Direct palladium-mediated on-resin disulfide formation from Allocam protected peptides

Article abstract of DOI:10.1039/c7ob00536a

The synthesis of disulfide-containing polypeptides represents a long-standing challenge in peptide chemistry, and broadly applicable methods for the construction of disulfides are in constant demand. Few strategies exist for on-resin formation of disulfides directly from their protected counterparts. We present herein a novel strategy for the on-resin construction of disulfides directly from Allocam-protected cysteines. Our palladium-mediated approach is mild and uses readily available reagents, requiring no special equipment. No reduced peptide intermediates or S-allylated products are observed, and no residual palladium can be detected in the final products. The utility of this method is demonstrated through the synthesis of the C-carboxy analog of oxytocin.

Full text of DOI:10.1039/c7ob00536a

View Article Online
View Journal
Organic &
Biomol ec ular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: T. D.
Kondasinghe, H. Y. Saraha, S. B. Odeesho and J. L. Stockdill, Org. Biomol. Chem., 2017, DOI:
1
0.1039/C7OB00536A.
Volume 14 Number
1
7
January 2016 Pages 1–372
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Page 1 of 10
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00536A
Organic and Biomolecular Chemistry
COMMUNICATION
Direct Palladium-Mediated On-Resin Disulfide Formation from
Allocam Protected Peptides
Received 00th January 20xx,
Accepted 00th January 20xx
a,†
a,†
a
a
Thilini D. Kondasinghe, Hasina Y. Saraha, Samantha B. Odeesho, and Jennifer L. Stockdill
DOI: 10.1039/x0xx00000x
The synthesis of disulfide-containing polypeptides represents a long-standing challenge in peptide chemistry, and broadly
applicable methods for the construction of disulfides are in constant demand. Few strategies exist for on-resin formation
of disulfides directly from their protected counterparts. We present herein a novel strategy for the on-resin construction
of disulfides directly from Allocam-protected cysteines. Our palladium-mediated approach is mild and uses readily
available reagents, requiring no special equipment. No reduced peptide intermediates or S-allylated products are
observed, and no residual palladium can be detected in the final products. The utility of this method is demonstrated
through the synthesis of the C-carboxy analog of oxytocin.
www.rsc.org/
9
both implicated in social interactions in mammals. Lanreotide
Introduction
(3) is a somatostatin analog used in the treatment of
1
0
acromegaly.
Disulfide linkages improve the thermal, chemical, and
enzymatic stability of polypeptides, as well as enforcing a
1
defined tertiary structure. Nature has capitalized on these
The rapid preparation of disulfide-containing peptide
targets and their analogs is currently limited by the lengthy
nature of solution-phase folding protocols, which require resin
cleavage and removal of all side chain protecting groups,
purification of the reduced peptide, oxidative folding, and then
a final purification. This approach is even more problematic for
hydrophobic peptides or those with complex solubility profiles,
where significant amounts of material can be lost during
properties, in combination with the diversity of amino acids, to
produce vast libraries of stable, selective polypeptides with
2
defined structures and biological activities. Meanwhile,
peptide-based pharmaceuticals are becoming increasingly
3
important because of their greater specificity, potency and
lower toxicity profile over traditional small molecule
b,
pharmaceuticals.
1
1
3
4
solution-phase manipulations. Additionally, the need for
multiple purifications increases costs. To address these
challenges, we report herein a mild new single-step strategy
for on-resin disulfide formation beginning from Allocam-
protected peptides.
Additionally, new biological entities
benefit from a higher regulatory approval rate than traditional
c
small molecule targets. There are currently over 500 peptides
3
in preclinical development, and 140 were in clinical trials as of
3
a
2015.
Several important single-disulfide containing
5
6
In addition to avoiding the aforementioned problems, an
on-resin approach to disulfide formation is advantageous
pharmaceuticals are illustrated in Figure 1. Oxytocin (
1
) is
used clinically to induce parturition and lactation in nursing
7
1
1
because it favors intramolecular disulfide formation. Much
effort has been devoted to the development of new cysteine
protecting groups with the goal of improving the available
mothers and to decrease bleeding postpartum. Desmopressin
(2) is a vasopressin analog used in the treatment of nocturnal
8
enuresis and blood clotting. Oxytocin and vasopressin are
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 1

Organic & Biomolecular Chemistry
Page 2 of 10
View Article Online
DOI: 10.1039/C7OB00536A
Communication
Organic & Biomolecular Chemistry
1
2
toolbox for folding disulfide-rich polypeptides. Although reagents and have the potential to be orthogonal to other
some of these groups can be removed while the peptide is on cysteine protecting groups as well as be compatible with all
resin and in the presence of related protecting groups, several resins. In this context, we viewed the little-used
have practical limitations preventing their widespread use. For allyloxycarbonyl-aminomethyl group (Allocam,
6, Scheme 2A)
example, known photolabile groups are incompatible with as potentially enabling. From a mechanistic perspective,
13a
piperidine, and reductive removal of protecting groups can Allocam, which is cleaved with Pd(0)
π-allyl conditions, should
interfere with existing disulfide linkages, and sometimes be orthogonal to most common cysteine protecting groups. A
13
b
cannot be removed at all. Furthermore, the oxidation step is few key limitations have likely prevented the general use of
generally a separate operation that is conducted in the Allocam and the related Fsam and Fnam ( ) groups. These
solution phase after resin cleavage and purification of the include acid lability, incomplete conversion due to catalyst
reduced peptide. The relatively few methods for on-resin poisoning, the use of Bu SnH as an allyl scavenger, and the
observation of significant amounts of S-allylated side
9
3
1
4
disulfide formation
traditional approach to access disulfides on solid support (
involves treatment with excess iodine, usually for peptides required to convert the initial deprotection products (
bearing trityl (Trt), acetamidomethyl (Acm), or 4-methoxytrityl desired disulfide ( ). The acid stability of Allocam was
This is a convenient improved via the installation of an electron-withdrawing group
are summarized in Scheme 1. A
1
9
5)
products. Additionally, a separate oxidation step was
) to the
7
8
12
15, a
(
Mmt) protected cysteines (4a).
