 6b is necessary. Introduction of a 4-methyl group into the 3,4-
didehydropyroglutamate framework could be achieved simply by quenching
the a-phenylseleno enolate, the reaction intermediate in the phenylselenenyla-
tion of the pyroglutamate derivative, with iodomethane followed by the usual
oxidative deselenenylation reaction using hydrogen peroxide. Olefin 6b was
obtained as the sole product and the isomeric exomethylene compound could
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M. OBA ET AL.
not be detected in the reaction mixture. As compared with 6a, the methylated
olefin 6b was less stable and should be immediately used in the next step
without further purification. Thus, a catalytic deuteration of olefin 6b in the
presence of palladium(II) oxide in MeOD was carried out using the slow-
addition technique to give 4-methyl[3,4-2H2]pyroglutamate 7b in quantitative
yield. According to the procedure described in our previous paper,5,6
chemoselective ring-opening of pyroglutamate 7b, reductive deuteration of
the terminal carboxyl group, and standard deprotection procedure afforded
[3,4,5,5,5-2H5]leucine ð10Þ in 27% yield for 5 steps.
As with [3,4-2H2]glutamic acid ð8Þ, the relative stereochemistry of deuterium
substitution in [3,4,5-2H3]proline ð9Þ and [3,4,5,5,5-2H5]leucine ð10Þ was
established; however, their optical purities determined by HPLC were 14
and 18%ee, respectively, and the values were not reproducible on repeated
runs. It seems that the extent of the racemization was varied according to the
reaction conditions employed in the olefin synthesis.
Conclusion
We have demonstrated
a
simple route to [3,4-2H2]glutamic acid,
[3,4,5-2H3]proline, and [3,4,5,5,5-2H5]leucine via a catalytic deuteration of
novel 3,4-didehydropyroglutamate derivatives. Although the obtained deut-
erated amino acids are partially racemized, the protocol described here still has
some advantages. We are now seeking alternative protective groups for the
carboxyl functionality which improve the stability of the unsaturated
pyroglutamate derivatives.
Experimental
1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. All
chemical shifts are reported as d values (ppm) relative to residual chloroform
(dH 7.26), sodium 3-(trimethylsilyl)[2,2,3,3-2H4]propionate (dH 0.00), or the
central peak of CDCl3 (dc 77.0). High-resolution mass spectra (HRMS) were
determined using perfluorokerosene as an internal standard. Optical purity
was determined on an HPLC system equipped with a chiral MCIGEL
CRS10W column using 2 mM CuSO4 solution as an eluent.
tert-Butyl (2S)-N-tert-Butoxycarbonyl-3,4-didehydropyroglutamate (6a)
A solution of 1 M NaN(SiMe3)2 in THF (39.0 ml, 39.0 mmol) was treated with
N,N0-dimethylpropyleneurea (5 ml) at 08C under an argon atmosphere for
10 min, cooled to ꢀ788C, and treated with a solution of tert-butyl (2S)-(N-tert-
butoxycarbonyl)pyroglutamate (4, 5.06 g, 17.7 mmol) in THF (40 ml). After
0.5 h, a solution of PhSeCl (3.64 g, 19.0 mmol) in THF (30 ml) was added and
the mixture was stirred for 2 h. The reaction was quenched with saturated
aqueous NH4Cl and the mixture was extracted with ethyl acetate. The organic
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CONVENIENT SYNTHESIS OF DEUTERATED GLUTAMIC ACIDS
233
layer was washed with water and brine, dried over MgSO4, and concentrated.
The residue was chromatographed on silica gel (hexane/ethyl acetate=88/12)
to give the corresponding 4-phenylselenopyroglutamate 5a (7.81 g) as a
mixture of diastereomers in quantitative yield.
