Enzyme-catalyzed Henry (nitroaldol) reaction

Article abstract of DOI:10.1016/j.molcatb.2009.12.005

Transglutaminase was first used to catalyze Henry reactions of aliphatic, aromatic and hetero-aromatic aldehydes with nitroalkanes. The reactions were carried out at room temperature, and the corresponding nitro alcohols were obtained in yields up to 96%.

Full text of DOI:10.1016/j.molcatb.2009.12.005

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 62–67
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzyme-catalyzed Henry (nitroaldol) reaction
Rong-Chang Tang, Zhi Guan, Yan-Hong He∗, Wen Zhu
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Transglutaminase was first used to catalyze Henry reactions of aliphatic, aromatic and hetero-aromatic
aldehydes with nitroalkanes. The reactions were carried out at room temperature, and the corresponding
nitro alcohols were obtained in yields up to 96%.
Received 1 September 2009
Received in revised form 4 December 2009
Accepted 4 December 2009
Available online 16 December 2009
© 2009 Elsevier B.V. All rights reserved.
Keywords:
Henry reaction
Transglutaminase
Enzyme
␤-Nitroalcohols
Biocatalytic promiscuity
1. Introduction
their enzymatic and physiological properties have been extensively
studied [26,27]. It is well known that TGase catalyses an acyl trans-
The Henry (nitroaldol) reaction is a powerful and atom economi-
cal C–C bond forming reaction in synthetic organic chemistry [1–3].
The resulting ␤-nitro alcohols are often used as valuable synthetic
intermediates in the synthesis of numerous products [4], such
as 2-aminoalcohols, 2-nitroketones and nitroalkenes [5–8], which
are useful for the synthesis of biologically important compounds
[9,10]. In recent years, the development of new chiral catalysts for
this important reaction has attracted the interest of many groups
[11–18]. On the other hand, enzymes as practical catalysts have
been increasingly exploited for synthetic transformations due to
their simple processing requirements, high selectivity and mild
reaction conditions [19,20]. However, to the best of our knowl-
edge, there was only one group that had reported the asymmetric
catalytic Henry reaction using Hydroxynitrile Lyase from Hevea
brasiliensis (HbHNL) (EC 4.1.2.39) [21,22]. Therefore, the develop-
ment of new enzymatic catalysts is still in great demand. Herein,
we wish to report a novel discovery that the cheap and read-
ily available transglutaminase (TGase) efficiently promotes the
Henry reaction (nitroaldol) of nitroalkanes with aliphatic, aro-
matic and hetero-aromatic aldehydes resulting in moderate to good
yields.
fer reaction between a ␥-carboxyamide group of glutamine residue
and ␧-amino group of lysine residue or other primary amines. In this
report, however, TGase was first used to catalyze Henry (nitroaldol)
reaction. It shows the ability of the enzyme to catalyze synthetic
reaction which varies from its natural role [28]. This is known
as biocatalytic promiscuity, a new frontier which has emerged
recently and largely extended the application of enzymes. Some
elegant reports have addressed the importance and wide appli-
cation of biocatalytic promiscuity in organic synthesis [29–46].
Herein, we report this TGase-catalyzed Henry reaction as another
example of biocatalytic promiscuity.
2. Experimental
2.1. General remarks
Immobilized lipase from Thermomyces lanuginosus (0.25 U/mg.
One unit corresponds to the amount of enzyme producing 1 ␮mol
methyl oleate from triolein per minute at 35 ◦C) was pur-
chased from Novozymes (China) Investment Co., Ltd. TGase from
Streptorerticillium griseoverticillatum (0.06 U/mg. The activity was
determined by the colorimetric hydroxamate procedure. One unit
generates 1 ␮mol hydroxamic acid per minute at 37 ◦C), Pancre-
atin from porcin pancreas (4 U/mg. One unit of activity was defined
as the amount of the enzyme to produce TCA-soluble hydrolysis
products from casein, which gives an absorbance value equiva-
lent to 1 ␮g of tyrosine at 275 nm per minute at 40 ◦C and pH
7.5), Papain from fruit jam from chaenomeles (650 U/mg. One unit
TGase (protein-glutamine ␥-glutamyltransferase; EC 2.3.2.13)
are a family of enzymes that are widely distributed in mam-
mals [23], plants [24], fishes [25] and micro-organisms [26], and
∗
Corresponding author. Fax: +86 23 68254091.
