Synthesis of 13C/D doubly labeled L-leucines: Probes for conformational analysis of the leucine side-chain

Full text of DOI:10.1021/jo010263r

J . Org. Chem. 2001, 66, 5919-5922
5919
Syn th esis of 13C/D Dou bly La beled
L-Leu cin es: P r obes for Con for m a tion a l
An a lysis of th e Leu cin e Sid e-ch a in
Makoto Oba,* Masahito Kobayashi,
Fumiyo Oikawa, and Kozaburo Nishiyama*
Department of Material Science and Technology,
Tokai University, 317 Nishino, Numazu,
Shizuoka, 410-0395, J apan
Masatsune Kainosho
Department of Chemistry, Faculty of Science,
Tokyo Metropolitan University, 1-1 Minami-Ohsawa,
Hachioji, Tokyo 192-0397, J apan
makoto@wing.ncc.u-tokai.ac.jp
Received March 9, 2001
F igu r e 1. Newman projections for three staggered rotamers
about the CR-Câ (I, II, III) and Câ-Cγ (IV, V, VI) bonds of
leucine.
with 13CH3 and another methyl group and one diastereo-
topic methylene proton at the â-position are labeled with
deuteriums. The NMR spectra of these samples are
expected to provide two pairs of vicinal spin coupling
constants J (H2-H3S) and J (H2-H3R) about the CR-Câ
bond and J (C5R-H3S) and J (C5S-H3R) about the Câ-Cγ
bond straightforwardly. From these values, we can
unambiguously determine the dominant conformation of
the leucine side-chain, the ø1 and ø2 angles in peptides,
as depicted in Figure 1.
In tr od u ction
Recently, the combination of stable isotope labeling and
advanced multidimensional NMR spectroscopy has be-
come a well-established method for determining the
detailed three-dimensional structures of proteins in solu-
tion.1 In particular, the vicinal spin couplings are valu-
able carriers of structural information on the backbone
and side-chain conformation.2 To obtain precise dihedral
angle information on the protein side-chain, the stereo-
selective isotope labeling of only one of the diastereotopic
protons and methyl groups is essential. In light of the
above background, we here focus on the amino acid
L-leucine because the amino acid has been recognized as
an important residue which participates in the inter- and
intramolecular hydrophobic interactions based on its
lipophilic side-chain in polypeptides.
Resu lts a n d Discu ssion
We previously reported the stereoselective synthesis
of L-leucine, in which both the diastereotopic methyl and
methylene protons were chirally labeled with deuterium,
from trans-4-hydroxy-L-proline.6 However, the introduc-
tion of the methyl group using the Gilman reagent, the
key step in the synthesis, used the methyl sources
excessively. We, therefore, planned to devise a novel
protocol to access the labeled leucine in order to incor-
porate a carbon-13 label efficiently into one of the
diastereotopic methyl groups.
Recently, we envisioned the use of pyroglutamic acid
as a chiral template for the asymmetric synthesis of
deuterium-labeled amino acids. Our previous paper
demonstrated the stereoselective synthesis of [3,4,5-D3]-
proline and [4,5,5,5-D4]isoleucine using the template.7
Scheme 1 shows the synthetic course of (2S,3S,4S)-[5-
13C;3,4,5′,5′,5′-D5]leucine (7) starting from L-pyroglutamic
acid.
There are some methods for the synthesis of L-leucine
stereoselectively labeled in either the diastereotopic
methyl group with carbon-13 or deuterium;3 however,
reports on the synthesis of L-leucine labeled with deute-
rium in only one of the diastereotopic methylene protons
are limited to those by us.4,5
The target molecules in this study are novel 13C/D
doubly labeled L-leucines (2S,3S,4S)-7 and (2S,3R,4R)-
7, in which one diastereotopic methyl group is substituted
(1) Kainosho, M. Nature Struct. Biol. 1997, 858-861. Roberts, G.
