'Watching' lipase-catalyzed acylations using 1H NMR: Competing hydrolysis of vinyl acetate in dry organic solvents

Article abstract of DOI:10.1016/S0957-4166(99)00274-8

Lipase-catalyzed acetylations of 1-phenylethanol with vinyl acetate were monitored in situ by ¹H NMR spectroscopy. Surprisingly, even under dry conditions (no added water) the major reaction was hydrolysis of the vinyl acetate, not acetylation of the substrate. Because this competing hydrolysis consumes water and releases acetic acid, the reaction conditions in lipase-catalyzed acylations are not constant, but vary with the reaction time. Addition of a chiral shift reagent reveals the enantiomeric purity of the starting alcohol and allows calculation of the enantiomeric ratio, E, for the reaction.

Full text of DOI:10.1016/S0957-4166(99)00274-8

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2635–2638
‘Watching’ lipase-catalyzed acylations using 1H NMR:
competing hydrolysis of vinyl acetate in dry organic solvents
Hedda K. Weber,a,∗ Hansjörg Weber a and Romas J. Kazlauskas b
aInstitut für Organische Chemie, Technische Universität Graz, Stremayrgasse 16, 8010 Graz, Austria
bDepartment of Chemistry, McGill University, 801 Sherbrooke Street W., Montréal, Québec H3A 2K6, Canada
Received 19 May 1999; accepted 1 July 1999
Abstract
Lipase-catalyzed acetylations of 1-phenylethanol with vinyl acetate were monitored in situ by 1H NMR
spectroscopy. Surprisingly, even under dry conditions (no added water) the major reaction was hydrolysis of the
vinyl acetate, not acetylation of the substrate. Because this competing hydrolysis consumes water and releases
acetic acid, the reaction conditions in lipase-catalyzed acylations are not constant, but vary with the reaction time.
Addition of a chiral shift reagent reveals the enantiomeric purity of the starting alcohol and allows calculation of
the enantiomeric ratio, E, for the reaction. © 1999 Elsevier Science Ltd. All rights reserved.
Many new applications of lipases to organic synthesis involve reactions in organic solvents.1 The most
common are enantioselective acetylations of racemic or prochiral alcohols using vinyl acetate. These
reactions are fast, the lipase is easy to recover by filtration, and the enantioselectivity is sometimes higher
than for hydrolysis of the corresponding acetate in water. To maximize the rate and enantioselectivity of
these reactions researchers vary the solvent type, the acyl donor type, and water activity.2 However,
optimization remains a largely trial and error process.
1
To speed up reaction optimization in organic solvents, we used H NMR spectroscopy to monitor
reactions in situ. This monitoring allowed us to ‘watch’ all the components of the reaction, especially
vinyl acetate and acetaldehyde, which are difficult to monitor by HPLC or GC. This experiment revealed a
surprisingly large amount of hydrolysis even under dry conditions. Furthermore, addition of a chiral shift
reagent revealed the enantiomeric purity of the remaining starting material thereby allowing calculation
of the enantiomeric ratio, E, for the reaction.
As a test reaction, we used the lipase-catalyzed acetylation of 1-phenylethanol (1-PE) with vinyl
acetate (Fig. 1).3 As expected, the resonances for the starting materials (1-PE and vinyl acetate)
decreased, while the resonances for the products (1-PE acetate and acetaldehyde) increased as a function
of time. However, the amount of acetaldehyde (aldehyde proton resonance at 9.2 ppm) increased three
times faster than the amount of ester (methine resonance at 5.9 ppm) indicating that hydrolysis competes
∗
Corresponding author. E-mail: hedda@orgc.tu-graz.ac.at
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00274-8

