Asymmetric synthesis of [2,3-13C2,15N]-4-benzyloxy-5,6-diphenyl-2, 3,5,6-tetrahydro-4H-oxazine-2-one via lipase TL-mediated kinetic resolution of benzoin: General procedure for the synthesis of [2,3-13C2</s

Article abstract of DOI:10.1021/jo015725f

Lipase TL-mediated kinetic resolution of benzoin proceeded to give the corresponding optically pure (R)-benzoin (R)-1. On the other hand, (S)-benzoin O-acetate (S)-7 could be hydrolyzed without epimerization to give (S)-benzoin (S)-1 under alkaline condit

Full text of DOI:10.1021/jo015725f

8010
J . Org. Chem. 2001, 66, 8010-8014
Asym m etr ic Syn th esis of
[2,3-13C2,15N]-4-Ben zyloxy-5,6-d ip h en yl-2,3,5,6-tetr a h yd r o-4H-oxa zin e-2-on e
via Lip a se TL-Med ia ted Kin etic Resolu tion of Ben zoin : Gen er a l
P r oced u r e for th e Syn th esis of [2,3-13C2,15N]-L-Ala n in e
Yutaka Aoyagi* and Ai Iijima
School of Pharmacy, Tokyo University of Pharmacy & Life Science,
4132-1 Horinouchi, Hachioji, Tokyo 192-0392, J apan
Robert M. Williams*
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
rmw@chem.colostate.edu
Received May 1, 2001
Lipase TL-mediated kinetic resolution of benzoin proceeded to give the corresponding optically
pure (R)-benzoin (R)-1. On the other hand, (S)-benzoin O-acetate (S)-7 could be hydrolyzed without
epimerization to give (S)-benzoin (S)-1 under alkaline conditions. Furthermore, both enantiomers
of benzoin (1) were converted to [15N]-(1R,2S)- and (1S,2R)- 2-amino-1,2-diphenylethanol (3a and
3b), respectively, according to the procedure reported previously. [2,3-13C2,15N]-(5S,6R)-4-benzyloxy-
5,6-diphenyl-2,3,5,6-tetrahydro-4H-oxazine-2-one (10) was synthesized from ethyl [1,2-13C2]-
bromoacetate and (1R,2S)-2-amino-1,2-diphenylethanol (3b) in three steps. Finally, [2,3-13C2,15N]-
L-alanine (12) was prepared via alkylation of the lactone 10 and hydrogenation of the alkylated
product 11.
In tr od u ction
in multidimensional heteronuclear correlation NMR
spectroscopic analyses.6 In our own work,7 we have
extensively demonstrated that (5S,6R)- and (5R,6S)-4-
CBz- and -t-BOC-5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-
oxazin-2-ones (4a and 4b) are useful as chiral, nonrace-
mic glycine templates for the synthesis of structurally
diverse R-amino acids (Scheme 1).
Amino acids1 serve a central role in biology and
chemistry. They are the fundamental constituents of
proteins and chemical mediators of nitrogen metabolism
and provide the raw materials from which a large
number of biologically important primary and secondary
metabolites are constructed.2 Application of NMR3 and
mass spectrometric analysis to stable isotopically labeled
compounds has proven useful for research on amino acid
and protein metabolism.4 In particular, the use of mul-
tiply labeled amino acids has become important as an
internal standard for the study of metabolic transforma-
tion using mass spectrometric analysis.5 In addition,
multiply labeled amino acids serve as starting materials
for the preparation of uniformly labeled peptides for use
To synthesize oxazinones 4a and 4b, it is necessary to
prepare erythro-2-amino-1,2-diphenylethanol 3a and 3b
(6) (a) Rienstra, C. M.; Hatcher, M. E.; Leonard, J . M.; Sun, B.;
Fesik, S. W.; Griffin, R. G. J . Am. Chem. Soc. 1998, 120, 10602-10612.
(b) Ishii Y.; Tycko, R. J . Am. Chem. Soc. 2000, 122, 1443-1455.
(7) (a) Sinclair, P. J .; Zhai, D.; Reibenspies, J .; Williams, R. M. J .
Am. Chem. Soc. 1986, 108, 1103-1104. (b) Williams, R. M.; Sinclair,
P. J .; Zhai, D.; Chen, D. J . Am. Chem. Soc. 1988, 110, 1547-1557. (c)
Williams, R. M.; Sinclair, P. J .; Zhai, W., J . Am. Chem. Soc. 1988, 110,
482-483. (d) Williams, R. M.; Im, M.-N., Tetrahedron Lett. 1988, 29,
6075-6078. (e) Williams, R. M.; Im, M.-N. J . Am. Chem. Soc. 1991,
113, 9276-9286. (f) Williams, R. M.; Zhai, W., Tetrahedron, 1988, 44,
5425-5430. (g) Williams, R. M.; Hendrix, J . A. J . Org. Chem. 1990,
55, 3723-3728. (h) Williams, R. M.; Im, M.-N.; Cao, J . J . Am. Chem.
Soc. 1991, 113, 6976-6981. (i) Williams, R. M.; Fegley, G. J . J . Am.
Chem. Soc. 1991, 113, 8796-8806. (j) Williams, R. M.; Yuan, C. J . Org.
