Lipase catalyzed 1,2-addition of thiols to imines under mild conditions

Article abstract of DOI:10.1039/c7nj03387g

This is the first report of enzymes used as biocatalysts for a 1,2-sulfur addition. In this study, we describe the synthesis of N,S-acetals using an environmentally friendly process with low catalyst loading and in short reaction times using porcine pancreatic lipase, chymosin, and bovine serum albumin (BSA). The hydrogen bond between the enzyme and the N-Boc imine is a key factor in this reaction.

Full text of DOI:10.1039/c7nj03387g

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ARTICLE
Lipase catalyzed 1,2-addition of thiols to imines under mild
conditions
Tábata B. Albuquerque,a Caren D. G. da Silva, Aline R. de Oliveira,a Beatriz F. dos Santos,a Beatriz
A. L. da Silva,a Ramesh Katla,a Mariana P. D. Rochaa and Nelson L. C. Dominguesa
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
This is the first report of enzymes used as a biocatalyst for a 1,2-sulfur addition. In this study, we describe the N,S-acetals
synthesis using an environmentally friendly process with low catalyst loading and in short reaction times using lipase
pancreas porcine, chymosin, and bovine serium albumin (BSA). The hydrogen bond between the enzyme and the N-Boc
imine is a key factor in this reaction.
www.rsc.org/
several infectious processes and its complications with high
efficacy and low cost.9 Another known antibiotic is
Fuzaperazine A, which is naturally produced and broadly used
Introduction
Biocatalysis research has exponentially grown over the last
past decades owing to discoveries related to the synthesis of
many compounds, mainly with biological properties, which are
beneficial for the society.1,2 In addition, environmental
concerns have driven many research groups around the world,
and even industries, to describe new synthetic protocols that
are not harmful for the environment and to consider the
subproducts formed by side reactions. This results in the use of
catalysts that allow faster reactions using low cost reactants
and avoid the wastage of solvents, which have a great impact
in the environmental factor (E factor).3 Thus, many protocols
that use enzymes (e.g., lipases) have been reported. Enzymes
possess great catalytic characteristics such as high efficiency,
biodegradation (unlike heavy metals); in some cases, the
chemical transformations occur in aqueous solutions, avoiding
the use of polluting solvents4. Moreover, such enzymes have
great specificity and can be used both in free form or
immobilized; they are usually cheap and stable catalysts and
can be inserted as heterogeneous catalysts in organic reaction
media4. Many examples of enzymatic catalyzed reactions are
described in the literature such as Mannich5, aldolic6,
decarboxylative aldol and Knoevenagel7, Morita-Baylis–
Hillman8, and sulfa-Michael4b reaction.
against viruses, bacteria, and carcinogenic cells.9 Owing to
their important applications, over the years, a great number of
synthetic strategies aimed to produce new N,S-acetals
molecules have been developed.9 Thiols were added to imines
using thiourea quaternary ammonium salts as catalysts to
obtain N,S-acetals derivatives.9a N,S-acetals were synthesized
using isatin and thiols, as well as isoindolinones and thiol
derivates with BINOL-phosphoric acid derivative as
catalyst9b,9c. The synthesis of N,S-acetals in high yields and
excellent enantiomeric excess using chiral phosphoric acids
were also reported9d. We published a synthetic protocol to
obtain some N,S-acetals using Zn(Pro)2 as a heterogeneous
catalyst10. However, to the best of our knowledge, no
procedures are described for the synthesis of N,S-acetals using
a biological catalyst such as enzymes. Since we have developed
synthetic methods that use enzymes as catalyst, for example in
the sulfa-Michael reaction4b, we propose employing enzymes
to synthesize some N,S-acetals under mild and green reaction
conditions herein (Figure 1).
NHBoc
NHBoc
SH
PPL
S
SO2Ph
+
The N,S-acetals, which are commonly found in a great range of
biological products with medical and biochemical applications,
have attracted great attention.9 An excellent example is
DME, K2CO3, rt
1a
2a
3a
penicillin, which belongs to the β-lactamics antibiotic family
characterized by the therapeutic treatment. Penicillin prevents