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Figure 1. 1H NMR spectrum of a Burkholderia cepacia lipase-catalyzed acetylation of 1-phenylethanol (1-PE) in C6D6 at ∼15%
conversion. The relative area of the signals at 9.2 ppm (acetaldehyde) and at 4.2 and 4.7 ppm (vinyl acetate) reveals consumption
of acyl donor, while the relative areas of the signals at 5.9 ppm (methine proton of 1-PE acetate) and 4.6 ppm (methine proton of
1-PE) reveals the conversion of alcohol to acetate. This reaction consumed three times more acyl donor than alcohol indicating
competing hydrolysis of the acyl donor
with acetylation of 1-PE even under these dry conditions.4 A similar reaction under dry conditions using
CAL-B5 (Novo SP525) showed that hydrolysis occurred twice as fast as acetylation (Fig. 2a). In another
reaction, the solution was equilibrated with a salt/salt hydrate pair Na2SO4/Na2SO4·10H2O for one
hour to adjust the thermodynamic water activity6 to a relatively high value (aw=0.76).7 Hydrolysis now
occurred six times faster than acetylation (Fig. 2b). The rate of conversion was similar in both cases.
Figure 2. 1H NMR spectra of Candida antarctica lipase B-catalyzed acetylation of 1-PE as a function of time. The stacked plots
show spectra from 0.6–4.6 h under dry conditions (a), and at a water activity of 0.76 in (b). Both reactions show hydrolysis of
vinyl acetate as well as acetylation of 1-PE. Hydrolysis is detected because the signal at 9.2 ppm (acetaldehyde) increases faster
than the signal at 5.9 ppm (methine of 1-PE-acetate). Hydrolysis is three times faster than acetylation under dry conditions and
six times faster in the wetter reaction mixture (aw=0.76)
Although hydrolysis of the product ester is also conceivable, we observed no ester hydrolysis even after
32 h (∼50% conversion, CAL-B, aw=0.76). Under some conditions researchers observed spontaneous
acetylation of alcohols with vinyl acetate,8 but we observed no spontaneous reaction under our conditions
(Fig. 3a). Omitting the substrate, 1-PE, increased the rate of hydrolysis of vinyl acetate by a factor
of 1.2 at aw=0.76 (Fig. 3b). Both the lack of hydrolysis without lipase and the apparent competition
between the hydrolysis and acylation reactions strongly suggests that the hydrolysis of vinyl acetate is a
lipase-catalyzed reaction. Thus, the major reaction in lipase-catalyzed acetylations with vinyl acetate
is not the desired acetylation, but the competing hydrolysis of vinyl acetate. This hydrolysis is 2–3
times faster than acetylation even under dry conditions.9 This hydrolysis changes the reaction conditions
because it consumes water and releases acetic acid. This phenomenon may account for changes in

H. K. Weber et al. / Tetrahedron: Asymmetry 10 (1999) 2635–2638
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Figure 3. Control experiments with the CAL-B-catalyzed acetylation of 1-phenylethanol with vinyl acetate at aw=0.76. (a) No
reaction, neither hydrolysis nor ester formation, occurs in the absence of the enzyme. (b) In the absence of substrate alcohol,
the hydrolysis of vinyl acetate is 1.2 times faster
the enantioselectivity at different conversions.10 We recommend that careful optimizations of lipase-
catalyzed acetylations with vinyl acetate include both a buffer for the water content (e.g. a salt/salt hydrate
pair) to minimize the effect of water consumption and an organic phase pH buffer to minimize the effect
of acetic acid release. Another possibility is the addition of a non-reactive organic base, e.g. Et3N, for
neutralizing any acid released as described by Rakels et al.11 and more recently by Parker et al.12
1
A simple extension of this H NMR monitoring technique also permits measurement of the enan-
tiomeric ratio. We added a chiral shift reagent, praseodymium–tris(heptafluorocamphorato) complex
[Pr(hfc)3] to an aliquot of the reaction mixture. This reagent shifted the resonances for the methyl group
of 1-PE (1.3 ppm), which is already furthest upfield, even further upfield while separating the signals
for both enantiomers. The more commonly used europium shift reagents were not suitable because
they shifted resonances downfield, leading to overlapping signals. The conversion of 1-PE into the
corresponding acetate was calculated from the resonances at 4.6 ppm and 5.9 ppm. The enantiomeric
ratio was calculated from the conversion and the enantiomeric purity of the remaining starting material.
The measured enantiomeric ratios agreed with those in the literature although they were measured for
slightly different reaction conditions (Table 1).13
Table 1
Enantioselectivity of several lipases measured by an extension of the 1H NMR monitoring technique
The main strength of this method is the ability to monitor all reactants and products at the same time
without derivatization and without repeated sample taking. It is very close to actually ‘watching’ the
reaction. This method could also be applied to study the influence of different solvents or acyl donors.

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Currently it is used in our laboratory in the development of a method to synthesize enantiomerically pure
drug precursors. This method for determination of the enantiomeric ratio for reactions in organic media
is complementary to existing methods.
Acknowledgements
We thank Dr. Françoise Sauriol (McGill University) for perfect technical support. H. K. Weber thanks
the Austrian Science Foundation (FWF) for an Erwin-Schrödinger-Auslandsstipendium as a postdoctoral
fellow at McGill University.
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