Letter
Detection of Thiol Functionality and Disulfide Bond Formation by
ACCESS
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ABSTRACT: The detection of thiol functionality and intramolecular disulfide bond
formation of peptides using the α-Keggin type polyoxometalate molybdenum−oxygen
cluster (H PMo O ·nH O) is described. Our method entails the addition of this
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2
polyoxometalate to solutions of thiol, whereupon the color of the solution changes
from colorless to deep blue. Reduction of the polyoxometalate from Mo(VI) to
Mo(V) occurs with concomitant oxidation of the thiol functionality, to form disulfide
bonds. To exemplify the utility this phenomenon, we accomplished the oxidation of
glutathione, reduced linear oxytocin, bactenecin, and α-conotoxin SI; all of which proceeded smoothly and in good conversion in 24
h to less and were accomplished by a change in the color of the reaction solutions.
KEYWORDS: polyoxometalate, thiol, disulfide bond formation, blue staining, detection of cysteine residue, cyclic peptides
olyoxometalates (POMs) are a versatile class of negatively
the colorimetric analyses of thiols, and a wide variety of
11,12
P
charged early transition metal−oxygen nanoclusters
fluorescent probes for thiols are also available.
In addition,
whose unique catalytic, electrochemical, magnetic, and
luminescence properties have stimulated research in broad
disulfide bonds play a key role in stabilizing protein structures,
the disruption of which is strongly associated with a loss of
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,2
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fields of science. They can be categorized based on their
chemical structures: Lindqvist-type POMs are iso-polyoxome-
talates; α-Keggin and Wells-Dawson POMs have tetrahedrally
coordinated heteroatoms; and Anderson−Evans POMs in-
corporate an octahedral central atom. Modification of all types
of POMs can be accomplished by replacement of their MO
moieties with other transition metals (ruthenium, rhodium,
palladium, etc.). In addition, giant POMs with nanosized
cavities can be obtained by the aggregation of simple clusters in
water the presence of reducing reagents and templates and
protein function and activity. Disulfide bond formation is
therefore one of the most important steps in peptide/protein
synthesis. Many methods for disulfide bond formation have
been reported to date and include the use of oxidizing reagents
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such as dimethyl sulfoxide, iodine, silyl chloride-sulf-
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oxide, thallium trifluoroacetate, silver trifluoromethansul-
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fonate, and 3-nitro-2-pyridinesulfenate (Npys). Regiose-
lective disulfide bond formation has also been accomplished in
the synthesis of multidisulfide peptide/proteins, such as
insulin, the defensins, and the cyclotides, by using these
reagents in combination with product precursors bearing
orthogonal thiol-protecting groups on the side chains of their
cysteine residues. Herein, we describe our study of the POM
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with control of pH.
POMs are of particular interest in the
context of heterogeneous catalysis: for example, porous
materials based on POMs have been recently studied as
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catalysts in asymmetric dihydroxylation of olefins and the
(
1) H PMo O ·nH O and our discovery of its utility as a
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allylation of benzaldehyde with allyl tributyltin, and Hill
mild and self-indicating oxidizing reagent for thiols to give the
corresponding disulfides. The utility was exemplified first using
glutathione (2) and, then, in the synthesis of the disulfide-
containing bioactive peptides, such as oxytocin (3),
bactenecin (4), and α-conotoxin SI (5).
−
reported that the Keggin-type POM [CuPW O ] catalyzed
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the rapid chemo- and shape-selective oxidation of thiols to
disulfides, and the rapid and sustained removal of toxic H S by
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the oxidation of S . Therefore, POMs have huge potential in
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organic synthesis and the life sciences. The detection and
measurement of free thiols (i.e., free cysteine, glutathione, and
cysteine residues on proteins) is an essential task in the study
of biological processes and events in many biological systems.
First, we screened eight different POMs for their ability to
oxidize thiols to the corresponding disulfide and the degree to
which this change in redox state altered the color of the
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,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB or Ellman’s
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Received: August 21, 2020
reagent) has been used widely for the quantification of thiols,
especially for assays of various enzymes including acetyl- and
butyrylcholinesterase, the substrates of which release thiols
through enzymatic reactions. However, other reagents have
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been used too: for example, 4,4′-bipyridyl disulfide and 5-(2-
10
aminoethyl)dithio-2-nitrobenzoic acid have proved useful for
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XXXX American Chemical Society
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ACS Comb. Sci. XXXX, XXX, XXX−XXX