Linum usitatissimum hydroxynitrile lyase cross-linked enzyme aggregates: A recyclable enantioselective catalyst

Article abstract of DOI:10.1002/adsc.200800309

An immobilized form of the hydroxynitrile lyase from Linum usitatissimum (LuHNL) as cross-linked enzyme aggregate (CLEA) with high specific activity (303.5 U/g) and recovery (33%) was developed. Molecular imprinting using 2-butanone as additive in the immobilization process improved the synthetic activity of the biocatalyst. LuCLEA could be partially recycled for the synthesis of (R)-2-butanone cyanohydrin on a preparative scale over two batches. The enantioenriched cyanohydrin obtained was further hydrolyzed to give (R)-2-hydroxy-2-methylbutyric acid in 85% yield (from 2-butanone) and 87% ee.

Full text of DOI:10.1002/adsc.200800309

FULL PAPERS
DOI: 10.1002/adsc.200800309
Linum usitatissimum Hydroxynitrile Lyase Cross-Linked Enzyme
Aggregates: A Recyclable Enantioselective Catalyst
Fabien L. Cabirol,a,b,c Pei Loo Tan,b Benson Tay,b Shiryn Cheng,b Ulf Hanefeld,a
and Roger A. Sheldona,*
a
Laboratory of Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology,
Julianalaan 136, 2628 BL Delft, The Netherlands
Fax : (+31)-15-278-1415; e-mail: r.a.sheldon@tudelft.nl
Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, SACTHRE(UNG 627833), Singapore
b
Fax : (+65)-6316-6182
c
Current address: Codexis Laboratories Singapore, 61 Science Park Road, #03-15/24, The Galen, Singapore Science Park
III, S(117525), Singapore
ACHTREUNG
Received: May 19, 2008; Revised: August 1, 2008; Published online: October 1, 2008
Abstract: An immobilized form of the hydroxynitrile batches. The enantioenriched cyanohydrin obtained
lyase from Linum usitatissimum (LuHNL) as cross- was further hydrolyzed to give (R)-2-hydroxy-2-
linked enzyme aggregate (CLEA) with high specific methylbutyric acid in 85% yield (from 2-butanone)
activity (303.5 U/g) and recovery (33%) was devel- and 87% ee.
oped. Molecular imprinting using 2-butanone as ad-
ditive in the immobilization process improved the
synthetic activity of the biocatalyst. LuCLEA could Keywords: biocatalysis; cross-linked enzyme aggre-
be partially recycled for the synthesis of (R)-2-buta- gate (CLEA); cyanohydrins; hydroxynitrile lyase;
none cyanohydrin on a preparative scale over two oxynitrilase; quaternary stereogenic carbon
Introduction
A[8,9] and germinalinine.[10] (2) The synthesis of I via
the enantioselective addition of cyanide to the prochi-
The enantioselective synthesis of quaternary stereo- ral butanone and subsequent hydrolysis involves a
genic carbon atoms is a major challenge in organic daunting stereodifferentiation between a methyl and
chemistry. In particular, chiral tertiary alcohols are at an ethyl group (Scheme 1). While chemical catalysis
the centre of attention. Due to their importance in has so far proven rather unsuccessful for this chal-
the pharmaceutical industry and the drive towards en- lenge,[11,12,13,14] enantioselective biocatalysis using hy-
vironmentally benign synthesis,[1,2] catalytic routes are droxynitrile lyases (HNL) is the way forward to pre-
the focus of current research.[3] 2-Hydroxy-2-methyl- pare both the R- and S-enantiomers of I.[11,12,15]
butyric acid (I) is such a building block. It is, for two
Recently, an HNL-based pathway to S-I was de-
reasons, a particularly interesting synthetic challenge. scribed in which butanone was “disguised”, that is, an
(1) Its S-enantiomer is used for the preparation of a auxiliary was introduced in order to ease stereodif-
COX-2 specific inhibitor,[4] while its R-enantiomer ferentiation.[16] This elegant approach has the draw-
forms part of several biologically active natural prod- back that its atom-economy is poor,[1,2] comparable to
ucts, such as the clerodendrins,[5,6,7] protoveratrine the chemical chiral auxiliary route.[17] For the R-enan-
Scheme 1. Synthetic approach towards (R)-enantioenriched 2-hydroxy-2-methylbutyric acid (R)-I.
Adv. Synth. Catal. 2008, 350, 2329 – 2338
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Fabien L. Cabirol et al.
