The first enzyme-promoted addition of nitromethane to imines (aza-Henry reaction)

Article abstract of DOI:10.1016/j.bioorg.2019.103377

Enzyme catalytic promiscuity is the ability of a single enzyme active site to catalyze several chemical transformations, among them those which are different from natural. We have attempted to use this feature of enzymes in the nucleophilic addition of nitromethane to aldimines (the aza-Henry reaction) whose chemically catalyzed version leads to synthetically useful β-nitroamines. We succeded in obtaining for the first time the desired products in the yields up to 81%. The most efficient proved lipase TL (from Pseudomonas stutzeri) and oxynitrilase from Arabidopsis thaliana. However, all the reactions investigated were non-stereoselective.

Full text of DOI:10.1016/j.bioorg.2019.103377

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The first enzyme-promoted addition of nitromethane to imines (aza-henry re-
action)
Ignacy Janicki, Piotr Łyżwa, Piotr Kiełbasiński
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S0045-2068(19)31128-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103377
YBIOO 103377
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Bioorganic Chemistry
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19 October 2019
Please cite this article as: I. Janicki, P. Łyżwa, P. Kiełbasiński, The first enzyme-promoted addition of
nitromethane to imines (aza-henry reaction), Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103377
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THE FIRST ENZYME-PROMOTED ADDITION OF NITROMETHANE TO IMINES
(AZA-HENRY REACTION)
Ignacy Janicki, Piotr Łyżwa and Piotr Kiełbasiński*
Division of Organic Chemistry, Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland
E-mail: piokiel@cbmm.lodz.pl
ABSTRACT
Enzyme catalytic promiscuity is the ability of a single enzyme active site to catalyze several
chemical transformations, among them those which are different from natural. We have attempted
to use this feature of enzymes in the nucleophilic addition of nitromethane to aldimines (the aza-
Henry reaction) whose chemically catalyzed version leads to synthetically useful β-nitroamines.
We succeded in obtaining for the first time the desired products in the yields up to 81 %. The most
efficient proved lipase TL (from Pseudomonas stutzeri) and oxynitrilase from Arabidopsis
thaliana. However, all the reactions investigated were non-stereoselective.
1. Introduction
Enzyme catalytic promiscuity, i.e. the ability of a single active site of the enzyme to catalyze
more than one reaction, particularly those which are different from that designed by Nature, has
been a subject of increasing research interest in the recent few years [1-6]. As enzyme catalytic
promiscuity is highly advantageous to chemists, since it broadens the applicability of enzymes in
chemical synthesis and particularly in the stereoselective transformations, it is of great importance
to search for new applications of this feature of enzymes. Therefore, and in connection with our
previous work on this subject [7], we have decided to make an attempt to discover new, unknown
thus far, enzyme-promoted transformations which will be based on this phenomenon. The
transformation of choice has been the nucleophilic addition of nitroalkanes 2 to imines 1, called
the “aza-Henry” or “nitro-Mannich” reaction (Scheme 1) which is known to lead (in its chemical
version [8,9]) to the formation of synthetically useful substituted β-nitroamines 3 with potentially
possible two stereogenic centres. These compounds may be used as interesting building blocks
(routes a, b and c). The biocatalytic version of the aza-Henry reaction has not been reported in the
literature.

Scheme 1. The aza-Henry reaction
A chance of finding new examples of enzyme catalytic promiscuity is based on numerous
literature data. So far, several interesting examples of this phenomenon have been reported. Among
them, a very important case seems the ability of hydrolytic enzymes to catalyze a carbon-carbon
[10] and carbon-heteroatom bond [11] formation , i.e. to exhibit a lyase activity. Lipases are those
hydrolytic enzymes for which numerous examples of such a promiscuous activity have been
described. In addition to their original activity comprising hydrolysis of lipids and, generally,
catalysis of the hydrolysis or formation of carboxylic esters [12], lipases are able to catalyze,
among others, also Michael additions of C-acids to nitroalkenes, aldol condensation [13,14] and
Knoevenagel condensation [15]. From the point of view of the present account, the most inspiring
example is the ability of various hydrolytic enzymes to catalyze the analogous reaction, namely the
nucleophilic addition of nitroalkanes 2 to carbonyl compounds (the Henry reaction, Scheme 2).
Scheme 2. The Henry reaction
This reaction was found to be efficiently (i.e. giving products in good yield and with good to
high enantiomeric excess, ee) catalyzed by esterases (esterase from Thermotoga naphthophila and
from Aeropyrum pernix), lipases (lipase from Candida antarctica, CAL-B, and from porcine
pancreas) [16] and proteases (aminopeptidase from Sulfolobus tokodaii [16], but also by selected
lyases, e.g. hydroxynitrilases (hydroxynitrilase from Hevea brasiliensis [17] and from Arabidopsis
thaliana [18]). Moreover, application of protease from Streptomyces griseus [19] or acylase I from
Aspergillus melleus [20] as catalysts in the Mannich reaction gave the products with up to 92/2
diastereomer ratio and up to 89% ee. However, in certain cases the enzymatic reactions were non-

