Enzyme-mediated domino synthesis of 2-alkylbenzimidazoles in solvent-free system: A green route to heterocyclic compound

Article abstract of DOI:10.1016/j.molcatb.2010.06.013

Solvent-free domino acylation/cyclization reactions between fatty acid esters and o-phenylenediamine mediated by immobilized lipase from Mucor miehei (MML) were found to be an efficient way in the synthesis of 2- alkylbenzimidazole. Compared with other substrates, methyl fatty acid esters with moderated chain length exhibited best activity with yield up to 95%. The mechanism of the domino process was clarified deeply through some detail experiments, including separation of intermediates. This efficient enzymatic domino process provides an attractive procedure for heterocyclic compound synthesis and could be regarded as a potential synthetic method in modern organic chemistry.

Full text of DOI:10.1016/j.molcatb.2010.06.013

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 16–20
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzyme-mediated domino synthesis of 2-alkylbenzimidazoles in solvent-free
system: A green route to heterocyclic compound
Li Wang, Chao Li, Na Wang∗, Kun Li, Xi Chen, Xiao-Qi Yu∗
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, No 29, Wangjiang Road, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 April 2010
Received in revised form 9 June 2010
Accepted 25 June 2010
Available online 1 August 2010
Solvent-free domino acylation/cyclization reactions between fatty acid esters and o-phenylenediamine
mediated by immobilized lipase from Mucor miehei (MML) were found to be an efficient way in the
synthesis of 2-alkylbenzimidazole. Compared with other substrates, methyl fatty acid esters with mod-
erated chain length exhibited best activity with yield up to 95%. The mechanism of the domino process
was clarified deeply through some detail experiments, including separation of intermediates. This effi-
cient enzymatic domino process provides an attractive procedure for heterocyclic compound synthesis
and could be regarded as a potential synthetic method in modern organic chemistry.
Keywords:
Lipase
Domino reaction
Solvent-free system
2-Alkylbenzimidazole
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
is the unclear mechanism of the process. In the present work, we
focused on a facile and useful enzymatic strategy for the synthe-
The development of efficient synthesis of bioactive compounds
in an ecologically and economically favorable way is a great chal-
lenge in modern chemistry [1]. Benzimidazole derivatives are
important scaffolds, presenting in a variety of biologically active
molecules, such as anti-hepatitis B virus agents and omeprazole
of peptic antiulcer, etc. [2]. Among the approaches to synthesis of
benzimidazoles [3], domino synthesis has received much attention
in recent years, owing to the reduction of intermediate purification
and isolation steps, which can minimize the waste and consump-
tion of solvents [4]. However most domino reactions are promoted
by conventional chemical catalysts, which are always not envi-
ronmentally friendly [5]. As a mild and facile protocol in organic
synthesis, biocatalysis, especially domino biocatalysis, presents a
valuable alternative to conventional chemical methods due to its
high selectivity and simplicity [6].
It is surprising that only a few enzymatic routes to heterocyclic
compound such as benzimidazole have been reported. To the best
of our knowledge, Renard and co-workers reported the one and
only example of enzymatic synthesis of 2-alkylbenzimidazole [7].
However, moderate yield could be achieved only for long chain
fatty acid. Additionally, the use of organic solvent and large con-
sumption of lipase obviously enlarged the E factor of the enzymatic
process [8]. Another critical problem which has not been solved
sis of 2-alkybenzimidazoles by a enzymatic acylation/cyclization
domino reactions between 1,2-arylenediamine and fatty acid esters
(Scheme 1). A series of 2-alkybenzimidazoles with different chain
length had been synthesized with moderate to good yields. The
immobilized biocatalyst MML could be easily recycled and the
activity did not decrease significantly after two cycles. In addition,
a two-step mechanism was proposed based on the experiments,
including the separation of intermediate a: Firstly, MML-mediated
mono-acylation of 1,2-arylenediamine occurred, and sequentially
the product b can be formed through cyclization promoted by a
fatty acid, which produced from MML-catalyzed hydrolysis.
2. Experimental
2.1. Materials and methods
1H NMR and 13C NMR spectra were recorded on a Bruker DMX
400 or 600. Chemical shifts are given in ı relative to tetramethyl-
silane (TMS). HPLC was carried out using a Shimadzu organizer
consisting of a LC-2010AHT Integrator, a UV/VIS Detector. Dai-
cel Chiralpak OJ column and Welchrom-C18 (5 ␮m, 4.6 × 150 mm)
column were used in the HPLC experiments. Electrospray ion-
ization mass spectrometry (ESI-MS) experiments were performed
on Bruker Daltonics Bio TOF mass spectrometer. MJL (lipase
from Mucor javanicus, 10,000 U/mg, Aldrich); MML (lipase from
Mucor meihei, 140 U/mg, Fluka), CRL (lipase from Candida rugosa,
700–1500 U/mg, Sigma); CAL-B (lipase acrylic resin from Can-
∗
Corresponding authors. Tel.: +86 28 85460576; fax: +86 28 85415886.