C. K. NMR of Macromolecules. A Practical Approach; Oxford University
Press: Oxford, 1993. Wuthrich, K. NMR of Proteins and Nucleic Acids;
J ohn Wiley & Sons: New York, 1986.
(2) Bystrov, V. F. Prog. Nucl. Magn. Reson. Spectrosc. 1976, 10, 41-
81.
The synthesis of leucine 7 was begun by introduction
of the [13C]methyl group into γ-lactam 18 readily acces-
sible from L-pyroglutamic acid. Treatment of the lactam
1 with two equivalents of sodium hexamethyldisilazane
(NaHMDS) and phenyl selenenyl chloride followed by
iodo[13C]methane afforded the [13C]methylated phenyl
(3) Cardillo, R.; Fuganti, C.; Ghiringhelli, D.; Grasselli, P.; Gatti,
G. J . Chem. Soc., Chem. Commun. 1977, 474-476. Sylvester, S. R.;
Lan, S. Y.; Stevens, C. M. Biochemistry 1981, 20, 5609-5611. Aberhart,
D. J .; Weiller, B. H. J . Labeled Compds. Radiopharm. 1983, 20, 663-
668. Kelly, N. M.; Reid, R. G.; Willis, C. L.; Winton, P. L. Tetrahedron
Lett. 1995, 8315-8318. Hill, R. K.; Abacherli, C.; Hagishita, S. Can.
J . Chem. 1994, 72, 110-113. August, R. A.; Khan, J . A.; Moody, C.
M.; Young, D. W. Tetrahedron Lett. 1992, 4617-4620. August, R. A.;
Khan, J . A.; Moody, C. M.; Young, D. W. J . Chem. Soc., Perkin Trans.
1 1996, 507-514.
(4) Oba, M.; Ueno, R.; Fukuoka, M.; Kainosho, M.; Nishiyama, K.
J . Chem. Soc., Perkin Trans. 1 1995, 1603-1609.
(5) Oba, M.; Nakajima, S.; Nishiyama, K. J . Chem. Soc., Chem.
Commun. 1996, 1875-1876. Oba, M.; Terauchi, T.; Owari, Y.; Imai,
Y.; Motoyama, I.; Nishiyama, K. J . Chem. Soc., Perkin Trans. 1 1998,
1275-1281.
(6) Oba, M.; Terauchi, T.; Miyakawa, A.; Kamo, H.; Nishiyama, K.
Tetrahedron Lett. 1998, 39, 1595-1598.
(7) Oba, M.; Miyakawa, A.; Nishiyama, K.; Terauchi, T.; Kainosho,
M. J . Org. Chem. 1999, 64, 9275-9278. Oba, M.; Miyakawa, A.;
Shionoya, M.; Nishiyama, K. J . Labeled Compds. Radiopharm. 2001,
44, 141-147.
(8) Ohfune, Y.; Tomita, M. J . Am. Chem. Soc. 1982, 104, 3511-3513.
10.1021/jo010263r CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/31/2001

5920 J . Org. Chem., Vol. 66, No. 17, 2001
Notes
Sch em e 1a
a
Reagents and Conditions: (a) NaN(TMS)2, PhSeCl, 13CH3I, quant.; (b) H2O2, 78%; (c) D2 (5 kgf/cm2), PtO2, MeOD, 98%; (d) TsOH,
MeOH, 88%; (e) RuO2, NaIO4, 89%; (f) Me2NCH(OCH2CMe3)2, tert-BuOH, 55%; (g) 1 M LiOH, quant.; (h) ClCO2Bui, Et3N, then, NaBD4,
64%; (i) PPh3 (polystyrene-supported), I2, 88%; (j) Bu3SnD, AIBN; (k) 1 M HCl, 110 °C, then Dowex 50W-X8, 65%.