E-mail address: heyh@swu.edu.cn (Y.-H. He).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.005

R.-C. Tang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 62–67
63
of activity was defined as the amount of the enzyme to produce
TCA-soluble hydrolysis products from casein, which gives an
absorbance value equivalent to 1 ␮g of tyrosine at 275 nm/min at
37 ◦C and pH 7.0), Lysozyme from hen egg white (20,000 U/mg. The
activity determination was according to the procedure described
by Shugar [47]. One unit of activity was defined as the amount of
enzyme that lowers 0.001 absorbance at 450 nm/min), Chymosin
from fruit jam from chaenomeles (20 U/mg. The activity determi-
nation was according to the procedure described by Lowry et al.
[48]. One unit of milk-clotting activity was defined as the amount
of enzyme required to clot 1 ml of milk in 40 min at 35 ◦C), Nuclease
from Penicillium citrinum (5 U/mg. The activity determination was
according to the procedure described by Eaves and Jeffries [49]. One
unit of activity was defined as the amount of the enzyme which
liberates the digestion product not precipitated by the ammonium
molybdate-perchloric acid reagent and gives an extinction change
of 1 at 260 nm), Bromelain from pineapple peduncle (500 U/mg.
One unit of activity was defined as the amount of the enzyme to
produce TCA-soluble hydrolysis products from casein, which gives
an absorbance value equivalent to 1 ␮g of tyrosine at 275 nm/min
at 37 ◦C and pH 7.0), and Cellulase from Trichoderma (10 U/mg.
One unit of activity was defined as the amount of enzyme which
released 1.0 mg of glucose from cellulose in 1 h at 40 ◦C and pH
4.8) were purchased from Guangxi Nanning Pangbo Biological Engi-
neering Co. Ltd. (Nanning, China). Alkaline proteinase from Bacillus
licheniformis No 2709 (200 U/mg. One unit of activity is the amount
of enzyme that liberates 1.0 ␮equiv. of tyrosine from casein per
minute at 40 ◦C and pH 10.5), Acidic proteinase from Aspergillus
usamii No 537 (50 U/mg. One unit of activity is the amount of
enzyme that liberates 1.0 ␮equiv. of tyrosine from casein per
minute at 40 ◦C and pH 3.0), Neutral proteinase from Bacillus subtilis
A.S.1.398 (130 U/mg. One unit of activity is the amount of enzyme
that liberates 1.0 ␮equiv. of tyrosine from casein per minute at 30 ◦C
and pH 7.5), and Trypsin from porcin pancreas (4 U/mg. One unit of
activity was defined as the amount of the enzyme to produce TCA-
soluble hydrolysis products from casein, which gives an absorbance
value equivalent to 1 ␮g of tyrosine at 275 nm/min at 40 ◦C and pH
8.0) were purchased from Wuxi Xuemei Enzyme Co. Ltd. (WuXi,
China). Unless otherwise noted, all reagents were obtained from
commercial suppliers and were used without further purification.
All reactions were monitored by thin-layer chromatography (TLC)
with Haiyang GF254 silica gel plates. Flash column chromatogra-
phy was carried out using 100–200 mesh silica gel at increased
pressure.
Scheme 1. Enzyme-catalyzed Henry reaction of 4-nitrobenzaldehyde and
nitromethane.
3. Results and discussion
Initial studies were undertaken using 4-nitrobenzaldehyde and
nitromethane as a model reaction. We chose a cyclohexane/water
10:1 (v/v) system as the reaction medium. The reaction was per-
formed at room temperature (20–30 ◦C) (Scheme 1). In order to
select the appropriate enzymes, by using the model system several
commercially available enzymes were screened (Table 1).
As shown in Table 1, the best result (93% yield) was achieved by
using TGase as catalyst after 32 h (Table 1, entry 1). Immobilized
lipase and Pancreatin also showed moderate catalytic activities
(Table 1, entries 2 and 3), while some tested enzymes including
Papain, Lysozyme, Chymosin, Alkaline proteinase, and Nuclease
presented low activities in this reaction (Table 1, entries 4–8). In
addition, in the presence of Acidic proteinase, Neutral proteinase,
Trypsin, Bromelain and Cellulase, respectively, only trace product
was observed on TLC (Table 1, entries 9–13).