E-mail address: xqyu@tfol.com (X.-Q. Yu).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.06.013

L. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 16–20
17
2.2.4. N-(2-Aminophenyl)palmitamide
1H NMR (CDCl3, 400 MHz): ı 7.28 (s, 1H, NHCO), 7.20 (m, 1H,
ArH), 7.08 (m, 1H, ArH), 6.82 (m, 2H, ArH), 2.41 (t, J = 7.2 Hz, 2H,
CH2–CO), 1.75 (m, 2H, CH2–CH2CO), 1.23 (m, 24H, (CH2)n), 0.90 (t,
J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz): ı 171.96, 140.69,
127.11, 125.24, 124.51, 119.64, 118.26, 37.10, 31.91, 29.68, 29.64,
29.60,29.48, 29.44, 29.34, 29.30, 29.25,29.10, 25.87, 24.78, 22.67,
14.08. ESI-MS: m/z = 329.3 [M+H]+. IR (KBr, cm−1): 3399.7, 3331.3,
3265.5, 2919.7, 2850.4, 1642.9, 1534.9, 1462.7, 1254.7, 766.8, 718.3.
2.2.5. 2-Tridecyl-benzimidazole
Scheme 1. Process of the enzyme-mediated domino synthesis of 2-
1H NMR (CDCl3, 400 MHz): ı 7.31 (m, 2H, ArH), 7.12 (m, 2H,
ArH), 2.28 (t, J = 7.2 Hz, 2H, CH2–Ar), 1.64 (m, 2H, CH2–CH2Ar),
1.21 (m, 20H, (CH2)n), 0.81 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (CDCl3,
100 MHz): ı 155.22, 122.14, 31.29, 29.67, 29.65, 29.61, 29.49, 29.41,
29.38, 29.36, 29.34, 28.28, 22.69, 14.12. ESI-MS: m/z = 301.2 [M+H]+.
IR (KBr, cm−1): 3442.8, 2922.3, 2852.0, 1460.6, 761.1, 717.8. Dai-
cel Chiralpak OJ column, hexane/i-PrOH (85/15, v/v), 0.5 mL/min,
7.3 min.
alkylbenzimidazoles.
dida antarctica, 10,000 U/mg, Sigma); PCL (lipase from Penicillium
camemberti, 30,000 U/mg, Fluka); BSNP (proteinase N from Bacil-
lus subtilis, 6.67 U/mg, Sigma); and AMA (acylase I from Aspergillus
melleus, 0.72 U/mg, Fluka).
2.2. A typical enzymatic procedure of synthesis
2.2.6. N-(2-Aminophenyl)tetradecanamide
1H NMR (CDCl3, 400 MHz): ı 7.49 (s, 1H, NHCO), 7.06 (m, 1H,
ArH), 6.95 (m, 1H, ArH), 6.68 (m, 2H, ArH), 4.01 (s, 2H, NH2), 2.26
(t, J = 7.6 Hz, 2H, CH2–CO), 1.61 (m, 2H, CH2–CH2CO), 1.19 (m, 20H,
(CH2)n), 0.81 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz):
ı 172.29 (–C O), 140.88, 127.15, 125.47, 124.32, 119.44, 118.07,
36.97, 31.93, 29.70, 29.67, 29.53, 29.40, 29.37, 29.33, 25.89, 22.70,
14.13. ESI-MS: m/z = 341.2 [M+Na]+. IR (KBr, cm−1): 3397.4, 3330.1,
3263.7, 2919.3, 2850.0, 1643.2, 1535.2, 1467.5, 1453.4, 1258.7,
766.5, 717.8.
The reaction was initiated by adding 20 mg MML to a mixture
of 20 mg (0.19 mmol) o-phenylenediamine and 1.11 mmol corre-
sponding ester. The mixture was stirred at 50 ◦C for 60 h (formation
of products was detected by TLC). Then ether was added and the
residue was filtered off. After washing with NaOH aqueous solu-
tion and ethanol mixture (1N NaOH in an EtOH:H2O (1:5, v/v)
mixture) and water, the solution was finally dried on Na2SO4. A
single product was prepared by silica gel chromatograph with an
eluent consisting petroleum ether/ethyl acetate (from 5:1 to 2:1,
v/v). Samples detected with OJ column were dissolved in the mix-
ture of isopropanol and hexane and others were dissolved in the
mixture of isopropanol and methanol. UV detection was carried
out at 254 nm.
