A study to develop platinum(iv) complex chemistry for peptide disulfide bond formation

Article abstract of DOI:10.1039/c9dt04738g

Platinum(iv) complexes with a heterocyclic ligand and an ancillary ligand have been investigated and applied for treating various tumour cell lines. Another application of the Pt(iv) complexes in forming peptide disulfide bonds was investigated in this work. For development of Pt(iv) complex chemistry for disulfide bond formation in peptides, two Pt(iv) complexes, [PtCl₂(phen)(en)]Cl₂ and [PtCl₂(bpy)(en)]Cl₂, were synthesized and characterized using elemental analysis, ESI-MS and NMR. Subsequently, they were investigated as oxidants for the formation of disulfide bonds in various peptides. Excellent purities and yields of disulfide-containing peptides were achieved when the reactions were carried out in aqueous solution. The reactions were completed rapidly in a wide range of pH values even in acidic medium at room temperature. An intramolecular disulfide bond was formed in each of the peptides in a solution containing two dithiol-containing peptides, making the Pt(iv) complexes useful for generating disulfide-containing peptide libraries. In addition, the two Pt(iv) complexes can be used as oxidants for the synthesis of disulfide bonds on a resin, which is a more convenient method to synthesize disulfide-containing peptides through automation.

Full text of DOI:10.1039/c9dt04738g

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A study to develop platinum(IV) complex chemistry for peptide disulfide bondsView Article Online
DOI: 10.1039/C9DT04738G
formation
Changying Song, Jingjing Sun, Xiaowei Zhao, ShuyingHuo* and Shigang Shen*
College of Chemistry and Environmental Science, Key Laboratory of Analytical
Science and Technology of Hebei Province, and MOE Key Laboratory of Medicinal
Chemistry and Molecular Diagnostics, Hebei University, Baoding 071002, Hebei
Province, P. R. China
Revised MS for: Dalton Transactions (ID: DT-COM-12-2019-004738)
MS Type:
Communication
Correspondence to
Dr. Shuying Huo and Dr. Shigang Shen
College of Chemistry and Environmental Science
Hebei University, Baoding 071002, Hebei Province,
P. R. China
E-mail:
Fax:
shuyinghuo@hbu.edu.cn; shensg@hbu.edu.cn
int-(86)-312-5079386

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Abstract: Platinum(IV) complexes with a heterocyclic ligand and ancillarDyOIl:i1g0.a10n3d9/C9DT04738G
have been investigated and applied in treating various tumour cell lines. Another
application of the Pt(IV) complexes in forming peptide disulfide bonds was
investigated in this work. For development of the Pt(IV) complex chemistry for
disulfide bonds formation in peptides, two Pt(IV) complexes, [PtCl2(phen)(en)]Cl2
and [PtCl2(bpy)(en)]Cl2, were synthesized and characterized using elemental analysis,
ESI-MS and NMR. Subsequently, they were investigated as the oxidants for the
formation of disulfide bonds in various peptides. Excellent purities and yields of
disulfide-containing peptides were achieved when the reactions were carried out in
aqueous solution. The reactions were completed rapidly in a wide range of pH even in
acidic medium at room temperature. Intramolecular-disulfide bond was formed in
each of peptides in the solution containing two dithiol-containing peptides, making
the Pt(IV) complexes are useful for disulfide-containing peptide libraries.
Additionally, the two Pt(IV) complexes can be used as oxidants for the synthesis of
disulfide bonds on resin, making the synthesis more automatically in manufacture.
Keywords: platinum(IV) complexes, peptide, disulfide bond, oxidation

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The platinum(IV) complexes with a heterocyclic ligand (1,10-phenanthrolinDeO(I:p10h.1e0n39)/C9DT04738G
or methyl substituted phen) and ancillary ligand (S,S or R,R isomer of
1,2-diaminocyclohexane or ethylenediamine) have raised interest due to their
encouraging cytotoxicity results in vitro.1 Pt(IV) complexes possess six-coordinate
octahedral geometry and are kinetically inert. Their anticancer activities are triggered
by a two-electron reduction.1 The reductants involve cysteine, homocysteine,
glutathione, ascorbic acid et al. Among these reductants, thiol-containing compounds
can be oxidized to their disulfide forms.1,2 Therefore, the anticancer active Pt(IV)
complex may be used as an oxidant for synthesis of disulfide bonds in peptides.
Recently,
we
synthesized
a
phen-liganded
Pt(IV)
complex
trans-[Pt(OH)2(phen)(en)]2+, it was successfully used as an oxidant for formation of
peptide disulfide bonds in a aqueous solution.3 An additional two Pt(IV) complexes,
trans-[PtCl2(CN)4]2- and trans-[PtCl2(en)2]2+, have also been investigated for the
purpose of forming peptide disulfides.4 However, trans-[PtCl2(CN)4]2- can oxidize
methionine residue to methionine sulfoxide, making it unsuitable for the formation of
disulfide bond in a methionine-containing peptide. trans-[PtCl2(en)2]2+ is a very
efficient reagent in an optimal pH 4-5 buffer solution. 5 The reaction time is increased
dramatically with the decreasing of pH.
Peptide-based drugs containing disulfide bonds have been successfully used for
the treatment of various diseases. Moreover, a great many of peptide candidates are
developed in clinical trials. Disulfide bond helps contribute to stabilizing a peptide’s
secondary structure, which can promote its activity, selectivity and proteolytic
stability.6 Hence, the construction of disulfide bonds is one of the critical steps in
peptide synthesis. Generally, disulfide bonds formation can be achieved through
oxidation of thiol moieties of two cysteine residues via homogeneous or