Sch em e 2a
a
Reagents and Conditions: (a) Ac2O, 2 M NaOH, 70 °C; (b) Aspergillus Acylase, CoCl2, pH 8, 37 °C, 29% (2 steps).
selenide as a mixture of diastereisomers (syn:anti )
64:36). The selenide was treated with 30% hydrogen
peroxide to give [13C]methylated unsaturated lactam 2
in 78% yield. Only a trace amount of the isomeric
exomethylene compound, which could be removed by
column chromatography, was detected in the reaction
[5-13C;3,4,5′,5′,5′-D5]leucine (7) in 65% yield. The enantio-
meric purity (97% ee) at the R-position was determined
by HPLC analysis using a chiral stationary phase
column.
To obtain the isotopomeric (2S,3R,4R)-[5-13C;3,4,5′,5′,5′-
D5]leucine (7), we next examined epimerization of
(2R,3R,4R)-[5-13C;3,4,5′,5′,5′-D5]leucine (7) at the R-posi-
tion followed by enzymatic resolution as shown in Scheme
2. Thus, treatment of the (2R,3R,4R)-7, prepared from
D-pyroglutamic acid according to the Scheme 1, with an
excess of acetic anhydride at 70 °C gave a mixture of
(2S,3R,4R)- and (2R,3R,4R)-N-acetyl[5-13C;3,4,5′,5′,5′-D5]-
leucine (8). The mixture was then subjected to stereo-
specific hydrolysis using Aspergillus acylase to afford the
(2S,3R,4R)-7. The enantiomeric purity (100% ee) at the
R-position was checked by a chiral HPLC.
1
mixture by H NMR spectroscopy, indicating the regio-
selective syn elimination of the selenenic acid occurred.
Catalytic deuteration of the olefin 2 was carried out
using deuterium gas at medium pressure (5 kgf/cm2) in
MeOD. The use of PtO2 as a catalyst proved advanta-
geous, resulting in clean formation of 3,4-dideuterated
γ-lactam 3 as a single diastereomer. In this case, pal-
ladium on carbon was not suitable, causing considerable
H-D scrambling and olefin isomerization. After removal
of the silyl protecting group of the lactam 3 by treatment
with p-toluenesulfonic acid in MeOH, RuO4-oxidation of
the resulting primary alcohol and subsequent esterifica-
tion with dimethylformamide di-tert-butyl acetal afforded
tert-butyl 4-[13C]methyl[3,4-D2]pyroglutamate 4.
Conversion of the pyroglutamate 4 into the desired
leucine 7 was carried out using the procedure previously
reported by us.6 Thus, the pyroglutamate 4 was treated
with 1 M LiOH and the resulting carboxylic acid was
reduced to the deuterated alcohol 5 using NaBD4 via a
mixed anhydride with isobutyl chloroformate in 64%
yield. Iodination of the alcohol 5 was carried out using
iodine and polystyrene-supported triphenylphosphine to
give 5-iodoleucine 6 in 88% yield.9 Finally, radical-based
reduction of the iodide 6 with Bu3SnD-AIBN followed by
the standard deprotection procedure furnished (2S,3S,4S)-
1
Figure 2 shows the 400 MHz H NMR spectra of the
labeled leucines (2S,3S,4S)-7 (upper) and (2S,3R,4R)-7
(middle) along with the unlabeled leucine (bottom). It is
evident that the regio- and stereoselective incorporation
of the stable isotopes into the leucine framework has been
accomplished. From these spectra, we can easily obtain
the vicinal spin coupling constants between the R- and
â-positions and between the â- and γ-positions. By taking
J (H2-H3S) ) 5.6 Hz and J (H2-H3R) ) 8.4 Hz, the
approximate rotamer populations PI (0.53), PII (0.20), and
PIII (0.27) about the CR-Câ bond can be given by Pachler’s
equations: PI ) [J (H2-H3R) - J g]/(J t - J g), PII ) 1 - (PI
+ PIII), PIII ) [J (H2-H3S) - J g]/(J t - J g), in which J t
)
13.6 Hz and J g ) 2.6 Hz.10 These results are in good
agreement with the reported data.4 Using the hetero-
nuclear vicinal spin coupling constants J (C5R-H3S) ) 3.9
(9) Quagliato, D. A.; Andrae, P. M.; Matelan, E. M. J . Org. Chem.
