Esterase activity of cyclodextrin dithiocarbamates

Article abstract of DOI:10.1016/j.tetlet.2004.03.156

The catalytic activity of dithiocarbamate appended α- and β-cyclodextrins in the hydrolysis of p-nitrophenyl acetate (PNPA) was studied at alkaline pH. The values of k_cat/k_uncat for the hydrolysis of PNPA ranged from 8 to 165 in the

Full text of DOI:10.1016/j.tetlet.2004.03.156

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 4069–4071
Esterase activity of cyclodextrin dithiocarbamates
*
~
Alex Fragoso, Roberto Cao and Maysa Banos
Laboratory of Bioinorganic Chemistry, Faculty of Chemistry, University of Havana, Havana 10400, Cuba
Received 22 March 2004; accepted 25 March 2004
Abstract—The catalytic activity of dithiocarbamate appended a- and b-cyclodextrins in the hydrolysis of p-nitrophenyl acetate
(PNPA) was studied at alkaline pH. The values of kcat=kuncat for the hydrolysis of PNPA ranged from 8 to 165 in the presence of the
catalyst and are pH independent in the range 7.5–9.0. Addition of Cu(II) increases the activity by factors of 75–515 and kcat values
increase with increasing pH in the same range. The differences in the obtained kinetic parameters are interpreted on the basis of two
different mechanisms.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Cyclodextrins (CDs) are a class of cyclic oligosaccha-
rides composed of a-(1 fi 4)-linked -glucopyranose
complexes with Cu(II).7;8 In the present letter we wish to
report on the esterase activity of several monosubsti-
tuted CD-dithiocarbamates (Scheme 1) and their Cu(II)
complexes. The role of the metallic centre on the
hydrolysis mechanism is also discussed.
D
units featuring an essentially hydrophobic central cavity.
Such a structure allows them to form stable inclusion
complexes with a wide variety of guests.1 This remark-
able property has been successfully applied in the past to
study biomimetic reactions in which CDs behave as
artificial enzymes.2
Table 1 shows the kinetic parameters obtained for the
studied systems. As can be seen, aC2DTC, bC2DTC
and bC6DTC accelerate the hydrolysis of PNPA with
respect to the buffer alone in factors of 165, 88 and 8.2,
respectively. The dithiocarbamate moiety is responsible
for a 100-, 51- and 4.1-fold activity increase with respect
to the corresponding native CD. At the same time, the
role of the CD cavity in assisting the catalysis is evident
considering that it causes up to a 121-fold increase in the
activity. On the other hand, 1=Km also decreases in the
order aC2DTC > bC2DTC > bC6DTC indicating that
PNPA is bounded more tightly to the smaller cavity of
aCD.
Structural and functional models of several enzymes
have been prepared so far by attaching a catalytic
moiety to one of the rims of CD, which acts as a sub-
strate recognition site.3 Among them, hydrolase models
have attracted the attention of many research groups in
the past years and several elegant examples can be found
in the literature.3;4 Research on artificial hydrolases has
been mainly focused on CDs attached to nitrogen con-
taining residues such as amines and imidazole but there
are only few reports on sulfur-containing CDs.5 On the
other hand, substituents capable to coordinate transi-
tion metals have been used to construct metalloenzyme
models.6 The presence of the metal ion usually changes
the classical acyl-transfer mechanism of CDs by coor-
dination to the substituent donor atoms. Therefore,
these systems are also of theoretical interest to study
hydrolysis reactions.
Figure 1 shows the UV–vis spectrum of 6.25 · 10ꢀ5 M
aC2DTC in the presence of 10ꢀ3 M p-nitrophenolate
We have been interested in the past on the biological
activity of dithiocarbamates and their coordination
Keywords: Cyclodextrin; Dithiocarbamate; Esterase activity; Copper.
* Corresponding author. Tel.: +53-78792145; fax: +53-78735863;
e-mail: fragoso@fq.uh.cu
Scheme 1.
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.03.156

