Synthesis and evaluation of a novel bleomycin A2 analogue: Continuing assessment of the linker domain

Article abstract of DOI:10.1016/S0040-4039(00)01633-6

The synthesis and evaluation of a bithiazole ester analogue of deglycobleomycin A₂ that addresses the importance of the bithiazole-L-threonine amide for efficient DNA cleavage is described. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01633-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9493–9498
Synthesis and evaluation of a novel bleomycin A2 analogue:
continuing assessment of the linker domain†
Dale L. Boger,* Brian M. Aquila, Winston C. Tse and Mark Searcey
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 28 August 2000; revised 18 September 2000; accepted 21 September 2000
Abstract
The synthesis and evaluation of a bithiazole ester analogue of deglycobleomycin A2 that addresses the
importance of the bithiazole–L-threonine amide for efficient DNA cleavage is described. © 2000 Elsevier
Science Ltd. All rights reserved.
The bleomycins are a group of glycopeptide antibiotics isolated from the microorganism
Streptomyces verticillus by Umezawa and co-workers.1 Bleomycin A2,2–11 differing from other
bleomycins only at the C-terminus, is the major component of the anticancer drug Blenoxane
which has found clinical use in combination chemotherapy for the treatment of Hodgkin’s
lymphoma, carcinomas of the skin, head and neck, and testicular cancers.12 Its properties are
thought to arise from its ability to mediate the catalytic oxidative cleavage of duplex DNA,13
and possibly RNA,14,15 in the presence of a redox-active metal cation, oxygen, and a reductant.
3,16,17
Through extensive structural, physical, chemical, and biological studies on bleomycin A2
and its derivatives,18 four functional domains of the active molecule and their roles have been
identified: the N-terminus domain, the C-terminus domain, the carbohydrate domain, and the
linker domain. NMR solution structures of bleomycin–oligonucleotide complexes as well as
point mutations in the structure of deglycobleomycin revealed that the substituents on the
valerate and
a single rigid compact conformation necessary for efficient DNA cleavage.19 Among them, the
-threonine subunit substituent restricts the valerate–threonine amide to a single orientation,
L
-threonine subunits composing the linker domain induce bleomycin A2 to adopt
L
inducing a turn in the linker domain characteristic of the compact conformation observed in free
and DNA-bound bleomycin A2. In these prior studies, we also demonstrated that N-methylation
of the valerate–threonine amide decreased DNA cleavage efficiency 10–15 fold and the DNA
* Corresponding author. Fax: (+1) 858-784-7550; e-mail: boger@scripps.edu
†
Dedicated to Harry Wasserman on the occasion of his 80th birthday.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01633-6

9494
cleavage sequence selectivity of bleomycin A2 was nearly abolished.20 Herein we describe the
synthesis and evaluation of a bithiazole ester analogue replacing the adjacent
L
-
threonineꢀbithiazole amide bond with a more flexible ester linkage (1) removing the hydrogen
bond donor amide yet minimizing perturbation of the natural system.
1. Synthesis
The first stage of the synthesis of 1 was the construction of the modified bithiazole subunit 2
(Scheme 1). Conjugate addition of benzyl alcohol to acrylonitrile (3, 1 equiv. BnOH, Na, 80°C,
80%) followed by conversion to the thioamide (1.2 equiv. Et2NH, sat. with H2S, DMF, 45°C,
74%) produced 4. Its coupling with the a-bromo ketone 518 to provide the bithiazole 6 was
effected in two stages first by reaction in dioxane followed by elimination of water (2 equiv.
MsCl, 2 equiv. iPr2NEt, CH2Cl2, 79%). LiOH hydrolysis of the ethyl ester, followed by EDCI
(1.5 equiv.) and HOAt (1.5 equiv.) mediated coupling of 7 (2 equiv. NaHCO3, 3:1 CH2Cl2–
DMF, 0–25°C, 88%) with 8 afforded the protected bithiazole subunit 2. Benzyl deprotection (3
equiv. BCl3, 0°C, 58%), coupling of the suitably protected -threonine 10 with the free alcohol
L
9 (1.5 equiv. EDCI, 1.5 equiv. DMAP, DMF, 0°C, 71%), and formation of the sulfonium salt
(1.1 equiv. MeI, MeOH, in the dark, >95%) afforded 11. Stepwise deprotection of the OTBS and
N-BOC protecting groups provided the modified tripeptide S sulfonium salt (13).
Scheme 1.
Dipeptide 14 was obtained from the hydrolysis (LiOH, 72%) of the coupling product between
1518 and 1618 (3 equiv. EDCI, 3 equiv. HOAt, 1.5 equiv. NaHCO3, 3:1 CH2Cl2–DMF, 0°C 69%).
Coupling of 13 with 14 utilizing BOP (3 equiv.) and Hunig’s base (3 equiv.) yielded the
pentapeptide 18 (DMF, 25°C, 70%, Scheme 2). Deprotection of the silyl ether (Bu4NF) followed
by simultaneous removal of the N-BOC and trityl protecting groups (20% TFA–CH2Cl2) set the
stage for the final coupling step. In the presence of DPPA, N-BOC-pyrimidoblamic acid (21),
prepared as previously described,21 was reacted with 20 in high yield (1.1 equiv. DPPA, 2 equiv.
iPr2NEt, DMF, 0°C, 85%). Acid-catalyzed deprotection afforded the deglycobleomycin bithia-
zole ester analogue 1.22