Chem. 1992, 57, 6519-6527. (k) Williams, R. M.; Fegley, G. J .
Tetrahedron Lett. 1992, 33, 6755-6758. (l) Williams, R. M.; Zhai, W.;
Aldous, D. J .; Aldous, S. C. J . Org. Chem. 1992, 57, 6527-6532. (m)
Williams, R. M.; Fegley, G. J . J . Org. Chem. 1993, 58, 6933-6935. (n)
Williams, R. M.; Yuan, C. J . Org. Chem. 1994, 59, 6190-6193. (o)
Williams, R. M.; Colson, P.-J .; Zhai, W. Tetrahedron Lett. 1994, 35,
9371-9374. (p) Bender, D. M.; Williams, R. M. J . Org. Chem. 1997,
62, 6690-6691. (q) Williams, R. M.; Aoyagi, Y. Tetrahedron 1998, 54,
10419-10433. (r) Scott, J . D.; Tippie, T. N.; Williams, R. M. Tetrahedron
Lett. 1998, 39, 3659-3662. (s) Aoyagi, Y.; Williams, R. M. Synth. Lett.
1998, 1099-1101. (t) Aoyagi, Y.; Williams, R. M. Tetrahedron 1998,
54, 13045-13058. (u) Williams, R. M. In Peptidomimetics Protocols;
Kazmierski, W., Ed.; Humana Press: Totowa, 1999; Vol. 23, Chapter
19, pp 339-356. (v) Sebahar, P.; Williams, R. M. J . Am. Chem. Soc.
2000, 122, 5666-5667. (w) Aoyagi, Y.; J ain, R. P.; Williams, R. M., J .
Am. Chem. Soc. 2001, 123, 3472-3477. (x) DeMong, D. E.; Williams,
R. M. Tetrahedron Lett. 2001, 42, 183-185. (y) DeMong, D. E.;
Williams, R. M. Tetrahedron Lett. 2001, 42, 3529-3532. (z) J ain, R.
P.; Williams, R. M. Tetrahedron 2001, 57, 6505-6509.
* To whom correspondence should be addressed. E-mail: (R.M.W.)
rmw@chem.colostate.edu and (Y.A.) aoyagiy@ps.toyaku.ac.jp.
(1) For a review, see: (a) Williams, R. M. Synthesis of Optically
Active R-Amino Acids. In The Organic Chemistry Series; Baldwin, J .
E., Ed.; Pergamon Press: Oxford, 1989. (b) Barrett, G. C., Ed.
Chemistry and Biochemistry of the Amino Acids; Chapman and Hall:
London, 1985. (c) Wagner, I.; Musso, H. Angew. Chem., Int. Ed. Engl.
1983, 22, 816. (c) Greenstein, J . P.; Wintz, M. Chemistry of the Amino
Acids; Robert E. Krieger: FL, 1984; Vols. 1-3.
(2) Dewick, P. M. Medicinal Natural Products: A Biosynthetic
Approach (Natural Product Chemistry) ‘97; Wiley: New York, 1997
and cited references therein.
(3) (a) Coggan, A. R. Proc. Nutr. Soc. 1999, 58, 953-961. (b)
Scrimgeour, C. M. Spec. Publ. R. Soc. Chem. 1999, 244, 15-31. (c)
Koletzko, B.; Demmelmair, H.; Hartl, W.; Kindermann, A.; Koletzko,
S.; Sauerwald, T.; Szitanyi, P. Early Hum. Dev. 1998, 53 (Suppl. 1),
S77-S97. (d) Brenna, J . T. Prostaglandins, Leukotrienes Essent. Fatty
Acids 1997, 57, 467-472.
(4) (a) Simpson, T. J . Topics in Current Chemistry (Biosynthesis:
Polyketides and Vitamins); Springer-Verlag: Berlin, 1998; Vol. 195,
pp 1-48. (b) Simpson, T. J . Isot. Phys. Biomed. Sci. 1991, 2, 431-474.
(c) Leete, E.; Morris, G. J .; Rao, H. S. Prakash Rev. Latinoam. Quim.
1986, 17, 24-32.
(5) Duncan, M. W.; Poljak, A. Anal. Chem. 1998, 70, 890-896 and
the references therein.
