Discovery of a highly selective and efficient reagent for formation of intramolecular disulfide bonds in peptides

Article abstract of DOI:10.1021/ja0008618

We have discovered that trans-[Pt(en)₂Cl₂]²⁺ (en = ethylenediamine) is a highly selective and efficient reagent for the quantitative formation of intramolecular disulfide bonds in peptides. A series of 14 dithiol peptides which form disulfide-containing rings ranging in size from 14 to 53 atoms were used to characterize the reagent. The dithiol peptides are cleanly and rapidly converted to their disulfide forms by a slight excess of the platinum complex under mild reaction conditions (slightly acidic and neutral media). For all the dithiol peptides studied, including a penicillamine-derived peptide, the oxidation yields range from 97% to 100%. No side reactions were observed, including no oxidation of the methionine side chain. The reaction kinetics for oxidation of reduced pressinoic acid were found to be second order overall: rate = k'[Pt(IV)][dithiol peptide], where k' is a pH-dependent second-order rate constant. Values of 0.60 ± 0.01, 3.5 ± 0.2, and 22 ± 1 M^-1 s^-1 were determined for k' at pH 3.0, 4.0, and 5.0, respectively (25 °C and 0.45 M ionic strength). A reaction mechanism for oxidation of dithiol peptides by [Pt(en)₂Cl₂]²⁺ is proposed. [Pt(en)₂Cl₂]²⁺ and its reduction product [Pt(en)₂]²⁺ are essentially substitution inert under the conditions used for disulfide formation, they are nontoxic, and they are readily separated from peptides by HPLC. The characteristics of [Pt(en)₂Cl₂]²⁺ and its reaction properties with dithiol peptides suggest that [Pt(en)₂Cl₂]²⁺ is a universal reagent for the rapid and quantitative formation of intramolecular disulfide bonds in peptides.

Full text of DOI:10.1021/ja0008618

VOLUME 122, NUMBER 29
J ULY 26, 2000
© Copyright 2000 by the
American Chemical Society
Discovery of a Highly Selective and Efficient Reagent for Formation
of Intramolecular Disulfide Bonds in Peptides
Tiesheng Shi and Dallas L. Rabenstein*
Contribution from the Department of Chemistry, UniVersity of California, RiVerside, California 92521
ReceiVed March 9, 2000
Abstract: We have discovered that trans-[Pt(en)2Cl2]2+ (en ) ethylenediamine) is a highly selective and efficient
reagent for the quantitative formation of intramolecular disulfide bonds in peptides. A series of 14 dithiol
peptides which form disulfide-containing rings ranging in size from 14 to 53 atoms were used to characterize
the reagent. The dithiol peptides are cleanly and rapidly converted to their disulfide forms by a slight excess
of the platinum complex under mild reaction conditions (slightly acidic and neutral media). For all the dithiol
peptides studied, including a penicillamine-derived peptide, the oxidation yields range from 97% to 100%. No
side reactions were observed, including no oxidation of the methionine side chain. The reaction kinetics for
oxidation of reduced pressinoic acid were found to be second order overall: rate ) k′[Pt(IV)][dithiol peptide],
where k′ is a pH-dependent second-order rate constant. Values of 0.60 ( 0.01, 3.5 ( 0.2, and 22 ( 1 M-1 s-1
were determined for k′ at pH 3.0, 4.0, and 5.0, respectively (25 °C and 0.45 M ionic strength). A reaction
mechanism for oxidation of dithiol peptides by [Pt(en)2Cl2]2+ is proposed. [Pt(en)2Cl2]2+ and its reduction
product [Pt(en)2]2+ are essentially substitution inert under the conditions used for disulfide formation, they are
nontoxic, and they are readily separated from peptides by HPLC. The characteristics of [Pt(en)2Cl2]2+ and its
reaction properties with dithiol peptides suggest that [Pt(en)2Cl2]2+ is a universal reagent for the rapid and
quantitative formation of intramolecular disulfide bonds in peptides.
Introduction
A variety of oxidants have been explored for formation of
disulfide bonds; however, all have drawbacks and some limita-
tions in practical use, including formation of dimers/oligomers
and/or side reactions on the side chains of methionine, tyrosine,
and tryptophan.1,6 For instance, thallium(III) trifluroacetate is a
mild oxidant that can be employed as an alternative to iodine,
and it gives a higher efficiency in some cases.1,7 The major
drawback of this reagent is its high toxicity. Also, thallium can
be difficult to remove from sulfur-containing peptides due to
its high affinity for sulfur and the oxidation conditions need to
be optimized (solvent, reaction temperature, and the concentra-
tion of Tl(III)).1,7
Although formation of intrapeptide disulfide bonds can
generally be achieved by oxidation of the free thiol or sulfur-
protected precursors, a continuing challenge in peptide synthesis
has been to develop more efficient and selective methods.1-6
(1) For reviews see: (a) Andreu, D.; Albericio, F.; Sole´, N. A.; Munson,
M. C.; Ferrer, M.; Barany, G. In Peptide Synthesis Protocols; Pennington,
M. W., Bunn, B. M., Eds.; Humana Press: Totowa, NJ, 1994; pp 91-169.
(b) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Bo¨hner, S.; Siedler, F.