method that cannot always be employed because of issues on the carbonate nitrogen (i.e. Fsam and Fnam, Scheme 2B).
with oxidation of sensitive amino acids (Met/Trp) or undesired However, this functional group often requires a subsequent
2
0
1
6
cleavage from the popular TGT and CTC resins. On-resin acidic treatment to convert intermediate 10 to the free thiol
oxidation of free thiols can be conducted using N- 11). Again, a separate oxidation step is required to access the
disulfide ( ). For an on-resin approach, acid lability is not a
to significant concern. We envisioned that a re-investigation of
(
1
7
12
b
chlorosuccinimide or acidic DMSO (4b, HX= HCl or HOAc).
A variety of protecting groups (4c) can be cleaved by Tl(tfa)
8
3
directly yield the disulfide containing product. This approach is the conditions for the cleavage of the Allocam group would
not orthogonal to other cysteine protecting groups or acid- enable in situ oxidation and potentially minimize S-allylation.
labile side chain protecting groups, and it will also induce
1
8
cleavage from TGT and CTC resins. Benzyl derived protecting
groups (4d) require a 2-step conversion to the disulfide. First,
the benzyl group is removed with low concentrations of TFA
Results and Discussion
Although Fmoc-Cys(Allocam)-OH was known, its synthesis
19
(again causing incompatibility with Cys(Mmt), Cys(Trt), and
was not readily reproducible. Thus, we developed an
CTC resins), then the free thiols are oxidized with CCl /TEA to
4
alternative approach as described in Scheme 3. First, ammonia
was bubbled through a solution of allyl chloroformate (12) to
access the intermediate carbamate in 95% yield. Treatment
give the corresponding disulfide bond. Historically, solution
phase disulfide formation has yielded cleaner peptides than
1
1
these on-resin approaches. The final approach (4e) was
developed to improve peptide purity when disulfide
formation is conducted on the resin. This strategy entails 1)
reductive cleavage of an StBu group, 2) oxidation with 2,2’-
dithiobis(5-nitropyridine) (DTNP) to form RS–SNP, 3)
acidolysis of Mmt, and 4) microwave-assisted displacement of
2
with paraformaldehyde and Ba(OH) generated hydroxymethyl
1
1
the SNP group. This approach, while effective, is rather
operationally intensive.
We endeavored to develop a single-step method for
on-resin disulfide formation that would employ common
2
| Org. Biomol. Chem, 2017, 00, 1-3
This journal is © The Royal Society of Chemistry 2017

Page 3 of 10
Organic & Biomolecular Chemistry
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00536A
Communication
carbamate 13 in 48% yield. Trimethylsilyl chloride activation of Allocam, reduced, and mono- and bis-allylation products
the hydroxymethyl group followed by addition of Fmoc-Cys(H)- entry ). Switching the catalyst to Pd(dppf)Cl •CH Cl
(
3
2
2
2
OtBu provided fully protected cysteine 14 in 70% yield. Finally, completely shut down the reaction (entry 4). However,
acidolysis of the t-Bu ester with 30% TFA produces Fmoc- repeating this reaction in the presence of 20 equiv
2
1
Cys(Allocam)-OH (15) in 99% yield.
With a convenient, scalable route to Fmoc-Cys(Allocam)- desired disulfide with no partially reacted products and no S-
OH in hand, we began investigations to develop a new set of allylation (entry 5). By contrast, PhSiH was not needed for
deprotection conditions that would affect both deprotection Pd(OAc) . In the presence of 7 equiv Pd(OAc) , 10% NMM, and
phenylsilane resulted in nearly complete conversion to the
3
2
2
and disulfide formation in a single reaction. We envisioned 5% AcOH in DMSO, we observed <5% starting material within 2
that the reaction would proceed according to the mechanism h as well as a small amount of mono-Allocam peptide (entry
in Scheme 4. If a Pd(II) pre-catalyst is employed, initial in situ
reduction would convert Pd(II) to Pd(0). Coordination of to time to 4 h led to complete disulfide formation with no side
affords 18, then oxidative addition leads to cationic -allyl products (entry 7). Further reducing the loading to only 1
Pd(II) species 19 and intermediate 20 which then equiv resulted in significantly reduced conversion and
decarboxylates, releases an equivalent of formimine, and increased side product formation (entry 8). An increase to 1.5
liberates the thiol. -Allyl Pd(II) intermediate 19 undergoes equiv (entry 9) led to nearly complete conversion, which was
6). Lowering the loading to 5 equiv and extending the reaction
6
17
π
,
π
nucleophilic attack to turn over the catalyst (21 then 17) and improved to 100% conversion when less NMM was used (entry
release the allylated nucleophile. Meanwhile, the thiol would 10). These were taken as the optimized conditions for the
be oxidized in situ to the disulfide using a compatible oxidant.