10.1021/jo015725f CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/30/2001

Synthesis of [2,3-13C2,15N]-L-Alanine
J . Org. Chem., Vol. 66, No. 24, 2001 8011
Sch em e 1
in optically pure form. The preparation of the optically
pure amino alcohols 3a and 3b has recently received
widespread attention due to the use of these amino
alcohols as chiral auxiliaries in asymmetric synthesis,8
chiral stationary phase for HPLC applications,9 and
ligands in asymmetric catalysis.10 Several methods have
been described in the literature for the synthesis of both
antipodes of amino alcohols 3a and 3b. The classical
synthesis of these substances involves the resolution of
racemic erythro-2-amino-1,2-diphenylethanol, obtained by
the hydrogenation of benzoin oxime (2), with glutamic
acid as Tishler and co-workers reported in 1951 (Scheme
1).11 More recently, the asymmetric dihydroxylation (AD)
of trans-stilbene followed by amination has been de-
scribed by Sharpless and co-workers.12 Fujisawa and co-
workers reported the asymmetric reduction of 1,2-diaryl-
2-benzyloxyiminoethanones13 and Davis and co-workers
reported an asymmetric synthesis of benzoin oxime by
the asymmetric enolate oxidation of deoxybenzoin.14
Despite the existence of these procedures, there remains
a need for a practical and convenient preparation of
optically pure benzoin, from which a host of important
optically active compounds, including the erythro-2-
amino-1,2-diphenylethanols, can be accessed. The Tishler
procedure,11 which involves a rather time-consuming
crystallization procedure, has limited large (>10 kg)
throughput. This limitation has prompted us to investi-
gate an alternative approach. Several synthetic proce-
dures have been reported for the preparation of pure
benzoin.14,15 Demir and co-workers reported an enzymatic
synthesis of chiral benzoin in aqueous medium, but this
procedure gave low yields and moderate enantiomeric
ratios (er’s) of benzoin.16 Lipase-mediated kinetic resolu-
tions of secondary alcohols in nonaqueous medium are
well-known to be a powerful tool in organic synthesis to
prepare chiral synthetic intermediates.17 Adam and co-
workers have reported lipase-mediated kinetic resolu-
tions18 of R-hydroxy ketones by using Amano PS and
Amano AK, but these procedures gave low conversion and
required relatively long reaction times. Despite these
reports, there is relatively little known about the kinetic
enzymatic resolution of benzoins. In a preliminary pa-
per,19 we have recently reported the efficient enzymatic
optical resolution of benzoin 1 and the preparation of
optically pure (1R,2S)- and (1S,2R)-2-amino-1,2-diphe-
nylethanol. In this paper, we wish to describe the lipase
TL-mediated kinetic resolution of benzoin in detail and
its conversion to [2,3-13C2,15N]-(5S,6R)-4-CBz-5,6-di-
phenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-one (10), which
should prove to be a useful template for the synthesis of
15N,13C-multiply labeled optically active R-amino acids.
Finally, the synthesis of [2,3-13C2,15N]-L-alanine (12) was
conducted to demonstrate the utility of the doubly labeled
oxazinone.
(8) (a) Williams, R. M. Aldrichim. Acta 1992, 25, 11-25. (b)
Williams, R. M. In Advances in Asymmetric Synthesis; Hassner, A.,
Ed.; J AI Press: Greenwich, CT, 1995; Vol. 1, pp 45-94 and references
therein. (c) The requisite diphenyloxazinones are commercially avail-
able from Aldrich Chemical Co.: (2S,3R) R1 ) Cbz catalog no. 33185-6
(CAS Registry no. 105228-46-4), R1 ) t-Boc catalog no. 33181-3 (CAS
Registry no. 112741-50-1); antipode (2R,3S) R1 ) Cbz catalog no.
33187-2 (CAS Registry no. 100516-54-9), R1 ) t-Boc catalog no. 33184-8
(CAS Registry no. 112741-49-8).
(9) Yuki, Y.; Saigo, K.; Tachibana, K.; Hasegawa, M. Chem. Lett.
1986, 1347-1350.
(15) (a) Enders, D.; Bhushan, V. Tetrahedron Lett. 1988, 29, 2437-
2440. (b) Baburi, F.; Fiandanese, V.; Marchese, G.; Punzi, A. Tetra-
hedron 1999, 55, 2431-2440.
(16) Demir, A. S.; Hamamci, H.; Tanyeli, C.; Akhmedov, I. M.;
Doganel, F. D. Tetrahedron: Asymmetry 1998, 9, 1673-1677.
(17) Hanson, J . R. Hydrolytic Reactions. In An Introduction to
Biotransformation in Organic Chemistry; Mann, J ., Ed.; Biochemical
& Medicinal Chemistry Series; W. H. Freeman: New York, 1995; pp
11-22.
(18) Adam, W.; Diaz, M. T.; Fell, R. T.; Saha-Mo¨ller, C. R. Tetra-
hedron: Asymmetry 1996, 7, 2207-2210.
(19) Aoyagi, Y.; Agata, N.; Shibata, N., Horiguchi, M.; Williams, R.
M. Tetrahedron Lett. 2000, 41, 10159-10162.
(10) (a) Brown, B.; Hegedus, L. S. J . Org. Chem. 1998, 63, 8012-
8018. (b) Hegedus, L. S. Acc. Chem. Res. 1995, 28, 299-305.
(11) Weijlard, J .; Pfister, K.; Swanezy, E. F.; Robinson, C. A.; Tishler,
M. J . Am. Chem. Soc. 1951, 73, 1216-1218.
(12) (a) Gao, Y.; Sharpless, K. B. J . Am. Chem. Soc. 1988, 110, 7538-
7539. (b) Chang, H.-T.; Sharpless, K. B. Tetrahedron Lett. 1996, 37,
3219-3222.
(13) Shimizu, M.; Tsukamoto, K.; Matsutani, T.; Fujisawa, T.
Tetrahedron 1998, 54, 10265-10274.
(14) Davis, F. A.; Haque, M. S.; Przeslawski, R. M. J . Org. Chem.
1989, 54, 2021-2024.

