Pyrazine-derived disulfide-reducing agent for chemical biology

Article abstract of DOI:10.1039/c4cc04491f

For fifty years, dithiothreitol (DTT) has been the preferred reagent for the reduction of disulfide bonds in proteins and other biomolecules. Herein we report on the synthesis and characterization of 2,3-bis(mercaptomethyl)pyrazine (BMMP), a readily accessible disulfide-reducing agent with reactivity under biological conditions that is markedly superior to DTT and other known reagents. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc04491f

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Pyrazine-derived disulfide-reducing agent for
chemical biology†
John C. Lukesh, III,a Kelly K. Wallinb and Ronald T. Raines*ab
Cite this: Chem. Commun., 2014,
50, 9591
Received 13th June 2014,
Accepted 20th June 2014
DOI: 10.1039/c4cc04491f
www.rsc.org/chemcomm
For fifty years, dithiothreitol (DTT) has been the preferred reagent for
the reduction of disulfide bonds in proteins and other biomolecules.
Herein we report on the synthesis and characterization of
2,3-bis(mercaptomethyl)pyrazine (BMMP), a readily accessible disulfide-
reducing agent with reactivity under biological conditions that is
markedly superior to DTT and other known reagents.
The redox state of cysteine residues can have a profound effect
on protein structure and function.1–4 Consequently, reagents
that reduce disulfide bonds to thiols can be crucial to progress
in chemical biology.1,5,6 Necessarily, the reduction of disulfide
bonds within biomolecules must be accomplished under mild
conditions: in water, at neutral pH, and at room temperature.1,7–10
Thiols can accomplish these goal and do so (unlike phosphines) in
a reversible manner. Their reduction mechanism entails thiol–
disulfide interchange initiated by the attack of a thiolate.11–18 The
use of monothiols such as L-glutathione or b-mercaptoethanol
(bME) can lead to the trapping of the resulting intermediate as a
mixed disulfide. In 1964, Cleland reported that dithiothreitol (DTT
or Cleland’s reagent; Fig. 1), a racemic dithiol, readily completes
the reduction reaction by forming a stable six-membered ring.7
The potency of DTT is evident from the low reduction potential
(E10= ꢀ0.327 V) of its oxidized form.19 As a result, DTT has achieved
widespread use for the quantitative reduction of disulfide bonds
in proteins and other biomolecules.
Fig. 1 Physicochemical properties of dithiol reducing agents. a Values are
from ref. 11. b Value is from ref. 19. c Values are from this work. d Values are
from ref. 23.
result, several attempts have been made to create water-soluble
DTT has, however, a serious limitation. As thiolates but not reducing agents that sport depressed thiol pKa values.9,21,22
thiols are nucleophilic in aqueous solution,20 the observed rate
Recently, we reported on dithiobutylamine (DTBA; Fig. 1), a
of disulfide reduction is dependent on the thiol pKa of the dithiol reducing agent derived from L-aspartic acid.23 Like DTT,
reducing agent. With thiol pKa values of 9.2 and 10.1, o1% DTBA is a potent disulfide-reducing agent. Moreover, the amino
of DTT resides in the reactive thiolate form at neutral pH.8 As a group of DTBA confers depressed thiol pKa values of 8.2 and 9.3 and
facile functionalization.23,24 That amino group, however, appeared
to deter the ability of the molecule to reduce certain disulfide
a Department of Chemistry, University of Wisconsin-Madison, 1101 University
Avenue, Madison, WI 53706-1322, USA. E-mail: rtraines@wisc.edu
b Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive,
Madison, WI 53706-1544, USA
bonds due to unfavorable Coulombic interactions.23
We were determined to improve upon DTBA. Dithiols (like
DTBA and DTT) that form six-membered cyclic disulfides are
potent reducing agents, reflecting a balance between the high
enthalpic stability of the incipient ring and the low entropic
loss for its formation.19,25 We reasoned that the entropic loss
† Electronic supplementary information (ESI) available: Experimental proce-
dures, computational data, and spectral data for novel compounds. See DOI:
10.1039/c4cc04491f
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could be diminished further by limiting rotation around one of both DTT and DTBA, and presumably results from the decreased
the bonds between the two sulfhydryl groups. We also sought enthalpic stability imparted by the two sp2-hybridized carbons in its
an electron-withdrawing group that could lower a thiol pKa to a six-membered ring.25 BMMP is, however, a much more potent
value close to physiological pH (which is pH 7.365 in human reducing agent than common monothiols such as b-mercapto-
blood). Then, an optimal balance is achieved between the ethanol (bME), cysteamine, and L-glutathione.16,30 To probe
concentration of the thiolate nucleophile (low pKa is better) and this difference, we equilibrated oxidized bME (bMEox) with a
its incipient nucleophilicity (high pKa is better).11,26 Accordingly, slight excess of reduced BMMP for 24 h. Analysis by HPLC
we suspected that incorporating a highly electron-deficient moiety revealed the complete reduction of bMEox (Fig. S3, ESI†).
