C-C Bond Formation: Cyanohydrin Formation/Henry Reaction

1
2.1 C—C Bond Formation
2.1.1 Cyanohydrin Formation/Henry Reaction
K. Steiner, A. Glieder, and M. Gruber-Khadjawi
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General Introduction
Enantiopure cyanohydrins 1 and 2-nitro alcohols 2 (Scheme 1) are versatile building

blocks and intermediates that serve as starting materials for many enzymatic and chem-
ical follow-up reactions that find application in the pharmaceutical, agrochemical, and
cosmetic industries.
Scheme 1 Asymmetric Cyanohydrins and

2-Nitro Alcohols
OH
HO CN R3
R1 ∗ R2 R1 ∗ ∗ R2
NO2
1 2
R1 = alkyl, cycloalkyl, aryl, hetaryl; R2 = R3 = H, Me
Although various chemical methods for the preparation of these compounds, such as
metal catalysis with chiral ligands and the use of chiral organocatalysts, have been report-
ed and reviewed,[1–4] biocatalysis has gained more and more attention in recent years ei-
ther through direct synthesis using hydroxynitrile lyases (HNLs) or by kinetic resolution
of racemic mixtures by hydroxynitrile lyases, lipases, and esterases.[5–10] Biocatalytic reac-
tions are usually carried out under mild conditions, thereby avoiding unwanted side reac-
tions. Moreover, the selectivity of enzymes minimizes the need for protecting group
chemistry and thus the number of reaction steps.
In nature, hydroxynitrile lyases (EC 4.1.2.10, EC 4.1.2.11, EC 4.1.2.46, and EC 4.1.2.47;
alternative names: oxynitrilase, hydroxynitrilase, mandelonitrile lyase, hydroxymandelo-
nitrile lyase) catalyze the enantioselective cleavage of cyanohydrins. Almost two centu-
ries ago, in 1837, hydroxynitrile lyase activity was reported for the first time by Liebig
and Wçhler, when they observed the formation of hydrogen cyanide during the cleavage
of amygdalin using “emulsin”, a protein extract derived from bitter almonds (Prunus
amygdalus).[11]
For synthetic purposes, the C—C bond-forming reaction is significantly more valu-
able compared to the reverse cyanogenesis reaction: the carbon chain is prolonged by
one carbon atom, a chiral center is formed, and an additional versatile functional group,
the nitrile, is introduced into the molecule.[12–16] At the beginning of the last century,
Rosenthaler used emulsin for the first synthesis of (R)-mandelonitrile.[17]
for references see p 26

2 Biocatalysis 2.1 C—C Bond Formation
About a decade ago, it was discovered that hydroxynitrile lyases are not only able to
use cyanide as nucleophile, but also nitroalkanes, resulting in the formation of 2-nitro al-
cohols (nitroaldol or Henry reaction)[18] with up to two new chiral centers.[19]
This chapter will focus on the application of hydroxynitrile lyases for the synthesis of
chiral cyanohydrins and 2-nitro alcohols including enzyme production and stabilization,
and mechanistic aspects.
2.1.1.1 Hydroxynitrile Lyases Used in Synthesis
As mentioned above, the first hydroxynitrile lyase (HNL) used in synthesis was the (R)-se-
lective enzyme from Prunus amygdalus (PaHNL).[17] The first (S)-selective hydroxynitrile
lyase was discovered in Hevea brasiliensis (HbHNL) in 1989.[20] Since then, a number of
other hydroxynitrile lyases, both (R)- and (S)-selective, have been discovered (Table 1).
Cyanogenesis has not only been reported in many plant species such as Rosaceae,
Linaceae, Clusiaceae, Olacaceae, Euphorbiaceae, Gramineae, and Polypodiaceae, in which it
serves as a defense mechanism against herbivores and microbial attack,[21] but also in
other organisms such as bacteria, fungi, lichen, arthropods, and insects.[22–25] Interesting-
ly, hydroxynitrile lyases have also been discovered in non-cyanogenic plants such as Ara-
bidopsis thaliana.[26]
Hydroxynitrile lyases from Rosaceae are a quite conserved group of (R)-selective en-
zymes that are flavin adenine dinucleotide (FAD) containing monomeric glycoproteins.
Their natural substrate is (R)-mandelonitrile, but they have a broad substrate spectrum ac-
cepting aromatic aldehydes as well as aliphatic aldehydes and ketones. All other known
hydroxynitrile lyases are FAD independent. They are more versatile and vary in their
amino acid sequence, molecular weight, structural fold, oligomerization state, glycosyla-
tion pattern, enantioselectivity, and substrate range. Whereas some hydroxynitrile ly-
ases, e.g. the (R)-selective PaHNL from P. amygdalus and the (S)-selective HbHNL from H.
brasiliensis, have a broad substrate tolerance toward aliphatic, aromatic, and heteroaro-
matic aldehydes and ketones, others prefer either aromatic or aliphatic substrates (see
Sections 2.1.1.1.1 and 2.1.1.1.2).
Interestingly, although they all catalyze the same reaction, hydroxynitrile lyases be-
long to at least four different structural folds: Æ/-hydrolase fold proteins, oxido-
reductases, cupins, and alcohol dehydrogenases (see Section 2.1.1.1.4). Thus, hydroxyni-
trile lyases are considered a perfect example of convergent evolution.[27]
For a long time crude preparations of plant parts or purified enzymes from plants
were mainly used, which limited large-scale applications. However, in the past 20 years,
several genes encoding hydroxynitrile lyases have been identified and nowadays hydroxy-
nitrile lyases are also produced by heterologous expression in Escherichia coli or eukaryotic
expression systems such as Pichia pastoris (see Table 1). This enables the production of suf-
ficient quantities of proteins with constant quality and batch-to-batch reproducibility at
low cost.
Although E. coli is a common expression host, especially for prokaryotic proteins, be-
cause it is safe and easy to handle, has a fast generation time, and is cost efficient,[28] it has
some disadvantages for the expression of eukaryotic proteins, such as the insufficient
folding of complex proteins from higher organisms and the lack of posttranslational mod-
ifications, which results in insoluble and inactive proteins. P. pastoris has turned out to be
an ideal complementary expression system for eukaryotic proteins as it has a high growth
rate reaching high cell densities and can grow on inexpensive media.[29] It is also possible
to secrete the overexpressed protein to the medium and thus to reduce the purification
effort.[30] So far, PaHNL, which is glycosylated and contains a disulfide bridge, can only be
efficiently expressed in P. pastoris as soluble and active protein. Although hydroxynitrile
lyases that contain an Æ/-hydrolase fold can be intracellularly expressed in soluble form
in E. coli (Table 1), the expression levels are sometimes not high enough for industrial ap-
2.1.1 Cyanohydrin Formation/Henry Reaction 3
plication and the P. pastoris expression system is used instead. Alternatively, the hydroxy-
nitrile lyase from Manihot esculenta (MeHNL), which was originally expressed mainly in in-
clusion bodies in E. coli, is one example, for which the expression level was significantly
improved simply by codon optimization and optimizing the expression conditions.[31]
Table 1 Heterologously Expressed Hydroxynitrile Lyases[22,26,32–48]
Enzyme Source HNL Substrate Range Selectivity Expression Refa

System
Prunus amygdalus PaHNL aliphatic, aromatic, heteroaromat- R,Sb P. pastoris [32,33]
(almond) ic, and Æ,-unsaturated aldehydes