8012 J . Org. Chem., Vol. 66, No. 24, 2001
Ta ble 1. Lip a se TL-Med ia ted Kin etic Resolu tion of Ben zoin
Aoyagi et al.
amount (mg)
(S)-7
yield (%)
(R)-1
entry
(+)-1
Lipase TL
solventa
acyl donord
reaction time (h)
er
yield (%)
er
1
2
3
4
5
100
100
100
100
100
100
100
500
500
3760
10
50
100
250
100
100
100
1250
1250
9400
THFa
VA
VA
VA
VA
VA
VA
IA
VA
VA
VA
20
20
42
20
20
20
43
20
20
20
9
29
40
52
2
18
3
42
46
50
79
35
53
46
95
69
88
40
THFa
THFa
THFa
EtOAca
benzenea
THFa
6
7
8e
9f
10g
THFb
THFb
THFc
>99:1
>99:1
>99:1
97:3
96:4
48
a
b
d
5 mL of solvent was used. 25 mL of solvent was used. c 188 mL of solvent was used. VA, vinyl acetate; IA, isopropenyl acetate; 12
equiv of the acyl donor was employed. e 2.5 g of Celite 545 was used. f Reaction in the absence of Celite. The enantiomeric ratios (er’s)
g
were determined by means of HPLC with a Chiralcel OD column.
Sch em e 2a
Resu lts a n d Discu ssion
Initially, we examined the kinetic resolution of (()-
benzoin (1) in the presence of various commercially
available lipases, including PPL, Amano PS, Amano I,
Amano II, and Lipase MY, UL, TL, SC, AL, OF, and QL
in a mixture of vinyl acetate and THF at room temper-
ature. Among these lipases, Lipase TL20 was found to be
the most effective (conversion rate up to 50%). Next, the
reaction parameters were varied including the acyl donor,
reaction time, and solvent; the results are shown in Table
1. The reaction did not proceed in hexane as a solvent.
The best results were obtained when the reaction was
a
Reagents and conditions: (a) K2CO3, MeOH, H2O, 0 °C, 91%;
conducted in a mixture of THF as a solvent and vinyl
(b) (i) 15NH2OH-HCl, NaOAc, EtOH, H2O, quant; (ii) 5% Pd-C,
H2 (3 atm), 3% HCl in EtOH.
acetate as an acyl donor at room temperature (Table 1,
entry 4). The products were easily separated by flash
column chromatography on silica gel. On the basis of
these results, the conditions employed in entry 4 were
scaled up to 500 mg and 4000 mg of racemic benzoin, in
both the presence and absence of Celite. The best results
obtained (Table 1, entries 9 and 10) gave (R)-1 in 41∼48%
yield and 96:4 er plus (S)-7 in 46∼47% yield and >99:1
er. The enantiomeric ratios of benzoin acetate and the
recovered benzoin were determined by means of chiral
HPLC analysis (Chiralcel OD). When the kinetic resolu-
tion of benzoin was conducted in the presence of recov-
ered lipase TL, the exact same results were obtained and
thus, although a 2.5 gm/gm excess of enzyme was found
to be optimal, the enzyme was easily recovered and
reused without loss of enzymatic activity. Hydrolyis21 of
the optically pure benzoin O-acetate (S)-7 proceeded at
0 °C under alkaline conditions to give the corresponding
benzoin (S)-1 in 91% yield on 100 mg scale by means of
flash column chromatography (Scheme 2). On the other
hand, on a 1 gram scale, separation was conducted
without column chromatography to give (S)-7 in 76%
yield. The er’s of the products were >98:2.
With both enantiomers of approximately optically pure
benzoin in hand, we examined the preparation of the
amino alcohols. Namely, the condensation of the chiral
benzoins (R)-1 and (S)-1 with [15N]-NH2OH‚HCl in a
refluxing mixture of water and ethanol gave a mixture
of the E- and Z-isomers of the corresponding oxime (ca.
53:47 on the basis of 1H NMR analysis), which were used
directly as an E/Z mixture.11 Without further purification
of the oximes, hydrogenation was performed in ethanol
containing 3% HCl under an atmosphere of hydrogen (3
atm) to give the erythro-amino alcohols 3a and 3b in 50%
and 62% yields, respectively (Scheme 2). The enantio-
meric ratios were determined to be 99:1 to 99:2 by means
of chiral HPLC analysis with a Chiral CD-Ph column.
Mass spectral analysis showed that these compounds
contained 99% of 15N.
Next, following our published procedure,7 transforma-
tion of the chiral 15N-labeled amino alcohol 3b to [2,3-
13C2,15N]-(5S,6R)-4-CBz-5,6-diphenyl-2,3,5,6-tetrahydro-
4H-1,4-oxazin-2-one (10) was conducted as shown in
Scheme 3. Namely, the reaction of compound 3b with
ethyl [1,2-13C2]bromoacetate proceeded to give compound
8, which was employed in the next reaction without
further purification. Crude 8 was easily transformed into
the corresponding CBz derivative 9. In the presence of
p-TsOH, the lactonization of 9 with azeotropic distillation
of benzene was conducted to give the lactone 10 in 62%
overall yield for the three steps. To demonstrate the
(20) This lipase can be purchased from Meito Sangyo Co. Ltd (Tokyo,
J apan).
(21) There are some papers that describe the hydrolysis of benzoin
O-acetate. (a) In acidic medium: Harada, K.; Shiono, S. Bull. Chem.