Figure 1. Reaction scheme for the N,S-acetal synthesis
biocatalyzed by Porcine pancreas lipase (PPL).
a.Organic Catalysis and Biocatalysis Laboratory (LACOB), Federal University of
Grande Dourados, Itahúm rod. km 12 s/n°, Dourados, MS, Brazil. E-mail:
nelsondomingues@ufgd.edu.br or nelsonluis.domingues@hotmail.com
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Experimental
The reaction in ethyl acetate did not afford the desired
compound. We concluded that this is likely because of the
insolubility of compound 1a in that solvent. Therefore, we also
attempted to perform the N,S-acetal synthesis in DCM;
however, the yield obtained was moderate (50%). To our
surprise, when the solvents were DME and DMSO, we
achieved a 100% yield. To further evaluate the biocatalyst
effectiveness, a blank reaction (without biocatalyst) was
performed in those two solvents. The reaction in DMSO
occurred without PPL, affording 50% of the desired compound
after 2 h. However, reaction in DME did not afford N,S-acetal
even after 24 h. Therefore, DME was chosen as the solvent for
the biocatalyzed reactions. The catalyst loading effect on the
yield obtained was studied later (Table 1).
General methods:
All the chemicals, reagents, and solvents were used as
received unless otherwise stated. The respective reactions
were monitored via thin layer chromatography (TLC) using
MACHEREY-NAGEL silica plates (SIL G/UV254). The compounds
were purified via column chromatography on silica gel. 1H and
13C NMR spectra were recorded in CDCl3 on a Bruker
spectrometer (300 and 75 MHz, respectively). The infrared
spectra were recorded on
spectrometer.
a FT/IR 4100 type A Jasco
General procedure for the N-Boc imines synthesis:
The mixture of aldehyde (5.0 mmol), sodium benzenesulfinate
(5.0 mmol), tert-butyl carbamate (5.0 mmol), and HCO2H (217
µL) was placed in a 50-mL round-bottom flasks. CH3OH (5 mL)
and water (10 mL) were added, and the mixture was
magnetically stirred at room temperature for 24 h. When the
reaction was complete, the solid obtained was filtered,
washed with ethyl ether, and dried overnight.
Table 1. PPL catalyst loading and yields for N,S-acetal
synthesis*.
NHBoc
NHBoc
SO2Ph
SH
X mg PPL
S
+
DME, K2CO3, RT
General procedure for N,S-acetal synthesis:
1a
3a
2a
The mixture of thiol (0.2 mmol), amidosulfones (0.1 mmol),
K2CO3 (0.5 mmol), and enzyme (0.01 g) in DME (4 mL) was
placed in a tube at room temperature. The reaction was stirred
using a magnetic bar and monitored using TLC (hexane/EtOAc,
90:10). After the reaction, the catalyst was separated via
Entry Catalyst loading Yield (%)
(mg)
1
2
3
4
5
-
-
filtration, and the mixture was extracted with DCM (3
× 10
2.5
5.0
7.5
10.0
23
52
73
>99
mL). The organic phases were combined and dried over
anhydrous Na2SO4; the salt was filtered before concentrating
the organic phase under reduced pressure. The product was
purified via column chromatography on silica gel
(hexane/EtOAc, 90:10).
* Reaction conditions: 1a (0.1 mmol), 2a (0.2 mmol), K2CO3
(0.5 mmol), solvent (4 mL), 2 h.
Results and discussion
We initially evaluated the effectiveness of the biocatalyst in
different organic solvents such as ethyl acetate, THF, DCM,
DME, and DMSO (Figure 2).
The yield increased with the catalyst loading. As seen in Table
1, increasing the catalyst proportion from 0.0 to 10.0 mg
resulted in a yield raise from 0.0 to >99%. These results
indicate that a high yield can be obtained even at low catalyst
loading (10.0 mg), which clearly demonstrates the
effectiveness of the catalyst. Based on these deductions, the
following standard protocol was established: N-Boc
sulfonylamine (0.1 mmol), thiol derivatives (0.2 mmol), K2CO3
(0.5 mmol), and DME (4 mL) for 2 h. This procedure was used
for several other reactions involving different N-Boc
sulfonylamines and thiols (Table 2).
NHBoc
NHBoc
SO2Ph
SH
10 mg PPL
S
+
solvent, K2CO3, RT
2a
1a
3a
>99
>99
100
85
70
)
50
%
(
55
40
25
10
-5
s
33
d
l
e
i
Y
0
Ethyl
Acetate
THF
DCM
Solvents
DM E
DM SO
Figure 2. Yields for the tert-butyl (phenyl (phenylthio) methyl)
carbamate (3a) catalyzed by PPL [reaction conditions: 1a (0.1
mmol), 2a (0.2 mmol), K2CO3 (0.5 mmol), solvent (4 mL), 2 h].
2 | J. Name., 2012, 00, 1-3
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Table 2. Yields for the N,S-acetal synthesis biocatalyzed using Owing to this bond, the nucleophilicity of sulfur species
