Asymmetric synthesis of L-[4-13C]glutamic acid and glutamine

Article abstract of DOI:10.1002/jlcr.1055

L-[4-¹³C]Glutamic acid (1) and L-[4-¹³C]glutamine (2) were synthesized from sodium [2-¹³C]acetate (5) and Dellaria's oxazinone 3 as a chiral glycine equivalent. Sodium [2-¹³C]acetate (5) was converted to [2-

Full text of DOI:10.1002/jlcr.1055

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 445–453.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1055
Research Article
Asymmetric synthesis of l-[4-13C]glutamic acid
and glutamine
Kazuhiko Takatori, Takuya Sakamoto and Masahiro Kajiwara*
Department of Medicinal Chemistry, Meiji Pharmaceutical University, 2-522-1 Noshio,
Kiyose-shi, Tokyo 204-8588, Japan
Summary
l-[4-l3C]Glutamic acid (1) and l-[4-13C]glutamine (2) were synthesized from sodium
[2-13C]acetate (5) and Dellaria’s oxazinone 3 as a chiral glycine equivalent. Sodium
[2-13C]acetate (5) was converted to [2-13C]acrylate 4. Diastereoselective Michael
addition of the enolate of 3 to the acrylate 4 proceeded with high diastereoselectivity
to give the adduct 12. Reductive cleavage of the C–S bond, ethanolysis, hydrogen-
olysis and hydrolysis gave l-[4-13C]glutamic acid (1). l-[4-13C]Glutamine (2) was
synthesized from 1 in 4 steps. Copyright # 2006 John Wiley & Sons, Ltd.
Key Words: l-[4-13C]glutamic acid; l-[4-13C]glutamine; labeled amino acid; chiral
glycine equivalent; oxazinone
Introduction
Glutamic acid is an important chemical mediator in the brain, and metabo-
tropic glutamate receptors modulate neuronal function. Glutamic acid dis-
charged from the neurons is collected by astrocytes, and metabolized to
glutamine. Glutamine is then returned to the neurons, where it is reconverted
into glutamic acid, and used for neurotransmission.1 l-[4-13C]Glutamic acid
(1) and l-[4-13C]glutamine (2) are required for detailed NMR studies of this
glutamate–glutamine cycle. We have already reported asymmetric syntheses of
various specifically 13C-labeled amino acids from Dellaria’s oxazinone 32 or its
13C-labeled form as a chiral optically active glycine equivalent.3–6 In this
paper, we describe an asymmetric chemical synthesis of l-[4-13C]glutamic acid
(1) and l-[4-13C]glutamine (2) by diastereoselective Michael addition of the
oxazinone 3 to l3C-labeled acrylate 4 bearing a phenylsulfonyl group as an
*Correspondence to: Masahiro Kajiwara, Department of Medicinal Chemistry, Meiji Pharmaceutical
University, 2-522-1 Noshio, Kiyose-shi, Tokyo 204-8588, Japan. E-mail: kajiwara@my-pharm.ac.jp
Contract/grant sponsor: Ministry of Education, Culture, Sports and Technology of Japan
Copyright # 2006 John Wiley & Sons, Ltd.
Received 7 December 2005
Revised 10 December 2005
Accepted 20 December 2005

446
K. TAKATORI ET AL.
electron-withdrawing group to inhibit polymerization in the Michael reaction.
Enzymatic or chemo-enzymatic syntheses of 1 have already been reported by
other groups.7,8
Results and discussion
The 13C-labeled acrylate 4 corresponding to C3–C5 of glutamic acid was pre-
pared from sodium [2-13C]acetate (5) (Scheme 1). Sodium [2-13C]acetate
(5, 99 at.% 13C) was converted to ethyl bromo[2-13C]acetate by using the
method previously reported,4,9 and then treatment with sodium benzenesul-
finate gave ethyl 2-(phenylsulfonyl)[2-13C]acetate (6) in 78% yield from 5.
Conversion from 6 to 4 was performed in three steps: methylation, selenylation
and oxidative elimination of the selenyl group. The order of methylation and
selenylation was investigated using non-labeled 6. Initially, selenylation of the
non-labeled form of 6 was carried out by treatment with phenylselenyl
bromide and NaH in THF-HMPA, and gave 7 in quantitative yield. Subse-
quent methylation with NaH and methyl iodide in THF unfortunately
afforded the ylide 8, methylated at the selenyl group, in quantitative yield.
