Synthesis of aliphatic and α-halogenated ketone cyanohydrins with the hydroxynitrile lyase from Manihot esculenta

Article abstract of DOI:10.1002/cctc.201300965

The potential of the hydroxynitrile lyase from Manihot esculenta towards ketone substrates was investigated. It was observed that the length of the aliphatic chain is a key parameter for the conversion of aliphatic, non-branched ketones. Smaller substrates are readily converted with high enantioselectivites, but the elongation of the chain length causes a significant loss in enzyme activity. For a number of halogenated, herein especially fluorinated, acetophenone derivatives the corresponding cyanohydrins have been synthesized with good to moderate enantioselectivities. Overcoming limitations: Ketone cyanohydrins are extremely useful intermediates for the synthesis of unusual tertiary alcohols. In this contribution, the limits of the hydroxynitrile lyase from Manihot esculenta towards such substrates are investigated and evaluated. High enantioselectivities are obtained in a simple two-phase system, even with very challenging fluorinated acetophenone derivatives.

Full text of DOI:10.1002/cctc.201300965

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DOI: 10.1002/cctc.201300965
Synthesis of Aliphatic and a-Halogenated Ketone
Cyanohydrins with the Hydroxynitrile Lyase from Manihot
esculenta
Johannes Diebler,[a, c] Jan von Langermann,[a] Annett Mell,[a] Martin Hein,[b] Peter Langer,[b]
and Udo Kragl*[a]
The potential of the hydroxynitrile lyase from Manihot esculen-
ta towards ketone substrates was investigated. It was observed
that the length of the aliphatic chain is a key parameter for
the conversion of aliphatic, non-branched ketones. Smaller
substrates are readily converted with high enantioselectivites,
but the elongation of the chain length causes a significant loss
in enzyme activity. For a number of halogenated, herein espe-
cially fluorinated, acetophenone derivatives the corresponding
cyanohydrins have been synthesized with good to moderate
enantioselectivities.
and higher applicability.[6] Thus, these biocatalysts were repeat-
edly applied in large-scale productions within the pharmaceuti-
cal and agrochemical industry.[20] Examples include (S)-3-phe-
noxybenzaldehyde cyanohydrin, which is a precursor of various
synthetic pyrethroids, and (R)-2-hydroxy-4-phenylbutyronitrile
for the production of angiotensin-converting enzyme inhibi-
tors.[21–23]
The vast majority of successful HNL-catalyzed cyanohydrin
formations include the synthesis of aldehyde cyanohydrins,
which are typically obtained in high yield and enantiomeric
purity. In contrast to this, ketone cyanohydrins are considered
as less easily achievable owing to steric hindrance, lower stabil-
ity, and the limitation of substrate acceptance of HNLs.[4,24–26]
However, ketone cyanohydrins are considered as even more
Hydroxynitrile lyases (HNLs) catalyze selectively the CÀC cou-
pling of hydrogen cyanide to carbonyl compounds to yield
enantiopure cyanohydrins.[1–3] Owing to the formation of two
new functional groups (one hydroxyl and one nitrile group),
these enantiopure substances are considered as valuable inter-
mediates for a variety of building blocks for pharmaceuticals,
including a-hydroxy carboxylic acids, b-hydroxyamines, and
a-aminonitriles.[4–8] Since the first experiments of Wçhler in
1837 and Rosenthaler in 1909, HNLs became extremely valua-
ble biocatalysts for the synthesis of enantiopure cyanohy-
drins.[9–11] Important examples of highly enantioselective en-
zymes are the (R)-selective HNLs from Prunus amygdalus
(PaHNL, bitter almond) and Arabidopsis thaliana (AtHNL, thale
cress) and the (S)-selective HNLs from Hevea brasiliensis
(HbHNL, rubber tree) and Manihot esculenta (MeHNL, cassa-
va).[12–15] Within the HNL-catalyzed synthesis of cyanohydrins,
the natural defense mechanism (release of hydrogen cyanide)
is simply reversed.[16–19] Furthermore, in comparison to other
chemical cyanohydrin synthesis routes, the enzymatic method
offers higher enantioselectivities, moderate reaction conditions,
valuable chemicals, as they possess
a tertiary alcohol
group.[13,27–29] For example, the MeHNL-catalyzed synthesis of
unsubstituted acetophenone cyanohydrin, a precursor for atro-
lactic acid, was found to be thermodynamically limited with an
equilibrium cyanohydrin yield of 2%.[26] Medium engineering
can be used to overcome, at least partially, the thermodynamic
restrictions, but it is rather limited to liquid reactants and re-
quires a significantly higher amount of biocatalyst. In contrast,
several successful examples of enantioselective conversions of
ketones into the respective cyanohydrins were reported in the
past. Examples include the AtHNL-catalyzed synthesis of 2-hex-
anone cyanohydrin with 48% conversion and 95% (R) enantio-
meric excess (ee),[11] selected (branched) aliphatic ketone cya-
nohydrins with enantioselectivities up to 91%ee(S) (MeHNL),[24]
and the synthesis of heterocyclic tertiary cyanohydrins with
HbHNL and PaHNL.[30]
Table 1. HNL-catalyzed synthesis of aliphatic methyl ketone cyanohydrins
and a-halogenated acetophenone cyanohydrins.