We also performed some control experiments to demonstrate
the specific catalytic effect of the TGase. Just as we expected, the
Henry reaction of 4-nitrobenzaldehyde and nitromethane in the
absence of enzyme showed low yield adduct (10%) even after 5
days (Table 1, entry 14). Moreover, the reaction catalyzed by non-
enzyme protein bovine serum albumin (B.S.A.) gave product only
Table 1
The catalytic activities of different enzymes in Henry reaction between 4-
nitrobenzaldehyde and nitromethane.a.
Entry
Catalyst
Time (h)
Yield
(%)b
1
2
TGase from Streptorerticillium
griseoverticillatum
32
48
93
64
Immobilized lipase from
Thermomyces lanuginosus
Pancreatin from porcin pancreas
Papain from fruit jam from
chaenomeles
3
4
48
48
41
15
5
6
Lysozyme from hen egg white
Chymosin from fruit jam from
chaenomeles
48
48
13
13
2.2. Representative procedure for enzyme-catalyzed Henry
reactions (products 2a–s, 3a–d, 4a–d)
7
Alkaline proteinase from Bacillus
licheniformis No 2709
48
12
For 2a–s: a 25 ml round-bottomed flask was charged with the
enzyme (200 mg), deionized water (3 ml) and CH2Cl2 (5 ml), to
which the aldehyde (200 mg) and nitromethane (2 g, 32 mmol)
were introduced. The resulting solution was stirred for the specified
amount of time at rt (20–30 ◦C).
8
9
Nuclease from Penicillium citrinum
Acidic proteinase from Aspergillus
usamii No 537
Neutral proteinase from Bacillus
subtilis A.S.1.398
48
48
11
Trace
10
48
Trace
11
12
Trypsin from porcin pancreas
Bromelain from pineapple
peduncle
Cellulase from Trichoderma
No enzyme
Bovine serum albumin (B.S.A.)
TGase denatured with EDTA c
TGase inhibited with NBSd
48
48
Trace
Trace
For 3a–d and 4a–d: a 25 ml round-bottomed flask was charged
with the enzyme (200 mg), deionized water (3 ml) and CH2Cl2
(5 ml), to which the aldehyde (1 mmol) and nitroalkane (25 mmol)
were introduced. The resulting solution was stirred for the specified
amount of time at 30 ◦C.
The reaction was terminated by filtering the enzyme. CH2Cl2
was used to wash the filter paper to assure that products obtained
were all dissolved in the filtrate. The filtrate was extracted three
times with 20 ml of CH2Cl2. The combined extracts were dried
over anhydrous Na2SO4 and the solvent was then removed under
reduced pressure. The crude products were purified by column
chromatography with petroleum ether/ethyl acetate as eluent.
13
14
15
16
17
48
120
48
120
48
Trace
10
16
0
12
a
All reactions were carried out using 4-nitrobenzaldehyde (200 mg, 1.3 mmol),
nitromethane (2 g, 32 mmol), deionized water (0.5 ml), cyclohexane (5 ml) and
enzyme (200 mg) at rt (20–30 ◦C).
b
Yield of the isolated product after chromatography on silica gel.
c
Pre-treated with EDTA at 100 ◦C for 24 h.
d
Pre-treated with NBS at 25 ◦C for 24 h.

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R.-C. Tang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 62–67
Table 2
Henry reaction of 4-nitrobenzaldehyde and nitromethane catalyzed by TGase in
different solvents.a.
Entry
Solvent
Time (h)
Yield (%)b
1
2
3
4
5
6
7
8
DMSO
DMF
THF
TBME
CH2Cl2
Cyclohexane
H2O
59
59
59
59
59
59
59
59
73
85
80
90
90
93
32
92
Solvent-free
a
All reactions were carried out using 4-nitrobenzaldehyde (200 mg, 1.3 mmol),
TGase (200 mg), nitromethane (2 g, 32 mmol), deionized water (0.5 ml) and organic
solvent (5 ml) at rt (20–30 ◦C).
b
Yield of the isolated product after chromatography on silica gel.
