N-chlorosuccinimide, an efficient peptide disulfide bond-forming reagent in aqueous solution

Article abstract of DOI:10.1039/c3ra43149e

A novel method has been developed for the efficient formation of peptide disulfide bonds under aqueous conditions using N-chlorosuccinimide. Complete disulfide bond formation is achieved in 15 min with solvent mixtures containing water and acetonitrile. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra43149e

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N-chlorosuccinimide, an efficient peptide disulfide
bond-forming reagent in aqueous solution3
Cite this: RSC Advances, 2013, 3, 14277
Tobias M. Postmaab and Fernando Albericio*abcd
Received 4th May 2013,
Accepted 2nd July 2013
DOI: 10.1039/c3ra43149e
www.rsc.org/advances
A novel method has been developed for the efficient formation of
peptide disulfide bonds under aqueous conditions using
N-chlorosuccinimide. Complete disulfide bond formation is
achieved in 15 min with solvent mixtures containing water and
acetonitrile.
Subsequently, we reported NCS as a highly efficient on-resin
disulfide-forming reagent for solid-phase peptide synthesis.10
Quantitative disulfide formation was achieved in 15 min using 2
equiv. of NCS in DMF, which we demonstrated in the synthesis of
several peptides, including an on-resin regioselective synthesis of
an a-conotoxin using 2 sequential NCS oxidations (Fig. 1).
Shechter and co-workers reported that NCS is compatible with
all amino acids except the oxidation-sensitive Trp and Met.11
However, we found that under our conditions both Trp and Met
are compatible with NCS.{ This finding shows that all amino acids
can be used with the NCS method on the solid-phase. In addition,
NCS was found to be compatible with the amino acid protecting
groups Trt and Mmt, while the common disulfide-forming
oxidants such as iodine and thallium salts are not. These results
allowed us to conclude that NCS is the most widely applicable
disulfide-forming reagent for solid-phase peptide synthesis.
However, solid-phase peptide synthesis has several disadvantages
Disulfide bonds are widespread in peptides and proteins where
they strengthen conformation and lead to increased rigidity and
proteolytic stability.1–3 The number of disulfide-rich therapeutics
currently available is increasing, such as the FDA-approved peptide
drugs Prialt (ziconotide) and Linzess (linaclotide), both of which
contain 3 disulfide bonds.4–6 In order to prepare disulfide-rich
peptides, efficient disulfide-forming reagents must be used that
are compatible with all amino acids, and also preferably with the
associated amino acid protecting groups. Many oxidants are used
for disulfide bond formation in peptide chemistry (e.g. air, DMSO,
iodine, thallium salts, mercury salts, and carbon tetrachloride).7,8
However, given that there is no universal protocol for the
formation of these bonds, this process is empirical and needs to
be assessed on a case to case basis. Robust and widely applicable
disulfide-forming reagents are required in order to quickly
determine the conditions that will allow rapid and quantitative
disulfide bond formation with minimal side-reactions.
Recently, we used N-chlorosuccinimide (NCS) to form a mixed
disulfide between Fmoc-Cys-OH and trimethoxythiophenol under
anhydrous conditions.9 In comparison to most common oxidants
for disulfide formation, NCS is an easy to handle, cheap, and shelf-
stable crystalline solid that is soluble in both organic and aqueous
solvent mixtures. NCS was found to be an efficient reagent for the
formation of mixed cysteine disulfides in organic solvents.
aInstitute for Research in Biomedicine, Baldiri Reixac 10, Barcelona, 08028, Spain.
E-mail: fernando.albericio@irbbarcelona.org; Fax: +34 93 40 37126; Tel: +34 93 40
37088
bCIBER-BBN, Barcelona, 08028, Spain
c
´
`
Department of Organic Chemistry, Martı i Franques 1, Barcelona, 08028, Spain
dSchool of Chemistry University of KwaZulu Natal, Durban, 4001, South Africa
Electronic supplementary information (ESI) available: Detailed experimental
3
procedures, characterization, spectroscopic and chromatographic data. See DOI:
10.1039/c3ra43149e
Fig. 1 Regioselective on-resin NCS oxidation.
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over solution phase such as: limited scalability and dilution, and
generally slower reaction kinetics. For instance, when pseudo-
dilution on the solid-phase is not sufficient and leads to
intermolecular side-reactions, dilution is not possible and would
necessitate resynthesis on a resin with a lower loading. To
overcome these disadvantages with the solid-phase NCS method
we initiated a study of NCS oxidation in solution. Herein, we
report on the formation of peptide disulfide bonds in aqueous
solution with NCS. This achievement significantly broadens the
applicability of the NCS method.