[a] J. Diebler, Dr. J. von Langermann, A. Mell, Prof. Dr. U. Kragl
University of Rostock
Department of Chemistry, Industrial Chemistry
Albert-Einstein-Strasse 3A, 18059 Rostock (Germany)
Fax: (+49)381-498-6452
E-mail: udo.kragl@uni-rostock.de
Aliphatic
R1
Aromatic
R1
1, 2
R2
1, 2
R2
[b] Dr. M. Hein, Prof. Dr. P. Langer
University of Rostock
a
b
c
d
e
f
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
h
i
j
k
l
Ph
Ph
Ph
Ph
Ph
CH3
Department of Chemistry, Organic Chemistry
Albert-Einstein-Strasse 3A, 18059 Rostock (Germany)
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
CH2F
CHF2
CF3
[c] J. Diebler
Leibniz Institute for Catalysis (LIKAT) Rostock, Organocatalysis
Albert-Einstein-Strasse 29a, 18059 Rostock (Germany)
CF2Cl
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300965.
g
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The aim of this study was to deepen the understanding of
the synthesis of ketone cyanohydrins (Table 1). Herein, the bio-
catalyst MeHNL was chosen to study the conversion of a series
of nonbranched aliphatic methyl ketones and a-halogen-sub-
stituted acetophenone cyanohydrins within industrially pre-
ferred two-phase systems consisting of a buffer and an organic
solvent such as diisopropyl ether (DIPE).[31,32]
verted by MeHNL at pH 4.0 in high yields with moderate to
high enantioselectivities, for example, 1a and 1b. Unfortunate-
ly, racemization of the product occurred aside the non-enzy-
matic reaction (see also Figure S7, Supporting Information),
which gradually decreased the enantiomeric excess of the
product over time, especially for product 2b. Lowering the pH
value in the aqueous phase to pH 3.0 or even lower was not
successful as a result of a significant loss of enzyme activity
under the resulting reaction conditions.
Corresponding cyanohydrins 2a–l are known as highly val-
uable products.[4,33] In particular, fluorinated aromatic com-
pounds are of great interest, as they facilitate a different lipo-
philicity in comparison to non-halogenated pharmaceuti-
cals.[34,35] For example, enantiopure 2k was used as a key inter-
mediate in the synthesis of an anticonvulsant. Another applica-
tion is its use as a precursor for potential inhibitors of a HIV
protease and reverse transcriptase.[36] Within the related litera-
ture, Nguyen et al. presented an alternative approach to 2k,
which was obtained through esterase-catalyzed kinetic resolu-
tion from a racemic solution.[37] Unfortunately, this procedure
required multiple reaction steps and yielded only a maximum
conversion of 50% within the hydrolysis step, which resulted
in a significance decrease in overall atom efficiency.[38] There-
fore a direct HNL-catalyzed route to enantioenriched ketone
cyanohydrins is preferred.
Substrates with medium-sized chain lengths (i.e., 1c–e) still
allowed moderate to high yields at pH 4.0, but a significant
loss of reactivity was observed. For example, 2c was obtained
with 94% conversion after 5 h, whereas the synthesis of 2e re-
quired 22.5 h for a conversion of 63%. High enantioselectivities
of approximately 90% ee(S) were obtained, which represent
the perfect balance of reactivity and high enantioselectivity of
the biocatalyst. Substrates with even longer side chains exhib-
ited significantly reduced reactivity with MeHNL. At pH 4.0, 1 f
was only converted with 19% and 1g with 10% conversion
even after prolonged reaction times. Fortunately, a further in-
crease in the pH value to pH 5.0 enhanced the conversion of
1 f and 1g to 88 and 75% with 87 and 81%ee(S), respective-
ly.[12,40] The varying initial enantiomeric excess values of the
products possibly depend on steric differences of both side
chains flanking the carbonyl functionality. Thus, a better ste-
reoselectivity is probably achieved for larger differences, as the
substrate fits better into the active site.