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heterogeneous reactions. The oxidants including iodine, oxygen, thallDiOuIm: 10(.I10I3I9)/C9DT04738G
trifluoroacetate, carbon tetrachloride, dimethyl sulfoxide (DMSO),
trans-3,4-dihydroxyselenolane oxide (DHS), N-chlorosuccinimide (NCS) and
3-nitro-2-pyridinesulfenates (NPys) have been used for the preparation of peptide
disulfide bonds. However, Drawbacks such as low yield, long reaction time and the
formation of side products are often observed in the oxidation reactions.7 Therefore,
the research and development of an efficient oxidant has grown steadily.
In order to develop the Pt(IV) complex chemistry for the preparation of disulfide
bonds in peptides. 1,10-Phenanthroline (phen) and 2,2′-dipyridine (bpy) substituted
one of the ethylenediamine ligands of trans-[PtCl2(en)2]2+, affording two Pt(IV)
complexes trans-[PtCl2(phen)(en)]2+ and trans-[PtCl2(bpy)(en)]2+. The two Pt(IV)
complexes were synthesized according to our previous work.1f,1g
To investigate whether methionine can be oxidized to methionine sulfoxide by
the Pt(IV) complex. The reaction between trans-[PtCl2(phen)(en)]2+ and methionine
was firstly studied by NMR. As shown in Figure S1, the initial 2.07 and 2.58 ppm
resonances of the CH3 and CH2 groups bonded to sulfur, respectively, were replaced
by resonances at 2.68 and 3.10 ppm, respectively. These changes in the chemical shift
are in the direction of formation of methionine sulfoxide, that is observed in the
reaction of trans-[PtCl2(CN)4]2- with methionine (see Fig. S2).8 However, no
methionine
sulfoxide
was
observed
when
peptide
1
(Sequence:
CGYCHKLHQMK-NH2) was reacted with trans-[PtCl2(phen)(en)]2+ and
trans-[PtCl2(bpy)(en)]2+ (Fig. 1). Disulfide bond was formed immediately at room
temperature. Excellent purity of disulfide-containing peptide 1 was achieved in
aqueous solution. Moreover, no side reaction on tyrosine residue was observed,
confirming that the Pt(IV) complexes can not oxidize the tyrosine residue in peptide.

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DOI: 10.1039/C9DT04738G
In order to investigate whether the two Pt(IV) complexes can oxidize tryptophan
residue in peptide. Reduced somatostatin was reacted with the Pt(IV) complexes, no
tryptophan related oxidation product was observed after the reaction.
The oxidation reactions between each of the Pt(IV) complexes and peptide 2
(Sequence: CGYCHKLHQGK-NH2) were investigated in various solvents. The
results are shown in Table 1. Excellent purities of oxidized peptide 2 are achieved in
various solvents. Therefore, the Pt(IV) complexes tolerate a range of hydrogen ion
concentration upon the formation of peptide disulfide bonds, and the reaction time
increased with the increasing of the concentration of hydrogen ion.
Eight dithiol-containing peptides with variable lengths were selected for
demonstrating the efficiency of the two Pt(IV) complexes for disulfide bonds
formation; HPLC chromatograms and ESI-MS spectra are displayed in the Supporting
Information. The results are summarized in Table 2. As can be seen, excellent purities
and yields were obtained for all disulfide-containing peptides. By comparison,
formation of disulfide bond in oxytocin via oxgen oxidation was also studied in this
work. It needs about 22 h to convert dithiol group to disulfide bond with a yield of
97% in pH 7.95 buffer solution (Fig. S62). Formation of disulfide bond in oxytocin
via the Pt(IV) complex oxidation was completed in less than one minute, while no
disulfide bond was formed in 24 h via oxygen oxidation in 10.0 mM of HCl solution.
Moreover, the oxidation products are easily separated by HPLC, and the Pt(IV)
complexes and their corresponding reduction product, Pt(II) complexes, can be
collected, regenerated and reused for the formation of disulfide bond.
To investigate the applicability of the Pt(IV) complex oxidation method with
multiple-disulfide-containing peptide, reduced α-conotoxin SI (a 13-residue peptide
from a piscivorous cone snail, and it contains two disulfide bonds and a C-terminal