Our model system was based on the well-studied oxytocin, a
nonapeptide currently used as a drug in obstetric medicine to
facilitate childbirth.12 Oxytocin contains one disulfide bond and a
C-terminal amide. The peptide was synthesized on the solid-phase
using standard Fmoc/tBu chemistry on a Rink Amide resin. The
reducing agent-labile protecting group S-Tmp was used for the
protection of the cysteine thiol.9 Prior to cleavage, S-Tmp was
removed by treatment with dithiothreitol under mildly basic
conditions. As a proof of concept for the NCS oxidation of oxytocin
under aqueous conditions, experiments were set up using H2O,
and H2O/acetonitrile mixtures. The fully deprotected peptide was
dissolved in H2O or H2O/acetonitrile mixtures, and a solution
containing 1.5 equiv. of NCS was added. The mixtures were
shaken for 15 min at room temperature, lyophilized and
subsequently analyzed by chromatographic and spectroscopic
techniques. Upon analysis of the reactions, we found that in all
cases oxidized oxytocin (2) was formed in high purity and all the
starting material (1) was consumed (Scheme 1).
We next studied the effects of excess NCS on the efficiency of
disulfide bond formation in H2O/acetonitrile. The peptide was
dissolved in H2O/acetonitrile (1 : 1) and 1.1, 1.5 or 2 equiv. of NCS
were added. This mixture was then shaken for 15 min, and
lyophilized. In all cases, oxidized oxytocin was formed in high
purity and no starting material was left (Table 1).
This observation shows that NCS can cleanly form peptide
disulfide bonds in an aqueous solution under a wide variety of
conditions. Moreover, we observed that an excess of NCS was not
detrimental to the purity of the final peptide. The variety of
aqueous solvent mixtures and NCS equiv. allows the tailoring of
the reaction conditions to the peptide, thus ensuring optimal
solubility while retaining highly efficient disulfide bond forma-
tion.
To further demonstrate the applicability of aqueous NCS
disulfide bond formation, we synthesized the octapeptide
octreotate. This compound is a somatostatin analog used for
targeting somatostatin receptor-positive tumors, and for radio-
nuclide treatment when conjugated to a radionuclide chelator.13,14
Octreotate, contains 1 disulfide bond and 2 D-amino acids (D-Phe
and D-Trp). The D-Trp-Lys sequence forms the key b-turn
pharmacophore, which causes the high affinity of octreotate for
somatostatin receptors.15
Octreotate was synthesized on a chlorotrityl resin using
standard Fmoc/tBu chemistry and subsequently cleaved from
the resin. The deprotected peptide was dissolved in H2O/
acetonitrile (1 : 1), a solution containing 1.5 equiv. of NCS was
added, and the mixture was shaken for 15 min at room
Scheme 1 Aqueous NCS oxidation of oxytocin.
temperature, lyophilized, and subsequently analyzed. Oxidized
cyclic octreotate (4) was formed in high purity, and no linear
octreotate was present (Scheme 2).
As a final example, we prepared a linear a-conotoxin from a
piscivorous conesnail. Oxidative folding of multiple disulfide-
containing peptides is widely used for disulfide-rich peptides.16,17
However, due to the multiple Cys residues present, several
disulfide connectivities can arise, leading to complex mixtures of
peptides. The desired disulfide connectivity can be achieved by
careful tweaking of the oxidation conditions.18 In this case, we
Table 1 Results of oxytocin NCS oxidation under distinct conditions
Oxytocin
H2O/CH3CN
NCS equiv.
Purity (%)a
1
n/a
n/a
1.5
1.5
1.5
1.1
1.5
2.0
98
88
94
94
94
95
94
2-1
2-2
2-3
2-4
2-5
2-6
H2O
1 : 1
1 : 3
1 : 1
1 : 1
1 : 1
a
Determined by the peak area of a HPLC chromatogram at 220 nm.
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Fig. 2 HPLC chromatograms of linear and cyclic Si conotoxin.
Scheme 2 Aqueous NCS oxidation of octreotate.
Conclusions
prepared linear Si conotoxin, a peptide containing 13 residues
with 4 Cys and a C-terminal amide.19 The subsequent oxidative
folding was performed to demonstrate the feasibility of NCS to
fully oxidize disulfide-rich peptides under aqueous conditions,
without optimizing the oxidation conditions to obtain a specific
disulfide connectivity.
Si conotoxin was dissolved in H2O/acetonitrile (1 : 1) and a
solution containing 2.2 equiv. of NCS was added. The mixture was
then shaken for 15 min and subsequently lyophilized. The linear
peptide was completely consumed, and two major products were
formed with the same mass in a ratio 1 : 4 (Fig. 2). The mass
corresponds to the fully oxidized peptide and therefore two cyclic
peptides with different disulfide connectivities were formed (6a
and 6b). This result shows that NCS can be used to efficiently
oxidize disulfide-rich peptides in aqueous oxidative folding
strategies. Moreover, NCS oxidation is completed in 15 min
whereas air oxidation, the common approach in oxidative folding
strategies, is time-consuming. Thus, aqueous NCS oxidation
allows rapid screening of oxidation conditions.
