Reactive sulfur species: Aqueous chemistry of sulfenyl thiocyanates

Article abstract of DOI:10.1021/ja048585a

Sulfenyl thiocyanate (RSSCN) derivatives of penicillamine (PENSCN) and glutathione (GSSCN) have been synthesized in situ at pH = 0 from equilibrium mixtures that consists of hypothiocyanous acid (HOSCN), thiocyanogen ((SCN)₂), and trithiocyanate ((SCN)₃^-). The electrophilic thiocyanating agent N-thiocyanatosuccinimide (NTS) also reacts with PEN and GSH to yield the corresponding RSSCN derivatives. PENSCN and GSSCN were characterized by NMR, ES-MS, and IR spectroscopy. While stable at pH = 0, at higher pH the RSSCN derivatives decompose to give products that are consistent with hydrolysis and formation of reactive sulfenic acids. Copyright

Full text of DOI:10.1021/ja048585a

Published on Web 07/29/2004
Reactive Sulfur Species: Aqueous Chemistry of Sulfenyl Thiocyanates
Michael T. Ashby* and Halikhedkar Aneetha
Department of Chemistry and Biochemistry, UniVersity of Oklahoma, 620 Parrington OVal,
Norman, Oklahoma 73019
Received March 11, 2004; E-mail: mashby@ou.edu
Scheme 1
The familiar roles of thiols in cellular oxidant defense systems
are altered when reactive conjugates are formed. These so-called
reactive sulfur species (RSS)1,2 can exhibit beneficial or deleterious
biochemistry. While most RSS that have been studied to date are
organic compounds, it has been suggested that the inorganic RSS
hypothiocyanite (OSCN-), an antimicrobial agent that is produced
by myeloperoxidase,3,4 eosinophil peroxidase,5-8 lactoperoxidase,9
and salivary peroxidase,10 may target key protein sulfhydryl groups
in pathogens, perhaps vis-a`-vis sulfenyl thiocyanates (RSSCN).
Sulfenyl thiocyanates are relatively esoteric compounds due to their
mM) can be added 30 min later to produce quantitative yields of
general instability. Nonetheless, despite the aforementioned implica-
GSSCN (eq 3).
tion of RSSCN intermediates in the nonimmune defense system,5,11
and similar proposed intermediates in the synthesis of polypep-
SCN- + (SCN)2 + GSH pH ) 08 GSSCN
(3)
tides,12 there exist no bona fide examples of such compounds in
aqueous solution. We demonstrate herein that the equilibrium
mixture OSCN- a HOSCN a (SCN)2 a (SCN)3- 13-15 hereafter
,
We interpret the product distributions of experiments 1-3 in terms
of equilibria 4 and 5 and reaction 6:
collectively referred to as “hypothiocyanite”, reacts with cysteine
derivatives to yield sulfenyl thiocyanates. These products represent
the first organic species to be characterized that are deriVed from
hypothiocyanite in water and the first water-soluble sulfenyl
thiocyanates.
Following our realization that t-BuSSCN16 can be synthesized
from (SCN)2 in organic solvents and that it is sufficiently stable to
permit isolation, we prepared the tertiary sulfenyl thiocyanate
derivative of penicillamine (Scheme 1, PENSCN) in situ at low
pH (eq 1, RSH ) PEN; in this and subsequent formulas the
sequence of addition of the reagents to acidified solutions is the
order in which they appear in the equation).
(SCN)2 + H2O h SCN- + HOSCN + H+
(4)
(5)
(6)
(SCN)2 + SCN- h (SCN)3
-
2 HOSCN f SCN- + O2SCN- + 2 H+
We have performed preliminary stopped-flow kinetics measure-
ments that demonstrate equilibria 4 and 5 are much faster than the
kinetics of reaction 1 (RSH ) GSH). Accordingly, we believe the
higher yield of GSSCN at higher [SCN-] is due to the effect of
SCN- driving equilibrium 4 to the left, thereby diminishing the
[HOSCN] and the rate of disproportionation 6. Cyanosulfite
(O2SCN-) is generally believed to be a short-lived species in water
RSH + (SCN)2 pH ) 08 RSSCN + H+ + SCN-
(1)
2-
The thiocyanogen (10 mM, synthesized from Pb(SCN)2 + Br2
in CCl4)17 was extracted into 1 M HCl solutions of PEN (5 mM).
Details concerning the procedures that were employed to effect the
reactions we report in this communication are described in the SI.
In planning our experiments, we incorrectly assumed a tertiary thiol
was required to confer stability on RSSCN compounds. In fact,
the glutathione (GSH) analogues can be synthesized under similar
conditions (Scheme 1, eq 1). PENSCN and GSSCN were identified
in situ by characteristic NMR, IR, and ES-MS spectra.
While the yields are high for reactions 1, the order of addition
of the reagents is critical. Thus, if (SCN)2 (10 mM) is first extracted
into 1 M HCl and GSH (5 mM) is added immediately thereafter,
a 1:1 mixture of disulfide and unreacted GSH is observed (eq 2).
(eq 6) so that, presumably, it eventually decomposes to give SO4
and HNCO. The latter species, isocyanic acid, further reacts in 1
M HCl to give CO2 and NH4+ in a reaction sequence that ultimately
depletes the thiocyanating electrophile. It is clear that SCN- has
the effect of increasing the longevity of the thiocyanating elec-
trophile, since the amount of GSSCN from reactions such as 3 with
more modest concentrations of SCN- ([SCN-] ) 50 mM,
[(SCN)2] ) 15 mM, [GSH] ) 5 mM in 1 M HCl) vary with the
time that passes before addition of GSH (GSSCN ) 100, 69, 52,
47, and 14% and GSSG ) 0, 14, 25, 33, 71% for t ) 1, 5, 10, 15,
30 min, respectively).
The aforementioned experiments indicate (SCN)2, (SCN)3
-
,
HOSCN, and/or OSCN- are the thiocyanating electrophiles. Thio-
cyanogen is certainly capable of producing sulfenyl thiocyanates
in organic media, but what about OSCN- and HOSCN in aqueous
solution? This is an important issue because eq 4 lies to the right
under physiological conditions and only ca. 2% of the OSCN- is
protonated at pH ) 7. Since even small amounts of SCN- will
(SCN)2 + GSH pH ) 08 GSSG
(2)
It appears that the thiocyanating electrophile is short-lived in
reaction 2. Remarkably, the longevity of this species can be
increased substantially by adding SCN-. Thus, when SCN- (150
mM) is added to 1 M HCl, followed by (SCN)2 (15 mM), GSH (5
-
drive eq 4 to a mixture of (SCN)2 and (SCN)3 at pH ) 0,
conditions under which the hypothiocyanite equilibrium mixture
9
10216
J. AM. CHEM. SOC. 2004, 126, 10216-10217
10.1021/ja048585a CCC: $27.50 © 2004 American Chemical Society