Therefore, the conversion from 6 to 4 was conducted by applying methylation
first, then selenylation. Methylation of 6 was performed by enolization with
NaH in THF followed by addition of the resulting enolate to excess methyl
iodide in THF at 08C, to give the desired ethyl 2-(phenylsulfonyl)[2-13C]pro-
pionate (9) in 69% yield together with the over-methylated product 10 in 12%
yield and the recovered starting material 6 in 14% yield. Subsequent
selenylation of 9 with NaH and phenylselenyl bromide in HMPA-THF gave
11 in 88% yield.10 Oxidative elimination of the selenyl group with H2O2 gave
l3
the C-labeled acrylate 4 in quantitative yield.
Michael addition of Dellaria’s oxazinone 3 to the 13C-labeled acrylate 4 was
performed by enolization of excess 3 with sodium bis(trimethylsilyl)amide
(NaHMDS) in THF, followed by addition of 4 at ꢀ788C to give the
Scheme 1. Reagents and conditions: (a) (i) PhCOBr, PhCO2H, (ii) Br2, (iii)
EtOH, (iv) PhSO2Na, 78% from 5; (b) NaH, Mel, 69%; (c) NaH, PhSeBr,
HMPA, 88%; (d) H2O2, 08C quant
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SYNTHESIS OF L-[4-13C]GLUTAMIC ACID AND GLUTAMINE
447
corresponding Michael adduct 12 as a 1:1 diastereomeric mixture relative to
the phenylsulfonyl group in 49% yield from 11 (Scheme 2). Reductive cleavage
of the C–S bond of 12 with SmI2 gave 13 as a single isomer in 65% yield.
Ethanolysis of the oxazinone ring and removal of the Boc group of 13 were
simultaneously carried out by heating at 858C in ethanol-saturated HCl gas to
give 14. Hydrogenolysis for removal of the chiral auxiary of 14 with catalytic
Pd(HO)2 in ethanol gave the diethyl glutamate. Hydrolysis with HCl gave
l-[4-13C]glutamic acid hydrochloride (1) in 49% yield from 13. The enrichment
l3
ratio was 98 at.% C, determined from the integration ratio of the signals at
2.49 ppm, assigned to the 4-position of 1 and the residual non-labeled form, in
1
the H-NMR spectrum.
The resulting l-[4-13C]glutamic acid hydrochloride (1) was converted to
13C-glutamine 2 using the following sequence. Selective esterification of 1 in
dry methanol and protection of the amino group of 15 with Cbz-Cl gave 16 in
65% yield. Transamidation of 16 with ammonium hydroxide and removal of
the Cbz group of 17 by hydrogenolysis gave l-[4-13C]glutamine (2) in 48%
yield. The 13C-NMR spectrum of 2 showed the enriched signal at 33.6 ppm,
assigned to the 4-position of glutamine. The optical purity was >96% ee and
the absolute configuration was l, as determined by HPLC-CD analysis using a
chiral column.11
Scheme 2. Reagents and conditions: (a) NaHMDS then 4, THF, ꢀ788C, 49%
from 11; (b) SmI2, 65%; (c) saturated HCl-EtOH; (d) (i) H2, Pd(OH)2, (ii) 6M
HCl, 49% from 13; (e) MeOH, 97%; (f) Cbz-Cl, Na2CO3, 67%; (g) NH4OH,
62%; (h) H2, Pd-C, 78%
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448
K. TAKATORI ET AL.
In summary, we have synthesized l-glutamic acid (1) and l-glutamine (2)
l3C-labeled at the 4-position, from sodium [2-13C]acetate (5) by using diaster-
eoselective Michael addition of Dellaria’s oxazinone 3 to the acrylate 4. Com-
pounds 1 and 2 will be useful for biochemical experiments.
