Synthesis and DNA binding ability of cyclam-amino acid conjugates

Article abstract of DOI:10.1016/j.bmcl.2008.03.045

N-Substituted cyclam-amino acid conjugates have been synthesised both in solution and on the solid phase. The DNA binding affinity of these species has been studied: the nature of the amino acid strongly influences the change in melting temperature sugges

Full text of DOI:10.1016/j.bmcl.2008.03.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3007–3010
Synthesis and DNA binding ability of
cyclam–amino acid conjugates
A. Venkata Ramana,a Michael Watkinsonb and Matthew H. Todda,*
aSchool of Chemistry, University of Sydney, NSW 2006, Australia
bThe Walter Besant Building, School of Biological and Chemical Sciences, Queen Mary,
University of London, Mile End Road, London E1 4NS, UK
Received 19 December 2007; revised 13 March 2008; accepted 17 March 2008
Available online 20 March 2008
Abstract—N-Substituted cyclam–amino acid conjugates have been synthesised both in solution and on the solid phase. The DNA
binding affinity of these species has been studied: the nature of the amino acid strongly influences the change in melting temperature
suggesting that simple cyclam–peptide conjugates could interact with DNA in a highly selective manner.
Ó 2008 Elsevier Ltd. All rights reserved.
The interaction of small molecules1 and metal nucleases2
with DNA has attracted much attention in chemistry,
biology and medicine. In this context, tetraazamacrocy-
cles are of interest due to their ability to form stable
metal complexes which are able to hydrolytically cleave
DNA.3 There is even preliminary evidence that such
ligands are able to cleave DNA in the absence of metal
ions.4 The ligands also have potential applications as
anti-tumour and anti-HIV agents.5
Among the various azamacrocycles, cyclam (1,4,8,11-
tetraazacyclotetradecane) and its N-substituted deriva-
tives have attracted much attention due to their wide
range of applications in medicine.10 Peptide nucleic
acids with a terminal cyclam have been synthesised
and their interactions with DNA have been studied.11
We sought to apply our recent synthetic experiences in
the synthesis of cyclam analogues functionalised with
biotin12 to the synthesis of cyclam–amino acid conju-
gates, although there are other literature examples of
the synthesis of cyclam–oligopeptide conjugates both
in solution and on solid phase supports, primarily for
radiolabelling applications.13 Whilst there have been
syntheses of cyclam–amino acid conjugates in which
the linking bond is the amide arising from a cyclam
nitrogen and the amino acid carboxylic acid,14 we, and
others, have found the metal complexes of such species
to be unstable with respect to hydrolysis.15 As our ulti-
mate goal of this research is the synthesis of functional-
ised azamacrocyclic complexes for the sequence-selective
cleavage of DNA that will rely on the presence of the
peptide chain, such behaviour would preclude the use
of this linker. As the first step, we report here the synthe-
sis of several novel cyclam–amino acid conjugates and
their interaction with calf thymus (CT) DNA.
Nature exquisitely controls the site-selective cleavage of
DNA with metal nucleases by employing peptide-based
interaction domains, for example, in restriction
enzymes.6 The introduction of such peptidic side chains
into azamacrocycles could permit sequence selectivity in
their interaction with DNA, as well as allowing the
fine-tuning of other parameters such as lipophilicity
and bioavailability, leading to sequence-selective small
molecule nucleases or binders, of which there are still
few examples.7 Such an approach could generate a
new class of molecular therapeutics based on selective
interactions with problematic DNA sequences, for
example, the TT(N5)AA consensus sequence that binds
to Stat-5, a transcription factor implicated in prostate
and breast cancers,8 or the G-rich sequence of telomeres,
associated with ‘immortalised’ cancer cell lines.9
Synthesis of cyclam–amino acid conjugates. Solution
phase synthesis. Commercially available cyclam
1
was treated with di-tert-butyldicarbonate to give the
tri-Boc derivative 2 using standard literature methodol-
ogy (Scheme 1).12,16 This was further coupled with ethyl
bromoacetate to give 3, which on hydrolysis under
Keywords: Cyclam; Tetraazamacrocycle; DNA binding; Sequence
selectivity.
*
Corresponding author. Tel.: +61 2 9351 2180; e-mail: m.todd@chem.
usyd.edu.au
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.045