stereoselective, although the yields of the products were up to 95 %. This concerned the use of
lipases from Aspergillus niger, Rhizopus niveus, Mucor javanicus, Pseudomonas fluorescens,
Candida rugosa, Burkholderia cepacia [21], protease from Bacillus licheniformis [22], papain from
papaya latex [21], and transglutaminase from Streptorerticillium griseoverticillatum [23].
2. Results and discussion
In our preliminary work, we screened the reaction of N-arenesulfonylaldimines 1, synthesized
according to known literaturę procedures, with nitromethane 2a (Scheme 3).
Scheme 3. The enzyme-promoted aza-Henry reaction
In this case, only one stereogenic centre could be created in products 3 (R3 = H). We applied
various reaction conditions and a broad variety of enzymes, including lipases, proteases and
hydroxynitrilases. The results are collected in Table 1.
Table 1: Enzyme-promoted aza-Henry reaction
Entry N-arenesulfonylaldimine Enzyme Solvent Conversion
Product 3
[%]
1
R1
R2
Symbol
Isolated
yield [%]
1
2
3
p-TolSO2
p-TolSO2
p-TolSO2
2-thienyl
2-thienyl
2-thienyl
TL
MeNO2 No reaction
-
-
-
-
-
AtHNL MeNO2 No reaction
AtHNL
H2O
Hydrolysis
of imine
100
4
5
6
7
p-TolSO2
p-TolSO2
p-TolSO2
p-TolSO2
2-thienyl
2-thienyl
2-thienyl
2-thienyl
AtHNL
TL
EtOH
EtOH
MeCN
BF
74
43
81
45
3a
3a
3a
3a
60
TL
100
AtHNL
ND

8
p-TolSO2
2-thienyl
TL
AtHNL
TL
BF
ND
100
70
40
24
15
38
26
32
54
16
32
3a
3b
3b
3b
3c
3c
3c
3c
3d
9
p-TolSO2 1-naphthyl
p-TolSO2 1-naphthyl
p-TolSO2 1-naphthyl
EtOH
EtOH
MeCN
EtOH
MeCN
EtOH
MeCN
EtOH
(94%)
MeCN
MeCN
EtOH
EtOH
10
11
12
13
14
15
16
TL
70
p-TolSO2
p-TolSO2
p-TolSO2
p-TolSO2
PhSO2
phenyl
phenyl
phenyl
phenyl
phenyl
AtNHL
AtNHL
TL
50
70
70
TL
40
AtHNL
ND
17
18
19
20
PhSO2
PhSO2
PhSO2
PhSO2
phenyl
phenyl
phenyl
phenyl
AtHNL
TL
ND
60
20
41
30
5
3d
3d
3d
3d
TL
ND
ND
PPL
Enzyme: TL– lipase from Pseudomonas stutzeri; AtHNL – oxynitrilase from Arabidopsis thaliana;
PPL – porcine pancrease lipase; BF – ionic liquid: 1-butyl-3 methylimidazolium tetrafluoroborate;
Conversion calculated on the basis of the consumption of the starting imine 1
The results obtained need certain comments. First of all, from among a high number of
enzymes examined, only three enzymes proved suitable and gave the desired products 3 in the
yield up to 81%. These were: lipase TL from Pseudomonas stutzeri (entries 5, 6, 8, 10, 11, 14, 15,
18 and 19); oxynitrilase from Arabidopsis thaliana, AtHNL, (entries 4, 7, 9, 12, 13, 16 and 17)
and, to a certain extent, porcine pancrease lipase, PPL (entry 20). The products obtained were
isolated, purified, fully analyzed and characterized. Interestingly, in many cases the conversion
degree was quite high,. This was determined by NMR of the crude reaction mixtures, in which the
relative amount of the unreacted starting imine was measured. The lower yield of the isolated
products was due to the presence of some by-products, particularly the corresponding aldehydes
resulting form the hydrolysis of the imines. No reaction was observed in the absence of enzymes
and in the presence of the following enzymes: papain, lipases from: Candida antarctica B, Mucor
javanicus, Candida rugosa, Pseudomonas species and Pseudomonas fluorescens. No products
were formed in the control reactions in which those denatured enzymes, which earlier proved
efficient in the reaction investigated, were applied (see Experimental Section). This can be taken