Biopolymers 1996, 40, 207-234. (c) Annis, I.; Hargittai, B.; Barany, G.
Methods Enzymol. 1997, 289, 198-221.
(2) Tam, J. P.; Wu, C.-R.; Liu, W.; Zhang, J.-W. J. Am. Chem. Soc.
1991, 113, 6657-6662.
(3) Munson, M. C.; Barany, G. J. Am. Chem. Soc. 1993, 115, 10203-
10216.
(7) (a) Fujii, N.; Otaka, A.; Funakoshi, S.; Bessho, K.; Watanabe, T.;
Akaji, K.; Yajima, H. Chem. Pharm. Bull. 1987, 35, 2339-2347. (b)
Albericio, F.; Hammer, R. P.; Garcia-Echeverria, C.; Molins, M. A.; Chang,
J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Peptide Protein
Res. 1991, 37, 402-413.
(4) Shik, H. J. Org. Chem. 1993, 58, 3003-3008.
(5) Annis, I.; Chen, L.; Barany, G. J. Am. Chem. Soc. 1998, 120, 7226-
7238.
(6) Shi, T.; Rabenstein, D. L. J. Org. Chem. 1999, 64, 4590-4595.
10.1021/ja0008618 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

6810 J. Am. Chem. Soc., Vol. 122, No. 29, 2000
Shi and Rabenstein
Table 1. Dithiol Peptides Used in the Study To Characterize the Disulfide Bond Forming Properties of [Pt(en)2Cl2]2+
no.
sequence of dithiol peptides
ring sizea
oxidation yield,b,c
%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Ac-Cys-Gly-Pro-Cys-NH2
Ac-Cys-Pro-Phe-Cys-NH2
Ac-Thr-Cys-Pro-Phe-Cys-Arg-NH2
Ac-Pro-Thr-Cys-Pro-Phe-Cys-Arg-Lys-NH2
Ac-Lys-Pro-Thr-Cys-Pro-Phe-Cys-Arg-Lys-Thr-NH2
Ac-Thr-Asp-Ile-Thr-Cys-Gly-Tyr-Cys-His-Lys-Leu-His-NH2
Ac-Cys-Pro-Phe-Ala-Ala-Cys-NH2
Cys-Tyr-Phe-Gln-Asn-Cys (reduced pressinoic acid)
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (reduced oxytocin)
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 (reduced arginine-vasopressin)
Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (reduced somatostatin)
Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Pro-Leu-Met-NH2
Cys-Phe-Gly-Ser-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Met-Gly-Cys-Gly-Arg-Phe
14
14
14
14
14
14
20
20
20
20
38
14
53
20
100
97
100
100
100
100
100
100
98
100
99
99
100
97
d
HSC(Me)2CH2CO-Tyr(Me)-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
a Ring members for the oxidized forms. b Oxidation yield is reported for the single step of conversion of dithiol peptides to their disulfide forms
with slight excess of platinum(IV) complex. c The experimental errors for the yields are estimated to be (5%. d Reduced form of [1-deaminopeni-
cillamine, 2-(O-methyl)tyrosine]arginine-vasopressin.
In a previous report, we demonstrated that trans-[Pt-
(CN)4Cl2]2- is an efficient reagent for the rapid and quantitative
oxidation of dithiol peptides to their disulfide forms.6 Oxidation
takes place by a Cl+ atom transfer mechanism. Unfortunately,
[Pt(CN)4Cl2]2- is a sufficiently strong oxidizing agent (E°′ )
0.926 V8) that it also oxidizes methionine to methionine
sulfoxide.6,9 However, this was the only side reaction observed
for an otherwise excellent reagent for formation of intramo-
lecular peptide disulfide bonds, which encouraged us to
investigate other trans-dichloro-Pt(IV) complexes; by tuning
down the redox potential of the complex, it may be possible to
eliminate this side reaction while still maintaining the rapid and
quantitative nature of the reaction. A major design consideration
was that the equatorial coordination sites should be blocked to
avoid reaction of the product Pt(II) complex with other
functional groups of the peptide. In this paper, we report that,
by pursuing this design approach, we have discovered that trans-
[Pt(en)2Cl2]2+ (en ) ethylenediamine, E°′ for [Pt(en)2Cl2]2+
/
[Pt(en)2]2+ is 0.58 V10) is a highly selective and efficient reagent
for formation of intramolecular disulfide bonds in peptides
without any observed side reactions. Further, the reaction
conditions are mild and the Pt(IV) complex and its Pt(II)
reduction product [Pt(en)2]2+ are nontoxic and readily separable
from peptides, which are desirable properties for a disulfide
bond-forming reagent.1,2,5 These properties together with the
results presented here suggest that the discovery of [Pt(en)2Cl2]2+
as a disulfide bond-forming reagent represents a particularly
significant development in the search for efficient methods for
formation of intrapeptide disulfide bonds.
