Deoxysugars via microbial reduction of 5-acyl-isoxazolines: Application to the synthesis of 3-deoxy-D-fructose and derivatives

Article abstract of DOI:10.1021/jo001480f

5-Acylisoxazolines 3a-d were obtained by 1,3-dipolar cycloaddition from acetoxymethyl vinyl ketone and nitro precursors. Compounds 3a-d were biotransformed by Aspergillus niger into a 1:1 mixture of stereomers of 5-dihydroxyethyl isoxazolines (+)-4a-d (anti) and (-)-5a-d (syn). Both stereomers were obtained in good yields and with high optical purities. Carbonyl reduction by Aspergillus niger produces alcohols of R-configuration thus giving an access to D-sugar analogues: Compound (+)-4d was converted to 3-deoxy-D-erythro-hexulose and several protected derivatives. Total synthesis of 3-deoxy-D-fructose-6-phosphate was also achieved in two steps and 64% overall yield from (+)-4d.

Full text of DOI:10.1021/jo001480f

2296
J . Org. Chem. 2001, 66, 2296-2301
Deoxysu ga r s via Micr obia l Red u ction of 5-Acyl-isoxa zolin es:
Ap p lica tion to th e Syn th esis of 3-Deoxy-D-fr u ctose a n d Der iva tives
Thierry Gefflaut,* Christel Martin, Suzy Delor, Pascale Besse, Henri Veschambre, and
J ean Bolte
Universite´ Blaise Pascal, Laboratoire SEESIB, UMR 6504, 63177 Aubie`re Cedex, France
gefflaut@chimtp.univ-bpclermont.fr
Received October 16, 2000
5-Acylisoxazolines 3a -d were obtained by 1,3-dipolar cycloaddition from acetoxymethyl vinyl ketone
and nitro precursors. Compounds 3a -d were biotransformed by Aspergillus niger into a 1:1 mixture
of stereomers of 5-dihydroxyethyl isoxazolines (+)-4a -d (anti) and (-)-5a -d (syn). Both stereomers
were obtained in good yields and with high optical purities. Carbonyl reduction by Aspergillus
niger produces alcohols of R-configuration thus giving an access to D-sugar analogues: Compound
(+)-4d was converted to 3-deoxy-D-erythro-hexulose and several protected derivatives. Total
synthesis of 3-deoxy-D-fructose-6-phosphate was also achieved in two steps and 64% overall yield
from (+)-4d .
Sch em e 1
In tod u ction
2-Isoxazolines (4,5-dihydroisoxazoles) are easily ob-
tained by 1,3-dipolar cycloaddition to alkenes of nitrile-
oxides or silyl-nitronates generated in situ from primary
nitro-compounds or chloro-aldoximes. These heterocycles
have proven versatile intermediates for the synthesis of
different classes of functionalized molecules.1 Of special
interest is complete reduction of isoxazolines to γ-amino
alcohols as well as conversion to aldols through reductive
N-O bond cleavage followed by hydrolysis of the inter-
mediate imine. These transformations have been used
in the construction of carbohydrates, especially amino-
sugars2 and deoxysugars.3 Thus, the use of (S)-2-vinyl-
1,3-dioxolane as dipolarophile allowed the synthesis of
2-deoxy-D-ribose,3a D-lividosamine,2c or D-allosamine2f via
stereoselective hydroxylation at position 4 of the hetero-
cycle. In every case, diastereoselection was observed
during the cycloaddition, leading to an approximately 4:1
mixture of anti and syn isomers (Scheme 1).
valuable substrates to achieve stereoselective synthesis.
Alternatively, bioconversion processes have been reported
for the preparation of chiral isoxazolines including en-
zyme kinetic resolutions of ester-substituted isoxazolines5
or microbial reductions of isoxazolines bearing a carbonyl
group.6 Indeed, bakers’ yeast reduction of 5-acetyl-
isoxazolines has been shown to lead to both enantiomeri-
cally pure diastereomers in excellent yields (Scheme 2).6a
This nondiastereoselective approach allows for poten-
tial access to all four stereomers through an appropriate
choice of the biocatalyst and reaction conditions, giving
a unique configuration for the newly formed alcohol.
This anti-directing effect appears as a general feature
of nitrile-oxide cycloadditions4 and makes R-chiral olefins
(1) For selected reviews, see (a) Grundmann, C.; Gru¨nanger, P. The
Nitrile Oxides; Springer-Verlag: New York, 1971. (b) Caramella, P.;
Gru¨nanger, P. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.,
Wiley: New York, 1984; Vol. 1, p 291. (c) Kozikowski, A. P. Acc. Chem.
Res. 1984, 17, 410. (d) J a¨ger, V.; Mu¨ller, I.; Schohe, R.; Frey, M.; Ehrler,
R.; Ha¨fele, B.; Schro¨ter, D. Lect. Heterocycl. Chem. 1985, 8, 79. (e)
Curran, D. P. Adv. Cycloadd. 1988, 1, 129. (f) Torsell, K. B. G. Nitrile
Oxides, Nitrones and Nitronates in Organic Synthesis; VCH Publish-
ers: New York, 1988. (g) Kanemasa, S.; Tsuge, O. Heterocycles 1990,
30, 719. (h) Padwa, A. In Comprehensive Organic Synthesis; Trost, B.
B.; Fleming, I., Eds., Pergamon Press: Oxford, 1991; vol. 4, p 1069.
(2) (a) J a¨ger, V.; Schwab, W.; Buss, V. Angew. Chem., Int. Ed. Engl.
1981, 20, 601. (b) Mu¨ller, I.; J a¨ger, V. Tetrahedron Lett. 1982, 23, 4777.
(c) J a¨ger, V.; Schohe, R. Tetrahedron 1984, 40, 2199. (d) J a¨ger, V.;
Mu¨ller, I. Tetrahedron 1985, 41, 3519. (e) J a¨ger, V.; Mu¨ller, I.; Paulus,
E. F. Tetrahedron Lett. 1985, 26, 2997. (f) J a¨ger, V.; Schro¨ter, D.
Synthesis 1990, 556. (g) Schaller, C.; Vogel, P.; J a¨ger, V. Carboh. Res.
1998, 314, 25. (h) Zimmermann, P. P.; Blanarikova, I.; J a¨ger, V. Angew.
Chem., Int. Ed. 2000, 39, 910. (i) Wade, P. A.; D’Ambrosio, S. G.; Price,
D. J . J . Org. Chem. 1995, 60, 6302.
(4) (a) Houk, K. N.; Moses, S. R.; Wu, Y. D.; Rondan, N. G.; J a¨ger,
V.; Schohe, R.; Fronczek, F. R. J . Am. Chem. Soc. 1984, 106, 3880. (b)
Wade, P. A.; Singh, S. M.; Pillay, M. K. Tetrahedron 1984, 40, 601. (c)
Houk, K. N.; Duh, H. Y.; Wu, Y. D.; Moses, S. R. J . Am. Chem. Soc.