(e.g., a pyrazine ring) that also serves to preorganize the reagent
Singh and Whitesides put forth N,N0-dimethyl-N,N0-bis(mercapto-
for disulfide-bond formation could be advantageous. A compound acetyl)hydrazine (DMH; Fig. 1) as a faster disulfide-reducing agent
that fits this description is 2,3-bis(mercaptomethyl)pyrazine than DTT.21 Notably, their reported pKa = 7.6 (8.9) and E10 = ꢀ0.300 V
(BMMP; Fig. 1).
values for DMH are indistinguishable from those of BMMP (Fig. 1).
Prior to designing a synthetic route to BMMP, we calculated The E10 value of DMH was corrected subsequently by Lees and
the free energies for the optimized geometry of reduced BMMP Whitesides to be ꢀ0.272 V,19 which is more consistent with its
and oxidized BMMP and compared them to those of reduced forming an 8-membered ring upon oxidation. To our knowl-
DTT and oxidized DTT. We were aware of a prior study that edge, the pKa value of DMH had not been examined again.
predicted the stability of cyclic disulfides using molecular Accordingly, we synthesized DMH so as to reexamine its proper-
mechanics calculations,25 and sought to assess our design at ties and utility. Our observed value of E10 = (ꢀ0.262 ꢁ 0.003) V
a higher level of theory (B3LYP/6-311+G(2d,p),27 see ESI†). The for DMH was even higher than that of Lees and Whitesides, and
thiol pKa values of BMMP were calculated to be substantially confirms that DMH is a markedly weaker reducing agent than
lower than those of DTT. Moreover, the oxidation of BMMP was is BMMP, DTBA, or DTT (Fig. 1). Likewise, our value of pKa =
calculated to be slightly more favorable than that of DTT.
8.0 ꢁ 0.1 (Fig. 1) is higher than that reported by Singh and
Because of these encouraging computational results, we Whitesides, but consistent with values reported for mercapto-
synthesized BMMP from 2,3-dimethylpyrazine (1) via a simple acetamido groups.9,22,31
three-step route (Scheme 1). We isolated BMMP as an off-white/
Next, we analyzed the reactivity of BMMP with relevant
yellow powder in 22% overall yield. The compound has low disulfide bonds. At pH 7.0, BMMP reduced the disulfide bond
odor and aqueous solubility (64 mM reduced; 8 mM oxidized)
that is adequate for typical applications in chemical biology.
As predicted, BMMP has low thiol pKa values. A pH titration
monitored by UV spectroscopy revealed these values to be 7.6 ꢁ
0.1 and 9.0 ꢁ 0.1. These pKa values are significantly lower than
those of DTT and DTBA (Fig. 1).23,28 Moreover, the lower of its
two thiol pKa values is closer to physiological pH than any
known dithiol-based reducing agent (vide infra).8,21,29
BMMP was also found to be a potent reducing agent, albeit
slightly less than predicted by our calculations. We found that the
equilibrium reaction between reduced BMMP and oxidized DTT
favors those species (rather than oxidized BMMP and reduced
DTT) by B1.2 kcal molꢀ1, which corresponds to a reduction
potential of E10 = (ꢀ0.301 ꢁ 0.003) V (Fig. 1 and Fig. S2, ESI†)
for oxidized BMMP. This E10 value is slightly less negative than
Fig. 2 Time-course for the reduction of bMEox (5 mM) by BMMP, DMH,
DTBA, or DTT (5 mM) in buffered water. (A) In 50 mM potassium phosphate
BMMP DMH
obs
BMMP DTBA
obs
BMMP DTT
buffer, pH 7.0: k
/kobs = 1.8, k
/kobs = 3.2, k
obs
/kobs = 11.4. (B) In
BMMP DTBA
obs
BMMP DTT
obs
Scheme 1 Synthetic route to BMMP (4).
