Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

Article abstract of DOI:10.1002/cbic.201700487

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation

Full text of DOI:10.1002/cbic.201700487

Accepted Article
Title: Native Chemical Ligation Directed by Photocleavable Peptide
Nucleic Acid (PNA) Templates
Authors: Stephen Middel, Cornelia Panse, Swantje Nawratil, and Ulf
Diederichsen
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700487
Link to VoR: http://dx.doi.org/10.1002/cbic.201700487
A Journal of
www.chembiochem.org

10.1002/cbic.201700487
ChemBioChem
COMMUNICATION
Native Chemical Ligation Directed by Photocleavable Peptide
Nucleic Acid (PNA) Templates**
Stephen Middel, Cornelia H. Panse, Swantje Nawratil, and Ulf Diederichsen*
Abstract: A novel peptide-peptide ligation strategy is introduced that
has the potential to provide peptide libraries of linearly or branched
coupled fragments and will be suited to introduce simultaneous
protein modifications at different ligation sites. Ligation is assisted by
templating peptide nucleic acid (PNA) strands, and therefore, ligation
specificity is solely encoded by the PNA sequence. PNA templating in
general allows for various kind of covalent ligation reactions. As a
prove of principle, a native chemical ligation strategy was elaborated.
This PNA templated ligation includes easy on-resin procedures to
couple linker and PNA to the respective peptides as well as a
traceless photocleavage of the linker/PNA oligomer after the ligation
step. A 4,5-dimethoxy-2-nitrobenzaldehyde based linker that allows
the photocleavable linkage of two biooligomers was developed.
peptide ligation strategy that includes photocleavage of the PNA
helper strands following the ligation reaction. In addition, this
concept gives access to difficult to synthesize branched peptides,
which have therapeutical potential as multivalent binders.[10]
The general concept introduced herein is based on
biooligomer hybrids of the respective peptide to be ligated with
corresponding PNA recognition sequence. The PNA and peptide
oligomers are joined by a photocleavable linker providing hybrid
oligomers like 1 and 2 (Scheme 1). Hybrids with complementary
PNA sequences assemble due to PNA base pairing bringing the
ligation sites of peptides 1 and 2 in proximity, thereby, facilitating
the ligation reaction. For covalent linkage of the peptide fragments
a native chemical ligation (NCL) approach is used creating a
native amide bond.[11] Finally, the PNA recognition units can be
cleaved by irradiation of ligation product 3 yielding peptide 4.
Nature uses biomacromolecules as templates to facilitate efficient
and selective product formation out of countless different
reactants in low concentrations. These templates recognize
reactants, bind them non-covalently, bring their reactive centers
in close proximity, and therefore, increase the effective
concentration of the reaction partners.[1] Chemists adopted this
concept, and first template-directed ligation approaches were
already developed in 1966 by Naylor and Gilham.[2] Most ligation
approaches developed since focus on ligation of two
complementary lagging strand fragments or ligation of the leading
and the lagging strand.[3] This concept was expanded to template-
mediated transfer reactions[4] also allowing selective protein
modifications.[5] Mainly nucleic acid derived templates have been
used, but also peptidic templates are established like the self-
replication on amphiphilic β-sheet peptides,[6] protein templated
peptide ligations[1], and coiled-coil motifs as guiding units.[7] The
potential of template-directed reactions was demonstrated when
applied to in vivo modifications of proteins.[8]
The advantage of nucleic acid derived templates is the
sequence dependent recognition of reaction partners allowing for
multiple orthogonal ligation events. Peptide nucleic acids (PNA)
are well established as DNA surrogates with higher contribution
of base pair recognition to duplex stabilities and lower mismatch
tolerance compared to DNA and RNA.[9] The reduced solubility of
PNA in water can be compensated with terminal lysine or arginine
residues. We envisioned a PNA based template-directed peptide-
[*]
Dr. S. Middel, Dr. C. H. Panse, S. Nawratil, Prof. Dr. U.
Diederichsen
Georg-August-Universität Göttingen
Institut für Organische und Biomolekulare Chemie
Tammannstraße 2, 37077 Göttingen (Germany)
E-mail: udieder@gwdg.de
This work was supported by a priority program (SPP1623) of the
German Research Foundation (DFG).
Scheme 1. Concept of the template-directed ligation strategy applying
photocleavable PNA strands. Two PNA/peptide hybrids with complementary
PNA strands recognize each other by PNA base pairing. The reactive centers
of the peptides are brought in close proximity and the ligation takes place. After
the ligation, the PNA strands can be cleaved by irradiation. PCL
photocleavable linker.