1986, 108, 2754. (d) Curran, D. P.; Kim, B. H. Synthesis 1986, 312. (e)
Annunziata, R.; Cinquini, M.; Cozzi, F.; Raimondi, L. Tetrahedron
1988, 44, 4645. (f) De Amici, M.; De Micheli, C.; Ortisi, A.; Gatti, G.;
Gandolfi, R.; Toma, L. J . Org. Chem. 1989, 54, 793. (g) Annunziata,
R.; Benaglia, M.; Cinquini, M.; Cozzi, F.; Raimondi, L. J . Org. Chem.
1995, 60, 4697.
(5) (a) De Amici, M.; Magri, P.; De Micheli, C.; Cateni, F.; Bovara,
R.; Carrea, G.; Riva, S.; Casalone, G. J . Org. Chem. 1992, 57, 2825.
(b) Yang, S.; Hayden, W., Faber, K.; Griengl, H. Synthesis 1992, 365.
(c) Secundo, F.; Riva, S.; Carrea, G. Tetrahedron: Asymmetry 1992, 3,
267. (d) De Amici, M.; De Micheli, C.; Cateni, F.; Carrea, G.; Ottolina,
G. Tetrahedron: Asymmetry 1993, 4, 1063. (e) Carrea, G.; De Amici,
M.; De Micheli, C.; Liverani, P.; Carnielli, M.; Riva, S. Tetrahedron:
Asymmetry 1993, 4, 1063. (f) Yang, S.; Hayden, W.; Griengl, H.
Monatsh. Chem. 1994, 125, 469. (g) De Amici, M.; De Micheli, C.;
Carrea, G.; Riva, S. Tetrahedron: Asymmetry 1996, 7, 787. (h) Kappeler,
H.; J ary, W. G.; Hayden, W.; Griengl, H. Tetrahedron: Asymmetry 1997,
8, 245.
(3) (a) Kozikowski, A. P.; Ghosh, A. K. J . Am. Chem. Soc. 1982, 104,
5788. (b) Kozikowski, A. P.; Ghosh, A. K. J . Org. Chem. 1984, 49, 2762.
(c) Torsell, K. B. G.; Hazell, A. C.; Hazell, R. G. Tetrahedron 1985, 41,
5569.
(6) (a) Ticozzi, C.; Zanarotti, A. Tetrahedron Lett. 1988, 29, 6167.
(b) Ticozzi, C.; Zanarotti, A. Liebigs Ann. Chem. 1989, 1257.
10.1021/jo001480f CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/13/2001

Microbial Reduction of 5-Acyl-isoxazolines
J . Org. Chem., Vol. 66, No. 7, 2001 2297
Ta ble 1. Red u ction of r a c-3a -d by Asper gillu s n iger
Sch em e 2
Sch em e 3
a
4 and 5 were always isolated in approximately equal quanti-
b
ties. Determined by HPLC on a chiralcel OJ (4a -d , 5b) or by
comparison to optical rotations of authentic samples (5a , 5c, and
5d ).
observed during the course of the reaction results in the
presence of two different substrates for enzymatic reduc-
tion. Indeed, reduction of the free ketol derived from 3b
(data not shown) gave quite different results: a 3:1
mixture of (-)-4b and (+)-5b was obtained with 63% and
91% ee, respectively. These results clearly indicate that
reduction of isoxazolines 3 by bakers’ yeast is a complex
process likely involving more than one reductase.
We next turned to other microorganisms. Being pri-
marily interested in an access to the D-series of sugar
analogues, we focused our attention on biocatalysts giving
an alcohol of R configuration. After screening of a few
strains, the best results were obtained with the fungus
Aspergillus niger (ATCC 9142) and are summarized in
Table 1. The reductions of 3a -d (1.2-1.6 g‚L-1) were
performed at 27 °C in water with fresh resting cells (100
g‚L-1). Carbonyl reduction and acetate hydrolysis oc-
curred with equivalent rates and were complete within
24 h leading to a 1:1 mixture of diols 4 and 5.
Absolute configurations were determined by reference
to authentic samples of (+)-4a -d and (-)-5a -d obtained
by cycloaddition from (S)-2-vinyl-1,3-dioxolane (see Scheme
1). Here again, our results are different from those
concerning the reduction of 5-acetyl-isoxazolines by As-
pergillus niger which produced the syn isomer with low
enantioselectivity.6b Reduction of isoxazolines 3 leads to
both diastereomers in good yields and high ee, thus
offering an access to D-sugar analogues.
As shown in Scheme 4, 3-deoxy-D-fructose 6 and
derivatives 7-11 have been obtained from diol (+)-4d
formed with 96% ee after reduction of rac-3d by Aspergil-
lus niger. Catalytic hydrogenation of (+)-4d over Pd on
charcoal (10%) was achieved under hydrolytic conditions
to give 6 in 80% yield. Though an efficient synthesis of
this molecule from D-fructose has already been described
in three steps and 15% overall yield,12 the isoxazoline
route constitutes a competitive alternative and allows for
access to unusual protection patterns as shown by the
synthesis of 8 and 9. Hydrogenation over Raney Ni in
the presence of water and acetic acid thus allowed the
conversion of isoxazolines 4d and 7 to ketols in 88% and
76% yields, respectively, while preserving the benzyl and
acetonide protective groups. Finally, isoxazoline (+)-4d
was used as precursor of 3-deoxy-D-fructose-6-phosphate
(11), a molecule of special interest for the study or
inhibition of enzymatic reactions involving the biologi-
cally important D-fructose-6-phosphate. Phosphorylation
of (+)-4d was accomplished using dibenzylphosphoroio-
didate generated from tribenzyl phosphite and iodine.13
In this paper, we describe our first results concerning
the microbial reduction of 5-acyl-isoxazolines applied to
deoxysugar synthesis. Several chiral molecules were
obtained in good yields and with high optical purities
using the fungus Aspergillus niger. This microorganism
produces alcohols of R configuration, precursors of
D-sugar analogues. This strategy was applied to the
synthesis of 3-deoxy-D-fructose (3-deoxy-D-erythro-hexu-
lose) and several protected derivatives.
Resu lts a n d Discu ssion
Isoxazolines 3a -d were obtained by cycloaddition of
1,3-dipoles on acetoxymethyl vinyl ketone 1 easily pre-
pared from butyne-1,3-diol7 (Scheme 3).
