Hypervalent organochalcogenanes as inhibitors of protein tyrosine phosphatases

Article abstract of DOI:10.1039/c0ob01050b

A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0ob01050b

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Hypervalent organochalcogenanes as inhibitors of protein tyrosine
phosphatases†
Leandro Piovan,a Li Wu,b Zhong-Yin Zhang*b and Leandro H. Andrade*a
Received 18th November 2010, Accepted 22nd December 2010
DOI: 10.1039/c0ob01050b
A series of organochalcogenanes was synthesized and evalu-
ated as protein tyrosine phosphatases (PTPs) inhibitors. The
results indicate that organochalcogenanes inactivate the PTPs
in a time- and concentration-dependent fashion, most likely
through covalent modification of the active site sulfur-moiety
by the chalcogen atom. Consequently, organochalcogenanes
represent a new class of mechanism-based probes to modulate
the PTP-mediated cellular processes.
In this way, based on the reactivity of selenium- and tellurium-
containing compounds and their molecular interaction with
different enzymes, the investigation of hypervalent chalcogenanes
as inhibitors of other thiol-dependent enzymes is warranted.
Protein tyrosine phosphatases (PTPs) constitute a large family of
cysteine-dependent enzymes that catalyze the hydrolysis of phos-
photyrosine residues in proteins.13 PTPs, together with protein
tyrosine kinases, play a central role in cell signalling by regulating
the phosphorylation status and, in turn, the functional properties,
of target proteins in various signal transduction pathways.14
Dysfunction in PTP activity has been linked to the etiology of
several human diseases, including cancer, diabetes and obesity, and
autoimmune disorders.15 Consequently, there is intense interest in
developing small molecule PTP inhibitors that not only serve as
powerful tools to delineate the physiological roles of these enzymes
in vivo, but also as excellent leads for the development of new
therapeutic agents.
Herein, we described a study of the potential use of hypervalent
selenium(IV) and tellurium(IV) (organoselenanes and organotel-
luranes, respectively) as inhibitors of the PTPs (Fig. 1). Besides
the presence of chalcogen atoms (Se or Te), these compounds
were designed to contain halogen (Cl or Br) atoms and a chiral
center. The presence of a methyl group at the benzylic carbon
generates an asymmetric center, which offers the possibility of
differential recognition of the enantiomers by the enzyme (PTP)
and also a good comparison with the achiral congener. The
halogens bounded to chalcogen were chosen to evaluate the
possible influence of the leaving group stability to the activity of
these compounds. In this way, with simple structural modifications
a SAR could be established.
Introduction
The prospection of tellurium and selenium compounds exhibiting
biological activity has been increased in the last decades, especially
after a series of studies that have demonstrated the biological
potential of these exotic compounds.1 Antioxidant activity,2
anti-inflammatory properties,3,4 neuroprotective and convulsant
effects,5 cancer prevention,6 apoptotic events,7 and immunomodu-
lator activities8 are some of the biological properties that have been
documented for selenium and tellurium-containing compounds.
The development of small selenium- and tellurium-containing
molecules as enzymatic inhibitors is based on the reactivity and
high affinity of selenium and tellurium atoms towards thiol-
dependent enzymes such as caspases,9 tyrosine kinase10 and
cysteine (papain, cathepsins) proteases.11 A particular class of
selenium and tellurium compounds that has been less explored
in enzymatic inhibition is the hypervalent organochalcogenanes.
Recent investigations have shown that organoselenanes and organ-
otelluranes are very potent inhibitors of cysteine cathepsins, a
thiol-dependent enzyme.12 The affinity between the sulfur-moiety
from the catalytic site of these enzymes and chalcogen atom
(especially tellurium) makes favorable the formation of a sY–S-Enz
(Y = Se and Te, S-Enz = thiol-dependent enzyme) bound in the in-
hibitory process. Due to their distinct molecular arrangement and
charge distribution, the chalcogen, present in these hypervalent
compounds, accommodates a positive charge and consequently,
become more electrophilic than their chalcogenides congeners.