and methyl ketones
[34–36]
Hevea brasiliensis HbHNL aliphatic, aromatic, heteroaromat- S E. coli, P. pastoris, S.
(rubber tree) ic, and Æ,-unsaturated aldehydes cerevisiae
and methyl ketones
[37–39]
Manihot esculenta MeHNL aliphatic, aromatic, heteroaromat- S E. coli, P. pastoris, S.
(cassava) ic, and Æ,-unsaturated aldehydes cerevisiae, Leishma-
and methyl ketones, and aromatic nia tarentolae
ketones
[40,41]
Sorghum bicolor (mil- SbHNL aromatic and heteroaromatic alde- S E. coli (inactive)
let) hydes and methyl ketones
[42–44]
Linum usitatissimum LuHNL R: aliphatic aldehydes and methyl R,S E. coli, P. pastoris
(flax) ketones
S: benzyl ketones
[26]
Arabidopsis thaliana AtHNL aliphatic, aromatic, heteroaromat- R E. coli
(cress) ic, and Æ,-unsaturated aldehydes
and methyl ketones
[45]
Prunus mume PmHNL aliphatic, aromatic, and heteroaro- R P. pastoris
(Japanese apricot) matic aldehydes and methyl ke-
tones
[46]
Baliospermum monta- BmHNL aliphatic and aromatic aldehydes S E. coli
num (red physic nut) and ketones
[47,48]
Eriobotrya japonica EjHNL aliphatic, aromatic, and heteroaro- R P. pastoris
(loquat) matic aldehydes
[22]
Granulicella tundricola GtHNL aromatic aldehydes R E. coli
(acidobacterium)
a
Selected references describing the identification, isolation, and expression of the hydroxynitrile lyases; for their
application in synthesis refer to the schemes below.
b
Change of product configuration as a consequence of the CIP priority rules.
In addition to the hydroxynitrile lyases whose sequences are known and which can be
heterologously expressed (Table 1), hydroxynitrile lyase activity has been detected in
many other plants,[49–52] e.g. Prunus lyonii (Catalina cherry, PlyHNL),[53] Prunus persica
(peach),[50] Prunus domestica (plum),[50] Prunus avium (wild cherry),[50] Prunus serotina (black
cherry, PsHNL),[54] Prunus armeniaca (apricot, ParsHNL),[55] Prunus pseudoarmeniaca (wild
apricot),[56] Prunus communis L. var. dulcis Borkh (almond, PcHNL),[57] Prunus amygdalus turco-
manica [(Lincz.), a wild almond, PatHNL],[58] Malus communis (apple),[59] Chaenomeles speciosa
(quince, CsHNL),[60] Ximenia americana (yellow plum, XaHNL),[61] Passiflora edulis (passion
fruit, PeHNL),[62] Phlebodium aureum (fern, PhaHNL),[63] Vicia sativa (common vetch),[64] Sam-
bucus nigra (black elderberry),[49] Annona muricata (guanabana, AmHNL),[65] Annona cherimo-
la (cherimoya),[50] Annona squamosa (sugar apple),[50] Pouteria sapota (mamey),[66] Abrus pre-
catorius (rosary pea),[67] and bacteria Xylella fastidiosa (XfHNL),[25] Pseudomonas mephitica
(PsmHNL),[68] and Burkholderia phytofirmans strain PsJN (BpHNL).[68]

2.1.1.1.1 Applications of (R)-Selective Hydroxynitrile Lyases
One of the pioneering applications in the field of biocatalysis was the utilization of a
crude enzyme preparation from almonds, which was named “emulsin”, for the synthesis
of (R)-mandelonitrile [hydroxy(phenyl)acetonitrile; (R)-3] (Scheme 2).[17]
Scheme 2 Synthesis of (R)-Mandelonitrile Using

"Emulsin"[17]
emulsin, NaCN
O OH
acetate buffer, pH 4, rt, 24 h
Ph H 87%; 95% ee Ph CN
(R)-3
An (R)-selective enzyme found in emulsin catalyzes the formation of enantioenriched

(R)-cyanohydrins from a significant number of aromatic and aliphatic aldehydes and ke-
tones (see Sections 2.1.1.1.1.1 and 2.1.1.1.1.2). Meanwhile, many (R)-selective hydroxy-
nitrile lyases are available (Table 1).[5,10]
2.1.1.1.1.1 Hydrogen Cyanide Addition to Aldehydes
The first preparative application of an (R)-selective hydroxynitrile lyase (HNL) was the use
of the (R)-selective hydroxynitrile lyase from almond.[69,70] Brussee and co-workers used
the crude extract from almond meal in aqueous methanol with in situ generation of hy-
drogen cyanide.[71,72] Effenberger and co-workers showed for the first time the application
of the immobilized (R)-selective hydroxynitrile lyase from Prunus amygdalus.[73] Later, dif-
ferent research groups showed that the application of the natural support (unpurified al-
mond meal) in organic solvents containing a small amount of water leads to products
with high enantiomeric purities (up to 99% ee).[74] Performing media engineering, such
as “micro-aqueous systems”,[75,76] temperature, pH, and buffer choice,[77] as well as sub-
strate engineering,[78,79] (R)-cyanohydrins with high enantiomeric excesses could be ob-
tained. An (R)-selective hydroxynitrile lyase from apple seed meal catalyzes the transfor-
mation of sterically hindered aldehydes such as pivalaldehyde.[80] The suitability of the
(R)-specific hydroxynitrile lyase from Linum usitatissimum (flax) for the preparation of ali-
phatic cyanohydrins has been investigated and gave promising results (up to 99% ee).[44,81]
The (R)-selective hydroxynitrile lyase from Arabidopsis thaliana shows high activity toward
(R)-mandelonitrile [(R)-3] and the substrate range is similar to the (S)-selective hydroxy-
nitrile lyases from Hevea brasiliensis and Manihot esculenta, yielding cyanohydrins with
high enantiopurity.[26,82]
Until very recently, only (R)-selective hydroxynitrile lyases from plants have been ap-
plied for the biocatalytic synthesis of cyanohydrins. Interestingly, a new class of (R)-selec-
tive hydroxynitrile lyases has been discovered in bacteria.[22,68] A summary of selected cy-
anohydrin formations with various aldehydes as substrates catalyzed by (R)-selective hy-
droxynitrile lyases is collected in Scheme 3.[22,26,57,73,83–100]
Scheme 3 Selected Examples of the Enantioselective Synthesis of Cyanohydrins from

Aldehydes Catalyzed by (R)-Selective Hydroxynitrile Lyases[22,26,57,73,83–100]
O (R)-HNL OH
+ HCN
R1 H R1 CN
R1 (R)-HNL Sourcea Conversion (%) ee (%) Ref

Ph PaHNL 99b >99 [83]
[26]
Ph AtHNL >99 >99
[22]
Ph GtHNL 80 90
[73]
3-PhOC6H4 PaHNL 99 98
[26]
Bn AtHNL 97 96
[26]
(CH2)2Ph AtHNL 99 68
[84]
2-MeOC6H4 PaHNL 65 96
[85]
3-MeOC6H4 PaHNL 85 98
[86]
4-MeOC6H4 almond meal 47 99
[57]
4-MeSC6H4 PcHNL 98 96
[26]
2-FC6H4 AtHNL >99 99
[26]
3-FC6H4 AtHNL >99 >99
[26]
4-FC6H4 AtHNL >99 >99
[87]
3,4-F2C6H3 almond meal 71 84
[87]
[87]
[26]
2-ClC6H4 AtHNL >99 99
[26]
2-BrC6H4 AtHNL 99 98
[26]
2-IC6H4 AtHNL >99 >95
[26]
4-HOC6H4 AtHNL 96 97
[88]
3-O2NC6H4 PaHNL 89 89
[89]
2-PhC6H4 ParHNL 68 32
[90]
1-naphthyl PaHNL 89 90
[90]
2-naphthyl PaHNL >99 95
2-furyl PaHNL 96 99 (S)c [91]
PaHNL 60 97 (S)c [92]
O
[91]
3-furyl PaHNL 96 99
c [91]
2-thienyl PaHNL 71 99 (S)
[91]
3-thienyl PaHNL 95 99
[92]
3-pyridyl PaHNL 99 50
CMe=CH2 PmHNL 88b 32 [100]
[94]
(CH2)2CH=CH2 PaHNL 67 >99
[89]
(CH2)2CH=CH2 ParHNL 80 98
[89]
ParHNL 82 98

R1 (R)-HNL Sourcea Conversion (%) ee (%) Ref

[88]
(E)-CH=CHPh PaHNL 99 54
[95]
(E)-CH=CHMe PaHNL 99 98
(E)-CMe=CHEt PmHNL 58b 96 [100]
b [100]
(E,E)-CH=CHCH=CHMe PmHNL 32 96
PmHNL 54b 90 [100]
[96]
Cy PaHNL 90 99
cyclopentyl PmHNL 70b 94 [93]
b [100]
cyclobutyl PmHNL 78 92
[26]
(CH2)8Me AtHNL 56 >95
b [97]
(CH2)7Br PaHNL 40 97
[98]
(CH2)4OH PaHNL quant 46
[99]
iPr PaHNL 99 83
[84]
t-Bu PaHNL 58 92
PmHNL 62b 89 [100]
PmHNL 82b 99 [100]
a
See Section 2.1.1.1.
b
Yield (%) rather than conversion.
c
Change of product configuration because of the priority reassignment
according to CIP rules.
Cyanohydrin synthesis catalyzed by (R)-selective hydroxynitrile lyases has mostly been ap-
plied in biphasic systems.[94,101,102] An invention of Chen and co-workers[75] was the use of a
biphasic system in a continuous-flow reactor by pumping a premixed solution of alde-
hyde and hydrogen cyanide in aqueous diisopropyl ether through a column filled with
almond meal (for a comprehensive review see ref [103]).
2.1.1.1.1.2 Hydrogen Cyanide Addition to Ketones
The addition of hydrogen cyanide to ketones catalyzed by the (R)-selective hydroxynitrile

lyase from Prunus amygdalus (PaHNL) was first reported by Effenberger and co-workers in
1991.[104] The transformation was demonstrated in organic media. Cyanohydrins of alkyl
methyl ketones were obtained in moderate yields and high optical purities. In addition to
acyclic ketones, cyclic, bicyclic, heterocyclic, and silicon-containing ketones[105] have
been transformed into cyanohydrins since then.[106,107]
The (R)-selective hydroxynitrile lyase from Linum usitatissimum (LuHNL) catalyzes the
transformation of methyl ethyl ketone to give the corresponding (R)-cyanohydrin on a
preparative scale (Scheme 4).[81] This stereoselective conversion is remarkable as the two
substituents (ethyl and methyl) next to the carbonyl functionality do not differ much in
size. At that time, the enzyme LuHNL was believed to accept aliphatic substrates only.[81]
More recently, Roberge and co-workers have demonstrated that the (R)-selective LuHNL
converts benzylic ketones into optically active cyanohydrins with inverted selectivity
(Scheme 4).[108]
Scheme 4 Formation of Methyl Ketone Cyanohydrins Catalyzed by