Figure 1. Chromatograms of 80 µM 1 in water (top) and the products
from the reaction of 80 µM 1 and 150 µM [Pt(en)2Cl2]2+ in pH 7.3
phosphate buffer for 30 min (bottom). The mobile phase contained 5%
acetonitrile, 0.10 M NaH2PO4, and sufficient H3PO4 to adjust the pH
to 2.5. Peak assignments: A, solvent; B, solvent + [Pt(en)2Cl2]2+
[Pt(en)2]2+; C, 1; and D, oxidized form of 1.
+
Results and Discussion
The dithiol peptides used in the present study (Table 1) range
from 4 to 20 amino acid residues in length and the disulfide-
containing rings vary in size from 14 to 53 atoms.
Peptides 1-7. Peptides 1-6 are model compounds for the
active site of thioredoxin (1),11 glutathione thioltransferase (2-
5),12 and the disulfide bond-forming protein DsbC (6).13 Peptide
7 is derived from peptide 1, with two additional alanine residues
to increase the ring size in the oxidized form. Reaction of
peptides 1-7 with [Pt(en)2Cl2]2+ to form the intramolecular
disulfide bond was monitored by isocratic reversed phase HPLC.
To illustrate, Figure 1 gives results for the oxidation of 1. The
top chromatogram is for a freshly prepared 80 µM solution of
1; the bottom chromatogram is for the products obtained after
reaction of 80 µM 1 and 150 µM [Pt(en)2Cl2]2+ in pH 7.3
phosphate buffer for 30 min. The peak assignments given in
the legend to Figure 1 for [Pt(en)2Cl2]2+ and [Pt(en)2]2+ are
based on chromatograms for authentic samples. The broad peak
at 23.2 min is assigned to the disulfide form of 1 based on the
mass spectrometric results (theoretical 416.5, found 417). The
broad peak for the disulfide form is ascribed to the slow cis-
trans isomerization of the glycine-proline peptide bond and a
(8) Goldberg, R. N.; Helper, L. G. Chem. ReV. 1968, 68, 229-252.
(9) Shi, T.; Berglund, J.; Elding, L. I. J. Chem. Soc., Dalton Trans. 1997,
2073-2077.
(10) Bard, A. J.; Parsons, R.; Jordan, J. Standard Potentials in Aqueous
Solution; Marcel Dekker: New York, 1985; pp 345-365.
(11) Holmgren, A. Annu. ReV. Biochem. 1985, 54, 237-271.
(12) Gan, Z.-R.; Wells, W. W. J. Biol. Chem. 1986, 261, 996-1001.
(13) Zapun, A.; Missiakas, D.; Raina, S.; Creighton, T. E. Biochemistry
1995, 34, 5075-5089.

Formation of Intramolecular Disulfide Bonds in Peptides
J. Am. Chem. Soc., Vol. 122, No. 29, 2000 6811
Figure 2. Chromatograms of 100 µM 8 (top) and a reaction mixture
of 100 µM 8 and 150 µM [Pt(en)2Cl2]2+ in a pH 7.3 phosphate buffer
after reaction for 30 min (bottom). The mobile phase contained 12%
acetonitrile, 0.10 M NaH2PO4, and sufficient H3PO4 to adjust the pH
Figure 3. Chromatograms of 40 µM 13 (top) and the products from
the reaction of 40 µM 13 and 150 µM [Pt(en)2Cl2]2+ in pH 6.0
phosphate buffer for 30 min (middle) and 10 h (bottom). The mobile
phase contained 20% acetonitrile, 0.10 M NaH2PO4, and sufficient H3-
PO4 to adjust the pH to 2.5. Peak assignments: A, solvent; B, solvent
+ [Pt(en)2Cl2]2+ + [Pt(en)2]2+; C, 13; and D, oxidized form of 13.
to 2.5. Peak assignments: A, solvent; B, solvent + [Pt(en)2Cl2]2+
[Pt(en)2]2+; C, oxidized form of 8; and D, 8.
+
relatively high population of the cis isomer.14,15 The bottom
chromatogram was also run for a longer time than shown in
Figure 1; no late-eluting peaks due to dimers or higher oligomers
were observed. The chromatograms in Figure 1 indicate that a
slight excess of [Pt(en)2Cl2]2+ cleanly and rapidly transforms 1
to its intramolecular disulfide form. It was determined by using
the peak area and the HPLC response factor for the disulfide
form of the peptide, together with an HPLC detection limit of
3%, that the transformation is quantitative within experimental
error, cf. Table 1. Chromatographic patterns for the products
of the oxidation of peptides 2-7 are similar to that for 1 except
that peaks for the disulfide forms are sharp, cf. Supporting
Information.
Reduced Peptide Hormones 8-11. Peptides 8-11 are the
reduced forms of the disulfide-containing hormones pressinoic
acid, oxytocin, arginine-vasopressin, and somatostatin, respec-
tively. Figure 2 presents results for oxidation of peptide 8 by
[Pt(en)2Cl2]2+. The top chromatogram is for a freshly prepared
100 µM solution of 8. The bottom chromatogram is for a
reaction mixture of 100 µM 8 and 150 µM [Pt(en)2Cl2]2+ in
pH 7.3 phosphate buffer after 30 min of reaction. The peak at
15.7 min in the bottom chromatogram was assigned to the
oxidized form of 8 by comparison to the chromatogram of an
authentic sample of pressinoic acid. Peak area measurements
indicate that conversion of 8 to its disulfide form is quantitative.