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tiomer of I, Prunus amygdalus, Prunus mume, and length of exposure to a cosolvent prompted us to
Linum usitatissimum HNL have been employed. select conditions that allow fast precipitation at 08C
However, Prunus amygdalus and Prunus mume en- (data not shown). A screening procedure could then
zymes displayed moderate enantioselectivity[18,19] be developed to determine the best cosolvents from a
while the Linum usitatissimum HNL (LuHNL) was range of alcohols and saturated aqueous salt solutions
reported to give good to very good enantiopurities of in various amounts (Figure 1).
the cyanohydrin intermediate.[20,21] We recently de-
Ideally, the cosolvent should provide 100% activity
scribed that immobilizing the HNLs from Prunus recovery (no deactivation) in the formof an aggre-
amygdalus, Hevea brasiliensis and Manihot esculenta gate (no enzyme left in solution). One particular sol-
as cross-linked enzyme aggregates (CLEAs) can im- vent, tert-butanol, in amounts greater than twice (v/v)
prove their stability and ease their recycling.[22,23,24] In the volume of commercial LuHNL solution gave very
particular, their application in organic solvents satisfying results in this regard. Saturated aqueous
became possible. All these parameters are essential ammonium sulfate [sat. (NH4)2SO4] also provided a
for the repeated and sustainable application of en- fair amount of precipitate with little deactivation and
zymes in a green process.[1,2,25,26,27] Here we describe soluble enzyme left when it was used in amounts
the preparation of a CLEA from LuHNL (LuCLEA) greater than twice the initial volume of enzyme solu-
and its application in the synthesis of R-I.
tion. The other alcohols screened here did not pro-
vide efficient precipitation since a large amount of
enzyme was left in the solution. Brine had a severe
deleterious effect on the enzyme activity. We contin-
ued this screening of cosolvents with a range of
water-miscible organic solvents and PEG (Figure 2).
Glyme and diglyme stood out as relatively good can-
didates for precipitation in this screening. The extent
of deactivation was particularly significant with N,N-
Results and Discussion
Development of LuCLEA
Enzyme Precipitation
The selection of precipitation parameters (nature and dialkylamide-type solvents such as dimethylform-
amount of cosolvent, duration, and temperature) is a amide (DMF) and dimethylacetamide (DMAc) while
AHCTREUNG
critical step in the preparation of active CLEAs with PEG, DMSO and acetonitrile left a significant
a high recovery of enzyme activity.[24,28] A rapid evalu- amount of active enzyme in solution.
ation of LuHNL sensitivity towards temperature and
Figure 1. Activity recovery (% of commercial enzyme solution used) in the precipitate (black) and supernatant (white) after
enzyme precipitation using alcohols and saturated saline solutions [the amount of precipitant is expressed as a factor (in vol-
umes) of the commercial enzyme solution used].
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Linum usitatissimum Hydroxynitrile Lyase Cross-Linked Enzyme Aggregates
FULL PAPERS
Figure 2. Activity recovery (%) in the aggregate and the supernatant after enzyme precipitation using organic solvents and
PEG [the amount of precipitant is expressed as a factor of the volume of commercial enzyme solution used].
Interestingly the influence of precipitants on the ac- screened in order to fine tune this parameter when
tivity of LuHNL was consistent with earlier investiga- combined with the cross-linking step.
tions on suitable water-miscible cosolvents in the
When glyme was used as a precipitant, no satisfac-
LuHNL-catalyzed conversion of 2-butanone into its tory results could be obtained and this cosolvent was
corresponding cyanohydrin.[20] Based on the results not considered further. The optimized conditions
obtained in the precipitation study, four cosolvents using the other three cosolvents (Table 1) also indicat-
[tert-butanol, sat. (NH4)2SO4, glyme and diglyme] ed that diglyme was the least preferable and we did
were selected for the cross-linking study.
not consider this precipitant further.
The CLEAs prepared using optimized amounts of
tert-butanol and sat. (NH4)2SO4 as precipitants and
glutaraldehyde as cross-linker (Table 1) were selected
for further study. We also noticed that although
Cross-Linking of Aggregates
Although precipitation aims at shaping the physical
state of the aggregates while maintaining the enzyme
activity,[29] the cross-linking step is no less important
to the successful preparation of an active CLEA. Effi-
cient cross-linking will indeed “lock” the enzyme into
its active state and prevent redissolution (leaching)
during reaction.[26] Moreover, cross-linking determines
the particle size and contributes to making the biocat-
alyst more robust toward deleterious effects such as
substrate/product inhibition or organic solvent deacti-
vation during the reaction.[23] Traditionally, glutaralde-
hyde is preferred as a cross-linking agent as it is com-
mercially available and inexpensive. We investigated
the activity recovery in the CLEA after cross-linking
using 5%, 10%, 20% and 30% (v/v of the commercial
enzyme solution used) of glutaraldehyde for each of
the four cosolvents selected in the precipitation study.
The amount of each precipitant used was also
Table 1. Cross-linking study: optimized results for each pre-
cipitant.
Co-solvent[a]
Cross-linker[b]
Activity Recovery[c]
tert-Butanol (2)
Sat. (NH4)2SO4 (4)
Diglyme (3)
10
10
5
19.5 (0)
53 (8)
17 (3)
[a]
Precipitant and amount (parentheses) as a factor of the
volume of commercial enzyme solution used.
Amount of glutaraldehyde 25% aqueous solutions as a
percentage (v/v) of the volume of commercial enzyme
solution used.
Activity recovery (%) in the CLEA based on the initial
amount of enzyme used (in units). The number in paren-
theses indicates the percentage of activity present in the
buffer used to wash the CLEA (enzyme not immobi-
lized).