NovaSynTGT resin. Because PEG resins have superior swelling
4
2
For our investigations, we employed a model peptide properties relative to polystyrene resins, we hypothesized
derived from the first 9 amino acid residues of islet amyloid that ChemMatrix resin would allow for lower loadings of
polypeptide (IAPP1-9
,
22
disulfide linkage. We selected this model because the Pd(OAc)
residues between the two cysteines do not adopt any
,
Scheme 3), which has a native Pd(OAc)
2
. Unfortunately, upon exposure to 0.75 or 1.0 equiv
, only 11 or 50% conversion was observed after 4 h
2
2
2
(
entries 11, 12). However, under the previously optimized
secondary structure, thus, the peptide should be neither conditions, complete conversion was observed after 2 h
particularly biased toward nor against disulfide formation. In instead of 4h (entry 13). Overall, we were delighted with the
5
2
the supplementary information of a 1993 report detailing low loadings of palladium needed for the transformation.
cleavage conditions for an allyl aspartate, it was noted that a Rinsing the resin with a Pd scavenger prevents any detectable
2
6
mixture of DMSO, AcOH, and NMM led to undesired oxidation contamination of the cleaved products. Under the optimized
of cysteine and produced disulfides as byproducts, presumably conditions, we did not observe partially deprotected or un-
2
3
as a result of air oxidation. We took these conditions as the oxidized products, and no S-allylated products were
2
1
starting point for our investigations. Thus, IAPP1-9 was observed.
subjected to 5 equiv Pd(PPh in the presence of 3% NMM Next, we employed these conditions for direct, on-resin
and 5% AcOH in a DMSO/THF solvent mixture (Table 1, entry disulfide formation in the synthesis of the neurotransmitter,
). After 2 h, we observed a mixture of unreacted starting
3 4
)
1
peptide 23, desired disulfide 24, and the mono-Allocam
version of 23. Increasing the amount of NMM increased the
conversion to the disulfide, but ~1% mono-S-allylated product
was observed along with the mono-Allocam side product
3 4
(entry 2). With 7 equiv Pd(PPh ) , the desired disulfide was the
major product (80% conversion), but we also observed mono-
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 3

Organic & Biomolecular Chemistry
Page 4 of 10
View Article Online
DOI: 10.1039/C7OB00536A
Communication
Organic & Biomolecular Chemistry
6 27
,
oxytocin (
1),
a 9-mer peptide with one disulfide bond. We Instrument Center for spectroscopic support and Shimadzu for
synthesized carboxy-oxytocin to facilitate analysis of the a grant supporting the mass spectrometer. This manuscript is
th
disulfide forming reaction. Fully protected peptide 25 was dedicated to Prof. Samuel J. Danishefsky in honor of his 80
assembled by Fmoc-SPPS on a TGT-linked ChemMatrix resin birthday (March 2016).
(
Scheme 5A). Under the optimized IAPP1-9 conditions, ~80%
conversion was observed after 6 h. Increased Pd(OAc)
loadings (3.0 equiv) facilitated complete conversion of peptide
to protected carboxy-oxytocin (27). The resulting folded
2
Notes and references
25
peptide was cleaved from the resin, the side chain protecting
1
2
(a) M. G. Benitez, J. T. Puche, F. Albericio, Chem. Rev., 2014,
14, 901–926; (b) H. E. Swaisgood, Biotechnol. Adv., 2005, 23,
71–73.
(a) B. M. Olivera, J. Biol. Chem., 2006, 281, 31173–31177; (b) H.
Terlau, B. M. Olivera, Physiol. Rev., 2004, 84, 41–68; (c) A.
Rojas, A. Feregrino, C. Ibarra-Alvarado, M. B. Aguilar, A. Falcón,
E. Heimer de la Cotera, Venom. Anim. Toxins incl. Trop. Dis.,
groups were removed, and it was purified by RP-HPLC (Scheme
2
1
1
5B
) to afford carboxy-oxytocin (27) in 32% isolated yield.
Conclusions
In summary, we present a novel Pd-mediated method to form
disulfides on resin that proceeds directly from the protected
peptide without loss of any acid-labile side chain protecting
groups. This approach avoids the intermediacy of the reduced
peptide, avoids solution-phase manipulations of the peptide,
and minimizes the number of HPLC purifications needed to
access the desired peptide. The reaction employs bench-stable
reagents and requires no special equipment. No trace Pd or S-
allylated products are observed. We have demonstrated the
utility of this method in the solid phase synthesis and on-resin
folding of the carboxy-terminated analog of the
neurotransmitter oxytocin. Meanwhile, we anticipate that this
method will be of broad utility in the synthesis of more
complex multi-disulfide targets. Efforts to employ this reaction
in such contexts are actively being pursued in our laboratory.
Detailed investigations into the mechanistic nuances of this
chemistry are ongoing and will be reported in due course.
2
008, 14, 497–513.
(a) K. Fosgerau, T. Hoffman, Drug Disc. Today, 2015, 20, 122–
28; (b) T. Uhlig, T. Kyprianou, F. G. Martinelli, C. A. Oppici, D.
3
1
Heiligers, D. Hills, X. R. Calvo, P. Verhaert, EuPA Open
Proteom., 2014, 4, 58–69; (c) P. Vlieghe, V. Lisowski, J.
Martinez, M. Khrestchatisky, Drug Disc. Today, 2010, 15, 40–
56; (d) A. A. Kaspar, J. M. Reichert, Drug Disc. Today, 2013, 18,
8
07–817.
4
5
6
A. K. Sato, M. Viswanathan, R. B. Kent, C. R. Wood, Curr. Opin.
Biotechnol., 2006, 17, 638–642.
E. A. Ilardi, E. Vitaku, J. T. Njardarson, J. Med. Chem., 2014, 57,
2
832–2842.
(a) M. Muttenthaler, A. Andersson, A. D. de Araujo, Z. Dekan,
R. J. Lewis, P. F. Alewood, J. Med. Chem., 2010, 53, 8585–8596;
(
1
b) T. Wang, S. J. Danishefsky, J. Am. Chem. Soc., 2012, 134,
3244−13247.
