Design and synthesis of enediyne-based peptide with selective peptide-cleaving activity

Article abstract of DOI:10.1039/b923814j

A hybrid peptide-enediyne molecule was synthesised and shown to undergo selective intramolecular peptide chain cleavage by the 1,4-diyl radical, the potential intermediate of the enediyne system.

Full text of DOI:10.1039/b923814j

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Design and synthesis of enediyne-based peptide with selective
peptide-cleaving activityw
Snigdha Roy and Amit Basak*
Received (in Cambridge, UK) 12th November 2009, Accepted 17th December 2009
First published as an Advance Article on the web 25th January 2010
DOI: 10.1039/b923814j
A hybrid peptide–enediyne molecule was synthesised and shown
to undergo selective intramolecular peptide chain cleavage by the
1,4-diyl radical, the potential intermediate of the enediyne
system.
Some naturally occurring enediynes are protein complexes, the
Fig. 1 Design of peptide cleaving agent.
non-protein (i.e. enediyne) component of which has proteolytic
activity. In the case of kedarcidin, the non-protein enediyne
component recognises and cleaves histones.1,2 The enediyne
component of esperamicin has also been shown to cause
proteolysis and to have antitumour activity.3 Diradicals,
generated from the reactive enediyne moiety, abstract hydrogen
from proteins,4 including at backbone positions, resulting in a
captodatively stabilized radical as an intermediate,5 whose fate
depends on the environment. It may interact with molecular
oxygen and the resulting peroxy radical ultimately leads to the
cleavage of the peptide. Alternatively, the peptide radical may
cross-link or form an adduct with another radical source.
Inspired by this finding, we intended to synthesize a novel
enediyne–peptide hybrid with peptide cleavage activity at
tailor-made positions. For this we needed a molecular scaffold
where the basic template contains two long parallel chains in
the cisoid form. One of these is a polypeptide chain while the
other carries the enediyne frame connected to the template via
a suitable linker (Fig. 1). The cisoid conformation of these two
chains is essential to bring the enediyne moiety closer to the
peptide chain. In order to achieve such an arrangement, we
have selected linkers that can participate in hydrogen bonding
with the peptide chain. The purpose is to cut the peptide chain
selectively via the aid of the 1,4-diyl radicals. Based upon
our earlier experience with H-bonded enediyne containing
scaffolds,6,7 we have designed and synthesized a novel class
of peptide–enediyne conjugate 3 (Fig. 2) and subsequently
demonstrated its selective cleavage ability of the peptide
moiety by mass spectrometric studies. A peptide 4 with a
similar scaffold also followed a similar cleavage pathway, thus
supporting selectivity in the degradation pathway.
first converting the E to the Z form by photoirradiation (365 nm)
followed by a thermal re-isomerization experiment. The half
life for the re-isomerization was found to be B12 h at room
temperature. Since generation of diradicals from aryl fused
enediynes requires temperatures higher than 60 1C, a half life
of 12 h at B30 1C is not sufficient to keep the two arms of the
azo compound in the cis form. This result prompted us to
prepare the corresponding more reactive aliphatic enediyne-
based compound 2 following the same synthetic protocol as
for compound 1. It was found to be extremely reactive and
could be converted to the Z-form (half life B12 h) by
photoirradiation. Upon incubation of a d6-benzene solution
of compound 2 in predominant Z form, fragmentation of the
peptide chain could be observed, as revealed by the appearance
of peaks having smaller m/z values in the ESI mass spectrum
recorded on the incubation mixture. However, the fragmentations
could not be explained properly and the pattern did not match
that proposed by Jones and Warner10 or Hirama et al.11 for
the degradation of peptides or proteins (Scheme 1).
After the inconclusive results with the azo-based compounds,
we decided to change the basic template to a 1,2-dialkynyl
benzene in order to avoid the problem of thermal re-
isomerization. The reason for choosing the two alkyne moieties
Our first template, based on the azobenzene moiety, relied
upon the photochemically or thermally induced reversible
E–Z-isomerization8,9 that would have offered a new handle
to trigger cleavage activity. Thus the molecule 1 was first
synthesized and its half-life in the Z form was measured by
Indian Institute of Technology, Chemistry, IIT Main Road,
Kharagpur, West Bengal 721302, India.
E-mail: absk@chem.iitkgp.ernet.in
w Electronic supplementary information (ESI) available: Synthesis of
1, 2 and 4, 1H, 13C NMR of new compounds, VT NMR, COSY, DSC
and MS spectra. See DOI: 10.1039/b923814j
Fig. 2 Target enediyne–peptide hybrids.
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Chem. Commun., 2010, 46, 2283–2285 | 2283