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A. Venkata Ramana et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3007–3010
Scheme 1. Reagents and conditions: (a) R1 = Me; (b) R1 = Bn. (i) (Boc)2O, Et3N, DCM, À15 °C to rt, 12 h, 53%; (ii) BrCH2CO2Et, Na2CO3,
CH3CN, 85 °C, 16 h, 97%; (iii) NaOH, MeOH, rt, 5 h, 68%; (iv) NH2CHR1CO2Me, DCC, DMAP, DMF, DCM, rt, 12 h, 60–75%; (v) NaOH,
MeOH, rt, 6 h, 78–85%; (vi) TFA, DCM, rt, 12 h, 80–85%.
standard conditions gave acid 4. This acid was coupled
with alanine or phenylalanine amino acid esters to give
the novel compounds 5a–b. Direct saponification of
the methyl esters in 5 was readily achieved to provide
the acid intermediates 6a–b from which the free
macrocycles 7a–b, respectively, could be liberated using
TFA. Analogous esters 8 were obtained by direct Boc
deprotection of 5 using TFA. This sequence, though
requiring protection and deprotection steps, is
experimentally quite simple and proceeds in an overall
yield of approximately 15% for the six steps. However,
one of the low yielding steps is the peptide coupling.
In our previous work on a related reaction we discov-
ered that HATU gave yields of 80% or more for this
kind of coupling reaction,12 but if we were to iterate this
coupling step in the synthesis of cyclam ligands func-
tionalised with oligopeptide arms, the final yield would
become unacceptably low. We consequently examined
an alternative solid phase synthetic approach to these
molecules.
Scheme 2. Reagents and conditions: (a) R = Me; (b) R = Bn; (c)
R = CH2OBn. (i) Compound 4, DIPEA, HBTU, DCM, 12 h; (ii) TFA,
DCM, 10 h; (iii) H2SO4, MeOH, 5 h.
The solid phase synthesis of these compounds presents
considerable advantages over the solution phase as we
were able to prepare these compounds with high purity
and in higher yields than the corresponding solution
phase methods (Table 1, yields based on comparable
sequences for the two routes). The solid phase method
provided a significantly more expedient route to these
compounds than the corresponding solution phase
approach. All compounds synthesised by the solid phase
approach were consistently around 90% pure as judged
by HPLC analysis of the isolated material with no
further purification.17
Solid phase synthesis. Cyclam–amino acid conjugates
were synthesised on the solid phase by employing stan-
dard Fmoc methods using chlorotrityl resin. The resin
was loaded with an Fmoc protected amino acid, which
on deprotection gave the supported amine 9. The amine
could then be coupled to acid 4 to give the supported
conjugate 10. The free acid 7 could then be liberated
from the resin on treatment with TFA and converted
to their methyl esters 8 in solution on treatment with
H2SO4 in MeOH (Scheme 2). Using this method we
were also able to synthesise the serine analogues 7c
and 8c; these had given poor yields when synthesised
by the solution phase approach.
DNA binding experiments. The DNA-binding ability of
these new cyclam–amino acid conjugates was investi-

A. Venkata Ramana et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3007–3010
3009
Table 1. Comparative yields for solution versus solid phase synthesis
of cyclam–amino acid conjugates
and solid phase. The solid phase approach has a num-
ber of distinct advantages in terms of yield and oper-
ational simplicity, and will be a powerful method for
the synthesis of more elaborate cyclam–peptide conju-
gates. These compounds have been further studied for
their ability to bind to DNA. It was found that the
nature of the amino acid employed greatly affected
the increase in melting temperature. We observed a
large increase in melting temperature for the cyclam
attached to O-benzyl-protected serine. It is possible
that the stabilisation arises from the intercalation of
the benzyl group with the DNA bases. However, the
stabilisation observed with the phenylalanine analogue
was considerably lower in magnitude. It is possible
that the protected oxygen of the serine is involved in
a favourable hydrogen-bonding interaction with the
DNA. The deprotected form of 7c is therefore of
further interest. The assessment of the ability of the
corresponding metal complexes of these derivatives
to bind DNA and cleave simple phosphate esters is
currently underway.
Compound
% Yield in
solution phasea
% Yield on
solid phaseb
7a
8a
7b
8b
7c
8c
34
51
39
52
—
—
90
90
85
82
84
78
a Yields based on yield of final product from 4.
b Yields based on steps between 9 and isolated product after removal
from resin.
gated by thermal denaturation studies using calf thymus
(CT) DNA (Fig. 1). Melting studies show that these
compounds stabilize the thermal helix coil or melting
stabilization (DTm) for the CT-DNA duplex at pH 7.0.
The compound/DNA molar ratios measured were 1:3
and 1:5. For the acids (7a–c) the change in melting tem-
perature (DTm) is variable, showing that the extent of
stabilisation is governed by the amino acid employed.
The esters 8a–c in contrast exhibit far less stabilisation
of the helix. These results are particularly pleasing when
it is observed that cyclam itself does not appreciably sta-
bilise the helix, and the tri-Boc acid 4 does not stabilise
the helix at all. Further the magnitude of the increase in
melting temperature for compound 7c is approximately
8 °C, which is nearly as large as that observed for ethi-
dium bromide, our reference compound used for these
experiments.18 It is particularly surprising that a rela-
tively large increase in melting temperature can be gen-
erated with the addition of only one amino acid. This is
a significant result, and suggests it might be possible to
tune the stabilisation of the DNA helix with polypep-
tides on more elaborate cyclam-based structures. The
mode of interaction with DNA was electrostatic in nat-
ure, confirmed by a gradual lowering of the DTm when
the melting temperatures were measured after the addi-
tion of increasing quantities of 1 M NaCl solution to the
original buffer.
Acknowledgments
We thank the Sydney University Cancer Research Fund
for funding. We thank Taliesha Paine and Clement Pon-
toizeau for preliminary synthetic experiments towards
compounds 4 and 5.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.03.045.
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Figure 1. Changes in calf thymus DNA melting temperatures upon
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