Various (R)-Selective Hydroxynitrile Lyases[44,104,108,109]
O (R)-HNL HO
+ HCN
1
R R1 CN
R1 (R)-HNL Source Yield (%) ee (%) Ref

Et LuHNL quanta 95 (R) [44]
(CH2)3Cl PaHNL 73a 99 (R) [104]
a [104]
iPr PaHNL 54 90 (R)
(CH2)2CH=CH2 PaHNL 80a 97 (R) [104]
a [109]
Ph PaHNL 14 90 (R)
[108]
3-FC6H4CH2 LuHNL 20 83 (S)
[108]
3-ClC6H4CH2 LuHNL 24 89 (S)
[108]
3-BrC6H4CH2 LuHNL 37 99 (S)
[108]
3-TolCH2 LuHNL 40 97 (S)
[108]
3-F3CC6H4CH2 LuHNL 19 96 (S)
[108]
3-MeOC6H4CH2 LuHNL 31 99 (S)
[108]
4-MeOC6H4CH2 LuHNL 30 84 (S)
a
Conversion (%) rather than yield.
PaHNL has been used for the addition of hydrogen cyanide to alkyl- and alkoxy-substitut-
ed cyclohexanones,[106,110] as well as five- and six-membered heterocyclic ketones such as
dihydrofuran-3(2H)-one and dihydro-2H-pyran-3(4H)-one.[111]
2.1.1.1.1.3 Transhydrocyanation
Conversion of aliphatic and aromatic aldehydes into (R)-cyanohydrins using acetone cya-
nohydrin as transhydrocyanation reagent was reported for the first time in 1991 (Scheme
5).[84] The big advantage of this method is the avoidance of the highly toxic and explosive
liquid hydrogen cyanide. The enantiomeric excesses obtained by this method are slightly
lower than those obtained in conversions using free hydrogen cyanide. Huuhtanen and
Kanerva optimized this procedure in 1992.[99]
Scheme 5 Transhydrocyanation with Acetone Cyanohydrin as Cyanide

Donor[84]
O OH O
HO CN (R)-HNL
+ +
R1 H R1 CN
R1 = alkyl, cycloalkyl, aryl, hetaryl
Racemic 2-methyl-2-hydroxyhexanenitrile (4) can also act as cyanide donor.[97] Optically

active ø-bromo cyanohydrins (R)-5 are obtained from the corresponding aldehydes by
this method with high enantiomeric excesses (90–97% ee) (Scheme 6). ø-Hydroxyalde-
hydes are converted into optically active cyanohydrins by the same procedure.[98]

Scheme 6 2-Methyl-2-hydroxyhexanenitrile as Transhydrocyanation Reagent[97]
H HO CN (R)-HNL CN HO CN
Br + Br +
n Bu n Bu
O OH
4 (R)-5 (S)-4
Silicon-containing aliphatic ketones are subjected to transhydrocyanation with the (R)-se-

lective hydroxynitrile lyase from apple seed meal, yielding products with higher enantio-
meric excesses than those obtained using almond meal.[105,112] (R)-Selective hydroxynitrile
lyases from other sources [e.g., Eriobotrya japonica (loquat) and Prunus japonica] also accept
silicon-containing ketones in transhydrocyanation reactions.[113,114]
The transhydrocyanation approach with ethyl cyanoformate as cyanide donor leads
to simultaneous protection of the hydroxy group (Scheme 7).[115]
Scheme 7 PaHNL-Catalyzed Transhydrocyanation with Ethyl Cyanoformate as

Reagent[115]
O O OH O OEt
PaHNL
+ +
Ph H NC OEt Ph CN Ph CN
(R)-3
2.1.1.1.1.4 Nitroalkane Addition to Aldehydes (Henry Reaction)
The chemical addition of a nitroalkane to a carbonyl compound, also known as the nitro-
aldol or Henry reaction, was described in 1895.[18] The Henry (nitroaldol) reaction is a fre-
quently used reaction in organic synthesis[116,117] because vicinal nitro alcohols can, for ex-
ample, easily be transformed into nitroalkenes, 2-nitro ketones, Æ-hydroxycarboxylic
acids, and 1,2-amino alcohols. Stereoselective methods for the Henry reaction are avail-
able that cover transition-metal catalysis,[118–126] organocatalysis,[1,127–130] as well as bio-
catalysis.[8,19,131] The protein-mediated catalysis of the Henry reaction by the carrier pro-
tein bovine serum albumin (BSA) in water can be categorized as organocatalysis because
the observations of the scientists led to the conclusion of unspecific protein catalysis.[132]
Biocatalytic nitroaldol reactions have also been reported with enzymes such as a
transglutaminase,[133] a hydrolase,[134] a protease,[135] lipases,[136] and a glucoamylase[137]
but in all cases the reactions were not enantioselective or there were no comments re-
garding enantioselectivity. The first biocatalytic enantioselective nitroaldol reaction was
demonstrated in 2006[19] applying the (S)-selective hydroxynitrile lyase from Hevea brasi-
liensis (HbHNL; see Section 2.1.1.1.2.4). This was followed, in 2011, by an (R)-selective nitro-
aldol reaction catalyzed by an enzyme of the same fold, the (R)-selective hydroxynitrile
lyase from Arabidopsis thaliana (AtHNL).[131] By careful optimization of the reaction condi-
tions, 2-nitroethanol derivatives could be detected. The AtHNL works best in a biphasic
system comprising 20 mM potassium phosphate buffer (at pH 7) and butyl acetate in a
1:1 ratio at room temperature (Scheme 8). Only the conversion of aromatic aldehydes
led to 1,2-nitro alcohols.
Scheme 8 Nitroaldol Reaction Catalyzed by AtHNL[131]
AtHNL
20 mM potassium phosphate buffer/butyl acetate (1:1)
O pH 7, rt, 2 h OH
+ MeNO2 NO2
R1 H R1
R1 Yield (%) ee (%) Ref

[131]
Ph 30 91
[131]
2-Tol 12 95
[131]
4-MeOC6H4 2 79
[131]
2-ClC6H4 34 68
[131]
3-ClC6H4 17 91
[131]
4-ClC6H4 9 87
[131]
2-naphthyl 7 >99
2.1.1.1.2 Applications of (S)-Selective Hydroxynitrile Lyases
The hydroxynitrile lyase (HNL) from Sorghum bicolor (SbHNL) was the first (S)-selective en-
zyme utilized for the synthesis of (S)-cyanohydrins.[40] The natural substrate of this en-
zyme is (S)-4-hydroxymandelonitrile (see Scheme 9, Section 2.1.1.1.2.1).[138] The substrate
scope of SbHNL is limited to aromatic and heteroaromatic aldehydes. Hydroxynitrile ly-
ases from Hevea brasiliensis, first characterized in detail in 1996,[139] and Manihot esculenta,
first characterized in 1994,[38] are the two most extensively applied (S)-selective hydroxy-
nitrile lyases.
2.1.1.1.2.1 Hydrogen Cyanide Addition to Aldehydes
The substrate scope of the (S)-selective hydroxynitrile lyases from Manihot esculenta and
Hevea brasiliensis is broad, and is comparable to the substrate range of the (R)-selective hy-
droxynitrile lyase from Prunus amygdalus.
Scheme 9 shows a summary of selected applications of (S)-selective hydroxynitrile
lyases and the outcome of the biotransformations.[65,74,83,90,106,138,140–146]

Scheme 9 Aldehydes as Substrates for (S)-Selective Hydroxynitrile

Lyases[65,74,83,90,106,138,140–146]
O (S)-HNL OH
+ HCN
R1 H R1 CN
R1 (S)-HNL Sourcea Conversion (%) ee (%) Ref

