Thiols and selenols as electron-relay catalysts for disulfide-bond reduction

Article abstract of DOI:10.1002/anie.201307481

Pass them on! Dithiobutylamine immobilized on a resin is a useful reagent for the reduction of disulfide bonds. Its ability to reduce a disulfide bond in a protein is enhanced greatly if used along with a soluble strained cyclic disulfide or mixed diselenide that relays electrons from the resin to the protein. This electron-relay catalysis system provides distinct advantages over the use of excess soluble reducing agent alone. Copyright

Full text of DOI:10.1002/anie.201307481

Angewandte
Chemie
DOI: 10.1002/anie.201307481
Thiol–Disulfide Interchange
Hot Paper
Thiols and Selenols as Electron-Relay Catalysts for Disulfide-Bond
Reduction**
John C. Lukesh III, Brett VanVeller, and Ronald T. Raines*
For the proper function of many proteins, sulfhydryl groups
need to be maintained in a reduced state or disulfide bonds
need to be maintained in an oxidized state.[1] In cells, this
maintenance entails thiol–disulfide interchange reactions,
often initiated by a membrane-associated protein and medi-
ated by a soluble protein or peptide (e.g., glutathione).[2]
In vitro, small-molecule thiols and disulfides, such as those
in Scheme 1, can accomplish this task.[3]
DTBA; reduced 1), derived from l-aspartic acid.[4] As
dithiothreitol (DTT; reduced 2), DTBA is a dithiol capable
of adopting an unstrained ring upon oxidation.[5] A distinct
and untapped attribute of DTBA is the ability of its amino
group to act as a handle for facile conjugation. Small-
molecule reducing agents typically need to be maintained at
millimolar concentrations, and their removal diminishes
process efficiency and economy. We reasoned that attaching
DTBA to a solid support would enable its removal after
disulfide reduction by either filtration or centrifugation.[6]
To test our hypothesis, we choose TentaGel resin as the
solid support. This resin consists of hydrophilic poly(ethylene
glycol) units grafted onto low-cross-linked polystyrene.[7] We
found DTBA immobilized on TentaGel to be a potent
disulfide-reducing agent with E8’ = (ꢀ0.316 ꢁ 0.002) V (see
Figures S1 and S2 in the Supporting Information), a value
similar to that of soluble DTBA.[4] Immobilized DTBA
(10 equiv) was able to reduce cystamine (3) and oxidized b-
mercaptoethanol (4) completely (Figures S3 and S4). Immo-
bilized DTBA (10 equiv) was even able to reduce highly
stable disulfides, such as oxidized DTBA (1) and oxidized
DTT (2) with yields of 76% and 68%, respectively (Figur-
es S5 and S6). After each procedure, the resin was easily
isolated, regenerated, and reused without any observable loss
in activity. The latter are not attributes of immobilized
reducing agents derived from phosphines, which form recal-
citrant phosphine oxides.
Recently, we reported on a novel disulfide-reducing agent,
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or
Next, we assessed the ability of immobilized DTBA to
reduce a disulfide bond in a folded protein, which can be
a challenging task.[8] As the target protein, we choose papain,
a cysteine protease.[9] Upon treatment with S-methyl meth-
anethiosulfonate, the active-site cysteine of papain (Cys25)
forms a mixed disulfide that has no detectable enzymatic
activity.[10] When we incubated the oxidized enzyme with
100 equivalents of immobilized DTBA, we found that less
than half of papain-Cys25-S-S-CH3 had been reduced after
30 minutes (Figure 1). Moreover, the rate of reduction for this
heterogeneous reaction was slow, approximately 0.1% of that
provided by typical solution-phase reagents,[4,11] and activa-
tion ceased after 10 minutes. When papain was treated with
1000 equivalents of immobilized DTBA, full generation of
activity was observed within 10 minutes (Figure 1). These
data indicate that the inefficiency is likely due to a diminished
ability of the protein disulfide—in comparison to small-
molecule disulfides—to access the sulfhydryl groups of
immobilized DTBA.[8a,c]
Scheme 1. Disulfides (1–6) and diselenides (7–9) used in this work.