Fig. 1. Influence of water content in organic solvent on the yield of the
TGase-catalyzed Henry reaction. Conditions: TGase (200 mg), nitromethane (2 g,
32 mmol), 4-nitrobenzaldehyde (200 mg, 1.3 mmol) and dichloromethane (5 ml)
was stirred at rt (20–30 ◦C) for 48 h. Deionized water was added from 0 to 0.9
[water/dichloromethane, v/v].
in 16% yield (Table 1, entry 15), which excluded the possibility of
protein catalysis. When the reactants were incubated with EDTA-
denatured TGase (pre-treated with EDTA at 100 ◦C for 24 h), no
product was detected even after 5 days (Table 1, entry 16), sug-
gesting that the promiscuity of TGase was metal-dependent, and
the tertiary structure of the enzyme might also be necessary. In
addition, a strong inhibition of the catalytic activity of TGase in
Henry reaction was observed by using NBS (N-Bromosuccinimide)
(pre-treated with NBS at 25 ◦C for 24 h). The reaction catalyzed
by NBS-inhibited TGase gave product only in 12% yield after 48 h
(Table 1, entry 17). These results implied that the reaction must
take place in a specific fashion on the catalytic site of TGase. There-
fore, we chose TGase as catalyst for Henry reaction in our study.
TGase was from S. griseoverticillatum (0.06 U/mg. The activity was
determined by the colorimetric hydroxamate procedure. One unit
generates 1 ␮mol hydroxamic acid per minute at 37 ◦C).
The selection of organic solvents is also crucial in the enzyme-
catalyzed reactions, due to their effects on the enzyme stability and
the solubility of substrates. Thus, a preliminary solvent screen was
performed (Table 2). We found that the reaction using tert-butyl
methyl ether (TBME), dichloromethane or cyclohexane gave the
anticipant product in excellent yield (Table 2, entries 4–6), while
moderate yield was obtained with DMSO, DMF or THF (Table 2,
entries 1–3). It seems likely that less polar solvents favor the reac-
tion. In addition, the reaction in deionized water gave the product
in low yield (Table 2, entry 7). It may be attributed to the poor
solubility of substrates in water. Interestingly, even in solvent-free
conditions, the high yield was obtained (Table 2, entry 8). Based on
the results of solvent screen, in view of the good ability to resolve
the wide scope of substrates, we chose dichloromethane as the
optimum solvent for the Henry reaction.
We next investigated the effect of TGase loading on the reac-
tion. As shown in Table 4, evident difference in the chemical yields
(68% and 89%) was observed between TGase loading of 50 mg and
100 mg. However, once the quantity of TGase surpasses 100 mg, the
yield of product kept unchanged as 90%. The results showed that
100 mg of TGase was the optimum quantity for the Henry reaction
between 4-nitrobenzaldehyde and nitromethane. However, in con-
sideration of reaction rates of some less active substrates, we chose
200 mg of TGase as the optimum quantity for the Henry reaction.
We further studied the effect of temperature on the reac-
tion. From Table 5, there were no significant differences in yields
between room temperature (20–30 ◦C), 30 ◦C and 35 ◦C for the
model reaction of 4-nitrobenzaldehyde and nitromethane. In view
Table 3
Effect of molar equivalents of nitromethane on the yield of Henry reaction.a.
Entry
Nitromethane 4-Nitrobenzaldehyde
Time (h)
Yield (%)b
(mmol)
(mmol)
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
10
15
20
25
30
1
1
1
1
1
1
1
1
1
1
48
48
48
48
48
48
48
48
48
48
73
74
74
74
76
84
89
90
93
93
For the enzymes require a specific amount of water bound to
them to maintain activities, it is important to confirm the opti-
mal water content in reaction system. Therefore, we analyzed the
effects of the amount of added H2O on the reaction rate (Fig. 1).
From Fig. 1, it was found that the rate of the enzymatic
reaction can be accelerated by increasing the concentration of
water used, and reaches the highest rate at 0.6 water content
(water/dichloromethane, v/v). However, once the water content
surpasses 0.6, the yield of Henry product decreased evidently, prob-
ably due to the insolubility of the substrates. Thus, the optimum
water content for the reaction was 0.6 (water/dichloromethane,
v/v). The results indicated that water is obviously essential in the
enzyme-catalyzed Henry reaction.
a
All reactions were carried out using 4-nitrobenzaldehyde (151 mg, 1 mmol),
TGase (200 mg), deionized water (3 ml), CH2Cl2 (5 ml) and specified amount of
nitromethane at rt (20–30 ◦C).
b
Yield of the isolated product after chromatography on silica gel.
