Selectivity Paradigm in Lipase Reactions
J . Org. Chem., Vol. 64, No. 22, 1999 8107
P LAP -Ca ta lyzed Bip h a sic Hyd r olysis of 7. A solution
of diacetate 7 (5 mmol) in petroleum ether/benzene (2:1) (150
mL) was added to a suspension of PLAP (500 mg) in 50 mM
sodium phosphate buffer (75 mL), pH 8.0, at 25 °C, and the
pH was maintained at 8 using auto-stat with 0.1 M NaOH
solution. At the end of 2 h the reaction mixture was filtered
through Celite, and usual workup followed by silica gel column
chromatographic purification (elution with 20% ethyl acetate/
petroleum ether) first gave pure 9 as a thick oil (20% yield)
and then a mixture of 8 and 9 in 5% yield. 9: IR (neat) νmax
amount of label retained may depend on its position
(primary or secondary) in the substrate. These results
(Scheme 2) convincingly indicate that in AmanoPS-
catalyzed hydrolysis of (()-7, although the observed
regioselectivity from the final product composition ap-
pears to be 90%, the actual sn-1 selectivity is ∼100%,
since 10% of 9 is a result of intramolecular acyl migration
during the hydrolysis. In PLAP-catalyzed hydrolysis of
(()-7 at pH 8.0, the observed regioselectivity is very high
(∼90%) and the actual selectivity differs markedly (in-
tramolecular acyl migration/multiple point attachment
and hydrolysis).
In summary, we have demonstrated with the above two
examples the first simple method to correlate the actual
and observed regioselectivity in enzymatic hydrolysis of
unsymmetrical diacetates. By suitable manipulations in
reaction conditions (enzyme type, solvent system, pH
range, and temperature), it may be possible to obtain
both isomers with high regioselctivity by completely
preventing or forcing the acyl migration (Scheme 2). This
strategy may also be useful for assessing the actual and
observed regioselectivities in enzymatic hydrolysis of
polyacylated systems including sugars2k and stereoselec-
tivities in meso diacetates by exclusively labeling the
pro-R or pro-S acetate.
1
3440, 1720 cm-1; H NMR (CDCl3, 200 MHz) δ 2.08 (s, 3H),
2.60 (bs, 1H), 3.45-3.62 (m, 2H), 3.97-4.10 (m, 1H), 4.08-
4.26 (m, 2H), 4.58 (s, 2H), 7.20-7.48 (m, 5H); 13C NMR (CDCl3,
50 MHz) 21.0, 65.8, 68.9, 71.2, 73.6, 128.0 (2C), 128.7, 138.0,
178.3.
Similarly 11 upon PLAP-catalyzed hydrolysis furnished a
mixture of 9+12+13 as a thick oil in 47:8:45 proportion, while
10+11 (9:1) upon PLAP-catalyzed hydrolysis furnished a
mixture of 8+9+13 as a thick oil in 5:66:29 proportion.
La belin g of Hyd r oxya ceta te-2 w ith CD3COOD/DCC. To
a stirred solution of hydroxyacetate 2 (1 mmol), CD3COOD (72
mg, 1.2 mmol), and a catalytic amount of DMAP in EDC (5
mL) was added a solution of DCC (247 mg, 1.2 mmol) in EDC
(2 mL) in a dropwise fashion at room temperature. The
reaction mixture was further stirred at room temperature for
45 min and then filtered through Celite, the residue was
washed with EDC, and the organic layer was concentrated in
vacuo. The residue on usual workup followed by silica gel
column chromatographic purification (elution with 10% ethyl
acetate/petroleum ether) gave the corresponding monolabeled
compound 4 in 90-95% yield as a thick oil. 4: IR (neat) νmax
Exp er im en ta l Section
DCC and CD3COOD (99.5%) were obtained from Aldrich
Chemical Co. The substrate diacetates (()-1,8 (()-7,11,12a and
(2S,3R)-hydroxyacetate 28 were prepared as reported before.
The biphasic enzymatic hydrolyses of (()-1 and (-)-4 with
AmanoPS (800 U) and PLAP (20 U) were carried out using
known8 procedures. The activity of lipase powder has been
expressed in terms of units: 1 unit corresponds to micromoles
of butyric acid (estimation by GC) liberated from glyceryl
tributyrate per minute per milligram of enzyme powder.16 The
term usual workup refers to extraction with ethyl acetate,
washing the organic layer with water and brine, drying of the
organic layer over Na2SO4, and concentration in vacuo. Column
chromatographic purification was done on ACME silica gel.
