Enantioselective biocatalytic reactions on (±)-aryl alkyl ketones with native and modified porcine pancreatic lipase

Article abstract of DOI:10.3109/10242421003734704

Porcine pancreatic lipase (PPL), pre-incubated with acetophenone in tetrahydrofuran, fails to recognize ortho-and para-acyloxy functions with respect to the nuclear carbonyl group in (±)-2,4-diacyloxyphenyl alkyl ketones and produces novel aryl alkyl keto

Full text of DOI:10.3109/10242421003734704

Biocatalysis and Biotransformation, May–June 2010; 28(3): 172–184
ORIGINAL ARTICLE
Enantioselective biocatalytic reactions on (؎)-aryl alkyl ketones with
native and modified porcine pancreatic lipase
MOFAZZAL HUSAIN1,2,3, VINEET KUMAR1,4, RAJESH KUMAR1, NAJAM A. SHAKIL1,8,
SUNIL K. SHARMA1, ASHOK K. PRASAD1, CARL E. OLSEN5, RAJENDRA K. GUPTA2,
SANJAY V. MALHOTRA4, ERIK VAN DER EYCKEN3, ANTHONY L. DEPASS6,
KALLE LEVON7 & VIRINDER S. PARMAR1
1Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India, 2School of Biotechnology,
GGSIP University, Delhi, India, 3Laboratory for Organic & Microwave-Assisted Chemistry (LOMAC),
Department of Chemistry, Katholieke Universiteit Leuven, Leuven, Belgium, 4Laboratory of Synthetic Chemistry,
Development Therapeutics Program Support, National Cancer Institute–Frederick, Frederick, Maryland, USA,
5Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark,
6Department of Biology, Long Island University, Brooklyn, NewYork, USA, 7Department of Chemistry, Polytechnic
University, Brooklyn, NewYork, USA and 8Division of Agricultural Chemicals, Indian Agricultural Research
Institute, Pusa, New Delhi, India
Abstract
Porcine pancreatic lipase (PPL), pre-incubated with acetophenone in tetrahydrofuran, fails to recognize ortho- and para-
acyloxy functions with respect to the nuclear carbonyl group in (Ϯ)-2,4-diacyloxyphenyl alkyl ketones and produces novel
aryl alkyl ketones in moderate-to-highly optically active forms; this result supports the hypothesis on the mechanism of
action of PPL in deacylation reactions on peracylated polyphenolics.
Keywords: Lipase, aryl akyl ketones, enzymatic deacylation, Schiff's base complex
Introduction
2004; Faber 2004; Jestin & Kaminski 2004; Valetti
Polyphenolics are secondary metabolites of
plants and possess an array of biological activities
(Amakura et al. 2008; Bracke et al. 2008; Naithani
et al. 2008; Ghosh & Scheepens 2009). The synthe-
sis of a variety of natural polyphenolic compounds
such as coumarins (Ghate et al. 2005), chalcones
(Narender et al. 2007; Sashidhara et al. 2009), fla-
vones (Tang et al. 2005), flavanones (Wang & Cheng
2006; Pathak et al. 2008) and chromanones (Cham-
bers & Marfat 1994) requires polyhydroxyaryl alkyl
ketones as substrates. Synthesis of enantiomerically
pure heterocyclic polyphenolic compounds can be
achieved either by the use of enantiomerically pure
substrates or by the resolution of final racemic prod-
ucts. The use of enzymes in a synthetic sequence
provides unique advantages of efficiency, stereospeci-
ficity and environmental friendliness (Walsh 2001;
Drauz & Waldmann 2002; Bommarius & Riebel
& Gilardi 2004; Chica et al. 2005; Khersonsky
et al. 2006; Rubin-Pitel & Zhao 2006).We have pre-
viously demonstrated the capabilities of lipases from
porcine pancreas (PPL), Candida rugosa (CRL),
Candida antarctica (CAL) and Pseudomonas spp. for
carrying out the selective protection/deprotection of
hydroxyl groups in different classes of compounds,
such as aryl alkyl ketones (Kumar et al. 1998; Prasad
et al. 1999; Kumar et al. 2001), hydroxymethylated
phenolic compounds (Parmar et al. 1999), triazolyl
sugars (Prasad et al. 2002; Bhattacharya et al. 2003),
benzoxazines (Shakil et al. 2003), dihydrocoumarins
(Singh et al. 2003), chromanones (Poonam et al. 2001)
and modified sugar molecules (Prasad et al. 2007;
Maity et al. 2008). It has been observed by us that
PPL in tetrahydrofuran (THF) mediates the deacety-
lation of all other acetoxy groups except the one at
the ortho or peri position(s) to the nuclear carbonyl
Correspondence: V. S. Parmar, Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi–110 007, India. Tel/Fax: ϩ91-11-27667206.
E-mail: virparmar@gmail.com
(Received 28 May 2009; revised 21 December 2009; accepted 26 February 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/10242421003734704

Enantioselective biocatalytic reactions with native and modified PPL 173
group in chalcones (Parmar et al. 1992a), acetophe-
USA) at 300 and 75.5 MHz, respectively, using
TMS as an internal standard. The chemical shift
values are on the δ scale and the coupling constants
(J) are in Hz. Optical rotations were measured with
a Bellingham–Stanley (Kent, UK) AD 220 pola-
rimeter. The accurate masses were determined by
measuring FAB-HRMS spectra on a JEOL (Tokyo,
Japan) JMS-AX505W high-resolution mass spec-
trometer in positive mode using the matrix HEDS
(bishydroxydiethylsulfide) doped with sodium acetate
or on a Kratos (Manchester, UK) MS50TC instru-
ment in the EI mode at a resolution of 10 000. The
Raman spectra of different samples were recorded
on a Thermo Electron Corporation (Waltham, MA,
USA), data handling NXR FT-Raman module cou-
pled to Nicolet (Waltham, MA, USA) 5700 FT-IR
Omnic version 7.3 software using laser power of
1.0 W. The enzyme, PPL (porcine pancreatic lipase,
Type-II), was purchased from Sigma Chemical Co.
(St Louis, MO, USA) and used after storing in vacuo
over P2O5 for 12 h. THF was redistilled and dried
over molecular sieves (4 Å). TLC was carried out on
commercially available Merck (Darmstadt, Germany)
silica gel 60F254 plates.
nones (Parmar et al. 1992b), desoxybenzoins (Parmar
et al. 1998b) and flavones (Parmar et al. 1993). To
explain the mechanism of selective deacetylation
catalyzed by PPL, we postulated that the nuclear
carbonyl group present in the substrate is engaged
in the formation of a transient (dynamic) Schiff's
base-type complex with the ξ-amino function of the
lysine residue present in the active site of PPL (by
analogy to the human pancreatic lipase). The for-
mation of this complex causes the ortho/peri-acetoxy
function to be embedded under the hydrophobic bulk
of the active site of the enzyme and the serine-OH of
the lipase takes part in deacetylation of other more
suitably placed acetoxy function(s) in the same mol-
ecule (Bisht et al. 1996). However, no direct proof for
this mechanism could be given as the structure of the
active site of PPL is not known and also because the
Schiff's base formation, being a transient (dynamic)
process, could not be verified. However, a similar
formation of Schiff's base at the ξ-amino function of
the lysine residue present in the active site of CRL
was later reported by Weber et al. (1997). Esters and
amides of aromatic hydroxy acids do not form Schiff's
bases easily; consequently the incubation of esters and
amides of polyacetoxy aromatic acids with PPL leads
to the formation of ortho hydroxy compounds together
with other possible products (Parmar et al. 1997,
1998a). These findings substantiate our hypothesis
on the presence of lysine in the active site of the PPL
and the formation of the Schiff's base-type complexes
during deacetylation of peracetates of polyphenolic
compounds bearing a nuclear carbonyl group.
General procedure for preparation of (Ϯ)-2,
4-dihydroxyphenyl alkyl ketones, 2a–2d (Scheme 1)
The heterogeneous mixture of fused ZnCl2 (2 g, 15
mmol) and the racemic acid (1a–1d, 10 mmol) was
heated slowly with stirring to make the mixture
homogeneous. Resorcinol (1.1 g, 10 mmol) was
added and the reaction mixture was kept for about
2 h at 150°C, poured onto crushed ice containing
hydrochloric acid (1:1) and extracted with dichlo-
romethane (2ϫ50 mL).The composition is v/v.The
organic layer was washed with sodium bicarbonate
solution (5%), the % composition is w/v, dried over
anhydrous Na2SO4 and the solvent removed under
reduced pressure. The residue thus obtained was
purified by column chromatography using petro-
leum ether–ethyl acetate as eluting solvent to afford
racemic 2,4-dihydroxyphenyl alkyl ketones 2a–2d as
viscous oils in 50–55% yields (Shakil et al. 2001).
The composition of the petroleum ether-ethyl ace-
tate mixture is: ‘90/10 (v/v) throughout the text.
Earlier we reported the deacetylation of 2,4-
diacetoxyphenyl alkyl ketone catalyzed by modified
PPL (pre-incubated with acetophenone for 1 h) in con-
firmation of our hypothesis on dynamic Schiff's base
complex formation (Shakil et al. 2001). We report
herein the expanded study to prove our dynamic Schiff's
base complex formation hypothesis by studying the
Raman spectra of native and modified PPL. Further,
deacylation reactions on a series of novel (Ϯ)-2,4-
diacyloxyphenyl alkyl ketones by modified PPL have
been carried out, providing access to a large number
of enantiomerically enriched, useful novel synthons.
Experimental
General procedure for acetylation of (Ϯ)-2,
4-dihydroxyphenyl alkyl ketones, 2a–2d: preparation of
(Ϯ)-diacetates, 3a–3d (Scheme 1)
Melting points were determined either on a Mettler
FP62 (Columbus, OH, USA) instrument or in a sul-
furic acid bath and are uncorrected. The IR spectra
were recorded on a Perkin–Elmer (Waltham, MA,
USA) model 2000 FT-IR spectrophotometer. The
1H NMR and 13C NMR spectra were recorded on
a Bruker Avance 300 spectrometer (Billerica, MA,
(Ϯ)-2,4-Dihydroxyphenyl alkyl ketone (2a–2d, 3
mmol) was stirred with acetic anhydride and a cata-
lytic amount of N,N-dimethylaminopyridine (DMAP)
at 22–25°C.The reaction was monitored periodically