2.2.7. 2-Undecyl-benzimidazole
1H NMR (CDCl3, 600 MHz): ı 9.97 (s, 1H, NH), 7.53 (m, 2H,
ArH), 7.21 (m, 2H, ArH), 2.78 (m, 2H, CH2–Ar), 1.73 (m, 2H,
CH2–CH2Ar), 1.25 (m, 16H, (CH2)n), 0.88 (t, J = 7.2 Hz, 3H, CH3). 13
C
NMR (CDCl3, 100 MHz): ı 154.93, 136.39, 122.88, 114.34, 35.05,
31.90, 29.63, 29.52, 29.37, 29.30, 29.22, 28.03, 25.23, 22.67, 14.08.
ESI-MS: m/z = 273.2 [M+H]+. IR (KBr, cm−1): 3424.0, 2923.1, 2852.1,
1458.2, 1413.7, 1273.2, 749.2, 723.8. Welchrom-C18 column (5 ␮m,
4.6 × 150 mm), MeOH/H2O (90/10, v/v), 0.8 mL/min, 7.7 min.
2.2.1. 2-Heptadecyl-benzimidazole
1H NMR (CDCl3, 400 MHz): ı 7.44 (m, 2H, ArH), 7.12 (m, 2H, ArH),
2.72 (t, J = 7.6 Hz, 2H, CH2–Ar), 1.66 (m, 2H, CH2–CH2Ar), 1.18 (m,
28H, (CH2)n), 0.81 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz):
ı 155.06, 136.54, 136.48, 122.85, 114.35, 31.94, 29.73, 29.69, 29.65,
29.59, 29.51, 29.44, 29.39, 29.28, 29.27, 28.12, 22.70, 14.12. ESI-MS:
m/z = 357.3 [M+H]+. IR (KBr, cm−1): 3425.1, 2919.4, 2851.2, 1466.2,
1274.5, 726.4. Daicel Chiralpak OJ column, hexane/i-PrOH (85/15,
v/v), 0.5 mL/min, 7.1 min.
2.2.8. N-(2-Aminophenyl)dodecanamide
1H NMR (CDCl3, 400 MHz): ı 7.21 (s, 1H, NHCO), 7.17 (d, J 8.0, 1H,
ArH), 7.05 (t, J = 7.2 Hz, 1H, ArH), 6.80 (d, J = 6.4 Hz, 2H, ArH), 3.91
(s, 2H, Ar–NH2), 2.38 (t, J = 7.2 Hz, 2H, CH2–CO), 1.73 (d, J = 6.4 Hz,
2H, CH2–CH2–CO), 1.26 (m, 16H, (CH2)n), 0.88 (t, J = 6.4 Hz, 3H,
CH3). 13C NMR (CDCl3, 100 MHz): ı 171.96, 140.84, 127.13, 125.26,
124.41, 119.52, 118.21, 37.08, 31.91, 30.93, 29.61, 29.50, 29.37,
29.34, 29.32, 25.88, 22.69, 14.12. ESI-MS: m/z = 289.3 [M−H]−. IR
(KBr, cm−1): 3397.3, 3330.4, 3265.1, 2920.3, 2850.7, 1644.1, 1535.1,
1503.0, 1467.3, 1454.2, 1257.4, 767.3, 717.2
2.2.2. N-(2-Aminophenyl)stearamide
1H NMR (CDCl3, 400 MHz): ı 7.53 (s, 1H, NHCO), 7.04 (d,
J = 7.6 Hz, 1H, ArH), 6.97 (m, 1H, ArH), 6.67 (m, 2H, ArH), 3.76 (s,
2H, NH2), 2.22 (t, J = 7.6 Hz, 2H, CH2–CO), 1.61 (m, 2H, CH2–CH2CO),
1.23 (m, 28H, (CH2)n), 0.81 (t, J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3,
100 MHz): ı 172.18 (–C O), 140.93, 127.12, 125.43, 124.37, 119.41,
118.07, 36.95, 31.94, 29.72, 29.68, 29.54, 29.41 29.38, 29.34, 25.88,
22.70, 14.13. ESI-MS: m/z = 397.3 [M+Na]+, 413.2 [M+K]+. IR (KBr,
cm−1): 3413.4, 3382.4, 3273.6, 2920.0, 2850.7, 1646.13, 1532.5,
1460.2, 1261.0, 740.8.
