The study of biocatalyzed thio-Michael reaction: A greener and multi-gram protocol

Article abstract of DOI:10.1016/j.tetlet.2013.11.047

This Letter introduces a new, cheap and green protocol for the thio-Michael reaction. Here we applied three free enzymes such as lipase from pancreas porcine, chymosin and papain and an immobilized one: the Liposyme. The reactions were executed at room te

Full text of DOI:10.1016/j.tetlet.2013.11.047

Tetrahedron Letters 55 (2014) 430–434
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Tetrahedron Letters
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The study of biocatalyzed thio-Michael reaction: a greener
and multi-gram protocol
a
a
b
b
b
Paula V. S. Rizzo , Ligia A. Boarin , Ingridhy O. M. Freitas , Roberto S. Gomes , Adilson Beatriz ,
c
a,
⇑
Andrelson W. Rinaldi , Nelson Luís C. Domingues
a
Hybrid Materials Laboratory-LMH, FACET, Federal University of Grande Dourados—UFGD, Dourados/Itahúm rod. km 12 s/n°, Zip Code 79804-970, Dourados, MS, Brazil
Organic Synthesis Laboratory LP4, Federal University of Mato Grosso do Sul—UFMS, Senador Filinto Muller Av. 1555, Campo Grande, MS, Brazil
Department of Chemistry, State University of Maringá-UEM, Av. Colombo 5790, Zona 7, Maringá, PR, Brazil
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter introduces a new, cheap and green protocol for the thio-Michael reaction. Here we applied
three free enzymes such as lipase from pancreas porcine, chymosin and papain and an immobilized
one: the Liposyme . The reactions were executed at room temperature and resulted in the thio-Michael
adduct in good or excellent yields. The protocol describes the use of EtOH as solvent and a less percentage
of enzymes, which is in concordance with the green chemistry topics, so we can mention that chymosin
and papain were used as biocatalyst in an organic reaction for the first time in this Letter.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 5 August 2013
Revised 10 November 2013
Accepted 13 November 2013
Available online 20 November 2013
Ò
Keywords:
Thio-Michael
Biocatalysis
Free enzymes
Immobilized enzyme
Multi-gram scale
Introduction
catalysts, stringent dry conditions, moderate yields, use of stoi-
chiometric amount of catalysts, and expensive metal precursors
6
–8
Thio-Michael is one of the most important reactions when the
aim is to generate a CAS bond. This reaction is responsible for
many compounds described in the literature with biological
applications such as antibiotic, antimicrobial, analgesic, anti-
that are harmful to the environment.
On the other hand, one
methodology that emerges in contrast with all the disadvantages
previously described is the biocatalysis, which is also included in
green chemistry protocols and has attracted a lot attention now-
1
2
10
HIV, or in non-natural amino acids synthesis. The most cited
compound in the literature is Diltiazem, a calcium channel
blocker.3 Many inorganic catalysts (such as Lewis acid) were al-
adays. The biocatalysis applies as biocatalyst free enzymes,
1
0
whole cell of microorganisms or vegetables, and besides being
an eco-friendly protocol, it confers some advantages for example
the possibility of carrying out the reaction in mild conditions
(solvents, temperature and low amount of biocatalyst), which
sometimes is not possible when we use chemical methodologies.
Furthermore, another fact that directs the researchers to biocatal-
ysis is the property described by He for some free or immobilized
enzymes, named ‘catalytic promiscuity’ or ‘induced adjust-
ready reported to execute this reaction, for example, InCl
3
and
O. But there are also examples of organic
catalyst applied in thio-Michael reactions like -proline or other
4
5
InBr
3
or Cu(BF
4
)
2
ÂH
2
L
6
amino acids. We can also find other examples involving cin-
chona alkaloids and thioureas derivatives8 as Michael organo-
catalysts. Inasmuch as catalysis is very important nowadays,
especially the heterogenic one, there are other important topics
that make the organic reaction protocols more ecofriendly and
desirable as the elimination or reduction of hazardous solvents
and/or chemical catalysts.9 In this regard, the classical catalyzed
thio-Michael protocols presented some disadvantages such as
long reaction time, high reaction temperatures, use of expensive
7
1
1
ment’. This property allows the researchers to use some en-
1
2
zymes in several extensive reactions for example Mannich,
1
3
14
aldolic, decarboxylative aldol and Knoevenagel, and Morita-
Baylis–Hillman inter alia. Besides that, it is worth mentioning
15
16
that one biocatalyzed thio-Michael reaction has already been re-
1
7
ported in the literature but without producing a multi-gram
scale. Regarding to all the previously exposed papers and to the
absence of a multi-gram study of biocatalyzed thio-Michael reac-
tion, we described the use for the first time of some free and
immobilized enzymes as biocatalysts.
⇑
Corresponding author. Tel.: +55 (67) 3410 2081; fax: +55 (67) 3410 2072.
E-mail addresses: nelsondomingues@ufgd.edu.br, nelsonluis.domingues@hot-
mail.com (N.L.C. Domingues).
0
040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.11.047