174
M. Husain et al.
by TLC and, on completion, poured into ice-cold
water (50 mL). The compound was extracted with
dichloromethane (2ϫ25 mL), the combined organic
layer dried over anhydrous Na2SO4 and the solvent
removed under reduced pressure.The crude product
thus obtained was purified by column chromatogra-
phy using petroleum ether–ethyl acetate as eluent to
afford (Ϯ)-2,4-diacetoxyphenyl alkyl ketones 3a–3d
as viscous oils in 90–95% yields (Shakil et al. 2001).
optical rotation values are given in Table I.
All of the monoacetates 4a–4d were charac-
terized on the basis of their spectral data and
also by comparing their spectral data with
those given in the literature. The spectral
dataof(ϩ)-2,4-diacetoxyphenylalkylketones
3a–3d were found to be identical to the cor-
responding data of (Ϯ)-2,4-diacetoxyphenyl
alkyl ketones 3a–3d synthesized by acetyla-
tion of (Ϯ)-2,4-dihydroxyphenyl alkyl
ketones 2a–2d and also found to be identical
to spectral data given in the literature (Shakil
et al. 2001).
General procedure for deacetylation of (Ϯ)-2,
4-diacetoxyphenyl alkyl ketones (3a–3d) mediated by
modified porcine pancreatic lipase
(2) After 50% conversion of the starting materi-
als 3a–3d into the products, the reaction was
quenched by filtering off the enzyme, the sol-
vent was removed under reduced pressure
and the crude products were purified by col-
umn chromatography over silica gel using a
gradient solvent system of chloroform–
acetone to afford the optically enriched, (ϩ)-
2,4-diacetoxyphenyl alkyl ketones 3a–3d,
mono-deacetylated (–)-2-acetoxy-4-hydrox-
yphenyl alkyl ketones 4a–4d and the (ϩ)-2,
4-dihydroxyphenyl alkyl ketones 2a–2d;
yields and optical rotation values are given in
Table II. All of the monoacetates 4a–4d, dia-
cetates 3a–3d and dihydroxy ketones 2a–2d
were characterized on the basis of their spec-
tral data and also by comparison with spec-
tral data given in the literature (Shakil et al.
2001).
To a solution of acetophenone (0.25 g, 2 mmol) in
dry THF (30 mL), PPL (250 mg) was added and
the suspension was stirred at 40–42°C for 1 h in an
incubator
shaker.
A
solution
of
(Ϯ)-2,
4-diacetoxyphenyl alkyl ketone (3a–3d, 2 mmol) in
THF (10 mL) was added to the incubated suspen-
sion, followed by the addition of n-butanol (5 molar
equivalents) and stirring continued at the same tem-
perature.The reaction was stopped at three different
stages (Scheme 2).
(1) When the dihydroxy compounds just started
to appear; reaction was quenched by filtering
off the enzyme, solvent was removed under
reduced pressure and the crude product was
purified by column chromatography over
silica gel using a gradient solvent system of
chloroform–acetone (The composition of
the chloroform-acetone mixture is ‘85/15
(v/v) throughout the text) to afford the opti-
(3) After total conversion of starting materials
3a–3d into the products, the reaction mix-
ture was quenched by filtering off the enzyme
and the solvent removed under reduced pres-
sure; the crude products were purified by
cally
enriched,
recovered
(ϩ)-2,4-
diacetoxyphenyl alkyl ketones 3a–3d and the
mono-deacetylated (–)-2-acetoxy-4-hydrox-
yphenyl alkyl ketones 4a–4d; yields and
Table I.Time, optical rotation [α]D25 values, yields and estimated enantiomeric excess (ee) valuesa of modified PPL-catalyzed deacetylation
products (at the stage when the dihydroxy compounds just started to appear)b, Scheme 2.
(ϩ)-Diacetates
(–)-Partial acetates
3a–3d
4a–4d
(Ϯ)-Di-acetates
3
Time (min)
[α]D25 (CHCl3)
%Yieldc
[α]D25 (CHCl3)
%Yieldc
a
b
c
50
60
40
45
56
55
52
59
(–)24.44 (c 1.0)
(–)19.12 (c 1.0)
(–)17.28 (c 1.0)
(–)18.25 (c 1.0)
(–)Ϫ4d, estimated
ee valuea: 56%
20
28
25
21
(ϩ)11.33 (c 1.0)
(ϩ)15.18 (c 1.0)
(ϩ)12.15 (c 1.0)
(ϩ)13.15 (c 1.0)
(ϩ)Ϫ3d, estimated
ee valuea: 52%
d
aee values of compounds 3d and 4d were estimated by comparing the sign and specific rotation values of the corresponding optically pure
(R)- and (S)-ketones.
bAll these reactions, when performed under identical conditions, but without adding native or modified PPL, did not yield any
product.
cYields were calculated by assuming corresponding single enantiomer as 100% in the starting substrate.