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amide;9 Sequence: ICCNPACGPKYSC-NH2 ) was used as obtained and anaDlyOzI:e10d.10b3y9/C9DT04738G
HPLC (Figure 2a). The reaction between excess of trans-[PtCl2(phen)(en)]2+ and
reduced α-conotoxin SI was investigated. As shown in Figure 2b, isomers
(Cys2-Cys3/Cys7-Cys13
and
Cys2-Cys13/Cys3-Cys7)
and
α-conotoxin
SI
(Cys2-Cys7/Cys7-Cys13) were observed. The ratio of isomers to α-conotoxin SI is
47% : 53%, which was calculated by the peak area.
The reaction between trans-[PtCl2(phen)(en)]2+ and the mixture of two
dithiol-containing peptides (peptide 1 and reduced phenypressin) was studied by
HPLC (Fig. 3). The yields of peptide 1 and phenypressin are all almost 100%. No
intermolecular disulfide bond was formed between two peptides. Therefore, the Pt(IV)
complex is a very excellent oxidant for the synthesis of intra-molecular disulfide
bonds in a solution containing two peptides. Hence, the Pt(IV) complex is useful for
disulfide bond containing peptide libraries.
The applicability of the Pt(IV) complex oxidation method for on-resin disulfide
bonds formation was investigated in this work. On-resin oxytocin (sequence:
CYINQCPLG-NH2), isotocin (sequence: CYISNCPIG-NH2) and iRGD (sequence:
CRGDKGPDC-NH2) with S-Mmt-protected cysteine residues were used as obtained.
The Mmt protected group was removed by CH2Cl2 solution with 1% TFA, the
obtained peptidyl resin was reacted with trans-[PtCl2(phen)(en)]2+ or
trans-[PtCl2(bpy)(en)]2+. After the reaction, oxytocin, isotocin and iRGD peptide
cleaved from the resin were analyzed by HPLC (Fig. 4). As can be seen, peptide
disulfide bonds were successfully formed on resin with the yields of 85%, 85% and
82% for oxytocin, isotocin and iRGD, respectively. Moreover, the reactions can be
carried out automatically on a peptide synthesizer, making the peptide disulfide
synthesis more easily to manufacture.

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Reduction of excess of Pt(IV) complex trans-[PtCl2(phen)(en)]2+ by aDrOeI:d1u0.c10e3d9/C9DT04738G
iRGD peptide was studied by 1H NMR. NMR spectrum is shown in Figure S3. As can
be seen, trans-[PtCl2(phen)(en)]2+ was reduced to its correspondence Pt(II) complex
with the loss of two axial chloride ligands. Moreover, conversion the dithiol group of
reduced iRGD peptide to disulfide bond was best achieved when the mole ratio of
Pt(IV) complex to reduced iRGD peptide was 1:1. The reaction mechanism for
oxidative formation of disulfide bond by Pt(IV) complex has been studied, a
mechanism named chloride-mediated reductive elimination reaction has been widely
accepted as the main pathway for reduction of the Pt(IV) complex.2 Based on our
above results, the mechanism for the synthesis of disulfide bonds by
trans-[PtCl2(phen)(en)]2+ and trans-[PtCl2(bpy)(en)]2+ was proposed as displayed in
Scheme 1.
In summary, the Pt(IV) complexes, trans-[PtCl2(phen)(en)]2+ and
trans-[PtCl2(bpy)(en)]2+, are efficient reagents for the formation of disulfide bonds in
various solvents. Disulfide bonds formation can be achieved with excellent purities
and yields at room temperature. Formation of peptide disulfides were successfully
achieved in a solution containing two peptides, making the Pt(IV) complexes are
useful for disulfide-containing peptide libraries. Additionally, the two Pt(IV)
complexes can be used as excellent oxidants for the synthesis of disulfide bonds on
resin, making the synthesis of peptide disulfides more automatically in manufacture.
Acknowledgements
Financial support for this work through grants from the National Natural Science
Foundation of China (21406047) and the Natural Science Foundation of Hebei

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DOI: 10.1039/C9DT04738G
Province (B2016201014) is gratefully acknowledged.
Conflicts of interest
There are no conflicts to declare.

