Novel antibiotics: Second generation macrocyclic peptides designed to trap Holliday junctions

Article abstract of DOI:10.1016/j.tetlet.2004.09.084

Described are the syntheses of 15 macrocyclic peptides designed to trap Holliday junctions (HJs) in bacteria during site-specific and homologous recombination. This leads to inhibiting bacterial growth. These second generation macrocycles were based on th

Full text of DOI:10.1016/j.tetlet.2004.09.084

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 8447–8450
Novel antibiotics: second generation macrocyclic peptides
designed to trap Holliday junctions
Lisa A. Liotta,a, Irene Medina,a, Jennifer L. Robinson,a Chris L. Carroll,a Po-Shen Pan,a
Ricardo Corral,a Jennifer V. C. Johnston,a Kristina M. Cook,a Fiona A. Curtis,b
Gary J. Sharplesb and Shelli R. McAlpinea,*
aDepartment of Chemistry and Biochemistry, 5500 Campanile Dr., San Diego State University, San Diego, CA 92182-1030, USA
bCentre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen’s Campus, Stockton-on-Tees TS17 6BH, UK
Received 1 September 2004; revised 12 September 2004; accepted 13 September 2004
Available online 1 October 2004
Abstract—Described are the syntheses of 15 macrocyclic peptides designed to trap Holliday junctions (HJs) in bacteria during site-
specific and homologous recombination. This leads to inhibiting bacterial growth. These second generation macrocycles were based
on the C-2 symmetrical HJ. They were synthesized using a strategy that permits elucidation of the amino acid role in binding HJs.
The syntheses of these macrocycles are an important step in the development of a new class of antibiotics.
Ó 2004 Elsevier Ltd. All rights reserved.
Peptides rarely function well as drugs due to their low
bioavailability and rapid degradation within cells.1
However, they make convenient initial synthetic targets
due to ease of assembly. Moreover, a large number of
natural product cyclic peptides have interesting biologi-
cal activity.2 The conversion of these active peptides into
peptidomimetics has been a successful approach for
making new biologically active compounds.3
gene expression, amplify episome copy number, separate
chromosomes during bacterial cell division and create
genetic diversity. Homologous recombination also pro-
motes diversity, although its main function is to main-
tain genomic integrity by facilitating DNA repair and
replication restart.5 By trapping the HJ, we may inhibit
either or both of these pathways.6 Blocking these recom-
bination reactions in vivo has led to bacterial death.7–9
Segall and co-workers have shown that linear dodeca-
peptides successfully trap the HJ in nanomolar concen-
trations and cause bacteria cell death via this mechanism
of action.9 Compounds that successfully trap the HJ and
lead to bacterial cell death represent a new class of anti-
biotics. Such inhibitors are reminiscent of the quinolone/
fluoroquinolone class of antibiotics, which stabilize a
normally transient intermediate.
Antibiotic resistance has become a major public health
concern.4 As pathogenic bacteria become resistant to
first and second line antibiotics, easily treatable infec-
tious diseases are becoming life threatening. In order
to keep up with the evolutionary pressure from patho-
genic bacteria during the infectious disease process, we
must continually develop new antibiotics and aim at
new biological targets within bacteria. One such new
target is the Holliday junction (HJ) generated as an
intermediate in XerCD site-specific recombination, in
RecA-dependent genetic exchange and by replication
fork regression.5 Site-specific recombination can control
The initial peptide leads were linear hexapeptides, which
dimerize via a disulfide bridge giving the active dodeca-
peptide structure. These leads were problematic given
their size, solubility, and flexibility, making it difficult
to identify specific residues involved in the binding
event. To elucidate the biological mechanism of action,
and find soluble, effective compounds that trap this
unique target, we synthesized a first generation of macro-
cycles. These were based on the C-2 symmetrical HJ
binding site7,10 and SegallÕs lead linear dodecapeptides
(Fig. 1).8,9 The first generation was cyclized to maximize
Keywords: Macrocycles; Peptides; Antibiotics; Holliday junction;
Macrocyclic peptides; C-2 symmetrical.