[140]
Ph HbHNL 96 99
[141]
Ph MeHNL quant 98
[74]
Ph SbHNL 97 97
[138]
4-HOC6H4 SbHNL 99 87
[106]
3-PhOC6H4 HbHNL 99 87
[142]
3-PhOC6H4 MeHNL 47 96
[142]
Bn MeHNL 99 98
[140]
CH2OBn HbHNL 92 12
[142]
4-Tol MeHNL 50 99
[143]
2-MeOC6H4 HbHNL 61 77
[143]
[143]
O
[144]
MeHNL 84 86
O
[142]
2-ClC6H4 MeHNL 96 98
[142]
2-BrC6H4 MeHNL 96 96
[142]
2-HOC6H4 MeHNL 47 91
[90]
1-naphthyl HbHNL 97 73
b c [83]
2-furyl HbHNL 90 >99 (R)
[145]
3-furyl HbHNL 98 98
c [145]
2-thienyl HbHNL 98 99 (R)
[145]
CH=CH2 HbHNL 92 98
[141]
CH=CH2 MeHNL 70 56
b [83]
(CH2)2CH=CH2 HbHNL 99 >99
[145]
(E)-CH=CHPh HbHNL 93 98
[142]
(E)-CH=CHPh MeHNL 80 95
[65]
(E)-CH=CHPh AmHNL 11 82
[141]
(E)-CH=CHMe MeHNL quant 92
[145]
(E)-CH=CH(CH2)4Me HbHNL 87 99
[143]
HbHNL 87 99
[140]
Cy HbHNL 95 99
[142]
(CH2)7Me MeHNL 99 80
[140]
(CH2)4Me HbHNL 81 96
[141]
iPr MeHNL 91 95

[141]
t-Bu MeHNL 80 94
[90]
HbHNL 63 >99
[146]
Fc HbHNL 98 99
a
b
c
Change of product configuration because of the priority reassignment
according to CIP rules.
2.1.1.1.2.2 Hydrogen Cyanide Addition to Ketones
The hydroxynitrile lyases MeHNL and HbHNL both catalyze the synthesis of (S)-configured
cyanohydrins from aliphatic and aromatic ketones with excellent enantioselectivities
(Scheme 10).[108,141,142,145,147]
Scheme 10 Formation of (S)-Configured Methyl Ketone Cyanohydrins Catalyzed

by Various (S)-Selective Hydroxynitrile Lyases[108,141,142,145,147]
O (S)-HNL HO
+ HCN
R1 R1 CN

[141]
Et MeHNL 91 18
[145]
Pr HbHNL 99 74
[145]
iPr HbHNL 99 98
t-Bu HbHNL 49b 83 [145]
[141]
t-Bu MeHNL 81 28
[145]
Ph HbHNL 40 99
[147]
Ph MeHNL 22 96
[142]
Bn MeHNL 99 98
[142]
(CH2)2Ph MeHNL 90 67
b [108]
3-BrC6H4CH2 MeHNL 61 93
3-F3CC6H4CH2 MeHNL 67b 97 [108]
b [108]
4-MeOC6H4(CH2)2 MeHNL 68 14
a
b
With 4-substituted cyclohexanones, the (S)-selective MeHNL shows a different diastereose-

lectivity to the (R)-selective PaHNL. Whereas the latter catalyzes the formation of trans-iso-
mers (R1 and OH in anti configuration) almost completely, MeHNL catalysis leads to the cis-
isomers 6 (R1 and OH in syn configuration) (Scheme 11). The reaction rate for MeHNL is
also higher.[106]

Scheme 11 Formation of 4-Substituted Cyclohexanone Cyanohydrins

Catalyzed by MeHNL[106]
OH
O
MeHNL
+ HCN CN
R1
R1
6
R1 = alkyl, Ph
2.1.1.1.2.3 Transhydrocyanation
In addition to (R)-selective hydroxynitrile lyase (HNL) mediated transhydrocyanation de-
scribed in Section 2.1.1.1.1.3, the (S)-selective hydroxynitrile lyase from Manihot esculenta
(MeHNL) has been applied for transhydrocyanation of the silicon-containing ketone 7 (X =
Si) and compared to 3,3-dimethylbutan-2-one (7, X = C) transhydrocyanation (Scheme
12).[148]
Scheme 12 Transhydrocyanation of Ketones[148]
O
HO CN MeHNL HO O
Me + Me +
X X CN
Me Me Me Me
7
X = C, Si
2.1.1.1.2.4 Nitroalkane Addition to Aldehydes (Henry Reaction)
The first biocatalytic formation of 2-nitro alcohols was reported in 2006 using the (S)-se-
lective hydroxynitrile lyase from Hevea brasiliensis (HbHNL).[19] A broad range of aliphatic,
aromatic, and heteroaromatic aldehydes is converted into 2-nitro alcohols by the addition
of nitromethane (Scheme 13).[19,149] Even a ketone reacts, leading to a chiral quaternary
center, although the conversion is extremely low.[149]
Scheme 13 (S)-2-Nitroethanol Formation Catalyzed by HbHNL[19,149]
HbHNL
50 mM potassium phosphate buffer/t-BuOMe (1:1)
O rt, 48 h OH
+ MeNO2 NO2
R1 H R1
R1 pH Yield (%) ee (%) Ref

[19]
Ph 7.0 63 92
[149]
Ph 5.5 32 97
[19]
3-HOC6H4 7.0 46 18
[149]
4-MeOC6H4 5.5 20 99
[19]
4-O2NC6H4 7.0 77 28
[149]
2-ClC6H4 5.5 23 95
[149]
3-ClC6H4 5.5 36 98
[149]
4-ClC6H4 5.5 25 97
[149]
(CH2)5Me 5.5 34 96
[149]
Cy 5.5 18 99
R1 pH Yield (%) ee (%) Ref

[149]
(CH2)2Ph 5.5 13 66
[149]
2-furyl 5.5 43 88
[149]
3-furyl 5.5 16 89
[149]
2-thienyl 5.5 29 98
Nitroethane is also accepted by HbHNL and leads to the formation of two new chiral cen-
ters in the product. In addition to the high enantioselectivities obtained for the (S)-config-
ured C—OH functionality, the diastereoselectivity is high when using nitroethane (anti/syn
9:1). Even the bulky 2-nitropropane is accepted as nucleophile although the yield drops
significantly (Scheme 14).[149] The drawback of this stereoselective reaction is the low ac-
tivity of wild-type HbHNL.
Scheme 14 Biocatalytic Nitroaldol Reaction with Various Nitroalkanes Catalyzed by

HbHNL[149]
HbHNL
50 mM potassium phosphate buffer/t-BuOMe (1:1) OH
O R1 pH 7, rt, 48 h R1
+ Ph R2
Ph H R2 NO2
NO2
R1 R2 Yield (%) ee (%) Ref

[149]
H H 63 92
[149]
H Me 67 95 (anti)
[149]
Me Me 7 80
[149]
H Ph 0 0
The nitroaldol reaction is not known in nature thus far, so it is not surprising that nature
has not evolved a specialist system with high activity for this nonnatural transformation.
The homologous MeHNL, which shows high similarity to HbHNL (77% sequence identity)
and also shares the same fold (Æ/-hydrolase superfamily), gives only 20% conversion of
benzaldehyde into (S)-2-nitro-1-phenylethanol, whereas the same amount of HbHNL yields
up to 63% of this product (Scheme 14).
An esterase bearing mutations designed by an engineering strategy based on the
identification of potential ancestral enzymes resulted in very high activity for the nitro
aldol reaction[150] as well as for cyanohydrin synthesis.[151] However, the enantiomeric ex-
cess in the cyanohydrin synthesis was rather low (20% ee)[151] and not shown for the nitro-
aldol reaction.
During detailed investigations of the reaction kinetics, the pH optimum for the
HbHNL-catalyzed Henry reaction was found to be pH 6.0. At pH <6 the spontaneous unse-
lective (nonenzymatically catalyzed) reaction was almost completely suppressed, where-
as at pH >6.5 it enhanced exponentially. So pH 6 seems to be a good compromise for high-
est enzyme activity and low background reaction.[152]
A general procedure for the HbHNL catalyzed nitroaldol reaction at pH 6 to give
2-nitro alcohols 8 is shown in Scheme 15.[149]

Scheme 15 HbHNL Catalyzed Synthesis of 2-Nitro Alcohols at pH 6[149]
HbHNL
50 mM potassium phosphate buffer/t-BuOMe (1:2) OH
O R3 pH 6, rt, 48 h
R3
+ R1 R2
R1 H R2 NO2
NO2
8
R1 = alkyl, aryl, hetaryl; R2 = R3 = H, Me
2-Nitro Alcohols 8; General Procedure:[149]