The reaction of [Pt(en)2Cl2]2+ with peptides 9-11 was also
found to be rapid and quantitative for these reaction conditions,
cf. Supporting Information.
In previous syntheses of oxytocin and arginine-vasopressin
and their derivatives,16-19 it was reported that oxidation of the
dithiol groups by ferricyanide results predominantly, but not
exclusively, in formation of intramolecular disulfide bonds. This,
however, was not the case for the related hexapeptides,
tocinamide and pressinamide.19 Oxidation of the dithiol forms
of tocinamide and pressinamide by ferricyanide results mainly
in the formation of dimers and higher polymers.19 In contrast,
reaction with [Pt(en)2Cl2]2+ results in the quantitative formation
of intramolecular disulfide bonds, regardless of the peptide
sequence.
Methionine-Containing Peptides 12 and 13. Peptides 12
and 13 are the dithiol forms of the methionine-containing
peptides [Cys3,6, Tyr8, Pro9]-Substance P (selective NK-1
agonist) and Atrial Natriuretic Factor (ANF, 11-30, frog),
respectively.20,21 Figure 3 shows chromatograms for the oxida-
tion of 13 in a phosphate buffer at pH 6.0; peak assignments
(16) Hope, D. B.; Murti, V. V. S.; Vigneaud, V. D. J. Biol. Chem. 1962,
237, 1563-1566.
(17) Hruby, V. J.; Upson, D. A.; Agarwal, N. S. J. Org. Chem. 1977,
42, 3552-3556.
(18) Live, D. H.; Agosta, W. C.; Cowburn, D. J. Org. Chem. 1977, 42,
3556-3561.
(19) Moore, G. Biochem. J. 1978, 173, 403-409.
(20) Lavielle, S.; Chassaing, G.; Ploux, O.; Loeuillet, D.; Besseyre, J.;
Julien, S.; Marquet, A.; Convert, O.; Beaujouan, J.-C.; Torrens, Y.;
Bergstro¨m, L.; Saffroy, M.; Glowinski, J. Biochem. Pharmacol. 1988, 37,
41-49.
(14) (a) Larive, C. K.; Rabenstein, D. L. J. Am. Chem. Soc. 1993, 115,
2833-2836. (b) Rabenstein, D. L.; Shi, T.; Spain, S. J. Am. Chem. Soc.
2000, 122, 2401-2402.
(21) Lazure, C.; Ong, H.; McNicoll, N.; Netchitailo, P.; Chre´tien, M.;
De Le´an, A.; Vaudry, H. FEBS Lett., 1988, 238, 300-306.
(15) Shi, T.; Spain, S.; Rabenstein, D. L. Unpublished results.

6812 J. Am. Chem. Soc., Vol. 122, No. 29, 2000
Shi and Rabenstein
and reaction conditions are given in the figure legend. The top
chromatogram is for a fresh solution of 40 µM 13, and the
middle and bottom chromatograms are for a mixture of 40 µM
13 and 150 µM [Pt(en)2Cl2]2+ in pH 6.0 phosphate buffer after
30 min and 10 h of reaction, respectively. The peak at 23.7
min is assigned to the oxidized form based on its retention time
being identical with that for an authentic sample and the
molecular weight of the isolated peptide (theoretical 2116.6;
found 2116). As determined by HPLC analysis, 100% of the
dithiol peptide was converted to the disulfide form after reaction
for 30 min. Also, the excess [Pt(en)2Cl2]2+ and the Pt(II) product
in the reaction mixture did not interact with the oxidized peptide
including the methionine side chain, as evidenced by the fact
that the chromatogram obtained after 10 h of reaction was
identical with that obtained after 30 min of reaction. Similar
results were obtained for peptide 12, which also contains a
methionine residue. Formation of the 53-membered disulfide-
containing ring in the oxidized form of peptide 13, and also
the 38-membered ring in 11, was found to be as rapid and as
efficient as formation of the much smaller rings in peptides 1-7.
Oxidation yields, which range from 97% to 100%, are given
in Table 1. It is noteworthy that the peptides studied include
all the amino acids with readily oxidizable side chains (me-
thionine, tyrosine, and tryptophan). Thus, the oxidation of dithiol
peptides to their disulfide forms by [Pt(en)2Cl2]2+ is quantitative
within experimental error, with no known side reactions. The
oxidation reaction can be described by eq 1.
Figure 4. Chromatograms of 100 µM 14 (top) and the products from
the reaction of 100 µM 14 and 200 µM [Pt(en)2Cl2]2+ in pH 4.0
phosphate buffer for 1.5 h (bottom). The mobile phase contained 24%
acetonitrile, 0.10 M NaH2PO4, and sufficient H3PO4 to adjust the pH
to 2.5. Peak assignments: A, solvent; B, solvent + [Pt(en)2Cl2]2+
+
[Pt(en)2]2+; C, oxidized form of 14; and D, 14. Minor peaks at 11.0
and 16.7 min are probably from side reaction products, cf. text.
Peptide 14. The oxidation of dithiol peptides by [Pt-
(en)2Cl2]2+ was extended to penicillamine-derived peptide 14,22
the purpose being to examine the oxidation efficacy when steric
hindrance near the thiol groups is increased. Figure 4 shows
results for oxidation of 14; the reaction conditions are given in
the figure caption. The bottom chromatogram was also run for
a longer time; no late-eluting peaks due to dimers or higher
oligomers were observed. The peak at 12.3 min was assigned
to the disulfide form of 14 by comparison to the retention time
of an authentic sample. It is of interest to note that, although
steric hindrance due to the introduction of a deamino-penicil-
lamine increases the difficulty for ring closure, formation of
the disulfide form of 14 (yield 97%) is still the predominant
reaction. However, some minor peaks (ca. 3%) are observed in
Figure 4, which are probably due to side products as discussed
below.
Oxidation by [Pt(CN)4Cl2]2-. Oxidation of peptides 2-5
and 8-11 by [Pt(CN)4Cl2]2- to form the corresponding disul-
fides was found previously to also be essentially quantitative,
with no undesirable side reactions.6 Oxidation of the peptide
Ac-Asp-Ile-Thr-Cys-Gly-Tyr-Cys-His-Lys-Leu-His-Gly-Gln-
Met-Lys-NH2 by [Pt(CN)4Cl2]2- also resulted in quantitative
formation of the intrapeptide disulfide bond; however, the
methionine side chain was oxidized to the sulfoxide form.6 For
a direct comparison of [Pt(CN)4Cl2]2- and [Pt(en)2Cl2]2+ as
reagents for disulfide bond formation in methionine-containing
peptides, peptide 12 was also oxidized with [Pt(CN)4Cl2]2- in
the present study using the reaction conditions reported previ-
ously.6 The predominant reaction product was found to be the
disulfide peptide, but with the methionine side chain converted
to the sulfoxide form (M+: theoretical 1366.8; found 1366.4).
Reaction Conditions. The oxidation reaction was studied
under various conditions. To determine if it is necessary to
isolate the dithiol peptide in pure form before formation of the
disulfide bond, [Pt(en)2Cl2]2+ was reacted directly with the crude
product obtained by cleavage of peptide 6 from solid-phase
peptide synthesis resin and deprotection of side chain functional
groups (vide infra). Chromatograms obtained by semipreparative
HPLC for a reaction mixture containing ca. 2 mM 6 and 3 mM
[Pt(en)2Cl2]2+ (phosphate buffer at pH 6.0, reaction time 30 min)
indicate that 6 is rapidly oxidized to its disulfide form, as
identified by mass spectrometry, cf. Supporting Information.
No additional peaks due to dimer or polymers of 6 were
observed, which indicates that oxidation is still clean and
quantitative even at millimolar peptide concentrations.
The rate of formation of the intramolecular disulfide bond
by reaction with [Pt(en)2Cl2]2+ is pH dependent. Formation of
the disulfide bond by reaction of 100 µM 8 with 250 µM
[Pt(en)2Cl2]2+ was found to be complete in 3 h, 20 min, 3 min,
and 20 s at pH 4.0, 5.0, 6.0, and 7.0, respectively, at room
temperature. No peaks were detected from side products, even
for the pH 7.0 conditions where the reaction took place very
rapidly.
To determine if [Pt(en)2Cl2]2+ can be used to form disulfide
bonds in peptides which are only slightly water-soluble but are
soluble in aqueous-organic mixtures, the oxidation of 13 was
studied in aqueous/acetonitrile solution. Peptide 13 was found
to be cleanly and quantitatively oxidized in aqueous phosphate
buffer containing up to 50% acetonitrile. [Pt(en)2Cl2]Cl2 is
(22) Bankowski, K.; Manning, M. J. Med. Chem. 1978, 21, 850-853.

Formation of Intramolecular Disulfide Bonds in Peptides
J. Am. Chem. Soc., Vol. 122, No. 29, 2000 6813
Scheme 1
Figure 6. Plot of kobsd versus [Pt(IV)] according to eq 3 for reaction
between peptide 8 and [Pt(en)2Cl2]2+ at pH 3.0, 25 °C, and ionic strength
of 0.45 M.
Figure 5. Plot of ln A versus reaction time according to eq 2 for
oxidation of peptide 8 by [Pt(en)2Cl2]2+ in pH 3.0 phosphate buffer.
Reaction conditions: [8] ) 80 µM, [Pt(IV)] ) 2.00 mM, ionic strength
of 0.45 M, and 25 °C.
To determine the order of the reaction with respect to [Pt-
(en)2Cl2]2+, the concentration of [Pt(en)2Cl2]2+ in a pH 3.0
reaction mixture containing 30-80 µM 8 was varied from 0.30
to 2.50 mM. The dependence of kobsd on [Pt(en)2Cl2]2+ is linear
with a zero intercept, as shown in Figure 6, which demonstrates
that the oxidation reaction is also first order with respect to the
[Pt(en)2Cl2]2+. Thus, the oxidation reaction follows an overall
second-order rate law as described by eq 3, where k′ denotes
the pH-dependent second-order rate constant. Values of 0.60
( 0.01, 3.5 ( 0.2, and 22 ( 1 M-1 s-1 were determined for k′
soluble at the millimolar level in mixed aqueous/organic solvent
mixtures with methanol, ethanol, acetonitrile, and acetone.
Kinetics and Reaction Mechanism. The kinetics of the
reaction of peptide 8 with [Pt(en)2Cl2]2+ were studied. The
oxidation reaction was followed by isocratic HPLC by monitor-
ing the disappearance of 8 under pseudo-first-order conditions
with [Pt(en)2Cl2]2+ present in a 10-fold or larger excess. Because
8 can be totally converted to its disulfide form by reaction with
[Pt(en)2Cl2]2+ (Figure 2), the decrease in the area of the peak
for 8 vs time can be described by eq 2 if the reaction is first
order in 8, where A0 and A are the peak areas of 8 at the start
of the reaction and at time t, respectively. Plots of ln A versus
-d[8]/dt ) kobsd[8] ) k′[Pt(IV)][8]
(3)
at 25 °C and ionic strength 0.45 M at pH 3.0, 4.0, and 5.0,
respectively. The reaction is too fast to be followed by HPLC
at pH g6. The oxidation reaction was also studied in 1.00 M
HCl; the reaction of 60 µM 8 with 1.00 mM [Pt(en)2Cl2]2+
yielded ca. 4% and 10% oxidized peptide after 4 and 10 h,
respectively, at room temperature. The yields are comparable
to those obtained by air oxidation, which suggests that the
reaction of [Pt(en)2Cl2]2+ in 1.00 M HCl is negligibly slow.
ln A ) ln A0 - kobsd
t
(2)
time are linear, as shown in Figure 5 for pH 3 data, which
indicates that the reaction is indeed first order in 8; a pseudo-
first-order rate constant of kobsd ) 1.20 × 10-3 s-1 was obtained
from the linear plot in Figure 5.

6814 J. Am. Chem. Soc., Vol. 122, No. 29, 2000
Shi and Rabenstein
The rate law and k′ vs pH dependence for the reaction of
[Pt(en)2Cl2]2+ with 8 are similar to those found for reactions
between trans-dichloro-platinum(IV) complexes and monothiols
such as glutathione.23,24 By analogy with the reaction mecha-
nisms proposed previously for the oxidation of monothiols by
Pt(IV) complexes,23,24 a reaction mechanism for oxidation of
peptide 8 by [Pt(en)2Cl2]2+ is described in Scheme 1. The
reaction proceeds through a transition state in which Cl+ is
transferred from the Pt(IV) center to an incoming thiol or thiolate
nucleophile.6,9,23-26 The mechanism in Scheme 1 shows all
possible protonation states of 8 under the reaction conditions
used in the kinetic study.27 However, as described above, there
is little or no oxidation of peptide 8 by [Pt(en)2Cl2]2+ in 1.00
M HCl, which indicates that oxidation of the fully protonated
form of the peptide is negligibly slow. The reactions described
by k2 and k3 are the rate-determining steps and are assumed to
take place via parallel attack by thiol and thiolate on the
coordinated chloride, resulting in a Cl+ transfer from [Pt(en)2-
Cl2]2+ to the attacking group.6,9,23-26 The intermediates formed,
denoted by I and II in Scheme 1, then undergo an intramolecular
nucleophilic attack to form the disulfide bond. The rates of
formation of the disulfide bond from intermediates I and II are
expected to be largely controlled by conformational changes,
which are expected to be fast relative to the atom transfer step.28
This is similar to what has been found in detailed studies of
the kinetics of formation of intramolecular disulfide bonds in
peptides such as 9-11 by thiol-disulfide exchange,29,30 where
the nucleophilic displacement reaction in the second step of the
overall intramolecular disulfide bond forming reaction is
formally similar to the nucleophilic displacement of Cl- from
intermediates I and II. The relatively fast rates of ring closure
by formation of intramolecular disulfide bonds by thiol-
disulfide exchange reactions, even in the formation of the
disulfide bond to give the 38-membered ring of somatostatin,
are attributed to high effective concentrations of the attacking
thiolate. 29,30
The reaction rate increases almost exponentially as the
solution pH is increased, which indicates that the thiolate anion
is much more reactive than the protonated thiol forms.23,24 Thus,
at the pHs used in the kinetic studies, the reaction described by
k3 in Scheme 1 is the predominant oxidation pathway, while
the reaction path described by k2 contributes only slightly to
the overall reaction at pH 3-5.
