Lipase-catalysed kinetic resolutions of secondary alcohols in pressurised liquid hydrofluorocarbons

Article abstract of DOI:10.1016/j.tetlet.2009.03.037

Three model secondary alcohols have been subjected to enzymatic kinetic resolution using three common lipases and a typical acyl donor. The resolutions were performed in two pressurised low-boiling hydrofluorocarbons, which are novel media for enzymatic reactions, and five conventional organic solvents. In general, higher yields, ee's and rates of reaction were observed in the hydrofluorocarbons.

Full text of DOI:10.1016/j.tetlet.2009.03.037

Tetrahedron Letters 50 (2009) 3543–3546
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Lipase-catalysed kinetic resolutions of secondary alcohols in pressurised liquid
hydrofluorocarbons
Anthony J. Ball a, Stuart Corr b, Jason Micklefield a,
*
a School of Chemistry and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK
b INEOS Fluor Ltd, PO Box 13, The Heath, Runcorn, Cheshire WA7 4QF, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Three model secondary alcohols have been subjected to enzymatic kinetic resolution using three com-
mon lipases and a typical acyl donor. The resolutions were performed in two pressurised low-boiling
hydrofluorocarbons, which are novel media for enzymatic reactions, and five conventional organic sol-
vents. In general, higher yields, ee’s and rates of reaction were observed in the hydrofluorocarbons.
Ó 2009 Published by Elsevier Ltd.
Received 27 January 2009
Revised 2 March 2009
Accepted 5 March 2009
Available online 13 March 2009
Keywords:
Lipase
Kinetic resolution
Hydrofluorocarbon
Secondary alcohol
Biocatalysis
The kinetic resolution (KR) of secondary alcohols via enantiose-
lective acylation, catalysed by hydrolase enzymes, is a well known
procedure for production of chiral alcohols or esters for pharma-
ceutical synthesis and other applications.1–3 The most common
enzymatic method for performing kinetic resolutions of this type
(Fig. 1) is to use a non-aqueous solvent and an irreversible acyl do-
nor such as vinyl acetate, to drive the reaction in the synthetic
direction.4–6 The fact that common lipases tend to display opposite
stereoselectivity to some proteases means that both enantiomers
of the alcohol or ester product can in principle be obtained.7–9
Enzymatic acylation of secondary alcohols has also been used to
trigger enantioselective multistep (domino) reactions.10 In addi-
tion kinetic resolution of primary alcohols with remote stereocen-
tres has also been achieved.11,12 Despite this product yield for
kinetic resolution is limited to 50%. To overcome this problem, dy-
namic kinetic resolutions (DKRs)13,14 have been introduced, where
the unreacted secondary alcohol is racemised, for example, using a
ruthenium catalyst8,15,16 (e.g., 1, Fig 1).
on biocatalysis, the concept of solvent engineering1,2,17 was intro-
duced as a way to optimise and alter the course of enzymatic reac-
tions in non-aqueous media including conventional organic
solvents (COSs),1,2 supercritical fluids18 and ionic liquids.19 To this
end we introduced hydrofluorocarbon HFCs as alternative media
for biocatalysis.20 The use of pressurised liquid HFCs21,22 as media
A
L
L
L
M
M
M
Lipase
+
Acyl-donor
OH
OH
OAc
( )
Ph
Ru
H
Ph
N
Ph
Ph
OC
The selection of the non-aqueous solvent for enzymatic–kinetic
resolutions is of critical importance. Indeed early work with organ-
ic solvents showed that enzyme activity, yields and enantioselec-
tivities can all be affected by the solvent physical properties.1,2,17
Similarly with DKRs, the choice of a solvent that can afford both
high activity of the enzyme and the chosen racemisation catalyst
is an important factor. As a result of the critical role of the media,
Cl
OC
1
B
F
F
H
F
H
H
F
F
F
F
F
F
R-134a
R-227ea
F
F
Figure 1. (A) Lipase-catalysed kinetic resolution (KR) following the ‘Kazlauskas
rule’ and dynamic kinetic resolution (DKR) of secondary alcohols; (B) hydrofluoro-
carbons R-227ea and R-134a used in this study.