Mukaiyama’s procedure8 (method B) was employed to
generate nitrile-oxides from nitroethane and compound
2d obtained from nitroethanol and benzyltrichloroace-
timidate.9 Isoxazolines 3b and 3d were obtained in 57%
and 75% yields, respectively. However, this procedure
was not suitable with nitromethane10 or nitroethanol
used as dipole precursor. Silyl-nitronates3c,7 were gener-
ated instead (method A) and 2-silyloxy-isoxazolidines
obtained upon cycloaddition were converted into isox-
azolines 3a and 3c by acid treatment. Although 3a was
thus isolated in 61% yield, this direct synthesis of 3c from
nitroethanol still suffers from a low yield of 38% but in
agreement with previously reported results.11
Compounds 3b and 3c (1 g‚L-1) were first reduced by
freeze-dried bakers’ yeast (20 g‚L-1) under nonfermen-
tative conditions. In both cases, reduction was complete
within 48 h and accompanied by acetate hydrolysis
leading to a 1:1 mixture of diols 4 (anti) and 5 (syn). After
chromatographic separation, stereochemical analysis
showed these molecules to be nearly racemic. Our find-
ings greatly differ from the highly specific reduction of
5-acetyl-isoxazolines by bakers’ yeast6a described earlier.
However, the added acetoxy group is likely to alter the
reduction specificity. Furthermore, acetate hydrolysis
(7) Andersen, S. H.; Das, N. B.; J orgensen, G.; Kjeldsen, G.;
Knudsen, J . S.; Sharma, S. C.; Torsell, K. B. G. Acta Chem. Scand. B
1982, 36, 1.
(8) Mukaiyama, T.; Hoshino, T. J . Am. Chem. Soc. 1960, 82, 5339.
(9) (a) Iversen, T.; Bundle, D. R. J . Chem. Soc., Chem. Commun.
1981, 1240. (b) Eckenberg, P.; Groth, U.; Huhn, T.; Richter, N.;
Schmeck, C. Tetrahedron 1993, 49, 1619.
(10) Huisgen, R.; Christl, M. Chem. Ber. 1973, 106, 3291.
(11) Method B has already been used from nitroethanol. Reported
yields are 67% and 38%: Galley, G.; J ones, P. G.; Pa¨tzel, M. Tetrahe-
dron: Asymmetry 1996, 7, 2073.
(12) Dills, W. L. Carbohydr. Res. 1990, 208, 276.

2298 J . Org. Chem., Vol. 66, No. 7, 2001
Gefflaut et al.
Sch em e 4
D-sugar analogues. The strategy described is applicable
to the synthesis of aminosugar derivatives through
appropriate remodeling of isoxazolines 4 and 5. Finally,
microbial reductions producing an alcohol of S configu-
ration would allow for access to the L series of carbohy-
drates. Work is in progress.
Exp er im en ta l Section 14
2-Oxobu t-3-en yl a ceta te (1) was prepared in a yield of
ca. 65% from butyne-1,4-diol according to the modified proce-
dure of Andersen.6
1-Ben zyloxy-2-n itr oeth a n e (2d ). To a stirred solution of
2-nitroethanol (6.0 g, 66 mmol) and benzyl-trichloroacetimidate
(18.3 g, 72.5 mmol) in cyclohexane-CH2Cl2 (2:1, 200 mL) was
added triflic acid (300 µL, 3.3 mmol). After 1 h at room
temperature, the mixture was filtered, washed with aq satu-
rated NaHCO3 (50 mL) and with water (50 mL), dried over
MgSO4, and concentrated under reduced pressure. The residue
was purified by flash chromatography (eluent, cyclohexane-
EtOAc, 4:1, v/v) to give 2d (11.3 g, 94%) as a colorless liquid:
IR (neat film) 1556, 1377, 1215, 1117 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.35 (5H, m), 4.58 (2H, s), 4.55 (2H, t, J ) 5.0 Hz),
3.95 (2H, t, J ) 5.0 Hz)); 13C NMR (100 MHz, CDCl3) δ 137.1,
128.6, 128.1, 127.8, 75.2, 73.5, 65.4.
2-(4,5-Dih yd r oisoxa zol-5-yl)-2-oxoeth yl Aceta te (3a ).
Meth od A. To a stirred solution of 1 (1.28 g, 10 mmol),
nitromethane, (1.1 mL, 20 mmol) and Et3N (4.16 mL, 30 mmol)
in C6H6-CH2Cl2 (1:1, 80 mL) was added dropwise chlorotri-
methylsilane (3.8 mL, 30 mmol). The mixture was heated to
60 °C for 2 h. The solution was cooled, filtered, and concen-
trated under reduced pressure. The residue was dissolved in
Et2O (150 mL), and after filtration, p-TsOH (0.42 g, 2.2 mmol)
was added to the solution. After 1 h stirring, the solution was
washed with aq saturated NaHCO3 (25 mL), and the aqueous
layer was extracted with EtOAc (2 × 25 mL). The combined
organic layers were dried over MgSO4 and concentrated under
reduced pressure. Purification by flash chromatography (elu-
ent, cyclohexane-EtOAc, 1:1) gave 3a (1.5 g, 61%) as a
colorless liquid: IR (neat film) 1750, 1737, 1607, 1234, 1070,
825 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.19 (1H, s), 4.96 (1H,
dd, J ) 6.5, 8.5 Hz), 4.90 (2H, m), 3.27 (2H, m), 2.11 (3H, s);
13C NMR (100 MHz, CDCl3) δ 202.4, 170.2, 146.3, 80.2, 66.4,
38.6, 20.3. Anal. Calcd for C7H9NO4: C, 49.12; H, 5.30; N, 8.18.
Found: C, 48.90; H, 5.38; N, 8.28.
2-(3-Meth yl-4,5-d ih yd r oisoxa zol-5-yl)-2-oxoeth yl Ace-
ta te (3b). Mu k a iya m a ’s P r oced u r e: Meth od B. To a
solution of nitroethane (5.4 mL, 75 mmol) and Et3N (0.35 mL,
2.5 mmol) in dry C6H6 (50 mL) was added dropwise a mixture
of 1 (6.4 g, 50 mmol), phenyl isocyanate (8.1 mL, 75 mmol),
and Et3N (0.7 mL, 5 mmol) in C6H6 (100 mL). After 1 h stirring,
addition of phenyl isocyanate (2.7 mL, 25 mmol) and Et3N (0.35
mL, 2.5 mmol) was repeated. After 24 h, water (20 mL) was
added, and the mixture was stirred for an additional 2 h. The
precipitated material was removed by filtration and washed
with cyclohexane (50 mL). The aqueous layer was extracted
With 1.5 mol equiv of phosphorylating agent, 10 was
isolated in 80% yield. The reaction showed high regiose-
lectivity, as no monophosphorylated regioisomer and only
traces of diphosphorylated isoxazoline were detected in
the crude material. However, upon purification by column
chromatography, partial migration of the dibenzyl phos-
phate moiety was observed (up to 5%), leading to a
mixture of inseparable isomers. Ring opening and com-
plete deprotection was then achieved in one single step
by hydrogenation over Pd catalyst. Under these condi-
tions, hydrolysis of the intermediate imine was catalyzed
by the rapidly deprotected dihydrogenophosphate group
and required no external acid. Although this multistep
reaction appeared quite effective, we could not avoid the
formation of small quantities of amino derivatives (both
diastereomers) via competitive reduction of the interme-
diate imine formed upon N-O bond cleavage. These
impurities were removed by selective adsorption on a
cation exchange column (Dowex 50 W, H+), and 11 was
thus isolated in 80% yield, contaminated by less than 5%
of the 5-phosphate derivative. NMR analyses (0.5 mol‚L-1
,
(14) Gen er a l: IR spectra were determined on a Perkin-Elmer 881
spectrometer, and the resonances are expressed in frequency units (ν
cm-1). NMR spectra were recorded at 400 MHz for 1H and 100 MHz
for 13C on a Bruker AC 400 spectrometer. CHCl3 (δ ) 7.27), CDCl3 (δ
) 77.1) was used as the respective internal standard expressed in ppm.