Reaction mechanisms similar to that described in Scheme 1
can be derived for the other peptides in Table 1. However, for
peptide 14, reaction intermediates similar to I and II in Scheme
1 apparently undergo ring closure reactions more slowly due
to steric hindrance from the two methyl groups on the deamino-
penicillamine residue. Under such circumstances, the reaction
intermediates are sufficiently long-lived that hydrolysis to form
RSOH competes with ring closure by nucleophilic displacement
of Cl-. The RSOH formed can then be further oxidized to give
RSO2H and RSO3H, or it can undergo intermolecular reactions
with the thiol or thiolate groups of other molecules, resulting
in formation of dimers and higher polymers.6 This may explain
the presence of up to ∼3% side reaction products in the
oxidation of peptide 14, as indicated by the extra peaks in the
chromatograms shown in Figure 4.
It also is of interest to compare the properties of [Pt(en)2Cl2]2+
with those of [Pt(CN)4Cl2]2- as an oxidant for the formation of
intramolecular disulfide bonds in peptides. [Pt(CN)4Cl2]2- is a
somewhat stronger oxidizing agent, as indicated by its redox
potential, and as a result it can rapidly convert dithiol peptides
to their disulfide forms at lower pH (1-3) than [Pt(en)2Cl2]2+
.
The disadvantage, however, is that [Pt(CN)4Cl2]2- is a suf-
ficiently strong oxidizing agent that it can oxidize the sulfur of
the methionine side chain. Similar reaction rates can be achieved
with [Pt(en)2Cl2]2+ by carrying out the reaction at higher pH
(4-7) where a larger fraction is in the thiolate form; however,
oxidation of the methionine sulfur, which is a pH-independent
oxidation, does not occur.
The advantages and limitations of other reagents for forming
intrapeptide disulfide bonds, including the metal complexes
[Fe(CN)6]3- and Tl(tfa)3 and nonmetallic oxidants such as
oxygen, disulfides, dimethyl sulfoxide, iodine, and a mixture
of methyltrichlorosilane-diphenylsulfoxide, have been de-
scribed in previous publications, including the comprehensive
review by Annis et al.1c In general, these reagents are nonselec-
tive toward formation of intrapeptide disulfide bonds; dimers
and higher oligomers can also form and the oxidation conditions
need to be optimized with respect to pH, solvent, and concentra-
tions of peptide and oxidant. Also, depending on the oxidant,
side products can be formed by reaction with the side chains of
Met, Trp, and Tyr. In contrast, the results presented here show
that [Pt(en)2Cl2]2+ is a highly selective oxidant for the formation
of intramolecular disulfide bonds over a range of solution
conditions.
Conclusions. We have discovered a highly selective and
efficient reagent, trans-[Pt(en)2Cl2]2+, for formation of intra-
molecular disulfide bonds in peptides. It rapidly and quantita-
tively converts the dicysteine peptide precursor, even at milli-
molar concentrations, to its disulfide form in slightly acidic and
neutral media. As compared to [Pt(CN)4Cl2]2-, [Pt(en)2Cl2]2+
is a somewhat weaker oxidizing agent. No side reactions were
observed with [Pt(en)2Cl2]2+, including no oxidation of the
methionine side chain. [Pt(CN)4Cl2]2- is a sufficiently strong
oxidizing agent that it can rapidly oxidize dicysteine peptides
at pH 1-3. Similar rates can be achieved with [Pt(en)2Cl2]2+
by running the reaction at pH 4-7, where a larger fraction of
the thiol groups are in the thiolate form. The Pt(IV) complex is
also highly efficient for the oxidation of penicillamine-derived
dithiol peptides. Moreover, [Pt(en)2Cl2]2+ and its reduction
product [Pt(en)2]2+ are essentially substitution inert under the
conditions used for disulfide bond formation, nontoxic, and
readily separable from peptides by HPLC. Thus, [Pt(en)2Cl2]2+
should be widely useful for the rapid and quantitative formation
of intramolecular disulfide bonds in synthetic peptides.
(23) Shi, T.; Berglund, J.; Elding, L. I. Inorg. Chem. 1996, 35, 3498-
3503.
(24) Lemma, K.; Shi, T.; Elding, L. I. Inorg. Chem. 2000, 39, 1728-
1734.
(25) Shi, T.; Elding, L. I. Inorg. Chim. Acta 1998, 282, 55-60.
(26) Wilmarth, W. K.; Fanchiang, Y.-T.; Byrd, J. E. Coord. Chem. ReV.
1983, 51, 141-153.
(27) Noszal, B.; Guo, W.; Rabenstein, D. L. J. Org. Chem. 1992, 57,
2327-2334.
(28) Creighton, T. E. Proteins: Structures and Molecular Properties,
2nd ed.; W. H. Freeman and Company: New York, 1993; Chapter 5.
(29) Rabenstein, D. L.; Yeo, P. L. J. Org. Chem. 1994, 59, 4223-4229.
(30) Rabenstein, D. L.; Weaver, K. H. J. Org. Chem. 1996, 61, 7391-
7397.