* Corresponding author.
E-mail address: jason.micklefiled@manchester.ac.uk (J. Micklefield).
0040-4039/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2009.03.037

3544
A. J. Ball et al. / Tetrahedron Letters 50 (2009) 3543–3546
for biocatalysis was shown to give improved rates, yields and in
some cases enantioselectivities in lipase and protease-catalysed
model biotransformations.20 In addition, pressurised liquid HFCs,
which are used as refrigerants, propellants and as solvents for
extraction, offer several major operational benefits for potential
industrial processes, including ease of product recovery.20,22
Previously we showed that the kinetic resolution of racemic 1-
phenylethanol20 was particularly promising in HFCs. In light of this
we chose to explore the wider utility of these media for kinetic res-
olution of secondary alcohols. Initially we focused on the kinetic
resolution of 1-phenylpropan-2-ol (rac-2) with commercial Can-
dida antarctica lipase B on acrylic resin (Novozyme 435) and Pseu-
domonas cepacia lipase supported on ceramic (Amano PS-CII), with
vinyl acetate 3 as the acyl donor to give the acetylated product (R)-
4. For comparison with the HFCs R134a and R227ea (Fig. 1), a series
of conventional organic solvents (COSs) were selected which are
most commonly used for similar kinetic resolutions using lipases
and related enzymes. Through a series of preliminary experiments,
conditions were established so that the progress of reactions could
be monitored by GC allowing accurate initial rates to be deter-
mined during the early stages of the reaction when pseudolinear
kinetics are observed. At a set time point (24 h), when the reaction
has proceeded towards completion, the yield of the acetylated
product (R)-4 is recorded and it enantiomeric excess was deter-
mined by chiral HPLC. From this it can be seen (Table 1) that the
reaction in R134a, catalysed by Novozyme 435, exhibited the high-
est initial rate and yield compared with all other solvents. Of the
COSs hexane gave highest initial rates and yields, but slightly lower
ee. In light of this full time course experiments were run, under
optimised conditions, with higher quantities of enzyme in R134a
and hexane. This confirmed higher rates and yields in the HFC
and resulted in 42% yield of (R)-4 (99% ee) in R134a, compared with
36% yield of (R)-4 (99% ee) in hexane (see Supplementary data). In
the kinetic resolution of 1-phenylpropan-2-ol (rac-1), catalysed by
PS-CII, it is clear that the HFCs give rise to considerably higher ini-
tial rates compared with COSs. Again hexane gave significantly
higher rates than other COS, and interestingly initial rates in tolu-
ene are considerably higher for PS-CII than for Novozyme 435. A
full time course experiment was run in the best HFC (R227ea)
and best COS (hexane) with PS-CII, resulting in 45% yield of (R)-4
(99% ee) in R227ea, compared with 43% yield of (R)-4 (99% ee) in
hexane (see Supplementary data).