Recombinant HbHNL expressed in Pichia pastoris (1.0 mL, 4000 U • mL–1) was diluted with
50 mM phosphate buffer (pH 6.0, 0.5 mL) and mixed with t-BuOMe (3.0 mL). The mixture
was stirred for 10 min at 600 rpm. Freshly distilled aldehyde (1.0 mmol) was added to the
mixture and after it had been stirred for 5 min, nitroalkane (10.0 mmol) was added. The
mixture was stirred at rt for 48 h and extracted with EtOAc (3 20 mL). The combined ex-
tracts were dried (Na2SO4) and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography (cyclohexane/EtOAc 16:1). The re-
action was also performed on a scale of up to 500 mmol in a biphasic system composed of
phosphate buffer and t-BuOMe with nitromethane or nitroethane. The yields and enantio-
meric excesses were reproducible.
2.1.1.1.2.5 Resolution of Racemic 2-Nitro Alcohols
Prior to the discovery of the (R)-selective hydroxynitrile lyase from Arabidopsis thaliana
(AtHNL) in 2011, which enables the direct synthesis of (R)-configured 2-nitroethanol deriv-
atives,[131] the resolution of racemic 2-nitro alcohols by [(S)-selective] hydroxynitrile lyase
from Hevea brasiliensis (HbHNL) was performed in order to get access to (R)-configured
products.[153] Although the cleavage of the nitroaldol bond is much faster than the stereo-
selective C—C bond formation,[152] the resolution has not found application in organic syn-
thesis, because the enzyme activity is still too low for preparative use.
The concept of cascade reactions with one single enzyme (HbHNL) was demonstrated
by performing the fast cyanohydrin formation with the aldehyde formed during the race-
mic resolution[153] by adding hydrogen cyanide to the reaction mixture. In the best case,
50% (R)-2-nitro alcohol and 50% of the (S)-cyanohydrin would be obtained. A drawback of
the described approach was the complex and time-consuming separation of the products.
A brief summary of a variety of hydrolase applications for the resolution of racemic
2-nitro alcohols by hydrolysis, esterification, or transesterification can be found in ref [8].
2.1.1.1.3 Resolution of Racemic Cyanohydrins
As well as the addition of hydrogen cyanide to aldehydes and ketones catalyzed by hy-
droxynitrile lyases (HNLs), the resolution of racemic cyanohydrins is a further possibility
to obtain enantioenriched compounds.[109] Highly selective catalysts yield high enantio-
purities and maximal yields of 50%. The (R)-selective hydroxynitrile lyases from Prunus
amygdalus (PaHNL) was used for the resolution of racemic 3-phenoxybenzaldehyde cyano-
hydrin (9) in a biphasic system. A catalytic amount of aniline and semicarbazide were
added to capture the aldehyde (Scheme 16).[154]
Scheme 16 Resolution of Racemic 3-Phenoxybenzaldehyde Cyanohydrin Catalyzed by

PaHNL[154]
HO CN PaHNL HO CN O H
citrate buffer/iPr2O (40:1)
39 oC
+
50%
PhO PhO PhO
rac-9 (S)-9
Esterases and lipases can also act as catalysts for the resolution of racemic cyanohydrin
esters or acylation of racemic cyanohydrins. An esterase from Pseudomonas sp. has been
used for the hydrolysis of cyanohydrin acetates with enantiomeric excesses up to
98%.[155] The enzymatic acylation of racemic cyanohydrins with a lipase from Pseudomonas
sp. was one of the early examples for the dynamic kinetic resolution approach due to the
easy in situ racemization of the remaining (slowly reacting) cyanohydrins under basic
conditions (Scheme 17).[156]
Scheme 17 Application of the Dynamic Kinetic Resolution Approach for the Synthesis of
Enantiopure Acetylated Cyanohydrins[156]
AcO
anion-exchange resin lipase from
O OH OH OAc
(10 mol%) Pseudomonas sp.
+
R1 H CN R1 CN R1 CN
rac
R1 = aryl
Lipases from Arthrobacter and Pseudomonas sp. have been utilized to hydrolyze the racemic
acetate of 3-phenoxybenzaldehyde cyanohydrin (9),[157,158] whereas a racemic butanoate
derivative has been hydrolyzed by lipases from Candida cylindracea and Pseudomonas sp.
on a preparative scale to obtain (S)-3-phenoxybenzaldehyde cyanohydrin [(S)-9] with 97%
enantiomeric excess.[159] Resolution of racemic cyanohydrins from bicyclic aldehydes by
enantio- or diastereoselective acylation catalyzed by hydrolases is an appropriate method-
ology.[90]
2.1.1.1.4 Mechanistic Aspects
To date, the structures of seven hydroxynitrile lyases (HNLs) belonging to three different
folds have been published (Table 2). Whereas the structures of (S)-selective hydroxynitrile
lyases all belong to the Æ/-hydrolase fold, experimentally determined 3D structures of
(R)-selective enzymes can be found in all three fold classes. However, hydroxynitrile
lyase activity seems to be present in at least one more fold class, as the hydroxynitrile
lyase from Linum usitatissimum (LuHNL), which displays either R or S selectivity, depend-
ing on the substrate,[44,108] shows high sequence similarity to the Zn2+ alcohol dehydro-
genase superfamily. However, no structure has been published so far.

Table 2 Structures of Hydroxynitrile Lyases[22,160–175]
HNLa Selectivity Fold Structure PDB-ID Ref
1YB6, 1YB7,
1QJ4, 1SC9,
1SCI, 1SCK,
1SCQ, 1YAS,
HbHNL S Æ/-hydrolase 2YAS, 3YAS, [160–165]
4YAS, 5YAS,
6YAS, 7YAS,
3C6X, 3C6Y,
3C6Z, 3C7O
1EB8, 1EB9,
1E89, 1E8D,
1DWP,
MeHNL S Æ/-hydrolase [166–169]
1DWO,
1DWQ,
3RKS, 3RKT
SbHNL S Æ/-hydrolase 1GXS [170,171]
AtHNL R Æ/-hydrolase 3DQZ [172]
PaHNL iso- GMC oxido- 3GDN, 3GDP, [173–175]

R
enzyme 1 reductases 1JU2
PmHNL iso- GMC oxido- –

R 3RED
enzyme 1 reductases
[22]
GtHNL R cupin 4BIF
a
Thus, although hydroxynitrile lyases all catalyze the same reaction, unsurprisingly their
detailed reaction mechanisms differ significantly due to their different structures
(Schemes 18–20).[162,172,174,176]
Four of the hydroxynitrile lyases with known structure, those from Hevea brasiliensis
(HbNHL), Manihot esculenta (MeHNL), Sorghum bicolor (SbHNL), and Arabidopsis thaliana
(AtHNL), display an Æ/-hydrolase fold and are flavin adenine dinucleotide (FAD) inde-
pendent enzymes (Table 2). Due to high sequence similarity the hydroxynitrile lyase
from Baliospermum montanum (BmHNL) is also assigned to this group of hydroxynitrile
lyases.
The (S)-selective HbHNL and MeHNL share 77% sequence identity and are the two best
characterized hydroxynitrile lyases so far, with several structures solved of native and
mutant enzymes as well as enzyme–substrate complexes. Both of these are nonglycosylat-
ed hydroxynitrile lyases with a molecular mass of 29.2 kDa and acetone cyanohydrin is
the natural substrate. The active site is located in a deep cavity, which is accessible
through a narrow tunnel. The catalytic site contains a catalytic triad, formed by Ser80,
His235, and Asp207 in HbHNL and Ser80, His236, and Asp208 in MeHNL, in which the his-
tidine residues act as general base deprotonating the serine residues, which serve as me-
diator and are in direct contact with the cyanohydrin substrates (Scheme 18). The neces-
sity of a positive charge for the stabilization of the cyanide ion, and a general base for the
abstraction of a proton from the hydroxy group in the cyanohydrin substrates was already
suggested by Becker and Pfeil in 1965.[177] Complex structures of HbHNL with different
substrates [acetone cyanohydrin, mandelonitrile (3), and 2-hydroxy-2,3-dimethylbutane-
nitrile] reveal that the oxygen of the ketone or alcohol groups interacts with Ser80 and
Thr11 and the cyano group of the substrate is in close interaction with the Lys236 side
chain.[160,163] These structural data, together with mutagenesis studies, suggest that the
negative charge of the cyanide ion, which is released during the cleavage of the C—C
bond, is stabilized through protonation by the positive charge of Lys236.[162,165,178,179] For
the synthesis direction, crystal structures at atomic resolution in complex with substrates
as well as quantum chemical calculations give a deeper insight into the cyanohydrin syn-
thesis.[163] Ser80 and Thr11 induce electron withdrawal from the carbonyl carbon atom of
the substrate and thus facilitate the nucleophilic attack of cyanide. Although the proton-
ation state of the cyanide ligand during its entry and the order of substrate binding is still
a matter of discussion, these new data suggest that hydrogen cyanide enters in the neutral
state and is subsequently deprotonated by His235, with the proton finally shifted to
Ser80. Hydrogen cyanide would bind first, preparing the catalytic site for the binding of
the carbonyl compound,[163] which is in contrast to the originally proposed ordered uni-bi
mechanism in which the carbonyl compound binds first followed by hydrogen cya-
nide.[180,181] As all relevant residues are also conserved in MeHNL, it is assumed that it fol-
lows the same reaction mechanism.