Enantioselective biocatalytic reactions with native and modified PPL 175
Table II.Time, optical rotation [α]D25 values yields and estimated enantiomeric excess (ee) valuesa of modified PPL-catalyzed deacetylation
products (at the stage of 50% conversion of substrate)b, Scheme 2.
(ϩ)-Dihydroxy compounds
2a–2d
(ϩ)-Diacetates
3a–3d
(–)-Partial acetates
4a–4d
(Ϯ)-Di-acetates
3
Time (h)
[α]D25 (CHCl3)
%Yieldc
[α]D25 (CHCl3)
%Yieldc
[α]D25 (CHCl3)
%Yieldc
3a
3b
3c
3d
3.0
3.2
3.2
3.0
16
18
14
15
42
46
44
45
(–)26.62 (c 0.8)
(–)24.02 (c 0.8)
(–)20.12 (c 0.8)
(–)25.12 (c 0.8)
(–)-4d, estimated
ee valuea: 75%
24
23
28
29
(ϩ)19.14 (c 0.8)
(ϩ)18.25 (c 0.8)
(ϩ)25.76 (c 0.8)
(ϩ)31.57 (c 0.8)
(ϩ)-2d, estimated
ee valuea: 90%
(ϩ)15.18 (c 1.0)
(ϩ)16.78 (c 1.0)
(ϩ)16.18 (c 1.0)
(ϩ)17.25 (c 1.0)
(ϩ)-3d, estimated
ee valuea: 67%
aee values of compounds 2d, 3d and 4d were estimated by comparing the sign and specific rotation values of the corresponding optically
pure (R)- and (S)-ketones.
bAll these reactions, when performed under identical conditions, but without adding native or modified PPL, did not yield any
product.
cYields were calculated by assuming corresponding single enantiomer as 100% in the starting substrate.
column chromatography over silica gel using
a gradient solvent system of chloroform–
acetone to afford the optically enriched (ϩ)-
2,4-dihydroxyphenyl alkyl ketones 2a–2d
and the mono-deacetylated (–)-2-acetoxy-4-
hydroxyphenyl alkyl ketones 4a–4d; yields
and optical rotation values are given inTable
III. All of the monoacetates 4a–4d and the
dihydroxy ketones 2a–2d were characterized
on the basis of their spectral data and also by
comparison with spectral data given in the
literature (Shakil et al. 2001).
periodically by TLC and, on completion, poured
into ice-cold water (50 mL). The compound thus
obtained was extracted with dichloromethane
(2ϫ25 mL), the combined organic layer dried
over anhydrous Na2SO4 and the solvent removed
under reduced pressure. The products were puri-
fied by column chromatography using petroleum
ether–ethyl acetate as eluting solvent to afford the
(Ϯ)-2,4-diacyloxyphenyl (1-phenyl)propyl ketones
6a–6c.
(Ϯ)-2,4-Dibutanoyloxyphenyl
(1-phenyl)propyl
ketone (6a) was obtained as a viscous oil in 92% yield.
IR (Nujol, cm–1): 1768 (O–CϭO); 1703 (CϭO). 1H
NMR (300 MHz, CDCl3), δ (ppm): 0.87 (3H, t,
Jϭ7.3 Hz, C-3′H); 0.9–1.06 (6H, m, C-4′′′H); 1.68–
1.78 (5H, m, C-2′Ha and C-3′′′H); 2.09–2.16 (1H,
m, C-2′Hb); 2.40–2.50 (4H, m, C-2′′′H); 4.20 (1H,
t, Jϭ7.3 Hz, C-1′H); 6.88 (1H, d, Jϭ2.2 Hz, C-3H);
6.96 (1H, dd, Jϭ6.3 and 2.2 Hz, C-5H); 7.20–7.30
(5H, m, aromatic protons); 7.60 (1H, d, Jϭ8.6 Hz,
C-6H). 13C NMR (75.5 MHz, CDCl3), δ (ppm):
General procedure for preparation of (Ϯ)-2,
4-diacyloxyphenyl (1-phenyl)propyl ketones, 6a–6c
(Scheme 3)
(Ϯ)-2,4-Dihydroxyphenyl (1-phenyl)propyl ketone
(2d, 3 mmol) was stirred with the correspond-
ing acid anhydride 5a–5c (6.5 mmol) in pyridine
(10 mL) at 40–45°C. The reaction was monitored
Table III.Time, optical rotation [α]D25 values, yields and estimated enantiomeric excess (ee) valuesa of modified PPL-catalyzed deacetylation
products (at the stage of total conversion of substrate)b, Scheme 2.
(ϩ)-Dihydroxy compounds
2a–2d
(–)-Partial acetates
4a–4d
(Ϯ)-Diacetates
3
Time (h)
[α]D25 (CHCl3)
%Yieldc
[α]D25 (CHCl3)
%Yieldc
3a
3b
3c
3d
15
16
14
13
59
60
61
58
(–)40.00 (c 0.8)
(–)25.16 (c 0.8)
(–)28.30 (c 0.8)
(–)32.66 (c 0.8)
(–)-4d, estimated
ee valuea: 80%
7
8
8
8
(ϩ)24.81 (c 0.8)
(ϩ)20.68 (c 0.8)
(ϩ)23.20 (c 0.8)
(ϩ)28.18 (c 0.8)
(ϩ)-2d, estimated
ee valuea: 80%
aee values of compounds 2d and 4d were estimated by comparing the sign and specific rotation values of the corresponding optically pure
(R)- and (S)-ketones.
bAll these reactions, when performed under identical conditions, but without adding native or modified PPL, did not yield any
product.
cYields were calculated by assuming corresponding single enantiomer as 100% in the starting substrate.

