Novel solid-phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions

Article abstract of DOI:10.1021/ja981111p

The controlled formation of intramolecular disulfide bridges in peptides, while avoiding unwanted oligomerization, is a significant challenge. Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed originally in the context of an assay for measuring free thiol concentration under physiological conditions. The present studies demonstrate that this reagent, when bound through two sites to a suitable solid support (PEG-PS, modified Sephadex, or controlled-pore glass), is an effective mild oxidizing reagent that promotes the formation of disulfide bridges. Rates and yields of the reactions were determined as a function of pH, excess of oxidizing reagent, resin loading, and parent support, for the preparation of oxytocin and deaminooxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge between residues 3 and 14), α-conotoxin SI (13 residues, disulfide bridges between residues 2 and 7; 3 and 13), and apamin (18 residues, disulfide bridges between residues 1 and 11; 3 and 15). Cystine dimers of these peptide models formed, if at all, in relatively low amounts. Use of solid-phase Ellman's reagents to oxidize the linear precursors of conotoxin or apamin (tetrathiols) gave as the main products the correctly paired regioisomers. Particular advantages of the overall approach include fast reaction rates over a wide range of pH, from 2.7 to 6.6; easy purification of disulfide-containing products; and the specificity and reusability of the reagents.

Full text of DOI:10.1021/ja981111p

7226
J. Am. Chem. Soc. 1998, 120, 7226-7238
Novel Solid-Phase Reagents for Facile Formation of Intramolecular
Disulfide Bridges in Peptides under Mild Conditions1,2
Ioana Annis, Lin Chen, and George Barany*
Contribution from the Department of Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455
ReceiVed April 2, 1998
Abstract: The controlled formation of intramolecular disulfide bridges in peptides, while avoiding unwanted
oligomerization, is a significant challenge. Ellman’s reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed
originally in the context of an assay for measuring free thiol concentration under physiological conditions.
The present studies demonstrate that this reagent, when bound through two sites to a suitable solid support
(PEG-PS, modified Sephadex, or controlled-pore glass), is an effective mild oxidizing reagent that promotes
the formation of disulfide bridges. Rates and yields of the reactions were determined as a function of pH,
excess of oxidizing reagent, resin loading, and parent support, for the preparation of oxytocin and deamino-
oxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge
between residues 3 and 14), R-conotoxin SI (13 residues, disulfide bridges between residues 2 and 7; 3 and
13), and apamin (18 residues, disulfide bridges between residues 1 and 11; 3 and 15). Cystine dimers of these
peptide models formed, if at all, in relatively low amounts. Use of solid-phase Ellman’s reagents to oxidize
the linear precursors of conotoxin or apamin (tetrathiols) gave as the main products the correctly paired
regioisomers. Particular advantages of the overall approach include fast reaction rates over a wide range of
pH, from 2.7 to 6.6; easy purification of disulfide-containing products; and the specificity and reusability of
the reagents.
Introduction
lecular dimerization/oligomerization, scrambling, and/or modi-
fication of sensitive amino acid side chains (Tyr, Met, or Trp).4
Stimulated by the myriad applications of solid-phase method-
ologies in chemistry and biochemistry,5 we seek polymer-bound
reagents that can be used to overcome some of the aforemen-
tioned problems.6,7
Disulfide bridges represent significant structural motifs in
many biologically important peptides and proteins.3 Intramo-
lecular disulfides serve to covalently cross-link portions of the
polypeptide chain that are apart in the linear sequence but come
together in three dimensions. Introduction of disulfide bridges
into natural or designed peptides and small proteins is a valuable
tactic toward improving biological activities/specificities and
stabilities. Despite extensive research, the controlled formation
of intramolecular disulfide bridges from cysteine-containing
precursors remains a significant challenge.4 Thus, while cur-
rently available methods have facilitated progress, low yields
are frequently observed due to problems of solubility, intermo-
We report here how Ellman’s reagent, 5,5′-dithiobis-
(2-nitrobenzoic acid) (DTNB, 1), developed originally in the
(4) For reviews, see: (a) Ko¨nig, W.; Geiger, R. In PerspectiVes in Peptide
Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel,
1981; pp 31-44. (b) Bu¨llesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-
29. (c) Andreu, D.; Albericio, F.; Sole´, N. A.; Munson, M. C.; Ferrer, M.;
Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis
Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ,
1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-
Bo¨hner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.;
Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
(5) For monographs and reviews, see: (a) Biochemical Aspects of
Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971.
(b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sher-
rigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield,
R. B. Science 1986, 232, 341-347. (d) Fru¨chtel, J. S.; Jung, G. Angew.
Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A
Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S.
H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
(6) Initial efforts in this regard are reported in refs 2a-c and Chen, L.;
Barany, G. Lett. Pept. Sci. 1996, 3, 283-292.
(7) We are aware of two additional, possibly related, solid-phase
approaches to facilitate disulfide formation. In one approach, poly(thiol)
snake toxin substrates were passed over a commercially available agarose
glutathione-2-pyridyl disulfide support, and the oxidized products were
eluted by gradients of reduced and oxidized glutathione at pH 8.7. See:
(a) Smith, D. C.; Hider, R. C. Biophys. Chem. 1988, 31, 21-28. In the
second approach, a commercially available methacrylate resin, derivatized
in an unspecified manner, gave a support termed Ekathiox. This was
incubated with solutions of dihydrooxytocin and dihydrocalcitonin in dilute
acetic acid and said to promote rapid, high-yield transformations to the
corresponding intramolecular disulfides. See: (b) Clark, B. R.; Pai, M.
Presented at the 14th American Peptide Symposium, Columbus, OH, June
18-23, 1995; Poster 90. (c) Clark, B. R.; Pai, M. Chem. Abstr. (Selects
Plus: Amino Acids, Peptides and Proteins) 1996, 14, 23, 125: 12365k.
(1) Abbreviations and conventions are defined at first use in the text
and are summarized in full in the Supporting Information. Abbreviations
used repeatedly for this work include: CPG, controlled pore glass; DTNB,
5,5′-dithiobis(2-nitrobenzoic acid); El, 5-thio-2-nitrobenzoyl; PAC, p-
alkoxybenzyl ester (Peptide Acid Linker); PAL, 5-[[(4-amino)methyl]-3,5-
dimethoxyphenoxy]valeric acid handle (Peptide Amide Linker); PEG-PS,
poly(ethylene glycol)-polystyrene (graft resin support); TNB, 5-thio-2-
nitrobenzoic acid; and Xan, 9H-xanthen-9-yl.
(2) Portions of this work were reported in preliminary form: (a) Barany,
G.; Chen, L. In InnoVation and PerspectiVes in Solid-Phase Synthesis &
Combinatorial Libraries, 1996, Collected Papers, Fourth International
Symposium, Edinburgh, Scotland, September 12-16, 1995; Epton, R., Ed.;
Mayflower Scientific: Kingswinford, U.K., 1997; pp 181-186. (b) Annis,
I.; Barany, G. In Peptides-Chemistry, Structure and Biology: Proceedings
of the Fifteenth American Peptide Symposium, Nashville, TN, June 14-
19, 1997; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer: Dordercht, The
Netherlands, 1998, in press. (c) Annis, I.; Chen, L.; Barany, G. In InnoVation
and PerspectiVes in Solid-Phase Synthesis & Combinatorial Libraries, 1998,
Collected Papers, Fifth International Symposium, London, U.K., September
2-6, 1997; Epton, R., Ed.; Mayflower Scientific Ltd.: Kingswinford, U.K.,
1998, in press.
(3) For reviews, see: (a) Richardson, J. S. AdV. Prot. Chem. 1981, 34,
167-339. (b) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (c)
Creighton, T. E. BioEssays 1988, 8, 57-63. (d) Raines, R. T. Nat. Struct.
Biol. 1997, 4, 424-427.
S0002-7863(98)01111-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/09/1998

Intramolecular Disulfide Bridges in Peptides
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7227
context of an assay for measuring free thiol concentration under
physiological conditions,8,9 can be adapted successfully to the
new application of mediating intramolecular disulfide formation
in the solid-phase mode. (Schemes 1 and 3, both later in this
paper, present the overall approach and depict the structure of
1.) Our approach offers advantages described for other polymeric
reagents, including circumvention of potential problems due to
solubility characteristics of substrates as well as reagent, the
chance to carry out reactions under mild conditions conducive
to product formation and to drive reactions to completion with
excess reagent, the ready separation of reagent and concomitant
isolation of product by simple filtration, and the recovery of
reagent suitable for regeneration and reuse. In addition, the
pseudodilution principle for polymer-supported reactions10 is
expected to favor intramolecular reactions and to decrease the
extent of oligomerization, important considerations for the
desired application to the creation of disulfides.11 Finally, issues
unique to solid-phase oxidations, for example, compatible solid
supports and reaction milieus, loading and relative site isolation
of supports, and a yield-diminishing intermolecular side reaction
involving covalent adsorption to the support, have been
addressed with respect to optimizing intramolecular disulfide
formation.
TNB dianion chromophore at 412 nm. Depending on structural
and conformational features of the mono- or poly(thiol) substrate
that is reacted with DTNB, it is possible for the initially formed
mixed disulfide to undergo disproportionations and/or further
nucleophilic displacements. Such secondary transformations
may provide a combination of intra- and intermolecular disul-
fides, and will not change the thiol titer reported by TNB.
Studies with a range of organic thiols have shown that the rates
of bimolecular thiolytic displacements of TNB from mixed
aliphatic-aromatic disulfides are 1-2 orders of magnitude
slower than the rates of the original displacements from the
aromatic homodisulfide DTNB,12 but when the attacking thiol
is in the same molecule, an intramolecular cyclization step
ensues which is substantially faster than the reaction with DTNB
(i.e., no intermediate is observed from reaction of DTNB with
organic dithiols).12a Ellman’s chemistry can be harnessed for
the creation of intramolecular peptide disulfides in solution; this
approach is limited by the sparing solubility of DTNB at pH
below 7, and is inevitably accompanied by the formation of
S-TNB-containing byproducts.15-17
We envisaged that similar chemistry, but with important
differentiating features, would occur upon treating a peptide
substrate containing an even number of thiol groups with DTNB
bound covalently to a solid support through two points of
attachment (Scheme 1, top line). This allows the use of a much
wider pH range than in the solution precedents, because now
the reagent is on the solid phase and its solubility is no longer
an issue. At the same time, the reaction milieu and compatible
support can be optimized for proper folding and adequate
solubility of the peptide substrate. Our proposed oxidation
mechanism (Scheme 1) involves an initial “capture” step, that
is, reaction of one of the peptidyl-thiol groups with the solid-
phase reagent to provide a support-bound activated intermediate
(Scheme 1, middle). Next, this intermediate undergoes
intramolecular “cyclization” through attack by the other pep-
tidyl-thiol group, resulting in formation of the desired disulfide
bridge and concomitant release of the monomeric oxidized
peptide product back into solution (Scheme 1, bottom). During
this second step, the substrate is relatively sequestered (pseudodi-
lution) from other potential thiol nucleophiles in solution or at
other sites on the support, lessening the likelihood of competing
intermolecular attacks which would lead to dimeric and oligo-
meric byproducts.6,10,17 However, when intramolecular cycliza-
tion is retarded for conformational/steric reasons, an on-resin
intermolecular side reaction involving attack by the second
peptidyl-thiol group on a separate polymer-bound DTNB site
can occur, despite pseudodilution.11c,d This side reaction,
Results and Discussion
Concept, Experimental Design, and Overview. In aqueous
solution, at pH 6.8-8.2, thiol functions from low molecular
weight organic compounds, as well as from peptides or proteins,
react cleanly and rapidly with excess DTNB (1) to displace
5-thio-2-nitrobenzoic acid (TNB, 2) and form the corresponding
mixed aliphatic-aromatic disulfide intermediates.8,9,12,13 This
thiol-disulfide exchange reaction14 is driven by the stability
of the aromatic thiolate leaving group, as reflected by the low
pKa, ∼4.75,12a of the conjugate aromatic thiol; the original titer
of free aliphatic thiol groups is quantified on the basis of the
(8) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
(9) (a) Habeeb, A. F. S. A. Methods Enzymol. 1972, 25, 457-464. (b)
Riddles, P. W.; Blakeley, R. L.; Zerner, B. Methods Enzymol. 1983, 91,
49-61.
(10) This term, which implies the kinetic basis for minimizing intersite
reactions, was introduced along with experimental documentation for a
benzyne system by (a) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979,
101, 677-683. See also: (b) Barany, G.; Merrifield, R. B. In The Peptides-
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic:
New York, 1979; Vol. 2, pp 1-284, especially pp 27-29.
(11) The approach taken in the present study is reciprocal to other work
in which the peptide substrate for disulfide formation is attached to the
support, as reviewed in refs 4c and 4e and also covered in (a) Albericio,
F.; Hammer, R. P.; Garc´ıa-Echeverr´ıa, C.; Molins, M. A.; Chang, J. L.;
Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Pept. Protein Res.
1991, 37, 402-413. (b) Munson, M. C.; Barany, G. J. Am. Chem. Soc.
1993, 115, 10203-10216, and references therein. Due to pseudodilution,
intramolecular cyclization is favored, but intermolecular processes do
compete. Also, when intramolecular pathways are not available to the resin-
bound peptide substrates, conditions can often be found under which
intermolecular disulfide formation occurs in reasonably high yield, as shown
in (c) Bhargava, K. K.; Sarin, V. K.; Le Trang, N.; Cerami, A.; Merrifield,
R. B. J. Am. Chem. Soc. 1983, 105, 3247-3251. (d) Munson, M. C.; Lebl,
M.; Slaninova`, J.; Barany, G. Pept. Res. 1993, 6, 155-159.
(15) The first explicit application of DTNB to intramolecular disulfide
formation was reported in ref 11a, although in hindsight, several aspects of
the experiment were suboptimal and not all of the observed oxidation can
be attributed to DTNB. Thus, a linear oxytocin sequence assembled by solid-
phase peptide synthesis was selectively deblocked, and the resin-bound
peptide dithiol was treated with DTNB (0.5 equiv) in “buffered” DMF, pH
7.5, for 1 h. Cleavage from the support revealed monomeric oxytocin in
65% absolute yield, along with a major byproduct, detected by HPLC, which
was believed to contain TNB on the basis of UV absorbance at 220, 280,
and 340 nm.
(16) A preparative solution experiment used DTNB (10 equiv) to carry
out disulfide cyclization at pH 6.8 on a peptide (2 mM) that was labile and
sluggishly reactive under standard alkaline pH air-oxidation conditions. The
desired product was isolated in 36% yield, and an extra HPLC peak was
observed and postulated to be the peptide with both Cys thiol groups linked
to TNB groups through disulfide bonds. See: Engebretsen, M.; Agner, E.;
Sandosham, J.; Fischer, P. M. J. Peptide Res. 1997, 49, 341-346.
(17) Our own preliminary studies (see Experimental Section) showed
that solution oxidations with DTNB in the oxytocin, somatostatin, and
conotoxin families provide, at best, 50-85% monomeric intramolecular
disulfide, and identified significant experimental difficulties that limit the
practical usefulness of the method.
(12) (a) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem.
1977, 42, 332-338. (b) Wilson, J. M.; Bayer, R. J.; Hupe, D. J. J. Am.
Chem. Soc. 1977, 99, 7922-7926.
(13) (a) Butterworth, P. H. W.; Baum, H.; Porter, J. W. Arch. Biochem.
Biophys. 1967, 118, 716-723. (b) Riddles, P. W.; Blakeley, R. L.; Zerner,
B. Anal. Biochem. 1979, 94, 75-81. (c) Wilson, J. M.; Wu, D.; Motiu-
DeGrood, R.; Hupe, D. J. J. Am. Chem. Soc. 1980, 102, 359-363. (d) Li,
T.-Y.; Minkel, D. T.; Shaw, C. F., III; Petering, D. H. Biochem. J. 1981,
193, 441-446. (e) Kuwata, K.; Uebori, M.; Yamada, K.; Yamazaki, Y.
Anal. Chem. 1982, 54, 1082-1087. (f) Robey, F. A. Protides Biol. Fluids
1986, 34, 47-50. (g) Savas, M. M.; Shaw, C. F., III; Petering, D. H. J.
Inorg. Biochem. 1993, 53, 235-249.
(14) For a review, see: Gilbert, H. F. AdV. Enzymol. 1990, 63, 69-172.