Experimental
Sodium [2-13C]acetate (5, 99 at.% 13C) was supplied by Cambridge Isotope
Laboratories. Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. 1H- and 13C-NMR spectra were recorded on a
Varian Gemini-2000 (1H, 300 MHz: 13C, 75.4 MHz) Fourier-transform spec-
trometer. The chemical shifts are reported in d values relative to tetra-
methylsilane (TMS) at 0 ppm in CDCl3 and [1,l,l,3,3,3-D6]acetone, or sodium
1
3-(trimethylsilyl)[2,2,3,3-D4]propionate (TSP) at 0 ppm in D2O for H-NMR,
and relative to CDCl3, at 77.0 ppm, [1,1,1,3,3,3-D6]acetone at 29.8 ppm or TSP
at 0 ppm in D2O for l3C-NMR. IR spectra were recorded on a JASCO
VALOR-III Fourier-transform spectrometer. EI- and HR-FAB-MS were
obtained with a JEOL JMS-700 double-focusing spectrometer. CD spectra
were recorded on a JASCO J-720 CD spectrometer. HPLC-CD analysis was
carried out on a JASCO 800 Series HPLC system with a JASCO 875-UV
detector and a JASCO J-720 CD spectrometer as detectors. The column was a
Crown Pak CR(-) column (150 mm ꢁ 4 mm i.d.), purchased from Daicel.
Ethyl 2-(phenylsulfonyl)[2-13C]acetate ð6Þ
Ethyl bromo[2-13C]acetate was prepared from sodium [2-13C]acetate (5, 5.0 l g,
60.3 mmol) by using the method previously reported.4,9 To a solution of ben-
zenesulfinate dihydrate (13.29 g, 66.4 mmol) in DMF (50 ml) was added drop-
wise a solution of the crude ethyl bromo[2-13C]acetate in DMF (18 ml) at room
temperature, and the mixture was stirred for 3 d. The mixture was diluted with
ether, and the solution was washed with water 5 times. The combined aqueous
layers were extracted with ether 5 times. The combined organic layers were
washed with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (hexane : ethyl acetate=1:1) to give the ethyl
2-(phenylsulfonyl)[2-13C]acetate 6 (11.80 g) in 78% yield. 1H-NMR (CDCl3) d:
1.20 (t, J ¼ 7:1 Hz, 3H), 4.11 (d, JC2H ¼ 140:1 Hz, 2H), 4.15 (q, J ¼ 7:1 Hz,
2H), 7.59 (m, 2H), 7.70 (m, 1H), 7.96 (m, 2H). 13C-NMR (CDCl3) d: 61.0. IR
(neat) cmꢀ1: 3067, 2988, 2940, 1741, 1449, 1327, 1312, 1276, 1155, 1085, 1025.
HR-MS calculated for C193CH12O4S: m=z ¼ 229:0490. Found: m=z ¼ 229:0488
(M+). EI-MS m/z (%): 229 (M+, 0.4), 184 (21), 165 (100), 141 (97), 92 (88),
77 (76).
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF L-[4-13C]GLUTAMIC ACID AND GLUTAMINE
449
Ethyl 2-(phenylsulfonyl)[2-13C]propionate ð9Þ
To a stirred suspension of NaH (60% dispersion in mineral oil, 1.16 g,
29.00 mmol) in THF (70 ml) was added dropwise at 08C over 30 min a solution
of 6 (7.00 g, 30.55 mmol) in THF (15 ml). The suspension was stirred for
10 min, and the precipitate was dissolved by addition of further THF (42 ml).
The solution was added dropwise to a stirred solution of methyl iodide (48 ml,
0.77 mol) in THF (53 ml) over 1.5 h at 08C through a cannula. The suspension
was stirred at 48C overnight. The reaction was quenched with saturated
NH4Cl. The aqueous layer was extracted with ether. The combined organic
layers were washed with brine, dried over MgSO4, and evaporated. The res-
idue was chromatographed on silica gel (hexane : ethyl acetate=5:1) to give 9
1
(5.13 g) in 69% yield. H-NMR (CDCl3,) d: 1.20 (t, J ¼ 7:1 Hz, 3H), 1.58
2
(dd, JC2H ¼ 4:7 Hz, J ¼ 7:1 Hz, 3H), 4.05 (dq, JC2H ¼ 140:9 Hz, J¼ 7:1 Hz,
1H), 4.12 (q, J ¼ 7:1 Hz, 2H), 7.58 (m, 2H), 7.69 (m, 1H), 7.91 (m, 2H).