176
M. Husain et al.
(10 mL) at 30–35°C. The reaction was monitored
periodically by TLC and, on completion, poured
into ice-cold water (50 mL). The compound thus
obtained was extracted with dichloromethane (2ϫ25
mL), the combined organic layer dried over anhy-
drous Na2SO4 and the solvent removed under
reduced pressure; the product was purified by
column chromatography using petroleum ether–
ethyl acetate as eluting solvent to afford (Ϯ)-2,4-
dicyclohexanoylxyphenyl (1-phenyl)propyl ketone
(6d) as a viscous oil in 96% yield. IR (Nujol, cm–1):
1760 (O–CϭO); 1688 (CϭO). 1H NMR (300 MHz,
CDCl3), δ (ppm): 0.86 (3H, t, Jϭ7.35 Hz, C-3′H);
1.22–2.21 (22H, m, cyclohexyl rings protons
and C-2′H); 2.50–2.54 (2H, m, C-1′′′H); 4.19 (1H,
t, Jϭ7.21 Hz, C-1′H); 6.84 (1H, d, Jϭ2.11 Hz,
C-3H); 6.92 (1H, dd, Jϭ6.43 and 2.10 Hz, C-5H);
7.20–7.30 (5H, m, aromatic protons); 7.55 (1H, d,
Jϭ8.55 Hz, C-6H). 13C NMR (75.5 MHz, CDCl3),
δ (ppm): 12.08 (C-3′); 25.18 and 25.65 (2ϫC-3′′′,
2ϫC-4′′′ and 2ϫC-5′′′); 26.80 (C-2′); 28.73
(2ϫC-2′′′ and 2ϫC-6′′′); 43.08 (2ϫC-1′′′);
58.51 (C-1′); 115.32 (C-1); 117.13 (C-3); 118.51
(C-5); 127.03, 128.72 and 129.45 (C-2″, C-3″,
C-4″, C-5″ and C-6″); 130.17 (C-6); 138.76
(C-1″); 149.70 and 153.30 (C-2 and C-4); 173.41
and 173.79 (2ϫOCO); 199.57 (CϭO). FAB-
HRMS: 476.2570 found. Calc. for C30H36O5:
476.2563.
12.21 (C-3′); 13.52 (2ϫC-4′′′); 18.08 (2ϫC-3′′′);
26.96 (C-2′); 36.93 (2ϫC-2′′′); 58.38 (C-1′); 115.94
(C-1); 117.27 (C-3); 118.64 (C-5); 127.05, 128.48
and129.11(C-2″,C-3″,C-4″,C-5″andC-6″);130.39
(C-6); 138.84 (C-1″); 149.74 and 153.23 (C-2 and
C-4); 170.98 and 171.53 (2ϫOCO); 199.45 (CϭO).
FAB-HRMS, m/z: 419.1839 ([MϩNa]ϩ). Calc. for
C24H28O5ϩNa: 419.1834.
(Ϯ)-2,4-Diheptanoyloxyphenyl (1-phenyl)propyl
ketone (6b) was obtained as a viscous oil in 92%
yield. IR (Nujol, cm–1): 1768 (O–CϭO); 1706
1
(CϭO). H NMR (300 MHz, CDCl3), δ (ppm):
0.84–0.91 (9H, m, C-3′H and C-7′′′H); 1.25–1.35
(12H, m, C-3′′′H, C-4′′′H and C-5′′′H); 1.65–1.81
(5H, m, C-2′Ha and C-6′′′H); 2.11–2.16 (1H, m,
C-2′Hb); 2.48–2.58 (4H, m, C-2′′′H); 4.20 (1H, t,
Jϭ7.3 Hz, C-1′H); 6.87 (1H, d, Jϭ2.0 Hz, C-3H);
6.95 (1H, dd, Jϭ6.4 and 2.0 Hz, C-5H); 7.20–7.28
(5H, m, aromatic protons); 7.59 (1H, d, Jϭ8.5 Hz,
C-6H). 13C NMR (75.5 MHz, CDCl3), δ (ppm):
12.56 (C-3′); 14.40 (2ϫC-7′′′); 22.85 (2ϫC-6′′′);
24.98 (2ϫC-3′′′); 27.41 (C-2′); 29.17 (2ϫC-4′′′);
31.81 (2ϫC-5′′′); 34.65 (2ϫC-2′′′); 58.90 (C-1′);
115.50 (C-1); 117.70 (C-3); 119.07 (C-5); 127.51,
128.52 and 129.33 (C-2″, C-3″, C-4″, C-5″ and
C-6″); 130.83 (C-6); 139.30 (C-1″), 150.22 and
153.72 (C-2 and C-4); 171.62 and 172.15
(2ϫOCO); 199.95(CϭO). FAB-HRMS, m/z:
503.2771 ([MϩNa]ϩ ). Calc. for C30H40O5ϩNa:
503.2773.
(Ϯ)-2,4-Di-α-naphthoyloxyphenyl
(1-phenyl)
propyl ketone (6e) was prepared as 6d (except that
the acid chloride 5e was used). It was obtained as a
viscous oil in 93% yield. IR (Nujol, cm–1): 1738
(Ϯ)-2,4-Dibenzoyloxyphenyl
(1-phenyl)propyl
ketone (6c) was obtained as a white solid in 94%
yield;mp 87–89°C. IR (KBr, cm–1):1745 (O–CϭO);
1677 (CϭO). 1H NMR (300 MHz, CDCl3), δ
(ppm): 0.87 (3H, t, Jϭ7.0 Hz, C-3′H); 1.80–1.87
(1H, m, C-2′Ha); 2.14–2.23 (1H, m, C-2′Hb); 4.31
(1H, t, Jϭ6.9 Hz, C-1′H); 6.87 (1H, d, Jϭ2.0 Hz,
C-3H); 6.95 (1H, dd, Jϭ6.4 and 2.0 Hz, C-5H);
7.20–7.75 and 8.22 (16H, 2m, 12H and 4H each,
aromatic protons and C-6H). 13C NMR (75.5 MHz,
CDCl3), δ (ppm): 12.50 (C-3′); 27.16 (C-2′); 59.26
(C-1′); 115.45 (C-1); 117.82 (C-3); 119.23 (C-5);
127.51, 128.78, 129.04, 129.19, 130.42 and 130.62
(C-2″, C-3″, C-4″, C-5″, C-6″, 2ϫC-2′′′, 2ϫC-3′′′,
2ϫC-4′′′, 2ϫC-5′′′ and 2ϫC-6′′′); 130.84 (C-6);
134.17 and 134.33 (2ϫC-1′′′); 139.00 (C-1″);
150.13 and 153.82 (C-2 and C-4); 161.12 and
161.48 (2ϫOCO); 200.15 (CϭO). FAB-HRMS,
m/z:487.1560 ([MϩNa]ϩ).Calc.for C30H24O5ϩNa:
487.1521.
1
(O–CϭO); 1687 (CϭO). H NMR (300 MHz,
CDCl3), δ (ppm): 0.79 (3H, t, Jϭ7.32 Hz, C-3′H);
1.71–1.79 (1H, m, C-2′Ha); 2.12–2.14 (1H, m,
C-2′Hb); 4.32 (1H, t, Jϭ7.23 Hz, C-1′H); 7.17–
7.30 (7H, m, C-3H, C-5H, C-2″H, C-3″H, C-4″H,
C-5″H and C-6″H); 7.53–7.80 (6H, m, C-3′′′H,
C-6′′′H and C-7′′′H); 7.90–7.94 (2H, m, C-5′′′H);
8.09–8.19 (3H, m, C-6H and C-4′′′H); 8.48 (2H,
dd, Jϭ9.90 and 7.34 Hz, C-2′′′H); 9.01 (2H, d,
Jϭ8.52 Hz, C-8′′′H). 13C NMR (75.5 MHz,
CDCl3), δ (ppm): 12.03 (C-3′); 26.80 (C-2′); 58.70
(C-1′); 115.50 (C-1); 117.95 (C-3); 119.52 (C-5);
124.52, 125.73, 126.31, 127.03, 127.51, 128.12,
128.78, 129.04, 129.19, 130.42, 130.57, 130.62
and 131.61 (C-2″, C-3″, C-4″, C-5″, C-6″, 2ϫC-
1′′′, 2ϫC-2′′′, 2ϫC-3′′′, 2ϫC-4′′′, 2ϫC-5′′′, 2ϫC-
6′′′, 2ϫC-7′′′ and 2ϫC-8′′′); 131.84 and 133.86
(2ϫC-10′′′ and C-6); 134.17 (2ϫC-9′′′); 138.62
(C-1″); 150.33 and 153.57 (C-2 and C-4); 161.12
and 161.48 (2ϫOCO); 200.15 (CϭO). FAB-
HRMS: 564.1930 found. Calc. for C38H28O5:
564.1937.
(Ϯ)-2,4-Dicyclohexanoyloxyphenyl (1-phenyl)pro-
pyl ketone (6d). (Ϯ)-2,4-Dihydroxyphenyl (1-phe-
nyl)propyl ketone (2d, 3 mmol) was stirred with
cyclohexanoyl chloride 5d (6.5 mmol) and trieth-
ylamine (Et3N) (6.5 mmol) in dichloromethane

