Efficient chemoenzymatic synthesis of (2S,3S)-3-hydroxyleucine
mediated by immobilised penicillin G acylase†
Nitin W. Fadnavis,* Mohd. Sharfuddin, S. Kumara Vadivel and
Uday T. Bhalerao
Biotransformation Laboratory, Organic Division-I, Indian Institute of Chemical Technology,
Hyderabad-500 007, India
Immobilised penicillin G acylase (EC 3.5.1.11) has been
used in the key step to obtain optically pure (2S,3S)-(؉)-
hydroxyleucine (ee >99%).
The optical purity of 1 was determined by converting the
crude lyophilised product into its N-phenylacetyl derivative.
(Chiralcel OJ, Daicel, Japan; 5 × 250 mm, λ 254 nm; 15%
propan-2-ol in hexane containing 0.1% TFA; flow rate 0.7 ml
Ϫ1
min ; retention times (2R,3R): 7.3 and (2S,3S): 10.2 min). We
(
2S,3S)-(ϩ)-3-Hydroxyleucine 1 is an important constituent of
have found that this method is very reliable and especially useful
in cases of amino acids and amino alcohols whose enantiomers
do not separate well on a Crownpack CR (ϩ) column; or where
the detection wavelength is less than 230 nm and the presence
of impurities in the crude sample interfere in the analysis. Full
details about this methodology will be published in due course.
a range of naturally occurring cyclodepsipeptides that include
1
2
3
4
5
telomycin, azinothricin, citropeptin, variapeptin, A83586C,
6
7
L-156,602 and verucopeptin. Even though several elegant
8
asymmetric syntheses have been described in the literature, no
enzyme-catalysed resolution step has been reported for the syn-
thesis of optically pure 1. Here we describe a highly convenient
and efficient chemoenzymatic synthesis of (2S,3S)-(ϩ)-3-
hydroxyleucine where immobilised penicillin G acylase (EC
Experimental
3
.5.1.11) has been used in the key step for resolution of the
racemic hydroxy amino acid to obtain optically pure 1 (ee
99%). Although applications of penicillin acylase in the pro-
(
2S,3S)-3-Hydroxyleucine 1
N-Phenylacetyl-3-hydroxyleucine 4 (530 mg, 2 mmol)‡ was dis-
solved in water (10 cm ), the pH adjusted to 7.8 with 5 aque-
>
3
9
duction of 6-aminopenicillanic acid and kinetic resolution of
10
11
12
ous ammonia and immobilised penicillin G acylase (100 mg, 20
units) added. The reaction mixture was shaken in a conical flask
at 200 rpm (35 ЊC) and the reaction was followed by HPLC
analysis (Chrompack C8, 5 × 250 mm, λ 254 nm, 40%
acetonitrile–water containing 0.2% phosphoric acid, flow rate 1
racemic alcohols, amines and β-amino acids are well-
known, ours is the first example where penicillin acylase has
been used in the resolution of a β-hydroxy-α-amino acid.
A simple Doebner condensation between isobutyraldehyde
and malonic acid in pyridine solution affords the trans-α,β-
unsaturated acid 2 as a single isomer in 80% yield. Epoxidation
of 2 with dimethyldioxirane (prepared in situ by the reaction of
Oxone and acetone ) gave highly stable cis-epoxy acid 3 in
almost quantitative yield. In agreement with the results of
Caldwell and Bondy, opening of epoxy acid 3 with aqueous
13
Ϫ1
ml min ; retention times 4: 4.5 min, phenylacetic acid: 5.2
min). The reaction was stopped at 40% hydrolysis (1 h) and the
enzyme was filtered off and washed with water. The combined
aqueous layer was acidified with conc. HCl and then extracted
with dichloromethane to remove unreacted amide and phenyl-
acetic acid. The aqueous solution was lyophilised to get a white
14
15
ammonia occurred regioselectively in a trans fashion to give
racemic hydroxyleucine (95%). This was converted to its N-
phenylacetyl derivative 4 (95%) and subjected to enantioselec-
tive hydrolysis with immobilised penicillin G acylase in aqueous
solution at pH 7.8 to obtain optically pure (2S,3S)-(ϩ)-3-
hydroxyleucine 1 (ee >99%, 40% hydrolysis) (Scheme 1).