13C-NMR (CDC13) d: 65.4. IR (neat) cmꢀ1: 2986, 2943, 1737, 1449, 1323,
1311, 1150. HR-MS Calculated for C10 13CH14O4S: m=z ¼ 243:0647. Found:
m=z ¼ 243:0653 (M+). EI-MS m/z (%): 243 (M+, 0.3), 198 (20), 187 (20), 179
(81), 159 (22), 141 (72), 125 (22), 106 (34), 77 (100).
Ethyl 2-(phenylselenyl)-2-(phenylsulfonyl)[2-13C]propionate ð11Þ
To a stirred suspension of NaH (60% dispersion in mineral oil, 1.27 g,
31.64 mmol) in THF (64 ml) was added dropwise at 08C over 30 min a solution
of 9 (5.13 g, 21.09 mmol) in THF (29 ml). The mixture was stirred for 10 min,
HMPA (4.4 ml, 25.3 mmol) was added dropwise over 10 min, and the mixture
was stirred for 30 min. A solution of phenylselenyl bromide (5.47 g, 23.2 mmol)
in THF (29 ml) was added dropwise at 08C over 30 min, and the mixture was
stirred for 30 min. The reaction was quenched with saturated NH4Cl, and the
aqueous layer was extracted with ether. The combined organic layers were
washed with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (hexane : ethyl acetate=1:1) to give 11 (7.36 g)
in 88% yield, mp. 96.2–97.38C. 1H-NMR (CDCl3) d: 1.19 (t, J ¼ 7:1 Hz, 3H),
2
1.65 (d, JC2H ¼ 5:0 Hz, 3H), 4.08 (q, J ¼ 7:1 Hz, 2H), 7.33 (m, 2H), 7.44
(m, 1H), 7.55 (m, 2H), 7.65–7.73 (m, 3H), 7.98 (m, 2H). 13C-NMR (CDCl3)
d: 70.7. IR (KBr) cmꢀ1: 3010, 2976, 2933, 1723, 1447, 1325, 1307, 1233, 1153,
1083, 752, 696. HR-MS Calculated for C16 13CH18O4SSe: m=z ¼ 399:0125.
Found: m=z ¼ 399:0131 (M+). EI-MS m/z (%): 399 (M+, 0.01), 258 (100),
212 (32), 184 (51), 125 (13), 104 (10), 77 (30).
Ethyl 2-(phenylsulfonyl)[2-13C]acrylate ð4Þ
To a stirred solution of selenylpropionate 11 (4.26 g, 10.69 mmol) in
CH2Cl2 (107 ml) was added H2O2 (30%, 3.05 ml, 26.85 mmol) at 08C.
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K. TAKATORI ET AL.
The heterogeneous mixture was stirred vigorously 15 min and cold saturated
NaHCO3 was added at 08C. The aqueous layer was extracted with cold
CH2Cl2. The combined organic layers were dried over Na2SO4, and the solvent
was removed at 08C under reduced pressure to give the acrylate 4 (2.75 g) in
1
quantitative yield. H-NMR (CDCl3) d: 1.12 (t, J ¼ 7:1 Hz, 3H), 4.18 (q,
2
2
J ¼ 7:1 Hz, 2H), 7.03 (d, JC2H ¼ 0:8 Hz, 1H), 7.15 (d, JC2H ¼ 3:8 Hz, 1H),
7.55 (m, 2H), 7.64 (m, 1H), 7.98 (m, 2H). 13C-NMR (CDCI3) d: 143.4. IR
(neat) cmꢀ1: 1336, 3068, 2986, 2940, 1728, 1586, 1449, 1322, 1268, 1160, 1079,
748, 688.