Enantioselective biocatalytic reactions with native and modified PPL 177
13C NMR (75.5 MHz, CDCl3), δ (ppm):12.58 (C-3′);
General procedure of enzymatic deacylation of (Ϯ)-2,
14.39 (C-7′′′); 22.88 (C-6′′′); 24.90 (C-3′′′); 27.47
(C-2′); 29.17 (C-4′′′); 31.86 (C-5′′′); 34.71 (C-2′′′);
57.87 (C-1′); 111.67 (C-3); 113.28 (C-5); 123.77
(C-1); 127.33, 128.59 and 129.14 (C-2″, C-3″, C-4″,
C-5″ and C-6″); 132.35 (C-6); 140.01 (C-1″); 151.49
and 160.47 (C-4 and C-2); 173.53 (OCO); 199.19
(CϭO). FAB-HRMS, m/z: 391.1893 ([MϩNa]ϩ).
Calc. for C23H28O4ϩNa: 391.1885.
(R)-(–)-2-Benzoyloxy-4-hydroxyphenyl (1-phenyl)
propyl ketone (7c) was obtained as a viscous oil in
68% yield. [α]D25: –30.25 (c 0.4, CHCl3). IR (Nujol,
cm–1): 3345 (OH); 1750 (O–CϭO); 1670 (CϭO).
1H NMR (300 MHz, CDCl3), δ (ppm): 0.78 (3H,
t, Jϭ7.0 Hz, C-3′H); 1.65–1.75 (1H, m, C-2′Ha);
2.01–2.11 (1H, m, C-2′Hb); 4.21 (1H, t, Jϭ6.8 Hz,
C-1′H); 6.41–6.48 (2H, m, C-3H and C-5H); 7.18–
7.50 (8H, m, aromatic protons); 7.60 (1H, d, Jϭ6.9
Hz, C-6H); 8.13–8.18 (2H, m, aromatic protons).
13C NMR (75.5 MHz, CDCl3), δ (ppm): 12.54
(C-3′); 27.31 (C-2′); 58.17 (C-1′); 111.74 (C-3);
113.64 (C-5);124.09 (C-1);127.35, 128.75, 129.02,
129.61, 130.57 and 130.84 (C-2″, C-3″, C-4″, C-5″,
C-6″, C-2′′′, C-3′′′, C-4′′′, C-5′′′ and C-6′′′); 132.27
(C-6); 134.21 (C-1′′′;, 139.78 (C-1′); 151.28 and
160.67 (C-4 and C-2); 166.31 (OCO); 199.59
(CϭO). FAB-HRMS, m/z: 383.1245 ([MϩNa]ϩ).
Calc. for C23H20O4ϩNa: 383.1259.
(R)-(–)-2-Cyclohexanoylxy-4-hydroxyphenyl
(1-phenyl)propyl ketone (7d) was obtained as a vis-
cous oil in 63% yield. [α]D25: –25.78 (c 0.4, CHCl3).
IR (Nujol, cm–1): 3350 (OH); 1761 (O–CϭO);
1700 (CϭO). 1H NMR (300 MHz, CDCl3), δ
(ppm): 0.86 (3H, t, Jϭ7.31 Hz, C-3′H); 1.25–2.11
(12H, m, cyclohexyl ring protons and C-2′H); 2.50–
2.54 (1H, m, C-1′′′H); 4.19 (1H, t, Jϭ7.2 Hz,
C-1′H); 6.34–6.38 (2H, m, C-3H and C-5H); 7.21–
7.30 (5H, m, aromatic protons); 7.45 (1H, d, Jϭ8.4
Hz, C-6H). 13C NMR (75.5 MHz, CDCl3), δ (ppm):
12.13 (C-3′); 25.18 and 25.56 (C-3′′′, C-4′′′ and
C-5′′′); 26.76 (C-2′); 28.74 (C-2′′′ and C-6′′′); 43.09
(C-1′′′); 54.86 (C-1′); 111.16 (C-3); 112.69 (C-5);
116. 97 (C-1); 127.23, 128.02 and 128.92 (C-2″,
C-3″, C-4″, C-5″ and C-6″); 131.17 (C-6); 139.12
(C-1″); 156.70 and 164.67 (C-4 and C-2); 173.44
(OCO); 205.21 (CϭO). FAB-HRMS: 366.1845
found. Calc. for C23H26O4: 366.1831.
4-diacyloxyphenyl (1-phenyl)propyl ketones 6a–6e by
native porcine pancreatic lipase (Scheme 4)
To a solution of the (Ϯ)-2,4-diacyloxyphenyl (1-phe-
nyl)propyl ketones (6a–6e, 2 mmol) in anhydrous
THF (20–25 mL), n-butanol (5 equivalents) was
added, followed by the addition of native PPL (300
mg). The suspension was incubated in a shaker at
40–42°C and the progress of the reaction was mon-
itored periodically by HPLC and/or TLC examina-
tion. After about 50% conversion of the starting
material into the product, the reaction mixture was
quenched by filtering off the enzyme and the solvent
removed under reduced pressure to afford a thick oil
that was purified by column chromatography over
silica gel using a gradient solvent system of chloro-
form–acetone to afford optically enriched, (S)-(ϩ)-
2,4-diacyloxyphenyl (1-phenyl)propyl ketones 6a–6d
and the mono-deacylated (R)-(–)-2-acyloxy-4-hy-
droxyphenyl (1-phenyl)propyl ketones 7a–7d in
65–70% and 60–68% yields, respectively (Table V).
The spectral data of (S)-(ϩ)-2,4-diacyloxyphenyl
(1-phenyl)propyl ketones 6a–6d were found to be
identical with the data of (ϩ)-2,4-diacyloxyphenyl
(1-phenyl)propyl ketones 6a–6d given above.
(R)-(–)-2-Butanoylxy-4-hydroxyphenyl (1-phenyl)
propyl ketone (7a) was obtained as a viscous oil in
60% yield. [α]D25 : –31.42 (c 0.4, CHCl3). IR (Nujol,
cm–1): 3305 (OH); 1731 (O-CϭO); 1670 (CϭO).
1H NMR (300 MHz, CDCl3), δ (ppm): 0.84 (3H,
t,Jϭ7.3 Hz,C-3′H);1.01 (3H,t,Jϭ7.3 Hz,C-4′′′H);
1.72–1.79 (3H, m, C-2′Ha and C-3′′′H); 2.07–2.09
(1H, m, C-2′Hb); 2.58 (2H, t, Jϭ7.4 Hz, C-2′′′H);
4.20 (1H, t, Jϭ7.2 Hz, C-1′H); 6.88 (1H, d, Jϭ2.2
Hz, C-3H); 6.99 (1H, dd, Jϭ6.4 and 2.14 Hz,
C-5H); 7.18–7.30 (5H, m, aromatic protons); 7.42
(1H, d, Jϭ9.0 Hz, C-6H). 13C NMR (75.5 MHz,
CDCl3), δ (ppm): 12.45 (C-3′); 14.01 (C-4′′′); 18.43
(C-3′′′); 27.46 (C-2′); 36.50 (C-2′′′); 57.81 (C-1′);
111.7 (C-3); 113.77 (C-5); 123.42 (C-1); 127.36,
128.57 and 129.17 (C-2″, C-3″, C-4″, C-5″ and
C-6″); 132.04 (C-6); 139.94 (C-1″); 151.45 and
160.89(C-4andC-2);173.70(OCO);199.57(CϭO).
FAB-HRMS, m/z: 349.1439 ([MϩNa]ϩ). Calc. for
C20H22O4ϩNa: 349.1416.
(R)-(–)-2-Heptanoyloxy-4-hydroxyphenyl (1-phenyl)
propyl ketone (7b) was obtained as a viscous oil in 62%
yield. [α]D25: –26.32 (c 0.4, CHCl3). IR (Nujol, cm–1):
3367 (OH); 1734 (O–CϭO); 1675 (CϭO). 1H NMR
(300 MHz, CDCl3), δ (ppm): 0.83–0.93 (6H, m,
C-3′H and C-7′′′H); 1.25–1.43 (6H, m, C-3′′′H,
C-4′′′H and C-5′′′H); 1.69–1.78 (3H, m, C-2′Ha and
C-6′′′H); 2.06–2.13 (1H, m, C-2′Hb); 2.60 (2H, t,
Jϭ7.5 Hz, C-2′′′H); 4.20 (1H, t, Jϭ7.2 Hz, C-1′H);
6.37–6.40 (2H, m, C-3H and C-5H); 7.19–7.29 (5H,
m, aromatic protons); 7.49 (1H, d, Jϭ8.3 Hz, C-6H).
General procedure for chemical acylation of (R)-(–)
-2-acyloxy-4-hydroxyphenyl (1-phenyl)propyl ketones,
7a–7d
Compounds (–)-6a–6d were prepared by acylation of
thecorrespondingmonohydroxycompounds(–)-7a–7d
as discussed above in 98–99% yields.The spectral data
of the (R)-(–)-diacylates 6a–6d were identical to the
data of the (Ϯ)-diacylates 6a–6d reported above.