Experimental Section
Materials. [Pt(en)2]Cl2 and dithiothreitol (DTT) were obtained from
Aldrich. Phosphoric acid, sodium dihydrogen phosphate, sodium
monohydrogen phosphate, trifluoroacetic acid (TFA), and HPLC-grade
acetonitrile were purchased from Fisher Scientific Co. [Pt(en)2Cl2]Cl2
was prepared from [Pt(en)2]Cl2 according to a literature method;31,32
the UV-visible spectrum was in good agreement with that reported.
(31) Basolo, F.; Bailar, J. C.; Tarr, B. P. J. Am. Chem. Soc. 1950, 72,
2433-2438.
(32) Poe¨, A. J. J. Chem. Soc. 1963, 183-188.

Formation of Intramolecular Disulfide Bonds in Peptides
J. Am. Chem. Soc., Vol. 122, No. 29, 2000 6815
The peptide hormones pressinoic acid, oxytocin, arginine-vasopressin,
and somatostatin, [Cys3,6, Tyr8, Pro9]-Substance P,20 and [1-deami-
nopenicillamine, 2-(O-methyl)tyrosine]arginine-vasopressin22 were ob-
tained from Bachem Inc. (Torrance, CA). The peptide ANF (11-30,
frog)21 was supplied by PepSyn Co. (Dublin, CA). Water was purified
with a Millipore water purification system.
Peptide Synthesis and Purification. Peptides 1-7 in Table 1 were
synthesized on a Millipore Model 9050 Plus peptide synthesizer using
FMOC solid-phase peptide synthesis methods. A reagent of 88% TFA,
5% phenol, 5% water, and 2% triisopropylsilane was used to cleave
the peptide chain from the resin and remove side chain protecting
groups; crude peptide product was obtained after lyophilization. Peptides
1-7 were isolated from the crude mixture by reversed-phase gradient
HPLC on a 100 mm × 250 mm C18 column with an acetonitrile-
water mobile phase containing 0.1% TFA. The identities of the peptides
were confirmed by molecular weight. Peptides 8-14 were prepared
by reduction of their disulfide forms with a large excess of DTT at pH
7.0 and were isolated from the reaction mixture using the same HPLC
system.
Analysis of the Oxidation Reactions by HPLC. Oxidation reaction
mixtures were analyzed by isocratic HPLC on a 3.2 mm × 100 mm
C18 reversed-phase column (particle size 3 µm). The UV-detector was
set at 215 nm. Mobile phases were prepared by addition of NaH2PO4
(0.10 M final concentration) and acetonitrile to water, and the pH was
then adjusted to 2.5 with phosphoric acid. Chromatographic conditions
were optimized for separation of the reduced dithiol and oxidized forms
of the peptides by varying the percentage of acetonitrile in the mobile
phase.33 HPLC mobile phases were filtered through a 0.45 µm cellulose
nitrate filter, sparged with helium for ca. 15 min just before the
experiment, and were used for less than a week.
solution with a slight excess of [Pt(en)2Cl2]2+. We have found it
convenient to use a 2-10 mM stock solution of [Pt(en)2Cl2]2+ in 1.00
M NaCl and phosphate buffer (pH 4-7) containing 1 mM EDTA. A
solution of ca. 2 mM or less dithiol peptide (or crude peptide product
cleaved from synthesis resin) is mixed with the [Pt(en)2Cl2]2+ reagent
in phosphate buffer, with [Pt(en)2Cl2]2+ in slight excess. The mixture
is allowed to react from 2 h (pH 4) to 30 min (pH 7), after which
oxidation of the dithiol peptide to its disulfide form is complete.
Kinetic Measurements. Phosphate buffer solutions (0.10 M) at pH
3.0, 4.0, 5.0, and 6.0 that contained 1 mM EDTA were prepared. Stock
solutions of ca. 1 mM peptide 8 were prepared by dissolving 8 in water
and flushed with helium for 10 min. Each solution was only used for
1 h. Kinetic experiments were conducted by combining stock solutions
of the reactants and buffer which were equilibrated at 25 °C before
mixing. Ionic strength was adjusted to 0.45 M with stock solutions of
1.00 M NaCl. After initiation, the reaction mixture was kept at 25 °C
throughout the experiment. Aliquots were removed as a function of
time and quenched by adding an equal volume of 2.0 M HCl. The
quenched solutions were then analyzed by isocratic HPLC within 4 h.
Control experiments showed that the oxidation of 8 in 1.0 M HCl
solution is negligibly slow, confirming that addition of the reaction
mixture to 2.0 M HCl quenches the reaction.
Acknowledgment. Dr. J. Gu and Mr. M. Fernandez are
thanked for synthesis of some of the peptides used in this work.
Financial support from National Institutes of Health (GM 37000)
is gratefully acknowledged.
Supporting Information Available: Chromatograms for the
oxidation of 2, 6, 7, 10, and 11 by trans-[Pt(en)2Cl2]2+ in
phosphate buffer (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Conditions for Oxidation of Dicysteine Peptides with [Pt-
(en)2Cl2]2+. The optimum conditions for quantitative formation of
intramolecular peptide disulfide bonds are slightly acidic to neutral
(33) Yeo, P. L.; Rabenstein, D. L. Anal. Chem. 1993, 65, 3061-3066.
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