2.2.9. 2-Nonyl-benzimidazole
1H NMR (CDCl3, 600 MHz): ı 7.45 (m, 2H, ArH), 7.12 (m, 2H, ArH),
2.76 (t, J = 7.8 Hz, 2H, CH2–Ar), 1.70 (t, J = 6.6 Hz, 2H, CH2–CH2Ar),
1.23 (m, 12H, (CH2)n), 0.80 (t, J = 10.8 Hz, 3H, CH3). 13C NMR (CDCl3,
100 MHz): ı 155.19, 137.47, 137.37, 122.51, 114.45, 31.87, 29.69,
29.48, 29.39, 29.25, 28.79, 28.17, 22.63, 14.08. ESI-MS: m/z = 245.1
[M+H]+. IR (KBr, cm−1): 3428.5, 2925.2, 2853.9, 1453.3, 1270.2,
748.0. Welchrom-C18 column (5 ␮m, 4.6 × 150 mm), MeOH/H2O
(73/27, v/v), 0.8 mL/min, 26.0 min.
2.2.3. 2-Pentadecyl-benzimidazole
1H NMR (CDCl3, 400 MHz): ı 7.46 (m, 2H, ArH), 7.22 (m, 2H, ArH),
2.83 (t, J = 7.6 Hz, 2H, CH2–Ar), 1.701 (m, 2H, CH2–CH2Ar), 1.30 (m,
24H, (CH2)n), 0.90 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz):
ı 155.05, 137.42, 122.52, 114.48, 34.87, 31.94, 29.71, 29.67, 29.64,
29.54, 29.49, 29.38, 29.30, 28.79, 28.14, 25.18, 22.70, 14.13. ESI-
MS: m/z = 345.3 [M−H]−. IR (KBr, cm−1): 3429.4, 2919.5, 2849.8,
1464.6, 1417.7, 1271.1, 750.0, 722.4. Daicel Chiralpak OJ column,
hexane/i-PrOH (85/15, v/v), 0.5 mL/min, 7.2 min.
2.2.10. N-(2-Aminophenyl)decanamide
1H NMR (CDCl3, 600 MHz): ı 7.43 (s, 1H, NHCO), 7.19 (m,
1H, ArH), 7.06 (t, J = 7.2 Hz, 1H, ArH), 6.80 (m, 2H, ArH), 4.30
(s, 2H, Ar–NH2), 2.37 (t, J = 7.2 Hz, 2H, CH2–CO), 1.73 (m, 2H,

18
L. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 16–20
Table 1
CH2–CH2–CO), 1.28 (m, 12H, (CH2)n), 0.88 (t, J = 7.2 Hz, 3H, CH3).
13C NMR (CDCl3, 100 MHz): ı 172.11(–C O), 140.70,127.10, 125.35,
124.49, 119.59, 118.19, 37.03, 34.16, 25.86, 22.63, 14.05. ESI-MS:
m/z = 261.1 [M−H]−. IR (KBr, cm−1): 3397.3, 3330.7, 3262.8, 2956.4,
2922.6, 2852.0, 1643.1, 1534.1, 1501.2, 1458.9, 1259.6, 767.3, 717.0.
Synthesis of 2-undecyl-benzimidazole with different enzymesa.
Entry
Catalyst
Yield (%)
1
2
3
4
5
6
7
8
9
No enzyme
MML
CAL-B
MJL
CRL
PCL
–
25.7
6.3
6.0
3.2
–
–
–
–
2.2.11. 2-Heptyl-benzimidazole
1H NMR (CDCl3, 400 MHz): ı 7.53 (t, J 2.8, 2H, ArH), 7.19
(m, 2H, ArH), 2.87 (d, J = 4.0 Hz, 2H, CH2–Ar), 1.75 (t, J = 6.8 Hz,
2H,CH2–CH2Ar), 1.18 (m, 8H, (CH2)n), 0.80 (t, J = 6.4 Hz, 3H, CH3).
13C NMR (CDCl3, 100 MHz): ı 155.34, 122.12, 31.66, 29.40, 29.33,
28.98, 28.32, 22.58, 14.03. ESI-MS: m/z = 217.0 [M+H]+. IR (KBr,
cm−1): 3417.5, 2958.7, 2927.0, 2856.4, 1455.6, 1419.1, 1272.7,
745.6. Welchrom-C18 column (5 ␮m, 4.6 × 150 mm), MeOH/H2O
(90/10, v/v), 0.8 mL/min, 4.0 min.
BSNP
AMA
Denaturedb
a
Reaction conditions: lipase 20 mg, o-phenylenediamine 20 mg, methyl dode-
canoate: diamine = 6:1 (mol), n-hexane 1 mL, 50 ◦C, 24 h.
b
Pretreated with urea at 100 ◦C for 8 h.