Scheme 18 Proposed Reaction Mechanism of HbHNL[162]
Thr11 Thr11
Lys236
H R1 H Lys236
R1
O H C N O H
O H3N O N
−C H 3N
H
H
O H H
O
N N
N N
Ser80 H Ser80 H
O− O−
His235 His235
O Asp207 O Asp207
Thr11
R1 H Lys236
O H
O N
HC H3N
H
O
N
N
Ser80 H
O−
His235
O
Asp207
Initially, it was believed that Æ/-hydrolase fold hydroxynitrile lyases display exclusively
(S)-selectivity and thus that the stereopreference of hydroxynitrile lyases can be predicted
by sequence similarity to a certain protein fold. However, the discovery of an (R)-selective
hydroxynitrile lyase from the noncyanogenic plant A. thaliana (AtHNL) with high se-
quence similarity to the (S)-selective enzymes from H. brasiliensis and M. esculenta proved
this assumption wrong.[26,82] A detailed structural analysis confirmed the high similarity
of the structure to the structures of HbHNL and MeHNL including the catalytic Ser–His–
Asp triad (Ser81, His236, and Asp208) in the active site, but also revealed several differen-
ces in the active site, which are responsible for the diverging binding of the substrate and
thus result in a different reaction mechanism (Scheme 19).[172] In AtHNL, the hydroxy
group of the cyanohydrin is directly deprotonated by the His236 residue of the catalytic
triad. In addition, the hydroxy group interacts with Asn12 to facilitate the deprotonation.
Interestingly, the lysine residue, which stabilizes the negative charge of the released
cyano group and is crucial in HbHNL, is replaced by Met237 in AtHNL. It was proposed
that in the case of AtHNL, the cyano group could interact with the backbone amide groups
of Ala13 and Phe82 and thereby be stabilized by an oxyanion hole similar to those de-
scribed in serine hydrolases. Finally, the cyanide is protonated indirectly by His236 with
Ser81 as a mediator.[172]
Scheme 19 Proposed Reaction Mechanism of AtHNL[172]
Ala13
N H
H 1
H R Asn12
N
N C H
O
H O
N H H
Phe82 N
O
N
H
O−
Ser81
His236
O Asp208
Ala13
N
H H
H R1 Asn12
N C− N
H
H O
H O
N
Phe82 H
O
N
N
Ser81 H
O−
His236
O Asp208
Ala13
N
H H
H R1
N CH N Asn12
H
H O
N O
Phe82 H
O N
N
H
Ser81 O−
His236
O Asp208
The N-glycosylated flavin adenine dinucleotide (FAD) dependent hydroxynitrile lyases of

Prunus amygdalus (PaHNL, 61 kDa)[173–175] and Prunus mume (PmHNL, 58.1 kDa) resemble a
glucose-methanol-choline (GMC) oxidoreductase (Table 2). Other hydroxynitrile lyases
with known sequence from Rosaceae, such as PsHNL from Prunus serotina and EjHNL
from Eriobotrya japonica, also belong to this family.
By analyzing the crystal structure of PaHNL isoenzyme 1 in complex with benzalde-
hyde in combination with mutagenesis data, Dreveny and co-workers proposed the fol-
lowing reaction mechanism (Scheme 20):[174] as in Æ/-hydrolase fold hydroxynitrile ly-
ases, in a general acid/base catalysis His497 might act as a general base and abstract the
proton from the mandelonitrile hydroxy group (its natural substrate), which is within hy-
drogen-bonding distance of His497, Cys328, and Tyr457. The cyanide could be stabilized
by several positively charged residues surrounding the active site, which cause an overall
positive charge in the active site. The released cyanide ion might be protonated by a sec-
ond histidine residue, His459, which is interacting with Lys361, but the exact mechanism
is still under discussion. Although the catalytic site is located close to the flavin adenine

dinucleotide, the hydroxy group is too far away for a direct involvement of flavin adenine
dinucleotide in the reaction. Thus, it seems to be necessary for the structural stability of
the protein.
Scheme 20 Proposed Reaction Mechanism of PaHNL[174]
Tyr457
+ +
+
+ O
H H
N
C O H Cys328
N O
N S
FAD
H
N NH H
N N
O
N N His497
H
His495
+ + Tyr457
+
+ O
N H
H O H Cys328
N O
S
FAD C− H
N
N NH
N
H
O N
N His497
H
N
His495
Recently, the discovery of new bacterial hydroxynitrile lyases with cupin fold has been
reported (Table 2).[22,68] One of the new enzymes, GtHNL from Granulicella tundricola, has
been characterized in detail and its structure has been solved.[22,182] It is a small metal-de-
pendent monocupin with a molecular weight of ~15 kDa that forms a tetramer. Although
metal exchange, metal analysis, and site-directed mutagenesis clearly prove that the
metal is indispensable for the activity of the enzyme, but not for its stability, and that ad-
ditional histidine residues in the active site are crucial, a detailed reaction mechanism has
not been proposed so far.
2.1.1.1.5 Optimization of the Reaction Systems
2.1.1.1.5.1 Enzyme Engineering
Enzyme engineering, by either random or directed methods, can be used to increase the
stability (at elevated temperature, at acidic or basic pH, or in organic solvents), broaden
the substrate range, and improve the enantioselectivity of an enzyme (Table 3).[183,184] A
prerequisite for the screening of large libraries is the availability of high-throughput
screening systems based on colorimetric reactions. Several screening systems are avail-
able for hydroxynitrile lyase activity both in the cyanogenesis and synthesis direction,
which either detect the released hydrogen cyanide on colony level or in microtiter
plates,[185,186] or the carbonyl compound.[187,188]
Table 3 Overview of Various Improved Hydroxynitrile Lyase Variants[33,37,142,151,169,187,189–198]
HNLa Mutationb Propertyc Ref

[189,190]
HbHNL Trp128Ala, Trp128Phe higher conversion and better selec-
tivity with bulky substrates
[189]
HbHNL His103Leu/Trp128Ala, Trp128Ala/ further increased activity and selec-
Pro187Leu, Trp128Ala/Gln215His tivity compared to the starting var-
iant Trp128Ala
[142,169]
MeHNL Trp128Ala, Trp128Leu, Trp128Cys, higher conversion and better selec-
Trp128Tyr tivity with bulky substrates
[191]
MeHNL Gly113Ser enhanced thermal stability, slightly
higher activity in an acidic environ-
ment
MeHNL Gly165acidic aa,d Val173Leu, and in enhanced thermal stability and in- [192]
combination creased stability toward organic sol-

vent
[193]
MeHNL Lys176Pro, Lys199Pro, Lys224Pro, higher in vivo solubility and expres-
and combinations thereof sion in E. coli
[37,193]
MeHNL His103Met, His103Leu, His103Val, higher in vivo solubility and expres-
His103Ile, His103Cys sion in E. coli
[194]
MeHNL Val2Lys, Val2Ile, Val2Asn, Val2Arg, higher in vivo solubility and expres-
Val2Gln sion in E. coli
[194]
MeHNL Val2Ile/His103Leu higher in vivo solubility and expres-
sion in E. coli
[195]
AtHNL Pro48Gln/Gln50Glu/Ala51Gln/ increased stability and thus activity
Glu53Asn/Lys60Glu/Glu64Thr/Lys67- at acidic pH
Glu/Ile93Arg/Glu138Thr/Arg140Ile/
Asn141Thr
[33]
PaHNL Leu1Gln/Ala111Gly improved yield and ee for (R)-2-chlo-
romandelonitrile
[196]
PaHNL Leu1Gln/Ile108Met/Ala111Gly, improved yield and ee for (R)-2-chlo-
Leu1Gln/Asn3Ile/Ala111Gly, Ile108- romandelonitrile
Met/Ala111Gly/4E10,e Leu1Gln/As-
n3Ile/Ile108Met/Ala111Gly/4E10
[187]
PaHNL Val317Ala increased yield and ee for hydroxy-
pivaldehyde cyanohydrin
[197]
PaHNL Val360Ile improved ee for the synthesis of
(R)-2-hydroxy-4-phenylbutanenitrile
[151]
SABP2 Gly12Thr/Met239Lys switch of an esterase to an HNL, low
enantioselectivity
[198]
EstC Ser276Lys switch of an esterase to an HNL, low
enantioselectivity
a
b
Many other variants were constructed during the investigation of the reaction mechanisms of
various hydroxynitrile lyases and/or yielded in reduced activity.
c
Compared to wild-type enzyme, if not indicated otherwise.
d
aa = amino acid.
e
The variant also contained two silent mutations at positions 85 and 432.
Some hydroxynitrile lyases display low activity toward industrially relevant substrates,
which is rather unfortunate, as the required high amount of the commonly used crude
enzyme preparation results in an increased chemical background reaction and lower
enantioselectivities. Thus, several enzyme engineering approaches have been applied to
broaden the substrate scope of these enzymes (Table 3). In HbHNL and MeHNL a trypto-