178
M. Husain et al.
Table IV. Time, yields and estimated enantiomeric excess (ee) valuesa of modified PPL-catalyzed deacylation products of (Ϯ)-6a–6d
(at the stage of 50% conversion of substrate)b, Scheme 3.
Dihydroxy compound
Diacylates
Partial acylates
2d
6a–6d
7a-7d
(Ϯ)-Diacyloxy
ketones
6
Estimated
ee valuea (%)
Estimated
ee valuea (%)
Estimated
ee valuea (%)
Time (h)
%Yieldc
%Yieldc
%Yieldc
6a
6b
6c
6d
2.2
50
1.3
65
74
86
83
90
20
21
21
23
80
93
85
90
46
45
47
46
86
90
90
97
27
26
26
25
aee values of compounds 2d, 6a–6d and 7a–7d were estimated by comparing the sign and specific rotation values of the corresponding
optically pure (R)- and (S)-ketones.
bAll these reactions, when performed under identical conditions, but without adding native or modified PPL, did not yield any product.
cYields were calculated by assuming corresponding single enantiomer as 100% in the starting substrate.
General procedure for deacylation of (Ϯ)-2,4-
diacyloxyphenyl (1-phenyl)propyl ketones, 6a–6e,
mediated by modified porcine pancreatic lipase
(Scheme 3)
7a–7d and (S)-(ϩ)-2,4-dihydroxyphenyl (1-phenyl)
propyl ketone 2d; the yields, optical rotation values
and enantiomeric excess values of all the products are
given in Table IV.
To a solution of acetophenone (0.25 g, 2 mmol) in
dryTHF (30 mL), PPL (250 mg) was added and the
suspension was stirred at 40–42°C for 1 h in an incu-
bator shaker. A solution of (Ϯ)-2,4-diacyloxyphenyl
(1-phenyl)propyl ketones 6a–6e (2 mmol) in THF
(10 mL) was added into the incubated suspension,
followed by the addition of n-butanol (5 molar equiv-
alents) and stirring continued at the same tempera-
ture.The reaction was stopped after 50% conversion
of substrate into products by filtering off the enzyme
and the solvent was removed under reduced pressure.
The crude product was purified by column chroma-
tography over silica gel using a gradient solvent sys-
tem of chloroform–acetone to afford optically
enriched, (S)-(ϩ)-2,4-diacyloxyphenyl (1-phenyl)
propyl ketones 6a–6d, mono-deacylated (R)-(–)-2-
acyloxy-4-hydroxyphenyl (1-phenyl)propyl ketones
Results and discussion
The racemic aryl alkyl ketones 2a–2d were prepared
by the Nencki reaction (Nencki & Sieber 1881) of
resorcinol with the corresponding racemic aliphatic
acids 1a–1d in the presence of fused ZnCl2 at 150°C
in 50–55% yields; the diacetates 3a–3d were pre-
pared by acetylation of 2a–2d using the acetic
anhydride/DMAP method in quantitative yields
(Scheme 1). All of the (Ϯ)-dihydroxyphenyl alkyl
ketones 2a–2d and the (Ϯ)-diacetoxyphenyl alkyl
ketones 3a–3d were characterized by comparing
their spectral data with those given in the literature
(Shakil et al. 2001). We have earlier reported the
lack of selectivity of modified PPL during the
deacetylation of 2,4-diacetoxyphenyl methyl ketone
TableV.Time, yields and estimated enantiomeric excess (ee) valuesa of native PPL-catalyzed deacylated products and chemically acylated
products (50% conversion of substrate)b, Scheme 4.
(R)-(–)-2,4-Diacyloxyphenyl
(1-phenyl)propyl ketones
6a–6d obtained by chemical
(–)-2-Acyloxy-4-
hydroxyphenyl (1-phenyl)
propyl ketones 7a–7d
Recovered (S)-(ϩ)-2,4-
diacyloxyphenyl-(1-phenyl)
propyl ketones 6a–6d
acylation of enzymatically
catalyzed deacylation reaction
product (–)-7a–7d
Racemic substrate/
time (h) for 50%
conversion
Estimated
Estimated
Estimated
ee valuea (%)
%Yieldc
ee valuea (%)
%Yieldc
ee valuea (%)
%Yieldc
6a/5
6b/68
6c/2
96
94
97
60
62
68
63
92
89
93
65
66
70
65
96
94
97
98
99
98
98
6d/72
Ͼ99
Ͼ99
Ͼ99
aee values of compounds 6a–6d, 7a–7d and (–)-7a–7d and were estimated by comparing the sign and specific rotation values of the
corresponding optically pure (R)- and (S)-ketones.
bAll these reactions, when performed under identical conditions, but without adding native or modified PPL, did not yield any product.
cYields were calculated by assuming corresponding single enantiomer as 100% in the starting substrate.

Enantioselective biocatalytic reactions with native and modified PPL 179
HO
OH
HO
OH
Fused ZnCl2
R
R
R1 CH-COOH
+
0
R1
150 C
(+)-1a-1d
O
(+)-2a-2d
R1
R
1, 2 & 3
Ac2O
DMAP
-CH3
-CH3
-CH2CH3
a
b
c
-CH2CH2CH2CH3
AcO
OAc
R
-C6H5 -CH3
-C6H5 -CH2CH3
d
R1
O
(+)-3a-3d
Scheme 1. Synthesis of racemic diacyloxyphenyl alkyl ketones.
In order to study the changes occurring at the
active site of PPL on incubation with acetophenone,
we have recorded the Raman spectra of native PPL
and the modified PPL (i.e. PPL pre-treated with
acetophenone). A band at 3347.9 cm–1 (presumably
for the NH2 group of the lysine residue of the enzyme)
was observed in the Raman spectrum of native PPL,
which disappeared in the Raman spectrum of modi-
fied PPL and a new band at 1599.7 cm–1 was observed
due to the CϭN bond of the Schiff's base complex
(Dollish et al. 1974). Also a band at 1665.1 cm–1 in
the case of native PPL shifted to 1682.2 cm–1 in the
Raman spectrum of modified PPL. These observa-
tions indicate modifications at the active site of PPL
by involvement of acetophenone and form ation of a
Schiff's base complex between the nuclear carbonyl
group of the aryl alkyl ketone and the lysine residue
of the enzyme. As the structure of the active site of
PPL is not known, it is difficult to delineate which
lysine residue in PPL may be involved in Schiff's base
formation. However, the structure of human pancre-
atic lipase is known and contains an active-site lysine
residue which would be capable of forming a tran-
sient Schiff's bases with ketonic compounds as previ-
ously postulated (Stryer 1981; Bisht et al. 1996).
Interestingly, in the case of deacetylation reac-
tions on 3a–3d with modified PPL, there was no
indication of the formation of ortho-hydroxy-para-
acetoxy derivatives throughout the reaction.This may
be attributed to the dipole–dipole interactions
between the carbonyl groups of the ortho-acetoxy
moiety and that of the keto group attached to the
benzenoid ring (Figure 1), which resists deacylation
(Shakil et al. 2001). Herein, PPL was incubated
with acetophenone in THF at 40–42°C in an incu-
bator shaker for 1 h and this modified PPL was
used to catalyze the deacetylation of (Ϯ)-2,4-
diacetoxyphenyl alkyl ketones 3a–3d, leading to the
formation of completely deacetylated compounds
2a–2d, mono-deacetylated compounds 4a–4d
together with the remaining diacetoxy compounds
3a–3d at the stage of 50% conversion of the reac-
tants (Scheme 2).
The formation of dihydroxy ketones 2a–2d
indicates that PPL, which in its native form exclu-
sively mediates the deacetylation of the para-acetoxy
function of 2,4-diacetoxyphenyl alkyl ketones 3a–3d
over the ortho-acetoxy group with respect to the car-
bonyl group (Parmar et al. 1992a,b, 1993), does not
show any preference for the ortho- or para-acetoxy
functions when it is pre-incubated with acetophe-
none.This observation supports our proposed mech-
anism of action of PPL in THF involving formation
of a dynamic Schiff’s base complex between the
ξ-amino group of the lysine residue in the active site
of PPL and the keto group directly attached to the
benzenoid ring (Bisht et al. 1996). In the modified
PPL (pre-incubated with acetophenone), the ξ-amino
group of its lysine residue gets engaged in Schiff’s
base formation with the carbonyl group of acetophe-
none and therefore the PPL–acetophenone system
fails to differentiate between the ortho- and para-ace-
toxy functions of 2,4-diacetoxyphenyl alkyl ketones
3a–3d as the active-site lysine residue(s) of PPL may
no longer be available for Schiff's base formation with
the carbonyl group(s) of the substrates 3a–3d.