(3S,5S)-tert-Butyl 3-(3-ethoxy-3-oxo-2-(phenylsulfonyl)[2-13C]propyl)-2-oxo-
5-phenylmorpholine-4-carboxylate ð12Þ
To a solution of oxazinone 3 (8.94 g, 32.22 mmol) in THF (110 ml) was added
dropwise NaHMDS (1 M solution in hexane, 31.1 ml, 31.1 mmol) at ꢀ788C
under nitrogen. The mixture was stirred for 1 h, then a solution of the acrylate
4 (2.75 g, 11.40 mmol) in THF (34 ml) was added dropwise over 30 min
through a cannula. The reaction mixture was stirred at ꢀ788C for 1 h. The
reaction was quenched with saturated NH4Cl, and the mixture was extracted
five times with ether. The combined organic layers were washed with saturated
NaCl, dried over MgSO4, and evaporated. The residue was chromatographed
on silica gel (hexane : ethyl acetate=5:1) to give 12 (2.75 g) as a 1:1 di-
1
astereomeric mixture in 49% yield from 11. H-NMR (CDCl3, 558C) d: 1.07–
1.34 (m, 12H), 2.50–3.03 (m, 2H), 3.95–4.60 (m, 4H), 4.80–5.09 (m, 3H), 7.04
(m, 2H), 7.25–7.39 (m, 3H), 7.57 (m, 2H), 7.69 (m, 1H), 7.89 (m, 2H). mp.
119.2–120.48C. 13C-NMR (CDCI3, 558C) d: 66.8, 67.61. IR (KBr) cmꢀ1: 2980,
2932, 1753, 1701, 1450, 1371, 1326, 1261, 1149, 1125, 1084, 1047, 1028.
HR-MS Calculated for C25 13CH31NO8S: m=z ¼ 518:1804. Found: m=z ¼
518:1799 (M+). EI-MS m/z (%): 518 (M+, 1), 418 (36), 231 (43), 104 (100),
77 (13).
3-(3-ethoxy-3-oxo[2-13C]propyl)-2-oxo-5-phenylmorpholine-4-carboxylate ð13Þ.
To a 0.1 M solution of SmI2 in THF (700 ml) was added dropwise over 1 h
through a cannula a solution of 12 (2.99 g, 5.77 mmol) in a mixture of THF
(42 ml) and MeOH (4.8 ml) at 08C. The mixture was stirred for 30 min, and
20% K2CO3, (70 ml) was added. The mixture was filtered through Celite. The
aqueous layer of the filtrate was separated and extracted with ether. The com-
bined organic layers were washed with brine, dried over MgSO4, and evap-
orated. The residue was chromatographed on silica gel (hexane : ethyl
1
acetate=2:1) to give 13 (1.43 g) in 65% yield, mp. 135.2–136.08C. H-NMR
(CDCl3, 558C) d: 1.26 (t, J ¼ 7:1 Hz, 3H, and s, 9H), 2.17 (m, 1H), 2.32–2.57
(m, 1H), 2.56 (dt, JC2H ¼ 129:6 Hz, J ¼ 7:1 Hz, 2H), 4.07–4.23 (m, 2H), 4.40
(dd, J ¼ 1:1, 11.8 Hz), 4.80–4.88 (m, 1H), 4.86 (dd, J ¼ 3:0, 11.8 Hz, 1H),
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SYNTHESIS OF L-[4-13C]GLUTAMIC ACID AND GLUTAMINE
451
5.02 (m, 1H), 7.07 (m, 2H), 7.23–7.36 (m, 3H). 13C-NMR (CDCl3, 558C) d:
30.6. IR (KBr) cmꢀ1: 2978, 2934, 1741, 1684, 1486, 1471, 1457, 1450, 1375,
1336, 1301, 1250, 1218, 1194, 1122, 1098, 1080, 888. HR-MS Calculated for
C19 13CH27NO6: m=z ¼ 378:1872. Found: m=z ¼ 378:1866 (M+). EI-MS m/z
(%): 378 (M+, 5), 278 (51), 233 (22), 104 (100), 57 (39).