7
8
9
(a) G. Gimpl, F. Fahrenholz, Physiol. Rev., 2001, 81, 629–683.
(b) H. H. Zingg, S. A. Laporte, Trends. Endocrinol. Metabol.
2
R. A. Janknegt, M. M. Zweers, K. P. J. Delaere, A. G. Kloet, S. G.
S. Khoe, H. J. Arendsen, J. Urol. (Baltimore) 1997, 157, 513–
003, 14, 222–227.
Acknowledgements
5
17.
The authors would like to thank the National Institutes of
Health (R00-GM097095) and Wayne State University for
generous financial support (startup funds to JLS, Rumble-
(a) M. Gozzi, E. M. Dashow, A. Thurm, S. E. Swedo, C. F. Zink,
Neuropsychopharm., 2016, 1–11; (b) Z. R. Donaldson, L. J.
Young, Science 2008, 322, 900–904.
Schaap Fellowship to TDK, Knoller Fellowship to HYS). We also 10 (a) F. Roelfsema, N. R. Biermasz, A. M. Pereira, J. A. Romijn,
gratefully acknowledge the staff of the WSU Lumigen
Biologics: Targets & Therapy, 2008, 3, 463–479; (b) S. Manjila,
O. C. Wu, F. R. Khan, M. M. Khan, B. M. Arafah, W. R. Selman,
Neurosurg. Focus., 2010, 4, E14.
1
1
1
2
A. S. Galanis, F. Albericio, M. Grøtli, Pept. Sci., 2009, 92, 23–34.
(a) T. M. Postma, F. Albericio, Eur. J. Org. Chem., 2014, 3519–
3
2
530; (b) A. Cuthbertson, B. Indrevoll, Org. Lett. 2003, 5, 2955–
957; (c) M. Mochizuki, S. Tsuda, K. Tanimura, Y. Nishiuchi,
Org. Lett., 2015, 17, 2202–2205; (d) E. Calce, R. M. Vitale, A.
Scaloni, P. Amodeo, S. De Luca, Amino Acids, 2015, 47, 1507–
1
515.
1
1
1
3
4
5
(a) N. Kotzur, B. T. Briand, M. Beyermann, V. Hagen, J. Am.
Chem. Soc., 2009, 131, 16927–16931; (b) M. Góngora-Benítez,
J. Tulla-Puche, M. Paradís-Bas, O. Werbitzky, M. Giraud, F.
Albericio, Pept. Sci., 2011, 96, 69–80.
D. Andreu, F. Albericio, N. A. Sole, M. C. Munson, M. Ferrer,
G. Barany, Methods in Molecular Biology: Peptide Synthesis
Protocols (Eds.: M. W. Pennington, B. M. Dunn), Humana
Press, Inc., Totowa, NJ, 1994, vol. 45, p. 91–169.
J. Eichler, R. A. Houghten, Protein Pept. Lett., 1997, 4, 157–164.
4
| Org. Biomol. Chem, 2017, 00, 1-3
This journal is © The Royal Society of Chemistry 2017

Page 5 of 10
Organic & Biomolecular Chemistry
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00536A
Communication
16
(a) K. Barlos, D. Gatos, S. Kutsogianni, G. Papaphotiou, C.
Poulos, T. Tsendenidis, Chem. Biol. Drug Design, 1991, 38, 562–
68; (b) EMD Millipore Catalog page for TGT resin.
5
http://www.emdmillipore.com/US/en/product/NovaSynTGT-
alcohol-resin,MDA_CHEM-855010 (accessed 5 January 2017).
T. M. Postma, F. Albericio, Org. Lett., 2013, 15, 616–619.
N. Fujii, A. Otaka, S. Funakoshi, K. Bessho, T. Watanabe, K
AkaJi, H. Yajima, Chem. Pharm. Bull., 1987, 35, 2339–2347.
(a) A. M. Kimbonguila, A. Merzouk, F. Guibé, A. Loffet,
Tetrahedron, 1999, 55, 6931–6944; (b) A. M. Kimbonguila, A.
Merzouk, F. Guibé, A. Loffet, Tetrahedron Lett., 1994, 35,
1
1
7
8
19
9035–9038; (c) F. Guibé, Tetrahedron, 1998, 54, 2967–3042.
2
0
(a) P. Gomez-Martinez, A. M. Kimbonguila, F. Guibé,
Tetrahedron, 1999, 55, 6945–6960; (b) P. Gomez-Martinez, F.
Guibe, F. Albericio, Lett. Pept. Sci., 2000, 7, 187–194.
See Supporting Information for details.
(a) P. Westermark, A. Andersson, G. T. Westermark, Physiol.
Rev., 2011, 91, 795–826; (b) P. W. R. Harris, R. Kowalczyk, D. L.
Hay, M. A. Brimble, Int. J. Pept. Res. Ther., 2013, 19, 147–155.
S. A. Kates, S. B. Daniels, F. Albericio, Anal. Biochem., 1993,
2
2
1
2
2
3
4
212, 303–310.
2
(a) G. Comellas, Z. Kaczmarska, T. Tarragó, M. Teixidó, E. Giralt,
PLoS One, 2009, 4, e6222; (b) M. Junkers, SigmaAldrich
ChemFiles 2011, 11, article 1; (c) S. Frutos, J. Tulla-Pucha, F.
Albericio, E. Giralt, Intern. J. Pept. Res. Ther., 2007, 13, 221–
227; (d) Y. García-Ramos, M. Paradís-Bas, J. Tulla-Puche, F.
Albericio, J. Pept. Sci., 2010, 16, 675–678.