7228 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Annis et al.
Scheme 1. Proposed Mechanism for Intramolecular
Disulfide Formation Mediated by Solid-Phase Ellman’s
Reagentsa
the fact that conversion can reach completion and provides the
expected intramolecular cyclic disulfides as the predominant
products found in solution. Moreover, the observed absolute
yields are a gauge of how readily the peptide structure can
assume a structure leading to cyclization, because the principal
alternative pathways result in byproducts that are retained on
the support.
Solid Supports. Our approach should be viable using, as
parent supports, materials with a variety of chemical composi-
tions and physical characteristics and architectures; experimental
results as a function of choice of support are expected to reflect
the extent of site isolation in the solid phase. Initial studies
were focused on three types of commercially available supports
that have been well established for synthesis and/or chroma-
tography applications involving peptides, small proteins,
and/or oligonucleotides: (1) microporous poly(ethylene glycol)-
polystyrene (PEG-PS) grafts,18 (2) cross-linked dextran with a
molecular weight exclusion of 1500 (Sephadex G-15),19 and
(3) controlled pore glass (CPG).20 PEG-PS has advantageous
physical and mechanical properties for batchwise and continuous
flow reactions, and its grafted PEG chains impart good swelling
in a range of organic solvents as well as in water. Sephadex is
more fragile, but has excellent compatibility with aqueous
systems and exhibits significant swelling in water. CPG
provides a rigid (nonswelling) support, and its functional sites
are located on the walls of uniformly distributed pores.
DTNB derivatives contain pendant carboxyl groups which
are suited for attachment, via amide bond forming reactions, to
complementary amino sites on appropriate functionalized
supports. Amino-PEG-PS (0.15-0.38 mmol/g loading) and
aminopropyl-CPG (1000 Å pore size, 35 m2/g, 0.02-0.09
mmol/g loading) are commercially available. However, intro-
duction onto Sephadex G-15 of amino groups at a sufficiently
high loading level required development of a new procedure
(Scheme 2).21 Bromoethylamine hydrobromide (3) was neutral-
ized and treated with di-tert-butyl dicarbonate to provide the
Boc-protected derivative tert-butyl N-(2-bromoethyl)carbamate
(4).22 Subsequently, Sephadex G-15 was activated with base
a Solid supports
demonstrated in this work include poly(ethylene
®
glycol)-polystyrene (PEG-PS) graft, Sephadex G-15, and controlled
pore glass (CPG). For preparative experiments, DTNB was attached
directly to the amino-functionalized supports, whereas for some
mechanistic experiments, appropriate spacers and/or cleavable linkers
were used between DTNB and the support backbone. Details in text.
documented later in this paper (Scheme 6 and accompanying
discussion), is the solid-phase equivalent of bis(TNB) byproduct
formation presumed to occur in solution;16,17 it leads to covalent
retention of the peptide on the support, and lowers the overall
yield of product obtained in solution without affecting its purity.
(18) (a) Barany, G.; Albericio, F.; Sole´, N. A.; Griffin, G. W.; Kates, S.
A.; Hudson, D. In Peptides 1992. Proceedings of the Twenty-Second
European Peptide Symposium; Schneider, C. H., Eberle, A. N., Eds.;
ESCOM Science: Leiden, 1993; pp 267-268. (b) Zalipsky, S.; Chang, J.
L.; Albericio, F.; Barany, G. React. Polym. 1994, 22, 243-258. (c) Barany,
G.; Albericio, F.; Kates, S. A.; Kempe, M. In Chemistry and Biological
Application of Polyethylene Glycol, ACS Symposium Series 680; Harris,
J. M., Zalipsky, S., Eds.; American Chemical Society Books: Washington,
DC, 1997; pp 239-264.
(19) (a) Ko¨ster, H.; Heyns, K. Tetrahedron Lett. 1972, 16, 1531-1534.
(b) Vlasov, G. P.; Bilibin, A. Y.; Skvortsova, N. N.; Kalejs, U.; Kozhevni-
kova, N. Y.; Aukone, G. In Peptides 1994. Proceedings of the Twenty-
Third European Peptide Symposium; Maya, H. L. S., Ed.; ESCOM
Science: Leiden, 1995; pp 273-274.
(20) (a) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D.
R.; Fisher, E. F.; McBride, L. J.; Matteuci, M.; Stabinski, Z.; Tang, J.-Y.
Methods Enzymol. 1987, 20, 2571-2580. (b) Beaucage, S. L.; Caruthers,
M. H. In Bioorganic Chemistry: Nucleic Acids; Hecht, S. M., Ed.; Oxford
University: New York, 1996; pp 36-74. (c) Blackburn, G. M.; Gait, M. J.
In Nucleic Acids in Chemistry and Biology, 2nd ed.; Blackburn, G. M.,
Gait, M. J., Eds.; Oxford University: Oxford, 1996; pp 83-145.
(21) Several methods for activating Sephadex have been reported
previously, but they give maximal loadings of 0.1-60 µmol/g. See: (a)
Axe´n, R.; Porath, J.; Ernback, S. Nature 1967, 214, 1302-1304. (b) Parikh,
I.; March, S.; Cuatrecasas. Methods Enzymol. 1975, 34, 77-102. (c)
Brocklehurst, K.; Carlsson, J.; Kierstan, M. P. J.; Crook, E. M. Methods
Enzymol. 1975, 34, 531-547. As this work was being readied for
publication, a method for converting the commercially available HiTrap
Sepharose derivative to a form with 0.2 mmol/g of amino functionalization
was reported; see: (d) Tegge, W.; Frank, R. J. Peptide Res. 1997, 49, 355-
362.
Implementation of our concept required first the development
of efficient methods for preparation of solid-phase Ellman’s
reagents. Note the significance of attaching DTNB through two
sites; if this were not the case, approximately half of the capture
events (Scheme 1, first step) would result in a mixed disulfide
intermediate released back into solution, and hence the suggested
pseudodilution benefit would be forfeited. As shown later,
creation of an aromatic disulfide cross-link as part of the
structure of the polymer-bound reagent was achieved in both
inter- and intramolecular fashion; the intermolecular cross-
linking is more difficult because of the relative site isolation
implicit in pseudodilution.10,11 In the next phase of this research,
solid-phase Ellman’s reagents made by several methods and on
different types of parent supports were evaluated for their
capability to mediate disulfide formation in a representative
menu of peptide substrates (e.g., Table 1). Transformations
were monitored by chromatographic analysis of species remain-
ing in solution; polymer-bound intermediates and byproducts
were also quantified (Figure 1). The data do not permit a
complete kinetic description of heterogeneous multistep reac-
tions with competing pathways, but a good idea of relative rates
is provided by t1/2app, the time at which the material remaining
in solution comprises equal amounts of starting reduced substrate
and oxidized product(s). Our overall concept is validated by
(22) This compound has been prepared previously by a similar method:
Beylin, V. G.; Goel, O. P. Org. Prep. Proced. Int. 1987, 19, 78-80.