l-[4-13C]Glutamic acid hydrochloride ð1Þ
A solution of 13 (1.43 g, 3.77 mmol) in saturated HCl in ethanol (18 ml) was
stirred at 858C overnight, and the mixture was evaporated. The residual
diethyl ester 14 was dissolved in EtOH (18 ml), and 20% Pd(OH)2 (2.65 g,
3.77 mmol) was added. The mixture was stirred at room temperature overnight
under a hydrogen atmosphere. The catalyst was removed by filtration through
Celite, and the filtrate was evaporated to give diethyl glutamate. The gluta-
mate was dissolved in 6 M HC1 (60 ml). The mixture was heated at 808C for
12 h, then washed with chloroform and ether. The aqueous layer was evap-
orated, and the residue was chromatographed on Amberlite IRA904 resin
1
(OH form) with 1 M HCl to give 1 (343 mg) in 49% yield from 13. H-NMR
(D2O) d: 1.99–2.16 (m, 2H), 2.49 (dt, JC2H ¼ 128:6 Hz, J ¼ 7:1 Hz, 2H), 3.89
3
(dt, JC2H ¼ 4:4 Hz, J ¼ 6:6 Hz, 1H). 13C-NMR (D2O) d: 32.4. IR (KBr)
cmꢀ1: 3379, 3200–2200 (br), 1725, 1671, 1612, 1509, 1421, 1275, 1254, 1213,
1078, 996, 824. HR-FAB-MS (glycerol) Calculated for C4 13CH10NO4:
m=z ¼ 149:0643. Found: m=z ¼ 149:0637 (MH+).
l-[4-13C]Glutamic acid 5-methyl ester hydrochloride ð15Þ
A solution of l-[4-13C]glutamic acid hydrochloride (1, 220 mg, 1.19 mmol) in
dry MeOH (5 ml) was stirred at room temperature for 42 h. The mixture was
1
evaporated to give the 5-methyl ester 15 (230 mg) in 97% yield. H-NMR
(D2O) d: 2.02–2.15 (m, 2H), 2.49 (dt, JC2H ¼ 129:4 Hz, J ¼ 7:4 Hz, 2H), 3.62
(s, 3H), 3.73 (m, 1H). 13C-NMR (D2O) d: 32.5. IR (KBr) cmꢀ1: 3700–2300
(br), 1725, 1637, 1509, 1441, 1221. HR-FAB-MS (glycerol) Calculated for
C5 13CH12NO4,: m=z ¼ 162:0800. Found: m=z ¼ 162:0799 (MH+).
l-N-Benzyloxycarbonyl[4-13C]glutamic acid 5-methyl ester ð16Þ
Cbz-Cl (0.19 ml. 1.32 mmol) was added to a solution of 15 (218 mg,
1.10 mmol) in water (l ml) and acetonitrile (1.5 ml). To the mixture was add-
ed dropwise a solution of Na2CO3 (181 mg, 1.70 mmol) in water (1.5 ml) over
45 min at room temperature. The mixture was vigorously stirred for 1 h, then
further Cbz-Cl (0.15 ml, 1.05 mmol) and a solution of Na2CO3 (135 mg,
1.27 mmol) in water (2.0 ml) was added. The mixture was stirred for 1 h,
and evaporated to remove acetonitrile. The mixture was washed with
ether. The organic layer was extracted with water 3 times. The combined
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K. TAKATORI ET AL.
aqueous layers were acidified with 3 M HC1, and extracted with ethyl acetate 5
times. The combined organic layers were washed with brine, dried over
Na2SO4, and evaporated to give 16 (216 mg) in 67% yield. 1H-NMR (CDCl3)
d: 1.90–2.70 (m, 4H), 3.67 (s, 3H), 4.43 (m, 1H), 5.12 (s, 2H), 5.54
(d, J ¼ 7:1 Hz, 1H), 7.25–7.37 (m, 5H). 13C-NMR (CDCl3) d: 30.1. IR (neat)
cmꢀ1: 3333, 3034, 2955, 1736, 1531, 1440, 1256, 1251, 1177, 1050. HR-MS
Calculated for C13 13CH17NO6: m=z ¼ 296:1089. Found: m=z ¼ 296:1095
(M+). EI-MS m/z (%): 296 (M+, 5), 251 (6), 207 (12), 108 (48), 91 (100), 85
(19), 79 (17).