[b]
[c]
Adv. Synth. Catal. 2008, 350, 2329 – 2338
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LuHNL shares several structural homologies with the The specific activity and activity recovery for CLEAs
Zn2+-containing alcohol dehydrogenases (ADHs)[21,30] of PaHNL and MeHNL were also measured for com-
efficient cross-linking could be achieved using gluta- parative purposes (Table 2).
raldehyde while dextran polyaldehyde had to be used
Better recovery was obtained when the CLEA was
to prepare CLEAs of the ADH from Lactobacillus prepared fromsat. (NH )2SO4 than from tert-butanol
4
brevis since glu
This observation further highlights the need to opti- LuEA
mize the conditions for CLEA preparation independ- ed to a large excess of salt [(NH4)2SO4] in the catalyst.
ACHTREUNG
AHCTREUNG
ently of results obtained for similar enzymes.
The EA was indeed washed with acetonitrile since an
aqueous buffer would redissolve the enzyme aggre-
gate. As a result some of the salt precipitated upon
addition of MeCN resulting in low specific activity
Enzyme Aggregates (EAs) and other HNLs
AHCTREUNG
The results obtained so far in the study seemto indi-
ACHTREUNG
cate that the relatively low activity recovery in the as for LuCLEAACHTREUNG
CLEA is mostly due to the cross-linking step. For in- also improved. PaCLEA was not active in the condi-
stance, when tert-butanol was used as precipitant the tions of the standard activity test which was attributed
recovered activity was greater than 80% after precipi- to the relatively low pH (4.1). On the other hand, the
tation and below 20% after cross-linking. Since we very high specific activity and activity recovery mea-
aimed at developing an immobilized version of sured for MeCLEA are consistent with an earlier
LuHNL that can performin buffer-saturated organic
report on the robustness and high activity of this bio-
solvent as we reported earlier for MeCLEA,[23]
simple aggregate of LuHNL (without cross-linking)
could also be considered as an immobilized catalyst
a
catalyst.[23]
as it would be insoluble in the reaction media. Molecular Imprinting
Enzyme aggregates of LuHNL (LuEA) were there-
fore prepared using tert-butanol and sat. (NH4)2SO4 in The use of surfactants and crown ethers as additives
a scale-up (factor 10) of the optimized conditions for in the preparation of CLEAs has been reported[28]
precipitation. The preparation of CLEAs fromthese
and very significant improvements of the biocatalyst
two precipitants was also scaled-up accordingly and a activity could be observed in some instances. These
significant drop in activity recovery was observed additives were selected in order to stabilize the
(Table 2) when compared to the results obtained on a enzyme leading to a more robust biocatalyst and to
smaller scale as described in the cross-linking study. modify the enzyme conformation into a more active
state that could be “locked” by immobilization. Our
approach to additives for the development of immobi-
lized LuHNL was closer to the concept of molecular
imprinting[32,33,34] where the enzyme could potentially
be immobilized in its enzyme-substrate complex con-
formation. A cyanohydrin as substrate for this pur-
pose would be problematic since its decomposition
into the corresponding carbonyl compound and HCN
would be difficult to prevent without complete inhibi-
tion of the enzyme. However, in the absence of HCN,
a carbonyl compound that can bind to the active site
of LuHNL in solution and be washed away easily
after immobilization would be suitable. Moreover, the
additive should be water-soluble, preferably inexpen-
sive and a low boiling point would be an advantage in
order to remove traces under vacuum. Considering
these requirements, we selected 2-butanone as an ad-
ditive and studied its influence on the preparation of
CLEAs and EAs (Table 3).
Table 2. Specific activity and activity recovery of CLEAs
and EAs.
Biocatalyst Precipitant
X-link- Activity[a] Recovery[b]
ing
LuCLEA
(Am.Sulf) (NH4)2SO4
LuEA Sat.
(Am.Sulf) (NH4)2SO4
Sat.
Yes
No
180.5 U/g 20%
9.9 U/g 5%
LuCLEA
(t-Bu)
tert-Butanol Yes
tert-Butanol No
110.9 U/g 6%
221.9 U/g 9%
LuEA
(t-Bu)
PaCLEA[c] Glyme
Yes
Yes
Not
nd[d]
active
847.1 U/g 36%
MeCLEA[c] Sat.
(NH4)2SO4
[a]
[b]
Specific activity per g of solid measured according to the
standard procedure for LuHNL.
We used a standard 1 mL of 2-butanone per unit of
LuHNL as a reference and observed significant differ-
Activity recovery (%) in the catalyst based on the initial
amount of enzyme used (in units).
ences in the specific activities of LuEA
LuCLEA(t-Bu). A drop in activity was indeed ob-
served for the EA whereas the CLEA specific activity
ACHTRE(UGN t-Bu) and
[c]
[d]
Prepared according to literature procedure.[22,24]
nd: not determined.
AHCTREUNG
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Linum usitatissimum Hydroxynitrile Lyase Cross-Linked Enzyme Aggregates
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Table 3. Influence of 2-butanone as additive on the immobi-
lization process.
fore decided to screen the immobilized forms of
LuHNL for the synthesis of 2-butanone cyanohydrin
in buffer-saturated DIPE as suggested in Scheme 1.