180
M. Husain et al.
OAc
AcO
R
R1
O
(+)-3a-3d
Modified PPL/THF
n-BuOH
40-42 oC
50 % conversion
of substrate
Total conversion
of substrate
Dihydroxy compound
started to appear
OH
OAc
HO
HO
OAc
R
AcO
HO
R
R
R
*
1
R1
*
* R1
O
(+)-3a-3d
O
O
(+)-2a-2d
(-)-4a-4d
+
+
OAc
R
AcO
+
OH
R
HO
OAc
R
* R1
R1
*
*
O
R1
O
(+)-3a-3d
O
(+)-2a-2d
(-)-4a-4d
OAc
R
HO
R1
*
R
R1
2, 3 & 4
O
-CH3 -CH2CH3
a
b
c
(-)-4a-4d
-CH3 -CH2CH2CH2CH3
-C6H5 -CH3
d
-C6H5 -CH2CH3
Scheme 2. Modified PPL catalyzed deacylation of racemic diacyloxyphenyl alkyl ketones stopped at different stages of reaction.
at the ortho position to some extent. However, these
interactions are not as strong as the covalent Schiff's
base; thus after the formation of monoacetates 4a–4d,
the ortho-acetoxy function also gets deacylated to give
the dihydroxy derivatives 2a–2d. The ketonic com-
pounds 2a–2d, 3a–3d and 4a–4d obtained during the
deacetylation of (Ϯ)-3a–3d catalyzed by modified
PPL were found to be optically active, which revealed
that the rates of deacetylation of acetoxy functions of
both enantiomers are not equal.
The deacetylation reactions on (Ϯ)-3a–3d were
terminated at three different stages to observe the
regio- and enantioselective control exhibited by
modified PPL (Scheme 2). At the stage when the
dihydroxy compounds had just started to form, two
products were obtained, i.e. monoacetates 4a–4d
(21–28%) and the recovered diacetates 3a–3d
(52–59%); all compounds of both series were opti-
cally active (Table I). In another set of experiments,
the reactions were stopped after 50% conversion of
the substrate; three products were obtained (Scheme
2), i.e. dihydroxy ketones 2a–2d (14–18%), mono-
acetylated ketones 4a–4d (23–29%) and the recov-
ered diacetates 3a–3d (42–46%). This result shows
that the modified PPL loses its regioselective con-
trol, but retains its enantioselective control as is
evident from the observation that all compounds of
the three series are optically active (Table II). In the
third set of experiments, we allowed the reaction to
O
δ+
O
O
O
O
O
O
O
δ-
O
δ-
δ+
O
Figure 1. Different orientations of carbonyls in 3a, indicating the
dipole–dipole interactions between the two carbonyl moieties.

Enantioselective biocatalytic reactions with native and modified PPL 181
R
O
4
R
O
HO
OH
C H
6
5
3
O
Pyridine/TEA, DCM
2
O
C H
6
5
1
(RCO)2O/RCOCl
5
3'
1'
O
6
5a-5e
2'
(+)-2d
O
(+)-6a-6e
R
5, 6 & 7
n-BuOH, 40-42 0C
3'''
4'''
2'''
Modified PPL, THF
-CH CH CH
a
2
2
3
R
O
R
O
R
-CH (CH ) CH
O
b
2
2 4
3
HO
OH
O
HO
C H
O
O
2''' 3'''
6
5
O
O
O
C H
6
5
1'''
4'''
c
C H
6
5
+
+
*
5'''
6'''
*
*
d
e
(S)-(+)-2d
(R)-(-)-7a-7d
(S)-(+)-6a-6d
2''' 3'''
1'''
4'''
10'''
5'''
9'''
8'''
6'''
7'''
Scheme 3. Modified PPL catalyzed deacylation of racemic diacyloxyphenyl alkyl ketones.
proceed to complete conversion of the substrate; at
this stage, only two products were obtained (Scheme
2), i.e. the dihydroxy ketones 2a–2d (58–61%) and
the monoacetylated ketones 4a–4d (7–8%) (Table
III). In the case of 3d, the enantiomeric excess (ee)
values were estimated by comparing the sign and
specific rotation values of the optically pure (R)-
and (S)-ketones for the reactions that were stopped
at three different stages, i.e. when the dihydroxy
compound just started to form, at the 50% con-
version stage and by running the reaction to total
conversion of the substrate. We noted that the esti-
mated ee value of the dihydroxy compound 2d (ee
90%) obtained at 50% conversion of 3d was higher
than the estimated ee value of dihydroxy compound
2d (ee 80%) obtained after complete deacylation
of the diacetate 3d. But the estimated ee value of
the monoacetate 4d was higher when we allowed
the reaction to proceed to complete conversion of
the diacetate 3d.
We have further extended this work to analyze
the enantio- and regioselectivity of modified PPL
R
R
R
O
R
O
R
O
O
O
HO
O
O
O
O
O
Native PPL, THF
C H
6
C H
6
C H
6
5
5
5
0
*
n-BuOH, 40-42 C
*
O
(R)-(-)-7a-7d
O
O
(S)-(+)-6a-6d
(+)-6a-6d
R
6 & 7
Pyridine/
TEA, DCM
(RCO) O/RCOCl
2
-CH2CH2CH3
a
-CH2(CH2)4CH3
b
R
O
R
O
c
O
O
C H
6
5
d
*
O
(R)-(-)-6a-6d
Scheme 4. Native PPL catalyzed deacylation of racemic diacyloxyphenyl alkyl ketones.