Soc. J pn. 1984, 57, 1040-1045. (b) Using scandium triflate: Kajiro,
H.; Mitamura, S.; Mori, A.; Hiyama, T. Tetrahedron Lett. 1999, 40,
1689-1692. Kajiro, H.; Mitamura, S.; Mori, A.; Hiyama, T. Bull. Chem.
Soc. J pn. 1999, 72, 1553-1560.

Synthesis of [2,3-13C2,15N]-L-Alanine
J . Org. Chem., Vol. 66, No. 24, 2001 8013
Sch em e 3a
mined on a Fisons VG Auto Spec instrument. High-perfor-
mance liquid column chromatography (HPLC) was conducted
using a J ASCO UV-970 UV detector, a J ASCO PU-980 pump,
and a J ASCO 807-IT integrator. The chiral columns Chiralcel
OD and Chiral CD-Ph were purchased from Daicel Co. Ltd.,
Tokyo, and Shiseido Co. Ltd., Tokyo, respectively. Medium-
pressure column chromatography (MPLC) was conducted
using a UVILOG 5III spectrometer as the UV detector (Oyo
Bunko Kiki Co., Ltd., Tokyo) and Kieselgel 60 (Merck AG,
Darmstadt) as the packing material. Lipase TL was purchased
from Meito Sangyo Co., Ltd., J apan. The lipase must be dried
at room temperature under reduced pressure (1-2 mmHg) for
24 h before use. All other reagents were employed in the
reaction without further purification. 15N-Hydroxylamine hy-
drochloride (catalog no. T85-70214, min 99 atom %) and 1,2-
13C2-ethyl bromoacetate (catalog no. T83-02517, min 99 atom
%) were purchased from ISOTEC Co. Ltd. (USA).
Kin etic Resolu tion of Ben zoin 1. (a ) Colu m n Sep a r a -
tion . A mixture of benzoin 1 (100 mg, 0.47 mmol), THF (5
mL), Lipase TL (250 mg), and vinyl acetate (0.5 mL, 5.64
mmol) was stirred at room temperature for 15 h. After
filtration, the filtrate was concentrated in vacuo to give an oily
residue. The residue was purified with MPLC (hexane-AcOEt
solvent system) to give (S)-acetate 2 [[R]D +217° (c ) 0.35,
CHCl3) (lit.21a (R)-benzoin acetate [R]D -214.3° (c ) 1.0,
CHCl3))] and recovered (R)-benzoin 1. [[R]D -118° (c ) 0.2,
acetone) (lit.21a [R]D -111.6° (c ) 1.0, acetone))]; physical data
for this compound were identical with those reported in the
literature.21 The er’s were determined by means of HPLC
analysis with a chiral column, as described below.
(b) Usin g Recycled Lip a se TL: Sem i-La r ge Sca le. Th e
lipa se m u st be d r ied a t r oom tem per a tu r e for 24 h u n d er
r ed u ced pr essu r e befor e u se. A mixture of benzoin 1 (3.76
g, 17.7 mmol), THF (188 mL), Lipase TL (9.40 g), and vinyl
acetate (19.6 mL, 213 mmol) was stirred at room temperature
for 20 h. After filtration, the filtrate was concentrated in vacuo
to give a pale yellow residue (5.3 g). i-Pr2O (100 mL) was then
added to the residue, which was then heated at 40 °C for
several minutes and filtered to give optically pure (R)-benzoin
1 (0.62 g, 17%). The filtrate was evaporated under reduced
pressure to give a residue. A second trituration with i-Pr2O
(80 mL) gave more optically pure (R)-benzoin 1 (0.45 g, 12%).
Finally, the filtrate was evaporated and purified with MPLC
(hexane-AcOEt solvent system) to give the (S)-acetate (S)-7
(2.26 g, 50%) as a less polar component and recovered (R)-
benzoin (R)-1 (1.0 g, 27%). As a result, total recovered (R)-1
was 1.80 g (48%).
Reagents and conditions: (a) Br13CH213COOEt, Et3N, THF;
a
(b) CBzCl, NaHCO3, CHCl3; (c) p-TsOH, benzene; (three steps
overall yield 63%); (d) NaN(TMS)2, MeI, THF, 91%; (e) PdCl2, H2
(3 atm), 84%.
potential utility of the doubly labeled oxazinone thus
obtained, [15N,13C2]-labeled L-alanine was prepared. Alky-
lation of the lactone 10 with methyl iodide in the presence
of sodium bis(trimethylsilyl)amide at -78 °C gave a
single diastereomer (11) in 82% yield. Hydrogenation of
11 with palladium chloride in a mixture of tetrahydro-
furan and ethanol under a hydrogen atmosphere (3 atm)
gave the corresponding [15N,13C2]-L-alanine 12.6 The er
of amino acid 12 was determined to be higher than 99:1
by means of 1H and 19F NMR analysis of the correspond-
ing Mosher amide.
In summary, the synthesis of [2,3-13C2,15N]-(5S,6R)-4-
CBz-5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-
one (10) has been described. The requisite amino alcohols
3 have been obtained in high optical purity via the lipase
TL-mediated kinetic resolution of benzoin, which are both
commercially available starting materials. The kinetic
resolution of benzoin 1 using lipase TL proceeded to give
(S)-benzoin-O-acetate (S)-7 and (R)-benzoin (R)-1 in good
yields. Hydrolysis of the acetate (S)-7 gave the corre-
sponding (R)-benzoin 1. Both enantiomers were converted
to erythro-amino alcohols 3a and b in moderate yields.