phan residue in the entrance tunnel seems to limit the access of bulky substrates. Muta-
tions of Trp128 to smaller amino acids enhanced the conversion of bulky substrates in
both enzymes.[142,169,189,199] However, some of the MeHNL variants showed unspecific or in-
verted stereoselectivity on aldehydes with two stereogenic centers.[200]
Several PaHNL variants with improved activity and enantioselectivity toward chal-
lenging substrates were also made available in recent years by removing steric hindrance
for bulky substrates or altering the hydrophobic interaction with the substrate (Table
3).[33,187,196,197]
AtHNL is significantly less stable at acidic pH (below pH 5.4) than the similar (S)-selec-
tive hydroxynitrile lyases HbHNL and MeHNL.[82] In a protein-engineering approach to sta-
bilize AtHNL, eleven amino acids at its surface were exchanged to the corresponding
amino acids from MeHNL (Table 3).[195] The resulting variant is active and more stable at
pH 4.5 [23.6 U • mg–1 initial rate activity in the cleavage of mandelonitrile (3), a half-life of
about 13 h at 20 8C, and 70% conversion with 99.3% ee at pH 4.5 and 0 8C in a citrate phos-
phate buffer/t-BuOMe two-phase system].[195] Interestingly, the fusion of AtHNL to the bac-
terial flavin-based fluorescent protein also stabilized the enzyme at pH below 5.[201]
In addition to changing the expression host and optimizing the expression condi-
tions (see Section 2.1.1.1), improvement of the expression level and solubility of several
hydroxynitrile lyases has also been achieved by protein engineering. For MeHNL, several
positions have been identified, which have an influence on the solubility and the expres-
sion level in E. coli (Table 3).[37,193,194]
Optimizing the codon usage of PaHNL for expression in Pichia pastoris, the exchange
of the first amino acid leucine to glutamine and replacement of the native plant secretion-
signal peptide with the alpha-mating factor sequence from Saccharomyces cerevisiae have a
major impact on the protein expression.[33,202]
2.1.1.1.5.2 Reaction Engineering
Reaction conditions have a significant impact on the asymmetric cyanohydrin reac-

tion.[203] Enantiopurity is the outcome of the ratio of the enzymatic to the nonenzymatic
cyanohydrin formation. Low pH values suppress the nonenzymatic reaction significantly
and the enzymatic reaction gains the upper hand thus leading to higher enantiomeric ex-
cess values.[204] On the other hand, enzyme activity decreases upon lowering the pH which
leads to lower reaction rates as enzyme deactivation increases. The choice of the optimal
buffer system is also very important and might have a significant impact on the enzyme
activity.[82] In the case of the 3-hydroxy-2,2-dimethylpropanal substrate, dimerization
competes with the enzyme-catalyzed cyanohydrin formation and can be a major cause
of low yields and enantiomeric excess. This can be avoided by conducting reactions at
very low pH.[187]
Temperature is another important factor that influences the ratio of the enzymatic to
the nonenzymatic cyanohydrin formation. At low temperatures, the rate of the diffusion-
limited nonenzymatic reaction is decreased to a larger extent than the enzymatic trans-
formation,[77] thus enabling the synthesis of cyanohydrins with higher enantiopurities.
Substrate engineering attempts in hydroxynitrile lyase catalyzed transformations
have been reported by Griengl and co-workers.[78] An example of a coupled approach of
substrate and enzyme engineering was published by the same group showing impressive
results regarding both activity (10–20 times less enzyme amount) and selectivity (ee in-
creased from 10 to about 90%).[189]
Currently, the most widely applied methodology for hydroxynitrile lyase (HNL) cata-
lyzed transformations is the use of biphasic systems.[101] High substrate concentrations
can be employed which lead to better space-time yields and have no significant impact
on enzyme stability by substrate and/or product inhibition because substrates and prod-
ucts are mainly soluble in the organic phase whereas the biocatalyst remains in the aque-
ous phase.
A typical procedure for the synthesis of (R)-mandelonitrile (3) is shown in Scheme
21,[102] and a general procedure for the synthesis of ketone cyanohydrins 10 using hy-
droxynitrile lysases from Hevea brasiliensis (HbHNL) or Prunus amygdalus (PaHNL) is illus-
trated in Scheme 22.[78]
In many cases, water-free reaction conditions are favorable either to shift the equili-
brium or to suppress the chemical background reaction mentioned above. For this pur-
pose, the enzymes need to be stabilized. From a reaction engineering point of view, the
use of immobilized enzymes has advantages over reactions in homogeneous systems: im-
mobilized enzymes are easily removed from the reaction mixture, can be reused, and fa-
cilitate product purification, thus reducing waste and the number of steps in multistep
syntheses. For hydroxynitrile lyases, many different methods of immobilization have
been applied:[101,205] (i) noncovalent or covalent immobilization on solid supports (hydro-
phobic, hydrophilic, ionic, or functionalized carriers); (ii) encapsulation or entrapment in
a carrier; or (iii) carrier-free immobilization by precipitation from aqueous buffer and sub-
sequent cross-linking with a bifunctional reagent, such as glutaraldehyde, to afford so-
called cross-linked enzyme aggregates (CLEAs). Because of the different properties of the
hydroxynitrile lyases and requirements of the different applications, no general immobi-
lization protocol for hydroxynitrile lyases exists and comparison between results is diffi-
cult. Thus, the Hanefeld group set out to systematically investigate and compare the activ-
ity and recyclability of differently immobilized hydroxynitrile lyase from Manihot esculen-
ta (MeHNL).[206] The best recyclability was obtained for MeHNL immobilized on Celite. In
another recent approach for optimization, hydroxynitrile lyase from Arabidopsis thaliana
(AtHNL) was used in buffer-saturated (pH 6.5) tert-butyl methyl ether as precipitated pro-
tein, immobilized on Celite, or entrapped in sol-gel.[207] Whereas the most prominent im-
provement in activity and enantiomeric excess in the first round of reaction was obtained
by fresh sol-gel entrapped AtHNL, Celite-immobilized AtHNL was much more stable dur-
ing recycling (in a solvent-resistant, nylon mesh “tea-bag”) and storage. This “tea-bag” ap-
proach was subsequently also applied for E. coli whole cells expressing AtHNL in a mono-
phasic micro-aqueous reaction system with tert-butyl methyl ether, enabling the recy-
cling of the cells.[208]
Co-immobilization of two or more enzymes is often used to overcome problems with
incompatibility of the optimal reaction conditions of the involved enzymes, thereby en-
abling one-pot reactions.[209] In the most recent triple cross-linked enzyme aggregate
(CLEA) approach combining MeHNL with a nitrilase from Pseudomonas fluorescens EBC
191 and an amidase from Rhodococcus erythropolis, (S)-mandelic acid could be obtained as
the sole product in 90% yield and >99% ee.[210] The introduction of the amidase in this tri-
ple CLEA overcomes the previous drawback of significant side product (amide) formation
observed with the hydroxynitrile lyase/nitrilase double CLEA.[211–213]

Scheme 21 Synthesis of (R)-Mandelonitrile Catalyzed by an (R)-Selective

Hydroxynitrile Lyase[102]
(R)-HNL
50 mM citrate buffer/t-BuOMe (1:1)
O OH
pH 5.5, 22 oC, 20 min
+ HCN
Ph H 97%; 98% ee Ph CN
(R)-3
Scheme 22 Enantioselective Synthesis of Ketone Cyanohydrins Cata-