2000, 65, 5037-5042.
(10) Pachler, K. G. R. Spectrochim. Acta 1964, 20, 581-587.

Notes
J . Org. Chem., Vol. 66, No. 17, 2001 5921
1
F igu r e 2. 400 MHz H NMR Spectra of labeled leucines (2S,3S,4S)-7 (upper) and (2S,3R,4R)-7 (middle) and unlabeled leucine
(bottom) in 5% NaOD/D2O.
Hz and J (C5S-H3R) ) 5.2 Hz, the fractional populations
at 0 °C under an argon atmosphere for 10 min, cooled to -78
°C, and treated with a solution of the 2-pyrrolidone 1 (4.20 g,
PIV (0.46), PV (0.15), and PVI (0.31) about the Câ-Cγ bond
12.8 mmol) in THF (20 mL). After 0.5 h, a solution of PhSeCl
can be also obtained by the following equations: PIV
)
)
(2.69 g, 14.1 mmol) in THF (15 mL) was added and the mixture
was stirred for 2 h. Then, 13CH3I (2.01 g, 14.1 mmol) was added.
The reaction was quenched with saturated aqueous NH4Cl and
the mixture was extracted with ether. The organic layer was
washed with saturated aqueous NH4Cl, dried over MgSO4, and
evaporated to give 3-[13C]methyl-3-phenylseleno-2-pyrrolidone
in quantitative yield.
[J (C5S-H3R) - J g]/(J t - J g), PV ) 1 - (PIV + PVI), PVI
[J (C5R-H3S) - J g]/(J t - J g), J t ) 9.8 Hz, J g ) 1.3 Hz.11
To the best of our knowledge, this is the first example of
the estimation of the rotamer population about the Câ-
Cγ bond of leucine with the help of selective isotope
labeling.
To a solution of the obtained crude phenylselenide (6.21 g,
12.88 mmol) in THF (51 mL) was added dropwise H2O2 (14.5 g,
12.8 mmol) at 0 °C and the reaction mixture was stirred at room
temperature for 1 h. The mixture was extracted with ether and
the organic layer was washed with saturated aqueous NaHCO3
and dried over MgSO4. After removal of the solvent, the residue
was chromatographed on silica gel. Elution with a mixture of
hexane and ethyl acetate (90:10) afforded the title compound 2
In conclusion, we have completed the synthesis of
L-leucine in which both the diastereotopic methyl and
methylene protons were chirally labeled with carbon-13
and deuterium. This approach has a further advantage
in that it enables the preparation of uniformly carbon-
13 and nitrogen-15 labeled samples starting from the
commercially available [13C5;15N]glutamic acid, which
should prove useful for structural determination of larger
proteins.
1
(3.29 g, 78%) as an oil. H NMR (CDCl3) δ 0.02 (s, 3 H), 0.04 (s,
3 H), 0.86 (s, 9 H), 1.55 (s, 9 H), 1.88 (ddd, J ) 128, 2 and 2 Hz,
3 H), 3.65 (dd, J ) 10 and 4 Hz, 1 H), 4.13 (dd, J ) 10 and 7 Hz,
1 H), 4.45 (m, 1 H), 6.88 (m, 1 H). 13C NMR (CDCl3) δ -5.52,
-5.60, 10.81 (enhanced), 16.08, 25.65, 28.07, 61.21, 62.49, 82.64,
134.98 (d, J ) 48 Hz), 142.39, 149.71, 170.11. HRMS m/z
343.2122 [(M + H)+, calcd for C1613CH32NO4Si 343.2134].
Exp er im en ta l Section
Gen er a l. 1H and 13C NMR spectra were measured at 400 and
100 MHz, respectively. All chemical shifts are reported as δ
values (ppm) relative to residual chloroform (δH 7.26), sodium
3-(trimethylsilyl)[2,2,3,3-D4]propionate (δH 0.00), the central
peak of deuteriochloroform (δC 77.0), or dioxane (δC 66.5). High-
resolution mass spectra (EI) were obtained at an ionization
potential of 70 eV unless otherwise noted. Enantiomeric purity
was determined on an HPLC system (monitored at 254 nm)
equipped with a chiral column (MCIGEL CRS10W) using 2 mM
CuSO4 solution as an eluent. Solvents and reagents were of
commercial grade and were purified if necessary.