*
Corresponding author. Tel.: +1 6195945580; fax: +1 6195944634;
e-mail: mcalpine@chemistry.sdsu.edu
These two authors contributed equally to this paper.
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.084

8448
L. A. Liotta et al. / Tetrahedron Letters 45 (2004) 8447–8450
3
2
H3CO
HN
1
O
O
1
O
O
HN
NH
O
O
NH
1
O
O
NHBoc
3
O
NH
2
2
3
Fragment 1
3
2
HN
O
H3CO
O
Figure 1. A co-crystal structure of the Holliday junction. The co-
crystal structure was obtained by mixing a mutant Cre protein with
loxS DNA substrates. The crystal was dependent on blocking
catalysis. A similar co-crystal structure was obtained between wild
type Cre protein and lox substrates, but only in the presence of the
linear lead peptides. This second crystal contains additional electron
density in the HJ center, consistent with bound peptide in the central
ÔholeÕ. 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. Bacterial RuvA, RuvC, and
RecG target similar DNA structures.
O
4
HN
HN
O
1
O
1
NH
NHBoc
4
O
NH
O
H
1
O
N
4
O
2
NH
O
O
H N
O
3
Fragment 2
N
2
3
2 =
N
c
1 =
a
a
b
HN
inherent rigidity of the compounds,11 identify the
contacts between the compounds and HJ, and decrease
degradation inside cells.
b
N
N
H
d
N
H
H
N
e
This first generation of macrocycles contained six amino
acid residues, estimated to fit the approximate size of the
4 =
3 =
˚
HJ binding site, which is approximately 25A by
a
c
a
b
b
10,12
˚
10A.
N
H
These compounds also mimicked the symme-
NH(2-Cl-Z)
NH(2-Cl-Z)
OH
OH
try of the HJ and contained residues found in the lead
compounds. Like the linear lead dodecapeptides, a num-
ber of first generation macrocycles successfully trapped
the HJ.12 However, unlike the linear lead peptides, they
were not bactericidal. This led us to design and synthe-
size a second generation of macrocycles that we antici-
pate will be more effective antibiotics.
Figure 2. Synthesis strategy.
cially available natural and unnatural amino acids, this
generation incorporates hydrophobic residues shown
to trap the HJ, while including hydrophilic residues
not present in our first generation.
Hydrophobic residues may play an important role in
intercalating into the DNA involved in the HJ interme-
diate, and/or the proteins involved in the HJ formation.
They are known to be important for binding to the
DNA because they appear in all lead compounds8,9
and are thought to stack with DNA nucleotides that sur-
round the center of the HJ. The first generation peptides
contained only hydrophobic residues, making them rel-
atively insoluble. Since they contained no hydrophilic
residues, the side chains were unable to hydrogen bond
with HJs. As a result, this second generation of macro-
cycles has incorporated hydrophilic residues to increase
the solubility and hydrogen bonding properties. In addi-
tion, the minimal number of residues involved in bind-
ing to the HJ is not known. It is estimated that six to
eight amino acid residues will fit into the HJ binding
site.10 Therefore, our addition of macrocyclic octapep-
tides in this synthesis will examine an ideal ÔfitÕ in the HJ.
Using 2(1-H-benzotriazole-1-yl)-1,1,3-tetramethyl-uro-
nium tetrafluoroborate (TBTU) as a coupling reagent,
and diisopropylethylamine (DIPEA), acid protected resi-
due 1(a,b) and N-Boc protected residue 2(a–e) (Fig. 3)
were coupled to give the dipeptide 1-2-Boc (80–94%
yield). Deprotection of the amine on residue 2 using
TFA gave the free amine 1–2 (ꢀquantitative yields).
Coupling of this dipeptide to monomer 3(a–c) gave the
desired tripeptide (Fragment 1) in good yields (65–
94%).13
Fragment 1 was separated into two equal aliquots (Fig.