The conversion of 2-butanone (II) and the ee of the
cyanohydrin formed (III) were monitored to evaluate
the synthetic activity and selectivity of the catalyst, re-
spectively (Figure 3).
Biocatalyst
LuCLEA(Am.Sulf)
Activity difference[a]
A
+8%
nd[b]
+44%
À68%
LuEA(Am.Sulf)
LuCLEA(t-Bu)
(t-Bu)
ACHTREUNG
A
LuEAACHTREUNG
[a]
Results are presented as the difference in specific activity
between the biocatalyst prepared with and without 2-bu-
tanone as additive (see experimental section).
nd: not determined (catalyst not active when 2-butanone
was used as additive).
[b]
increased very significantly. Since the EA redissolves
in the aqueous buffer of the standard activity test
thereby restoring the free enzyme conformation (non-
complexed) one would not expect a significant differ-
ence in activity. In contrast, a CLEA retains the struc-
tural features imposed during immobilization even in
an aqueous buffer. Hence, we conclude that the
enzyme-substrate complex was indeed formed but the
benefits of the additive could only be retained in the
biocatalyst after “locking” the structure by cross-link-
ing. The additive had little effect on the activity of
LuCLEAACHTREUNG(Am.Sulf) which suggests that tert-butanol
also plays a role in coordinating to the active site
during immobilization but the enzyme conformation
obtained with tert-butanol can be modified further to
the enzyme-2-butanone complex allowing immobiliza-
tion in an even more active form. The deleterious
effect of the additive was observed on LuEA-
ACHTREUNG
ence immobilization of LuHNL in the formof an EA
or CLEA led us to select five immobilized forms for
synthetic applications: LuCLEA
tive), LuCLEA(t-Bu) (with and without additive),
and LuEA(t-Bu) (with and without additive).
MeCLEA was also selected as a standard for a robust
and active biocatalyst.
ACHTRE(UNG Am.Sulf) (no addi-
Figure 3. LuHNL- and MeCLEA-catalyzed conversions of
2-butanone (A) and ee of the formed cyanohydrin (B). Re-
action conditions: 2-butanone (0.50 mmol), HCN (4 equiv.),
biocatalyst (2.2 U), roomtemperature, in buffer-saturated
ACHTREUNG
ACHTREUNG
*
*
DIPE (1.00 mL). : LuCLEA
(Am.Sulf), : LuCLEA
U
~
: LuCLEAACHTREU(NG t-Bu) prepared using 2-butanone as additive,
~
&
: MeCLEA, : LuEA
ACHRTUNEG(t-Bu), &: LuEACAHTRE(UGN t-Bu) prepared
Synthetic Application
Catalyst Selection
using 2-butanone as additive.
The catalysts could be clearly divided into two cate-
The stereodifferentiation in 2-butanone is a challenge, gories based on their synthetic activity (Figure 3, A).
in particular since the racemic non-catalyzed addition LuCLEA(t-Bu) (with and without additive) per-
AHCTREUNG
of HCN to the ketone strongly depends on the reac- formed exceptionally well under the above selected
tion conditions. In an uncatalyzed control experiment conditions, matching the 2-butanone conversion pro-
using a biphasic system(DIPE:buffer pH 4.1) the
file obtained with MeCLEA. LuCLEAACHTREU(NG t-Bu) pre-
conversion of 2-butanone into its racemic cyanohydrin pared in the presence of 2-butanone performed even
was indeed 16% in 2 h. When the control experiment better than the standard MeCLEA (Figure 3, A).
was performed in buffer-saturated DIPE the conver- LuCLEA
ACHTRE(UGN Am.Sulf) and the EAs performed poorly
sion of 2-butanone was still 9% after 48 h. We there- under these conditions, the EA prepared with 2-buta-
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Fabien L. Cabirol et al.
FULL PAPERS
none being slightly more active. We investigated the
This arrangement is typical of CLEAs of HNLs
(Am.Sulf) by such as the highly active MeHNL.[24] The structure
effect of organic solvents on LuCLEAACHTREUNG
washing the freeze-dried catalyst with acetonitrile, maximizes the catalyst surface available for reaction
ethyl acetate and then diethyl ether and only 31% of (amalgams of small aggregates separated by pores)
the activity could be recovered. The specific activity while minimizing the diffusion effect within the cata-
of LuCLEAACHTREUNG(Am.Sulf) had also decreased greatly to lyst (branch-like sub-structure).
39.8 U/g after washing. These results indicated a
severe deleterious effect of the media for this catalyst.