50 mM sodium acetate buffer, pH 5.0: k
/kobs = 3.6, k
/kobs = 14.1.
9592 | Chem. Commun., 2014, 50, 9591--9594
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in bMEox 11-fold faster than did DTT and 3-fold faster than did near neutral pH. (For example, the pKa of the conjugate acid of
DTBA (Fig. 2A; Table S1, ESI†). Commensurate with their pKa 2,5-dimethylpyrazine is 2.1.38) Indeed, unfavorable Coulombic
values, DMH reduced bMEox faster than did DTT or DTBA interactions were not apparent with BMMP, which was found to
but slower than did BMMP. At pH 5.0, BMMP reduced bMEox reduce the mixed disulfide in creatine kinase 6-fold faster than
14-fold faster than DTT and 4-fold faster than did DTBA did DTT and 7-fold faster than did DTBA (Fig. 3B; Table S1, ESI†).
(Fig. 2B; Table S1, ESI†). These data highlight the broad
pH-range at which BMMP can be utilized effectively.
In conclusion, we have designed, synthesized, and characterized
BMMP, a novel disulfide-reducing agent with high reactivity under
Finally, we assessed the ability of BMMP to reduce disulfide biological conditions. The pyrazine ring of BMMP fuels its enhanced
bonds in two proteins. Papain is a cysteine protease that contains performance without Coulombic consequences. In a variety of
an active-site sulfhydryl group (Cys25) that needs to be in a reduced relevant assays, BMMP reduces disulfide bonds B10-fold faster
state for catalysis.32 Treatment with S-methyl methanethiosulfonate than does DTT. Notably, the depressed thiol pKa values of
generates an active-site mixed disulfide that results in complete BMMP extended the pH range at which disulfide bonds can
loss of enzymatic activity.33 This loss in activity, however, is be reduced efficiently. These attributes render BMMP as an
reversible upon treatment with a disulfide-reducing agent. attractive reagent for the reduction of the disulfide bonds
We found that BMMP reduced the mixed disulfide in papain encountered in chemical biology.
13-fold faster than did DTT and at a rate comparable to that of
This paper is dedicated to the memory of our colleague,
W. W. (Mo) Cleland (1930–2013), on the 50th anniversary of his
DTBA (Fig. 3A; Table S1, ESI†).23
Creatine kinase, like papain, is an enzyme that contains a seminal publication on DTT (ref. 7). We are grateful to Robert
thiol group (Cys283) that needs to be in a reduced state for W. Newberry for advice on computations. This work was
catalytic function.34–37 When treated with oxidized L-glutathione, supported by grant R01 GM044783 (NIH), and made use of
the resulting mixed disulfide eliminates its enzymatic activity. the National Magnetic Resonance Facility at Madison, which is
Previously, we reported that the ability of DTBA to reduce this supported by grant P41 GM103399 (NIH).
disulfide bond was compromised-presumably by unfavorable
Coulombic interactions-resulting in a low reaction rate compar-
Notes and references
able to that of DTT.23 In contrast to DTBA, BMMP is not cationic
1 Biochemistry of the SH Group: The Occurrence, Chemical Properties,
Metabolism and Biological Function of Thiols and Disulfides,
ed. P. C. Jocelyn, London, UK, 1972.
2 C. Jacob, G. I. Giles, N. M. Giles and H. Sies, Angew. Chem., Int. Ed.,
2003, 42, 4742–4758.
3 Oxidative Folding of Peptides and Proteins, ed. J. Buchner and
L. Moroder, The Royal Society of Chemistry, Cambridge, UK, 2009.
4 M. Lindahl, A. Mata-Cabana and T. Kieselbach, Antioxid. Redox
Signaling, 2011, 14, 2581–2642.
5 H. F. Gilbert, Adv. Enzymol. Relat. Areas Mol. Biol., 1990, 63, 69–172.
6 P. S. Kim and R. L. Baldwin, Annu. Rev. Biochem., 1990, 59, 631–660.
7 W. W. Cleland, Biochemistry, 1964, 3, 480–482.
8 R. P. Szajewski and G. M. Whitesides, J. Am. Chem. Soc., 1980, 102,
2011–2026.
9 G. V. Lamoureux and G. M. Whitesides, J. Org. Chem., 1993, 58,
633–641.
10 K. Van Laer, C. J. Hamilton and J. Messens, Antioxid. Redox Signaling,
2013, 18, 1642–1653.
11 G. M. Whitesides, J. E. Lilburn and R. P. Szajewski, J. Org. Chem.,
1977, 42, 332–338.