l-N-Benzyloxycarbonyl[4-13C]glutamine ð17Þ
The 5-methyl ester 16 (214 mg, 0.712 mmol) was dissolved in concentrated
ammonium hydroxide (5 ml), and the solution was stirred for 4 h at room
temperature. The solution was evaporated, the residue was dissolved in water,
and the solution was acidified with 3 M HCl. The mixture was extracted with
ethyl acetate 5 times. The combined organic layers were washed with brine,
dried over Na2SO4, and evaporated to give the amide 17 (125 mg) in 62%
1
yield. H-NMR ([l,l,l,3,3,3-D6]acetone) d: 1.90–2.70 (m, 4H), 4.26 (m, 1H),
5.08 (s, 2H), 6.29 (brs, 1H), 6.77 (d, J ¼ 7:4 Hz, 1H), 6.91 (brs, 1H), 7.25–7.40
(m, 5H). 13C-NMR ([l,l,l,3,3,3-D6]acetone) d: 32.1. IR (KBr) cmꢀ1: 3331, 3067,
3034, 2959, 1711, 1655, 1541, 1455, 1418, 1348, 1248, 1055. HR-MS Calcu-
lated for C12 13CH16N2O5: m=z ¼ 281:1093. Found: m=z ¼ 281:1105 (M+).
EI-MS m/z (%): 281 (M+, 4), 263 (7), 236 (6), 174 (18), 129 (13), 108 (55), 91
(100), 85 (25), 79 (21).
l-[4-13C]Glutamine ð2Þ
A mixture of 17 (118 mg, 0.418 mmol) and 10% Pd-C (44 mg, 0.042 mmol) in
dry MeOH (5 ml) was stirred for 4 h at room temperature under a hydrogen
atmosphere. The catalyst was removed by filtration through Celite, and the
Celite pad was washed with water. The combined filtrates were evaporated.
The residue was treated with activated carbon in water, then evaporated to
give crude l-[4-l3C]glutamine (2). Crystallization from water : EtOH=1:5 gave
1
2 (48 mg) in 78% yield, mp. 172.4–173.18C. H-NMR (D2O) d: 1.99–2.19 and
3
2.54–2.62 (m, 4H), 3.67 (dt, JC2H ¼ 4:9 Hz, J ¼ 6:0 Hz, 1H). 13C-NMR
(D2O) d: 33.6. IR (KBr) cmꢀ1: 3411, 3300–2200 (br), 1686, 1638, 1630, 1483,
1412, 1333, 1315, 1160. HR-FAB-MS (glycerol) Calculated for
C4 13CH11N2O3: m=z ¼ 148:0803. Found: m=z ¼ 148:0800 (MH+). CD
(pH 1.5 HClO4) l nm (De): 208 (+1.61). HPLC-CD analysis: the eluent was
pH 2.0 HClO4, the flow rate was 0.8 ml/min, the detection wavelength was
208 nm (UV and CD), the temperature was 08C, and other conditions were as
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SYNTHESIS OF L-[4-13C]GLUTAMIC ACID AND GLUTAMINE
453
described in a previous report,11 Rt ¼ 2:0 min (authentic D-form gave
Rt ¼ 4:3 min).
Acknowledgements
This work was supported in part by a grant for the promotion of the
advancement of education and research in graduate schools from the Ministry
of Education, Culture, Sports and Technology of Japan.
References
1. Zwingmann C, Butterworth R. Neurochem Int 2005; 47: 19–30.
2. Dellaria Jr JF, Santarsiero BD. J Org Chem 1989; 54: 3916–3926.
3. Takatori K, Nishihara M, Nishiyama Y, Kajiwara M. Tetrahedron 1998; 54:
15861–15869.
4. Takatori K, Nishihara M, Kajiwara M. J Label Compd Radiopharm 1999; 42:
701–708.
5. Takatori K, Toyama S, Narumi S, Fujii S, Kajiwara M. J Label Compd Radio-
pharm 2004; 47: 91–94.
6. Takatori K, Hayashi A, Kajiwara M. J Label Compd Radiopharm 2004; 47:
787–795.
7. Cappon JJ, Baart J, Van der Walle GAM, Raap J, Lugtenburg J. Recl Trav Chim
Pay-B 1991; 110: 158–166.
8. Goux WJ, Rench L, Weber DS. J Label Compd Radiopharm 1993; 33: 181–193.
9. Kurumaya K, Okazaki T, Seido N, Akasaka Y, Kawajiri Y, Kajiwara M. J Label
Compd Radiopharm 1989; 27: 217–235.
10. Gipstein E, Willson CG, Sachdev HS. J Org Chem 1980; 45: 1486–1489.
11. Takatori K, Toyama S, Fujii S, Kajiwara M. Chem Pharm Bull 1995; 43:
1797–1799.
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J Label Compd Radiopharm 2006; 49: 445–453