specific recombination can control gene expression, amplify
episome copy number, create genetic diversity, and separate
chromosomes at bacterial cell division. By trapping the HJ
intermediate, we may inhibit site-specific recombination.7
Blocking this recombination reaction should eventually lead
to bacterial death. Thus, compounds that successfully trap
the HJ will lead to new antibiotics. Such inhibitors are
reminiscent of the quinolone/fluoroquinolone class of anti-
biotics, which stabilize a normally transient intermediate.
Herein we describe the synthesis of eight potential
antibiotics. We based our design on the cocrystal structure8
of the hexapeptide that dimerizes to give the symmetrical
active lead structure9 and the C-2 symmetrical Holliday
junction (Figure 1). The previously identified peptide leads
fit the approximate size of the HJ binding site, which is
estimated to be ∼25 Å by 10 Å.8 Because symmetrical linear
peptides are known to trap the HJ intermediate, we designed
C-2 symmetric cyclic peptides that mimic the symmetry of
the HJ binding site and contain residues found in these lead
compounds (Figure 2).8,9
Figure 2. Synthesis strategy.
Our synthetic approach was chosen to simplify the
synthesis of C-2 symmetrical macrocyclic hexapeptides for
rapid biological assessment. By cyclizing the peptides, we
aimed to increase their rigidity10 so that we could identify
the exact nature of contacts between the compounds and the
HJ. At the same time this should decrease their degradation
rate inside cells.
The minimal number of residues involved in binding to
the Holliday junction is not known, but several important
interactions have been identified.9 Hydrophobic residues such
as tryptophan and phenylalanine are known to be important
for binding to the DNA because they appear in all of the
lead compounds9 and are thought to stack with the DNA
nucleotides that surround the center of the HJ. Hydrophilic
residues such as arginine and lysine may form hydrogen
bonds either with the protein that assembles the HJ or with
the DNA substrate itself, or both. Starting from commercially
available natural and unnatural amino acids, we have
synthesized cyclic hexameric peptides. Initially, we chose
to use a number of hydrophobic residues in the macrocyclic
peptides and sequentially exchange hydrophobic residues
with hydrophilic residues.
Figure 1. Cocrystal structure was obtained by mixing a mutant
Cre protein with loxS DNA substrates. The crystal was dependent
on blocking catalysis.8 A similar cocrystal structure was obtained
between wild-type Cre protein and lox substrates, but only in the
presence of the linear lead peptides. This latter crystal contains
additional electron density in the HJ center, consistent with bound
peptide in the central “hole”.8 Note the C-2 symmetry of the
structure. One lead linear hexapeptide structure is Lys-Trp-Trp-
Cys-Arg-Trp, where the active peptide is a dimer of this linear
peptide.
are linear dodecapeptides, and their flexibility presumably
prevents accurate pinpointing of the specific residues in-
volved in the binding event. However, electron density
attributed to the peptides binding to the HJ is detected in
the center of the crystal structure.
With the goal of gaining insight into the biological
mechanism of action and finding new antibiotics for this
unique target, we describe the synthesis of macrocycles that
were designed from the C-2 symmetric HJ binding site and
Segall’s discovery of lead compounds.9 These macrocycles
should be more rigid than the linear lead peptides, and they
A great deal of literature has documented the various
advantages and disadvantages associated with peptide chem-
istry.3 The eight compounds described represent an initial
set of potential antibiotics that lay the groundwork for our
(7) Nash, H. A. Cellular and Molecular Biology, 2nd ed.; Neidhardt, F.
H. C., Ed.; ASM Press: Washington, DC, 1996; pp 2363-2376.
(8) (a) Ghosh, K.; Cassell, G. C.; Segall, A.; Van Duyne, G. D.
Unpublished results with the dodecapeptide binding in the center of the
C-2 symmetric Holliday junction site. (b) Gopaul, D. N.; Guo, F.; Duyne,
G. D. V. EMBO J. 1998, 17, 4175-4187.
(9) (a)Cassell, G.; Klemm, M.; Pinilla, C.; Segall, A. M. J. Mol. Bio.
2000, 299, 1193-1202. (b) Klemm, M.; Cheng, C.; Cassell, G.; Shuman,
S.; Segall, A. M. J. Mol. Bio. 2000, 299, 1203-1216.
(10) (a) Tyndall, J. D. A.; Fairlie, D. P. Curr. Med. Chem. 2001, 8, 893.
(b) Fairlie, D. P.; Tyndall, J. D. A.; Reid, R. C.; Wong, A. K.; Abbenante,
G.; Scanlon, M. J.; March, D. R.; Bergmann, D. A.; Chai, C. L. L.; Burkett,
B. A. J. Med. Chem. 2000, 43, 1271.
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Org. Lett., Vol. 5, No. 2, 2003