Intramolecular Disulfide Bridges in Peptides
Table 1. Overview of Rates and Absolute Yields of Oxidation of Representative Peptide Substrates by Solid-Phase Ellman’s Reagenta
pH 2.7 pH 6.6
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7229
substrate
t1/2app (min)
yield (%)
t1/2app (min)
yield (%)
H-CYIQNCPLG-NH2 (oxytocin)
Mpa-YIQNCPLG-NH2 (deamino-oxytocin)
H-AGCKNFFWKTFTSC-OH (somatostatin)
5
10
330
150
330
255
54
72
62
70
80
70
86
79
96
<1
<1
5
3
4
3
3
5
77
73
75
90
90
89
90
92
H-ICC(Acm)NPACGPKYSC(Acm)-NH2 (conotoxin SH 2&7)
H-IC(Acm)CNPACGPKYSC(Acm)-NH2 (conotoxin SH 3&7)
H-IC(Acm)CNPAC(Acm)GPKYSC-NH2 (conotoxin SH 3&13)
H-ICCNPACGPKYSC-NH2 (R-conotoxin SI)
H-CNCKAPETALCARRCQQH-NH2 (apamin)
120
a Reaction conditions: 15-fold excess of oxidizing reagent El-Lys(El)-PEG-PS disulfide (prepared by route of Scheme 5, right side), 0.21 mmol/g
loading, and reduced peptide substrate 0.5 µmol, at a concentration of ∼1 mg/mL in buffer-CH3CN-CH3OH (2:1:1). The apparent half-time,
t1/2app, represents the time when the soluble peptide comprises equal amounts of reduced and oxidized species. Reactions were carried out until
negligible amounts of reduced substrate were observed by HPLC (a time referred to in later tables as tend; this proved empirically to be 2- to 5-fold
longer than the reported t1/2app for most of the peptides studied). The absolute yields reported represent monomeric oxidized product(s); in the cases
of apamin and R-conotoxin SI they are the sum of yields of all disulfide regioisomers formed (refer to Table 7 for regioselectivity studies). With
oxytocin and deamino-oxytocin, formation of dimers accounts for an additional 3-19% conversion of the initial reduced substrate (details in Table
8). With other peptides, dimers were not observed, and the major yield-diminishing side reaction was covalent adsorption to the support (details
later).
Scheme 2. Amination of Sephadex G-15
trolled by the reaction time and reagent excess in the earlier
alkylation step (∼50-70% of 4 becomes incorporated).
Each of the three families of functionalized supports selected
for study was transformed further to create dually attached solid-
phase DTNB, as described later (Schemes 4 and 5, and
accompanying discussion). Some of the routes that were worked
out required an on-resin oxidation step, and a number of reagents
were tested for this purpose. Iodine oxidation was the most
convenient, and the method of choice when the support was
Figure 1. Conversion of reduced somatostatin (4) to its disulfide form
()), as mediated by El-Lys(El)-PEG-PS disulfide at pH 2.7. Conditions
Sephadex or CPG. However, when iodine treatment was applied
to PEG-PS, either loaded with oxidizable functions or (as a
control) unsubstituted, it proved to be difficult to wash out all
of the excess reagent. The resultant (presumably noncovalent)
I2‚‚‚PEG-PS complex itself promoted disulfide formation from
peptide dithiol substrates, hence complicating interpretations of
experiments involving covalently bound Ellman’s reagent.
Consequently, oxidations to form DTNB attached to PEG-PS
were carried out with aqueous ferricyanide; controls showed
that ferricyanide-treated PEG-PS did not have residual oxidizing
properties.
DTNB Derivatization and Formation of Solid-Phase Ell-
man’s Reagents. The plan to use amide bond formation
involving the DTNB carboxyl and amino-functionalized supports
has been described in the preceding section. However, DTNB
(1) is bifunctional, and we expected that coupling of a
monofunctional DTNB derivative might provide more control.
Toward this end, 1 was conveniently reduced by treatment with
aqueous mercaptoethylamine in the presence of dimethylami-
noethanol to provide aromatic thiol 2 in quantitative yield
(Scheme 3, top line; reagents chosen to facilitate workup).
Subsequently, acid-catalyzed reaction of 2 with 9H-xanthen-9-
ol23 gave the S-9H-xanthen-9-yl (Xan)-protected derivative 5
and data correspond to entry 3 in Table 1. The zero timepoint for starting
reduced substrate (prior to addition of the solid-phase reagent), as well
as the endpoints for all species, were calibrated by HPLC and amino
acid analysis. The amounts of soluble species during the time course
of the reaction were monitored by HPLC analysis, and the amount
adsorbed to the support (O), either covalently or physically, was
app
calculated to add to 100%. Dotted lines mark and define t1/2 (330
min for this example), the time at which the curves for loss of dithiol
peptide and formation of disulfide peptide overlapped; t1/2 (230 min
for this example), the time at which half of the dithiol peptide has
been lost from solution [this is calculated by ln 2/k, where the apparent
pseudo-first-order rate constant k is the negative of the slope of a graph
of ln(% reduced somatostatin) vs time]; and tend, the time for complete
conversion. The final yield of disulfide peptide is typically 10-15%
higher, because upon completion of the oxidation, draining of the
Ellman’s reagent-resin under positive air pressure results in release of
additional product that was contained within the swollen reaction
volume and hence not sampled by the HPLC assay of soluble
components. The residual drained resin still contains covalently bound
peptide (30% for this example), as elaborated later (Scheme 6 and
accompanying discussion).
(wash with 2 N aqueous NaOH), washed, and subjected to an
alkylation reaction with 4 in the presence of a slight excess of
KOH. Finally, acidolytic removal of the Boc group with
trifluoroacetic acid (TFA)-CH2Cl2 (1:3) generated aminated
Sephadex, the loading of which (0.2-1.0 mmol/g) was con-
(23) Han, Y.; Barany, G. J. Org. Chem. 1997, 62, 3841-3848.

7230 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Scheme 3. Derivatization of DTNBa
Annis et al.
Scheme 4. Initial Approaches for Preparation of Solid-Phase
Ellman’s Reagentsa
a Compounds 6 and 7 were somewhat more difficult to prepare with
respect to 5, and they require a higher acid concentration to remove
the S-protecting group. Nevertheless, all three derivatives were taken
further to provide solid-phase Ellman’s reagents (Schemes 4 and 5),
which gave equivalent performance in mediating intramolecular dis-
ulfide formation.
(Scheme 3, second line); similarly 2 could be protected as its
S-2,4,6-trimethoxybenzyl (Tmob)24 (6) and S-triphenylmethyl
(Trt)25 (7) derivatives.
a K3Fe(CN)6 oxidation used for
) PEG-PS, and I2 oxidation used
®
for
) Sephadex G-15 or CPG. Acidolytic removal of S-Xan also
The most straightforward way to immobilize Ellman’s reagent
functionalities is by coupling of DTNB (1) to amino-function-
alized supports (Scheme 4, left side).26 Initial experiments
started with amino-PEG-PS, and coupling of 1 was mediated
by N,N′-diisopropylcarbodiimide (DIPCDI) in N,N-dimethyl-
formamide (DMF), in the presence of 1-hydroxybenzotriazole
(HOBt). This provided a mixture of DTNB with one site and
with two sites of attachment to the support. The ratio of these
species ranged from ∼1:3 to ∼1:1, depending on the original
degree of excess of DTNB carboxyls over resin amines (as
expected, more cross-linking was achieved with a smaller
excess). Because our intended application requires that DTNB
have two attachment sites, it was necessary to carry out a
reduction stepsproviding the 5-thio-2-nitrobenzoyl-resin
intermediatesfollowed by reoxidation. Alternatively, the mono-
functional S-Xan protected derivative 5 was coupled, followed
by acidolytic deblocking to give again a resin-bound aromatic
thiol, and finally oxidation (Scheme 4, right side).27 However,
in both of these initial routes, the intermolecular oxidation to
provide the final reagent bound through two sites went, at best,
®
possible with substantially less TFA, i.e., TFA-CH2Cl2-Et3SiH
(3:94:3, 2 × 1 h); this was done when the support included an acid-
labile PAL linker. Oxidative removal of S-Xan with concurrent disulfide
formation is discussed in ref 27. Experiments were also carried out
replacing 5 by 6 [S-Tmob removal conditions the same] or 7 [S-Trt
removal by TFA-CH2Cl2-Et3SiH (50:50:3, 3 × 10 min)].
to ∼80-90%. Restated, ∼10-20% or more of the aromatic
thiol sites in PEG-PS could not pair up and form disulfides due
to site isolation effects (the thiols were still reactive, since
alkylation with iodoacetamide gave the corresponding
S-carboxamidomethyl derivatives).
The difficulty in carrying out on-resin intermolecular disulfide
formation to completion led us to consider an intramolecular
alternative (Scheme 5, right side). Thus, incorporation of a
lysine spacer was followed by coupling of monofunctional
S-Xan-protected derivative 5 to both the R- and ꢀ-amino groups.
Subsequent acidolytic deprotection generated two aromatic thiols
in close proximity to each other, and these were easily bridged
in quantitative yield to form the final solid-phase reagent
(Scheme 5, bottom structure). Another way to access the same
lysine-containing solid-phase reagent started with some solution
reactions (Scheme 5, left side): DTNB (1) was converted to its
(24) Munson, M. C.; Garc´ıa-Echeverr´ıa, C.; Albericio, F.; Barany, G. J.
Org. Chem. 1992, 57, 3013-3018.
(25) (a) Hiskey, R. G.; Mizoguchi, T.; Igeta, H. J. Org. Chem. 1966,
31, 1188-1192. (b) Photaki, I.; Taylor-Papadimitriou, J.; Sakarellos, C.;
Mazarakis, P.; Zervas, L. J. Chem. Soc., Chem. Commun. 1970, 2683-
2687.
(26) It is instructive to compare our approach to the work of Fridkin
and collaborators, whose goal was to prepare solid-phase aromatic thiols.
Both symmetrical and unsymmetrical carboxyl-containing disulfides were
coupled, following which borohydride reduction gave the desired products.
Information about the ratio of single and dual attachments at the disulfide
stage was not reported. See: (a) Stern, M.; Warshawsky, A.; Fridkin, M.
Int. J. Pept. Protein Res. 1981, 17, 531-538. (b) Stern, M.; Fridkin, M.;
Warshawsky, A. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 1469-1487.
(27) It is known (ref 23) that pairwise oxidation of S-Xan-protected thiols
with iodine gives disulfides directly, raising the possibility to save a step
in the chemistry of Scheme 4, right side. However, this conversion was
ambiguous for ® ) PEG-PS due to problems already discussed with iodine,
and for ® ) Sephadex G-15, the reaction was slow and difficult to carry
out to completion. The residual (unoxidized) S-Xan-protected sites on the
support led to a new problem at the next stage, when such supports were
used in experiments to oxidize peptide substrates with two (or for that matter,
one) thiol function(s). Thus, under the semiaqueous reaction conditions,
the S-Xan group apparently transferred from the resin-bound aromatic thiol
to the aliphatic peptide thiol.

Intramolecular Disulfide Bridges in Peptides
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7231
Scheme 5. Optimized Approaches for Preparation of
threitol to strip any covalently bound byproducts and simulta-
neously restore the aromatic thiol (TNB) functions on the
support. Oxidation by the same methods already described then
provides again resin-bound DTNB. We were able to complete
three cycles of use/regeneration; after each stage, the overall
rates and yields of peptide disulfide formation with regenerated
reagent were unchanged.
Solid-Phase Ellman’s Reagentsa
Peptide Substrates. Several peptide substrates, the linear
reduced precursors of which were obtained readily by
9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis,30,31
were chosen to study the efficiency of intramolecular disulfide
bridge formation mediated by solid-phase Ellman’s reagents
(Table 1 and subsequent tables): (a) oxytocin (9 residues,
disulfide bridge between residues 1 and 6),32 (b) deamino-
oxytocin (same as oxytocin except for N-terminal â-mercap-
topropionyl residue),32c (c) somatostatin (14 residues, disulfide
bridge between residues 3 and 14),33 (d) R-conotoxin SI (13
residues, disulfide bridges between residues 2 and 7; 3 and
13),11b and (e) apamin (18 residues, disulfide bridges between
residues 1 and 11; 3 and 15).34 Oxytocin is considered to be a
particularly easy sequence to oxidize, while oxidation to
deamino-oxytocin is somewhat more difficult, and oxidation to
somatostatin is particularly sluggish under acidic and neutral
conditions. Dimers of oxytocin and deamino-oxytocin are
known from previous work,11d,35 allowing us to evaluate the
relative occurrence of inter- and intramolecular oxidations in
the present system. Conotoxin and apamin were chosen to study
the selectivity of our proposed method in forming the correctly
paired disulfide regioisomers. In the case of conotoxin, we also
prepared several differentially protected derivatives [e.g., “cono-
toxin SH 2&7” has free thiols at residues 2 and 7, while 3 and
13 are protected by acetamidomethyl, Acm] in order to invest-
igate selective formation of a predetermined (single) bridge.
a K3Fe(CN)6 oxidation used for
) PEG-PS, and I2 oxidation used
®
for
) Sephadex G-15 or CPG. Acidolytic removal of S-Xan also
®
possible with substantially less TFA, i.e., TFA-CH2Cl2-Et3SiH
(3:94:3); this was done when the support included an acid-labile PAL
linker.
Intramolecular Disulfide Formation Mediated by Solid-
Phase Ellman’s Reagents. Oxidations were carried out with
peptide substrates dissolved in aqueous buffer-CH3CN-
CH3OH (2:1:1) at a concentration of 0.7-2.0 mM, on scales of
0.5-5 µmol. Excess solid-phase Ellman’s reagent resins, 2-
to 50-fold over the amount of peptide, with loadings of
0.03-1.0 mmol/g, were added, and reactions were carried out
at 25 °C until HPLC analysis of the soluble portion indicated
an endpoint in the conversion of starting to product peptide(s)
bis(succinimidyl) ester 8, which was added under high dilution
conditions to lysine in aqueous bicarbonate to provide analyti-
cally pure DTNB-lysine macrocyclic adduct 9. The preparation
concluded by the quantitative coupling of 9 to amino-function-
alized supports. Both of these improved routes (Scheme 5) were
repeated on supports that included a tris(alkoxy)benzylamide
(PAL) handle28 as well as two protected aspartyl residues (to
help with solubility during the subsequent analysis). After all
indicated transformations had been conducted, the final deriva-
tized supports were cleaved with Reagent B29 [TFA-CH2Cl2-
H2O-Et3SiH-PhOH (92:5:1:1:1)] and were shown in each case
to be the expected model peptide amide [lysine derivative
substructure corresponding to 9 residue, followed by Asp-Asp,
and concluding with C-terminal (CdO)NH2], homogeneous by
analytical HPLC and providing the proper mass upon FABMS.
Such experiments prove the efficacy of these preparative
methods. Moreover, we document later in this paper that when
applied to the overall goal of this work, that is, to mediate
intramolecular disulfide bridge formation, the lysine-containing
solid-phase Ellman’s reagents (Scheme 5) provided significantly
better performance by comparison to that of any of the earlier
formulations (Scheme 4).
(30) For reviews, see: (a) Barany, G.; Kneib-Cordonier, N.; Mullen, D.
G. Int. J. Pept. Protein Res. 1987, 30, 705-739. (b) Fields, G. B.; Noble,
R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (c) Fields, G. B.; Tian,
Z.; Barany, G. In Synthetic Peptides: A User’s Guide; Grant, G. A., Ed.;
W. H. Freeman: New York, 1992; pp 77-183.
(31) The reduced linear precursors were 64-92% pure (<5% disulfide)
upon cleavage from the support by appropriate TFA-scavanger mixtures
and workup. For much of our work, these materials were used in oxidation
reactions directly, that is, without any preliminary purification. Portions of
both reduced and oxidized substrates were purified to homogeneity,
quantified by amino acid analysis, and used to determine accurate HPLC
response factors.
(32) (a) du Vigneaud, V.; Ressler, C.; Swan, J. M.; Roberts, C. W.;
Katsoyannis, P. G.; Gordon, S. J. Am. Chem. Soc. 1953, 75, 4879-4880.
(b) Live, D. H.; Agosta, W. C.; Cowburn, D. J. Org. Chem. 1977, 42, 3556-
3561. (c) Chen, L.; Zoul´ıkova´, I.; Slaninova´, J.; Barany, G. J. Med. Chem.
1997, 40, 864-876, and references therein.
(33) Rivier, J.; Kaiser, R.; Galyean, R. Biopolymers 1978, 17, 1927-
1938, and references therein.
The solid-phase Ellman’s reagents made in any of the ways
described in this section can be readily regenerated and recycled.
Thus, after the polymeric reagent has been used to transform a
suitable peptide substrate, there follows reduction with dithio-
(34) To the best of our knowledge, this is the first successful synthesis
of apamin by an Fmoc strategy. For stepwise synthesis or segment
condensation with Boc chemistry, see: (a) Van Rietschoten, J.; Granier,
C.; Rochat, H.; Lissitzky, S.; Miranda, F. Eur. J. Biochem. 1975, 56, 35-
40. (c) Sandberg, B. E. B.; Ragnarsson, U. Int. J. Pept. Protein Res. 1978,
11, 238-245. (d) Albericio, F.; Granier, C.; Labbe´-Juillie´, C.; Seagar, M.;
Couraud, F.; Van Rietschoten, J. Tetrahedron 1984, 40, 4313-4326.
(35) Chen, L.; Bauerova´, H.; Slaninova´, J.; Barany, G. Pept. Res. 1996,
9, 114-121.
(28) (a) Albericio, F.; Barany, G. Int. J. Pept. Protein Res. 1987, 30,
206-216. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.;
Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-
3743.
(29) Sole´, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403.

7232 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Annis et al.
Table 2. Oxidation of Conotoxin SH 3&7 by Solid-Phase
Table 4. Oxidation of Several Peptide Substrates by Solid-Phase
Ellman’s Reagent as a Function of pHa
Ellman’s Reagent as a Function of Reagent Excessa
soluble resin-
pH
t1/2app (min)
tend (min)
resin-bound (%)
yield (%)
app
excess t1/2
tend reduced bound yield
2.7
3.4
4.0
5.0
6.6
330
55
22
4
1320
150
53
20
15
25
17
18
16
6
70
80
78
81
90
substrate
pH (fold) (min) (min)
(%)
(%)
(%)
somatostatin
6.6
6.6
5
15
3
15
15
40
3
15
15
30
3
13
5
8
60
25
13
0
9
15
25
2
72
75
89
90
80
75
90
90
70
70
88
89
conotoxin SH 2&7 6.6
35
20
420
210
45
3.5
6.6
2.7
2.7
3
4
6
a Reaction conditions and definitions for these studies are the same
as in Table 1; in addition, the column labeled “resin-bound” reports
the amount of peptide substrate that becomes attached covalently to
the solid-phase Ellman’s reagent.
150
60
10
4
330 1320
150
8
8
8
6
12
17
4
conotoxin SH 3&7 6.6
6.6
2.7
15
4
6
5
4
10
1
25
26
2
2.7
conotoxin SH 3&13 6.6
6.6
480
40
20
Table 3. Comparison of Solid-Phase Ellman’s Reagents Prepared
by Different Routes for Efficacy in Promoting Oxidation of Several
Peptide Substrates at pH 2.7a
15
3
10
a Reaction conditions and definitions for these studies are the same
as in Tables 1 and 2, except the number of equivalents of El-Lys(El)-
PEG-PS disulfide has been varied. In addition, the column labeled
“soluble reduced” reports the amount of peptide substrate remaining
Scheme 4 (right) route
(0.4 mmol/g,
Scheme 5 (right) route
(0.2 mmol/g,
on PEG-PS)
on Lys-PEG-PS)
app
app
t1/2
yield
resin-
t1/2
yield
resin-
at tend
.
substrate
(h)
(%) bound (%) (h)
(%) bound (%)
somatostatin
15
7
21
9
50
78
54
80
50
14
40
15
5.5
2.5
5.5
4.3
70
80
70
86
23
12
25
14
made in the original way starting with PEG-PS at 0.2 mmol/g
gave even slower reactions and lower yields (data not shown),
and the reproducibility of results was tempered by the incom-
plete oxidation to create DTNB functions. Our conclusion from
these studies was to use the Lys-containing variants for all
further mechanistic and preparative work.
conotoxin SH 2&7
conotoxin SH 3&7
conotoxin SH 3&13
a Reaction conditions and definitions for these studies are the same
as in Tables 1 and 2. Loadings of the starting supports were chosen so
that solid-phase Ellman’s reagents prepared by the two routes have
comparable loadings of DTNB sites.
Our standard conditions use a 15-fold molar excess of
Ellman’s functions over the linear peptide to be oxidized. As
might be expected, the reaction rate varied linearly with the
reagent excess, while the overall yield was more or less the
same (Table 4). At the low end of the pH scale, i.e., pH 2.7,
rates were faster in proportion to the use of a greater excess of
solid-phase Ellman’s reagent. Conversely, at pH 6.6, it was
possible to use lower excesses and still achieve reasonable rates
and excellent absolute yields. However, these latter reactions
apparently reached an equilibrium short of completion, as
reflected by the relatively low amounts of starting reduced
peptide substrate that did not oxidize even upon substantially
longer incubation. Control experiments at pH 6.6 involved
treatment of the various peptide substrates under study with
acetylated PEG-PS; at most 15% background disulfide formation
was observed during the time frame required to achieve full
conversion with the El-Lys(El)-PEG-PS disulfide reagent.
Next, the effect of support loading was examined, using again
the Lys-PEG-PS variant of the solid-phase Ellman’s reagent
(Table 5). The amount of polymeric reagent introduced to the
reaction was reduced concordantly, so as to maintain the same
overall excess of DTNB functions. While for a number of solid-
phase applications higher loading is a benefit, for our goal of
mediating intramolecular disulfide formation the higher loading
led to slower reaction rates and dramatically lower absolute
yields of desired solubilized monomeric disulfide. Over a 2-fold
loading range, substantially more peptide was adsorbed co-
valently to the higher-loaded solid-phase reagent. These results,
and similar findings with Sephadex and CPG (footnote to Table
5 and the next paragraph of this paper), are entirely consistent
with our understanding of the principal side reactionsan
intermolecular process occurring within the swollen volume of
the solid-phase reagentswhich competes with the desired
intramolecular cyclization. The side reaction is favored in the
higher-loaded supports, which have a greater effective concen-
tration of DTNB sites. In addition, the slower overall rate
observed with the higher-loaded support may be plausibly
explained by noting that less resin was used (rationale earlier
in this paragraph) and that the rate-determining “capture” step
(e.g., Figure 1). Rates and yields of oxidation were found to
depend on the linear sequence of the peptide substrate and on
a number of factors which were investigated systematically as
reported in the following paragraphs.
Incubation of reduced peptides with solid-phase Ellman’s
reagents resulted in the desired formation of disulfides over a
wide pH range (Table 1). The capability of achieving complete
reactions under acidic conditions is one of the major advantages
of our approach, since more basic conditions can promote
scrambling and disproportionation. However, to maximize rates
as well as yields by this method, a higher pH for reactions was
preferred (Table 2, determined with conotoxin SH 3&7 as
substrate). In the pH range 2.7-5.0, rates varied linearly with
hydrogen ion concentration (data from Table 2 was graphed),
demonstrating the involvement of thiolate ions in the rate-
determining step. At pH closer to neutral, rates were very fast
and had leveled off, reflecting complete ionization of the TNB
leaving group. As expected, the peptides which oxidize more
rapidly at a given pH with solid-phase Ellman’s reagents are
those known to have conformations more compatible with
disulfide formation (as described in the previous section).
Moreover, these same peptides give higher yields of disulfide
product in solution (more so at higher pH), and correspondingly,
lower amounts of resin-bound byproducts.
The low pH conditions allowed a critical test of how the
method by which the solid-phase Ellman’s reagent is prepared
influences its eventual usefulness in the disulfide-forming
application (Table 3). PEG-PS at 0.4 mmol/g, transformed by
coupling of protected precursor 5, followed by deblocking and
on-resin oxidation (Scheme 4, right side) contains ∼85%
oxidized DTNB functions. This level of functionality is
comparable to El-Lys(El)-PEG-PS disulfide derived from
PEG-PS at 0.2 mmol/g (Scheme 5, right side). Nevertheless,
the applications of the Lys-containing formulation gave reaction
rates that were 2- to 4-fold faster; in addition, absolute yields
were somewhat better and the amounts of resin-bound
byproducts were similarly lower. Solid-phase Ellman’s reagent

Intramolecular Disulfide Bridges in Peptides
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7233
Table 5. Oxidation of Several Peptide Substrates by Solid-Phase
Scheme 6. Proposed Mechanism of Formation and
Analytical Proof of Covalently Bound Byproduct Leading to
Lower Overall Yield during Oxidation with Solid-Phase
Ellman’s Reagentsa
Ellman’s Reagent as a Function of Support Loadinga
0.21 mmol/g
0.38 mmol/g
app
app
t1/2
pH (min)
resin-bound t1/2
resin-bound
peptide
(%)
(min)
(%)
somatostatin
conotoxin SH 3&7 2.7 150
conotoxin SH 3&7 3.4 28
2.7 145
30
26
19
290
690
105
80
50
30
a Reaction conditions and definitions for these studies are the same
as in Tables 1 and 2, except a 30-fold excess of the El-Lys(El)-PEG-
PS disulfide reagent was used for this set of data. Additional
experiments to oxidize some of these substrates were also carried out
with El-Lys(El)-PEG-PS disulfide (0.08 mmol/g), (El)2-PEG-PS dis-
ulfide (loading range of DTNB sites: 0.1-0.2 mmol/g), El-Lys(El)-
CPG disulfide (loading range: 0.02-0.05 mmol/g), and El-Lys(El)-
Sephadex G-15 disulfide (loading range: 0.2-1.0 mmol/g). The results
were qualitatively in accord with the ones shown, i.e., slower rates
and greater levels of resin adsorption with higher loading (see Tables
3 and 6 for baseline values).
a The shorthand for the Ellman’s reagent is self-explanatory in
context, and the wavy line represents Lys-PAL [after TFA treatments,
the PAL handle is cleaved to provide the derivatized lysine C-terminal
amides]. This Scheme does not intend to imply that the two thiol-
disulfide exchange steps depicted with the first set of structures are
concerted. Further explanation in text.
Table 6. Oxidation of Several Peptide Substrates by Solid-Phase
Ellman’s Reagent as a Function of Parent Support at pH 2.7a
Lys-PEG-PS Lys-Sephadex G-15 Lys-CPG
described next; it is disappointing with regard to the prospects
for CPG being a useful support with our method. The results
with CPG indicate that despite the very low loadings on this
material, the density of functional groups on the active surfaces
is exceedingly high, ∼12 Å apart on a square lattice. By way
of comparison, functional groups in swollen PEG-PS or Sepha-
dex are distributed in three dimensions and the separation
between sites can be estimated as ∼30-40 Å. The comparison
among supports also suggests that reaction rates are affected
by diffusion into microporous materials, for example, PEG-PS
and Sephadex, whereas diffusion to functional sites on CPG is
rapid and does not limit the rates.
app
app
app
t1/2
(h)
yield
(%)
t1/2
(h)
yield t1/2
(%)
yield
substrate
(h) (%)
somatostatin
conotoxin SH 2&7
conotoxin SH 3&7
5.5
2.5
5.5
70
80
70
86
18
3.5
13
11
50
76
45
63
7
11
3.5 57
15
7.5 30
15
conotoxin SH 3&13 4.3
a Reaction conditions and definitions for these studies are the same
as in Table 1. The loadings for Lys-PEG-PS and Lys-Sephadex G-15
were 0.2 mmol/g, and the excess of oxidizing reagent was 15-fold. In
the case of Lys-CPG, the loading was 0.03 mmol/g, and a 2-fold excess
of oxidizing reagent was used (rates were faster with greater excesses,
consistent with observations on PEG-PS reported in Table 4). For each
yield reported in this table, the amount of resin-bound byproduct was
determined separately and the two values, that is, yield + resin-bound,
were found to add to nearly 100%. For the CPG experiments, because
Intermolecular Side Reaction to Application of Solid-
Phase Ellman’s Reagents: Formation of Covalently Bound
Byproducts. A yield-diminishing side reaction, involving
covalent retention of peptide on the solid-phase Ellman’s
supports, has been mentioned and documented throughout this
paper. Adsorption to the support clearly involved one or more
disulfide bonds, since (a) the bound peptide was released upon
reduction with dithiothreitol, and (b) negligible physical adsorp-
tion was observed with control peptides of the same sequence
but with the cysteine residues blocked or replaced. To elucidate
further the chemical structures involved and develop a corre-
sponding mechanistic understanding, we designed an experiment
where conditions were chosen to maximize the level of the side
reaction (Scheme 6). When reduced somatostatin was treated
with El-Lys(El)-PAL-CPG disulfide (0.05 mmol/g; 5 equiv) at
pH 2.7, ∼95% of the peptide became bound to the support.
Cleavage of the PAL handle with Reagent B29 released into
solution a peptide-reagent conjugate which was analyzed by
MALDI mass spectrometry and shown to comprise two
molecules of Ellman’s spacer bound through two disulfide bonds
to one molecule of peptide. Formation of this species is
consistent with a mechanism for the side reaction in which the
second peptidyl-thiol group attacks a second Ellman’s reagent
site on the solid phase (Scheme 6), rather than the desired
intramolecular attack to displace Ellman thiolate from the solid-
phase activated intermediate (Scheme 1). The competition
between intra- and intermolecular processes correlates with those
cases where yields are lower due to peptide conformation, and
also explains earlier reported loading and support effects. We
also rule out the possibility that this same activated intermediate
does not undergo complete reaction, because in such a case we
would expect to detect a conjugate comprised of one molecule
app
the amount of material bound to the resin was so high, t1/2 is 4- to
6-fold longer than t1/2 (both of these terms are defined in Figure 1); the
values for t1/2 were 1.6, 0.8, 2.5, and 1.6 h for somatostatin, conotoxin
SH 2&7, conotoxin SH 3&7, and conotoxin SH 3&13, respectively.
(defined in Scheme 1) is the heterogeneous reaction of a soluble
peptide with a solid phase that contains DTNB functions. This
point of view assumes that the most accessible, and hence most
reactive, sites on the polymeric support are occupied first, in
the course of preparing solid-phase reagents of lower loading,
and that the further sites introduced to achieve higher loading
contribute less to the overall kinetics.
Our findings with PEG-PS extended qualitatively to Sephadex
and CPG, although there were meaningful quantitative differ-
ences (Table 6). With Sephadex, under otherwise comparable
conditions (pH, loading, and substrate), rates were 1.5- to 4-fold
slower, and overall yields were lower compared with those of
PEG-PS. Thus, there is no advantage to using Sephadex when
working with the kinds of peptide substrates that were the topic
of the present studies, but it is still conceivable that our approach
may be of value for Sephadex in conjunction with protein
substrates and/or more aqueous media (the current work uses
mixed aqueous-polar organic milieus). With CPG, we noted
that reactions were 1-2 orders of magnitude faster than those
with the other supports examined, and hence the excesses of
El-Lys(El)-CPG disulfide needed to achieve complete conver-
sion were as low as 2-fold. However, even with such a low
excess of DTNB functions, we found that surprisingly little of
the peptide substrate was oxidized to the disulfide; instead, much
of the material was adsorbed covalently onto the CPG support.
This observation was exploited in the mechanistic experiment

7234 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Annis et al.
Table 8. Distribution of Soluble Products Formed during
Table 7. Yields and Regiochemistry in Oxidation of Tetrathiol
Substrates by Solid-Phase Ellman’s Reagenta
Oxidation of Oxytocin and Deamino-oxytocin with Solid-Phase
Ellman’s Reagenta
resin- natural
app
t1/2
tend bound isomer isomer isomer
soluble
parallel antiparallel
substrate
pH
(min) (min) (%)
(%) 1 (%) 2 (%)
concn time reduced yield dimer
substrate pH (mg/mL) (min) (%)
dimer
(%)
(%)
(%)
conotoxin SI 6.6
2.7
3
20
54 180
30
120 600
10
21
8
4
20
81
63
76
70
28
5
5
4
1
19
4
11
12
25
33
oxytocin
2.7
0.5
15
60
15
90
330
15
60
5
14
1
17
2
0
11
2
7
2
0
69
81
58
72
72
63
65
71
67
77
1
1
3
4
5
4
6
5
10
8
1
2
7
7
9
7
12
5
9
7
apamin
6.6
2.7
5
2.7
1.0
2.7(Gd‚HCl) 160 900
a Reaction conditions and definitions for these studies are the same
as in Tables 1 and 2. In the case of conotoxin SI, mispaired isomer 1
has disulfide bridges connecting Cys 2&3 and 7&13; mispaired isomer
2 has disulfide bridges connecting Cys 2&13 and 3&7. In the case of
apamin, the two mispaired regioisomers have not been assigned; they
are listed in order of retention upon HPLC. The oxidation reported on
the bottom line of the table was carried out in the presence of 8 M
guanidinium hydrochloride.
2.7
3.4
6.6
2.0
1.0
45
1
1.0
1.0
deamino- 2.7
oxytocin
3.4
15
45
5
45
3
22
4
13
1
42
62
62
69
73
4
2
2
6
4
2
2
2
3
10
1.0
1.0
of Ellman’s spacer bound through a single disulfide bond to
one molecule of peptide.
6.6
1
a Reaction conditions and definitions for these studies are the same
as in Tables 1 and 4. The excess of El-Lys(El)-PEG-PS disulfide reagent
was 15-fold in all cases. The heterogeneous reaction mixture was
sampled at various points in time (not all data shown) for HPLC
analysis. Resin-bound material, determined separately, ranged from 8
to 30% and accounts for the remaining starting reduced substrate (values
in table, plus resin-bound, add up to 100%).
As a practical matter, the side reaction just discussed does
not affect the purity of the product from oxidation with solid-
phase Ellman’s reagents. Moreover, the covalently bound
peptide is readily released from the support in reduced form by
treatment with dithiothreitol, and can be recycled through the
oxidation procedure. Regeneration and recycling of the poly-
meric reagent can also be done, as described in a previous
section.
Regioselectivity in the Use of Solid-Phase Ellman’s
Reagents to Oxidize Tetrathiol Substrates. When our method
was applied to the linear precursors of conotoxin SI or apamin,
the naturally occurring regioisomers were the major products,
formed in 63-81% absolute yields (Table 7). A further 16-
26% (for apamin) and 9-16% (for conotoxin SI) of mispaired
isomers36 were observed, with relatively little material covalently
adsorbed. Improved selectivities were observed at higher pH
values. For both of these peptides, control oxidations carried
out in solution at the same pH, in the presence of 1-20% (v/v)
DMSO (dimethyl sulfoxide),37 required considerably more time
to reach completion and gave substantially higher levels of
mispaired regioisomers.
pH values and under more dilute conditions [compare results
at 0.5 mg/mL of peptide substrate with those at 1-2 mg/mL
concentration; n.b., these concentrations are about an order of
magnitude higher than what is typically used for air oxidation].
Dimerization in the oxytocin family might be correlated to their
flexible conformation, which readily forms disulfides. Even
after the desired monomeric product is obtained, it may undergo
further equilibration in solution. The present results show that
the pseudodilution advantage of our solid-phase approach is not
absolute. However, it should be pointed out that other than
oxytocin and deamino-oxytocin, none of the peptides examined
in our studies gave evidence for dimer formation upon oxidation
with solid-phase Ellman’s reagents.
A hint that dynamic disulfide exchange equilibria are involved
during solid-phase oxidation may be found by comparing the
overall rates, at pH 2.7, in the conotoxin SI system. Thus,
formation of both disulfide bridges in the natural regioisomer
actually occurs 3- to 6-fold faster than oxidation of the single
disulfide bridge in models of the same sequence (Table 1).
Another instructive result, in the apamin system, compares
oxidation at pH 2.7 in the absence and in the presence of the
denaturant guanidinium hydrochloride (Table 7). When the
peptide is unfolded during disulfide formation, the distribution
of regioisomers is relatively statistical, rather than conforma-
tionally driven to favor the naturally occurring disulfide array.
Dimerization. A complete accounting of the course of
reaction of reduced oxytocin or deamino-oxytocin with the solid-
phase Ellman’s reagent (Table 8) revealed that 3-19% of the
peptide was converted to dimers (mixture of both parallel and
antiparallel). The amounts of dimers formed were less at lower
Summary and Conclusions
The solid-phase reagents presented here offer an attractive
route to the formation of disulfide bridges in a variety of peptide
substrates. The methodology may be particularly beneficial for
substrates that are challenging to oxidize for reasons of
conformational restrictions, limited solubility at pH values above
neutral, and/or the presence of labile amino acid residues. The
procedure is easy to carry outsparticularly with regard to the
facile isolation of the desired disulfide products by simple
filtration; it is selective over a wide pH range; and it allows
recycling and reuse of both the solid-phase reagent and of any
linear substrate that becomes covalently bound to the support.
In addition, studies related to the generation and the application
of the reagents on a variety of supports with differing morphol-
ogies have provided qualitative insights into the pseudodilution
phenomenon that governs the relative levels of intra- and
intermolecular processes in solid-phase systems.
(36) In the case of conotoxin, authentic standards of unambiguously
synthesized mispaired regioisomers were available; see: Hargittai, B.;
Barany, G. In Peptides-Chemistry, Structure and Biology: Proceedings
of the Fifteenth American Peptide Symposium; Tam, J. P., Kaumaya, P. T.
P., Eds.; Kluwer: Dordercht, The Netherlands, 1998, in press.
(37) The DMSO oxidation procedure was developed by Tam, J. P.; Wu,
C.-R.; Liu, W.; Zhang, J.-W. J. Am. Chem. Soc. 1991, 113, 6657-6662.
For applications to the conotoxin system, see ref 11b and Experimental
Section of this paper.
Experimental Section
General. Most of the materials, solvents, instrumentation, and
general methods have been described and summarized in our previous
publications,6,11a,b,d,28,29,32c,35 and are described further in the Supporting
Information. Fmoc-PAL-PEG-PS and HCl‚H2N-PEG-PS supports
(initial loading 0.14-0.4 mmol/g) were from the Biosearch Division

Intramolecular Disulfide Bridges in Peptides
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7235
of PerSeptive Biosystems (Framingham, MA), Sephadex G-15 was from
Aldrich (Milwaukee, WI), and controlled pore glass (50-100 µmol/g,
1000 Å pore size, 35 m2/g surface area) was from Solid Phase Sciences
(San Rafael, CA). Peptide chain assembly was carried out either
manually or on a PerSeptive (formerly MilliGen/Biosearch) model 9050
continuous-flow synthesizer, as detailed in the Supporting Information.
Completed peptide-resins were treated with piperidine-DMF (1:4)
to remove the terminal Fmoc group, washed with CH2Cl2, and then
cleaved with appropriate acid/scavenger cocktails (1 mL/100 mg of
resin) for 1.5-4.5 h at 25 °C. The cleavage mixtures were then filtered
and washed with the same cocktails, and the combined filtrates were
concentrated under a stream of N2. Next, cold Et2O was added, and
the resultant precipitated peptides were collected by low-speed cen-
trifugation and washed with cold Et2O. The peptides were dissolved
in 1% aqueous HOAc for analytical HPLC and related evaluations,
and then lyophilized for storage as powders at 4 °C.
Loadings of PEG-PS, Sephadex, and CPG supports were calculated,
after BOP/NMM/HOBt-mediated coupling of Fmoc-Gly-OH, by quan-
titative spectrophotometric monitoring30c following Fmoc deblocking
with piperidine-DMF (1:4, 2 + 2 + 8 min), and by amino acid
analysis, and both methods agreed closely. (In the case of Sephadex,
the process started with a resin suspension, and the weight was
determined after completion of Fmoc deprotection by appropriate
washing and drying in vacuo.) UV measurements were at 301 nm (ꢀ
) 7800 M-1 cm-1 for the piperidine-fulvene adduct). Analytical
HPLC was performed using a Vydac analytical C-18 reversed-phase
column (218TP54; 5 µm, 300 Å; 0.46 × 25 cm), and linear gradients
of 0.1% aqueous TFA and 0.1% TFA-CH3CN were run at 1.2
mL/min. When appropriate, crude peptide products, both reduced and
oxidized, were purified by preparative HPLC using a Vydac semi-
preparative C-18 reversed-phase column (218TP1010; 10 µm, 300 Å;
1.0 × 25 cm) and elution of aqueous TFA-CH3CN gradients at 5
mL/min. Fractions with the correct peptide were pooled and lyophilized
to provide white powders. The final isolated yields were calculated
by comparison of amino acid analyses of the purified peptide to those
on the initial loaded resin.
For oxidation studies (Tables 1-8), the crude lyophilized powders
were redissolved in one of the following buffer milieus: (a) 1% aqueous
HOAc at pH 2.7; (b) pH 3.4 buffer, obtained by adding aqueous HCl
(0.1 N, 55.2 mL) to disodium citrate solution (44.8 mL, in turn prepared
from 0.1 M aqueous citric acid (100 mL) and 1 N aqueous NaOH (20
mL)); (c) pH 4.0 buffer, obtained by adding aqueous HCl (0.1 N, 44.9
mL) to aqueous disodium citrate (55.1 mL, prepared as described
above); (d) pH 5.0 buffer, prepared by mixing aqueous potassium
hydrogen phthalate (0.1 N, 50 mL), NaOH (0.1 N, 22.6 mL), and H2O
(27.4 mL); (e) pH 6.6, 7.3, 7.5, or 8.0 buffers, prepared by titrating an
aqueous solution of Na2HPO4 (0.1 M) with concentrated H3PO4 (drops)
while monitoring with a pH meter.
2-Nitro-5-thiobenzoic Acid [TNB or El(H)] (2). The Ellman’s
reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, or El2) (1) (1.0 g,
2.5 mmol) was suspended in distilled, degassed H2O (25 mL), and
N,N-dimethylethanolamine (1.0 mL, 7.7 mmol) was added to form a
homogeneous solution. A solution of â-mercaptoethylamine hydro-
chloride (1.2 g, 10.4 mmol) in H2O (25 mL) was added next, and the
solution immediately became red. After 1 h of stirring at 25 °C under
N2, completion of the reaction was verified by TLC. The reaction
mixture was acidified with 1 N aqueous HCl (47 mL), and the product
was extracted rapidly with EtOAc (3 × 60 mL). The organic phase
was dried (Na2SO4) and concentrated in vacuo to provide orange
crystals, which were stored in a desiccator at <5 mmHg and used within
3 days of preparation. Yield 1.0 g (quantitative); mp 136-137 °C (lit.13b
mp 137-137.5 °C). TLC, 1H and 13C NMR results are in the
Supporting Information.
with EtOAc; upon in vacuo removal of solvent, 4 was a clear, viscous
liquid suitable for further use (white crystals formed after several days’
refrigeration). Yield 2.62 g (80%); mp 33-35 °C (lit.22 mp 33-
1
35 °C). TLC, H NMR, and FABMS results are in the Supporting
Information.
2-Nitro-5-S-(9H-xanthene-9-yl)thiobenzoic Acid [El(Xan)] (5). A
solution of 2-nitro-5-thiobenzoic acid (2) (1.5 g, 7.5 mmol) in anhydrous
CH2Cl2 (75 mL) was treated with TFA (0.58 mL, 7.5 mmol) and stirred
for 5 min. Next, 9H-xanthen-9-ol (1.5 g, 7.5 mmol) was added in small
portions over a 10 min period, and the homogeneous yellow reaction
mixture was stirred for 2 h at 25 °C under N2 (to prevent competing
reoxidation of 2 to 1). The reaction was quenched by addition of hexane
(300 mL), whereupon off-white needles formed; these were collected
after standing overnight at -20 °C. Yield 2.55 g (90%). Recrystal-
lization was carried out by dissolving the product in a minimum amount
of hot CH2Cl2, cooling the solution to 25 °C, adding an excess of
hexane, and cooling the mixture to -20 °C. Fluffy light yellow crystals;
1
mp 172 °C (dec). TLC, H and 13C NMR, FABMS, and EA results
are in the Supporting Information.
2-Nitro-5-S-(2,4,6-trimethoxybenzyl)thiobenzoic Acid [El(Tmob)]
(6). A solution of 2-nitro-5-thiobenzoic acid (2) (0.9 g, 4.5 mmol) in
anhydrous CH2Cl2 (42 mL) was treated with TFA (0.35 mL, 4.5 mmol),
following which a solution of 2,4,6-trimethoxybenzyl alcohol24 (0.9 g,
4.5 mmol) in anhydrous CH2Cl2 (45 mL) was added dropwise over a
5 min period. The reaction was stirred under N2; precipitation of the
yellow product started within 1 h. After 5 h total reaction, TLC
monitoring indicated complete reaction, and the precipitate was collected
and washed with hot CH2Cl2 (30 mL). Yield 1.03 g (60%); dark yellow
amorphous crystals; mp 182-184 °C. TLC, 1H and 13C NMR,
FABMS, and EA results are in the Supporting Information.
2-Nitro-5-S-(triphenylmethyl)thiobenzoic Acid [El(Trt)] (7). On
a 4.5 mmol scale, the same procedure as for 5 was followed, but using
triphenylmethanol (1.2 g, 4.5 mmol). Yield 1.77 g (89%); flaky light
yellow plates; mp 165 °C. TLC, 1H and 13C NMR, FABMS, and EA
results are in the Supporting Information.
5,5′-Dithiobis(2-nitrobenzoic Acid N-Hydroxysuccinimide Ester)
[DTNB(OSu)2] (8). N-Hydroxysuccinimide (0.58 g, 5.1 mmol) was
added to a solution of DTNB (1) (1.0 g, 2.5 mmol) in CH2Cl2-DMF
(10:1, 55 mL). The solution was chilled to
4 °C, and
N,N′-dicyclohexylcarbodiimide (DCC) (1.05 g, 5.1 mmol) was added.
The reaction mixture became cloudy immediately, and was stirred
overnight at 4 °C. TLC [EtOAc-CH3OH (7:3)] showed the disap-
pearance of 1, Rf 0.28, and formation of the product 8, Rf 0.9. The
reaction mixture was filtered twice, once with suction and once with
gravity, to remove the white precipitate, N,N′-dicyclohexylurea (DCU).
The filtrate was concentrated in vacuo at <40 °C, hence removing the
CH2Cl2 but not all of the DMF. The resulting thick, yellow liquid
was diluted with dry EtOAc (40 mL), filtered to remove the remaining
urea, and concentrated again to remove EtOAc. The concentrate was
diluted to the needed volume with dioxane, and used for the subsequent
reaction later on the same day, without purification.
Nr,NE-Bis(5-thio-2-nitrobenzoyl)-L-lysine Disulfide [Ellman’s Re-
agent-Lysine Adduct] (9). The bis(N-hydroxysuccinimide ester) 8
derived from 1 (2.5 mmol), prepared as described above, was dissolved
in dioxane (150 mL), and added dropwise, over a period of 4-5 h
(1 drop/4-5 s) with Vigorous stirring to a solution of L-lysine
hydrochloride (0.46 g, 2.5 mmol) in 5% aqueous NaHCO3 (160 mL).
Almost at once, the reaction mixture became cloudy, and took on an
increasingly dark yellow coloration. At a later stage of the addition,
the reaction mixture was orange, and an orange precipitate had started
to form. The reaction mixture was stirred overnight at 25 °C, and the
precipitated product (first crop) was collected the next day. A second
crop was collected after partial concentration of the filtrate to remove
dioxane. Yield 0.65 g (51%, combined two crops); mp 270 °C, with
significant darkening and possible decomposition occurring at
∼120 °C; HPLC, gradient, started after 3 min and conducted over 40
min, of 0.1% aqueous TFA and 0.1% TFA-CH3CN from 1:0 to 1:1,
tR ) 31.1 min, purity ∼87%. TLC, 1H NMR, FABMS, and EA results
are in the Supporting Information.
tert-Butyl N-(2-Bromoethyl)carbamate (4). A solution of 2-bro-
moethylamine hydrobromide (3) (4.7 g, 22.9 mmol) in saturated aqueous
Na2CO3 (30 mL) was extracted with a solution of di-tert-butyl
dicarbonate (Boc2O, 3.2 g, 14.7 mmol) in Et2O (40 mL), followed by
further extractions with Et2O (3 × 30 mL). The combined organic
phases were dried (Na2SO4), partially concentrated in vacuo to ∼40
mL, and stirred for 24 h at 25 °C. The product 4 was then separated
from excess unreacted free amine by silica gel chromatography, eluting
Amination of Sephadex G-15 (Scheme 2). The procedure which
follows is typical; reaction conditions may need to be adjusted

7236 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Annis et al.
somewhat to achieve target loadings with different batches of support.
Sephadex G-15 (0.5 g, powder) was weighed into a 6-mL plastic syringe
fitted with a polypropylene frit. The Sephadex was activated by
washing with 2 N aqueous NaOH (3 mL, applied 4 × 2 min) and rinsed
with deionized H2O (∼10 mL) until the pH of the filtrate was neutral.
The activated Sephadex was next washed with DMF (4 × 1 min) and
drained, and a mixture of THF-DMF (1:1, 0.5 mL) was added. Freshly
ground solid KOH (13 mg, 0.23 mmol) was then added, with aid of
further THF-DMF (1:1, 0.5 mL), and the suspension was stirred gently.
Amination was commenced by the addition of a solution of tert-butyl
N-(2-bromoethyl)carbamate (4) (45 mg, 0.2 mmol) in THF-DMF
(1:1, 1 mL) and was carried out with gentle stirring for 2 h at 25 °C.
Subsequently, the liquid was drained, and the Sephadex was washed
thoroughly with DMF (∼10 mL) and H2O (∼15 mL) until the pH of
the filtrate was neutral. The Boc-protected Sephadex G-15 was stored
at 4 °C as a suspension in DMF or H2O. Just before use, the Boc
group was removed by treatment with TFA-CH2Cl2 (1:3, 2 mL, 3 ×
5 min), at 25 °C, followed by washing with CH2Cl2 (3 mL, 2 × 1 min)
and DMF (2 mL, 2 × 1 min). Loading, determined as described in
“General”, was 0.20 mmol/g of Sephadex, with 50 mg of Sephadex
corresponding to a settled bed volume of 0.3 mL. The same experiment,
but using a larger amount of 4 (90 mg, 0.4 mmol) and a reaction time
of 3 h gave a loading of 0.44 mmol/g; use of 4 (225 mg, 1.0 mmol)
and a 20 h reaction time gave a loading of 1.0 mmol/g.
96 µmol) was extended by coupling DTNB (153 mg, 0.39 mmol) using
DIPCDI (60 µL, 0.39 mmol) and HOBt (52 mg, 0.39 mmol) in DMF
(0.8 mL), also overnight. Aliquots (40 mg) from both of these
polymeric reagents were cleaved with Reagent B and evaluated as
described in A. The first case (use of 1 equiv DTNB) showed a 1:2
ratio of El-El-Gly-âAla-Gly-NH2 (corresponding to a single site of
attachment, tR4 ) 25.0 min) to (El-Gly-âAla-Gly-NH2)2 (homodimeric
disulfide corresponding to two sites of attachment, tR2 ) 19.0 min); in
the second case (use of 4 equiv DTNB), the ratio was 1:1.2. A further
aliquot (150 mg) of the resin from the second case was treated with
DTT (180 mg, 1.16 mmol, 20 equiv) in NMM-DMF (1:9, 1.5 mL) at
25 °C for 1 h. This treatment gave El(H)-Gly-âAla-Gly-PAL-
PEG-PS with properties (including an HPLC-homogeneous monomeric
thiol derivative upon cleavage) intersecting with the resin formed after
S-Xan removal described in A. Furthermore, this reduced resin was
reoxidized with potassium ferricyanide by the already-described
procedure, again giving results overlapping with the previous.
(C) Starting with Fmoc-PAL-PEG-PS (0.4 g, 0.2 mmol/g), manual
DIPCDI/HOBt coupling protocols were used to add in turn Fmoc-Asp-
(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Fmoc)-OH, and El(Xan)
(5), to create the resin El(Xan)-Lys(El(Xan))-Asp(OtBu)-Asp(OtBu)-
PAL-PEG-PS. Further transformations and analyses were as described
in A; only (a) iodine and (b) potassium ferricyanide were tested for
oxidation (same number of equivalents with respect to each El residue
in the model). HPLC analysis, under the same conditions as in A,
showed a single peak, tR ) 24.7 min, assigned to the disulfide of El-
Lys(El)-Asp-Asp-NH2 (confirmed by ESMS (m/z): calcd for
C28H30N7O13S2, 736.71; found, 738.0 [MH]+). The corresponding
reduced model peptide had tR ) 25.0 min, known from a separate DTT
reduction experiment; importantly, none of the reduced peptide was
observed in the described preparation of lysine-containing solid-phase
Ellman’s reagent, thus establishing that the oxidation had reached
completion. As further confirmation of the described oxidation route,
and proof of the structure of the resin-bound species, the tR ) 24.7
min peak was the only one noted (confirmed by co-injection) when
Reagent B cleavage followed by HPLC analysis was performed on the
derivatized peptide-resin obtained by an alternative route, the coupling
of compound 9 (4 equiv) to H-Asp(OtBu)-Asp(OtBu)-PAL-PEG-PS
by a DIPCDI/HOBt protocol.
Preparation of Solid-Phase Ellman’s Reagents: Model Studies
(Schemes 4 and 5). The results of these exploratory experiments
suggested optimized procedures, which are presented subsequently, to
prepare the desired solid-phase Ellman’s reagents on larger scales.
Hence, specific details that overlap the preparative procedures are
described in the present section only in outline form.
(A) Starting with Fmoc-PAL-PEG-PS (0.4 g, 0.45 mmol/g), manual
DIPCDI/HOBt coupling protocols were used to add in turn Fmoc-Gly-
OH, Fmoc-âAla-OH, Fmoc-Gly-OH, and El(Xan) (5) to create the resin
El(Xan)-Gly-âAla-Gly-PAL-PEG-PS. This resin was divided into 50
mg portions [23 µmol of El(Xan) per experiment], and each portion
was treated with TFA-CH2Cl2-Et3SiH (3:94:3, 2 × 1 h) to remove
the S-Xan group. Oxidation was carried out for 20 h at 25 °C with
each of (a) iodine (58 mg, 0.23 mmol, 10 equiv) in DMF (0.6 mL), (b)
potassium ferricyanide (76 mg, 0.23 mmol, 10 equiv) in DMF-H2O
(1:1, 0.46 mL) in the dark, and (c) Et3N (6 µL, 43 µmol) in
N-methylpyrrolidone (NMP) (1 mL). Subsequently, the treated poly-
meric reagent was washed several times each with the original reaction
milieu minus reagent, followed by DMF and CH2Cl2, and then Reagent
B, TFA-PhOH-H2O-Et3SiH (88:5:5:2, 1 mL), was added for 1.5 h
to achieve cleavage. The cleavage mixtures were worked up as
described under “General” and subjected to HPLC analysis using a
gradient, started after 3 min and conducted over 40 min, of 0.1%
aqueous TFA and 0.1% TFA-CH3CN from 1:0 to 1:1. In general,
two peaks were observed: tR1 ) 12.1 min assigned to El(H)-Gly-âAla-
Gly-NH2 (monomeric thiol) and tR2 ) 19.0 min assigned to (El-Gly-
âAla-Gly-NH2)2 (homodimeric disulfide). Consistent with these
assignments, the tR1 ) 12.1 min peak disappeared while the tR2 ) 19.0
min peak increased upon overnight stirring in pH 8 buffer; alternatively,
when DTT (∼50 mM final concentration) was added to the same
solution at pH 8, the tR2 ) 19.0 min peak was reduced completely
while the tR1 ) 12.1 min peak increased. For the three oxidation
conditions cited, the ratios of thiol:disulfide were (a) 1:9, (b) 1:6, and
(c) 1:4. In other experiments designed to determine why oxidation
had not gone to completion, the polymeric reagent (50 mg) was
alkylated, after the oxidation step, with iodoacetamide (42 mg, 0.23
mmol) in the presence of NMM (25 µL, 0.23 mmol) in DMF (0.5 mL)
for 5 h. Cleavage and workup gave again the tR2 ) 19.0 min peak
upon HPLC analysis, as well as a tR3 ) 10.1 min peak assigned to
El(CH2CONH2)-Gly-âAla-Gly-NH2, in relative amounts consistent with
the earlier analyses.
Solid-Phase Ellman’s Reagent: El-Lys(El)-PEG-PS Disulfide.
The procedure which follows is representative; similar ones were used
with other loadings of starting support and with other TNB precursors
(i.e., 6 or 7 in place of 5). HCl‚H2N-PEG-PS (1.0 g, 0.2 mmol/g) was
weighed into a 36-mL plastic syringe fitted with a polypropylene frit.
All wash volumes were 10 mL, and wash times were 1 min. The resin
was swelled by washing with CH2Cl2 (3 min), rinsed with DMF, treated
with piperidine-DMF (1:4, 10 mL, 2 × 1 min) to neutralize the
hydrochloride salt, and washed again with DMF (3×). Fmoc-Lys-
(Fmoc)-OH (473 mg, 0.8 mmol, 4 equiv) and HOBt (108 mg, 0.8 mmol,
4 equiv) were combined and dissolved in DMF (3 mL), DIPCDI (125
µL, 0.8 mmol, 4 equiv) was added, and this mixture was added
immediately to the resin. The coupling was allowed to proceed at
25 °C for 1 h, at which time the resin was negative to the Kaiser
Ninhydrin test.38 The resultant Fmoc-Lys(Fmoc)-PEG-PS was washed
with DMF (3×), and treated with piperidine-DMF (1:4, 10 mL, 3 ×
6 min) to remove the Fmoc groups. Next, El(Xan) (5) (608 mg, 1.6
mmol, 4 equiv with respect to free NH2) and HOBt (216 mg, 1.6 mmol,
4 equiv) were combined and dissolved in DMF (4 mL), DIPCDI (250
µL, 1.6 mmol, 4 equiv) was added, and this mixture was added
immediately to the H-Lys(H)-PEG-PS resin. The coupling was allowed
to proceed at 25 °C for 1 h, at which time the resin was negative to the
Kaiser Ninhydrin test. After washes with DMF (3×) and CH2Cl2 (2×),
the S-Xan group was removed with TFA-CH2Cl2-Et3SiH (25:75:3,
10 mL, 3 × 5 min), followed by further washes with CH2Cl2 (2×) and
DMF (3×). On-resin oxidation was accomplished by treatment with
potassium ferricyanide (1.3 g, 4 mmol) in DMF-H2O (1:1, 8.0 mL)
for 20 h at 25 °C in the dark. The polymeric reagent was then washed
thoroughly with H2O (3×), DMF (2×), again H2O (2×), DMF (2×),
(B) Starting with Fmoc-PAL-PEG-PS (0.4 g, 0.38 mmol/g), an
H-Gly-âAla-Gly-PAL-PEG-PS resin was assembled as described in A.
One portion of this resin (0.15 g, 56 µmol) was extended by coupling
DTNB (23 mg, 56 µmol) using DIPCDI (9 µL, 56 µmol) and HOBt (8
mg, 56 µmol) in DMF (0.3 mL) overnight; a second portion (0.25 g,
(38) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal.
Biochem. 1970, 34, 595-598.

Intramolecular Disulfide Bridges in Peptides
J. Am. Chem. Soc., Vol. 120, No. 29, 1998 7237
and Et2O (1×), following which it was dried in vacuo for 30 min and
stored at 4 °C.
aliquots (20 µL) from the liquid phase for eventual HPLC analysis
(done in real time for relatively slow reactions; for fast reactions at
higher pH, aliquots were frozen in liquid N2 and thawed just prior to
HPLC injection). Upon completion of oxidation, as judged by
disappearance of the reduced substrate peak in the HPLC trace, the
liquid phase was drained into a vial, using positive air pressure, and
aliquots were taken for hydrolysis/amino acid analysis and endpoint
HPLC. The consumed solid-phase reagent was washed thoroughly with
DMF, H2O, and DMF again, rinsed with Et2O, and dried in vacuo; a
portion (∼10 mg) was then taken for hydrolysis/amino acid analysis
to determine the extent of covalently bound material.
Regeneration of Solid-Phase Ellman’s Reagents. The regeneration
process was usually carried out by combining the recovered resins from
several oxidations performed by the above procedure. The used solid-
phase reagents (up to 0.5 g) were washed thoroughly with DMF, H2O,
and CH2Cl2, and dithiothreitol (∼20 equiv with respect to the original
loading of DTNB functions; final concentration 0.8 M) dissolved in
NMM-DMF (1:9) was added. Treatment for 2 h resulted in reduction
of all on-resin disulfide bonds. Following washes with DMF and
CH2Cl2, the resin-bound TNB functions were oxidized, using either
iodine or K3Fe(CN)6 as appropriate for the parent solid support, in the
same ways as previously described for the creation of resin-bound
DTNB functions.
Controls to Rule Out Noncovalent Adsorption during Applica-
tion of Solid-Phase Ellman’s Reagents. The standard procedures and
analyses as described were carried out using a variety of solid-phase
Ellman’s reagents (40 mg; Scheme 4 and Scheme 5 routes, on
PEG-PS, Sephadex G-15, and/or CPG), together with the cysteine-
lacking model peptide [Abu3,Ala14]-somatostatin (1 mg) dissolved in
1% aqueous HOAc-CH3CN-CH3OH (2:1:1, 1 mL). The amount of
peptide in the liquid phase at different points in time was monitored
by HPLC (gradient, conducted over 25 min, of 0.1% aqueous TFA
and 0.1% TFA-CH3CN from 4:1 to 1:1; tR ) 12.3 min) and was never
more than 20% (typically 8-15%) reduced from the level before any
resin had been added. Upon draining the resins under positive air
pressure, typically 95-98% of the peptide was recovered. Hydrolysis/
amino acid analysis on the washed and dried resin did not reveal any
peptide content (<1%) in the solid phase.
Determination of Covalently Bound Byproduct Formed with
Solid-Phase Ellman’s Reagents (Scheme 6). Starting with CPG (70
mg, loading 0.09 mmol/g) and using procedures analogous to those
reported in “Solid-Phase Ellman’s Reagent: El-Lys(El)-CPG Disulfide”,
an El-Lys(El)-PAL-CPG disulfide support was assembled. BOP/HOBt/
NMM (4:4:8) coupling protocols were used. The initial coupling of
Fmoc-PAL-OH (16 mg, 0.032 mmol, 5 equiv) required 18 h; S-Xan
removal was with TFA-CH2Cl2-Et3SiH (3:94:3) for 2 × 1 h; and the
final loading was determined experimentally to be 0.05 mmol/g. The
solid-phase reagent was washed thoroughly with DMF (3 mL, 4 × 1
min), CH2Cl2 (3 mL, 3 × 1 min), drained, and a solution of reduced
somatostatin (1.1 mg, 0.7 µmol) in 1% aqueous HOAc-CH3CN-
CH3OH (2:1:1, 2 mL) was added. After 15 h of gentle shaking, HPLC
(gradient, conducted over 25 min, of 0.1% aqueous TFA and 0.1%
TFA-CH3CN from 4:1 to 1:1) indicated that only ∼5% of the initial
amount of peptide was present in solution (fully converted to the
monomeric cyclic disulfide of somatostatin). Hydrolysis followed by
amino acid analysis indicated that ∼95% of the peptide had became
bound to the support. The reaction mixture was drained, and the solid
support was washed thoroughly. Treatment with Reagent B, TFA-
CH2Cl2-H2O-PhOH-Et3SiH (92:5:1:1:1, 3 mL), for 1.5 h cleaved
the acid-labile handle PAL and provided a residue which was subjected
to MALDI analysis: calcd for the structure outlined on the lower right
side of Scheme 6, C116H148N28O33S6, 2652.9; found, 2646.0 (within
experimental error of the analytical procedure).
Solid-Phase Ellman’s Reagent: El-Lys(El)-Sephadex G-15
Disulfide. Procedures were carried out in a 12-mL plastic syringe fitted
with a polypropylene frit; wash volumes were 5 mL, and wash times
were 1 min. Aminated Sephadex (0.5 g, 0.2 mmol/g) that had been
freshly deprotected as described earlier (present as trifluoroacetate salt)
was washed with DMF (3×). Fmoc-Lys(Fmoc)-OH (237 mg, 0.4
mmol, 4 equiv), BOP (177 mg, 0.4 mmol, 4 equiv), and HOBt (54
mg, 0.4 mmol, 4 equiv) were combined and dissolved in DMF (3 mL),
NMM (90 µL, 0.8 mmol, 8 equiv) was added, and the mixture was
added immediately to the solid support. The coupling was allowed to
proceed for 1.5 h, at which time the resin was negative to the Kaiser
Ninhydrin test. The resultant Fmoc-Lys(Fmoc)-Sephadex was washed
with DMF (3×) and treated with piperidine-DMF (1:4, 5 mL, 3 × 6
min) to remove the Fmoc groups. Next, El(Xan) (5) (304 mg, 0.8
mmol, 4 equiv with respect to free NH2), BOP (354 mg, 0.8 mmol, 4
equiv), and HOBt (108 mg, 0.8 mmol, 4 equiv) were combined and
dissolved in DMF (4 mL), NMM (180 µL, 1.6 mmol, 8 equiv) was
added to the solution, followed by immediate addition of the mixture
to the H-Lys(H)-Sephadex. The coupling was allowed to proceed at
25 °C for 1 h, at which time the resin was negative to the Kaiser
Ninhydrin test. After washes with DMF (3×) and CH2Cl2 (1×), the
S-Xan group was removed with TFA-CH2Cl2-Et3SiH (25:75:3, 5 mL,
3 × 5 min), followed by further washes with CH2Cl2 (2×) and DMF
(3×). On-resin oxidation was accomplished with iodine (0.5 g, 2 mmol,
10 equiv based on each El residue) in DMF (6 mL) for 20 h at 25 °C.
The polymeric reagent was washed thoroughly with DMF (2×), CHCl3
(2×), and again DMF (2×) and stored in DMF at 4 °C.
Solid-Phase Ellman’s Reagent: El-Lys(El)-CPG Disulfide. Wash
volumes were 6 mL, and wash times were 1 min. CPG (500 mg,
loading 0.05 mmol/g) was weighed into a 12-mL plastic syringe fitted
with a polypropylene frit and washed with DMF. Fmoc-Lys(Fmoc)-
OH (74 mg, 0.125 mmol, 5 equiv), BOP (56 mg, 0.125 mmol, 5 equiv),
and HOBt (17 mg, 0.125 mmol, 5 equiv) were combined and dissolved
in DMF (1.2 mL), NMM (28 µL, 0.250 mmol, 10 equiv) was added,
and the mixture was added immediately to the CPG. The coupling
was allowed to proceed for 18 h. The resultant Fmoc-Lys(Fmoc)-CPG
was washed with DMF (3×) and treated with piperidine-DMF (1:4,
6 mL, 2 + 2 + 8 min) to remove the Fmoc group. Quantitative UV
measurement of the fulvene-piperidine adduct revealed a loading of
0.03 mmol of Lys/g of CPG. Next, El(Xan) (5) (46 mg, 0.12 mmol,
4 equiv with respect to free NH2), BOP (53 mg, 0.12 mmol, 4 equiv),
and HOBt (17 mg, 0.12 mmol, 4 equiv) were combined and dissolved
in DMF (1.2 mL), NMM (27 µL, 0.24 mmol, 8 equiv) was added to
the solution, followed by immediate addition of the mixture to the
H-Lys(H)-CPG. The coupling was allowed to proceed at 25 °C for
1.5 h. After washes with DMF (3×) and CH2Cl2 (1×), the S-Xan group
was removed with TFA-CH2Cl2-Et3SiH (25:75:3, 6 mL, 3 × 5 min),
followed by further washes with CH2Cl2 (2×) and DMF (3×).
On-resin oxidation was accomplished with iodine (77 mg, 0.3 mmol,
10 equiv based on each El residue) in DMF (3 mL) for 20 h at 25 °C.
The polymeric reagent was washed thoroughly with DMF (4 x),
CH2Cl2 (3×), and Et2O (1×), following which it was dried in vacuo
for 30 min and stored at 4 °C.
Oxidation of Peptide Substrates by Solid-Phase Ellman’s
Reagents. In a typical procedure, the reduced peptide (∼1 mg) was
dissolved in buffer-CH3CN-CH3OH (2:1:1, 1 mL). An aliquot (50
µL) was taken for hydrolysis and amino acid analysis, and another
aliquot (20 µL) was taken for HPLC analysis of the zero timepoint.
Separately, the solid-phase reagent (e.g., ∼50 mg at 0.2 mmol/g
corresponding to 15-fold excess based on DTNB functions) was
weighed into a plastic syringe fitted with a polypropylene frit. When
the parent support was PEG-PS, the resin was first swelled in CH2Cl2
(2 min), washed with DMF (2 × 1 min) and again with CH2Cl2
(1 min), and drained. In the cases of Sephadex and CPG, only DMF
washes were used prior to draining. The syringe tip was plugged, using
a small septum or a plastic lock cap, and the solution of peptide substrate
was added to the syringe already containing the solid-phase Ellman’s
reagent. The reaction mixture was stirred magnetically or agitated on
a rotary shaker, gently, at 25 °C. Monitoring was carried out by taking
Somatostatin (Disulfide). The oxidations of the reduced linear
somatostatin to monomeric somatostatin disulfide as mediated by solid-
phase Ellman’s reagents are covered in Figure 1 and Tables 1 and 3-6.
In addition, linear somatostatin (8 mg, 4.9 µmol) treated with (El)2-
Sephadex G-15 disulfide (390 mg, 0.2 mmol/g) in pH 6.6 buffer-
CH3CN-CH3OH (2:1:1, 8 mL) was oxidized completely in 90 min.
Preparative HPLC (gradient, conducted over 40 min, of 0.1% aqueous
TFA and 0.1% TFA-CH3CN from 77:23 to 2:3) gave somatostatin