=
[**]
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700487
ChemBioChem
COMMUNICATION
The design of the photocleavable linker was derived from 1-
(2-nitrophenyl)propargyl alcohol used as photolytically cleavable
linker of two peptide fragments.[12] Nitrobenzene derivatives are
appropriate linkers based on their photochemical properties[13]
and use as peptide caging groups.[12, 14] In order to avoid
interference of deprotection with the nucleobase absorption the
wavelength for uncaging was shifted to 347 nm applying the 3,4-
methoxy nitrobenzene derivative.[15] The respective peptide was
bound to the N,N'-disuccinimidyl carbonate (DSC) activated linker
followed by attachment of the PNA oligomer by copper-catalyzed
[2+3] cycloaddition between an azide in the linker and an alkyne
modified PNA strand. This linker unit allows efficient N-terminal
on-resin functionalization of the peptide and on-resin click-
chemistry to attach the peptide with linker to the resin-bound PNA
oligomer.
group introduced to the glutamic acid side chain.[16] After
synthesis of hybrid 14, the SEA group was transformed into its
corresponding 3-mercaptopropionic acid (MPA) thioester 15 in
order to obtain reasonable reaction rates during ligation.
The preparation of the second hybrid 16 with the cysteine
containing peptide and complementary PNA sequence followed
the same strategy as for hybrid 14. The cysteine was introduced
coupled to the side chain of 2,3-diamino propionic acid (Dap)
being the second last N-terminal amino acid of the peptide
sequence. The availability of thiol and amino functions for the
ligation allows the irreversible S,N-shift reaction and provides a
stable peptide bond. Dap was used instead of lysine because less
background reaction was expected when the reactive center is
closer to the backbone.
The photocleavable linker was synthesized starting from
4,5-dimethoxy-2-nitrobenzaldehyde (5, Scheme 2). Wittig
reaction provided the corresponding alkene
6 which was
converted into epoxide by treatment with meta-
7
chloroperoxybenzoic acid (mCPBA). Nucleophilic ring-opening
provided azide 8 in a 2:1 mixture with the non-desired regioisomer.
Finally, the alcohol was activated with DSC to give the
photocleavable linker 9.
Scheme 2. Four-step synthesis of the photocleavable linker 9.
NaHMDS = sodium bis(trimethylsilyl)amide.
The respective peptide was prepared by solid phase peptide
synthesis (SPPS) on a Wang resin and as final coupling step the
activated linker 9 was attached to the N-terminus of peptide 12
followed by cleavage from solid support (Scheme 3). The linker
was efficiently attached and not affected by the conditions applied
for cleavage from the resin. Also, PNA 10 was obtained by SPPS
on a rink amide MBHA resin and was N-terminally functionalized
with propiolic acid applying only a small excess of activator N-
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in DMF.
Full conversion to PNA 11 was obtained using base-free coupling
conditions in order to avoid side-reactions upon deprotonation of
the alkyne. The peptide-linker construct was attached to the resin
bound alkyne functionalized PNA strand 11 by copper-catalyzed
azide-alkyne cycloaddition. The resulting triazole 14 was obtained
in high purity as indicated by HPLC and MS analysis.
Scheme 3. Convergent strategy for the peptide/PNA hybrid synthesis.
Modification of the PNA strand (11), attachment of the caging group (13) and
subsequent PNA/peptide synthesis by CuAAC (14) are equal for all hybrids. For
thioester hybrids an additional transthioesterification step is necessary.
Since peptide-peptide ligation should be facilitated by
recognition of complementary PNA sequences, the PNA duplex
needed to be formed at room temperature and the ligation being
applicable in aqueous buffer at neutral pH. The PNA sequences
were taken from literature[17] and it was ensured that the melting
temperature of 46 °C remains also for the PNA/peptide hybrids
For covalent NCL a thioester and a cysteine are required.
As thioesters are labile under basic conditions applied for Fmoc
deprotection during SPPS, a thioester precursor was incorporated
into the peptide using Melnyk's bis(2-sulfanylethyl)amido (SEA)
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700487
ChemBioChem
COMMUNICATION
(Supporting Information). The native chemical ligation
approaches were performed in degassed NaH2PO4 buffer
(pH 7.0) with tris(2-carboxyethyl)phosphine (TCEP) as a reducing
agent at room temperature. The cysteine hybrid 16 was allowed
to equilibrate for 30 min to remove the StBu protecting group
before the thioester hybrid 15 was added. No additional additives,
such as thiols were required since the template effect ensured a
fast reaction even with the moderately reactive MPA thioester.
The reaction was monitored by LC-MS. After 2 h 53% of the
thioester was consumed and after 8 h a conversion of 67% was
observed (Scheme 4). This result was reproduced in a second
ligation reaction differing in peptide sequences (Supporting
Information). In case the thioester containing amino acid is
sequentially flanked by glycines, aspartimide formation-like
cyclization was observed. Nevertheless, cyclization was
significantly slower than the ligation reaction and in case of
alanines or other sterically more demanding amino acids no
cyclization was observed (Supporting Information).
was obtained forming the capped oligomer 18; only
monoalkylated product was observed.