3.2. Effect of reaction media
2.2.12. N-(2-Aminophenyl)octanamide
Reaction media is an important factor in the enzymatic reaction
since a given lipase may show distinct activities in different media
[11]. Some conventional organic solvents with different log P val-
ues were screened for the enzymatic synthesis and the results were
shown in Table 2. The alkane solvents such as n-hexane (yield up
to 25.7% in 24 h) and cyclohexane were more effective than the
hydrophilic solvents such as THF, dioxane and acetone. Remark-
ably, this domino reaction gave the best result under solvent-free
condition, providing yield up to 37.8% after 24 h. Compared with
the reactions employing organic solvents, the solvent-free domino
reaction permitted cost saving, high efficiency and less E factor [12].
Therefore solvent-free condition was chosen for all the subsequent
studies.
1H NMR (CDCl3, 400 MHz): ı 7.15 (s, 1H, NHCO), 7.10 (d,
J = 7.6 Hz, 1H, Ar), 6.98 (t, J = 7.6 Hz, 1H, ArH), 6.71 (d, J = 7.2 Hz, 2H,
ArH), 3.86 (s, 2H, Ar–NH2), 2.31 (t, J = 7.6 Hz, 2H, CH2–CO), 1.66 (t,
J = 6.4 Hz, 2H, CH2–CH2–CO), 1.22 (m, 8H, (CH2)n), 0.81 (t, J = 6.4 Hz,
3H, CH3). 13C NMR (CDCl3, 100 MHz): ı 171.97, 140.84, 127.16,
125.26, 124.40, 119.55, 118.23, 37.08, 31.69, 29.27, 29.03, 25.87,
22.61, 14.07. ESI-MS: m/z = 257.0 [M+Na]+. IR (KBr, cm−1): 3397.8,
3330.9, 3267.3, 2956.3, 2924.3, 2853.0, 1642.8, 1534.0, 1499.9,
1452.4, 1255.1, 766.0, 716.0.
2.2.13. 2-Pentyl-benzimidazole
1H NMR (CDCl3, 400 MHz): ı 10.57 (s, 1H, NH), 7.53 (t, J = 3.6 Hz,
2H, ArH), 7.19 (t, J = 3.6 Hz, 2H, ArH), 2.88 (t, J = 7.6 Hz, 2H, CH2–Ar),
1.81 (m, 2H, CH2–CH2Ar), 1.25 (t, J = 2.8 Hz, 4H, (CH2)n), 0.80 (t,
J = 6.8 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz): ı 155.69, 122.05,
31.50, 29.35, 28.11, 22.35, 13.87. ESI-MS: m/z = 188.9 [M+H]+. IR
(KBr, cm−1): 3426.9, 2951.7, 2928.3, 2864.4, 1453.6, 1419.4, 1268.0,
747.2. Welchrom-C18 column (5 ␮m, 4.6 × 150 mm), MeOH/H2O
(70/30, v/v), 0.8 mL/min, 6.9 min.
3.3. Effect of acyl donors
Further experiments were focused on the influence of differ-
ent acyl donors in activity. The alcohol part of esters was firstly
investigated (Table 3). For the same chain length of fatty acid,
methyl decanoate exhibited obviously higher activity than both
ethyl decanoate and butyl decanoate (entries 2, 3 and 4), due to
the steric effect and the feasibility of releasing alcohols from the
system. The results can be explained by the principle that alcohol
with lower boiling point was generally easier to be released from
a reaction system. The methyl decanoate (71.3%) got higher yield
than acid (64.1%) after 60 h, which obviously showing the reaction
was accelerated owing to the formation of MeOH. While vinyl ester
as a widely used acyl donor was also studied, none of the desired
product was observed after 24 h (entry 5). It might be caused by the
acetaldehyde formed in the active site quickly reacted with amine
in the system, leading to a hindrance to the acylation process.
2.2.14. N-(2-Aminophenyl)hexanamide
1H NMR (CDCl3, 400 MHz): ı 7.19 (s, 1H, NHCO), 7.09 (d,
J = 8.0 Hz, 1H, ArH), 6.97 (m, 1H, ArH), 6.720 (m, 2H, ArH), 3.81 (s,
2H, Ar–NH2), 2.32 (t, J = 7.6 Hz, 2H, CH2–CO), 1.67 (t, J = 7.2 Hz, 2H,
CH2–CH2–CO), 1.31 (m, 4H, (CH2)n), 0.85 (t, J = 6.4 Hz, 3H, CH3). 13
C
NMR (CDCl3, 100 MHz): ı 172.03, 140.78, 127.17, 125.29, 124.41,
122.17, 119.60, 118.24, 37.04, 31.46, 25.55, 22.42, 13.95. ESI-MS:
m/z = 229.0 [M+Na]+. IR (KBr, cm−1): 3410.3, 3274.6, 2957.4, 2929.8,
2863.2, 1642.3, 1532.7, 1458.1, 1299.8, 747.9, 716.2.