182
M. Husain et al.
by carrying out reactions on racemic diacyl deriva-
tives of 2d, i.e. (Ϯ)-2,4-dibutanoyloxyphenyl (1-phe-
nyl)propyl ketone (6a), (Ϯ)-2,4-diheptanoyloxyphenyl
i.e. (R)-(–)-2-butanoyloxy-4-hydroxyphenyl (1-phe-
nyl)propyl ketone (7a), (R)-(–)-2-heptanoyloxy-4-
hydroxyphenyl (1-phenyl)propyl ketone (7b), (R)-
(–)-2-benzoyloxy-4-hydroxyphenyl (1- phenyl)pro-
pyl ketone (7c) and (R)-(–)-2-cyclohexanoyloxy-4-
hydroxyphenyl (1-phenyl)propyl ketone (7d), and
the corresponding recovered diacyloxy compounds
6a–6d were separated by column chromatography.
The monoacyloxy and diacyloxy compounds 7a–7d
and 6a–6e are new compounds and were fully char-
acterized on the basis of their spectral data (IR,
1H NMR, 13C NMR and HRMS). All of the bio-
catalytic reaction products were found to be opti-
cally active (Tables IV and V) as indicated by their
observed optical rotations. In order to know their
absolute configuration and the extent of enanti-
oselectivity during the enzymatic deacylation reac-
tions, we synthesized the optically pure (R)- and
(S)-2,4-dihydroxyphenyl (1-phenyl)propyl ketone
through the nuclear acylation of resorcinol with the
(R)- and (S)-2-phenylbutanoic acids; both of these
were acylated by the corresponding anhydrides or
acid chloride/DMAP or Et3N methods to afford
the diacylated compounds. The ee values of enzy-
matically deacylated compounds were estimated by
comparing the sign and specific rotation values of
the optically pure (R)- and (S)-ketones and were
found to be in the range of 89–99% (Table V).
The acyl derivatives of (R)-/(S)-2,4-dihydroxy-
phenyl (1-phenyl)propyl ketone, i.e. dibutanoyl,
diheptanoyl, dibenzoyl and dicyclohexanoyl (R)-/(S)-
6a–6d, were prepared by using the corresponding
acid anhydride or cyclohexanoyl chloride in dichlo-
romethane in the presence of pyridine and Et3N,
(1-phenyl)propyl
ketone
(6b),
(Ϯ)-2,4-
dibenzoyloxyphenyl (1-phenyl)propyl ketone (6c),
(Ϯ)-2,4-dicyclohexanoyloxyphenyl (1-phenyl)propyl
ketone (6d) and (Ϯ)-2,4-dinaphthanoyloxyphenyl
(1-phenyl)propyl ketone (6e) (Scheme 3).
These diacyloxy compounds were then subjected
to enzymatic deacylation with modified PPL at 40–
42°C and the reaction stopped after 50% conversion
of the substrates 6a–6d, affording the corresponding
monoacylated, diacylated and dihydroxy ketones
(Scheme 3), thus supporting our hypothesis of the
formation of a Schiff’s base complex. In all these
cases, we found similar results to those seen in the
case of diacetates 3a–3d (Scheme 2). The ee values
were estimated by comparing the sign and specific
rotation values of the corresponding optically pure
(R)- and (S)-ketones and were found to be moder-
ate to high (Table IV).
The diacyloxy compounds 6a–6e were then sub-
jected to enzymatic deacylation by suspending them
in THF (containing n-butanol) with native PPL at
40–42°C, affording the corresponding monoacylated
ketones 7a–7d along with the recovered diacylated
ketones6a–6d(Scheme4).Theprogressofdeacylation
reaction was monitored byTLC and/or HPLC exami-
nation. In the case of (Ϯ)-2,4-dinaphthoyloxyphenyl
(1-phenyl)propyl ketone (6e), the reaction did not
proceed with either native or modified PPL.All other
reactions were worked up at about 50% conversion
of the starting compounds into the products by filter-
ing off the enzyme.The enzymatic reaction products,
OAc
AcO
C6H5
(S)-(+)-3d
Ac2O-AcOH
O
OH
HO
[α]25D +25.00 (c 1.00, CHCl3)
OH
O
C6H5
HO
C6H5
OAc
C6H5
AcO
MeOH-HCl
O
Ac2O-pyridine
(S)-(+)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone (2d)
[α]25D +35.00 (c 0.80, CHCl3)
(S)-(+)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone (2d)
[α]25D +34.38 (c 0.80, CHCl3)
O
(S)-(+)-3d
C6H11COCl-Et3N
[α]25D +25.51(c 1.00, CHCl3)
COC6H
O
11COC6H11
OH
HO
O
MeOH-HCl
(S)-(+)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone (2d)
C6H5
C6H5
O
O
[α]25D +35.63 (c 0.80, CHCl3)
(S)-(+)-6d
Scheme 5. Synthesis and chemical deacylation of optically pure diacyloxyphenyl (1-phenyl)propyl ketone.

Enantioselective biocatalytic reactions with native and modified PPL 183
respectively. The acyl derivatives have been used
native PPL and modified PPL (PPL pre-treated
with acetophenone). The results prove our earlier
hypothesis regarding the formation of a transient
Schiff's base-type complex between nuclear carbo-
nyl groups present in the substrate (aryl alkyl ketone)
and the ξ-amino function of the lysine residue pres-
ent in the active site of PPL, which governs the regi-
oselectivity of these reactions. Furthermore, the
novel optically active aryl alkyl ketones obtained in
this study may find application in the synthesis of
bioactive, chiral, naturally occurring chromanones,
flavanones and dihydrocoumarins, which are cum-
bersome to make in optically enriched forms by
chemical routes.
as standards to estimate the ee values of the prod-
ucts obtained during lipase-catalyzed deacylation
reactions carried out on (Ϯ)-2,4-diacyloxyphenyl
(1-phenyl)propyl ketones (Schemes 2, 3 and 4).
A few chemical transformations were carried out
in one case to confirm that there was no racemization
during acylation of (R)-/(S)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone with acid anhydrides or acid
chloride in dichloromethane in the presence of bases
like pyridine or Et3N (Scheme 5).Thus, (S)-(ϩ)-2,4-
dihydroxyphenyl (1-phenyl)propyl ketone (2d) was
converted into (S)-(ϩ)-2,4-diacetoxyphenyl (1-phe-
nyl)propyl ketone (3d) by refluxing with a mixture of
acetic anhydride–acetic acid (Ac2O–AcOH). The
rotation value and the sign of rotation of 2,4-diace-
toxyphenyl (1-phenyl)propyl ketone (3d) obtained by
the Ac2O–AcOH and Ac2O–pyridine methods were
compared and found to be identical, thereby indicat-
ing that there was no racemization during preparation
of different acylates of (R)-/(S)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone under acid anhydride–pyri-
dine condition (Scheme 5). Further, the (S)-(ϩ)-2,4-
diacetoxyphenyl (1-phenyl)propyl ketone (3d)
prepared from enantiomerically pure (S)-(ϩ)-2,4-
dihydroxyphenyl (1-phenyl)propyl ketone (2d) by the
acid anhydride–pyridine method was deacetylated
under acidic conditions (MeOH–HCl) and the rota-
tion value and the sign of rotation of the compound
thus obtained were compared with those of the
enantiomerically pure (S)-(ϩ)-2,4-dihydroxyphenyl
(1-phenyl)propyl ketone (2d); the rotations of both
these hydroxy compounds were identical, which fur-
ther indicated that no racemization occurs during the
acid anhydride–pyridine acylation procedure of the
enantiomerically pure hydroxy ketone.
Acknowledgements
The authors thank the University of Delhi (Delhi,
India), Polytechnic University (New York, USA)
and the Department of Biotechnology (DBT, Gov-
ernment of India, New Delhi) for financial assis-
tance for this work. We also thank Dr Hemant
Sharma (APL Research Center, Aurobindo Pharma
Ltd., Hyderabad, India) for his help in recording
the Raman spectra of several of our samples.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
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