Compounds 2, 5, 6, and 9 are racemic mixtures.
[*] J. C. Lukesh III, Dr. B. VanVeller, Prof. R. T. Raines
Department of Chemistry, 1101 University Avenue
University of Wisconsin-Madison
Madison, WI 53706 (USA)
E-mail: rtraines@wisc.edu
Homepage: http://www.biochem.wisc.edu/faculty/raines/lab
Prof. R. T. Raines
Department of Biochemistry, 433 Babcock Drive
University of Wisconsin-Madison
Madison, WI 53706 (USA)
[**] We are grateful to Prof. H. J. Reich for contributive discussions. B.V.
was supported by postdoctoral fellowship 289613 (CIHR). This work
was supported by grant R01 GM044783 (NIH). This work made use
of the National Magnetic Resonance Facility at Madison, which is
supported by grants P41 RR002301 and P41 GM066326 (NIH), and
the Biophysics Instrumentation Facility, which was established with
grants BIR-9512577 (NSF) and S10 RR13790 (NIH).
Taking inspiration from cellular thiol–disulfide inter-
change reactions,[2,12] we reasoned that the utility of immo-
bilized DTBA could be enhanced by a soluble molecule that
could “relay” electrons from the resin to the protein
Supporting information for this article (including experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201307481.
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Communications
Figure 1. Time course for the reactivation of papain-Cys25-S-S-CH3 by
immobilized DTBA (100 or 1000 equiv) in imidazole-HCl buffer
(0.10m, pH 7.0) containing EDTA (2 mm).
(Scheme 2).[13] To test this hypothesis, we incubated papain-
Cys25-S-S-CH3 with 100 equivalents of immobilized DTBA
and 30 mol% of disulfides 1–4 (relative to oxidized protein).
Unfortunately, we observed only a slight rate enhancement
(Figure 2A).
Suspecting that the rate of the heterogeneous reaction
between immobilized DTBA and unstrained disulfides 1–4
was slow (A!B in Scheme 2),[15] we turned to disulfides 5 and
6, believing that their incipient strain would accelerate
the turnover of the soluble catalyst. BMCox (5) is a ten-
membered cyclic disulfide. Rings of this size suffer trans-
Figure 2. Time course for the reactivation of papain-Cys25-S-S-CH3 by
immobilized DTBA (100 equiv) and a solution-phase disulfide catalyst
(30 mol%). Reactions were performed in imidazole-HCl buffer (0.10m,
pH 7.0) containing EDTA (2 mm). A) Unstrained disulfide catalysts.
ꢀ
ꢀ
obs
cat
obs
obs
cat
obs
Cystamine (3): k
k
¼1.9; DTBAox (1): k
k
¼1.6; DTTox (2):
uncat
uncat
ꢀ
ꢀ
obs
cat
obs
obs
cat
obs
uncat
k
k
ꢂ1.0; bMEox (4): k
k
ꢂ1.0. B) Strained disulfide cata-
uncat
ꢀ
ꢀ
lysts. BMCox (5): k
k
¼4.3; lipoic acid (6): k
k
¼1.9. Data
obs
cat
obs
obs
cat
obs
uncat
uncat
for immobilized DTBA alone are shown in both panels.
annular strain.[5b,16a,b] Similarly, cyclic five-membered disul-
fides (i.e., 1,2-dithiolanes), such as 6, place significant
distortion on the preferred CSSC dihedral angle.[5b,17]
Hence, the rate constant for the reaction between 1,3-
propanedithiol and 1,2-dithiolane is around 650 times greater
than that for the homologated exchange reaction between 1,4-
butanedithiol and 1,2-dithiane.[18]
Consistent with our expectations, we found that disulfide 5
provided a significant enhancement in the rate of papain-
Cys25-S-S-CH3 reduction. Disulfide 6 was somewhat less
effective, as its mixed disulfide (B in Scheme 2) has a higher
tendency to partition back to the disulfide (A).[5b] Moreover,
in the absence of immobilized DTBA, we found that the
reduced form of DTBA regenerates activity faster than does
the reduced form of BMC (Figure S8), affirming that the
reduction of the soluble disulfide catalyst (A!C in
Scheme 2) limits the rate of electron-relay catalysis.