The kinetic resolution of 1-(2-furyl)ethanol rac-5 was next ex-
plored, with the widely used Pseudomonas fluorescens lipase (PFL)
supported on Eupergit C, in addition to PS-CII. Again the produc-
tion of the acetyl product, (R)-6, was monitored by GC and chiral
GC to give initial rates as well as yields and ee after 24 h (Table
2). In this case TBME gave the highest initial rate and yield, fol-
lowed by R134a. With PS-CII hexane gave the highest initial rates,
followed by R134a, although after 20 h the reaction with R227ea
had progressed furthest. In this series a number of reactions had
proceeded beyond the 50% optimum yield, due to additional acyl-
Table 1
Initial rates, yields, ee and enantiomeric ratio (E) for acetylation of rac-2 to give (R)-4 in various solvents, catalysed by Novozyme 435 and PS-CII
O
Enzyme
Ph
Ph
Ph
+
+
OAc
)-4
OH
)-2
OH
rac-2
O
3
(
R
(S
Solvent
Novozyme 435
Yield 4 (%)
PS-CI
Yield 4 (%)
Initial rate (nmol minÀ1 UÀ1
)
ee 4 (%)
E
Initial rate (nmol minÀ1 UÀ1
)
ee 4 (%)
E
R134a
R227ea
Hexane
Toluene
Chloroform
TBME
7.40 0.07
2.00 0.08
3.50 0.01
n.d.a
0.90 0.02
2.80 0.15
2.00 0.02
34
27
20
17
5
97
97
108
93
1419 19.7
2229 30.4
1088 13.6
736 1.39
255 1.65
354 9.06
440 7.38
13
25
15
8
2
5
99
99
>200
>200
>200
n.d.b
>200
>200
>200
93
35
99
n.d.b
98
n.d.b
104
62
n.d.b
99
20
18
96
95
99
99
Vinyl acetate
48
11
Yields and ee for each reaction are recorded after 24 h. Conditions: 0.5 mmol rac-2, 10 mmol 3, 95U Novozyme 435 or 0.6U PS-CII, 5 mL solvent.
a
The rate was too low to be measured.
The toluene peak overlapped with those of the product.
b
Table 2
Initial rates, yields, ee and enantiomeric ratio (E) for acetylation of rac-5 to give (R)-6 in various solvents, catalysed by PFL and PS-CII
O
Enzyme
O
O
O
+
+
OAc
)-6
OH
)-5
OH
rac-5
O
3
(R
(S
Solvent
PFL
PS-CII
Yield 6 (%)
Initial rate (nmol minÀ1 UÀ1
)
Yield 6 (%)
ee 6 (%)
E
Initial rate (nmol minÀ1 UÀ1
)
ee 6 (%)
E
R134a
R227ea
Hexane
Toluene
Chloroform
TBME
9.10 0.48
n.d.
11
n.d.a
10
3
95
44
556 14.2
319 10.8
695 40.8
489 12.4
230 5.50
287 8.7
50
61
57
54
8
99
80
80
92
99
97
99
>200
34
39
118
>200
>200
>200
n.d.a
97
n.d.a
73
7.10 0.25
1.80 0.05
0.60 0.02
10.90 0.13
6.00 0.17
98
102
n.d.a
59
n.d.a
17
7
n.d.a
96
52
46
Vinyl acetate
93
30
133 15.6
Yields and ee for each reaction are recorded after 24 h. Conditions (5 mL total volume): 10U PFL, 2.0 mmol rac-5, 2.5 mmol 3; 3U PS-CII, 0.5 mmol rac-5, 5 mmol 3.
a
Yields too low to be measured.

A. J. Ball et al. / Tetrahedron Letters 50 (2009) 3543–3546
3545
Table 3
Initial rates, yields, ee and enantiomeric ratio (E) for acetylation of rac-7 to give (R)-8 in various solvents, catalysed by PS-CII and Novozyme 435
O
Enzyme
+
+
OAc
(R)-8
OH
(S)-7
OH
rac-7
O
3
Solvent
PS-CII
Novozyme 435
Initial rate (nmol minÀ1 UÀ1
)
Yield 8 (%)
ee 8 (%)
E
Initial rate (nmol minÀ1 UÀ1
)
Yield 8 (%)
ee 8 (%)
E
R134a
R227ea
Hexane
Toluene
Chloroform
TBME
3.60 0.12
6.20 0.21
7.30 0.27
n.d.a
53
53
55
55
4
89
89
82
82
>99
99
>99
123
123
73
9.6 0.53
10.8 0.21
4.5 0.20
n.d.a
49
51
55
15
4
>99
96
82
>99
>99
>99
>99
>200
>200
73
>200
>200
>200
>200
73
n.d.b
>200
>200
>200
n.d.b
0.80 0.01
31
24
1.10 0.04
13
10
Vinyl acetate
n.d.b
n.d.b
Yields and ee for each reaction are recorded after 24 h. Conditions: 0.5 mmol rac-7, 5 mmol 3, 120U PS-CII or 118U Novozyme 435, 5 mL solvent.
a
(R)-8 Peak obscured by solvent.