Photocleavage of ligated hybrid 18 was performed at 347
nm, 400 Watt (Scheme 5). After 45 min of irradiation 91% of the
oligomer was converted providing 68% of ligated peptide 19, the
templating oligomers 20 and 21, and only minor amounts of by-
product as indicated by ESI-MS of the crude reaction mixture
(Scheme 5). Interestingly, the caging group was cleaved at two
different positions. As expected, cleavage occurred at the
carbamate function and tracelessly released the unprotected
peptide. Nevertheless, photolytic cleavage also appeared at the
triazole breaking the C-N bond since the triazole is located at the
β-carbon with respect to the nitrophenyl moiety and triazole being
a good leaving group.[20] Despite these two cleavage sites the
overall deliberation of the ligated peptide was successfully
obtained.
Scheme 4. Template-directed native chemical ligation between thioester hybrid
15 and cysteine hybrid 16. HPLC chromatograms after 0, 1, 2, 4, 6, and 8 h
indicate consumption of hybrids 15 and 16 and formation of ligation product 17.
Conversion of thioester hybrid 15: 38% (1 h), 53% (2 h), 62% (4 h), 64% (6 h),
67% (8 h).
In order to ensure the impact of templating PNAs a
comparable ligation was conducted with hybrids containing non-
complementary PNA oligomers (identical sequences). We found
that non-templated ligation reactions proceed more than three
times slower than templated ligation reactions. (Supporting
Information).
Finally, photochemical cleavage of the linker/PNA was
attempted to deliberate the ligated peptide. First attempts
provided massive by-product formation most likely caused by the
free thiol group of the ligation product since this group is prone to
formation of radicals leading to side reactions such as hydrogen
abstraction, decarboxylation or fragmentation reactions.[18]
Therefore, prior to photocleavage the thiol was capped with
iodoacetamide in the ligation solution immediately following NCL.
In case the peptide sequence contains additional cysteins, NCL
based on selenocysteines will be considered which allows
selective deselenization afterwards.[19] Quantitative conversion
Scheme 5. a) Photocleavage of compound 18 provided the template units 20
and 21 and the released peptide 19. b) Left-hand side: HPLC chromatogram of
18 before irradiation. Right-hand side: HPLC chromatogram after 45 min of
irradiation. c) ESI-MS spectrum of the crude reaction mixture after irradiation.
In conclusion, an easily applicable and efficient protocol is
described for a new template-directed ligation strategy using
photocleavable guiding units. The PNA/peptide hybrids, linked by
a novel photocleavable linker, were readily synthesized, the
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700487
ChemBioChem
COMMUNICATION
[8]
[9]
U. Reinhardt, J. Lotze, S. Zernia, K. Mörl, A. G. Beck-Sickinger, O. Seitz,
Angew. Chem. 2014, 116, 10402-10406; Angew. Chem. Int. Ed. 2014, 53,
10237–10241.
S. Shakeel, S. Karim, A. Ali, J. Chem. Technol. Biotechnol. 2006, 81,
892–899.
ligation proceeded smoothly with good conversions and a
template effect was demonstrated. Finally, photolysis provided
traceless cleavage of the templating strands providing ligated
peptide 19. The strategy offers the possibility to synthesize
branched peptides with therapeutical potential.[10b] The template-
[10] a) C. Falciani, M. Fabbrini, A. Pini, L. Lozzi, B. Lelli, S. Pileri, J. Brunetti,
S. Bindi, S. Scali, L. Bracci, Mol. Cancer Ther. 2007, 6, 2441–2448; b) J.
Brunetti, C. Falciani, B. Lelli, A. Minervini, N. Ravenni, L. Depau, G. Siena,
E. Tenori, S. Menichetti, A. Pini, BioMed Res. Int. 2015, 2015, 1–7.
[11] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science 1994, 266,
776–779.
[12] J. A. Baccile, M. A. Morrell, R. M. Falotico, B. T. Milliken, D. L. Drew, F.
M. Rossi, Tetrahedron Lett. 2012, 53, 1933–1935.
[13] a) H. Schupp, W. K. Wong, W. Schnabel, J. Photochem. 1987, 36, 85–
97; b) P. Klán, T. Šolomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina,
V. Popik, A. Kostikov, J. Wirz, Chem. Rev. 2013, 113, 119–191; c) C. G.
Bochet, J. Chem. Soc. Perkin 1 2002, 125–142.
mediated ligation of C- to N-terminal linked and N-terminally
21]
linked peptides has been explored,[6,
here a strategy was
introduced for template-directed synthesis of branched peptides.