To a solution of the obtained phenylselenide 5a (4.39 g, 9.97 mmol) in THF
(30 ml) was added dropwise 30% H2O2 (20 ml) at 08C and the reaction
mixture was stirred at room temperature for 2 h. The mixture was extracted
with ethyl acetate and the organic layer was washed with saturated aqueous
NaHCO3 and brine and dried over MgSO4. Concentration of the solution
gave quantitative yield of the title compound 6a (2.82 g) as an oil, which is
1
used in the next step without further purification. H NMR (CDCl3) d 1.47
(s, 9 H), 1.53 (s, 9 H), 5.04 (dd, J=2.4 and 2.2 Hz, 1 H), 6.21 (dd, J=3.7
and 2.2 Hz, 1 H), 7.05 (dd, J=3.7 and 2.4 Hz, 1 H). 13C NMR (CDCl3)
d 27.75, 27.82, 65.31, 83.23, 83.34, 128.46, 143.47, 148.40, 165.09, 168.43.
HRMS (EI, 30 eV) m/z 284.1519 [(M + H)+, calculated for C14H22NO5
284.1498].
tert-Butyl (2S)-N-tert-Butoxycarbonyl-3,4-didehydro-4-methylpyroglutamate (6b)
A solution of 1 M NaN(SiMe3)2 in THF (21.0 ml, 21.0 mmol) was treated with
N,N0-dimethylpropyleneurea (3 ml) at 08C under an argon atmosphere for
10 min, cooled to ꢀ788C, and treated with a solution of tert-butyl (2S)-(N-tert-
butoxycarbonyl)pyroglutamate (4, 2.87 g, 10.1 mmol) in THF (20 ml). After
0.5 h, a solution of PhSeCl (2.11 g, 11.0 mmol) in THF (15 ml) was added and
the mixture was stirred for 2 h. Then, CH3I (2.01 g, 14.2 mmol) was added. The
reaction was quenched with saturated aqueous NH4Cl and the mixture was
extracted with ethyl acetate. The organic layer was washed with water and
brine, dried over MgSO4, and concentrated. The residue was chromato-
graphed on silica gel (hexane/ethyl acetate = 50/50) to give the corresponding
4-methyl-4-phenylselenopyroglutamate 5b (3.54 g) as a mixture of diastereo-
mers in 77% yield.
To a solution of the obtained phenylselenide 5b (1.00 g, 2.20 mmol) in THF
(10 ml) was added dropwise 30% H2O2 (2.5 ml) at 08C and the reaction
mixture was stirred at room temperature for 1 h. The mixture was extracted
with ethyl acetate and the organic layer was washed with saturated aqueous
NaHCO3 and brine and dried over MgSO4. Concentration of the solution gave
quantitative yield of the title compound 6b (778 mg) as an oil, which is used in
1
the next step without further purification. H NMR (CDCl3) d 1.47 (s, 9 H),
1.53 (s, 9 H), 1.90 (dd, J=2.1 and 1.7 Hz, 3 H), 4.89 (dq, J=2.5 and 2.1 Hz,
1 H), 6.68 (dq, J=2.5 and 1.7 Hz, 1 H). 13C NMR (CDCl3) d 11.12, 28.00,
28.07, 63.30, 83.14, 83.20, 136.20, 136.84, 148.86, 166.07, 169.30.
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M. OBA ET AL.
tert-Butyl (2S,3S,4R)-N-tert-Butoxycarbonyl-[3,4-2H2]pyroglutamate (7a)
A suspension of PdO (120 mg) in MeOD (10 ml) was stirred at room
temperature for 30 min under an atmosphere of deuterium gas. To the mixture
was added dropwise a solution of olefin 6a (510 mg, 1.80 mmol) in MeOD
(20 ml) and the mixture was stirred for 30 min. After removal of the catalyst
using a Celite pad, evaporation of the solvent afforded quantitative yield of the
1
title compound 7a (513 mg) as an oil. H NMR (CDCl3) d 1.47 (s, 9 H), 1.49
(s, 9 H), 1.96 (dd, J=10 and 2 Hz, 1 H), 2.57 (d, J=10 Hz, 1H), 4.46
(d, J=2 Hz, 1 H). 13C NMR (CDCl3) d 21.19 (t, J=21 Hz), 27.85 (6 C), 30.64
(t, J=21 Hz), 59.47, 82.10, 83.09, 149.37, 170.32, 173.11. HRMS (EI, 70 eV)
m/z 288.1778 [(M+H)+, calculated for C14H22D2NO5 288.1780].