Optical rotations were determined on a J ASCO polarimeter. HPLC
experiments for enantiomeric excess determination were performed
with a chiralcel OJ column and monitored at 220 nm [eluents, hexane-
iPrOH, 9:1 (4d , 7), hexane-EtOH, 97:3 (4b), 98:2 (5b), 94:6 (4c),
hexane-MeOH, 97:3 (4a )]. Column chromatographies were performed
on Merck Kieselgel 60 (0.040-0.063 mm) and commercial Kieselgel
60 F254 plates were used for thin-layer chromatography. Aspergillus
niger ATCC 9142 was laboratory-grown in the following medium: yeast
extract (5 g), soyoptim (Roquette, 5 g), glucose (20 g), NaCl (5 g), KH2-
PO4 (5 g), and H2O (1 L), preculture 24 h and culture 24 h. (S)-But-
3-ene-1,2-diol was purchased from Interchim, France. All solvents were
distilled before use following usual procedures. Satisfactory analytical
data (( 0.3%) were obtained for all new compounds at the Service
Central d’Analyse du CNRS, Solaize, France.
D2O, pH 7.6) showed 11 to be a mixture of furanoses (35%
and 36%) and open (29%) forms.
Con clu sion
The biotransformation of acylisoxazolines 3a -d by
Aspergillus niger gives both diastereomers (+)-4a -d and
(-)-5a -d in good yields and high enantiomeric excess.
As exemplified in the case of (+)-4d , these molecules
are useful intermediates for the construction of several
(13) (a) Stowell, J . K.; Widlanski, T. S. Tetrahedron Lett. 1995, 36,
1825. (b) Gefflaut, T.; Lemaire, M.; Valentin, M. L.; Bolte, J . J . Org.
Chem. 1997, 62, 5920.

Microbial Reduction of 5-Acyl-isoxazolines
J . Org. Chem., Vol. 66, No. 7, 2001 2299
with CH2Cl2 (2 × 25 mL). The combined organic layers were
dried over MgSO4 and concentrated under reduced pressure.
Purification by flash chromatography (eluent, cyclohexane-
EtOAc, 1:1) gave 3b (5.3 g, 57%) as a pale yellow oil: IR (neat
obtained as a white solid (mp 40 °C): IR (CHCl3) 3585, 3412,
1634, 1047, 868 cm-1; 1H NMR (400 MHz, CD3OD) δ 5.10 (2H,
s), 4.72 (1H, ddd, J ) 5.5, 8.5, 10.5 Hz), 3.82 (1H, ddd, J )
4.5, 6.0, 10.0 Hz), 3.78 (1H, dd, J ) 4.0, 11.5 Hz), 3.70 (1H,
dd, J ) 5.5, 11.5 Hz), 3.20 (2H, m), 2.16 (3H, s); 13C NMR (100
1
film) 1750, 1737, 1633, 1234, 1071, 849 cm-1; H NMR (400
MHz, CDCl3) δ 4.95 (1H, dd, J ) 7.2, 10.8 Hz), 4.90 (2H, m),
3.18 (2H, m), 2.10 (3H, s), 1.96 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 203.0, 170.1, 155.9, 82.0, 66.5, 41.5, 20.3, 12.6. Anal.
Calcd for C8H11NO4: C, 51.89; H, 5.99; N, 7.56. Found: C,
51.78; H, 6.13; N, 7.51.
MHz, CD3OD) δ 158.2, 82.0, 73.6, 64.5, 40.6, 13.2; [R]25
)
D
+112.4 (c 2.9, MeOH); ee > 98%. 5b was obtained as a colorless
1
oil: IR (neat film) 3369, 1637, 1046, 869 cm-1; H NMR (400
MHz, CDCl3) δ 4.60 (1H, ddd, J ) 4.0, 8.0, 12.0 Hz), 3.67 (3H,
m), 3.35 (2H, s), 3.02 (1H, dd, J ) 10.5, 17.0 Hz), 2.92 (1H,
dd, J ) 8.0, 17.0 Hz), 1.98 (3H, s); 13C NMR (100 MHz, CD3-
2-(3-H yd r oxym et h yl-4,5-d ih yd r oisoxa zol-5-yl)-2-oxo-
eth yl Aceta te (3c). 3c was prepared according to method A
from 1 (1.88 g, 14.7 mmol), nitroethanol (1.6 mL, 22.3 mmol),
Et3N (6.1 mL, 43.7 mmol), and Me3SiCl (5.6 mL, 44.1 mmol).
Purification by flash chromatography (eluent CH2Cl2-MeOH,
97:3) gave 3c (1.13 g, 38%) as a pale yellow oil: IR (neat film)
OD) δ 158.1, 81.7, 74.4, 64.4, 41.4, 13.1; [R]25 ) -153.6 (c
D
1.5, MeOH); ee > 98%.
(5S)-5-[(1R)-1,2-Dih yd r oxyeth yl]-3-h yd r oxym eth yl-4,5-
d ih yd r oisoxa zole (4c) a n d (5R)-5-[(1R)-1,2-Dih yd r oxy-
eth yl]-3-h yd r oxym eth yl-4,5-d ih yd r oisoxa zole (5c). From
3c (390 mg, 1.93 mmol) according to general procedure. Flash
chromatography (eluent, CH2Cl2-MeOH, 8:2) afforded a 1:1
mixture of 4c and 5c (208 mg, 67%) as a colorless liquid. Anal.
Calcd for C6H11NO4: C, 44.72; H, 6.88; N, 8.69. Found: C,
44.62; H, 6.97; N, 8.47. 4c and 5c were further separated by
a second column chromatography (eluent Et2O-MeOH-AcOH,
88:10:2). 4c was obtained as a white solid (mp 47 °C): 1H NMR
(400 MHz, CD3OD) δ 5.08 (3H, s), 4.78 (1H, ddd, J ) 6.0, 8.5,
10.5 Hz), 4.49 (2H, s), 3.85 (1H, ddd, J ) 4.5, 6.0, 10.5 Hz),
3.80 (1H, dd, J ) 4.5, 11.5 Hz), 3.73 (1H, dd, J ) 6.0, 11.5
Hz), 3.29 (2H, m); 13C NMR (100 MHz, CD3OD) δ 161.2, 82.4,
73.5, 64.5, 58.2, 37.2; [R]25D ) +120.1 (c 1.2, MeOH); ee ) 97%.