P. V. S. Rizzo et al. / Tetrahedron Letters 55 (2014) 430–434
431
Results and discussion
The aim of our project was to perform a biocatalyzed thio-
Michael reaction. As starting point, we studied the reaction involv-
ing the cinnamaldehyde (1) with thiophenol (2) to describe the
best conditions only for the thio-addition on the b-carbon without
the carbonyl attack (Scheme 1). The thio-Michael adduct (3) was
reduced to the corresponding alcohol (4). So we firstly used the
lipase from pancreas porcine type II (LPP) because it is a cheap
enzyme and presented biocatalytic application in other organic
reactions.
In this context, we examined the catalytic efficiency of LPP in
some solvent systems (Fig. 1) using tetrahydrofuran (THF), ethanol
Figure 1. Yields obtained from thio-Michael reaction using LPP as biocatalyst.
(
EtOH), water (H
2
O), and a mixture of 50% v/v of dimethylsulfoxide
13
and water (DMSO/H
2
O).
For this procedure, the reaction was carried out using 0.02 g of
À4
LPP or 4 Â 10 mmol or 0.05 mol % using the molecular weight of
LPP around 50,000 Da. Interestingly, we observed a great activity
of this enzyme even in this percentage. We also observed in Fig-
ure 1 that the dielectric constants of the solvents had a remarkable
effect on the biocatalyzed thio-Michael reaction. We found out the
2
highest yields were obtained using EtOH and DMSO/H O (Fig. 1),
so, we concluded that LPP presented most affinity with the highest
polar solvents. In all reactions the corresponding (L)-thio-Michael
adducts (ee 97% obtained by 1H RMN via shift reagent; see Sup-
porting information) were isolated in considerable yields without
any ketone protected as by-product. In order to prove the biocat-
alyst activity of LPP we executed the same reaction using the same
conditions as described above but without the biocatalyst and it
did not produce the thio-Michael adduct. So, we decided to de-
scribe a standard protocol using EtOH since it is an eco-friendly
solvent.
Figure 2. Effect of the amount of enzyme (g) in yields in thio-Michael reaction
involving LPP.
In the best of our knowledge we proposed the reaction mecha-
nism involving the triad composed of serine, histidine and aspar-
tate (or glutamate) in the enzyme. As described by Hult and
Bergulund, the mechanism involving the enzyme (lipase) is
consistent with an activated interaction of serine portion with
the carbonyl group and the interaction of the active-site histidine,
as base, which actives the eletrophile (Fig. 3).
cinnamaldehyde/g of enzyme or ca. 97 mg of enzyme/mmol of
cinnamaldehyde). After that, we stabilized all the conditions (EtOH
as solvent system, 2 h and 48 mg of enzyme/mmol of cinnamalde-
hyde) as the standard procedure.
1
8
Ò
To expand this protocol, we decided to use Liposyme , an
Additionally, we analyzed the effect of the amount of enzyme
on thio-Michael (Fig. 2) and we concluded that the yields increased
when we increased the amount of enzyme within the range of
immobilized lipase, as biocatalyst in the same thio-Michael reac-
tion. The reaction was executed the same way described for LPP
involving cinnamaldehyde and thiophenol using the best ratio ob-
tained for LPP and cinnamaldehyde (10 mmol of cinnamaldehyde/g
of enzyme). We observed that the yields of the reaction using Lipo-
symeÒ performed in 2 h were not good enough. For this reason, we
increased the reaction time to 4 h using only the previous promis-
ing results to obtain a better result, so we had them as being 0.08,
0.16, and 0.24 g (Fig. 4).
0
.04–0.08 g (0.09–0.19 mol %).
The increase provided by the addition of 0.02 g, goes from the
first experiment to the second one (Fig. 2), improving in 17 units
the percentage of the previous yields. The best result was obtained
when we used 0.08 g (0.02 mol %) of LPP with an increase of yields
in 21 units of percentage in comparison with the second experi-
ment and 38 units in comparison with the first one. However, sur-
prisingly, when we kept on increasing the amount of enzyme
loading, the yield decreased. So, we concluded that the best molar
ratio is ca. 515:511:1 of cinnamaldehyde, thiophenol, and LPP in
For LiposymeÒ the best molar ratio was 5.15 mmol of
cinnamaldehyde/g of enzyme (or 193.93 mg of enzyme/mmol of
cinnamaldehyde). In this case, as observed for LPP, we obtained
the (L)-thio-Michael adduct (see Supporting information). To
extend the study, we also verified the catalytic activity of another
thio-Michael
reaction,
respectively
(ca.
10 mmol
of
O
O
OH
SH
LPP
Solvents/ r.t.
NaBH
U.S; EtOH
4
+
Ph
(R)
(R)
Ph
Ph
SPh
Ph
SPh
Levo adduct
(1)
(2)
(3)
(4)
Scheme 1. Thio-Michael reaction using the LPP as biocatalyst.