25
M. Jbara, S. K. Maity, M. Seenaiah, A. Brik, J. Am Chem. Soc.,
2016, 138, 5069–5075.
2
2
6
7
The Pd complex is visible by MS prior to this rinse.
(a) M. Bodanszky, V. Du Vigneaud, Nature, 1959, 183, 1324–
1
1
325; (b) M. Bodanszky, V. Du Vigneaud, J. Am. Chem. Soc.
959, 81, 5688–5691.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 5

Organic & Biomolecular Chemistry
Page 6 of 10
View Article Online
DOI: 10.1039/C7OB00536A
Organic and Biomolecular Chemistry
COMMUNICATION
Direct Palladium-Mediated On-Resin Disulfide Formation from
Allocam Protected Peptides
Received 00th January 20xx,
Accepted 00th January 20xx
a,†
a,†
a
a
Thilini D. Kondasinghe, Hasina Y. Saraha, Samantha B. Odeesho, and Jennifer L. Stockdill
DOI: 10.1039/x0xx00000x
The synthesis of disulfide-containing polypeptides represents a long-standing challenge in peptide chemistry, and broadly
applicable methods for the construction of disulfides are in constant demand. Few strategies exist for on-resin formation
of disulfides directly from their protected counterparts. We present herein a novel strategy for the on-resin construction
of disulfides directly from Allocam-protected cysteines. Our palladium-mediated approach is mild and uses readily
available reagents, requiring no special equipment. No reduced peptide intermediates or S-allylated products are
observed, and no residual palladium can be detected in the final products. The utility of this method is demonstrated
through the synthesis of the C-carboxy analog of oxytocin.
www.rsc.org/
9
both implicated in social interactions in mammals. Lanreotide
Introduction
(3) is a somatostatin analog used in the treatment of
1
0
acromegaly.
Disulfide linkages improve the thermal, chemical, and
enzymatic stability of polypeptides, as well as enforcing a
1
defined tertiary structure. Nature has capitalized on these
The rapid preparation of disulfide-containing peptide
targets and their analogs is currently limited by the lengthy
nature of solution-phase folding protocols, which require resin
cleavage and removal of all side chain protecting groups,
purification of the reduced peptide, oxidative folding, and then
a final purification. This approach is even more problematic for
hydrophobic peptides or those with complex solubility profiles,
where significant amounts of material can be lost during
properties, in combination with the diversity of amino acids, to
produce vast libraries of stable, selective polypeptides with
2
defined structures and biological activities. Meanwhile,
peptide-based pharmaceuticals are becoming increasingly
3
important because of their greater specificity, potency and
lower toxicity profile over traditional small molecule
b,
pharmaceuticals.
1
1
3
4
solution-phase manipulations. Additionally, the need for
multiple purifications increases costs. To address these
challenges, we report herein a mild new single-step strategy
for on-resin disulfide formation beginning from Allocam-
protected peptides.
Additionally, new biological entities
benefit from a higher regulatory approval rate than traditional
c
small molecule targets. There are currently over 500 peptides
3
in preclinical development, and 140 were in clinical trials as of
3
a
2015.
Several important single-disulfide containing
5
6
In addition to avoiding the aforementioned problems, an
on-resin approach to disulfide formation is advantageous
pharmaceuticals are illustrated in Figure 1. Oxytocin (
1
) is
used clinically to induce parturition and lactation in nursing
7
1
1
because it favors intramolecular disulfide formation. Much
effort has been devoted to the development of new cysteine
protecting groups with the goal of improving the available
mothers and to decrease bleeding postpartum. Desmopressin
(2) is a vasopressin analog used in the treatment of nocturnal
8
enuresis and blood clotting. Oxytocin and vasopressin are
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 1

Page 7 of 10
Communication
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00536A
Organic & Biomolecular Chemistry
1
2
toolbox for folding disulfide-rich polypeptides. Although reagents and have the potential to be orthogonal to other
some of these groups can be removed while the peptide is on cysteine protecting groups as well as be compatible with all
resin and in the presence of related protecting groups, several resins. In this context, we viewed the little-used
have practical limitations preventing their widespread use. For allyloxycarbonyl-aminomethyl group (Allocam,
6, Scheme 2A)
example, known photolabile groups are incompatible with as potentially enabling. From a mechanistic perspective,
13a
piperidine, and reductive removal of protecting groups can Allocam, which is cleaved with Pd(0)
π-allyl conditions, should
interfere with existing disulfide linkages, and sometimes be orthogonal to most common cysteine protecting groups. A
13
b
cannot be removed at all. Furthermore, the oxidation step is few key limitations have likely prevented the general use of
generally a separate operation that is conducted in the Allocam and the related Fsam and Fnam ( ) groups. These
solution phase after resin cleavage and purification of the include acid lability, incomplete conversion due to catalyst
reduced peptide. The relatively few methods for on-resin poisoning, the use of Bu SnH as an allyl scavenger, and the
observation of significant amounts of S-allylated side
9
3
1
4
disulfide formation
traditional approach to access disulfides on solid support (
involves treatment with excess iodine, usually for peptides required to convert the initial deprotection products (
bearing trityl (Trt), acetamidomethyl (Acm), or 4-methoxytrityl desired disulfide ( ). The acid stability of Allocam was
This is a convenient improved via the installation of an electron-withdrawing group
are summarized in Scheme 1. A
1
9
5)
products. Additionally, a separate oxidation step was
) to the
7
8
12
15, a
(
Mmt) protected cysteines (4a).
method that cannot always be employed because of issues on the carbonate nitrogen (i.e. Fsam and Fnam, Scheme 2B).
with oxidation of sensitive amino acids (Met/Trp) or undesired However, this functional group often requires a subsequent
2
0
1
6
cleavage from the popular TGT and CTC resins. On-resin acidic treatment to convert intermediate 10 to the free thiol
oxidation of free thiols can be conducted using N- 11). Again, a separate oxidation step is required to access the
disulfide ( ). For an on-resin approach, acid lability is not a
to significant concern. We envisioned that a re-investigation of
(
1
7
12
b
chlorosuccinimide or acidic DMSO (4b, HX= HCl or HOAc).