4070
A. Fragoso et al. / Tetrahedron Letters 45 (2004) 4069–4071
Table 1. Kinetic parameters for the hydrolysis of PNPA by cyclodextrin dithiocarbamates (in 10 mM Tris buffer pH ¼ 9)
Catalyst
kcat·10ꢀ3 sꢀ1
Km·10ꢀ3 M
k
cat=Km Mꢀ1 Sꢀ1
k
cat=kuncat
kcat=kMorDTC
aC2DTC
bC2DTC
bC6DTC
MorDTCa
49.5
26.3
2.58
0.41
154
1.34
2.11
2.44
––
36.9
12.5
165
88
121
64
1.05
8.2
1.3
515
6.3
––
––
Cu(aC2DTC)2
Cu(bC2DTC)2
Cu(bC6DTC)2
3.21
5.11
5.68
48.0
18.8
376
234
55
96.1
22.5
320
75
3.96
a Morpholyldithiocarbamate.
Figure 1. UV–vis spectra at pH 9 of 6.25 · 10ꢀ5 M aC2DTC (––), in the
presence of 10ꢀ3 M p-nitrophenolate (- - - -) and
mixture of
6.25 · 10ꢀ5 M aC2DTC and 10ꢀ3 M PNPA 12 min after reaction (....).
a
Figure 3. kcat Versus pH profile for the hydrolysis of PNPA by
aC2DTC (j) and Cu(aC2DTC)2 (d) in Tris buffer pH 7.5–9.0 at
25 ꢁC. The solid curve for the Cu(II) system was calculated for
pKES ¼ 8:73 (see text for details).
paired with that obtained 12 min after mixing in the
same concentrations aC2DTC and PNPA. As can be
seen, both spectra are coincident and the bands of
aC2DTC are clearly visible at 253 and 282 nm, demon-
strating that aC2DTC acts catalytically in the hydrolysis
of PNPA. Figure 2 shows the spectral changes and the
time course of PNPA hydrolysis by aC2DTC. Under
hindered by the presence of the CuS4 moiety. These
systems constitute another example of the effectiveness
of metal coordinated CD derivatives as enzyme mim-
ics.4;6
these conditions, the acyl-CD recycle rate was 78 hꢀ1
.
The kcat versus pH profiles for aC2DTC and
Cu(aC2DTC)2 resulted to be very different, as shown in
Figure 3. The activity of aC2DTC is almost pH inde-
pendent suggesting that the hydrolysis takes place
through a nucleophilic attack of one of the sulfur atoms
of the dithiocarbamate moiety to the carboxyl atom of
PNPA without participation of the buffer. By contrast,
kcat increases with pH under the same conditions for
Cu(aC2DTC)2, suggesting a basic catalysis mechanism.
Since coordination of Cu(II) to the dithiocarbamate
moiety suppresses the possibility of a nucleophilic
catalysis carried out by the sulfur atoms of the dithio-
carbamate group, the catalytic function should be car-
ried out by an activated water molecule axially bounded
to the copper atom. In this case, the pH dependence of
When a Cu(II) salt is added to a solution of aC2DTC,
bC2DTC or bC6DTC, a complex of general formula
CuL2 is formed.8 These complexes show even higher
activity in the cleavage of PNPA than the corresponding
ligands (Table 1) and also obey LWB behaviour. The
highest acceleration is shown by Cu(aC2DTC)2 in a
factor of 515 with respect to the background buffer (3.1
with respect to aC2DTC). The values of 1=Km for the
complexes are higher that those of the corresponding
ligands, suggesting that the substrate interaction is now
kcat can be representꢀe1d by the equation:9 kcat
¼
max
ðKESkcat ÞðKES þ ½HþꢁÞ where KES is the ionization
constant of the bounded water. For Cu(aC2DTC)2,
max
pKES ¼ 8:73 and kcat
¼ 180 ꢂ 10ꢀ3 sꢀ1
. Figure 4
shows a schematic representation of the proposed
hydrolysis mechanisms.
In conclusion, we have demonstrated that dithiocarba-
mates attached to cyclodextrins and their Cu(II) com-
plexes possess hydrolase activity towards activated
esters.
Figure 2. (A) Spectral variations for the hydrolysis at pH 9 of 10ꢀ3 M
PNPA by 6.25 · 10ꢀ5 M aC2DTC recorded in 3 min intervals. (B) Time
course of the reaction.

A. Fragoso et al. / Tetrahedron Letters 45 (2004) 4069–4071
4071
Acknowledgements
Financial support from the Third World Academy of
Sciences (Grant 02-334 RG/CHE/LA to A.F.) is grate-
fully acknowledged.
References and notes
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Figure 4. Proposed mechanisms for the hydrolysis of PNPA by
aC2DTC (A) and Cu(aC2DTC)2 (B).
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110, 8553.
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Experimental: The CD derivatives aC2DTC, bC2DTC,
C6DTC and their Cu(II) complexes were prepared as
previously reported.8 PNPA (Aldrich) and Tris (Merck)
were used as received.
7. (a) Cao, R.; Fragoso, A.; Villalonga, R. Monatsh. Chem.
1996, 127, 775; (b) Cao, R.; Travieso, N.; Fragoso, A.;
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The kinetic studies were performed on a Ultrospec 2100
spectrophotometer (Amersham Biosciences) by mea-
suring the release of p-nitrophenolate at 400 nm in Tris
buffer pH 9.0 (10 mM) at 25 ꢁC. The catalyst concen-
tration was fixed at 6.25 · 10ꢀ5 M while PNPA concen-
tration was varied from 10ꢀ3 to 10ꢀ2 M. Lineweaver–
Burk approach was applied to evaluate the values of the
catalytic (kcat) and dissociation (Km) constants. The
influence of pH was studied in the same conditions but
using 10 mM Tris buffer of the appropriate pH value.
9. Ikeda, H.; Kojin, R.; Yoon, C.; Ikeda, T.; Toda, F.
J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 117.