Table 4
Effect of TGase loading on the yield of Henry reaction.a.
Entry
TGase (mg)
Time (h)
Yield (%)b
1
2
3
4
5
6
50
100
200
300
400
500
48
48
48
48
48
48
68
89
90
90
90
89
To optimize experimental conditions, we then examined the
effect of nitromethane stoichiometry on the reaction. From
Table 3, the best result was achieved by using 25 or 30 equiv. of
nitromethane. Thus, we chose 25 equiv. of nitromethane as the
optimum molar equivalent for the Henry reaction.
a
All reactions were carried out using 4-nitrobenzaldehyde (151 mg, 1 mmol),
nitromethane (1.5 g, 25 mmol), deionized water (3 ml), CH2Cl2 (5 ml) and specified
amount of TGase at rt (20–30 ◦C).
b
Yield of the isolated product after chromatography on silica gel.

R.-C. Tang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 62–67
65
Table 5
Effect of temperature on the yield of Henry reaction.a.
Table 7
TGase-catalyzed Henry reaction of aldehydes with nitroethane.a.
Entry
Temp. (◦C)
Time (h)
Yield (%)b
1
2
3
30
35
rtc
48
48
48
92
92
90
a
All reactions were carried out using 4-nitrobenzaldehyde (151 mg, 1 mmol),
TGase (200 mg), nitromethane (1.5 g, 25 mmol), deionized water (3 ml) and CH2Cl2
(5 ml) at specified temperature.
Entry
R
Time (h)
Yield (%)b
Anti:sync
b
1
2
3
4
4-MeOC6H4(1d)
4-CH3C6H4(1e)
4-NO2C6H4(1h)
Isobutyl(1s)
144
144
72
40
46
90
77
1:2.3
1:1.3
1:1.3
1:1
Yield of the isolated product after chromatography on silica gel.
c
20–30 ◦C.
72
Table 6
a
TGase-catalyzed Henry reaction of aldehydes with nitromethane.a.
All reactions were carried out using aldehyde (1 mmol), TGase (200 mg),
nitroethane (1.5 g, 25 mmol), deionized water (3 ml) and CH2Cl2 (5 ml) at 30 ◦C.
b
Yield of the isolated product after chromatography on silica gel.
c
Determined by HPLC on chiral stationary phase (AD-H) and 1H NMR.
Entry
R
Time (h)
96
Yield of 2 (%)b
gave corresponding product in 86% and 96% yield respectively
(Table 6, entries 6 and 8). In contrast, aromatic aldehydes contain-
ing an electron-donating group gave the products in relatively low
yields (Table 6, entries 2–5). This is because electron-withdrawing
groups enhance the electrophilicity of carbonyl carbons in aldehy-
des which facilitates the reaction, while electron-donating groups
render it less electrophilic. Moreover, it is clear that the reactions
using aliphatic aldehydes were faster and provided the correspond-
ing adducts with satisfied yields (Table 6, entries 17–19). This is
probably due to the relatively small steric hinderance of alkyls.
In the cases of hetero-aromatic aldehydes 1o and 1p, the
reaction was obviously affected by the heteroatom of the hetero-
aromatic ring, and both 1o and 1p gave anticipant product
␤-nitroalcohols in low yields (Table 6, entries 15 and 16). Inter-
estingly, the tandem Henry/Michael reaction was observed with
2-furaldehyde (1p), and the unexpected 1,3-dinitro compound 3
was obtained in 36% yield while expected ␤-nitroalcohol 2p was
only received in 12% yield (Scheme 2).