Aliphatic ketone cyanohydrins
The conversion of aliphatic methyl ketones into the corre-
sponding aliphatic ketone cyanohydrins within a two-phase
system revealed clearly that the length of the aliphatic chain is
a key parameter (Table 2). Smaller substrates were readily con-
The loss of reactivity of larger substrates may be related to
an increase in hydrophobicity of the long-chained aliphatic
substrates, which itself results in a decrease in substrate con-
centration within the aqueous phase. Thus, the apparent enzy-
matic activity of the biocatalyst, which is only located in the
aqueous phase, eventually drops significantly [the substrate
concentration in the aqueous phase is below the Michaelis-
Menten constant (KM) of the respective substrates]. In this case,
the KM value is required to be similar for all investigated sub-
strates. Alternatively, the transformation of larger substrates
may be hampered by steric effects in the active site of the
enzyme. This may also increase the value of KM for larger sub-
strates, which would result in a decrease in the apparent activi-
ty of the enzyme. Eventually, higher substrate concentrations
or enzyme loadings would be required.[41,42] Regardless, the ob-
served effect is at least partially compensated with the shown
increase in pH value from pH 4 to pH 5 in the aqueous phase.
This effect is related to increased enzyme activity at higher pH
values, as the pH optimum is found near pH 5.75.[43]
Table 2. Synthesis of aliphatic ketone cyanohydrins.[a]
Substrate
pH
t [h]
Conversion [%]
ee (S) [%]
1a[b]
1b
4[b]
1
5
1
5
1
5
1
22.5
1
22.5
1
22.5
1
22.5
1
91[b]
>99
>99
87
94
19
57
6
63
<1
19
59
88
<1
10
7
18[b]
78
19
93
87
>90
88
n.d.[c]
87
4
4
4
4
4
5
4
5
1c
1d
1e
1 f
–
In summary, two-phase systems are feasible reaction systems
for the conversion of aliphatic methyl ketones if the reaction
conditions are carefully adjusted. Herein, the loss of enantio-
meric excess throughout the reaction was almost completely
suppressed and high substrate concentrations were available
for the biocatalyst. Monophasic non-aqueous reaction systems,
for example, biocompatible tert-butyl methyl ether in combina-
tion with immobilized biocatalysts, may also be useful alterna-
tives for such substrates in the enzymatic synthesis of aliphatic
methyl ketone cyanohydrins.
n.d.
>90
75
–
87
1g
85
22.5
75
81
[a] Reaction conditions: Two-phase system consisting of a diisopropyl
ether/50 mm citrate buffer pH 4/pH 5 (1:1), substrate (50 mm), hydrogen
cyanide (250 mm), MeHNL (20 UmLÀ1), 108C. [b] Reported by Fçrster
et al.[39] (adsorbed MeHNL in monophasic diisopropyl ether). [c] n.d.=not
determined.
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Aromatic a-halogenated acetophenone cyanohydrins
In comparison to aliphatic substrates 1a–g even at the
chosen acidity of pH 5.0, a strong increase in the enzyme con-
centration was necessary to achieve the conversion of these
acetophenone derivatives. A further adjustment to pH 6.0
within the aqueous phase to compensate this effect was not
possible, as a parallel increase in the non-enzymatic addition
reaction and the racemization of the products occurred (see
also Figure 1). Prolonged reaction times were also observed
In contrast to the promising results of the HNL-catalyzed con-
version of aliphatic methyl ketones 1a–g, the use of the struc-
turally similar substrate acetophenone (1h) resulted in a signifi-
cant loss in equilibrium conversion. The conversion to 2h
dropped to an insufficient 2%, which prevented the efficient
synthesis of the corresponding cyanohydrins; this reaction is
also repeatedly given as a standard example of the limitations
in HNL-catalyzed reactions.[31] As mentioned above, medium
engineering can be used to overcome the restrictions, but it
was only partially successful with 22% yield [96%ee(S)]. A fur-
ther increase was only achievable by the additional introduc-
tion of electron-withdrawing groups at the aromatic ring of
acetophenone, preferably fluoro and nitro substituents in the
ortho position. Thereby, equilibrium concentrations were shift-
ed towards the product side by electronic effects.[26]
In the present study, the introduction of halogen substitu-
ents into the a-standing methyl group was chosen to further
tune the synthesis reaction towards high conversion and to
broaden the understanding of the conversion of a-substituted
ketones. A series of a-substituted acetophenone derivatives
1i–l was converted in the two-phase system at pH 5.0 and
compared to unsubstituted acetophenone 1h (Table 3). The
Figure 1. Conversion and enantiomeric excess values of the enzymatic syn-
thesis of a,a-difluoroacetophenone cyanohydrin. Reagents and conditions:
two-phase system consisting of diisopropyl ether/50 mm citrate phosphate
buffer pH 5 (1:1), substrate (50 mm), hydrogen cyanide (250 mm), MeHNL
(133 UmLÀ1), 108C.