4
32
P. V. S. Rizzo et al. / Tetrahedron Letters 55 (2014) 430–434
N
N
histidine portion
in enzyme
H
O
H
N
H
O
H
SPh
δ +
Ph
O
O
Ph
N
H
H
O
O
Hidrolysis
N
H
Ph
PhS
H
serine portion in enzyme
O
thio-Michael adduct
PhS
Ph
Figure 3. Mechanism proposed by biocatalysed thio-Michael.
Figure 5. Yields obtained from thio-Michael reaction using chymosin as
biocatalyst.
Figure 4. Effect of the amount of enzyme (g) in yields in thio-Michael reaction
involving Liposyme .
Ò
Study of the LPP reuse
enzyme: chymosin (ca. 50,000 Da), an aspartyl protease used
mainly in cheese manufacturing. So, we decided to introduce this
biocatalyst in thio-Michael reaction which was carried out with
We executed five cycles of thio-Michael reaction aiming to
verify the reuse of LPP (Fig. 6). We can observe that five recycling
steps were carried out, and, noteworthy, a high amount of enzyme
could not be recovered in each process. We observed that the per-
centage lost on the first three cycles was very high. This fact is due
the solubility of the enzyme in EtOH confirmed by the yields
0
.1 g of biocatalyst (Fig. 5). Unfortunately, the data presented by
chymosin were not as we expected. The highest yield from this
biocatalyst was obtained in THF media (23%). Noteworthy, it was
the first application of this enzyme in an organic reaction.

P. V. S. Rizzo et al. / Tetrahedron Letters 55 (2014) 430–434
433
Biocatalyzed thio-Michael using cyclic enones
To further expand the scope of the biocatalyzed thio-Michael
multi-gram methodology, we next applied our optimized proce-
dure to cyclic enones. The results exposed in Figure 8 showed that
LPP presented the highest yield. Noteworthy all free enzymes pre-
sented higher yields in comparison with the immobilized ones.
This fact proved that support, a macroporous ion-exchange resin
Ò
present in Liposyme , also decreased the yield in the case of cyclic
a
,bÀunsaturated enones. Another important data were the surpris-
ing activity presented mainly by chymosin (0.08 mol %) and the
papain (I.U.B.: 3.4.22.2), a cysteine hydrolase, stable and active un-
der a wide range of conditions with molecular weight of 23,406 Da
Figure 6. Yields (%) and loss of enzyme (%) by cycle of thio-Michael reaction.
(
0.18 mol %). This test was the first application of papain in thio-
Michael reaction. The data are promising for the application of
papain and chymosin as biocatalysts on other organic reactions.
Conclusion
We have documented a new, simple and efficient catalytic
enzymatic protocol for the thio-Michael addition involving LPP,
LiposymeÒ and, in some cases, chymosin and papain. All the sug-
gested protocol had moderate to excellent yields, are greener and
low cost effective. Short reaction time, room temperature and high
yields make this protocol a useful alternative to the existing meth-
ods for the synthesis of thio-Michael adducts. By the way, this
protocol is the first application of some free and immobilized en-
zymes in thio-Michael reaction and in the case of chymosin and
papain, it was the first use already described in the literature.
Figure 7. Yields and their variations obtained from the reaction in multi-gram scale
involving LPP as catalyst in thio-Michael reaction.
Acknowledgments
Brazilian authors thank to Fundação de Apoio ao Desenvolvi-
mento de Ensino, Ciência e Tecnologia do Estado do Mato Grosso
do Sul (Fundect) and to Conselho Nacional de Desenvolvimento
Científico e Tecnológico for the financial support offered for this re-
search (N.L.C.D.–FUNDECT N° 14/2009, FUNDECT/CNPq N° 08/2009
and A.W.R. CNPq–310820/2011-1 and 577527/2008-8) and the
authors P.V.S.R. I.O.M.F. and R.S.G. thank to Coordenação de Aper-
feiçoamento de Pessoal de Nível Superior (CAPES). The authors also
thank to CR Hansen Brazil for the chymosin donation.
Supplementary data
Supplementary data (general experimental procedures and
spectral data) associated with this article can be found, in the on-
line version, at http://dx.doi.org/10.1016/j.tetlet.2013.11.047.
Figure 8. Yields obtained from biocatalyzed thio-Michael reaction involving
cyclohex-2-enone and thiophenol.
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