PPL
decreased, and the yields for these reactants were low.
For the PPL, the mechanism shown in Figure 3, which is to the
one described by Rizzo,11 is proposed. Therein, the serine
portion of the enzyme (hydrogen bond plus imine), which is
obtained after the elimination of the sulfinic group in basic
media, can activate the interaction for the 1,2-sulfur addition.
As seen for the N-Boc sulphonamides derivatives, the
hydrogen bond between the imine nitrogen and the enzymatic
hydrogen (intermediate #3) made this intermediate more
reactive toward the 1,2-sulfur attack. Hence, the derived
conclusion is consistent with the data presented in entries 7–9
(Table 2). Moreover, when the imine had an electron
withdrawing group, such as nitro, it enabled the attack of the
sulfur species easier because of the mesomeric effect. This
effect increased the electrophilicity of the imine carbon.
Contrastingly, an electron donor group such as OCH3 bonded
at the phenyl group (entry 8, Table 2) or a simple alkyl group
such as butyl (entry 9, Table 2) decreased the electrophilicity
and resulted in a less effective hydrogen bond in the
intermediate #3. It is important to note that certain reports in
literature agree with this conclusion5,7
.
The feasibility to reuse the PPL in the N,S-acetal synthesis was
also studied herein. However, as informed by Rizzo11, we could
not reuse the enzyme because of the presence of sulfinic base
was inside the crude reaction. In addition, water could not be
used to remove only the sulfinic base because the enzyme was
also soluble in water.
O
O
HN
O
N
O
SO2Ph
K2CO3
2
1
n
o
i
t
r
o
p
O
e
m
y
N
H
O
3
z
n
H
N
H
E
6
O
N
O
H
+H+
n
o
i
t
r
o
p
a.
O
S
e
Yield calculated for purified compound after column
m
O
y
z
n
chromatography.
HN
O
SH
N
H
O
E
H
N
O
The reactions involving thiols containing either H or electron
withdrawing groups at the para-position (entries 1–3) afforded
the highest yields compared to the ones with electron donor
groups (entries 4–5). This fact was not expected; however, this
is believed to be caused by the hydrogen bond between the
nucleophiles and the acid hydrogen in the enzymes. We
concluded that more basic sulfur species resulted in stronger
hydrogen bond; thus, the addition of benzenethiol (entry 6), p-
OCH3 (entry 5) or p-CH3 (entry 4) enables these nucleophiles to
create strong hydrogen bond with enzyme’s hydrogen acids.
S
5
N,S Acetal derivative
4
Figure 3. Proposed mechanims for the N,S-acetal synthesis
biocatalyzed by PPL
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To obtain a more comprehensive analysis of the N,S-acetal not been reported for this important reaction. Certain
biosynthesis, we extended the study using two more enzymes important aspects make this protocol a useful alternative to
such as chymosin and bovine serium albumin (BSA) (Table 3).
the existing methods for the synthesis of N,S-acetals: the
reaction was achieved at short reaction times and room
Table 3. Yields for the N,S-acetal synthesis biocatalyzed by temperature, and high yields were obtained.
chymosin (CHY) and albumin (ALB).
Amidosulfone
Yielda (%)
Acknowledgements
Product
Entry
Thiol
SH
NHBoc
S
NHBoc
SO2Ph
The authors (N. L. C. D and R.K.) thank to Conselho Nacional de
Desenvolvimento Cientifico e Tecnológico (CNPq/Brazil) for
financial support (Process: 314140/2014-0 and 400706/2014-8
CNPq - Brazil). The author (T. B. A and N. L. C. D) thank to
Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e
Tecnologia do Estado de Mato Grosso do Sul (FUNDECT/Brazil -
Process: 59/300.439/2015–PAPOS REDE PRÓ-CENTRO-OESTE
FASE III and Process: 59/300.138/2016- BIOTA/MS) and
Coordenação de Aperfeiçoamento de Pessoal de Nível
53 (CHY)
76 (ALB)
1
SH
NHBoc
SO2Ph
NHBoc
S
2
93 (CHY)
100 (ALB)
F
F
NHBoc
SO2Ph
NHBoc
SH
Superior
(Auxílio
Nº
2209/2015,
Processo
Nº
3
S
56 (CHY)
60 (ALB)
23038.008241/2014-89, Programa CAPES-FAPCO) for financial
support.
Cl
Cl
NHBoc
S
SH
NHBoc
SO2Ph
4
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86 (CHY)
96 (ALB)
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PPL, Chymosin and BSA as efficient biocatalysts (low catalyst loading, high yields) in an eco-
friendly synthesis of N,S-acetal.

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Graphical Abstract