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Scheme 1 Generation and fate of C-centered peptide radicals.
Scheme 2 Synthesis of enediyne–peptide hybrid 3. Reagents and
conditions: (a) MsCl, Et3N, DCM, 0 1C, 15 min, 96%; (b) NaN3,
DMF, rt. 7 h, 67%; (c) PPh3, THF, H2O, rt, 10 h, 70%; (d) 10,
Cs2CO3, DMF, rt, 10 h, 59%; (e) TFA, anisole, DCM, 0 1C to rt, 1 h;
(f) 8, EDCÁHCl, DMAP, DCM, rt, 10 h, 50%; (g) TFA, DCM, 0 1C
to rt, 1.5 h; (h) 15, EDCÁHCl, DMAP, DCM, 0 1C to rt, 10 h, 41%.
in the template was to allow more space between the adjacent
chains so that these do not suffer severe steric crowding.
A succinoyl moiety was used as a linker to connect the
enediyne while the tetrapeptide (Boc-gly-gly-gly-gly) was
connected directly to the aminopropargyl arm. The synthesis,
although quite straightforward, involved several steps and was
accomplished starting from 1,2-diiodobenzene (Scheme 2). The
compound 3 was characterized by NMR and mass spectro-
scopic studies. It showed an onset temperature of B65 1C for
Bergman cyclization as indicated by DSC.12
Fig. 3 MS-MS spectra.
The formation of 19 and 20 could be explained if one of the
radical centres in the diradical abstracts the a-H from the third
glycine (gly-gly-gly-gly-Boc) which then reacts with molecular
oxygen. The resulting peroxy radical then undergoes degradation
following pathways proposed by Jones and Warner10 and
Hirama et al.11 to generate the fragments 19 and 20 respectively.
The fragmentation pathway is shown in Scheme 3. If similar
abstraction had taken place from the second glycine (gly-gly-
gly-gly-Boc), peaks at m/z 615 and 631 were expected which
were not present in the mass spectrum (Scheme 4). Similarly, the
absence of peaks at m/z 729 and m/z 745 also ruled out the
degradation from abstraction of the H from the terminal glycine.
To reinforce this concept of selective cleavage, a pentapeptide–
enediyne hybrid 4 was also synthesized and was subjected to the
same incubation conditions. It also showed production of
fragments via Jones and Hirama pathways, as indicated by the
appearance of peaks at m/z 764 and 780 in the MALDI spectrum
(Scheme 5). The exact reason for this preference to abstract an
a-H from the third glycine unit may be due to its proximity to the
radical (generated via BC). This has been supported by the
energy minimized structure of the starting enediyne (included
in the ESIw) which showed the proximity of the terminal
acetylenic carbon and the a-H of the third glycine unit.
To examine the self fragmentation of 3, we incubated its
solution in d6-benzene (500 mL) containing 4 drops of DMSO
(for solubility) at 65 1C for 10 days. The degradation of the
peptide side chain by the diradical formed by the cyclisation of
enediyne was confirmed by MALDI-TOF-MS which showed
two strong new peaks at m/z 672.5 and 688.5, corresponding to
molecular ions (+Na) for 19 and 20, respectively. The mass
spectrum was recorded in ESI mode which also showed the
presence of 19 and 20 in the incubation mixture (appearance of
peaks at m/z 649 and 665 respectively). ESI-MS-MS studies on
molecular ion peaks at 856 (molecular ion for 3 + Na),
665 and 649 revealed that all are separate entities. The MS
on the molecular ion for the starting enediyne gave peaks at
800 and 756. The MS analysis on 665 and 649 produced
peaks that are shown in Fig. 3 along with possible structures.
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peptide chain cleavage by the 1,4-diyl radical. This reveals the
possibility of selective protein cleavage via a similar strategy.
Current studies are aimed towards that direction.
The authors AB and SR are grateful to CSIR, Government
of India, for research funding and a fellowship respectively.
DST is thanked for the funds for a 400 MHz NMR facility
under the IRPHA program.
Notes and references
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Scheme 3 Mass fragmentation for abstraction of H at C-3.
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Scheme 4 Possible fragmentation for abstraction of H at C-2.
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Scheme
5 Abstraction of a-H from third glycine unit of the
pentapeptide chain of compound 4.
In conclusion, we have successfully accomplished the
synthesis of enediyne–peptide hybrids which showed selective
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2283–2285 | 2285