tert-Butyl(2S,3S,4S)-N-tert-Butoxycarbonyl-4-methyl[3,4-2H2]pyroglutamate
(7b)
According to the procedure for the preparation of compound 7a, deuteration
of olefin 6b (1.09 g, 3.67 mmol) in MeOD (100 ml) in the presence of PdO
(193 mg) gave quantitative yield of the title compound 7b (1.14 g) as an oil. 1H
NMR (CDCl3) d 1.25 (s, 3 H), 1.48 (s, 9 H), 1.51 (s, 9 H), 1.57 (d, J=6 Hz,
1 H), 4.38 (d, J=6 Hz, 1 H). 13C NMR (CDCl3) d 16.66, 28.11 (6 C), 29.42
(t, J=21 Hz), 37.56 (t, J=18 Hz), 58.24, 82.32, 83.52, 149.81, 170.86, 176.29.
HRMS (EI, 70 eV) m/z 301.1876 (M+, calculated for C15H23D2NO5
301.1858).
(2S,3S,4R)-[3,4-2H2]Glutamic Acid (8)
A mixture of compound 7a (48.0 mg, 1.67 mmol) in 1 M HC1 (30 ml) was
heated to reflux overnight. The cooled aqueous solution was washed with
chloroform and concentrated to dryness. The residue was submitted to ion-
exchange column chromatography on Dowex 50W-X8 and elution with 1 M
NH4OH gave the ammonium salt of compound 8 (232 mg, 84%) as a colorless
solid, m.p. 183–1858C (lit,11 2058C dec). The optical purity (63%ee) was
1
determined by HPLC. H NMR (D2O) d 1.89 (dd, J=8 and 8 Hz, 1 H), 2.25
(d, J= 8 Hz, 1 H), 3.59 (d, J= 8 Hz, 1 H). 13C NMR (5% NaOD in D2O) d
34.06 (t, J=19 Hz), 36.42 (t, J=19 Hz), 58.50, 185.64, 185.79. MS (FAB,
glycerol) m/z 150 [(M+H)+].
(2S,3S,4R,5S)-[3,4,5-2H3]Proline (9)
According to the procedure described in our previous paper,3,4 reduction and
deprotection of compound 7a (2.99 g, 10.4 mmol) gave 46%
(3 steps) yield of the title compound 9 (565 mg) as colorless solids, m.p.
215–2208C dec (lit,12 2288C dec). The optical purity (14%ee) was determined
by HPLC. The spectral data were identical with those reported by us.4
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CONVENIENT SYNTHESIS OF DEUTERATED GLUTAMIC ACIDS
235
1H NMR (D2O) d 1.97 (dd, J=7 and 7 Hz, 1 H), 2.05 (dd, J=7 and 7 Hz, 1 H),
3.32 (d, J=7 Hz, 1 H), 4.13 (d, J=7 Hz, 1 H).
(2S,3S,4R)-[3,4,5,5,5-2H5]Leucine (10)
According to the procedure described in our previous paper,5,6 ring-opening,
reduction of the terminal carboxyl group, and deprotection of compound 7b
(1.14 g, 3.78 mmol) gave 27% (5 steps) yield of the title compound 10 (140 mg)
as colorless solids, m.p. >2508C (lit,13 >3008C). The optical purity (18%ee)
was determined by HPLC. 1H NMR (5% NaOD in D2O) d 0.89 (s, 3 H), 1.36
(d, J=8 Hz, 1 H), 3.26 (d, J=8 Hz, 1 H). 13C NMR (5% NaOD in D2O) d
24.80, 24.92 (m), 26.16 (t, J=19 Hz), 46.22 (t, J=19 Hz), 57.07, 187.12.
HRMS (EI, 70 eV) m/z 137.1317 [(M+H)+, calculated for C6H9D5NO2
137.1338].
Acknowledgements
This work has been supported by CREST (Core Research for Evolutional
Science and Technology) of Japan Science and Technology Corporation (JST).
We also thank Professor Masatsune Kainosho of Tokyo Metropolitan
University for helpful discussions.
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