Currently, we are applying this ligation strategy to combinatorial
chemistry approach for peptide libraries encoded by a variation of
PNA sequences. Furthermore, the PNA mediated ligation
strategy will be explored for simultaneous protein modification at
different ligation sites as recently described for a library with
different protein-conjugates.[22]
[14] N. Bindman, R. Merkx, R. Koehler, N. Herrman, W. A. van der Donk,
Chem. Commun. 2010, 46, 8935–8937.
Keywords: peptide ligation • templated reactions •
photocleavable linker • peptide nucleic acid • native chemical
ligation
[15] R. Beukers, W. Berends, Biochim. Biophys. Acta 1961, 49, 181–189.
[16] a) E. Boll, J. Dheur, H. Drobecq, O. Melnyk, Org. Lett. 2012, 14, 2222–
2225; b) J. Dheur, N. Ollivier, A. Vallin, O. Melnyk, J. Org. Chem. 2011,
76, 3194–3202.
[17] P. Wittung, P. E. Nielsen, O. Buchardt, M. Egholm, B. Nordén, Nature
1994, 368, 561–563.
[18] F. Dénès, M. Pichowicz, G. Povie, P. Renaud, Chem. Rev. 2014, 114,
2587–2693.
[19] a) R. J. Hondal, B. L. Nilsson, R. T. Raines, J. Am. Chem. Soc., 2001,
123, 5140–5141. b) N. J. Mitchell, S. S. Kulkarni, L. R. Malins, S. Wang,
R. J. Payne, Chem. Eur. J., 2017, 23, 946-952.
[20] a) S.-S. Tseng, E. F. Ullman, J. Am. Chem. Soc. 1976, 98, 541–544; b)
K. Qvortrup, T. E. Nielsen, Chem. Commun. 2011, 47, 3278.
[21] a) K. Severin, D. H. Lee, A. J. Kennan, M. R. Ghadiri, Nature 1997, 389,
706–709; b) R. R. Araghi, B. Koksch, Chem. Commun. 2011, 47, 3544-
3546.
[1]
N. Brauckhoff, G. Hahne, J. T.-H. Yeh, T. N. Grossmann, Angew. Chem.
2014, 126, 4425-4429; Angew. Chem. Int. Ed. 2014, 53, 4337–4340.
R. Naylor, P. T. Gilham, Biochemistry 1966, 5, 2722–2728.
[2]
[3]
a) C. Böhler, P. E. Nielsen, L. E. Orgel, Nature 1995, 376, 578–581; b) A.
Luther, R. Brandsch, G. von Kiedrowski, Nature 1998, 396, 245–248; c)
Z.-Y. J. Zhan, D. G. Lynn, J. Am. Chem. Soc. 1997, 119, 12420–12421;
d) Z. J. Gartner, M. W. Kanan, D. R. Liu, Angew. Chem. 2002, 114, 1874-
1878; Angew. Chem. Int. Ed. Engl. 2002, 41, 1796–1800; e) A. Roloff, O.
Seitz, Chem Sci 2013, 4, 432–436; f) X. Li, D. R. Liu, Angew. Chem. 2004,
116, 4956-4979 Angew. Chem. Int. Ed. 2004, 43, 4848–4870.
R. K. Bruick, P. E. Dawson, S. B. Kent, N. Usman, G. F. Joyce, Chem.
Biol. 1996, 3, 49–56.
[4]
[5]
[22] S. Dickgiesser, N. Rasche, D. Nasu, S. Middel, S. Hörner, O. Avrutina, U.
Diederichsen, H. Kolmar, ACS Chem. Biol. 2015, 10, 2158–2165.
G. Li, Y. Liu, Y. Liu, L. Chen, S. Wu, Y. Liu, X. Li, Angew. Chem. 2013,
125, 9723-9728; Angew. Chem. Int. Ed. 2013, 52, 9544–9549.
[6] B. Rubinov, N. Wagner, H. Rapaport, G. Ashkenasy, Angew. Chem. 2009,
121, 6811-6814; Angew. Chem. Int. Ed. 2009, 48, 6683–6686.
[7]
a) D. H. Lee, J. R. Granja, J. A. Martinez, K. Severin, M. R. Ghadiri,
Nature 1996, 382, 525–528; b) R. Issac, Y. W. Ham, J. Chmielewski, Curr.
Opin. Struct. Biol. 2001, 11, 458–463.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700487
ChemBioChem
COMMUNICATION
COMMUNICATION
S. Middel, C. H. Panse, S. Nawratil, U.
Diederichsen*
Page No. – Page No.
Photocleavable PNA Strands for
Template-Directed Native Chemical
Ligation
A novel template-directed native chemical ligation strategy applying photocleavable
PNA strands is presented. PNA/peptide hybrids, connected by a photocleavable
linker, recognize by PNA base pairing and the reactive centers are brought in close
proximity facilitating the ligation. Afterwards, the templating PNA oligomers can be
cleaved by irradiation.
This article is protected by copyright. All rights reserved.