4). The acid was deprotected in the first aliquot using
4equiv of sodium hydroxide, while the amine was
deprotected in the second aliquot using TFA. These
two trimer peptides were coupled using multiple cou-
pling agents12,14 yielding 11 examples of linear hexapep-
tides (36–94% yield). The synthesis of Fragment 2 was
completed by deprotecting the tripeptide using TFA
and coupling the free amine to residue 4(a,b). In a simi-
lar fashion to Fragment 1, Fragment 2 was separated
into two equal aliquots (Fig. 4), where upon the first
Herein we describe the synthesis of 13, second genera-
tion, macrocyclic peptides. Our synthetic approach for
the second generation was chosen to simplify the synthe-
sis of the macrocycles, while providing flexibility in
amino acid placement (Fig. 2). Starting from commer-

L. A. Liotta et al. / Tetrahedron Letters 45 (2004) 8447–8450
8449
OCH3
O
aliquot was acid deprotected and the second was amine
deprotected. The subsequent coupling of the tetrapep-
tide free acid and free amine using multiple coupling
agents gave eight examples of linear octapeptides (36–
68% yield).
O
OH
H
N
1. (a)
2. (b)
Boc
H3CO
O
+
O
NH2
1
2
1-2
O
OCH3
O
NHBoc
HO
The linear hexapeptides and octapeptides were amine
deprotected using HCl (pH < 3). Upon completion, the
reaction was concentrated in vacuo, and the acid was
deprotected by neutralizing the reaction with sodium
hydroxide, and then adding four additional equivalents
of sodium hydroxide in methanol to give pH = 11. Fol-
lowing acid deprotection, the reaction was concentrated
in vacuo and subjected to HATU, TBTU, and DEPBT
coupling reagents (ꢀ1.0equiv each), and DIPEA
(ꢀ6equiv).12,14 The final macrocyclizations took
approximately 4 days due to the low concentration
(0.005–0.01M) required to maximize the yield.15 The
one-pot ring-closing yields varied from 8% to 25%.
The final compounds were then purified using reverse
phase HPLC and confirmed via LC–MS.16 These 15
macrocyclic compounds are currently being tested for
their ability to bind the HJ in the in vitro assay used
to test the first generation of compounds.12
3
NH
O
O
NHBoc
(a)
11 examples
69-85% yield
1-2-3
Fragment 1
OCH3
O
1. (b)
2. (a)
NHBoc
O
NH
O
H N
O
NHBoc
OH
8 examples
4
O
45-75% yield
1-2-3-4
Fragment 2
Figure 3. Synthesis of Fragment 1 and Fragment 2. Reagents and
conditions: (a) TBTU (1.2equiv), DIPEA (3equiv), CH2Cl2; (b) TFA
(20%), CH2Cl2, anisole (2equiv).
To establish that these second generation macrocycles
can function in blocking HJs we investigated their
impact on DNA binding by E. coli RuvC. RuvC is a
Holliday junction-specific endonuclease important in
eliminating joint molecules during homologous recom-
bination. All six of the compounds tested significantly
reduced the ability of RuvC to form complexes with a
radioactively labeled HJ substrate (Fig. 5). Most of
these were at least as effective as the first generation cyc-
lic peptides. The results confirm that these macrocycles
can bind to HJs and restrict access of a bacterial junc-
tion processing enzyme in vitro. All final macrocycles
are currently being tested in growth inhibition assays
and in vitro assays.