3. Results and discussion
Table 2
3.1. Selection of biocatalysts
Solvent effect on the yield of 2-undecyl-benzimidazolea.
Entry
Solvent
Log Pb
Yield (%)c
The enzyme source is one of the main factors influencing enzy-
matic reactions [9]. Methyl fatty acid esters with moderate chain
length were chosen for testing at 50 ◦C, and wide range of enzyme
were checked (Table 1). Results indicated that the MML, which is
widely used in esterification and acylation [10], was the best cata-
lyst among those enzymes (entry 2). Poor yields were obtained for
other hydrolases such as lipase acrylic resin from Candida antarc-
tica B (CAL-B), Amano lipase M from Mucor javanius (MJL) and
lipase from Candida rugosa (CRL). The reactions in the absence of
enzyme and incubated with denatured MML (pre-treated with urea
at 100 ◦C for 24 h) led to no product after 24 h. All the results reveal
that the reaction activity depends on the special spatial conforma-
tion of these lipases.
1
2
3
4
5
6
7
8
9
n-Heptane
n-Hexane
Cyclohexane
n-Pentane
Toluene
CH2Cl2
4.00
3.90
3.40
3.39
2.60
0.93
0.49
12.4
25.7
23.2
21.8
2.5
–
–
–
–
37.8
THF
1,4-Dioxane
Acetone
None
−0.50
−0.23
10
–
a
Reaction conditions: MML 20 mg, o-phenylenediamine 20 mg, methyl
dodecanoate:o-phenylenediamine = 6:1 (mol), solvent 1 mL, 50 ◦C, 24 h.
b
According to the literature.
Determined by HPLC.
c

L. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 16–20
19
Table 3
Synthesis of 2-alkybenzimidazoles with different decanoatea.
Entry
Ester
Time (h)b
Yield (%)c
1
2
3
4
5
Capric acid
24
24
24
24
24
46.2
20.7
12.8
9.8
Methyl decanoate
Ethyl decanoate
Butyl decanoate
Vinyl decanoate
C. M.b
6
7
8
9
Capric acid
60
60
60
60
64.1
71.3
34.4
28.7
Methyl decanoate
Ethyl decanoate
Butyl decanoate
a
Reaction conditions: MML 20 mg, o-phenylenediamine 20 mg, ester: o-
phenylenediamine = 6:1 (mol), 50 ◦C.
b
C.M.: complicated mixture, did not detect the aim compound by TLC.
Determined by HPLC.
c
Various fatty acid esters with alkyl chain length from two to
eighteen carbon atoms were incubated with o-phenylenediamine
under the optimized condition. As shown in Fig. 1, the esters with
moderate aliphatic chain (entries 6, 8, 10 and 12) were more fitted,
and the best result was the methyl dodecanoate (entry 10). Esters
with too short lipophilic group (R) were naturally unsuitable to the
active site of enzyme and reflected this on low yields (esters with
less than six carbon atoms were almost unreactive). Compared to
the literature previously reported by Renard, most of the substrates,
especially the short chain substrates (entries 4–8), can get higher
yields efficiently with lower amount of enzyme [7]. Like the results
in Table 3, all the methyl esters were more active than the ethyl
esters.
Fig. 1. Effect of chain length on the yield of 2-alkylbenzimidazoles, MML 20 mg,
ester: o-phenylenediamine = 6:1 (mol), 50 ◦C, 60 h, yields were determined by HPLC.
arated the intermediate (a) from the enzymatic reaction system.
Structure analysis (1H NMR, 13C NMR, MS and IR) revealed that it
was the mono-amide. Controlled experiments were performed to
get more information about the mechanism using intermediate (a)
as substrate: Benzimidazole product was detected in the presence
of fatty acid, whether the lipase was added or not. Interestingly, no
product was detected only in the presence of lipase. In addition, the
sample incubated with both lipase and fatty acid did not show any
difference to the one with only acid added. It was clearly demon-
strated that the acid accelerated the cyclization and the lipase did
not catalyze the cyclization directly. But where did the acid come
from? Since the lipase could efficiently hydrolyze ester into acid
and alcohol [13], further study was focused on the ester systems.