C O MMU N I C A T I O N S
is relatively stable and can be studied, it is necessary to synthesize
HOSCN in the absence of SCN- to address this issue. We believe
we have achieved such a reaction using the electrophilic thiocy-
anating agent, N-thiocyanatosuccinimide (NTS), a compound that
has been proposed as an intermediate but heretofore never isolated.18
We have developed a procedure for synthesizing NTS, apparently
the first chemical species with an N-SCN bond to be structurally
characterized by X-ray crystallography (SI). We have observed that
NTS reacts with t-BuSH in organic solvents to yield t-BuSSCN.
We have also observed that NTS dissolves in water to produce
succinimide and what we assume to be initially OSCN-/HOSCN
(eq 7). Hydrolysis of NTS affords the first method of generating
OSCN- at neutral pH in the absence of excess SCN-.
(eq 6) or by the condensation of GSSCN and GSH. Solutions of
GSSCN (5 mM) in 1 M HCl react cleanly with one molar equivalent
of GSH to yield GSSG (eq 12).
GSSCN + GSH f GSSG + SCN- + H+
(12)
Preliminary stopped-flow kinetic measurements demonstrate that
reaction 12 is slow relative to reaction 1. Nonetheless, it is
conceivable that reaction 12 becomes important if the reactants are
depleted before all of the GSH is consumed. After carrying out
several dozen measurements under different reaction conditions,
we believe the production of GSSG in some cases is the result of
a one-electron oxidant such as O2SCN- (or other decomposition
products of HOSCN or (SCN)2).
Therefore, what is the nature of the thiocyanate electrophile?
For NTS, since no unreacted NTS, and instead succinimide, is
observed in experiments 7, 9, and 10 prior to addition of GSH, we
assume the hydrolysis of eq 7 has occurred. However, we are
currently exploring the possibility that NTS reacts directly with
GSH during reactions 8 and 11 (rather than proceeding via the
hydrolysis of eq 7). When (SCN)2 is the reagent, it is conceivable
that HOSCN, (SCN)2, or (SCN)3- is the electrophile, or even some
combination of these species. We are presently determining the
rate law for reaction 3 to establish whether SCN- facilitates
formation of GSSCN or is in fact an inhibitor.
Provided GSH (5 mM) is already present in 1 M HCl, NTS (15
mM) added as a solid produces GSSCN quantitatively (eq 8).
GSH + NTS pH ) 08 GSSCN
(8)
At pH ) 0, 1, and 3, the lifetimes of GSSCN are ca. days, hours,
and minutes, respectively. The mixture of products that are observed
upon raising the pH, including disulfides, are consistent with the
formation of intermediate sulfenic acids (RSOH).19-21 We are
currently exploring the product distributions of these reactions.
It is conceivable that NTS reacts directly with GSH during reaction
8 (rather than proceeding via the hydrolysis of eq 7). However, we
note that the hydrolysis of eq 7 and the equilibrium of eq 4 are
evidenced by the fact that GSSCN is formed cleanly when NTS
(15 mM) is added to a 1 M HCl solution containing SCN- (150
mM) followed by GSH (5 mM), cf. eq 3. We note that there is no
unreacted NTS; instead, succinimide is observed in these experi-
ments prior to addition of GSH.
Acknowledgment. We are grateful to the Petroleum Research
Fund (35088-AC3) and the Oklahoma Center for the Advancement