(3S,4S,5S)-1-ter t-Bu toxyca r bon yl-5-ter t-bu tyld im eth yl-
siloxym eth yl-3-[13C]m eth yl-2-[3,4-D2]pyr r olidon e (3). A mix-
ture of the olefin 2 (3.29 g, 9.92 mmol) and PtO2 (160 mg) in
MeOD (100 mL) was stirred at room temperature for 1 h under
medium pressure (5 kgf/cm2) of deuterium gas. After removal
of the catalyst using a Celite pad, evaporation of the solvent gave
1
the title compound 3 (3.27 g, 98%) as an oil. H NMR (CDCl3) δ
0.03 (s, 3 H), 0.04 (s, 3 H), 0.87 (s, 9 H), 1.24 (d, J ) 127 Hz, 3
H), 1.53 (s, 9 H), 1.66 (dd, J ) 6 and 6 Hz, 1 H), 3.70 (dd, J ) 10
and 2 Hz, 1 H), 3.91 (dd, J ) 10 and 5 Hz, 1 H), 4.05 (m, 1 H).
13C NMR (CDCl3) δ -5.50, 16.51 (enhanced), 18.22, 25.80, 27.19
(t, J ) 20 Hz), 28.00, 36.80 (dt, J ) 37 and 20 Hz), 56.81, 63.37,
82.56, 150.37, 177.24. HRMS (30 eV) m/z 347.2410 [(M + H)+,
calcd for C1613CH32D2NO4Si 347.2416].
(5S)-1-ter t-Bu toxyca r bon yl-3-[13C]m eth yl-5H-2-p yr r oli-
n on e (2). A solution of 1 M NaHMDS in THF (26.9 mL, 26.9
mmol) was treated with N,N′-dimethylpropyleneurea (3.3 mL)
ter t-Bu t yl
m eth yl-[3,4-D2]p yr oglu ta m a te (4). To a solution of the com-
(2S,3S,4S)-N-ter t-Bu t oxyca r b on yl-4-[13C]-
(11) Cowburn, D.; Live, D. H.; Fischman, J .; Agosta, W. C. J . Am.
Chem. Soc. 1983, 105, 7435-7442.

5922 J . Org. Chem., Vol. 66, No. 17, 2001
Notes
pound 3 (2.79 g, 8.29 mmol) in MeOH (80 mL) was added
p-toluenesulfonic acid (140 mg, 0.829 mmol) and the resulting
solution was stirred at room-temperature overnight. After
removal of the solvent, the residue was extracted with ethyl
acetate. The organic layer was washed with saturated aqueous
NaHCO3 and dried over MgSO4. Evaporation of the solvent
afforded the desilylated product (1.67 g, 88%).
16.35 (enhanced), 27.92, 28.27, 31.81 (m), 36.25 (m), 52.19, 66.53
(m), 79.66, 81.73, 155.68, 172.51. HRMS (30 eV) m/z 309.2360
[(M + H)+, calcd for C1413CH26D4NO5 309.2409].