A variety of protecting groups (4c) can be cleaved by Tl(tfa)
8
3
directly yield the disulfide containing product. This approach is the conditions for the cleavage of the Allocam group would
not orthogonal to other cysteine protecting groups or acid- enable in situ oxidation and potentially minimize S-allylation.
labile side chain protecting groups, and it will also induce
1
8
cleavage from TGT and CTC resins. Benzyl derived protecting
groups (4d) require a 2-step conversion to the disulfide. First,
the benzyl group is removed with low concentrations of TFA
Results and Discussion
Although Fmoc-Cys(Allocam)-OH was known, its synthesis
19
(again causing incompatibility with Cys(Mmt), Cys(Trt), and
was not readily reproducible. Thus, we developed an
CTC resins), then the free thiols are oxidized with CCl /TEA to
4
alternative approach as described in Scheme 3. First, ammonia
was bubbled through a solution of allyl chloroformate (12) to
access the intermediate carbamate in 95% yield. Treatment
give the corresponding disulfide bond. Historically, solution
phase disulfide formation has yielded cleaner peptides than
1
1
these on-resin approaches. The final approach (4e) was
developed to improve peptide purity when disulfide
formation is conducted on the resin. This strategy entails 1)
reductive cleavage of an StBu group, 2) oxidation with 2,2’-
dithiobis(5-nitropyridine) (DTNP) to form RS–SNP, 3)
acidolysis of Mmt, and 4) microwave-assisted displacement of
2
with paraformaldehyde and Ba(OH) generated hydroxymethyl
1
1
the SNP group. This approach, while effective, is rather
operationally intensive.
We endeavored to develop a single-step method for
on-resin disulfide formation that would employ common
2
| Org. Biomol. Chem, 2017, 00, 1-3
This journal is © The Royal Society of Chemistry 2017

Organic & Biomolecular Chemistry
Page 8 of 10
View Article Online
DOI: 10.1039/C7OB00536A
Organic & Biomolecular Chemistry
Communication
carbamate 13 in 48% yield. Trimethylsilyl chloride activation of Allocam, reduced, and mono- and bis-allylation products
the hydroxymethyl group followed by addition of Fmoc-Cys(H)- entry ). Switching the catalyst to Pd(dppf)Cl •CH Cl
(
3
2
2
2
OtBu provided fully protected cysteine 14 in 70% yield. Finally, completely shut down the reaction (entry 4). However,
acidolysis of the t-Bu ester with 30% TFA produces Fmoc- repeating this reaction in the presence of 20 equiv
2
1
Cys(Allocam)-OH (15) in 99% yield.
With a convenient, scalable route to Fmoc-Cys(Allocam)- desired disulfide with no partially reacted products and no S-
OH in hand, we began investigations to develop a new set of allylation (entry 5). By contrast, PhSiH was not needed for
deprotection conditions that would affect both deprotection Pd(OAc) . In the presence of 7 equiv Pd(OAc) , 10% NMM, and
phenylsilane resulted in nearly complete conversion to the
3
2
2
and disulfide formation in a single reaction. We envisioned 5% AcOH in DMSO, we observed <5% starting material within 2
that the reaction would proceed according to the mechanism h as well as a small amount of mono-Allocam peptide (entry
in Scheme 4. If a Pd(II) pre-catalyst is employed, initial in situ
reduction would convert Pd(II) to Pd(0). Coordination of to time to 4 h led to complete disulfide formation with no side
affords 18, then oxidative addition leads to cationic -allyl products (entry 7). Further reducing the loading to only 1
Pd(II) species 19 and intermediate 20 which then equiv resulted in significantly reduced conversion and
decarboxylates, releases an equivalent of formimine, and increased side product formation (entry 8). An increase to 1.5
liberates the thiol. -Allyl Pd(II) intermediate 19 undergoes equiv (entry 9) led to nearly complete conversion, which was
6). Lowering the loading to 5 equiv and extending the reaction
6
17
π
,
π
nucleophilic attack to turn over the catalyst (21 then 17) and improved to 100% conversion when less NMM was used (entry
release the allylated nucleophile. Meanwhile, the thiol would 10). These were taken as the optimized conditions for the
be oxidized in situ to the disulfide using a compatible oxidant.
NovaSynTGT resin. Because PEG resins have superior swelling
4
2
For our investigations, we employed a model peptide properties relative to polystyrene resins, we hypothesized
derived from the first 9 amino acid residues of islet amyloid that ChemMatrix resin would allow for lower loadings of
polypeptide (IAPP1-9
,
22
disulfide linkage. We selected this model because the Pd(OAc)
residues between the two cysteines do not adopt any
,
Scheme 3), which has a native Pd(OAc)
2
. Unfortunately, upon exposure to 0.75 or 1.0 equiv
, only 11 or 50% conversion was observed after 4 h
2
2
2
(
entries 11, 12). However, under the previously optimized
secondary structure, thus, the peptide should be neither conditions, complete conversion was observed after 2 h
particularly biased toward nor against disulfide formation. In instead of 4h (entry 13). Overall, we were delighted with the
5
2
the supplementary information of a 1993 report detailing low loadings of palladium needed for the transformation.
cleavage conditions for an allyl aspartate, it was noted that a Rinsing the resin with a Pd scavenger prevents any detectable
2
6
mixture of DMSO, AcOH, and NMM led to undesired oxidation contamination of the cleaved products. Under the optimized
of cysteine and produced disulfides as byproducts, presumably conditions, we did not observe partially deprotected or un-
2
3
as a result of air oxidation. We took these conditions as the oxidized products, and no S-allylated products were
2
1
starting point for our investigations. Thus, IAPP1-9 was observed.
subjected to 5 equiv Pd(PPh in the presence of 3% NMM Next, we employed these conditions for direct, on-resin
and 5% AcOH in a DMSO/THF solvent mixture (Table 1, entry disulfide formation in the synthesis of the neurotransmitter,
). After 2 h, we observed a mixture of unreacted starting
3 4
)
1
peptide 23, desired disulfide 24, and the mono-Allocam
version of 23. Increasing the amount of NMM increased the
conversion to the disulfide, but ~1% mono-S-allylated product
was observed along with the mono-Allocam side product
3 4
(entry 2). With 7 equiv Pd(PPh ) , the desired disulfide was the
major product (80% conversion), but we also observed mono-
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 3

Page 9 of 10
Communication
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00536A
Organic & Biomolecular Chemistry
6 27
,
oxytocin (
1),
a 9-mer peptide with one disulfide bond. We Instrument Center for spectroscopic support and Shimadzu for
synthesized carboxy-oxytocin to facilitate analysis of the a grant supporting the mass spectrometer. This manuscript is
th
disulfide forming reaction. Fully protected peptide 25 was dedicated to Prof. Samuel J. Danishefsky in honor of his 80
assembled by Fmoc-SPPS on a TGT-linked ChemMatrix resin birthday (March 2016).
(
Scheme 5A). Under the optimized IAPP1-9 conditions, ~80%
conversion was observed after 6 h. Increased Pd(OAc)
loadings (3.0 equiv) facilitated complete conversion of peptide
to protected carboxy-oxytocin (27). The resulting folded
2
Notes and references
25
peptide was cleaved from the resin, the side chain protecting
1
2
(a) M. G. Benitez, J. T. Puche, F. Albericio, Chem. Rev., 2014,
14, 901–926; (b) H. E. Swaisgood, Biotechnol. Adv., 2005, 23,
71–73.
(a) B. M. Olivera, J. Biol. Chem., 2006, 281, 31173–31177; (b) H.
Terlau, B. M. Olivera, Physiol. Rev., 2004, 84, 41–68; (c) A.
Rojas, A. Feregrino, C. Ibarra-Alvarado, M. B. Aguilar, A. Falcón,
E. Heimer de la Cotera, Venom. Anim. Toxins incl. Trop. Dis.,
groups were removed, and it was purified by RP-HPLC (Scheme
2
1
1
5B
) to afford carboxy-oxytocin (27) in 32% isolated yield.
Conclusions
In summary, we present a novel Pd-mediated method to form
disulfides on resin that proceeds directly from the protected
peptide without loss of any acid-labile side chain protecting
groups. This approach avoids the intermediacy of the reduced
peptide, avoids solution-phase manipulations of the peptide,
and minimizes the number of HPLC purifications needed to
access the desired peptide. The reaction employs bench-stable
reagents and requires no special equipment. No trace Pd or S-
allylated products are observed. We have demonstrated the
utility of this method in the solid phase synthesis and on-resin
folding of the carboxy-terminated analog of the
neurotransmitter oxytocin. Meanwhile, we anticipate that this
method will be of broad utility in the synthesis of more
complex multi-disulfide targets. Efforts to employ this reaction
in such contexts are actively being pursued in our laboratory.
Detailed investigations into the mechanistic nuances of this
chemistry are ongoing and will be reported in due course.
2
008, 14, 497–513.
(a) K. Fosgerau, T. Hoffman, Drug Disc. Today, 2015, 20, 122–
28; (b) T. Uhlig, T. Kyprianou, F. G. Martinelli, C. A. Oppici, D.
3
1
Heiligers, D. Hills, X. R. Calvo, P. Verhaert, EuPA Open
Proteom., 2014, 4, 58–69; (c) P. Vlieghe, V. Lisowski, J.
Martinez, M. Khrestchatisky, Drug Disc. Today, 2010, 15, 40–
56; (d) A. A. Kaspar, J. M. Reichert, Drug Disc. Today, 2013, 18,
8
07–817.
4
5
6
A. K. Sato, M. Viswanathan, R. B. Kent, C. R. Wood, Curr. Opin.
Biotechnol., 2006, 17, 638–642.
E. A. Ilardi, E. Vitaku, J. T. Njardarson, J. Med. Chem., 2014, 57,
2
832–2842.
(a) M. Muttenthaler, A. Andersson, A. D. de Araujo, Z. Dekan,
R. J. Lewis, P. F. Alewood, J. Med. Chem., 2010, 53, 8585–8596;
(
1
b) T. Wang, S. J. Danishefsky, J. Am. Chem. Soc., 2012, 134,
3244−13247.
7
8
9
(a) G. Gimpl, F. Fahrenholz, Physiol. Rev., 2001, 81, 629–683.
(b) H. H. Zingg, S. A. Laporte, Trends. Endocrinol. Metabol.
2
R. A. Janknegt, M. M. Zweers, K. P. J. Delaere, A. G. Kloet, S. G.
S. Khoe, H. J. Arendsen, J. Urol. (Baltimore) 1997, 157, 513–
003, 14, 222–227.
Acknowledgements
5
17.
The authors would like to thank the National Institutes of
Health (R00-GM097095) and Wayne State University for
generous financial support (startup funds to JLS, Rumble-
(a) M. Gozzi, E. M. Dashow, A. Thurm, S. E. Swedo, C. F. Zink,
Neuropsychopharm., 2016, 1–11; (b) Z. R. Donaldson, L. J.
Young, Science 2008, 322, 900–904.
Schaap Fellowship to TDK, Knoller Fellowship to HYS). We also 10 (a) F. Roelfsema, N. R. Biermasz, A. M. Pereira, J. A. Romijn,
gratefully acknowledge the staff of the WSU Lumigen
Biologics: Targets & Therapy, 2008, 3, 463–479; (b) S. Manjila,
O. C. Wu, F. R. Khan, M. M. Khan, B. M. Arafah, W. R. Selman,
Neurosurg. Focus., 2010, 4, E14.
1
1
1
2
A. S. Galanis, F. Albericio, M. Grøtli, Pept. Sci., 2009, 92, 23–34.
(a) T. M. Postma, F. Albericio, Eur. J. Org. Chem., 2014, 3519–
3
2
530; (b) A. Cuthbertson, B. Indrevoll, Org. Lett. 2003, 5, 2955–
957; (c) M. Mochizuki, S. Tsuda, K. Tanimura, Y. Nishiuchi,
Org. Lett., 2015, 17, 2202–2205; (d) E. Calce, R. M. Vitale, A.
Scaloni, P. Amodeo, S. De Luca, Amino Acids, 2015, 47, 1507–
1
515.
1
1
1
3
4
5
(a) N. Kotzur, B. T. Briand, M. Beyermann, V. Hagen, J. Am.
Chem. Soc., 2009, 131, 16927–16931; (b) M. Góngora-Benítez,
J. Tulla-Puche, M. Paradís-Bas, O. Werbitzky, M. Giraud, F.
Albericio, Pept. Sci., 2011, 96, 69–80.
D. Andreu, F. Albericio, N. A. Sole, M. C. Munson, M. Ferrer,
G. Barany, Methods in Molecular Biology: Peptide Synthesis
Protocols (Eds.: M. W. Pennington, B. M. Dunn), Humana
Press, Inc., Totowa, NJ, 1994, vol. 45, p. 91–169.
J. Eichler, R. A. Houghten, Protein Pept. Lett., 1997, 4, 157–164.
4
| Org. Biomol. Chem, 2017, 00, 1-3
This journal is © The Royal Society of Chemistry 2017

Organic & Biomolecular Chemistry
Page 10 of 10
View Article Online
DOI: 10.1039/C7OB00536A
Organic & Biomolecular Chemistry
Communication
16
(a) K. Barlos, D. Gatos, S. Kutsogianni, G. Papaphotiou, C.
Poulos, T. Tsendenidis, Chem. Biol. Drug Design, 1991, 38, 562–
68; (b) EMD Millipore Catalog page for TGT resin.
5
http://www.emdmillipore.com/US/en/product/NovaSynTGT-
alcohol-resin,MDA_CHEM-855010 (accessed 5 January 2017).
T. M. Postma, F. Albericio, Org. Lett., 2013, 15, 616–619.
N. Fujii, A. Otaka, S. Funakoshi, K. Bessho, T. Watanabe, K
AkaJi, H. Yajima, Chem. Pharm. Bull., 1987, 35, 2339–2347.
(a) A. M. Kimbonguila, A. Merzouk, F. Guibé, A. Loffet,
Tetrahedron, 1999, 55, 6931–6944; (b) A. M. Kimbonguila, A.
Merzouk, F. Guibé, A. Loffet, Tetrahedron Lett., 1994, 35,
1
1
7
8
19
9035–9038; (c) F. Guibé, Tetrahedron, 1998, 54, 2967–3042.
2
0
(a) P. Gomez-Martinez, A. M. Kimbonguila, F. Guibé,
Tetrahedron, 1999, 55, 6945–6960; (b) P. Gomez-Martinez, F.
Guibe, F. Albericio, Lett. Pept. Sci., 2000, 7, 187–194.
See Supporting Information for details.
(a) P. Westermark, A. Andersson, G. T. Westermark, Physiol.
Rev., 2011, 91, 795–826; (b) P. W. R. Harris, R. Kowalczyk, D. L.
Hay, M. A. Brimble, Int. J. Pept. Res. Ther., 2013, 19, 147–155.
S. A. Kates, S. B. Daniels, F. Albericio, Anal. Biochem., 1993,
2
2
1
2
2
3
4
212, 303–310.
2
(a) G. Comellas, Z. Kaczmarska, T. Tarragó, M. Teixidó, E. Giralt,
PLoS One, 2009, 4, e6222; (b) M. Junkers, SigmaAldrich
ChemFiles 2011, 11, article 1; (c) S. Frutos, J. Tulla-Pucha, F.
Albericio, E. Giralt, Intern. J. Pept. Res. Ther., 2007, 13, 221–
227; (d) Y. García-Ramos, M. Paradís-Bas, J. Tulla-Puche, F.
Albericio, J. Pept. Sci., 2010, 16, 675–678.
25
M. Jbara, S. K. Maity, M. Seenaiah, A. Brik, J. Am Chem. Soc.,
2016, 138, 5069–5075.
2
2
6
7
The Pd complex is visible by MS prior to this rinse.
(a) M. Bodanszky, V. Du Vigneaud, Nature, 1959, 183, 1324–
1
1
325; (b) M. Bodanszky, V. Du Vigneaud, J. Am. Chem. Soc.
959, 81, 5688–5691.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem, 2017, 00, 1-3 | 5