as proof of the real participation of enzymes in the successful reactions. In search for the most
favourable reaction conditions, a variety of solvents were checked. No reaction was observed in
the case of common organic solvents such as dichloromethane, ethers, toluene or nitromethane
(used here both as reagent and solvent and for this reason visualized in Table 1). Water proved
useless as it caused hydrolysis of aldimines. Acetonitrile and ethanol proved to be the best solvents
although it is difficult at this point to explain unusual differences in the yield of particular products
when the same enzymes were used (e.g entries 5 and 6 versus 14 and 15). The use of an ionic liquid
1-butyl-3 methylimidazolium tetrafluoroborate, gave relatively good results (entries 7 and 8). It is
worth mentioning that ionic liquids are known as useful solvents in biotransformations [24] and
were successfully used also by us in hydrolase-catalyzed reactions [25]).
To our deep disappointment, all the reactions, performed thus far, were non-stereoselective.
Although precedents for such outcomes were cited in the literature (vide supra and in our paper
[7]), it seems necessary to seek an explanation for the lack of the stereoselectivity, as
stereoselectivity should be a common feature of enzyme-promoted transformations. In our opinion,
the substrates, which possess large electron-withdrawing substituents at the nitrogen atom, may not
be properly accommodated by the enzyme active site. On the other side, such substituents enhance
the addition of nitromethane to aldimines. Indeed, our experiments using the substrates which are
deprived of them, were thus far unsuccessful. Although these preliminary attempts were in certain
aspects disappointing, the investigations will be continued in the hope of finding proper substrates,
enzymes and reaction conditions.
3. Conclusions
In this preliminary study, the first successful enzyme-promoted addition of nitromethane to
N-arenesulfonylaldimines was accomplished, which was a result of the application of enzyme
catalytic promiscuity. Only three enzymes: lipase TL from Pseudomonas stutzeri; oxynitrilase
from Arabidopsis thaliana (AtHNL) and porcine pancrease lipase (PPL) proved suitable. The best
results were obtained using ethanol as solvent. However, all the reactions were non-stereoselective,
which is considered to be due to the large electron withdrawing substituents at the nitrogen atom
in the substrates. In case of the substrates deprived of such substituents, no reaction was observed.
Further investigations are in progress.

4. Experimental Section
All the N-sulfonyl imines 1 are known [26,27,28] and for our purposes were obtained
according to the procedure described by Garcia Ruano in the reaction of the corresponding aryl
aldehydes and p-toluenesulfonamide or benzenesulfonamide in the presence of catalytic amounts
of pyrrolidine [26].
Enzymes: Lipase TL – Lipase from Pseudomonas stutzeri; a generous gift by Meito Sangyo
Co., Ltd. AtHNL – oxynitrilase from Arabidopsis thaliana; PPL – porcine pancrease lipase, both
purchased from Sigma.
4.1.General procedure for enzymatic Aza-Henry reaction of imines 1 with nitromethane.
To the solution of an N-sulfonyl imine 1 (0.4 mmol) in the appropriate solvent (6mL),
enzyme [AtHNL (63μL), or TL (50 mg), or PPL (50 mg)] was added and the mixture was stirred
for 0.5 h. Nitromethane (1mL) was dropped and the reaction mixture was stirred for 24 h at ambient
temperature. Than the solvent was evaporated and CH2Cl2 (10 mL) was added and (in the case of
AtNHL) washed with water (2x5 mL) or (in the case of Lipase TL and PPL) filtered through Celite.
The organic fraction was dried over MgSO4 and evaporated. The crude reaction mixture was
purified using column chromatography to give pure products. All the products are known form the
literature [29,30,31]. Analytical and spectral data of the products obtained were compared with
those reported in the literature and proved correct.
Experiments with denatured enzymes
Two enzymes, namely AtHNL and lipase TL, were denatured by heating overnight in
boiling water at 100 ºC. The water was evaporated and the recovered denatured enzymes were
used in selected reactions according to the procedure described above. In no case was the formation
of the appropriate products 3 observed.
Acknowledgements
Financial support by the National Research Centre (NCN), Poland, grant OPUS
2016/23/B/ST5/02443 for P.K., is gratefully acknowledged.

Notes
The authors declare no competing financial interest.
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Highlights
 The first successful enzyme-promoted addition of nitromethane to sulfonylimines (aza-
Henry reaction) using enzyme catalytic promiscuity is presented.
 A series of various enzymes was screened to identify their promiscuous activity.
 Three enzymes proved efficient and led to the formation of the desired adduct in
up to 81% yield
 From among many solvents only three proved suitable for the reaction.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: