Porcine pancreatic lipase mediated regio- and stereoselective hydrolysis: Chemoenzymatic synthesis of (2S,3S)-2-amino-3,4-dihydroxybutyric acid

Article abstract of DOI:10.1016/S0957-4166(01)00099-4

Porcine pancreatic lipase was used in the chemoenzymatic hydrolysis of 2-azido-3-hydroxy-4-methylcarbonyloxybutyl acetate. The reaction occurred with high regio- and stereoselectivity to give enantiomerically pure (2S,3R)-3-azido-2,4-dihydroxy butyl aceta

Full text of DOI:10.1016/S0957-4166(01)00099-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 691–693
Porcine pancreatic lipase mediated regio- and stereoselective
hydrolysis: chemoenzymatic synthesis of
(2S,3S)-2-amino-3,4-dihydroxybutyric acid†
N. W. Fadnavis,* Mohd. Sharfuddin and S. Kumara Vadivel
Biotransformation Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 9 January 2001; accepted 13 February 2001
Abstract—Porcine pancreatic lipase was used in the chemoenzymatic hydrolysis of 2-azido-3-hydroxy-4-methylcarbonyloxybutyl
acetate. The reaction occurred with high regio- and stereoselectivity to give enantiomerically pure (2S,3R)-3-azido-2,4-dihydroxy
butyl acetate 5 (e.e. >99%) which was easily converted to (2S,3S)-2-amino-3,4-dihydroxybutyric acid 1, an important synthetic
intermediate in the synthesis of b-lactam antibiotics and phytosiderophores. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
an efficient preparation of the title compound. The
stereoselective porcine pancreatic lipase (PPL) mediated
hydrolysis of 2-azido-3-hydroxy-4-methylcarbonyloxy
butyl acetate was the key step in this synthesis (Scheme
1).
b-Hydroxy-a-amino acids are constituents of several
compounds, such as peptides, polyoxins and enzyme
inhibitors, covering a wide range of biological activity
from antibiotic to immunosuppressive properties,1
hence, their stereoselective synthesis is of great interest.
In particular, (2S,3S)-2-amino-3,4-dihydroxybutyric
acid 1 has been used as a synthetic intermediate in the
synthesis of b-lactam antibiotics and phytosiderophores.2
Commercially available cis-1,4-butenediol in pyridine
was treated with Ac2O to obtain the diacetate 2 in 90%
yield. Epoxidation of the olefin functionality of 2 was
achieved with dimethyldioxirane (prepared in situ by
the reaction of Oxone and acetone) at 25°C and pH
ꢀ7.2 to yield 3-methylcarbonyloxymethyl-2-oxiranyl-
methyl acetate 3, which was then opened cleanly with
NaN3 and NH4Cl in 80% aqueous ethanol at 80°C to
obtain the syn-2-azido-3-hydroxy compound 4 in high
86% overall yield from 2. The racemic azido alcohol (50
2. Results and discussion
In continuation of our studies3 on the chemoenzymatic
syntheses of biologically active compounds, we present
Scheme 1. (i) Ac2O/pyridine; (ii) Oxone, acetone, 27°C, pH 7.2; (iii) NaN3, NH4Cl, aq. EtOH; (iv) porcine pancreatic lipase, pH
7.2.
* Corresponding author. Fax: 91-40-7173387; e-mail: fadnavisnw@yahoo.com
†
IICT Communication No. 4699.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00099-4

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N. W. Fadna6is et al. / Tetrahedron: Asymmetry 12 (2001) 691–693
2.2. Stereoselectivity of enzymatic hydrolysis
The absolute stereochemistry at the centre generated in
enzyme mediated hydrolysis was determined as (S) by
preparing the title compound (2S,3S)-2-amino-3,4-
dihydroxybutyric acid 1 from 5. To determine the e.e.,
both 4 and 5 were completely acetylated with Ac2O.
The corresponding triacetyl derivative gave excellent
resolution on a Chiralcel OJ column (Daicel, Japan,
5×250 mm) with retention times as follows: 2-azido-3,4-
di(methylcarbonyloxy)-(2S,3R)-butyl acetate 30.1 min;
2-azido-3,4-di(methylcarbonyloxy)-(2R,3S)-butyl acet-
ate 34.5 min with propan-2-ol (10%) in hexane mobile
phase and a flow rate of 0.4 mL min−1. Detection of the
product was by eluate analysis at 215 nm. Product 5
was observed to be enantiomerically pure with an e.e.
of >99%, while recovered starting diacetate 4 was found
to have an e.e. of 55%.
Figure 1.
mmol) was subjected to enzymatic hydrolysis with
porcine pancreatic lipase (EC 3.1.1.3, 500 mg, 170
units/mg, Sigma, Type II) at 30°C in 0.01 M Tris–HCl
buffer containing 0.1 M aqueous NaCl (50 mL, pH
7.2). The pH of the reaction was maintained by the
constant addition of 0.2N aqueous sodium hydroxide
and the reaction was followed by monitoring the
amount of NaOH consumed. The reaction was stopped
after 1 hour when hydrolysis was 35% complete.
Extraction with ethyl acetate and purification of the
crude product by silica column chromatography with
hexane/ethyl acetate (4:1) afforded the 1,3-diol 5 in 30%
yield, whilst the unreacted azido alcohol 4 was returned
from this process in 62% yield.4
2.3. Synthesis of (2S,3S)-2-amino-3,4-dihydroxybutyric
acid
The synthesis of (2S,3S)-2-amino-3,4-dihydroxybutyric
acid 1 was successfully carried out in the following
manner (Scheme 2). Protection of the primary alcohol
functionality of the 1,3-diol 5 with TBDPSCl in the
presence of imidazole occurred with high chemoselec-
tivity and afforded (2S,3R)-3-azido-2-hydroxy-4-(tert-
butyldiphenyl)silyloxybutyl acetate 6 in 89% yield.
Deacetylation of 6 was accomplished with K2CO3 in
50% aq. methanol to furnish the 1,2-diol in 95%
yield, which was then cleanly protected as its ace-
tonide 7. Removal of the silyl ether protecting group
by treatment with TBAF in THF then gave alcohol
8. Oxidation7 of 8 with RuCl3·3H2O and NaIO4 at
0°C cleanly provided the requisite acid 9 in 85% yield
and the acetonide of 9 was cleaved using an acidic
resin (Amberlyst 15, Fluka) at 50°C in methanol to
afford a dihydroxyazide which was subjected to cata-
lytic hydrogenation with palladium on carbon catalyst
to give the target dihydroxy amino acid 1 in 70%
yield with >99% e.e. Additionally, 1 had identical
spectral and physical data to those reported in the
literature.2a,8
2.1. Regioselectivity of enzymatic hydrolysis
For the determination of regioselectivity in the enzy-
matic hydrolysis, diol 5 was subjected to periodate
oxidation. The absence of any cleavage product indi-
cated that 5 was a 1,3-diol. For further confirmation of
the structure, diol 5 was converted to its acetonide with
2,2-dimethoxypropane in the presence of catalytic CSA
in 92% yield.
Based on the work reported earlier by Rychnovsky et
al. for six-membered ring acetonides5a and Dana et al.
for five-membered ring acetonides (Fig. 1),5b using the
chemical shifts of the acetonide carbons in 13C NMR
spectroscopy allows the differentiation between 1,2-
and 1,3-diols.
A clear signal at l=99.46 and the absence of a peak in
the region of 107–110 ppm in the 13C NMR spectrum6
of acetonide confirmed that the enzymatic hydrolysis
reaction led to the formation of the 1,3-diol.
Scheme 2. (i) TBDPSCl/imidazole; (ii) K2CO3/CH3OH; (iii) 2,2-dimethoxypropane, CSA; (iv) TBAF/THF; (v) RuCl3·3H2O,
NaIO4, CCl4, CH3CN, H2O; (vi) Amberlyst 15, CH3OH, 50°C; (vii) Pd/C, H2, CH3OH.

N. W. Fadna6is et al. / Tetrahedron: Asymmetry 12 (2001) 691–693
693
3. Conclusion
(m, 1H, CHN3), 3.8 (br s, 1H, CHOH), 4.15 (m, 2H,
OCH2CHOH), 4.3 (m, 2H, OCH2CHN3); [h]2D5=−10.0 (c
1
1.0, CHCl3). 5: IR (neat) 3600–3250, 2110, 1740 cm−1; H
We have presented a simple and convenient approach
to the title compound and also demonstrated the prepa-
ration of the highly functionalised intermediate, 5, with
high enantiomeric purity using a simple PPL catalysed
hydrolytic reaction where high regiospecificity coupled
with high stereoselectivity is observed.9 Earlier reports
on the resolution of azido alcohols10 have been mainly
confined to the use of lipases from Candida cylindracea
and Pseudomonas sp. and although excellent enantio-
and diastereoselectivities have been observed in the
enzymatic reactions, high regioselectivities such as those
observed in the present case have not previously been
observed because both primary and secondary hydroxyl
groups are usually found to react under such condi-
tions.10g,h The methodology presented here is amenable
to further extension. Additionally, intermediate 5 can
be used in the synthesis of several biologically active
compounds.
NMR (CDCl3): l 2.1 (s, 3H, COCH3), 2.9 (br s, 1H,
OH), 3.15 (br s, 1H, OH), 3.5 (m, 1H, CHN3), 3.9 (m,
2H, OCH2CHOH); 4.0 (m, 1H, CHOH), 4.2 (d, 2H,
J=6.5 Hz, OCH2CHN3); mass: 189 M+; [h]D25=−24.7 (c
1
0.8, CHCl3). 6: IR (neat): 3450 (br), 2115, 1735 cm−1; H
NMR (CDCl3): l 1.1 (s, 9H, C(CH3)3), 2.05 (s, 3H,
COCH3), 2.3 (d, 1H, OH), 3.5 (m, 1H, CHN3), 3.9 (m,
3H, OCH2CHO, OCH2CHO and OCH2CHN3), 4.1 (m,
2H, OCH2CHO and OCH2CHN3), 7.4 (m, 6H, Ph), 7.65
(m, 6H, Ph); mass: 427 (M+); [h]D25=−16.4 (c 1, CHCl3).
1
8: IR (neat): 3550–3400 (br), 2120, 1690 cm−1; H NMR
(CDCl3): l 1.38 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.8 (br,
1H, OH), 3.4 (m, 1H, CHN3), 3.72 (m, 2H, CH2OH),
3.85 (m, 1H, OCH2), 4.05 (m, 1H, OCH2), 4.25 (m, 1H,
OCH); mass: 187 (M+); [h]D25=−12.6 (c 1.0, CHCl3). 9: IR
(neat): 2115, 1690 cm−1; 1H NMR (CDCl3): l 1.35 (s, 3H,
CH3), 1.5 (s, 1H, CH3), 3.75 (d, 1H, J=5 Hz, CHN3),
3.98 (m, 1H, OCH2CHO), 4.15 (m, 1H, OCH2CHO), 4.55
(m, 1H, (OCH2)CHO); mass: 201 (M+); [h]D25=−8.5 (c
1.0, MeOH).
Acknowledgements
5. (a) Rychnovsky, S. D.; Rogers, B. N.; Richardson, T. I.
Acc. Chem. Res. 1998, 31, 9; (b) Dana, G.; Danechpa-
jouh, H. Bull. Soc. Chim. Fr. 1980, II, 395.
We are thankful to CSIR, New Delhi and UGC, New
Delhi, for financial support.
6. 13C NMR (CDCl3): 18.35, 20.61, 28.38, 53.53, 63.77,
64.06, 69.60, 99.46, 170.42.
7. Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless,
K. B. J. Org. Chem. 1981, 46, 3936.
References
8. 1: mp 215°C; (lit2a 214°C); [h]D25=−13.4 (c 2, water), lit2a
1. Duthater, R. O. Tetrahedron 1995, 50, 1539 and refer-
ences cited therein.
1
−13.5 (c 2, water). H NMR (D2O): l 3.49–3.61 (m, 3H,
CHNH2, CH2OH), 3.98 (m, 1H, (HOCH2)CHOH).
9. (a) Schoffers, E.; Golebiowski, A.; Jonson, C. R. Tetra-
hedron 1996, 52, 3769; (b) Borreguero, I.; Sinisterra, J. V.;
Rumbero, A. J.; Hermoso, J. A.; Martinez-Ripoll, M.;
Alcantara, A. R. Tetrahedron 1999, 55, 14961.
2. (a) Okawa, K.; Hori, K.; Hirose, K.; Nakagawa, Y. Bull.
Chem. Soc. Jpn. 1969, 42, 2720; (b) Cativela, C.; Diaz-de-
Villegas, M. D.; Galvez, J. A.; Garcia, J. I. Tetrahedron
1996, 52, 9563; (c) Cativela, C.; Diaz-de-Villegas, M. D.;
Galvez, J. A. Tetrahedron Lett. 1995, 36, 2859; (d)
Palomo, C.; Cabre, F.; Ontoria, J. M. Tetrahedron Lett.
1992, 33, 4819; (e) Saito, S.; Takahashi, N.; Ishikawa, T.;
Moriwake, T. Tetrahedron Lett. 1991, 32, 667.
10. (a) Gru¨ber-Khadjavi, M.; Ho¨nig, H. Biotechnol. Lett.
1992, 14, 367; (b) Exl, C.; Ho¨nig, H.; Renner, G.; Rogi-
Kohlenprath, R.; Seebauer, V.; Seuter-Wasserthal, P.
Tetrahedron: Asymmetry 1992, 3, 1391; (c) Ho¨nig, H.;
Seufer-Wasserthal, P.; Weber, H. Tetrahedron 1990, 46,
3841; (d) Foelsche, E.; Hickel, A.; Ho¨nig, H.; Seufer-
Wasserthal, P. J. Org. Chem. 1990, 55, 1749; (e) Ho¨nig,
H.; Seufer-Wasserthal, P.; Fu¨lop, I. J. Chem. Soc., Perkin
Trans. 1 1989, 2341; (f) Faber, K.; Ho¨nig, H.; Seufer-
Wasserthal, P. Tetrahedron Lett. 1988, 29, 1903; (g)
Wang, Y. F.; Dumas, D. P.; Wong, C. H. Tetrahedron
Lett. 1993, 34, 403; (h) Ho¨nig, H.; Seufer-Wasserthal, P.;
Weber, H. Tetrahedron Lett. 1990, 31, 3011.
3. (a) Fadnavis, N. W.; Sharfuddin, M.; Vadivel, S. K.
Tetrahedron: Asymmetry 1999, 10, 4495; (b) Fadnavis, N.
W.; Vadivel, S. K.; Sharfuddin, M. Tetrahedron: Asym-
metry 1999, 10, 3675; (c) Fadnavis, N. W.; Sharfuddin,
M.; Vadivel, S. K.; Bhalerao, U. T. Tetrahedron: Asym-
metry 1997, 4003; (d) Fadnavis, N. W.; Vadivel, S. K.;
Sharfuddin, M.; Bhalerao, U. T. J. Chem. Soc., Perkin
Trans. 1 1997, 3577.
4. 4: IR (neat) 3600–3200, 2120, 1735 cm−1 1H NMR
;
(CDCl3): l 2.1 (s, 6H, (COCH3)2), 2.3 (br s, 1H, OH), 3.7
.
.