Table 3. Synthesis
derivatives.[a]
of
aromatic
a-halogenated
acetophenone
with an increasing degree of substitution at the a position.
Whereas a,a-difluoroacetophenone (1j) was converted with
74% after 31.5 h, a,a,a,-trifluoroacetophenone (1k) required
an additional 12 h (ꢀ44 h total) for comparable conversion
(76%). A further increase in size by exchange of one fluoro
group by a chloro substituent resulted in slightly higher reac-
tivity and required a total of 30 h for a conversion of 68%,
which represents equilibrium conversion. The loss of reactivity
for all derivatives can be explained by the nonoptimal fit
within the active site pocket as a result of the additional size
requirement from the introduced halogen atoms (as was de-
duced from examination of the active site of the enzyme, crys-
tal structure: PDB code: 1EB9).
Substrate
t [h]
Conversion [%]
2[b]
ee (S) [%]
1h[b]
1i
24[b]
–
no enzymatic conversion
1.75
31.5
1.5
44.25
1
1j
4
74
<1
76
17.5
68
79
61
–
1k
1l
60
n.d.[c]
58
30
[a] Reaction conditions: two-phase system consisting of diisopropyl
ether/50 mm citrate phosphate buffer pH 5 (1:1), substrate (50 mm), hy-
drogen cyanide (250 mm), MeHNL (133 UmLÀ1), 108C. [b] Obtained from
von Langermann et al.[26] [c] n.d.=not determined.
The obtained enantioselectivities of a-substituted cyanohy-
drins 2j and 2l were in general significantly lower than the
enantioselectivity of unsubstituted acetophenone cyanohydrin
2h. However, similar values were obtained for all investigated
halogen-substituted derivatives, which indicates again a general
limitation for substituents at the a-standing methyl group. The
tendency of the loss of enantiomeric excess was investigated
in detail for 2j (Figure 1). The enantiomeric excess of the prod-
uct steadily decreased with increasing product concentration
over time, and this highlights the initial relatively high enantio-
selectivity of 79% (after 2 h). This loss is mainly caused by the
parallel non-enzymatic reaction and only to an insignificant
degree by product racemization. As shown in Figure 1, after
31.5 h the enantiomeric excess dropped to 61%.
HNL-catalyzed synthesis of substituted products 2i–l has not
yet been described in the literature to the best of our knowl-
edge.
The results show clearly that in comparison to unsubstituted
acetophenone, significantly higher conversions (ꢀ75%) were
achievable by using a two-phase system. An exception was
a-fluoroacetophenone (1i), which was not converted by
MeHNL. Possibly, the substrate did not fit correctly into the
active site or a microkinetic step was significantly slowed
down. Further studies investigating the main kinetic parame-
ters (reaction velocity and KM values) would be required (for 1i
and similar substrates) to explain the observed effect.
In conclusion, a series of aliphatic methyl ketone and a-sub-
stituted acetophenone derivatives were successfully converted
into the corresponding cyanohydrins with the hydroxynitrile
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The non-enzymatic reaction was monitored by mixing the compo-
nents without the enzyme. Samples were drawn from the organic
phase and derivatized as mentioned below.
lyase from Manihot esculenta. Aliphatic ketone substrates 1a–g
were easily transformed into the corresponding cyanohydrins,
and the length of the aliphatic chain seemed to be of minor
importance. In contrast, introduction of substitutions at the
a-standing methyl group of acetophenone cyanohydrins
caused a strong drop in reactivity. This indicates a size limita-
tion within the active site at this position. The presented re-
sults help to evaluate and understand the MeHNL-catalyzed
synthesis of ketone cyanohydrins.
Derivatization
Owing to the thermal instability of ketone cyanohydrins, derivatiza-
tion prior to gas chromatography analysis was required. Samples
of aliphatic ketone cyanohydrins (or a 50 mL sample) were derivat-
ized in dichloromethane (700 mL), trifluoroacetic acid anhydride
(50 mL), and pyridine (50 mL) at room temperature. Derivatization of
a-substituted acetophenone cyanohydrins (or a 50 mL sample) re-
quired O-silylation with N,O-bis(trimethylsilyl)trifluoroacetamide
(50 mL) in dichloromethane (800 mL) and a reaction time of 12–20 h
at room temperature (Scheme 1).