Results and discussion
Synthesis of organoselenanes and organotelluranes (1–12)
The synthesis of organoselenanes involved a short and versatile
synthetic route, which employed classic reactions of selenium
chemistry (Scheme 1).12a
aInstituto de Qu´ımica, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes
748, SP 05508-900, Sa˜o Paulo, Brazil. E-mail: leandroh@iq.usp.br
Similar synthetic approaches were used to prepare the organ-
otelluranes congeners. The achiral organotelluranes 3 and 4
were synthesized from bromo-benzylic ether 1a. A bromo-lithium
exchange reaction of 1a followed by capture with an electrophilic
tellurium species (BuTeTeBu) led to telluride 1c in 79% yield.
The desired achiral organotelluranes 3 and 4 were obtained
bDepartment of Biochemistry and Molecular Biology, Indiana University
School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, 46202.
E-mail: zyzhang@iupui.edu
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob01050b
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Fig. 1 Selenanes and telluranes evaluated as inhibitors of Protein Tyrosin Phosphatase.
Scheme 1 Synthesis of organoselenanes 1–2, 5–6 and 9–10.12a
(Scheme 2-A) by a tellurium oxidation reaction with SO2Cl2
Assessment of organoselenanes and organotelluranes as PTP
inhibitors
or Br2. In order to acquire the chiral organotelluranes (7–8,
11–12) a chemo-enzymatic methodology was developed. In this
way, (RS)-1-phenylethanol 1g was submitted to enzymatic kinetic
resolution (EKR) through enantioselective transesterification re-
action mediated by Candida antartica lipase-B (CAL-B). This
reaction led to alcohol (S)-1g and the ester (R)-1h in high
enantiomeric excess (> 99%) and yield (45%), for each product.
After hydrolysis of (R)-1h, the BuTe- group was introduced to
each enantiomer of 1g. An ortho-lithiation reaction of (S)- or
(R)-1g followed by its capture with BuTeTeBu led to the telluro-
alcohols (S)- or (R)-1i in good yields (46%). O-Methylation of
the (S)- and (R)-1i and, finally, reaction of tellurides (S)- and
(R)-1j with SO2Cl2 or Br2 led to organotelluranes 7–8, 11–12
(Scheme 2-B).
The PTPs share a conserved active site and a common catalytic
mechanism that features a highly nucleophilic Cys residue.13 This
active site Cys displays an unusually low pKa of ~5,16 and is situated
at the bottom of the phosphotyrosine-binding pocket (i.e. the
˚
active site) such that its Sg atom is poised 3 A from the phosphorus
atom of phosphotyrosine.17 In the catalytic mechanism, the active
site Cys initiates a nucleophilic attack on the phosphorus atom,
leading to the formation of a thiophosphoryl enzyme intermediate.
Hydrolysis of this covalent enzyme intermediate then completes
the catalytic cycle.
Given the reactivity of selenium and tellurium atoms towards
thiol-dependent enzymes, we reasoned that the organochalco-
genanes may also display inhibitory activity against the PTPs.
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Scheme 2 Reagents and conditions: (i) a: t-BuLi, THF (-78-0 ◦C, 30 min); b: BuTeTeBu (r.t., 3 h); (ii) SO2Cl2 or Br2, THF (0 ◦C, 15 min); (iii) NaBH4,
MeOH (r.t., 1h); (iv) vinyl acetate, CAL-B, hexane (32 ◦C, 7 h); (v) K2CO3, MeOH, H2O (r.t. overnight); (vi) a: n-BuLi. TMEDA, pentane (reflux, 24 h),
b: BuTeTeBu (0 ◦C - r.t., 2 h); (vii) a: NaH, THF (0 ◦C, 30 min), b: MeI (r.t., 2 h).