ter t-Bu t yl (2S,3S,4S)-N-ter t-Bu t oxyca r b on yl-5-iod o[5-
13C;3,4,5′,5′-D4]leu cin e (6). To a mixture of polystyrene-sup-
ported Ph3P (1.20 g, 3.61 mmol) in CH2Cl2 (15 mL) was added
I2 (920 mg, 3.61 mmol) under an argon atmosphere and the
mixture was stirred at room temperature for 10 min. To the
reaction mixture was added imidazole (250 mg, 3.61 mmol)
followed by a solution of the alcohol 5 (510 mg, 1.64 mmol) in
CH2Cl2 (45 mL) and the resulting suspension was refluxed for
2 h. After filtration of the insoluble materials, the filtrate was
washed with dilute aqueous NaS2O3 solution and brine, and
dried over MgSO4. Evaporation of the solvent afforded the title
compound 6 (610 mg, 88%) as an oil. 1H NMR (CDCl3) δ 1.06 (d,
J ) 126 Hz, 3 H), 1.44 (s, 9 H), 1.47 (s, 9 H), 1.63 (dd, J ) 9 and
6 Hz, 1 H), 4.18 (dd, J ) 9 and 9 Hz, 1 H), 4.91 (d, J ) 9 Hz, 1
H). HRMS m/z 419.1453 [(M + H)+, calcd for C1413CH25D4NO4I
419.1426].
(2S,3S,4S)-[5-13C;3,4,5′,5′,5′-D5]Leu cin e (7). A solution of
the iodide 6 (264 mg, 0.630 mmol), Bu3SnD (280 mg, 0.950
mmol), and AIBN (10 mg) in dry benzene (10 mL) was heated
at 80 °C under an argon atmosphere for 1 h. After removal of
the solvent, the residue was treated with 1 M HCl (20 mL) at
110 °C for 3 h. 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 title compound
7 (45.0 mg, 65%) as a colorless solid. 1H NMR (5% NaOD in D2O)
δ 0.87 (d, J ) 125 Hz, 3 H), 1.33 (dd, J ) 8.4 and 5.2 Hz, 1 H),
3.24 (d, J ) 8.4 Hz, 1 H). HRMS (30 eV) m/z 138.1338 [(M +
H)+, calcd for C513CH9D5NO2 138.1372].
To a suspension of sodium metaperiodate (15.8 g, 72.7 mmol)
and RuCl3‚nH2O (440 mg) in H2O (45 mL) was added the
obtained alcohol in acetone (38 mL). The resulting two-phase
mixture was vigorously stirred at room temperature for 1 h. The
layers were separated. To the organic phase was added 2-pro-
panol (30 mL) and the mixture was stirred for 1 h. After removal
of the precipitated RuO2 using a Celite pad, the filtrate was
concentrated, extracted with chloroform, and dried over MgSO4.
Evaporation of the solvent gave 4-[13C]methyl-[3,4-D2]pyro-
glutamic acid (1.58 g, 89%). 1H NMR (CDCl3) δ 1.27 (d, J ) 128
Hz, 3 H), 1.50 (s, 9 H), 1.69 (dd, J ) 6 and 6 Hz, 1 H), 4.53 (d,
J
) 6 Hz, 1 H). HRMS m/z 247.1366 [(M + H)+, calcd for
C1013CH16D2NO5 247.1344].
To a refluxing solution of the obtained pyroglutamic acid in
benzene (25 mL) was added a mixture of N,N-dimethylforma-
mide dineopentyl acetal (2.68 g, 11.6 mmol) and tert-butanol
(1.43 g, 19.3 mmol), and the reaction mixture was refluxed for
0.5 h. Then the cooled reaction mixture was diluted with ethyl
acetate, washed with saturated aqueous NaHCO3 and brine, and
dried over MgSO4. After removal of the solvent, the residue was
chromatographed on silica gel. Elution with a mixture of hexane
and ethyl acetate (80:20) afforded the title compound 4 (1.06 g,
55%) as an oil. 1H NMR (CDCl3) δ 1.25 (d, J ) 129 Hz, 3 H),
1.48 (s, 9 H), 1.51 (s, 9 H), 1.56 (dd, J ) 6 and 5 Hz, 1 H), 4.38
(d, J ) 6 Hz, 1 H). 13C NMR (CDCl3) δ 16.31 (enhanced), 27.79
(2 C), 29.10 (t, J ) 21 Hz), 36.91 (dt, J ) 37 and 20 Hz), 57.91,
81.97, 83.12, 149.48, 170.60, 176.03. HRMS m/z 303.1995 [(M
+ H)+, calcd for C1413CH24D2NO5 303.1970].
ter t-Bu tyl (2S,3S,4S)-N-ter t-Bu toxyca r bon yl-5-h yd r oxy-
[5-13C;3,4,5′,5′-D4]leu cin e (5). To a solution of the pyro-
glutamate 4 (772 mg, 2.56 mmol) in THF (15 mL) was added
dropwise 1 M LiOH (3.07 mL) at 0 °C over a period of 15 min.