Experimental Section
Chemicals
All required chemicals were obtained from Sigma–Aldrich and
were used as received. Mandelonitrile was aliquoted and stored at
À188C to reduce spontaneous decomposition to benzaldehyde
and hydrogen cyanide. The enzyme (S)-hydroxynitrile lyase from
Manihot esculenta, MeHNL, was a gift from Julich Chiral Solutions,
GmbH (now Codexis).
Hydrogen cyanide
All reactions involving the use of hydrogen cyanide or its forma-
tion should be executed within a well-ventilated fume hood. The
use of an electrochemical hydrogen cyanide detector is strongly
recommended. The desired amount of HCN was freshly distilled
before use. In a general approach, 5m H2SO4 (5 mL) was added
dropwise to a solution of NaCN (1.6 g) in H2O (5 mL), and subse-
quently, the mixture was heated to 708C. The formed free HCN
was condensed, cooled, dried with anhydrous Na2SO4, and stored
below 58C. An electrochemical HCN detector (Micro III G203 by
GfG-Gesellschaft fꢁr Gerꢂtebau, Dortmund, Germany) was placed
in the fume hood for continuous HCN monitoring.
Scheme 1. Derivatization of the acetophenone cyanohydrins with N,O-bis(tri-
methylsilyl)trifluoroacetamide (50 mL) to form O-silylated cyanohydrins.
For the identification of silylated cyanohydrins enantiomers 3 h–k
with the applied GC method, silylated racemic mixtures were syn-
thesized according to Cabriol et al.[44] NaCN (20 mmol) was dis-
solved in dry DMSO in a three-necked, round-bottomed flask
equipped with a magnetic stirrer under an argon atmosphere.
After the addition of an initial amount of trimethylsilyl chloride
(TMSCl, 3 mmol), a mixture of the substrate (10 mmol) and TMSCl
(12 mmol) in 5 mL dry n-hexane was added dropwise, and the re-
sulting mixture was heated to 608C. After 15 min, the reaction was
stopped, and the mixture was extracted with n-hexane. The organ-
ic layer was washed with DMSO, and the residual amount of DMSO
was removed by cooling with ice, filtering, and eluting over a silica
plug. The synthesis of 2l (and 3l) was not successful.
Enzyme assay
Enzymatic activity was determined photometrically with a UV/Vis
spectrometer SPECORD 200 (Analytik Jena, Jena, Germany) by
monitoring the occurrence of benzaldehyde at 280 nm from the
consumption of racemic mandelonitrile. Therein, 50 mm citrate-
phosphate buffer (pH 5, 700 mL) and the enzyme solution (100 mL)
were combined in a quartz cuvette, which was tempered at 258C.
The measurement started by the injection of 67 mm mandelonitrile
(200 mL) in 50 mm citrate buffer (pH 2). The enzyme activity was
eventually corrected by subtraction of the non-enzymatic mande-
lonitrile cleavage reaction, which was monitored in parallel without
the addition of enzyme solution (100 mL citrate phosphate buffer
pH 5 was used instead). One unit (1 U) of enzyme activity was de-
fined as the formation of 1 mmol benzaldehyde per minute under
standard assay conditions.
GC analysis
The enzyme-catalyzed conversion of the ketones into the corre-
sponding cyanohydrins and the resulting enantiomeric excess
values were determined by gas chromatographic analysis by using
a CP CHIRASIL-Dex CB capillary column equipped with a flame ioni-
zation detector (FID) in a VARIAN CP 3800 gas chromatograph.
n-Decane was used as the internal standard. Carrier gas=
2 mLminÀ1 (He), oven temperature=808C (isothermal), injector
temperature=2008C, detector temperature=2508C.
Synthesis reaction
The enzymatic synthesis of the cyanohydrins was performed in an
aqueous two-phase system (ATPS) consisting of diisopropyl ether
and 50 mm citrate (phosphate) buffer at 108C in a ratio of 1:1 v/v.
The pH of the buffer was adjusted to the apparent reactivity of the
enzyme towards the corresponding substrate. After the addition of
the substrate (50 mm) and the enzyme (400 U), the reaction mix-
ture was shaken horizontally and started by adding 250 mm HCN.
Acknowledgements
Financial support from the Federal Ministry of Education and Re-
search (Bundesministerium fꢀr Bildung und Forschung–BMBF) is
gratefully acknowledged (project: Biohydroform, grant number:
0313402E). Part of this work was financially supported by the
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Keywords: biocatalysis
cyanide · oxynitrilase
· chirality · enzymes · hydrogen
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Published online on February 26, 2014
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