Here we demonstrated the feasibility of using NCS for peptide
disulfide bond formation under aqueous conditions. Highly
efficient disulfide bond formation with NCS was achieved using
H2O or H2O/acetonitrile mixtures and a slight excess of NCS.
Aqueous NCS disulfide bond formation significantly extends the
applicability of the NCS method because it overcomes the
limitations of solid-phase disulfide formation and can be used
in oxidative folding. Moreover, aqueous NCS oxidation is a
powerful addition to the disulfide forming reagent repertoire.
Acknowledgements
This work was partially supported by Fellowship Marie Curie
Initial Training Networks (ITN) MEMTIDE Project: FP7-
PEOPLE_ITN08, CICYT (CTQ2012-30930), and the Generalitat
de Catalunya (2009SGR 1024).
Notes and references
{ Met was found not to be compatible with NCS in solution. When Met is
present we recommend using NCS oxidation on the solid-phase.
1 G. Bulaj, Biotechnol. Adv., 2005, 23, 87–92.
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¨
2 L. Moroder, H.-J. Musiol, M. Gotz and C. Renner, Biopolymers,
2005, 80, 85–97.
12 C. Viero, I. Shibuya, N. Kitamura, A. Verkhratsky, H. Fujihara,
A. Katoh, Y. Ueta, H. H. Zingg, A. Chvatal, E. Sykova and
G. Dayanithi, CNS Neurosci. Ther., 2010, 16, 138–156.
13 H.-P. Hsieh, Y.-T. Wu, S.-T. Chen and K.-T. Wang, Bioorg. Med.
Chem., 1999, 7, 1797–1803.
3 D. P. McGregor, Curr. Opin. Pharmacol., 2008, 8, 616–619.
4 A. P. Bryant, R. W. Busby, W. P. Bartolini, E. A. Cordero,
G. Hannig, M. M. Kessler, C. M. Pierce, R. M. Solinga, J.
V. Tobin, S. Mahajan-Miklos, M. B. Cohen, C. B. Kurtz and M.
G. Currie, Life Sci., 2010, 86, 760–765.
¨
14 M. Laznicek, A. Laznickova, H. R. Macke, K. Eisenwiener, J.
C. Reubi and S. Wenger, Cancer Biother. Radiopharm., 2002, 17,
527–533.
5 E. Dolgin, Nat. Med., 2012, 18, 1308–1309.
6 E. Prommer, Drugs Today, 2006, 42, 369–378.
15 J. Gardiner, D. Langenegger, D. Hoyer, A. K. Beck, R. I. Mathad
and D. Seebach, Chem. Biodiversity, 2008, 5, 1213–1224.
16 E. Fuller, B. R. Green, P. Catlin, O. Buczek, J. S. Nielsen, B.
M. Olivera and G. Bulaj, FEBS J., 2005, 272, 1727–1738.
17 N. L. Daly, R. J. Clark and D. J. Craik, J. Biol. Chem., 2003, 278,
6314–6322.
18 T. Kimura in Synthesis of cystine peptides, in Houben-Weyl:
Methods of Organic Chemistry, Synthesis of Peptides and
Peptidominetics, ed. M. Goodman, A. Felix, L. Moroder and C.
Toniolo, Thieme, Stuttgart and New York, 2002, pp. 142–161.
19 A. J. Benie, D. Whitford, B. Hargittai, G. Barany and R. W. Janes,
FEBS Lett., 2000, 476, 287–295.
7 K. Akaji and Y. Kiso in Synthesis of cystine peptides, in Houben-
Weyl: Methods of Organic Chemistry, Synthesis of Peptides and
Peptidominetics, ed. M. Goodman, A. Felix, L. Moroder and C.
Toniolo, Thieme, Stuttgart and New York, 2002, pp. 101–141.
8 D. Andreu, F. Albericio, N. A. Sole, M. C. Munson, M. Ferrer and
G. Barany, Methods in Molecular Biology: Peptide Synthesis
Protocols, ed. M. W. Pennington and B. M. Dunn, Humana
Press, Inc., Totowa, NJ, 1994, vol. 45, pp. 91–169.
9 T. M. Postma, M. Giraud and F. Albericio, Org. Lett., 2012, 14,
5468–5471.
10 T. M. Postma and F. Albericio, Org. Lett., 2013, 15, 616–619.
11 Y. Shechter, A. Patchornik and Y. Burstein, Biochemistry, 1976,
15, 5071–5075.
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