It can be observed that excess amount of ester could not cyclize
the mono-amide without lipase. In contrast benzimidazole prod-
uct was successfully investigated when reaction was carried out
with both ester and lipase. As expected, when the hydrolysis of
3.4. Reaction mechanism of enzymatic synthesis of
2-alkylbenzimidazole
However, how did the cyclization achieve after the acylation
was finished in the first step was still unclear, was it caused by a
catalytic promiscuity of lipase or just reacted spontaneously? To
gain further insight into the mechanism of the reaction, we sep-
Scheme 2. Proposed mechanism of the acylation/cyclization domino synthesis of 2-alkylbenzimidazole.

20
L. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 16–20
Program for Changjiang Scholars and Innovative Research Team
in University, the Key Project of Chinese Ministry of Education
in China and Scientific Fund of Sichuan Province for Outstanding
Young Scientist for financial support. We also thank the Sichuan
University Analytical & Testing Center for NMR analysis.
References
[1] L.F. Tietze, Chem. Rev. 96 (1996) 115–136.
[2] (a) A. Gellis, H. Kovacic, N. Boufatah, P. Vanelle, Eur. J. Med. Chem. 43 (2008)
1858–1864;
(b) P.L. Beaulieu, Y. Bousuet, J. Gauthier, J. Gillard, M. Marquis, G. Mckercher, C.
Pellerin, S. Valois, G. Kukolj, J. Med. Chem. 47 (2004) 6884–6892;
(c) Y.F. Li, G.F. Wang, P.L. He, W.G. Huang, F.H. Zhu, H.Y. Gao, W. Tang, Y. Luo,
C.L. Feng, L.P. Shi, Y.D. Ren, W. Lu, J.P. Zuo, J. Med. Chem. 49 (2006) 4790–
4794.
[3] (a) A. Minatti, S.L. Buchwald, Org. Lett. 10 (2008) 2721–2724;
(b) E. Airiau, T. Spangenberg, N. Girard, A. Schoenfelder, J. Salvadori, M. Taddei,
A. Mann, Chem. Eur. J. 14 (2008) 10938–10948;
(c) Y.X. Chen, L.F. Qian, W. Zhang, B. Han, Angew. Chem. Int. Ed. 47 (2008)
9330–9333;
Fig. 2. Progress curves of synthesis of 2-heptadecal-benzimidazole, 50 ◦C.
(d) G. Altenhoff, F. Glorius, Adv. Synth. Catal. 346 (2004) 1661–1664;
(e) G. Evindar, R.A. Batey, J. Org. Chem. 71 (2006) 1802–1808;
(f) R.D. Viirre, G. Evindar, R.A. Batey, J. Org. Chem. 73 (2008) 3452–3459;
(g) H. Ohno, Y. Ohta, S. Oishi, N. Fujii, Angew. Chem. Int. Ed. 46 (2007)
2295–2298.
ester was inhibited by molecular sieves, no 2-alkybenzimidazole
can be detected by TLC and HPLC. Additionally, a signal of fatty
acid can be found on the NMR spectrum. All of these results proved
that in the domino process the acylation was the first step, and
then the weak acid, which was provided by the hydrolyzation of
corresponding fatty acid esters, efficiently improved the cycliza-
tion of mono-amide to the 2-alkybenzimidazole in the second step
(Scheme 2).
The progress of this reaction was inspected by measuring the
concentration of intermediate (a) and benzimidazole (b) with HPLC.
As shown in Fig. 2, with the increasing concentration of acid in the
enzymatic system, the growth rate of benzimidazole (b) was quite
slow at the beginning and then accelerated. Meanwhile, the con-
centration of intermediate (a) increased to the maximum at about
40 h and then gradually decreased to the very low concentration at
about 60 h, while the benzimidazole (b) reached the max concen-
tration.
[4] (a) G. Altenhoff, F. Glorius, Adv. Synth. Catal. 346 (2004) 1661–1664;
(b) H. Sharghi, M. Aberi, M.M. Doroodmand, Adv. Synth. Catal. 350 (2008)
2380–2390;
(c) Y. Ohta, H. Chiba, S. Oishi, N. Fujii, H. Ohno, Org. Lett. 10 (2008) 3535–3538;
(d) A. Minatti, S.L. Buchwald, Org. Lett. 10 (2008) 2721–2724;
(e) C.S. Bryan, M. Lautens, Org. Lett. 10 (2008) 4633–4636;
(f) R.D. Viirre, G. Evindar, R.A. Batey, J. Org. Chem. 73 (2008) 3452–3459.
[5] G.C. Kelly, R.R. Chen, Biotechnol. Prog. 23 (2007) 52–54.