5c was obtained as a colorless oil: IR (neat film) 3350, 1652,
1
3347, 1747, 1737, 1633, 1235, 1065, 855 cm-1; H NMR (400
MHz, CDCl3) δ 5.08 (1H, dd, J ) 6.0, 11.5 Hz), 4.89 (2H, m),
4.33 (2H, m), 3.34 (2H, m), 2.75 (1H, s), 2.16 (3H, s); 13C NMR
(100 MHz, CDCl3) δ 202.7, 170.6, 159.5, 82.2, 66.5, 57.2, 48.5
38.2. Anal. Calcd for C8H11NO5: C, 47.76; H, 5.51; N, 6.96.
Found: C, 47.55; H, 5.59; N, 6.93.
2-(3-Ben zyloxym eth yl-4,5-d ih yd r oisoxa zol-5-yl)-2-oxo-
eth yl Aceta te (3d ). 3d was prepared according to method B
from 1 (5 g, 39 mmol) and 2d (6.4 g, 35.3 mmol). Purification
by flash chromatography (eluent, cyclohexane-Et2O, 1:1) gave
3d (7.7 g, 75%) as a pale yellow oil: IR (neat film) 1750, 1738,
1232, 1074, 856 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.34 (5H,
m), 5.05 (1H, dd, J ) 6.5, 12.0 Hz), 4.96 (2H, m), 4.52 (2H, m),
4.30 (2H, s), 3.34 (2H, m), 2.16 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 202.3, 170.1, 157.1, 137.0, 128.5, 128.0, 127.9, 82.2,
72.8, 66.4, 63.8, 38.4, 20.3. Anal. Calcd for C15H17NO5: C,
61.84; H, 5.88; N, 4.81. Found: C, 61.51; H, 5.87; N, 4.91.
Gen er a l P r oced u r e for th e Red u ction of 3a -d w ith
Asper gillu s n iger . The reactions were performed in 500 mL
conical flasks containing each 3a -d (60-80 mg) dissolved in
ethanol (1 mL), Aspergillus niger wet cells (5 g), and water
(50 mL). After incubation at 27 °C for 24 h on a rotating table
set at 200 rpm, the mixture was filtered, and the cells were
carefully washed with water (50 mL). The filtrate was con-
centrated to dryness under reduced pressure. The residue was
triturated with MeOH (20 mL), insoluble material was re-
moved by filtration, and the solution was concentrated under
reduced pressure. Products were purified by flash chromatog-
raphy as described below.
1
1046, 881 cm-1; H NMR (400 MHz, CD3OD) δ 5.16 (3Η, s),
4.85 (1H, ddd, J ) 2.5, 9.0, 11.0 Hz), 4.49 (2H, s), 3.81 (3H,
m), 3.33 (1H, dd, J ) 11.0, 17.0 Hz), 3.23 (1H, dd, J ) 8.5,
17.0 Hz); 13C NMR (100 MHz, CD3OD) δ 161.2, 82.2, 74.4, 64.3,
58.2, 37.9; [R]25 ) -135.1 (c 1.9, MeOH); ee ) 92%.
D
(5S)-5-[(1R)-1,2-Dih yd r oxyet h yl]-3-b en zyloxym et h yl-
4,5-dih ydr oisoxazole (4d) an d (5R)-5-[(1R)-1,2-Dih ydr oxy-
eth yl]-3-ben zyloxym eth yl-4,5-dih ydr oisoxazole (5d). From
3d (600 mg, 2.04 mmol) according to general procedure. Flash
chromatography (eluent, CH2Cl2-MeOH, 9:1) afforded a 1:1
mixture of 4d and 5d (451 mg, 88%) as a colorless liquid. Anal.
Calcd for C13H17NO4: C, 62.14; H, 6.82; N, 5.57. Found: C,
62.11; H, 6.86; N, 5.51. 4d and 5d were further separated by
a second column chromatography (eluent Et2O-MeOH-AcOH,
97:1:2). 4d was obtained as a white solid (mp 45 °C): IR (CCl4)
3602, 1216, 1092, 878 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.33
(5Η, m), 4.57 (1H, ddd, J ) 5.0, 8.0, 11.0 Hz), 4.53 (2H, s),
4.27 (2H, s), 3.81 (1H, m), 3.70 (1H, m), 3.70 (2H, s), 3.58 (1H,
m), 3.13 (1H, dd, J ) 8.0, 17.5 Hz), 3.02 (1H, dd, J ) 11.0,
17.5 Hz); 13C NMR (100 MHz, CDCl3) δ 157.4, 137.2, 128.6,
(5S )-5-[(1R )-1,2-D ih y d r o x y e t h y l]-4,5-d ih y d r o is o x -
a zole (4a ) a n d (5R)-5-[(1R)-1,2-Dih yd r oxyeth yl]- 4,5-d i-
h yd r oisoxa zole (5a ). From 3a (240 mg, 1.4 mmol) according
to general procedure. Flash chromatography (eluent, CH2Cl2-
MeOH, 9:1) afforded a 1:1 mixture of 4a and 5a (163 mg, 89%)
as a colorless liquid. Anal. Calcd for C5H9NO3: C, 45.79; H,
6.92; N, 10.68. Found: C, 46.10; H, 7.11; N, 10.47. 4a and 5a
were further separated by a second column chromatography
(eluent Et2O-MeOH-AcOH, 93:5:2). 4a was obtained as a
white solid (mp 75 °C): IR (CHCl3) 3686, 3596, 3435, 1603,
1276, 1074, 840 cm-1; 1H NMR (400 MHz, CD3OD) δ 7.42 (1H,
t, J ) 2.0 Hz), 5.10 (2H, s), 4.68 (1H, ddd, J ) 5.5, 8.5, 11.0
Hz), 3.77 (3H, m), 3.23 (2H, m); 13C NMR (100 MHz, CDCl3) δ
128.1, 128.0, 80.9, 72.9, 71.9, 64.5, 63.2, 36.4; [R]25 ) +81.1
D
(c 1, MeOH); ee ) 96%. 5d was isolated as a white solid (mp
59 °C): IR (CHCl3) 3562, 3451, 1627, 1097, 877 cm-1; 1H NMR
(400 MHz, CDCl3) δ 7.33 (5Η, m), 4.65 (1H, ddd, J ) 4.0, 8.0,
11.5 Hz), 4.52 (2H, m), 4.27 (2H, s), 3.70 (2H, m), 3.63 (1H,
m), 3.22 (1H, d, J ) 6.0 Hz), 3.12 (1H, dd, J ) 11.0, 17.5 Hz),
3.03 (1H, dd, J ) 8.0, 17.5 Hz), 2.93 (1H, t, J ) 5.0 Hz); 13C
NMR (100 MHz, CDCl3) δ 157.3, 137.2, 128.5, 128.0, 127.9,
80.8, 73.0, 72.7, 64.3, 63.6, 37.5; [R]25D ) -104.0 (c 1.9, MeOH);
ee ) 87%.
148.5, 80.1, 73.4, 64.5, 37.6; [R]25 ) +93.2 (c 1, MeOH); ee >
D
98%. 5a was obtained as a white solid (mp 103 °C): IR (CHCl3)
Syn th esis of 4a a n d 5a fr om (S)-Bu t-3-en e-1,2-d iol. A
mixture of (S)-but-3-ene-1,2-diol (0.25 mL, 3 mmol), dimethoxy-
propane (0.55 mL, 4.5 mmol), and amberlyst 15 (H+, 100 mg)
in C6H6 (20 mL) was heated to 55-60 °C in a distillation
apparatus. After 1 h, azeotropic distillation of MeOH was
complete, and the reaction mixture was cooled and filtered.
To the solution of vinyldioxolane in C6H6 (15 mL) thus obtained
were added CH2Cl2 (15 mL), nitromethane (0.325 mL, 6 mmol),
Et3N (1.25 mL, 9 mmol), Me3SiCl (1.14 mL, 9 mmol), and the
mixture was heated to reflux. After 90 h, addition of ni-
tromethane (0.325 mL, 6 mmol), Et3N (1.25 mL, 9 mmol), and
Me3SiCl (1.14 mL, 9 mmol) was repeated, and reflux was
continued until total disappearance of vinyldioxolane (140 h).
The mixture was cooled and filtered, and the solution was
concentrated under reduced pressure. The residue was dis-
solved in MeOH (20 mL), and p-TsOH (200 mg, 1.05 mmol)
1
3686, 3565, 3441, 1603, 1280, 1102, 840 cm-1; H NMR (400
MHz, CDCl3) δ 7.42 (1H, t, J ) 1.5 Hz), 5.09 (2H, s), 4.76 (1H,
ddd, J ) 3.0, 8.5, 11.5 Hz), 3.80 (3H, m), 3.27 (1H, ddd, J )
1.5, 11.0, 18.0 Hz), 3.18 (1H, ddd, J ) 2.0, 8.5, 18.0 Hz); 13C
NMR (100 MHz, CDCl3) δ 148.5, 79.8, 74.4, 64.4, 38.5; [R]25
) -150.2 (c 1, MeOH); ee ) 97%.
D
(5S)-5-[(1R )-1,2-Dih yd r oxye t h yl]-3-m e t h yl-4,5-d ih y-
d r oisoxa zole (4b) a n d (5R)-5-[(1R)-1,2-Dih yd r oxyeth yl]-
3-m eth yl-4,5-d ih yd r oisoxa zole (5b). From 3b (360 mg, 1.9
mmol) according to general procedure. Flash chromatography
(eluent, CH2Cl2-MeOH, 9:1) afforded a 1:1 mixture of 4b and
5b (276 mg, 98%) as a colorless liquid. Anal. Calcd for C6H11
-
NO3: C, 49.65; H, 7.64; N, 9.65. Found: C, 49.76; H, 7.76; N,
9.52. 4a and 5a were further separated by a second column
chromatography (eluent Et2O-MeOH-AcOH, 93:5:2). 4b was

2300 J . Org. Chem., Vol. 66, No. 7, 2001
Gefflaut et al.
was added. The solution was stirred 24 h at room temperature,
neutralized with Et3N, and concentrated in vacuo. The residue
was purified by flash chromatography as already described to
g, 46%) was obtained as a colorless liquid: IR (neat film) 1626,
1
1213, 1076 cm-1; H NMR (400 MHz, CDCl3) δ 7.33 (5H, m),
4.67 (1H, ddd, J ) 4.5, 7.5, 11.0 Hz), 4.53 (2H, m), 4.31 (2H,
s), 4.23 (1H, ddd, J ) 4.5, 6.5 Hz), 4.06 (1H, dd, J ) 6.5, 8.5
Hz), 3.85 (1H, dd, J ) 6.5, 8.5 Hz), 3.11 (1H, dd, J ) 11.0,
17.5 Hz), 2.97 (1H, dd, J ) 7.5, 17.5 Hz), 1.44 (3H, s), 1.37
(3H, s); 13C NMR (100 MHz, CDCl3) δ 156.4, 137.4, 128.6,
128.1, 128.0, 110.1, 79.9, 76.7, 72.6, 65.4, 64.4, 37.2, 26.3, 25.4.
Anal. Calcd for C16H21NO4: C, 65.96; H, 7.26; N, 4.81. Found:
give a 3:1 mixture of 4a and 5a (156 mg, 39%). 4a : [R]25
)
D
+93.8 (c 0.5, MeOH); ee > 98%; 5a : [R]25 ) -154.5 (c 0.4,
D
MeOH).
Syn th esis of 4b a n d 5b fr om (S)-Bu t-3-en e-1,2-d iol. 4b
and 5b were prepared according to method B from (S)-2,2-
dimethyl-4-vinyl-1,3-dioxolane prepared as previously de-
scribed (4a and 5a ) from (S)-but-3-ene-1,2-diol (240 µL, 2.84
mmol). To the solution of olefin in C6H6 (25 mL) were added
phenyl isocyanate (0.92 mL, 8.5 mmol), Et3N (66 µL, 0.47
mmol), and nitroethane (300 µL, 4.26 mmol), and the mixture
was stirred at room temperature. Additions of phenyl isocy-
anate (130 µL, 1.18 mmol) and Et3N (14 µL, 0.1 mmol) were
repeated at 2 h and 48 h, and stirring was continued until
total disappearance of the alcene (72 h). After addition of water
(10 mL), the mixture was further stirred for 2 h and then
filtered. The precipitate was washed with cyclohexane (50 mL),
and the aqueous layer was extracted with cyclohexane (3 ×
25 mL) after saturation with NaCl. The combined organic
layers were dried over MgSO4 and concentrated under reduced
pressure. The residue was dissolved in MeOH (20 mL), and,
after addition of amberlyst 15 (H+, 100 mg), the solution was
stirred at room temperature for 24 h and concentrated in
vacuo. The residue was purified by flash chromatography as
already described to give a 3.7:1 mixture of 4b and 5b (211
C, 65.98; H, 7.29; N, 5.02; [R]25 ) -89.4 (c 1.3, CHCl3); ee )
D
87%.
