Design, synthesis and DNA-cleaving efficiency of photoswitchable dimeric azobenzene-based C2-symmetric enediynes

Article abstract of DOI:10.1039/b801644e

Designed azobenzene-based enediyne-amino acid C₂-symmetric hybrids have been synthesized and the role of amino acid linker in stabilizing the Z form has been demonstrated; DNA-binding and cleavage studies have established higher reactivity of t

Full text of DOI:10.1039/b801644e

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Design, synthesis and DNA-cleaving efficiency of photoswitchable dimeric
azobenzene-based C2-symmetric enediynesw
Amit Basak,* Debarati Mitra, Moumita Kar and Kumar Biradha
Received (in Cambridge, UK) 30th January 2008, Accepted 28th March 2008
First published as an Advance Article on the web 25th April 2008
DOI: 10.1039/b801644e
Designed azobenzene-based enediyne-amino acid C2-symmetric
hybrids have been synthesized and the role of amino acid linker
in stabilizing the Z form has been demonstrated; DNA-binding
and cleavage studies have established higher reactivity of the
Z-isomers.
The complementarity of conformation between the Z-isomer
and the helical shape of DNA may ensure higher binding with
ds-DNA and hence higher cleaving efficiency. The stability
imparted by the p-stacking interaction12 between the benzene
rings of the phenylalanine-based system is expected to slow
down the kinetics of thermal Z to E re-isomerisation. It is
interesting to note that the energy minimization of the Z-form
of the non C2-symmetric molecule 2e containing D- and
L-phenylalanine showed very little p-stacking interactions in
the Z-isomer. For 2d, the isopropyl group of valine lies away
from the phenyl group.
Symmetry plays an important role in many chemical and
biological processes.1,2 Many key proteins and enzymes exist
as homo dimers with two-fold axis of symmetry namely the
C2-symmetry. These are biologically active only in this homo
dimeric form.3–5 Various research groups have designed
C2-symmetric DNA-cleaving agents6 and studied their clea-
vage efficiency.7 Enediynes have now been established as a
fertile ground for designing novel DNA-cleaving agents.8
Although many reports of ingenious design of enediynes are
there in the literature,9 including from our own laboratory,10
as such there is no report of how the biological activity of
enediynes depends upon their symmetry property. In order to
study such effects, we have designed novel hybrid molecules
consisting of azo benzene,11 enediyne and amino acid. These
molecules have different symmetry groups in the E and Z
forms. Their synthesis and chemical as well as biological
reactivities are reported herein which nicely demonstrated
the importance of geometric considerations while designing
new enediynes. Since C2-symmetry is our prime consideration,
two types of molecules Type I and Type II were targeted
(Fig. 1). Both the types consisted of azobenzene, enediyne and
amino acid which are joined by two carbon linkers. Use of
same amino acid in Type I systems retains the C2 while
incorporation of dissimilar amino acids or same amino acid
with different configuration in Type II molecules breaks the
C2-symmetry. The latter molecules were synthesized for com-
paring the activity of the C2 and non-C2 symmetric molecules.
The actual target molecules belonging to different classes are
shown in Fig. 1. We wanted to address the following question:
can perturbation of symmetry element affect the DNA-cleav-
ing potential of enediyne?
The synthetic endeavour towards 1a/1b started with the
preparation of the azobenzene-amino acid diesters 3a/3b.
These were hydrolysed to the free acids 4a/4b. The corre-
sponding dipotassium salts 5a/5c were esterified with 2-bro-
moacetylamine 6 in DMF.13 The target molecules 1a/1b were
isolated pure in 85% yield after column purification (Si-gel,
DCM : MeOH = 30 : 1). The corresponding aliphatic system
1c was also prepared via a similar route (Scheme 1). The
synthesis of Type II molecules was similarly carried out. The
structures of all the compounds were in agreement with
1H, 13C NMR as well as mass spectral data (HRMS).
The configuration around the N–N double bond was first
determined by recording the UV spectrum in CH3CN. For the
trans compounds a strong lmax at 363 nm appeared typical of
the trans azobenzenes.14 For the cis isomers a characteristic
lmax at higher wavelength of 440 nm was observed. The
We first carried out the MM2 calculation to find out the
energy-minimized conformation of Type I molecules 1a and
2a. The exercise revealed a crescent shape conformation for
the Z-isomer, while the E-isomer has a zigzag conformation.
Bioorganic Laboratory, Department of Chemistry, Indian Institute of
Technology, Kharagpur, India. E-mail: absk@chem.iitkgp.ernet.in;
Fax: +91 3222 282252; Tel: +91 322 283300
w Electronic supplementary information (ESI) available: 1H, 13C
NMR of new compounds, VT NMR, details of MM2 and DNA-
binding and cleavage studies. CCDC 676458. For ESI and crystal-
lographic data in CIF or other electronic format see DOI: 10.1039/
b801644e
Fig. 1 Structures of target enediynes.
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Fig. 2 X-Ray crystal structure of 3b (CCDC 676458, ESIw).
(CDCl3) it has a half life of 22 h at 30 1C as compared to 36 h
for the simple sulfonamide of same ring size.17 The non-
aromatic enediynes 1c/2c are the most reactive ones while
the aromatic ones, although quite stable at room temperature
did show slow cyclization and hence are candidates for study-