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DOI: 10.1039/C9DT04738G
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5. T. Shi and D. L. Rabenstein, trans-Dichlorobis(ethylenediamine)platinum(IV), In
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Table 1. Pt(IV) complexes reaction with peptide 2 in various solvents.
No.
1
Solvent
[H+]/(mol/L)
10-7
Pt(IV) complex
Purity of oxidized peptide a (%) Reaction time (min)
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
97
97
< 1
< 1
phosphate buffer
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
96
97
< 1
< 1
2
3
acetate buffer
10-4.2
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
96
97
< 1
< 1
HCl
0.1
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
95
96
～ 5
～ 5
4
HCl
0.5

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trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
97
96
～ 20
～ 20
5
6
HCl
H2O
1.0
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
97
96
< 1
< 1
a
Purities of oxidized peptides were calculated by the areas of the chromatographic products peaks recorded at 215 nm; Peak areas of
Pt(IV)/Pt(II) are not included when calculating the purities of oxidized peptides.

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Table 2. Synthesis of peptide disulfides by Pt(IV) complexes in 10.0 mM of HCl solution.
Purity of
reduced
Purity of
oxidized
peptide b
(%)
Yield of
oxidized
peptide (%)
Reaction time
(min)
No.
Peptide
Sequence of peptide
Pt(IV) complex
peptide a
(%)
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
99
99
< 1
< 1
99
99
1
2
3
4
5
peptide 1
peptide 2
CGYCHKLHQMK-NH2
CGYCHKLHQGK-NH2
CYINQCPLG-NH2
99
99
75
88
90
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
97
96
< 1
< 1
99
99
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
80
77
< 1
< 1
96
96
reduced oxytocin
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
98
96
< 1
< 1
99
99
reduced arginine
vasopressin
CYFQNCPRG-NH2
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
90
90
< 1
< 1
97
97
reduced
AGCKNFFWKTFTSC-
OH
somatostatin

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trans-[PtCl2(phen)(en)]2+
74
75
< 1
< 1
97
97
reduce brain
6
7
8
9
CLSSRLDAC-NH2
CYFQNC-OH
70
90
90
85
84
trans-[PtCl2(bpy)(en)]2+
binding peptide
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
92
94
< 1
< 1
99
99
reduced pressinoic
acid
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
95
94
< 1
< 1
99
99
reduced crustacean
cardioactive peptide
PFCNAFTGC-NH2
CFFQNCPRG-NH2
CYIQNCPKG-OH
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
88
88
< 1
< 1
98
98
reduced
phenypressin
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
90
90
< 1
< 1
96
97
10 reduced vasotocin
Reduced atriopeptin
trans-[PtCl2(phen)(en)]2+
trans-[PtCl2(bpy)(en)]2+
85
82
3
3
98
98
SSCFGGRIDRIGAQSG
LGCNSFR-OH
11
II (rat, rabbit,
mouse)
90

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a, b Purities of peptides were calculated by the areas of the chromatographic products peaks recorded at 215 nm; Peak areas of Pt(IV)/Pt(II) are
not included when calculating the purities of oxidized peptides.

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DOI: 10.1039/C9DT04738G
Figure Captions
Figure 1. HPLC chromatograms of a) peptide 1; b) the reaction between
trans-[PtCl2(phen)(en)]2+ (2.1 mM) and peptide 1 (1.0 mM) in 10.0 mM of HCl
solution; c) the reaction between trans-[PtCl2(bpy)(en)]2+ (2.1 mM) and peptide 1 (1.0
mM) in 10.0 mM of HCl solution; d) the reaction between trans-[PtCl2(CN)4]2- (2.2
mM) and peptide 1 (1.0 mM) in 10.0 mM of HCl solution.
Figure 2. HPLC chromatograms of a) reduced α-conotoxin SI; b) the reaction
between trans-[PtCl2(phen)(en)]2+ and reduced α-conotoxin SI in 10.0 mM of HCl
solution.
Figure 3. HPLC chromatograms of a) mixture of peptide 1 (1.0 mM) and reduced
phenypressin (1.0 mM) in 10.0 mM of HCl solution; b) the reaction between
trans-[PtCl2(phen)(en)]2+ (4.0 mM) and the mixture of peptide 1 (1.0 mM) and
eeduced phenypressin (1.0 mM) in 10.0 mM of HCl solution.
Figure 4. Formation of disulfide bond on resin via Pt(IV) complex oxidation.
Scheme 1. The proposed reaction mechanism for the formation of peptide disulfide
bond via Pt(IV) complex oxidation.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Scheme 1

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Graphical Abstract