To improve catalytic efficiency further, we considered the
use of selenium, which has physicochemical properties similar
to those of sulfur. Yet, selenols manifest several desirable
attributes as reducing agents in aqueous solution.[19] For
example, selenols have pKa values that are typically three
units lower than those of analogous thiols, significantly
enhancing their nucleophilicity near neutral pH and their
ability to act as a leaving group.[20] Diselenides also have E8’
values that are typically 0.15 V lower than those of analogous
Scheme 2. Cycle for electron-relay catalysis of disulfide-bond reduction
by soluble thiols (C, X=S) or selenols (C, X=Se). Papain was
depicted with the program PyMOL (Schrodinger, Portland, OR) using
PDB entry 1ppn.[14]
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disulfides, making selenols more potent reducing agents. In
addition, reactions with selenium as the electrophile can be
104 times faster than those with sulfur as the electrophile, and
might not require strain for efficient turnover. Indeed, there
are numerous reports of small-molecule diselenides being
used as catalysts for biochemical oxidation reactions.[21]
Enzymes, such as thioredoxin reductase,[19h] are known to
employ a selenol as a reducing agent. Yet, reported in vitro
reduction reactions rarely employ small-molecule selenols,
and never diselenols. A practical problem is the high
reactivity of selenols with molecular oxygen. We recognized
that this problem would be averted in our system, which
would generate catalytic selenols in situ (Scheme 2). Because
of the efficacy of disulfide 5 (Figure 2B), we were motivated
to investigate its seleno congener. Accordingly, we synthe-
sized selenoBMCox (9) as well as selenoDTBAox (7), and we
obtained selenocystamine (8), which is available commer-
cially and has demonstrated marked success in mediating
thiol–disulfide interchange reactions.[20,21a,b]
nide (A) rather than to the diselenol (C) needed for
catalysis.[18,22] Notably, diselenide 8 led to significant rate
enhancements even at low loadings of catalyst.
In summary, we have established that the amino group of
DTBA allows for its facile conjugation to a resin. This
supported reagent was effective at reducing disulfide bonds in
small molecules. Unlike soluble reducing agents, immobilized
DTBA was easy to recover and reuse. We also demonstrated
that the rate of reducing a disulfide bond in a protein can be
enhanced markedly when the reduced resin is used in
conjunction with a “relay”. In this biomimetic strategy, the
resin acts as a repository of electrons that are relayed to
a macromolecule via a small-molecule catalyst. The optimal
catalysts are strained cyclic disulfides and acyclic diselenides,
both of which react with excess immobilized DTBA to form
a covalent intermediate that partitions toward reduced
catalyst and oxidized resin. Finally, we note that a vast
excess of soluble reducing agent is typically used to preserve
proteins in a reduced state.[23] Instead, maintenance could
require a minute (e.g., sub-micromolar) amount of a soluble
catalyst along with immobilized DTBA. We anticipate that
the low level of soluble reducing agent would be advanta-
geous in common bioconjugation reactions entailing the S-
alkylation of cysteine residues,[24] as well as in many other
experimental procedures.
We found that diselenide 7 is superior to its congener 1,
and that diselenide 9 performs comparably to its congener 5
(Figure 3A). These two cyclic diselenides were, however,
worse catalysts than was acyclic diselenide 8 (Figure 3B). This
finding is attributable to the selenylsulfide (B in Scheme 2)
generated by the reaction of 7 and 9 (but not 8) with
immobilized DTBA tending to partition back to the disele-
Received: August 26, 2013
Published online: October 10, 2013
Keywords: heterogeneous catalysis · proteins · redox chemistry ·
.
selenium · sulfur
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¼6.8; 1 mol%: k
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¼3.2.
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