The reaction was then extended to nitroethane and nitro-
propane. As shown in Tables 7 and 8, the electronic effects of
the substituent in the aromatic aldehydes affected the reactions
in the same way as using nitromethane. As expected, 4-
nitrobenzaldehyde, an aldehyde bearing an electron-withdrawing
group, gave high yields of 90% with nitroethane (Table 7, entry
3), and 94% with nitropropane (Table 8, entry 3). The aldehyde
with electron-donating group gave moderate to modest yields
(Tables 7 and 8, entries 1 and 2). Moreover, the aliphatic aldehy-
des also gave satisfied yields (Tables 7 and 8, entry 4). In addition,
anti/syn selectivity was observed in the reactions of nitroethane
and nitropropane with aromatic aldehydes, and the syn products
were obtained chiefly (Tables 7 and 8, entries 1–3). However, when
3-methyl-butanal (1s) was used, the adducts showed no anti/syn
selectivity (Tables 7 and 8, entry 4). Finally, it can be found that the
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Ph(1a)
58
51
59
50
60
86
62
96
68
68
77
64
64
79
21
12
61
70
71
2-MeOC6H4(1b)
3-MeOC6H4(1c)
4-MeOC6H4(1d)
4-CH3C6H4(1e)
2-NO2C6H4(1f)
3-NO2C6H4(1g)
4-NO2C6H4(1h)
4-FC6H4(1i)
4-BrC6H4(1j)
2-ClC6H4(1k)
3-ClC6H4(1l)
4-ClC6H4(1m)
4-CNC6H4(1n)
2-Thienyl(1o)
2-Furyl(1p)
120
120
120
120
120
120
48
120
120
120
120
120
120
120
120
48
Ethyl(1q)
n-Propyl(1r)
Isobutyl(1s)
48
48
a
All reactions were carried out using aldehyde 1 (200 mg), TGase (200 mg),
nitromethane (2 g, 32 mmol), deionized water (3 ml) and CH2Cl2 (5 ml) at rt
(20–30 ◦C).
b
Yield of the isolated product after chromatography on silica gel.
of the boiling point of CH2Cl2, we chose 30 ◦C as the optimum tem-
perature for the Henry reaction.
With the optimized conditions in hand, some other aldehydes
were used to expand upon this TGase-catalyzed Henry reaction
to show the generality and scope of this new enzymatic promis-
cuity. The results are summarized in Table 6. It can be seen that
a wide range of aromatic, hetero-aromatic and aliphatic aldehy-
des can effectively participate in the reaction. In general, aromatic
aldehydes bearing an electron-withdrawing substituent furnished
␤-nitro alcohols with yields well above 62% (Table 6, entries
6–14). Especially, 2-nitrobenzaldehyde and 4-nitrobenzaldehyde
Scheme 2. Tandem Henry/Michael reaction between 2-furaldehyde and nitromethane.

66
R.-C. Tang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 62–67
Table 8
TGase-catalyzed Henry reaction of aldehydes with nitropropane.a.
Entry
R
Time (h)
Yield (%)b
Anti:sync
1
2
3
4
4-MeOC6H4(1d)
4-CH3C6H4(1e)
4-NO2C6H4(1h)
Isobutyl(1s)
168
168
72
22
35
94
53
1:2.9
1:1.7
1:3.8
1:1
72
a
All reactions were carried out using aldehyde (1 mmol), TGase (200 mg), nitropropane (2.2 g, 25 mmol), deionized water (3 ml) and CH2Cl2 (5 ml) at 30 ◦C.
Yield of the isolated product after chromatography on silica gel.
b
c
Determined by HPLC on chiral stationary phase (AD-H) and 1H NMR.
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Scheme 3. Proposed mechanism of enzymatic Henry reaction.
length of carbon chain in the nitroalkane also obviously affected the
enzymatic Henry reaction. Generally, longer reaction times were
required, and lower yields were obtained with nitroethane and
nitropropane in comparison to nitromethane, due to steric hinder-
ance.
Based on the above results, we proposed the possible reac-
tion mechanism of TGase-catalyzed Henry reaction, depicted in
Scheme 3. From the experiments catalyzed by denatured enzyme,
inhibited enzyme and non-enzyme protein as well as blank experi-
ment (Table 1), it is reasonable to think that the reaction takes place
in a specific fashion on the catalytic site of TGase. Firstly, an amine
group on the reaction site assists the deprotonation of nitroalkane
to give nitronate. Secondly, nucleophilic attack of nitronate onto
carbonyl carbon in aldehyde forms C–C bond. Meanwhile, the
resulted aminium on enzyme provides a proton to carbonyl oxygen
to give ␤-nitro alcohol.
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In conclusion, we describe here the first TGase-catalyzed Henry
reaction. The cheap and readily available TGase efficiently catalyzed
the Henry reaction of nitroalkanes with aliphatic, aromatic and
hetero-aromatic aldehydes. The influence of reaction conditions
including solvents, water content, stoichiometry of nitroalkane,
loading of catalyst and temperature was also investigated. This
TGase-catalyzed Henry reaction provides a novel case of catalytic
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Financial support from the High-Tech Training Fund of South-
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