8a
powder which after recrystallisation from water–methanol
gave white crystalline (2S,3S)-3-hydroxyleucine 1: (105 mg, 71%
7
20
D
of theoretical), mp 221–223 ЊC (lit., 219–221 ЊC) ee >99%; [α]
1
ϩ26.0 (c 2.0 in water) (lit., [α]D ϩ22 (c 2.0 in water); δ (D O,
H
2
2
00 MHz) 3.90 (1H, d, J 2.5,§ CHNH), 3.50 (1H, dd, J 9.2, 3.1,
CHOH), 1.93 [1H, m, CH(CH ) ], 1.00 (3H, d, J 6.2, CH ), 0.97
3
2
3
(
3H, d, J 6.2, CH ). Compound 4 (300 mg, 94%); mp 189–
O
3
2
0
COOH
ii
COOH
i
191 ЊC, ee 45%; [α]D ϩ1.1 (c 1.0 in CH OH).
CHO
3
2
3
Acknowledgements
iii
We acknowledge UGC, New Delhi and CSIR, New Delhi for
awards of Senior Research Fellowships to M. S. and S. K. V.;
and SIRIS Bio-Tech Ltd, Hyderabad for the generous gift of
penicillin acylase.
OH
OH
OH
COOH
COOH
COOH
iv
+
1
NHCOCH2Ph
+)-4 (ee = 45%)
NH2
(+)-1 (ee >99%)
NHCOCH2Ph
‡ Selected H NMR spectral data for 4 and the intermediates leading
to 4: 2 δ (CDCl , 200 MHz) 10 (1H, br, COOH), 6.95 (1H, dd, J 15.5,
.5, CHCOOH), 5.70 [1H, d, J 15.5, CHCH(CH ) ], 2.51 [1H, m,
H
3
(
4
6
3 2
Scheme 1 Reagents and conditions: i, CH (COOH) , pyridine, 0 ЊC, 2
h, 80 ЊC, 8 h, 80%; ii, Oxone, acetone, NaHCO , 24 ЊC, 2 h, 98%; iii, aq.
CH(CH ) ], 1.01 [6H, d, J 6.45, (CH ) CH]; 3 δ (CDCl , 200 MHz) 8.65
2
2
3 2 3 2 H 3
3
(1H, br s, COOH), 3.28 (1H, d, J 2.4, CHCOOH), 2.95 [1H, dd, J 6.0,
2.4, CHCH(CH ) ], 1.65 [1H, m, CH(CH ) ], 1.06 (3H, d, J 6.7, CH ),
Ϫ
NH , reflux, 6 h, PhCH COCl, OH , 0 ЊC, 2 h, 95%; iv, immobilized
3
2
3
2
3
2
3
penicillin G acylase, pH 7.8–8.0, 1 h, 40% conversion, 71% of theor-
etical yield
1.01 (3H, d, J 6.7, CH ); 4 δ [CDCl ϩ (CD ) SO] 7.56 (1H, br d, NH),
3 H 3 3 2
7.28 (5H, m, Ph), 4.55 (1H, dd, J 6.2, 3.1, CHCOOH), 3.60 (2H, 2s,
CH Ph), 3.39 (1H, dd, J 6.2, 2.9, CHOH), 1.89 [1H, m, CH(CH ) ], 1.05
2
3 2
(
3H, d, J 6.2, CH ), 1.00 (3H, d, J 6.2, CH ).
3
3
†
IICT Communication No: 3858.
§ J Values are given in Hz.
J. Chem. Soc., Perkin Trans. 1, 1997
3577
Fadnavis, Nitin W.