Rates too low to be reliably measured.
b
ation of the (S)-5 enantiomer resulting in significantly lower ob-
served ee. After further optimisation, a full time course for the ki-
netic resolution of rac-5 with PS-CII was performed, which gave a
45% of (R)-6 (99% ee) in hexane and 43% of (R)-6 (99% ee) R134a,
under identical conditions (see Supplementary data).
Finally the kinetic resolution of 1-(1-naphthyl)ethanol rac-7
was studied using PS-CII and Novozyme 435 (Table 3). With PS-
CII, hexane exhibits highest initial rates and yields, closely fol-
lowed by R227ea. Novozyme 435, on the other hand, exhibits high-
est initial rates in R227ea and R134a, but a higher yield was
obtained in hexane. As with most of the other experiments chloro-
form proved to be the least effective solvent for these transforma-
tions. In order to rationalise the overall results from these kinetic
resolution experiments it is useful to compare the physical proper-
ties of HFCs22 with those of COSs.23,24 Of the many physical param-
eters available viscosity, polarity as indicated by dielectric
described here and previously.20 Most likely this is due to the fact
that HFCs, as well as being polar, are also hydrophobic.25 Generally
COSs of higher polarity tend to be more hydrophilic.
In summary kinetic resolutions of three model, secondary alco-
hols using three well-known lipases, have been investigated in two
HFC solvents and compared with five conventional organic sol-
vents that would typically be used in such transformations. On
the whole rates and yields for these reactions in HFCs were higher
than COSs. It is likely that this can be attributed to the unusual
physical properties of HFCs, which unlike COSs, possess both high
polarity and hydrophobicity,25 with low viscosity. The fact that
HFCs also offer improved operational properties such as very
low-boiling points20 allowing easier product recovery, low toxicity
and in many cases non-flammability, means that they offer signif-
icant advantages over COSs for enzymatic-production of chiral
alcohols or esters of industrial importance. Finally we have shown,
in a separate study, that HFCs are also good solvents for ruthe-
nium-complexes (e.g., 1) allowing fast racemisation of alcohols,
which could be beneficial in DKRs of secondary alcohols.8,15,16
Studies to explore the applications of HFCs as media for DKRs are
currently underway and will be presented shortly.
constants (e) or dipole moments (DM), and hydrophobicity as re-
flected by logP are probably the most informative (Table 4). Con-
sidering these parameters and the data presented here some
apparent trends can be observed. Firstly solvents that have lower
viscosity tend to exhibit faster initial rates, which can be due to in-
creased solute diffusivity and improved mass transfer, which is
particularly important with immobilised enzymes. Secondly
amongst COSs rates and yields tend to decrease with increasing
solvent polarity, which can in part be attributed to the propensity
of the more polar solvent to strip the enzyme of essential
water.1,2,17 In some cases the increased solubility of the substrate
alcohol in the more polar solvents can have the opposite effect. De-
spite this, HFCs and R134a in particular are highly polar, yet gener-
ally afford higher rates and yields for the lipase-catalysed reactions
Acknowledgements
We would like to thank INEOS Fluor for support. A.J.B. thanks
EPSRC for PhD case award. We are also grateful to Simon Gardner
and Simon Saul for useful discussions.
Supplementary data
Supplementary data (experimental procedures and additional
data) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2009.03.037.
Table 4
Physical properties of solvents used in this study22–24
Solvent
Viscositya
e b
DMc
LogP
References and notes
R134a
R227ea
Hexane
Toluene
Chloroform
TBME
0.21
0.26
0.29
0.56
0.53
0.34
0.41
9.5
4.1
1.9
2.4
4.8
1.4
3.0
2.05
0.93
0.08
0.31
1.01
1.37
1.42
2.17
4.00
2.73
1.97
0.94
0.73
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