lyzed by HbHNL or PaHNL[78]
HbHNL or PaHNL
50 mM citrate buffer/t-BuOMe
O pH 5.5, rt
HO CN
+
R1 ∗ R2
HCN
R1 R2
10
R1 = R2 = alkyl
(R)-Mandelonitrile [(R)-3]; Typical Procedure:[102]
CAUTION: Hydrogen cyanide can be absorbed through the skin and is extremely toxic.
Freshly distilled PhCHO (37.1 g, 0.35 mol), HCN (12.2 g, 0.42 mol), and (R)-HNL from al-
monds (78 mg) were dissolved in t-BuOMe (225 mL) and 50 mM citrate buffer (pH 5.5,
250 mL) at 22 8C. After the mixture had been stirred vigorously for 20 min, the phases
were separated. The aqueous phase was extracted with t-BuOMe (25 mL). The combined
organic solns were dried (MgSO4) and filtered, and the solvent was removed under re-
duced pressure; yield: 45.2 g (97%); 98% ee. The aqueous phase was reused four times
while applying the same amounts of reagents and solvents. In total 185.5 g (1.75 mol) of
PhCHO was converted into 226 g (1.70 mol) of (R)-mandelonitrile [(R)-3] with 78 mg of
(R)-HNL (0.035 wt%).
Ketone Cyanohydrins 10; General Procedure:[78]
CAUTION: Hydrogen cyanide can be absorbed through the skin and is extremely toxic.
All reaction equipment in which cyanides were used or produced was placed in a well-ven-
tilated hood. The required amount of HCN was freshly formed by dropping sat. aq NaCN
into 60% aq H2SO4 at 80 8C and condensing HCN at –12 8C in a cooling trap. For continued
control and warning, an electrochemical sensor for HCN detection was used. Waste solns
containing cyanides were treated with 10% aq NaOCl. The pH was adjusted to 7.0 with aq
H2SO4 prior to disposal.
An aq soln of HbHNL (700–1500 IU • mmol–1 ketone) or PaHNL (200–450 IU • mmol–1
ketone) was added to a soln of the ketone in t-BuOMe, and the resulting mixture was
stirred until an emulsion formed. The aqueous HbHNL preparation (ca. 5 kIU • mL–1) was
previously diluted with distilled H2O (1:2 v/v) and the pH was adjusted with 10% citric
acid. The pH of the aqueous PaHNL soln (ca. 3 kIU • mL–1) was previously adjusted with
10% citric acid. After addition of freshly generated HCN (2 equiv), the mixture was stirred
at rt until quantitative conversion had been obtained. The emulsion was broken with Cel-
ite 545, and the mixture was filtered and dried (Na2SO4). Evaporation of the solvent yield-
ed the crude cyanohydrins.
2.1.1.2 Conclusions and Outlook
A significant number of (R)- and (S)-selective hydroxynitrile lyases (HNLs) for the synthesis
of cyanohydrins are known and have been characterized to various extents in terms of
their substrate scope, specificity, structure, and reaction mechanisms;[5,49] some are even
commercially available (e.g., from ASA Spezialenzyme GmbH, Evocatal GmbH, Syncore
Laboratories).[214] Until now, only two enzymes with enantiocomplementary nitroaldol ac-
tivity are known. Both wild-type enzymes exhibit rather poor activities.
In the early years of hydroxynitrile lyase research and application, the focus was
mainly on condition optimization in order to obtain optimal product formation (high pro-
ductivity and enantioselectivity); today, however, enzyme engineering and optimization
is the state-of-the-art for improving biocatalytic transformations. High-throughput activi-
ty and selectivity screening assays allow enzyme improvements within reasonable time
frames.
Efficient recombinant expression systems and access to large amounts of enzyme
will lower the price of the catalysts and make biocatalytic approaches economically feasi-
ble. However, although several enzymes are available now as recombinant enzymes and
purification of these enzymes is straightforward, kinetic data on hydroxynitrile lyase re-
actions are scarce and available data usually show conversion rates and enantiomeric ex-
cess values of the resulting products rather than specific activities. This is especially the
case for the industrially interesting cyanohydrin synthesis reaction where organic sol-
vents or biphasic systems are applied. In addition, depending on the stability of the re-
spective enzymes, reactions are performed at different pH values and temperatures mak-
ing it difficult to compare catalytic activities of the available hydroxynitrile lyase biocata-
lysts.
There is still a need for new enzyme sources for stereoselective cyanohydrin synthe-
sis. Novel catalysts with different substrate scope to those known so far will open the way
for the synthesis of novel cyanohydrin building blocks and intermediates. Enzymes with
high activity and stability are especially interesting in view of the competing chemical
background reaction counteracting the stereoselective enzymatic conversion.
In the case of the enzymatic nitroaldol reaction, the investigations are in their infant
stage and there is a lot of room for improvement. The search for new enzymes with nitro-
aldol activity could be performed either randomly (screening of different organisms as
well as gene libraries) or by database mining. Comprehensive enzyme engineering ap-
proaches could be applied to enhance the activity as well as the substrate scope of the
known enzymes HbHNL and AtHNL. Investigations into the reaction mechanism of these
two enzymes, including kinetic and crystallographic studies, are highly desirable.

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C-C Bond Formation: Cyanohydrin Formation/Henry Reaction

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C-C Bond Formation: Cyanohydrin Formation/Henry Reaction

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1

2.1 C—C Bond Formation

2.1.1 Cyanohydrin Formation/Henry Reaction

K. Steiner, A. Glieder, and M. Gruber-Khadjawi

Enantiopure cyanohydrins 1 and 2-nitro alcohols 2 (Scheme 1) are versatile building

Scheme 1 Asymmetric Cyanohydrins and

R1 = alkyl, cycloalkyl, aryl, hetaryl; R2 = R3 = H, Me

for references see p 26

2.1.1.1 Hydroxynitrile Lyases Used in Synthesis

Table 1 Heterologously Expressed Hydroxynitrile Lyases[22,26,32–48]

Enzyme Source HNL Substrate Range Selectivity Expression Refa

(almond) ic, and Æ,-unsaturated aldehydes

for references see p 26

2.1.1.1.1 Applications of (R)-Selective Hydroxynitrile Lyases

Scheme 2 Synthesis of (R)-Mandelonitrile Using

An (R)-selective enzyme found in emulsin catalyzes the formation of enantioenriched

2.1.1.1.1.1 Hydrogen Cyanide Addition to Aldehydes

Scheme 3 Selected Examples of the Enantioselective Synthesis of Cyanohydrins from

R1 (R)-HNL Sourcea Conversion (%) ee (%) Ref

PaHNL 60 97 (S)c [92]

for references see p 26

R1 (R)-HNL Sourcea Conversion (%) ee (%) Ref

PmHNL 54b 90 [100]

PmHNL 62b 89 [100]

PmHNL 82b 99 [100]

2.1.1.1.1.2 Hydrogen Cyanide Addition to Ketones

The addition of hydrogen cyanide to ketones catalyzed by the (R)-selective hydroxynitrile

Scheme 4 Formation of Methyl Ketone Cyanohydrins Catalyzed by

R1 (R)-HNL Source Yield (%) ee (%) Ref

(CH2)3Cl PaHNL 73a 99 (R) [104]

Scheme 5 Transhydrocyanation with Acetone Cyanohydrin as Cyanide

R1 = alkyl, cycloalkyl, aryl, hetaryl

Racemic 2-methyl-2-hydroxyhexanenitrile (4) can also act as cyanide donor.[97] Optically

for references see p 26

Scheme 6 2-Methyl-2-hydroxyhexanenitrile as Transhydrocyanation Reagent[97]

Silicon-containing aliphatic ketones are subjected to transhydrocyanation with the (R)-se-

Scheme 7 PaHNL-Catalyzed Transhydrocyanation with Ethyl Cyanoformate as

2.1.1.1.1.4 Nitroalkane Addition to Aldehydes (Henry Reaction)

Scheme 8 Nitroaldol Reaction Catalyzed by AtHNL[131]

R1 Yield (%) ee (%) Ref

2.1.1.1.2 Applications of (S)-Selective Hydroxynitrile Lyases

2.1.1.1.2.1 Hydrogen Cyanide Addition to Aldehydes

for references see p 26

Scheme 9 Aldehydes as Substrates for (S)-Selective Hydroxynitrile

R1 (S)-HNL Sourcea Conversion (%) ee (%) Ref

R1 (S)-HNL Sourcea Conversion (%) ee (%) Ref

Scheme 10 Formation of (S)-Configured Methyl Ketone Cyanohydrins Catalyzed

R1 (S)-HNL Sourcea Conversion (%) ee (%) Ref

With 4-substituted cyclohexanones, the (S)-selective MeHNL shows a different diastereose-

for references see p 26

Scheme 11 Formation of 4-Substituted Cyclohexanone Cyanohydrins

Scheme 12 Transhydrocyanation of Ketones[148]

2.1.1.1.2.4 Nitroalkane Addition to Aldehydes (Henry Reaction)

Scheme 13 (S)-2-Nitroethanol Formation Catalyzed by HbHNL[19,149]

R1 pH Yield (%) ee (%) Ref

R1 pH Yield (%) ee (%) Ref

Scheme 14 Biocatalytic Nitroaldol Reaction with Various Nitroalkanes Catalyzed by

R1 R2 Yield (%) ee (%) Ref

for references see p 26

Scheme 15 HbHNL Catalyzed Synthesis of 2-Nitro Alcohols at pH 6[149]

2-Nitro Alcohols 8; General Procedure:[149]

2.1.1.1.2.5 Resolution of Racemic 2-Nitro Alcohols

2.1.1.1.3 Resolution of Racemic Cyanohydrins