3
OH
2
O
HN
O
NH
1
O
O
O
1. (a) *
2. (b)
3. (c)
4. (a)
HN
(c)
O
NHBoc
NH
O
O
1
O
Fragment 1
(b)
NH
OCH3
O
2
3
11 examples
Yields: 8-25%
Macrocyclic Hexapeptides
NH
O
O
NH2
1. 1=a, 2=a, 3=a 2. 1=a, 2=b, 3=a
3. 1=a, 2=c, 3=a 4. 1=a, 2=d, 3=a
5. 1=a, 2=e, 3=a 6. 1=b, 2=a, 3=a
7. 1=a, 2=a, 3=c 8. 1=b, 2=a, 3=c
9. 1=b, 2=c, 3=c 10. 1=a, 2=c, 3=c
1-2-3
11.1=b, 2=c, 3=a
OH
NHBoc
O
3
In summary, we describe the synthesis of a second gen-
eration of compounds for a unique target in bacteria,
the HJ. This new generation of compounds should help
elucidate structure–activity relationships of substrates
binding to HJs. In addition, these compounds will allow
isolation and study of this transient HJ intermediate via
co-crystal structure. Furthermore, this class of macrocy-
clic compounds may be viable leads for a novel class of
2
O
(c)
NH
O
O
HN
O
H N
O
O
1. (a) *
4
O
HN
Fragment 2
(b)
2. (b)
O
1
3. (c)
NH
2
OCH3
O
NH2
HN
4. (a)
1
O
O
NH
4
O
3
H N
NH
O
O
H N
O
1-2-3-4
4 examples
Yields: 18-25%
Macrocyclic Octapeptides
1. 1=a, 2=a, 3=a, 4=a 2. 1=a, 2=c, 3=a, 4=a
3. 1=a, 2=a, 3=b, 4=a 4. 1=a, 2=c, 3=b, 4=a
Figure 4. Synthesis of macrocycles. Reagents and conditions: (a)
coupling agentà, DIPEA (3equiv), CH3CN; (b) HCl, MeOH, anisole
(2equiv); (c) NaOH (4equiv), MeOH. *Linear hexapeptide precursor:
11 examples, yields 31–94%, linear octapeptide precursor: 8 examples,
yields 36–68%.
Figure 5. Macrocycle inhibition of E. coli RuvC binding to HJ.
Reagents and conditions: Junction DNA was prepared and binding
assays performed as described.17 RuvC (50nM) was mixed with 0.4nM
32P-labeled synthetic Holliday junction (J11) in the presence or absence
of second (1–6) or first (1st) generation macrocycles at 100 and
1000nM. Protein–DNA complexes were separated on 4% polyacryl-
amide gels.
à For the formation of linear peptides, typically two coupling reagents
were used: HATU and TBTU (ꢀ1equiv each). For cyclizations
HATU, DEPBT, and/or TBTU were used as coupling agents
(0.6equiv each).

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L. A. Liotta et al. / Tetrahedron Letters 45 (2004) 8447–8450
J. Bacteriol. 1999, 181, 5543–5550; (c) McGlynn, P.;
Lloyd, R. G. Nat. Rev. Mol. Cell Biol. 2002, 3, 859–
870.
antibiotics. Additional assays are currently being run,
and will be published in due course.
6. Nash, H. A. Site-specific Recombination: Integration
Excision Resolution and Inversion of Defined DNA Seg-
ments in Escherichia coli and Salmonella; ASM: Washing-
ton, DC, 1996.
Acknowledgements
7. Ghosh, K.; VanDuyne, G. 2002, unpublished results.
8. Cassell, G.; Klemm, M.; Pinilla, C.; Segall, A. M. J. Mol.
Biol. 2000, 299, 1193–1202.
9. Klemm, M.; Cheng, C.; Cassell, G.; Shuman, S.; Segall,
A. M. J. Mol. Biol. 2000, 299, 1203–1216.
We thank Pfizer, La Jolla, for equipment and financial
donations as well as their fellowships to I.M. (2003–
2005), R.C. (2003–2005), C.L.C. (SURF, Summer
2004), and L.A.L. (2004–2005). We thank the SDSU
McNair program for fellowships and support to
C.L.C. (Summer 2003 and 2004) and I.M. (Summer
2004). We thank the Howell Foundation for support
for C.L.C. (Spring 2004) and K.C. (Spring 2004). We
thank Merck Research Laboratories for support of
J.V.C.J. (Summer 2004). We thank the MIRT program
for their travel support of I.M. (2003–2004) and R.C.
(2004). We thank ARCS foundation for a fellowship
to J.L.R. (2003–2004). We thank San Diego State Uni-
versity and Boehringer-Ingelheim Pharmaceuticals for
financial support. We thank Professor Rodney Guy
(UCSF), and Dr. Greg Roth (Abbott) for helpful discus-
sions of this work. S.R.M. was supported by NIH
(AI058241-01) and F.A.C. by BBSRC (G15625).
10. Gopaul, D. N.; Guo, F.; VanDuyne, G. D. EMBO. J.
1998, 17, 4175–4187.
11. (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.
12. Bolla, M. L.; Azevedo, E. V.; Smith, J. M.; Taylor, R. E.;
Ranjit, D. K.; Segall, A. M.; McAlpine, S. R. Org. Lett.