Following our previously reported procedure, 3b was
converted to optically pure [2,3-13C2,15N]-(5S,6R)-4-CBz-
5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-one (10),
which was employed in the synthesis of [1,2-13C2,15N]-L-
alanine 12. This protocol should prove to be general for
the synthesis of multiply labeled amino acids. Our
laboratory has published extensively on the enolate
alkylation reactions and electrophilic substitutions of 10,
which amply demonstrates the general utility of this
simple glycine template for the synthesis of a wide array
of isotopically labeled amino acids and peptide isoteres.7
The single example of the synthesis of labeled alanine
reported herein, serves only to illustrate the synthetic
potential of the isotopically labeled glycine templates 10.
Additional applications of these labeled substrates shall
be reported on in due course from these laboratories.
Deter m in a tion of En a tiom er ic Ra tio (er ) of Recover ed
Ben zoin 1 a n d Ben zoin O-Aceta te 7 by Mea n s of HP LC
An a lysis w ith a Ch ir a l Colu m n . Column: Daicel CHIRAL-
CEL OD column (4.6 × 250 mm). Solvent: hexane/iPrOH )
9:1. UV wavelength: 270 nm. Flow rate: 1.0 mL/min. Pres-
sure: 20 kg/cm2. (R)-benzoin 1; tR ) 17.4 min. (S)-benzoin (S)-
1; tR ) 12.2 min. (R)-benzoin O-acetate (R)-7; tR) 6.5 min. (S)-
benzoin-O-acetate (S)-7; tR) 10.0 min.
Hyd r olysis of (S)-Ben zoin Aceta te (S)-7. A mixture of
(S)-acetate 2 (100 mg, 0.39 mmol), MeOH (30 mL), H2O (30
mL), and K2CO3 (54 mg, 0.39 mmol) was stirred at room
temperature for 10 min. After H2O (150 mL) was added to the
reaction mixture, the aqueous solution was extracted with
AcOEt (50 mL × 3). The organic layer was washed with
saturated aqueous NaCl (50 mL × 3), dried over MgSO4,
filtered, and evaporated under reduced pressure to give a
residue, which was purified with flash column chromatography
(hexane-AcOEt ) 6:1) to give (S)-benzoin (S)-1 (91%) as a
colorless solid: [[R]D +120° (c ) 0.2, acetone) ((R)-benzoin lit.21a
[R]D -111.6° (c ) 1.0, acetone))]. Physical data for this
compound were identical with those reported in the litera-
ture.21 The er of the product was determined by means of the
HPLC method mentioned above.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured on a Yanagimoto micromelting point apparatus and
are uncorrected. NMR spectra were obtained on Brucker DPX-
400 and DRX-500 spectrometers. IR spectra were recorded on
a J asco FT/IR-620 spectrometer. Mass spectra were deter-
P r ep a r a t ion of 15N-(1R,2S)-er yth r o-2-Am in o-1,2-d i-
p h en yleth a n ol 3b fr om (R)-Ben zoin 1. A mixture of (R)-
benzoin 1 (1.0 g, 4.7 mmol), NaOAc (0.95 g, 11.6 mmol),
15NH2OH‚HCl (0.8 g, 11.5 mmol), EtOH (40 mL), and H2O (8
mL) was refluxed for 2 h. After evaporation in vacuo, Et2O

8014 J . Org. Chem., Vol. 66, No. 24, 2001
Aoyagi et al.
(100 mL) was added to the residue. The organic phase was
washed with saturated aqueous NaHCO3 (40 mL × 3), dried
over anhydrous MgSO4, filtered, and evaporated under reduced
pressure to give a residue that was a mixture of E and Z oxime
(53:47). Without further separation, the mixture was employed
in the hydrogenation. A mixture of the oxime (1.0 g, 2.11
mmol), 5% Pd-C (0.117 g), and 3% HCl in EtOH (21 mL) was
hydrogenated under 3 kg/cm2 of H2 for 6 h. After the reaction,
H2O (100 mL) was added and the palladium catalyst was
filtered off through Celite 545. The pH of the aqueous solution
was made alkaline through addition of concentrated aqueous
NH3, which caused the appearance of a crystalline precipitate.
After filtration and drying under reduced pressure, compound
3b (0.61 g, 62%) was obtained as a colorless solid. The threo
1,4-oxazin-2-one 10 as colorless needles (0.98 g, 63%): mp
211-213 °C; [R]D -70.4° (c ) 0.14, CH2Cl2); 1H NMR (400
MHz, 383 K, DMSO-d6) δ 7.28-7.26 (m, 6H), 7.21-7.08 (m,
7H), 6.73 (d, 2H, J ) 7.3 Hz), 6.21 (brs, 1H), 5.32 (brs, 1H),
5.11 (d, 1H, J ) 12.7), 5.06 (d, 1H, J ) 12.7), 4.64 (ddd, 1H, J
) 143, 17.7, 6.5 Hz), 4.57 (ddd, 1H, J ) 143, 17.7, 6.5 Hz); 13
C
NMR (100 MHz, 300 K, CDCl3) δ 167.2 (d, J ) 56 Hz), 166.8
(d, J ) 56), 45.3 (dd, J ) 56, 11 Hz), 61.9 (d, J ) 4.7 Hz); 15
N
NMR (50 MHz, 300 K, CD3OD, CH3NO2 as internal standard)
δ ppm; IR (film) 1705 cm-1 (CdO); HRMS (FAB) calcd for
C2213C2H2215NO4 (M+ + H) 391.1586, found 391.1588. Anal.