Am a n oP S-Ca ta lyzed Bip h a sic Hyd r olysis of 7. A solu-
tion of diacetate 7 (1 mmol) in petroleum ether/benzene (2:1)
(20 mL) was added to a suspension of AmanoPS (125 mg) in
50 mM sodium phosphate buffer (10 mL), pH 7.0, at 25 °C.
After 2 h, the reaction mixture was filtered through Celite,
and usual workup followed by silica gel column chromato-
graphic removal of unreacted diacetate (elution with 20% ethyl
acetate/petroleum ether) furnished 8+9 (9:1) as a thick oil in
55% yield. In the spectral data only signals due to the major
component 8 have been listed below. 8: IR (neat) νmax 3440,
1730 cm-1; 1H NMR (CDCl3, 200 MHz) δ 2.12 (s, 3H), 3.67 (d,
J ) 5.5 Hz, 2H), 3.82 (d, J ) 5.5 Hz, 2H), 4.56 (d, J ) 2.5 Hz,
2H), 5.05 (quin, J ) 5.5 Hz, 1H), 7.20-7.45 (m, 5H); 13C NMR
(CDCl3, 50 MHz): 20.8, 62.0, 68.7, 73.2 (2C), 127.4, 127.6,
128.2, 137.6, 170.6; MS (m/e) 225, 207, 154, 137, 117, 105, 91.
Similarly 10+11 upon AmanoPS-catalyzed hydrolysis fur-
nished a mixture of 8+9+12 as a thick oil in 80:10:10
proportion.
1
1740, 1730, 1720 cm-1; H NMR (CDCl3, 200 MHz) δ 1.18 (t,
J ) 7.0 Hz, 3H), 2.10 (s, 3H), 3.80 (s, 3H), 4.15 (q, J ) 7.0 Hz,
2H), 5.30 (d, J ) 4.4 Hz, 1H), 6.22 (d, J ) 4.4 Hz, 1H), 6.88(d,
J ) 8.8 Hz, 2H), 7.32 (d, J ) 8.8 Hz, 2H); MS (m/e) 327, 264,
222, 179, 151, 137, 121.
Similarly 8+9 (9:1) gave a mixture of 10+11 (9:1) as a thick
oil in 90-95% yield. 10+11: 1H NMR (CDCl3, 200 MHz) δ 2.06
(s, 0.3H), 2.10 (s, 2.7H), 3.60 (d, J ) 5 Hz, 2H), 4.12-4.25 (dd,
J ) 12 and 7 Hz, 1H), 4.30-4.42 (dd, J ) 12 and 5 Hz, 1H),
4.54 (d, J ) 2 Hz, 2H), 5.23 (quin, J ) 6 Hz, 1H), 7.20-7.45
(m, 5H); MS (m/e) 270, 210, 162, 137, 91.
Similarly 9 gave 11 as a thick oil in 90-95% yield. 11: 1H
NMR (CDCl3, 200 MHz) δ 2.06 (s, 3H), 3.60 (d, J ) 5 Hz, 2H),
4.12-4.26 (dd, J ) 12 and 7 Hz, 1H), 4.28-4.40 (dd, J ) 12
and 5 Hz, 1H), 4.56 (d, J ) 2 Hz, 2H), 5.16-5.30 (m, 1H), 7.25-
7.45 (m, 5H); MS (m/e) 270, 207, 181, 162, 120, 91.
Ack n ow led gm en t. We thank Prof. D. Basavaiah,
University of Hyderabad, for the generous gift of PLAP
and Amano Pharmaceuticals Co., J apan, for providing
AmanoPS. S.B.D. thanks CSIR, New Delhi, for the
award of a research fellowship.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
2, 4, 7, 8+9, 9, 10+11, 11, 8+9+12, 9+12+13, and 8+9+13,
13C NMR spectra of 7, 8+9, and 9, mass spectra of 2, 4,
8+9+12, 10+11, 11, and 9+12+13. This material is available
(16) Dupus, C.; Corre, C.; Boyaval, P. Appl. Environ. Microbiol. 1993,
59, 4004.
J O990598X