Next to the excellent activity, LuCLEACAHTRE(UGN t-Bu) (with Catalyst Recycling
and without additive) also showed good selectivities
(Figure 3, B). 2-butanone as additive did not seemto
We investigated the recycling ability of LuCLEA on a
affect the ee profile. This indicates that the imprinting suitable scale to minimize catalyst loss during filtra-
strategy only affected the catalyst activity but not its tion between cycles. The catalyst loading was in-
selectivity. Difficult stereodifferentiation for this sub- creased to 8 U/mmol and the temperature set at
strate was reflected in the low selectivity observed for 308C. The reaction was monitored over 6 h under
MeCLEA (ee around 20% under the reaction condi- these conditions for four consecutive cycles
tions) which was consistent with earlier reports for (Figure 5).
this enzyme-substrate combination.[35]
When the reaction was catalyzed by fresh catalyst,
This evaluation of the synthetic activity highlighted good conversion and selectivity was achieved in a rel-
the importance of selecting an immobilized biocata- atively short time (6 h). LuCLEA activity decreased
lyst not only fromthe results in the standard activity
test but fromits performance under actual reaction
with each cycle and therefore it could not be reused
directly for repeated batches. In contrast, the selectiv-
conditions. The standard activity test proved to be a ity was maintained upon recycling (Table 4) consistent
fast and efficient method to narrow the range of suita- with racemization not being significant at 308C
ble conditions for the development of immobilized (Figure 5). In an attempt to further improve the over-
LuHNL but only the evaluation of the synthetic activ- all selectivity, the reaction was performed with fresh
ity allowed us to select LuCLEA
ACHTRE(UNG t-Bu) prepared catalyst at 58C under otherwise identical conditions
using 2-butanone as additive (referred to as LuCLEA (data not shown) but the reaction became extremely
fromhere on) as the best candidate for synthetic ap-
plication in buffer saturated organic solvent.
slow (7% conversion after 2 h) while the selectivity
improved only very slightly (84% ee after 2 h).
A straightforward filtration and evaporation under
SEM analysis of LuCLEA (Figure 4) revealed rela-
tively large particles of ca. 100 mm(Figure 4, A). The reduced pressure yielded the crude product which
internal structure of LuCLEA is organized as a net- contained less then 1% of residual substrate
work of “branches” (Figure 4, B) separated by pores (Table 4.)
of about 10 mm. Enlargement of these “branches”
showed aggregates in the range of 500 nm(Figure 4,
The loss of activity upon recycling without decrease
in selectivity was low enough to consider adding a
C). The amalgams of these aggregates created a fraction of fresh catalyst between batches in order to
second level of pores of about 1 mm. compensate for this effect. This approach is particu-
Figure 4. SEM photographs of LuCLEA; A: ”250, B: ”950, and C: ”8,000.
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FULL PAPERS
investigated this effect by measuring the specific ac-
tivity of the CLEA as a function of the freeze drying
duration and established that a ratio of 1 g CLEA ob-
tained per 12 mL of commercial LuHNL solution
gave a sufficiently dry catalyst with a very high specif-
ic activity (303.5 U/g) and recovery (33%).
LuCLEA was then used in the synthesis of (R)-2-
butanone cyanohydrin on an 80-mmol scale and the
reaction was complete in 3 h at 308C. Following the
conclusions of the recycling experiments, LuCLEA
was recycled for a second batch and a portion of fresh
catalyst (20% of the original loading) was added to
compensate for the loss of activity. The second batch
was equally fast, selective and high yielding thereby
allowing the preparation of (R)-2-butanone cyanohy-
drin in a 160-mmol scale over two 3 h batches using
an overall catalyst loading of 4.8 U/mmol. The ee of
the cyanohydrin formed was also improved signifi-
cantly to 87% which was attributed to the optimized
catalyst used here. This ee value comes close to a liter-
ature report for LuHNL immobilized on nitrocellu-
lose on an analytical scale (ee=95%). Due to the an-
alytical conditions, a ten times higher catalyst loading
(50 U/mmol) could be employed. This helped to sup-
press the fast racemic chemical reaction even better
than under the conditions described here.[21] The
crude cyanohydrin was easily obtained by filtration of
the catalyst and evaporation of the volatiles under re-
duced pressure and was subjected to acid hydrolysis
to give (R)-2-hydroxy-2-methylbutyric acid in 85%
isolated yield (from2-butanone) and 87% ee. The op-
tical purity of the acid can readily be improved by a
very efficient crystallization using enantiopure 1-(1-
naphthyl)ethylamine as a resolving reagent.[4]
Figure 5. LuCLEA recycling experiment in the preparation
of 2-butanone cyanohydrin (5 mmol scale). Reaction condi-
tions: 2-butanone (5.0 mmol), HCN (4 equiv.), LuCLEA
*
(40 U), 308C, in buffer-saturated DIPE (10 mL). : conver-
*
sion of 2-butanone (fresh catalyst), : ee of 2-butanone cya-
~
&
nohydrin (fresh catalyst). : conversion (cycle 1), : conver-
^
sion (cycle 2), : conversion (cycle 3).
Table 4. 2-Butanone cyanohydrin obtained upon recycling:
isolated yield after 6 h, ee, and residual substrate content.
Catalyst
Yield[a]
ee[b]
Residual Substrate[c]
Fresh CLEA
Cycle 1
Cycle 2
84
76
64
56
81
80
81
78
<1%
<1%
<1%
<1%
Cycle 3
[a]
Isolated yield (%).
ee of the isolated product.
In mol% as determined by H NMR.
[b]
[c]
1
The LuCLEA-based approach towards enantiopure
larly suitable for a carrier-free immobilized catalyst acid I described here is highly atom-efficient and gen-
with very high volumetric activity such as CLEAs erates little waste, even when a recrystallization is
since the catalyst accounts for a small percentage of taken into account. Moreover, LuCLEA can be recy-
the overall reaction volume.
cled without loss of enantioselectivity and modest loss
of activity. When compared to the substrate engineer-
ing approach described earlier[16] where overall yields
of 45% and 51% were reported for the preparation of
(S)-I in 99% ee, the direct synthesis of the cyanohy-
drin from2-butanone catalyzed by LuCLEA towards
Preparative Scale Synthesis of
(R)-2-Hydroxy-ACHTREUNG2-methylbutyric Acid
Our approach for the preparation of (R)-2-hydroxy-2- (R)-I proved to be high yielding with a good atom-
methylbutyric acid [(R)-I] in high enantiopurity economy. Moreover, relatively high catalyst loadings
(Scheme 1) involved the direct synthesis of the cyano- were required in the substrate engineering approach
hydrin (III) from2-butanone
( II) catalyzed by (>150 U/mmol) to achieve good conversion while our
LuCLEA followed by acid hydrolysis to the corre- approach required less than 5 U/mmol of LuCLEA,
sponding a-hydroxy acid (I) as reported for which corresponds to less than 15 U/mmol of initial
PaHNL.[18]
commercial LuHNL solution.
We first optimized LuCLEA preparation on a 90-
mL scale and noticed that freeze drying affected the
biocatalyst activity. Fromresults obtained in earlier
samples it was clear that the CLEA could not be
Conclusions
freeze-dried overnight and should be promptly stored The CLEA immobilization strategy was applied suc-
at À208C when the biocatalyst is sufficiently dry. We cessfully to the sole representative of the zinc-depen-
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FULL PAPERS
dent alcohol dehydrogenases group among HNLs: Standard Enzyme Activity Test
LuHNL. Various strategies were attempted to devel-
The activity of soluble enzyme was measured according to a
modified version of the literature procedure.[20] The reaction
was performed in a screw-cap vial using a dilute solution of
commercial LuHNL in phosphate buffer (5 mM, pH 6.5).
The dilute solution (50 mL) was added to citrate/phosphate
buffer (438 mL, 0.2M Na2HPO4, 0.1M citric acid, pH 4.1)
followed by 12 mL of a solution of acetone cyanohydrin
(10% v/v) in 0.1M citric acid. The reaction mixture was in-
cubated for 10 min at 308C and quenched with 0.01M aque-
ous HCl (500 mL). A sample (10 mL) was then diluted in
water (9.99 mL) and the cyanide concentration was mea-
sured using a commercially available cyanide test (Merck,
Spectroquant cyanide test). The test was calibrated using
standard aqueous solutions of sodiumcyanide. A reference
reaction (without enzyme) was performed in parallel and
the activity of the soluble enzyme was determined in mmol
of acetone cyanohydrin hydrolyzed per min. The CLEA and
EA activities were measured according to the same proce-
dure.
op this catalyst including the first report of molecular
imprinting as a tool to improve the activity of a
CLEA. LuCLEA with high specific activity
(303.5 U/g) could be prepared in good activity recov-
ery (33%) on a multigram scale.
(R)-2-Butanone cyanohydrin was synthesized on a
preparative scale over two batches upon the catalysis
of LuCLEA using only a small portion of fresh cata-
lyst (20%) between batches to compensate for the
loss of activity upon recycling. After hydrolysis, (R)-2-
hydroxy-2-methylbutyric acid was obtained in 85%
isolated yield (from2-butanone) and 87% ee.
Experimental Section
CAUTION: All procedures involving hydrogen cyanide
were performed in a well-ventilated fume-hood equipped
with an HCN detector. HCN-containing wastes were neu-
tralized using commercial bleach and stored independently
over a large excess of bleach for disposal.
Precipitation Study
Phosphate buffer (75 mL, 0.1M, pH 7) was added to a com-
mercial solution of LuHNL (225 mL, 114 U/mL) and the
mixture was kept shaking at 08C for 5 min. The cosolvent
(113 mL, 450 mL, or 900 mL depending on the experiment)
was then added and the mixture was kept shaking at 08C
for an additional 10 min. The aggregates were separated
from the supernatant by centrifugation (10 000 rpm, 5 min)
and the supernatant was diluted in 500 mL of phosphate
buffer (5 mM, pH 6.5) before measuring the activity. The ag-
gregates were dissolved in 1.50 mL of phosphate buffer
(5 mM, pH 6.5) and the activity was measured.
General Remarks
Enzymes: Solutions of the hydroxynitrile lyases from
Prunus amygdalus (PaHNL, 690 U/mL), Manihot esculenta
(MeHNL, 2.23 kU/mL), and Linum usitatissimum (LuHNL,
114 U/mL or 76.9 U/mL) were purchased from Codexis. The
CLEAs from PaHNL[22] and MeHNL[24] were prepared ac-
cording to literature procedures.
Chemicals: A standard solution of HCN (2M in DIPE)
was prepared as previously reported.[23] Acetone cyanohy-
drin was prepared according to a literature procedure.[36]
Racemic 2-butanone cyanohydrin was prepared via its TMS
derivative according to a literature procedure.[37] Other
chemicals and reagents were purchased from commercial
sources and used without further purification unless other-
wise specified.
Analytical Methods: 1H and 13C NMR spectra were re-
corded on a Bruker 400 Avance Ultrashield spectrometer.
Absorbance was measured using a Shimadzu Biospec-1601
UV spectrometer. For SEM analysis, the sample was coated
with gold and the pictures were taken on a Jeol JSM-6700M
field emission scanning electron microscope. Chiral gas
chromatography (GC) was performed using an Agilent
Cross-Linking Study
The cross-linking experiments were conducted as described
for the precipitation and after 10 min at 08C a 25% aqueous
glutaraldehyde solution (11 mL, 22.5 mL, 45 mL, 67.5 mL de-
pending on the experiments) was added to the mixture. The
suspension was kept shaking at 08C for 1 h and the CLEAs
were separated by centrifugation (10,000 rpm; 5 min). The
CLEAs were resuspended in 1.50 mL of phosphate buffer
(5 mM, pH 6.5) and after centrifugation the activity of the
supernatant was measured to determine the efficiency of the
cross-linking. The CLEAs were then suspended in 1.50 mL
of fresh phosphate buffer (5 mM, pH 6.5) and the activity
was measured accordingly.
Technologies 6890N chromatograph equipped with a b-Dex EAs and CLEAs Specific Activity and Recovery
225 column and an FID detector. High performance liquid
chromatography (HPLC) was performed on an Agilent
Technologies 1100 series chromatograph equipped with a
Enzyme Aggregates (EAs): Commercial LuHNL (900 mL,
69.2 U) was diluted in phosphate buffer (300 mL, 0.1M,
pH 7) and the solution was shaken in an ice/water bath.
diode array detector. Specific conditions for chromatogra-
After 5 min, saturated aqueous (NH4)2SO4 (3.6 mL) for
phy and retention times are given in the experimental sec-
LuEAACTHERNGU(Am.Sulf) or tert-butanol (1.8 mL) for LuEAACHTREUNG(t-Bu)
tion for the respective compounds.
was added and shaking in ice was continued for 10 min. The
suspension was then centrifuged (10,000 rpm, 5 min) washed
with acetonitrile and centrifuged again. The enzyme aggre-
gates were freeze dried and the activity was measured ac-
cording to the standard activity test.
2336
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 2329 – 2338

Linum usitatissimum Hydroxynitrile Lyase Cross-Linked Enzyme Aggregates
FULL PAPERS
Cross-Linked Enzyme Aggregates (CLEAs): Precipitation
of the enzyme was performed as described for the EAs. A
25% aqueous glutaraldehyde solution (90 mL) was added to
the suspension and shaking in ice was continued for 1 h. The
CLEAs were centrifuged (10,000 rpm, 5 min), washed with
phosphate buffer (5 mM, pH 6.5), and freeze dried. The
CLEA activity was measured according to the standard ac-
tivity test.
PaCLEA and MeCLEA: The biocatalysts were prepared
as reported.[22,24] The activity of commercial PaHNL,
MeHNL and the corresponding CLEA was measured ac-
cording to the standard activity test for LuHNL.
(10,000 rpm, 5 min), washed with phosphate buffer (5 mM,
pH 6.5), and freeze dried to give 392.5 mg of immobilized
biocatalyst (125.9 U/g, 7% recovery). LuCLEA (318.0 mg,
40 U) was then loaded in a jacketed flask and a 2M HCN
solution in DIPE (10 mL) was added. The temperature was
kept at 308C and biphenyl (ca. 2 mg) was added to the sus-
pension. 2-Butanone (448 mL, 5 mmol) was then added and
the reaction was monitored as described above. After 6 h of
reaction at 308C LuCLEA was filtered off, rinsed with fresh
DIPE (30 mL) and loaded back into the jacketed flask for
the next cycle. The combined DIPE phases were evaporated
under reduced pressure to give the crude 2-butanone cyano-
1
hydrin as a colourless liquid. H NMR (CDCl3, 400 MHz):
d=1.10 (t, 3H, J=7.5 Hz), 1.59 (s, 3H), 1.81 (mc, 2H), 3.77
(bs, 1H); 13C NMR (CDCl3, 100.65 MHz): d=8.4, 26.8, 34.6,
69.1, 121.8.
Molecular Imprinting Study
EAs and CLEAs with 2-butanone as additive were prepared
as describe above from a solution of commercial LuHNL
solution (900 mL, 69.2 U) in phosphate buffer (300 mL,
0.1M, pH 7) and 2-butanone (69.2 mL). The activity of the
biocatalysts obtained was measured according to the stan-
dard activity test and the results were expressed as the dif-
ference of specific activity (in percentage) between the cata-
lyst prepared with and without additive [Eq. (1)].