1-O-Ben zyl-3-d eoxy-5,6-O-isop r op ylid en e-D-th r eo-h ex-
u lose (8). To a solution of isoxazoline anti-7 (90 mg, 0.31
mmol) in 10:5:1 CH2Cl2-MeOH-H2O (10 mL) were added
AcOH (36 µL, 0.62 mmol) and a spatula tip of Raney Ni
(estimated to 100 mg). The reaction was placed under hydro-
gen using a balloon. The mixture was stirred vigorously for 2
h. After addition of NaHCO3 (100 mg), the mixture was stirred
for 10 min, filtered through Celite, and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (eluent, CH2Cl2-MeOH, 98:2) to give 8 (69 mg, 76%)
as a colorless liquid: IR (neat film) 3461, 1724, 1212, 1065
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.33 (5H, m), 4.60 (2H, s),
4.10 (2H, s), 4.09 (1H, m), 4.03 (1H, m), 3.95 (2H, m), 3.02
(1H, d, J ) 4.0 Hz), 2.85 (1H, dd, J ) 2.5, 17.5 Hz), 2.66 (1H,
dd, J ) 8.5, 17.5 Hz), 1.40 (3H, s), 1.34 (3H, s); 13C NMR (100
MHz, CDCl3) δ 209.2, 136.9, 128.6, 128.1, 128.0, 109.5, 77.6,
75.2, 73.4, 68.7, 66.7, 42.3, 26.6, 25.1. Anal. Calcd for
mg, 51%). 4b: [R]25 ) +112.2 (c 1, MeOH); ee > 98%. 5b:
D
C
16H22O5: C, 65.29; H, 7.53. Found: C, 65.00; H, 7.61; [R]25
[R]25 ) -153.0 (c 1.5, MeOH); ee > 98%.
D
D
) -20.8 (c 2.6, CHCl3).
Syn th esis of 4c a n d 5c fr om (S)-Bu t-3-en e-1,2-d iol. 4c
and 5c were prepared from (S)-but-3-ene-1,2-diol (238 µL, 2.83
mmol) according to method B as described for 4b and 5b using
tetrahydropyranyl ether of 2-nitroethanol15 (0.74 g, 4.25 mmol)
as the dipole precursor. Addition of PhNCO and Et3N were
repeated at 2, 48, and 64 h and vinyldioxolane was not detected
after 4 days at room temperature. Flash chromatography
3-Deoxy-D-th r eo-h exu lose (6). To a solution of 4d (100 mg,
0.4 mmol) in 9:1 MeOH-H2O (10 mL) were added AcOH (68
µL, 1.2 mmol) and 10% Pd/C (100 mg). The reaction was placed
under hydrogen using a balloon and stirred vigorously for 20
h. After addition of NaHCO3 (100 mg), the mixture was stirred
for 10 min, filtered through Celite, and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (eluent, CH2Cl2-MeOH, 8:2) to give 6 (48 mg, 73%)
as a semisolid substance which was not recrystallized. NMR
spectra showed the presence of five forms in equilibrium:
pyranoses (39% and 9%), furanoses (27% and 20%), and open
form (5%): 1H NMR (400 MHz, CD3OD) δ 4.53 (m), 4.39 (m),
4.31-4.05 (m), 3.93-3.54 (m), 2.93 (dd, J ) 3.5, 15.5 Hz), 2.80
(dd, J ) 9.0, 16.0 Hz), 2.60 (dd, J ) 7.5, 13.5 Hz), 2.37 (dd, J
) 7.0, 13.5 Hz), 2.31 (dd, J ) 7.0, 13.5 Hz), 2.18 (dd, J ) 4.0,
14.5 Hz), 2.10 (dd, J ) 12.3, 12.4 Hz), 2.05 (dd, J ) 4.0, 13.5
afforded a 2.9:1 mixture of 4c and 5c (181 mg, 40%). 4c: [R]25
D
) +129.7 (c 3.3, MeOH); ee > 98%. 5c: [R]25D ) -147.7 (c 1.6,
MeOH).
Syn th esis of 4d a n d 5d fr om (S)-Bu t-3-en e-1,2-d iol. 4d
and 5d were prepared from (S)-but-3-ene-1,2-diol (170 µL, 2
mmol) according to method B as described for 4c and 5c using
2d (0.78 g, 4.3 mmol) as the dipole precursor. Vinyldioxolane
was not detected after 6 days stirring at room temperature.
Purification by flash chromatography as already described
afforded a 3.5:1 mixture of 4d and 5d (270 mg, 54%). 4d : [R]25
D
Hz), 2.05 (dd, J ) 3.0, 14.5 Hz), 1.70 (dd, J ) 5.0, 12.5 Hz).13
C
) +84.5 (c 1, MeOH); ee > 98%; 5d : [R]25 ) -119.5 (c 1.7,
D
NMR (100 MHz, CD3OD) δ 212.2, 107.3, 107.1, 98.7, 97.7, 88.6,
87.6, 76.1, 73.1, 73.0, 69.9, 69.6, 69.4, 69.3, 68.7, 68.1, 67.5,
67.1, 66.9, 65.3, 64.4, 63.2, 60.7, 43.8, 43.5, 43.4, 36.3, 34.5;
MeOH).
(5S)-5-[(1R)-2,2-Dim et h yl-1,3-d ioxola n -4-yl]-3-b en zyl-
oxy-4,5-d ih yd r oisoxa zole (a n ti-7) a n d (5R)-5-[(1R)-2,2-Di-
methyl-1,3-dioxolan-4-yl]-3-benzyloxy-4,5-dihydroisoxazole
(syn -7). A 1:1 mixture of 4d and 5d (2 g, 8 mmol) was obtained
upon reduction of 3d by Aspergillus niger, followed by column
chromatography (eluent, CH2Cl2-MeOH, 9:1). The mixture of
isomers was dissolved in C6H6 (50 mL), and 2,2-dimethoxypro-
pane (1.5 mL, 12.2 mmol) and amberlyst 15 (H+, 500 mg) were
added. The solution was heated at 90 °C in a distillation
apparatus. After 2 h, azeotropic distillation of MeOH was
complete. The reaction mixture was cooled, filtered, and
concentrated under reduced pressure. Isomers anti- and syn-7
were separated by flash chromatography (eluent, Et2O-
cyclohexane, 1:1). Isoxazoline anti-7 (1.03 g, 42%) was obtained
as a colorless liquid: IR (neat film) 1625, 1212, 1074 cm-1; 1H
NMR (400 MHz, CDCl3) δ 7.33 (5H, m), 4.54 (1H, ddd, J )
6.5, 7.0, 10.5 Hz), 4.54 (2H, s), 4.30 (2H, s), 4.12 (1H, dd, J )
6.5, 8.5 Hz), 4.05 (1H, ddd, J ) 5.0, 6.5 Hz), 3.88 (1H, dd, J )
5.0, 8.5 Hz), 3.16 (1H, dd, J ) 10.5, 17.5 Hz), 3.07 (1H, dd, J
) 7.0, 17.5 Hz), 1.43 (3H, s), 1.35 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 156.7, 137.3, 128.6, 128.1, 128.0, 109.8, 80.9, 76.1,
72.8, 67.2, 64.5, 38.0, 26.8, 25.2. Anal. Calcd for C16H21NO4:
C, 65.96; H, 7.26; N, 4.81. Found: C, 65.79; H, 7.22; N, 5.02;
[R]25D ) +72.3 (c 2.4, CHCl3); ee ) 96%. Isoxazoline syn-7 (1.13
[R]25 ) -41.0 (c 1.0, water) [lit.9 -44 (c 1.0, water)].