7238 J. Am. Chem. Soc., Vol. 120, No. 29, 1998
Annis et al.
disulfide (2.4 mg, 1.5 µmol) in an overall purified yield of 30%. As
a control, linear somatostatin (1 mg, 0.6 µmol) in 0.1 M phosphate
buffer, pH 6.6 (1 mL), stirred open to air at 25 °C, was oxidized
completely in 26 h. However, the absolute amount of disulfide,
quantified by the analytical HPLC peak area (gradient, conducted over
25 min, of 0.1% aqueous TFA and 0.1% TFA-CH3CN from 4:1 to
1:1; tR ) 12.6 min) corresponded to only 30% of the initial amount of
precursor, tR ) 13.4 min. As another control, no oxidation occurred,
and the starting substrate remained unchanged, after 2 days in 1%
aqueous HOAc, pH 2.7.
dissolved in 1% aqueous HOAc, pH 2.7 (0.8 mL), and DMSO
(0.2 mL, 20% v/v) was added. After 48 h at 25 °C, the reaction mixture
was analyzed by HPLC and was shown to comprise 51% natural
regioisomer, 28% mispaired isomer SS 2&3, 7&13, and 21% mispaired
isomer SS 2&13, 3&7.
Apamin. Both solution (see following) and solid-phase oxidations
(Table 7) showed complicated kinetics, including the observation of
HPLC-detected intermediates at tR ) 14.6 min, tR ) 14.8 min (possibly
one-disulfide intermediates), and tR ) 18.2 min (not reduced by DTT
and not formed in the presence of 8 M guanidinium hydrochloride).
(A) Linear reduced apamin (1 mg, 0.5 µmol, tR ) 15.3 min), purified
by preparative HPLC (gradient, conducted over 40 min, of 0.1%
aqueous TFA and 0.1% TFA-CH3CN from 9:1 to 11:9) was dissolved
in 0.1 M phosphate buffer, pH 6.6 (1 mL). After 2 h at 25 °C, the
solution was analyzed by analytical HPLC (gradient, started after 2
min and conducted over 30 min, of 0.1% aqueous TFA and 0.1% TFA-
CH3CN from 9:1 to 7:3) and was found to contain no starting substrate,
76% of a peak at tR ) 11.2 min corresponding to apamin (verified by
co-injection with a standard), and several unidentified peaks.
(B) Linear reduced apamin (1 mg, 0.5 µmol), purified by preparative
HPLC, was dissolved in 1% aqueous HOAc, pH 2.7 (0.8 mL), and
DMSO (0.2 mL) was added. After 24 h at 25 °C, HPLC analysis
revealed 60% of desired apamin, tR ) 11.2 min; 1% of mispaired isomer
1, tR ) 13.7 min; and 39% of mispaired isomer 2, tR ) 13.8 min.
Oxidation of Selected Peptide Substrates by Ellman’s Reagent
(1) in Solution. For solubility reasons, reactions were carried out in
0.1 M phosphate buffer, pH 7.3. Peptide stock solutions were 0.6 mM
(200 µL used per experiment), and reactions were initiated by adding
aliquots from a 4.2 mM stock solution of DTNB (1) to achieve peptide:
DTNB ratios ranging from 10:1 to 0.5:1. Reactions were monitored
in real time by UV (after 15-fold dilution: full spectra from 190-540
nm and single wavelength measurements at 412 nm) and by HPLC.
The amount of TNB- formed was deduced from the UV data; the value
did not change after 5 min and was in close agreement with theoretical
expectation (for peptide in excess, the amount of DTNB added; for
DTNB in excess, the amount of SH groups in the reduced peptide
substrate). The highest absolute amounts of desired disulfide product
formed were observed within 15 min and with peptide:DTNB ratios
near to 1; these values were ∼85% for deamino-oxytocin, ∼50% for
somatostatin, and ∼75% for conotoxin SH 3&13. However, for all of
these peptides, up to three additional peaks were observed in the HPLC,
and the levels of these extra peaks increased with time while that for
the desired disulfide was reduced. The additional peaks absorbed at
330 nm and were reduced by DTT; they are tentatively assigned as
bis(TNB) derivatives of the parent peptide and/or its dimers. More
detailed investigations of the structures of these byproducts, and the
kinetics of their formation and interconversion, are beyond the scope
of this work.
One-Disulfide r-Conotoxin Analogues. Syntheses and character-
izations of the linear precursors are described in the Supporting
Information, and oxidations mediated by solid-phase Ellman’s reagents
are covered in Tables 1-6. In addition, a larger-scale experiment used
conotoxin SH 3&7 (3.7 mg, 2.5 µmol). Treatment with El-Lys(El)-
PEG-PS disulfide (190 mg, 0.2 mmol/g) in 1% aqeuous HOAc-
CH3CN-CH3OH (2:1:1, 3.8 mL) showed complete oxidation in ∼24
h (crude yield of 78%), and preparative HPLC (gradient, conducted
over 40 min, of 0.1% aqueous TFA and CH3CN from 19:1 to 3:2)
gave conotoxin SS 3&7 (1.4 mg, 0.9 µmol) in an overall purified yield
of 37%. Control oxidations in solution used reduced peptide substrate
(1 mg, 0.7 µmol) in buffer (1 mL), stirred at 25 °C, with the following
results: (a) oxidation of conotoxin SH 2&7 was complete and
quantitative in 10 h at pH 6.6, based on the analytical HPLC retention
(tR ) 17.5 min for SH, tR ) 17.8 min for SS) and area (gradient,
conducted over 25 min, of 0.1% aqueous TFA and 0.1% TFA-
CH3CN from 1:0 to 7:3); (b) the same finding for conotoxin SH 3&7
(tR ) 18.5 min for SH, tR ) 17.6 min for SS), except that a reaction
time of 30 h was required; (c) the same finding for conotoxin SH 3&13
(tR ) 18.6 min for SH, tR ) 17.4 min for SS), except that a reaction
time of 24 h was required; (d) at pH 2.7, none of these substrates
oxidized to any significant extent, over a 24-h period; and (e) incubation
of these peptides (1 mg), dissolved in pH 6.6 buffer-CH3CN-
CH3OH (2:1:1, 1 mL), with acetylated PEG-PS (40 mg, 0.2 mmol/g)
for 30 min gave the following relatively low amounts of oxidized
products formed: 10% for conotoxin SH 3&7; 12% for conotoxin SH
3&13, and 15% for conotoxin SH 2&7.
r-Conotoxin SI. Experiments using solid-phase Ellman’s reagents
are summarized in Table 7, and discussed in the text. There follow
descriptions of control oxidations in solution, and reference analytical
data. For both the solution and solid-phase oxidations, only the starting
reduced peptide (tetrathiol) and the final fully oxidized products
(naturally occurring as well as mispaired regioisomers) were observed
by HPLC.
(A) Crude linear reduced conotoxin (20 mg, 14.7 µmol) was
dissolved in 0.01 M phosphate buffer, pH 7.5 (19 mL), and DMSO
(0.2 mL) was added. After 20 h at 25 °C, the reaction mixture was
lyophilized, and the crude peptide was analyzed by HPLC, under
conditions identical to those just described for the one-disulfide
R-conotoxin analogues, and was shown to comprise 84% natural
regioisomer (tR ) 19.6 min), 8% mispaired isomer SS 2&3, 7&13 (tR
) 18.6 min), and 8% mispaired isomer SS 2&13, 3&7 (tR ) 18.8 min).
Purification of the main product by preparative HPLC, under conditions
identical to those just described for the one-disulfide R-conotoxin
analogues, gave 5 mg (3.7 µmol, 25%) of material that matched a
standard by co-injection and had FABMS (m/z): calcd for
C55H84N16O16S4: 1352.51; found, 1353.5 [MH]+.
Acknowledgment. I.A. is grateful to the National Science
Foundation for a graduate fellowship. We thank the National
Institutes of Health (GM 43552, 51628) for support of this
research.
Supporting Information Available: Additional experimen-
tal information, including details on general procedures and
instrumentation, characterizations of all organic compounds, and
methods for the assembly and characterizations of the linear
peptide precursors (9 pages, print/PDF). See any current
masthead page for ordering information and Web access
instructions.
(B) Crude linear reduced conotoxin (1.0 mg, 0.74 µmol) was
dissolved in 0.1 M phosphate buffer, pH 6.6 (0.9 mL), and DMSO
(0.1 mL, 10% v/v) was added. After 19 h at 25 °C, the reaction mixture
was analyzed by HPLC and was shown to comprise 86% natural
regioisomer, 6% mispaired isomer SS 2&3, 7&13, and 8% mispaired
isomer SS 2&13, 3&7.
(C) Crude linear reduced conotoxin (1.0 mg, 0.74 µmol) was
JA981111P