12 J. P. Snyder and L. Carlsen, J. Am. Chem. Soc., 1977, 99, 2931–2942.
13 R. E. Rosenfield, R. Parthasarathy and J. D. Dunitz, J. Am. Chem. Soc.,
1977, 99, 4860–4862.
14 Z. Shaked, R. P. Szajewski and G. M. Whitesides, Biochemistry, 1980,
19, 4156–4166.
15 J. Houk and G. M. Whitesides, J. Am. Chem. Soc., 1987, 109,
6825–6836.
16 D. A. Keire, E. Strauss, W. Guo, B. Noszal and D. L. Rabenstein,
J. Org. Chem., 1992, 57, 123–127.
17 D. M. Rothwarf and H. A. Scheraga, Proc. Natl. Acad. Sci. U. S. A.,
1992, 89, 7944–7948.
18 P. A. Fernandes and M. J. Ramos, Chem. – Eur. J., 2004, 10, 257–266.
19 W. J. Lees and G. H. Whitesides, J. Org. Chem., 1993, 58, 642–647.
20 R. A. Bednar, Biochemistry, 1990, 29, 3684–3690.
21 R. Singh and G. M. Whitesides, J. Org. Chem., 1991, 56, 2332–2337.
22 W. J. Lees, R. Singh and G. M. Whitesides, J. Org. Chem., 1991, 56,
7328–7331.
23 J. C. Lukesh, III, M. J. Palte and R. T. Raines, J. Am. Chem. Soc., 2012,
134, 4057–4059.
24 J. C. Lukesh, III, B. VanVeller and R. T. Raines, Angew. Chem., Int.
Ed., 2013, 52, 12901–12904.
Fig. 3 Time-course for the reduction of a mixed disulfide in proteins by
BMMP, DTBA, or DTT (7.8 mM) in 0.10 M imidazole–HCl buffer, pH 7.0,
BMMP
obs
containing EDTA (2 mM). (A) Papain Cys25–S–S–CH3 (4.4 mM): k
/
DTBA
obs
BMMP DTT
obs
k
=
1.2,
k
/kobs = 13.1. (B) Creatine kinase Cys283–S–S–L-
BMMP DTBA
obs
BMMP DTT
obs
glutathione (0.34 mM): k
/kobs = 6.8, k
/kobs = 5.8.
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25 J. A. Burns and G. M. Whitesides, J. Am. Chem. Soc., 1990, 112, 28 R. E. Benesch and R. J. Benesch, J. Am. Chem. Soc., 1955, 77, 5877–5881.
6296–6303. 29 A. S. Patel and W. J. Lees, Bioorg. Med. Chem., 2012, 20, 1020–1028.
26 W. P. Jencks, Catalysis in Chemistry and Enzymology, McrGraw–Hill, 30 K. K. Millis, K. H. Weaver and D. L. Rabenstein, J. Org. Chem., 1993,
New York, NY, 1969, pp. 79–85. 58, 4144–4146.
27 M. J. T. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, 31 K. J. Woycechowsky, K. D. Wittrup and R. T. Raines, Chem. Biol.,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, 1999, 6, 871–879.
G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, 32 I. Schechter and A. Berger, Biochem. Biophys. Res. Commun., 1967,
A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, 27, 157–162.
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, 33 D. J. Smith, E. T. Maggio and G. L. Kenyon, Biochemistry, 1975, 14,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, 766–771.
J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, 34 S. Putney, W. Herlihy, N. Royal, H. Pang, H. V. Aposhian,
J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,
L. Pickering, R. Belagaje, K. Biemann, D. Page, S. Kuby and
P. Schimmel, J. Biol. Chem., 1984, 259, 4317–4320.
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, 35 L. H. Chen, C. L. Borders, J. R. Vasquez and G. L. Kenyon, Biochemistry,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, 1996, 35, 7895–7902.
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, 36 J. K. M. Rao, G. Bujacz and A. Wlodawer, FEBS Lett., 1998, 439,
J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, 133–137.
G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, 37 A. M. Hurne, C. L. L. Chai and P. Waring, J. Biol. Chem., 2000, 275,
¨
A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski
25202–25206.
and D. J. Fox, Gaussian, Inc., Wallingford, CT, 2009.
38 D. A. Keyworth, J. Org. Chem., 1959, 24, 1355.
9594 | Chem. Commun., 2014, 50, 9591--9594
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