To determine whether organoselenanes and organotelluranes
(1–12) can function as mechanism-based PTP inhibitors, we first
examined for their effect on PTP activity using para-nitrophenyl
phosphate (pNPP) as a substrate (see ESI†). All 12 compounds
inactivated PTP1B and the PTP from Yersinia YopH in a time-
dependent first order process (Table 1).
These assays were very important to identify the relevance of the
chalcogen atom for the profile of the organochalcogenanes as in-
hibitor of PTPs. As we can see in Table 1, the values of kobs showed
that organotelluranes are more potent than organoselenanes for
inhibition of PTP1B and the YopH. However, the contributions
of the halogens and a possible stereochemistry discrimination of
these compounds were not clear from the observed SAR towards
the PTPs.
Inactivation of the PTPs by organoselenanes and organotel-
luranes appeared irreversible as extensive dialysis and/or buffer
exchange of the reaction mixture failed to recover enzyme activity.
Since organotelluranes displayed higher inhibitory profile than
organoselenanes, 3 was chosen as a model inhibitor, to perform
a more detailed kinetic analysis in the PTP1B inactivation.
Analysis of the pseudo-first-order rate constant as a function
of inhibitor concentration showed that compound 3-mediated
PTP1B inactivation displayed saturation kinetics (Fig. 2), yielding
values for the equilibrium binding constant KI and the inactivation
Fig. 2 Kinetic analysis of PTP1B inactivation by 3 at 25 ◦C and pH 7. Panel on the left: time and concentration dependence of inhibitor 3-mediated
PTP1B inactivation. Compound 3 concentrations were as follows: 6 mM, ꢀ 10 mM, ꢀ 18 mM, ꢀ 33 mM, ᭜ 59 mM, 106 mM, 190 mM, ꢁ 343 mM,
᭹
᭹
᭺
ꢀ 617 mM, ᭛ 1111 mM, and ꢁ 2000 mM. Panel on the right: Concentration dependence of the pseudo-first-order rate constants kobs for 3-mediated
PTP1B inactivation.
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Table 1 Rate constants for onset inactivation of the PTPs by
organochalcogenanes 1–12
whose mode of action likely involves at least two steps: binding to
the PTP active site followed by covalent modification of the active
site Cys residue. It is worthwhile to point out that the kinetic
parameters KI and ki for compound 3 compare very favorably to
those determined for previously described activity-based probes
for the PTPs, including a-bromobenzyl phosphonate18 and aryl
vinyl sulfonates.19
In summary, the results highlight the potential for develop-
ing hypervalent chalcogenated based small molecule probes to
modulate PTP activity in signaling and in diseases. Among the
organochalcogenanes used as inhibitor of PTPs, organotelluranes
showed to be more potent than organoselenanes for inhibi-
tion of PTP1B and the YopH. The general reactivity of the
organotelluranes toward the PTPs should facilitate the design
of novel activity-based PTP probes. Additionally, PTP isozyme-
specific organotelluranes based inhibitors could be developed by
introducing specificity determinants into the aryl group to increase
potency and selectivity.
Inactivator
Code
PTP1Ba
YopHb (kobs
,
Structure
(kobs, min-1)
min-1)
1
2
3
4
5
0.46 0.15
0.48 0.12
0.53 0.25
0.30 0.19
0.22 0.25
0.25 0.17
0.39 0.22
0.89 0.21
0.92 0.19
0.18 0.12
Acknowledgements
L.W. and Z.-Y.Z. were supported in part by the National Institutes
of Health Grants CA126937 and CA152194. L.P. and L.H.A.
thank CNPq, CAPES and FAPESP for financial support.
6
0.21 0.18
0.43 0.25
0.31 0.15
0.20 0.16
0.20 0.23
0.46 0.19
0.60 0.39
0.09 0.14
0.74 0.22
0.59 0.10
0.39 0.20
0.30 0.11
1.07 0.46
0.65 0.46
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