of Science and Technology (HR02-019) for their financial support.
Turning our attention to the nature of the species that produce
GSSG, as was the case for (SCN)2, no GSSCN is produced if NTS
is added first, followed by GSH (5 mM), but in this case the
principal species remaining is unreacted GSH (6% and 20% GSSG
were observed for 5 mM and 10 mM NTS, respectively) (eq 9).
Thus, the oxidizing species is shorter lived when NTS is used as a
precursor. This is consistent with the predomination of HOSCN in
reaction 8 and more facile disproportionation (eq 6).
Supporting Information Available: Experimental details and X-ray
crystal structure data for NTS. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Giles, G. I.; Tasker, K. M.; Jacob, C. Free Radical Biol. Med. 2001, 31,
1279-1283.
(2) Giles, G. I.; Jacob, C. Biol. Chem. 2002, 383, 375-388.
(3) van Dalen, C. J.; Whitehouse, M. W.; Winterbourn, C. C.; Kettle, A. J.
Biochem. J. 1997, 327, 487-492.
NTS + GSH pH ) 08 N.R.
(9)
(4) Kettle, A. J.; Winterbourn, C. C. Redox Rep. 1997, 3, 3-15.
(5) Arlandson, M.; Decker, T.; Roongta, V. A.; Bonilla, L.; Mayo, K. H.;
MacPherson, J. C.; Hazen, S. L.; Slungaard, A. J. Biol. Chem. 2001, 276,
215-224.
Lowering the concentration of HCl to 0.1 M is sufficient to
diminish the longevity of the thiocyanating species as evidenced
by lower yields of GSSCN and production of GSSG. For example,
solutions of SCN- (150 mM), NTS (15 mM), and GSH (5 mM) in
0.1 M HCl produce mainly GSSG (eq 10).
(6) Furtmueller, P. G.; Burner, U.; Regelsberger, G.; Obinger, C. Biochemistry
2000, 39, 15578-15584.
(7) Slungaard, A.; Mahoney, J. R., Jr. J. Biol. Chem. 1991, 266, 4903-4910.
(8) Van Dalen, C. J.; Kettle, A. J. Biochem. J. 2001, 358, 233-239.
(9) Thomas, E. L. Immunol. Ser. 1985, 27, 31-53.
(10) Pruitt, K. M.; Mansson-Rahemtulla, B.; Baldone, D. C.; Rahemtulla, F.
Biochemistry 1988, 27, 240-245.
SCN- + {(SCN)2 or NTS} + GSH pH ) 18
70-80% GSSG + 20-30% GSSCN (10)
(11) Aune, T. M.; Thomas, E. L. Biochemistry 1978, 17, 1005-1010.
(12) Hiskey, R. G.; Ward, B. F., Jr. J. Org. Chem. 1970, 35, 1118-1121.
(13) Barnett, J. J.; Stanbury, D. M. Inorg. Chem. 2002, 41, 164-166.
(14) Figlar, J. N.; Stanbury, D. M. J. Phys. Chem. A 1999, 103, 5732-5741.
(15) Figlar, J. N.; Stanbury, D. M. Inorg. Chem. 2000, 39, 5089-5094.
(16) Meijer, J.; Wijers, H. E.; Brandsma, L. Recl. TraV. Chim. Pays-Bas 1972,
91, 1423-1425.
However, as was the case in experiments 1 and 8, if GSH is already
present, high yields of GSSCN are produced. Thus, when NTS
(15 mM) is added to a solution of GSH (5 mM) in 0.1 M HCl,
only GSSCN is observed (eq 11).
(17) Wood, J. L. Org. React. 1946, 3, 240-266.
(18) Toste, F. D.; De Stefano, V.; Still, I. W. J. Synth. Commun. 1995, 25,
1277-1286.
(19) Patai, S., Ed. The Chemistry of Sulfenic Acids and Their DeriVatiVes;
Wiley: New York, 1990.
GSH + NTS pH ) 18 GSSCN
(11)
(20) Huang, K.-P.; Huang, F. L. Biochem. Pharmacol. 2002, 64, 1049-1056.
(21) Claiborne, A.; Yeh, J. I.; Mallett, T. C.; Luba, J.; Crane, E. J., III; Charrier,
V.; Parsonage, D. Biochemistry 1999, 38, 15407-15416.
The origin of GSSG is not certain in that it might be produced
by one-electron oxidation of GSH by species such as O2SCN-
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