ing the DNA-cleavage activity under biological conditions.
The formation of cycloaromatized products was confirmed by
mass spectrometric data which showed peaks at MH++4
corresponding to the addition of four hydrogens to the two
sets of diradicals.
The photoisomerization was done by irradiation (354 nm)
of CH2Cl2 solution of the E-enediynes for 4–5 h. The half lives
for the thermal re-isomerization of the Z-enediynes to the
corresponding E-forms are shown in the Table 1. Incorpora-
tion of amino acids as spacers has significantly slowed down
the Z to E conversion as compared to systems without amino
acid 9 and 10.
For the assessment of DNA-cleaving potential, the various
enediynes (both E and Z isomers at mmol concentrations) were
separately incubated with pBR322 plasmid DNA. The incuba-
tions were carried out at 20 1C (to suppress the re-isomeriza-
tion) and the other at 37 1C. For enediynes 1a/2a, the gel
pattern clearly indicated better cleaving efficiency (B2ꢁ) for Z
isomer 2a as compared to the E isomer 1a (Fig. 3). The valine-
based enediynes 1b/2b were, however, found to be poor DNA-
cleavers after 2 h incubation both at 20 and 37 1C thus making
it difficult to compare the relative cleaving efficiencies of the
E and Z isomers.
Scheme 1 Synthesis of azobenzene enediyne amino acid hybrids.
NOESY spectrum taken on a mixture of E and Z isomers
showed cross peaks between the proton at C-8 and the
benzylic methylene at C-50 for only the Z-isomer. It is also
to be noted that the aromatic protons in all the cis isomers
appeared upfield suggesting less conjugation due to non-
planarity of the azobenzene ring which is the case for the cis
form. Final confirmation about the geometry came from the
single-crystal X-ray structure of the valine based ester 3b
which clearly showed the trans disposition of the two arms
(Fig. 2). The structures of other azo systems were deduced
from a comparison of characteristic NMR signals with those
of 3b.
For the non-benzenoid enediynes 1c/2c, the gel pattern after
2 h clearly indicated higher cleaving potency for the Z-isomer
(B30%) as compared to the E-isomer. However, with time, as
more and more Z form is converted to the E-isomer, the extent
of cleavage became almost the same. The other interesting and
highly important observation is the generation of linear DNA
(form III) for the aliphatic enediynes; the Z-form showing
generation of form III after 2 h of incubation at 20 1C while at
37 1C both E and Z forms showed linear cuts. It is possible
that the linear cut observed for the E-isomer is due to the
presence of the Z-form (B15%) under equilibrating
The reactivity of the enediynes towards Bergman cyclization
(BC)15 as studied by differential scanning calorimetry (DSC)16
indicated similar onset temperatures (80–90 1C) for the aro-
matic enediynes which is very similar to what was observed for
isolated enediynyl amides.13 The solution phase kinetics per-
formed on 1a did show slow BC kinetics at 45 1C with a half
life of 7 days. For the non-aromatic enediyne, the E isomer 1c
showed an onset temperature of 50 1C for BC while in solution
Table 1 Kinetics of Z to E thermal isomerization
Compound
t1/2(20 1C)/h
Compound
t1/2(20 1C)/h
2a
2b
2c
30.0
19.0
7.2
2d
2e
10
12.0
19.0
2.3
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ing this idea to generate water-soluble cytotoxic agents. Since
E to Z isomerization is done photochemically, our work opens
up the possibility of enhancing the DNA-cleaving efficiency of
the C2-symmetric enediynes via photoirradiation.
A. B. thanks DST, Government of India for a research
grant. D. M. and M. K. are grateful to CSIR, Government of
India for a senior research fellowship. We are also grateful to
Prof. A. K. Ghosh and Prof. A. K. Das, Biotechnology
Department for their help in densitometric measurements
and MM2 calculations. The Department of Science and
Technology, Govt. of India, is thanked for the CCD-X-ray
(under FIST) and 400 MHz NMR facilities (under IRPHA).
Notes and references
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Fig. 3 pBR322 DNA cleavage experiment of compounds 1a–1e/
2a–2e; For A–H: lane 1: control DNA in TAE buffer (pH 8, 7 mL)
+ CH3CN (5 mL); lane 2: DNA in TAE buffer (pH 8, 7 mL) + E
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enediyne 8 is a much inferior cleaving agent as compared to 1c/
2c; the extent of cleavage even after 12 h incubation was much
less (only 50% as compared to 85%). It also failed to show
formation of any linear form under similar conditions.17 The
non-C2-symmetric molecules 1d/2d and 1e/2e have been found
to be very poor DNA-cleavers. All these point to the impor-
tance of having an azobenzene endowed with C2-symmetry in
the design.
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concentration of the probe18 indicated higher degree of hypo-
chromism19 for the Z-isomer 2a as compared to 1a. The
binding constant (using the Benesi–Hildebrand equation)
revealed greater binding affinity for enediyne 2a (2.5ꢁ as
compared to 1a). Thus the higher DNA-cleavage efficiency
for the Z-isomer is correlated to its binding efficiency.
Thus, we have successfully designed and synthesized azo-
benzene-based enediyne–amino acid C2-symmetric hybrids. A
new way of modulating the biological activity of this class of
molecules by introducing a symmetry element like C2 has been
firmly established. Future research will concentrate on exploit-
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