After being stirred for an additional 30 min, the mixture was
acidified to pH 4 with 10% aqueous citric acid and extracted with
ethyl acetate. The organic layer was dried over MgSO4 and the
solvent was evaporated to afford the 4-[13C]methyl[3,4-D2]-
glutamate (819 mg) in quantitative yield.
(2S,3R,4R)-[5-13C;3,4,5′,5′,5′-D5]Leu cin e (7). To a solution
of the (2R,3R,4R)-7 (183 mg, 1.33 mmol) in 1 M NaOH (1 mL)
was added dropwise acetic anhydride (228 mg, 2.24 mmol) over
3 h at 70 °C. The progress of the epimerization was monitored
by 1H NMR. After an additional heating for 30 min, the reaction
mixture was evaporated and the residue containing (2S,3R,4R)-
and (2R,3R,4R)-8 was directly subjected to enzymatic resolution.
The obtained N-acetylleucine and CoCl2‚6H2O (2.38 mg) were
dissolved in 2.5 M NaOH (10 mL) and the pH was adjusted to
8.0-8.5 using 1 M HCl. The solution was added a crude powder
of Aspergillus acylase (13 mg) and was incubated at 37 °C for
72 h. The reaction mixture was concentrated to dryness and
submitted to ion exchange column chromatography on Dowex
50W-X8 and the resin was washed with water. (2R,3R,4R)-8
was recovered from the aqueous washings. Elution with 1 M
NH4OH and evaporation of appropriate fractions (monitored by
ninhydrin spray) gave (2S,3R,4R)-7 (26.4 mg, 29%) as a colorless
The obtained acid and Et3N (340 mg, 3.33 mmol) were
dissolved in THF (25 mL) and the solution was cooled to -40
°C under an argon atmosphere. Isobutyl chloroformate (420 mg,
3.07 mmol) was added dropwise to the solution and the reaction
mixture was stirred for 1 h. The precipitated Et3N‚HCl was
filtered off and to the filtrate was added a mixture of NaBD4
(330 mg, 7.68 mmol) in THF (20 mL) and D2O (15 mL) at 0 °C
under an argon atmosphere. After being stirred for 1.5 h at room
temperature, the resulting suspension was extracted with ethyl
acetate, and the organic layer was washed successively with 10%
aqueous citric acid and brine, and dried over MgSO4. The solvent
was evaporated and the residue was chromatographed on silica
gel. Elution with a mixture of hexane and ethyl acetate (75:25)
1
solid. H NMR (5% NaOD in D2O) δ 0.89 (d, J ) 124 Hz, 3 H),
1.42 (dd, J ) 5.6 and 3.9 Hz, 1 H), 3.24 (d, J ) 5.6 Hz, 1 H).
HRMS (30 eV) m/z 138.1372 [(M + H)+, calcd for C513CH9D5-
NO2 138.1372].
Ack n ow led gm en t. This work has been supported
by CREST (Core Research for Evolutional Science and
Technology) of the J apan Science and Technology
Corporation (J ST).
1
afforded the title compound 5 (506 mg, 64%) as an oil. H NMR
(CDCl3) δ 0.96 (d, J ) 125 Hz, 3 H), 1.44 (s, 9 H), 1.46 (s, 9 H),
1.66 (dd, J ) 8 and 5 Hz, 1 H), 1.92 (br s, 1 H), 4.21 (dd, J ) 8
and 8 Hz, 1 H), 5.06 (d, J ) 8 Hz, 1 H). 13C NMR (CDCl3) δ
J O010263R