D
1-O-Ben zyl-3-d eoxy-D-th r eo-h exu lose (9). A solution of
4d (70 mg, 0.28 mmol) in 9:1 MeOH-H2O (10 mL) was treated
as described for the synthesis of 8. Purification by flash
chromatography (eluent, CH2Cl2-MeOH, 9:1) afforded 9 (62
mg, 88%) as a white solid (mp 88 °C): NMR spectra showed
the presence of five forms in equilibrium:1H NMR (400 MHz,
CDCl3) δ 7.32 (5H, m), 4.58 (2H, m), 4.35 (m), 4.24 (m), 4.02
(m), 3.88-3.50 (m), 3.35 (m), 2.66 (m), 2.32 (dd, J ) 8.0, 14.0
Hz), 2.25 (dd, J ) 7.0, 14.0 Hz), 2.09 (dd, J ) 5.0, 14.0 Hz),
2.04 (dd, J ) 4.0, 14.5 Hz), 1.92 (d, J ) 14.0 Hz), 1.79-1.65
(m); 13C NMR (100 MHz, CDCl3) δ 209.1, 137.5, 137.4, 137.2,
137.1, 128.6, 128.57, 128.51, 128.1, 128.06, 128.02, 127.9,
106.1, 105.6, 96.7, 95.8, 88.3, 88.1, 75.4, 74.7, 74.1, 74.0, 73.8,
73.6, 73.4, 72.7, 72.4, 68.5, 67.8, 66.5, 65.3, 63.9, 63.4, 63.1,
62.7, 59.4, 44.5, 42.5, 42.1, 35.5, 34.2, 29.7. Anal. Calcd for
C
13H18O5: C, 61.41; H, 7.13. Found: C, 61.44; H, 7.12; [R]25
D
) +1.4 (c 3.0, CHCl3, after 6 h).
Diben zyl 2(R)-2-(3-Ben zyloxym eth yl-4,5-d ih yd r oisox-
a zol-5-yl)-2-h yd r oxyeth yl P h osp h a te (10). I2 (0.76 g, 3
mmol) was added to a solution of tribenzyl phosphite (1.12 g,
3.18 mmol) in EtOH-free CH2Cl2 (20 mL) at -20 °C. After 20
min, the clear colorless solution was allowed to warm to 25
°C. It was then added dropwise over a period of 1 h to a
solution of 4d (0.5 g, 2 mmol) and pyridine (0.64 mL, 8 mmol)
(15) Kozikowski, A. P.; Adamczyk, M. J . Org. Chem. 1983, 48, 366.

Microbial Reduction of 5-Acyl-isoxazolines
J . Org. Chem., Vol. 66, No. 7, 2001 2301
in CH2Cl2 (20 mL) at -30 °C. The solution was then allowed
to warm to room temperature, filtered, and concentrated under
reduced pressure. The residue was diluted with Et2O (20 mL)
and water (10 mL). The organic layer was washed with aq
KHSO4 0.3 M (3 × 10 mL), aq saturated NaHCO3 (10 mL),
and brine (10 mL) and dried over MgSO4. After concentration,
the crude compound was chromatographed over silica gel
(eluent, cyclohexane-EtOAc, 4:6) to give 10 (0.74 g, 72%) as
a colorless oil which crystallized slowly after few weeks at -20
H2O (10 mL) was added 10% Pd/C (100 mg). The reaction was
placed under hydrogen using a balloon and stirred vigorously
for 18h. The catalyst was removed by filtration through Celite
and the solution concentrated under reduced pressure. The
residue was diluted with H2O (1 mL), applied onto a 10 × 1
cm column of Dowex 50W (H+, 200-400 mesh), and eluted
with H2O. Acidic fractions were combined and adjusted to pH
7.6 with 1 M NaOH. After lyophylization, 11 (136 mg, 80%)
was isolated as a white solid. NMR spectra showed the
presence of three forms in equilibrium: 1H NMR (400 MHz,
D2O) δ 4.61-3.54 (m), 2.88 (dd, J ) 3.5, 16.0 Hz), 2.74 (dd, J
) 9.0, 16.0 Hz), 2.55 (dd, J ) 7.0, 14.0 Hz), 2.38 (dd, J ) 7.0,
13.5 Hz), 2.23 (dd, J ) 7.0, 13.5 Hz), 2.00 (dd, J ) 3.5, 14.0
Hz); 13C NMR (100 MHz, D2O) δ 214.9, 108.7, 108.3, 88.1 (d,
J ) 8.0 Hz), 87.7 (d, J ) 8.0 Hz), 76.4 (d, J ) 5.5 Hz), 74.3,
74.0, 70.6, 70.1, 68.1, 67.8, 67.4 (d, J ) 3.5 Hz), 67.3 (d, J )
1
°C (mp 48 °C): IR (neat film) 3370, 1263, 1018, 879 cm-1; H
NMR (400 MHz, CDCl3) δ 7.33 (15H, m), 5.05 (4H, m),4.51
(1H, m), 4.50 (2H, s), 4.25 (2H, s), 4.18 (1H, d, J ) 5.0 Hz),
4.12 (2H, m), 3.76 (1H, m), 3.14 (1H, dd, J ) 7.5, 17.5 Hz),
3.02 (1H, dd, J ) 10.5, 17.5 Hz); 13C NMR (100 MHz, CDCl3)
δ 156.8, 137.1, 135.4 (d, J ) 6.0 Hz), 128.53, 128.49, 128.4,
128.3, 127.9, 79.5, 72.5, 70.2 (d, J ) 6.0 Hz), 69.5 (d, J ) 5.0
Hz), 68.7 (d, J ) 6.0 Hz), 64.2, 36.9. Anal. Calcd for C27H30
-
3.5 Hz), 66.6 (d, J ) 3.5 Hz), 44.3, 44.1, 43.7; [R]25 ) +3.4 (c
NO7P: C, 63.40; H, 5.91; N, 2.74; P, 6.05. Found: C, 63.61; H,
D
5.90; N, 3.00; P, 6.14; [R]25 ) +40.9 (c 1.5, CHCl3).
D
1, H2O, after 3 h).
3-Deoxy-D-th r eo-h exu lose-6-p h osp h a te Disod iu m Sa lt
(11). To a solution of 10 (300 mg, 0.5 mmol) in 9:1 MeOH-
J O001480F