Calcd for C2213C2H2115NO4: C, 74.33; H, 5.42; N, 3.84. Found:
C, 73.95; H, 5.48; N, 3.65.
Met h yla t ion of [2,3-13C2,15N]-(5S,6R)-4-CBz-5,6-d i-
p h en yl-2,3,5,6-tetr a h yd r o-4H-1,4-oxa zin -2-on e 10. To a
solution of compound 10 (0.31 g, 0.81 mmol), MeI (0.5 mL, 8.1
mmol), and dry THF (25 mL) was added 1.0 M NaN(TMS)2 in
THF (1.2 mL, 1.2 mmol) dropwise at -78 °C under an Ar
atmosphere. After the resulting solution was stirred at the
same temperature for 30 min, the reaction mixture was poured
into EtOAc (200 mL). The organic layer was washed with H2O
(100 mL × 2) and saturated aqueous NaCl (100 mL × 1), dried
over anhydrous MgSO4, filtered, concentrated, and purified by
means of silica gel flash chromatography (eluted with CHCl3/
EtOAc ) 25:1) to give compound 11 as a colorless solid (0.30
g, 91% yield): mp 188-190 °C; [R]D -49.8° (c ) 0.47, CH2Cl2);
1H NMR (400 MHz, 383 K, d6-DMSO) δ 7.29-7.22 (m, 6H),
7.20-7.15 (m, 1H), 7.12-7.08 (m, 6H), 6.61 (d, 1H, J ) 7.2
Hz), 6.21 (d, 1H, J ) 2.8 Hz), 5.31 (brs, 1H), 4.95 (dm, 1H, J
) 150), 5.07 (d, 1H, J ) 12.7), 5.00 (d, 1H, J ) 12.7 Hz), 4.95
(dm, 1H, J ) 150), 1.80-1.75 (m, 3H); 13C NMR (100 MHz,
300 K, CDCl3) δ 170.1 (d, J ) 56 Hz), 170.0 (d, J ) 55 Hz),
52.9 (dd, J ) 55, 9 Hz), 52.8 (dd, J ) 56, 11 Hz); 15N NMR (50
MHz, 300 K, CD3OD, CH3NO2 as internal standard) δ -283.98
(d, J ) 9.0 Hz), -284.46 (d, J ) 11 Hz) ppm; IR (film) 1709
(CdO) cm-1; FAB-MS 405 (M+ + H), 361 (M+ - CO2H); HRMS
(FAB) calcd for C2313C2H2415NO4 (M+ + H) 405.174278, found
405.174780.
1
isomer could not be detected in the H NMR spectrum. The er
of the product was determined by means of HPLC analysis
with a chiral column as described below: mp 142-144 °C; [R]D
1
-7.6° (c ) 0.6, EtOH); H NMR (400 MHz, 300 K, CDCl3) δ
7.33-7.21 (m, 10H), 4.75 (d, 1H, J ) 6.2 Hz), 4.17 (d, 1H, J )
6.2 Hz), 1.54 (brs, 3H); 13C NMR (100 MHz, 300 K, CDCl3) δ
141.5, 140.7, 128.3, 128.1, 127.8, 127.6, 127.6, 126.9, 78.4 (d,
J ) 3.1 Hz), 61.9 (d, J ) 4.7 Hz); 15N NMR (50 MHz, 300 K,
CD3OD, CH3NO2 as internal standard) δ -352.16 ppm; IR
(film) 3324, 3272 cm-1 (NH2, OH); HRMS (FAB) calcd for
C
14H1615NO 215.1202 (M+ + H), found 215.1202; er 98:2.
P r ep a r a t ion of 15N-(1S,2R)-er yth r o-2-Am in o-1,2-d i-
p h en yleth a n ol 3a fr om (S)-Ben zoin 1. Compound 3a was
prepared from (S)-1 (0.46 g, 2.19 mmol) in two steps, in a
manner similar to that for 3b: yield 0.22 g (50%); mp 142-
144 °C; [R]D +8.3° (c ) 0.6, EtOH); 1H NMR (400 MHz, 300 K,
CDCl3) δ 7.33-7.20 (m, 10 H), 4.74 (d, 1H, J ) 6.3 Hz), 4.16
(d, 1H, J ) 6.3 Hz), 1.72 (brs, 3H); 13C NMR (100 MHz, 300
K, CDCl3) δ 141.5, 140.7, 128.2, 128.1, 127.7 (overlap), 127.5,
126.9, 78.3 (d, J ) 3.0 Hz), 61.8 (d, J ) 4.4 Hz); 15N NMR (50
MHz, CD3OD, CH3NO2 as a internal standard) δ -352.08 ppm;
IR (film) 3324, 3271 cm-1 (NH2, OH); HRMS (FAB) calcd for
C
14H1615NO 215.1202 (M+ + H)., found 215.1201; er 98:2.