[6] (a) S.P. Yao, D.S. Lu, Q. Wu, Y. Cai, S.H. Xu, X.F. Lin, Chem. Commun. (2004)
2006–2007;
(b) M.R.G. Klaas, M. Kunz, S. Warwel, J. Mol. Catal. B: Enzym. 7 (1999) 283–289;
(c) Y. Kita, T. Naka, M. Imanishi, S. Akai, Y. Takebe, M. Matsugi, Chem. Commun.
(1998) 1183–1184;
(d) S. Akai, T. Naka, S. Omura, K. Tanimoto, M. Imanishi, Y. Takebe, M. Matsugi,
Y. Kita, Chem. Eur. J. 8 (2002) 4255–4264;
(e) S. Akai, K. Tanimoto, Y. Kita, Angew. Chem. Int. Ed. 43 (2004) 1407–1410.
[7] G. Renard, D.A. Lerner, New J. Chem. 31 (2007) 1417–1420.
[8] R.A. Sheldon, Chem. Ind. (1992) 903–906.
[9] (a) H.Y. Wang, C. Li, N. Wang, K. Li, X.W. Feng, T. He, X.Q. Yu, Bioorg. Med. Chem.
17 (2009) 1905–1910;
(b) J. Quan, Z.C. Chen, C.Y. Han, X.F. Lin, Bioorg. Med. Chem. 15 (2007)
1741–1748.
[10] (a) C. Li, H.Y. Wang, N. Wang, Y.G. Fang, X. Chen, X.Q. Yu, Bioorg. Med. Chem.
Lett. 17 (2007) 6687–6690;
The 2-alkylbenzimidazoles were good donors in Michael addi-
tion. They could be easily added to methyl propiolate without any
catalyst at room temperature. The potential use of the final product
in organic synthesis is still in progress.
(b) E. Santaniello, S. Casati, P. Ciuffreda, L. Gamberoni, Tetrahedron: Asymmetry
15 (2004) 3177–3179;
(c) E. Santaniello, P. Ciuffreda, S. Casati, L. Alessandrini, A. Repetto, J. Mol. Catal.
B: Enzym. 40 (2006) 81–85;
4. Conclusions
(d) P. Ciuffreda, S. Casati, E. Santaniello, Tetrahedron Lett. 44 (2003) 3663–3665.
[11] (a) G. Carrea, S. Riva, Angew. Chem. Int. Ed. 39 (2000) 2226–2254;
In conclusion, a facile biocatalysis to 2-alkybenzimidazole
has been developed. This lipase-mediated acylation/cyclization
domino reaction is carried out in solvent-free condition with less
enzyme loaded. Much higher yields are achieved for most of the
substrates in shorter reaction time, and a series of substrates are
tested. Methyl fatty acid esters with moderated chain length exhibit
the best reactivity. Meanwhile, the mechanism has been clarified
by detail experiments. This efficient domino process provides an
attractive procedure for heterocyclic compound synthesis and may
be a potential synthetic method for green organic chemistry.
(b) G.D. Gonzalo, I. Lavandera, K. Faber, W. Kroutil, Org. Lett.
2163–2166;
9 (2007)
(c) J. Lu, K. Nie, F. Wang, T. Tan, Bioresour. Technol. 99 (2008) 6070–6074;
(d) J.M. Xu, F. Zhang, B.K. Liu, Q. Wu, X.F. Lin, Chem. Commun. (2007)
2078–2080;
(e) J.M. Xu, F. Zhang, Q. Wu, Q.Y. Zhang, X.F. Lin, J. Mol. Catal. B: Enzym. 49
(2007) 50–54;
(f) N. Wang, Q. Wu, W.B. Wu, J. Quan, X.F. Lin, J. Mol. Catal. B: Enzym. 35 (2005)
14–18;
(g) C. Laane, S. Boeren, K. Vos, C. Veeger, Biotechnol. Bioeng. 30 (1987) 81–87.
[12] (a) G.W.V. Cave, C.L. Raston, J.L. Scott, Chem. Commun. (2001) 2159–2169;
(b) J.O. Metzger, Angew. Chem. Int. Ed. 37 (1998) 2975–2978;
(c) S.J. Jeon, H. Li, P.J. Walsh, J. Am. Chem. Soc. 127 (2005) 16416–16425.
[13] (a) U. Zutter, H. Iding, P. Spurr, B. Wirz, J. Org. Chem. 73 (2008) 4895–4902;
(b) E. Jurado, F. Camacho, G. Luzón, M. Fernández-Serrano, M. García-Román,
Biochem. Eng. J. 40 (2008) 473–484.
Acknowledgements
Authors gratefully acknowledge the National Natural Science
Foundation of China (Nos. 20972104, 20725206 and 20732004),