(R)-2-Hydroxy-2-methylbutyric Acid
Commercial LuHNL (90 mL, 6.92 kU) was diluted in phos-
phate buffer (30 mL, 0.1M, pH 7) at 08C and 2-butanone
(6.92 mL) was added. The solution was shaken at 08C for
5 min and tert-butanol (180 mL) was added. Shaking at 08C
was continued for 10 min and a 25% aqueous glutaralde-
hyde solution (9.00 mL) was added to the suspension. After
shaking at 08C for 1 h LuCLEA was centrifuged
(10,000 rpm, 5 min), washed with phosphate buffer (5 mM,
pH 6.5), and freeze dried until the weight of immobilized
biocatalyst was 7.59 g (303.5 U/g, 33% recovery). LuCLEA
(2.11 g, 640 U, 8.0 U/mmol) was then loaded in a jacketed
flask and a 2M HCN solution in DIPE (160 mL) was added.
The temperature was kept at 308C and biphenyl (ca. 10 mg)
was added to the suspension. 2-Butanone (7.2 mL, 80 mmol)
was then added and the reaction was monitored as described
above. After 3 h at 308C LuCLEA was filtered off, rinsed
with diethyl ether (3”100 mL), loaded back into the jacket-
ed flask, and resuspended in 2M HCN solution in DIPE
(160 mL). An additional loading of fresh LuCLEA
(421.9 mg, 128 U, 2.0 U/mmol) was added and a second
batch of 2-butanone cyanohydrin was prepared accordingly.
The combined ethereal phases from the first batch were
evaporated under reduced pressure to give the crude (R)-2-
butanone cyanohydrin (yield: 8.69 g, 86% ee). This crude cy-
anohydrin was stirred at room temperature in a mixture
(1:2) of water:conc. HCl while the enzymatic reaction for
the second batch proceeded (3 h). The crude (R)-2-butanone
cyanohydrin fromthe second batch (yield: 7.92 g, 87% ee)
was diluted in 60 mL of water:conc. HCl (1:2) and combined
with the reaction mixture of the first batch. The hydrolysis
was allowed to proceed upon vigorous stirring at 65–708C
Synthetic Activity
Selected CLEAs and EAs were loaded (2.2 U) in a screw-
cap vial and a solution of HCN 2M in DIPE (1.00 mL) was
added. The HPLC internal standard, biphenyl (2 crystals),
was added to the mixture upon stirring to ensure complete
dissolution. The reaction was started by addition of 2-buta-
none (44.8 mL, 0.50 mmol) and an analytical sample was
taken immediately after addition to determine the initial
conditions in HPLC. The conversion ratios were determined
by HPLC [10 mL reaction samples in 1.00 mL hexane; Chir-
alpak AD; mobile phase: hexanes:2-propanol (99:1); flow:
1.5 mLminÀ1
2.64 min, RtA(ketone)=3.45 min]. The ee of the cyanohydrin
; UV detection at 280 nm; RtACHTRE(UGN biphenyl)=
CHTREUNG
formed was monitored by chiral GC of the trifluoroacetate
derivative in a modified version of the literature proce-
dure[20] [20 mL reaction samples in 1.00 mL anhydrous di-
chloromethane, 10 mL trifluoroacetic acid anhydride, 10 mL
anhydrous pyridine; b-Dex 225 column; 808C; 10 psi;
Rt(R)=13.77 min , Rt(S)=14.20 min].
1
and monitored by H NMR of reaction samples (100 mL) ex-
tracted in ether (1.00 mL). After 20 h the reaction mixture
was cooled to roomtepmerature, diluted with water
(80 mL) and ether (300 mL) was added. After stirring for
10 min, the phases were separated and the aqueous layer
was extracted with ether (2”300 mL). The combined ethere-
al layers were dried (MgSO4) and the solvent was removed
under reduced pressure to give (R)-2-hydroxy-2-methylbuty-
ric acid as a white solid; yield: 16.08 g (136 mmol, 85%
Recycling Experiments
Commercial LuHNL (9.00 mL, 692 U) was diluted in phos-
phate buffer (3.00 mL, 0.1M, pH 7) at 08C and 2-butanone
(692 mL) was added. The solution was shaken at 08C for
5 min and tert-butanol (18 mL) was added. Shaking at 08C
was continued for 10 min and a 25% aqueous glutaralde-
hyde solution (900 mL) was added to the suspension. After
shaking at 08C for 1 h LuCLEA was centrifuged
1
yield, 87% ee). H NMR (CDCl3, 400 MHz): d=0.94 (t, 3H,
J=7.5 Hz), 1.47 (s, 3H), 1.73 (dq, J=7.5 Hz, 15.0 Hz, 1H),
1.86 (dq, J=7.5 Hz, 15.0 Hz, 1H), 5.79 (bs, 2H); 13C NMR
Adv. Synth. Catal. 2008, 350, 2329 – 2338
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2337

Fabien L. Cabirol et al.
FULL PAPERS
(CDCl3, 100.65 MHz): d=7.8, 25.4, 32.9, 75.1, 181.4. The ee
of the product was determined as described previously[4] [ca.
1 mg product dissolved in 2-propanol (100 mL) and diluted
in hexane (1.00 mL); Chiralpak AD; mobile phase: hex-
anes:2-propanol:TFA (96:4:0.1); flow: 1.5 mLminÀ1; UV de-
tection at 210 nm; Rt(R)=8.22 min, Rt(S)=9.10 min].
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