Deter m in a tion of En a tiom er ic Ra tio (er ) of Am in o
Alcoh ols 3a a n d 3b by Mea n s of HP LC An a lysis w ith a
Ch ir a l CD-P h Colu m n . Column: Shiseido Chiral CD-Ph
column (4.6 × 250 mm). Solvent: MeCN/0.5 M NaClO4 ) 6:4.
UV wavelength: 254 nm. Flow rate: 0.4 mL/min. Pressure:
32 kg/cm2. (2S,3R)-3b; tR ) 13.3 min. (2R,3S)-3a ; tR ) 17.2
min.
Hyd r ogen a tion of Com p ou n d 11. P r ep a r a tion of [1,2-
13C2,15N]-L-Ala n in e 12. A mixture of lactone 11 (0.20 g, 0.56
mmol), PdCl2 (0.025 g), EtOH (5 mL), and THF (5 mL) was
stirred at room temperature under H2 (3 kg/cm2) for 4 days.
After filtration through Celite 545, the filtrate was diluted with
H2O (30 mL). The solvent was washed with Et2O (15 mL × 2)
and evaporated in vacuo to a small volume (2 mL). After the
solution was adjusted to pH 2-3 by adding 5% HCl, the
solution was loaded onto a Dowex 50WX4-100 ion-exchange
resin and eluted with 0.5 N NH4OH. The elutant was lyoph-
ilized to give L-alanine as a colorless solid (0.038 g, 89%): mp
290-295 °C (EtOH-H2O); [R]D +2.5° (c ) 0.41, H2O); 1H NMR
(400 MHz, 300 K, D2O) δ 3.83 (ddq, 1H, J ) 145, 7.2, 5.2 Hz),
1.55-1.51 (m, 3H); 13C NMR (100 MHz, 300 K, D2O) δ 175.8
(d, J ) 54 Hz), 50.5 (dd, J ) 54, 5.5 Hz); 15N NMR (50 MHz,
300 K, D2O, CH3NO2 as a external standard) δ -337.24 (d, J
) 5.5 Hz) ppm; FAB-MS 93 (M+ + H); HRMS (FAB) calcd for
12C13C2H815NO2 93.059248, found 93.059217.
P r ep a r a t ion of [2,3-13C2,15N]-(5S,6R)-4-CBz-5,6-d i-
p h en yl-2,3,5,6-tetr a h yd r o-4H-1,4-oxa zin -2-on e (10). To a
mixture of 3b (0.85 g, 3.99 mmol) and 1,2-13C2-ethyl bromo-
acetate (1.0 g, 5.99 mmol) and dry THF solution (20 mL) was
added Et3N (1.1 mL, 8.0 mmol) at 0 °C under an Ar atmo-
sphere. The resulting mixture was stirred at room temperature
for 18 h. The mixture was filtered to remove Et3N‚HBr. The
filtrate was evaporated under reduced pressure to remove
excess Et3N, THF, and 1,2-13C2-ethyl bromoacetate to give
crude 8 as a solid (1.29 g), which was employed to the next
reaction without further purification.
To a mixture of crude 8, CH2Cl2 (20 mL), and saturated
NaHCO3 (20 mL) was added benzyl chloroformate (0.82 g, 4.8
mmol) at 0 °C under an Ar atmosphere. After the resulting
mixture was stirred vigorously at room temperature for 15 h,
the aqueous layer was separated and extracted with CH2Cl2
(20 mL × 3), and the combined organic phases were washed
with H2O (30 mL × 3), dried over anhydrous MgSO4, filtered,
and evaporated under reduced pressure to give crude 9 as a
colorless oil. To a stirred solution in benzene (200 mL) of the
crude N-CBz ethyl ester 9 obtained above in a 500 mL one-
neck round-bottom flask equipped with a Soxhlet extractor
packed with 60 g of CaCl2 was added p-TsOH‚H2O (0.07 g,
0.37 mmol). The mixture was brought to reflux for 8 h. The
solvent was evaporated under reduced pressure to give a solid.
After addition of CH2Cl2 (50 mL) to the residue, the resulting
solution was washed with 5% NaHCO3 (20 mL × 1) and H2O
(20 mL × 2). The solvent was dried over anhydrous MgSO4,
filtered, and evaporated under reduced pressure to give a
residue, which was recrystallized from EtOH to give [2,3-
13C2,15N]-(5S,6R)-4-CBz-5,6-diphenyl-2,3,5,6-tetrahydro-4H-
Ack n ow led gm en t. This work was supported by a
Grant for Private Universities Provided by the Ministry
of Education, Culture, Sports, Science and Technology
and the Promotion and Mutual Aid Corporation for
Private Schools of J apan (to Y.A.) and the National
Science Foundation (Grant No. CHE 9731947 to R.M.W.).
We thank Meito Sangyo Co., Ltd., for their generous gift
of Lipase TL. We are grateful to Dr. Hiroshi Hasegawa
(School of Pharmacy, Tokyo University of Pharm & Life
Science) for useful discussions. We are also grateful to
Prof. J uan F. Sanz-Cervera of the University of Valen-
cia, Spain, for assistance in preparing the manuscript.
J O015725F