2003, 5, 109–112.
13. All dipeptide and tripeptide structures were confirmed
using 1H NMR. All linear hexameric peptides were
confirmed using LC–MS and 1H NMR and cyclized
1
peptides were all confirmed using LC–MS, H NMR and
High Resolution Mass Spectrometry.
14. (a) Robinson, J. L.; Taylor, R. E.; Liotta, L. A.; Bolla, M.
L.; Azevedo, E. V.; Medina, I.; McAlpine, S. R. Tetrahe-
dron Lett. 2004, 45, 2147–2150; (b) Ring closing reactions
are slow and typically low yielding. Unpublished results
from the Guy lab at UCSF, and recently our lab, have
found that the use of several coupling reagents facilitates
ring-closing reactions by providing a choice of reagents for
the specific substrate. This is performed in lieu of
optimizing each individual reaction for each individual
coupling agent.
15. Macrocycles containing 2-Cl-Z protected lysine residues
(3c, 4b) are subjected to palladium catalyzed hydrogena-
tion for the final amine deprotection.
16. One representative example of a macrocyclic hexapeptide
and macrocyclic octapeptide in Figure 4 are as follows
(note: MS data is given as major peaks with +23[Na+], and
+1 being those peaks):
References and notes
1. Hirschmann, R.; Smith, A. B.; Sprengeler, P. A. New
Perspectives in Drug Design; Academic: New York, 1995.
2. (a) Vongvanich, N.; Kittakoop, P.; Isaka, M.; Trakulna-
leamsai, S.; Vitmuttipong, S.; Tanticharoen, M.; Thebtar-
anonth, Y. J. Nat. Prod. 2002, 65, 1346–1348; (b) Yano,
T.; Inukai, M.; Takatsu, T.; Tanaka, I. U.S. Patent
2,002,160,946, WO 2000-JP5937, 2002; (c) Aron, J. L.;
Parthun, M. R.; Marcucci, G.; Kitada, S.; Mone, A. P.;
Davis, M. E.; Shen, T.; Murphy, T.; Wickham, J.;
Kanakry, C.; Lucas, D. M.; Reed, J. C.; Grever, M. R.;
Byrd, J. C. Blood 2003, 102, 652–658; For general review
on peptides known to be biologically active see this
reference: (d) Izumiya, N.; Kato, T.; Aoyagi, H.; Waki,
M.; Kondo, M. Synthetic Aspects of Biologically Important
Active Cyclic Peptides; Halsted: New York, NY, 1979.
3. Bursavich, M. G.; West, C. W.; Rich, D. H. Org. Lett.
2001, 3, 2317–2320.
Macrocyclic Hexapeptides
1a-2b-3a (MW=992.5) MS: 1015.5, 993.4,
|_______| Yield: 15%
Macrocyclic Octapeptides
4. (a) Neu, H. C. Science 1992, 257, 1064; (b) Kaufman, M.
Worries Rise Over Effect of Antibiotics in Animal Feed,
Washington Post 2000, March 17th, p A1; (c) Shortridge,
V. D.; Doern, G. V.; Brueggemann, A. B.; Beyer, J. M.;
Flamm, R. K. Clin. Infect. Dis. 1999, 29, 1186–1188.
5. (a) Blakely, G.; May, G.; McCulloch, R.; Arciszewska, L.
K.; Burke, M.; Lovett, S. T.; Sherratt, D. J. Cell 1993, 75,
351–361; (b) Sharples, G. J.; Ingleston, S. M.; Lloyd, R. G.
1a-2c-3a-4a (MW=1264) MS:1265.4
Yield: 10%.
|__________|
17. Sharples, G. J.; Curtis, F. A.; McGlynn, P.; Bolt, E. L. J.
Mol. Biol. 2004, 340, 739–751, Compounds 1–6 are:
Compound 1 = 1a–2c–3a–4a, Compound 2 = 1a–2b–3a,
Compound 3 = 1a–2a–3a, Compound 4 = 1a–2a–3a–4a,
Compound 5 = 1a–2c-3a, Compound 6 = 1a–2d–3a.