9495
Scheme 2.
2. Evaluation
The ability of the Fe(II) complex of 1 to cleave duplex DNA was conducted first through
examination of single-strand and double-strand cleavage of supercoiled FX174 DNA (Form I)
to produce relaxed (Form II) and linear (Form III) DNA, respectively. The Fe(II) complex of
1 was found to cleave the FX174 DNA with a 2 fold decrease in efficiency as compared to
bleomycin A2 (Fig. 1) which is the identical reduction in cleavage efficiency shown by
deglycobleomycin A2.
Figure 1. Agarose gel illustrating the cleavage reactions of supercoiled FX174 DNA by Fe(II)-agents at 25°C for 1
h in buffer solutions containing 2-mercaptoethanol23
The sequence selectivity of DNA cleavage was examined with duplex w794 DNA24 by
monitoring strand cleavage of singly [32P]-5%-end labeled duplex DNA after exposure to the
Fe(III) complexes of the agents following activation with H2O2.25,26 This protocol is much more
sensitive to the distinctions in the relative efficiency of DNA cleavage than the supercoiled DNA
cleavage assays. Consequently, bleomycin and its analogues exhibit cleavage over a much
broader range of concentrations. As seen in Fig. 2, the bithiazole ester analogue performed with
the same trends observed in the supercoiled DNA cleavage assay, namely a minor 2–5 fold
decrease in cleavage efficiency relative to bleomycin analogous to the 2–5 reduction observed
with deglycobleomycin. By contrast, N-methylation of the adjacent valerate–threonine amide

9496
provided an analogue that cleaved w794 DNA only at a concentration of 128 mM20 versus the
1–2 mM observed with 1. Notably, the DNA cleavage selectivity is not compromised and 1
displayed the identical preference toward cleavage of 5%-GC and 5%-GT sites.
Figure 2. Cleavage of double stranded DNA by Fe(III)-agents (SV40 DNA fragment, 144 base pairs, nucleotide no.
27
5238–138, clone w794) in phosphate–KCl buffer containing H2O2
Analysis of the linker domain through single point changes in the structure of degly-
cobleomycin A2, such as the one detailed herein, have revealed many crucial elements necessary
Figure 3.

9497
for efficient DNA cleavage.19 With the synthesis of the bithiazole ester analogue 1, we have
shown that the replacement of the threonine–bithiazole amide with a depsipeptide ester linkage
does not effect the properties (Fig. 3) and does not alter the linker domain’s ability to adopt the
decisive compact DNA bound conformation. The absence of a substantial change in cleavage
efficiency and sequence selectivity indicates that neither the conformational or H-bond proper-
ties of the threonine–bithiazole amide nitrogen are critical.
Acknowledgements
We gratefully acknowledge the financial support of the National Institutes of Health
(CA42056, D.L.B.) and the Skaggs Institute for Chemical Biology.
References
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L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.; Stubbe, J. J. Am. Chem. Soc. 1998, 120, 9149.
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21. Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L.; Dang, Q.; Yang, W. J. Am. Chem. Soc. 1994, 116, 82.
22. All intermediates and final compounds were characterized by IR, NMR, and HRMALDI FTMS. Spectroscopic
data for 1: Rf=0.08 (SiO2, 10:9:1 CH3OH–10% aqueous CH3CO2NH4–10% aqueous NH4OH); [h]2D5 −20.7 (c
1
0.29, CH3OH); H NMR (CD3OD, 400 MHz) l 8.86 (s, 1H), 8.22 (s, 2H), 7.57 (s, 1H), 5.42 (d, J=4.4 Hz, 1H),
4.78 (d, J=9.2 Hz, 1H), 4.68 (dd, J=1.9, 4.7 Hz, 1H), 4.60 (d, J=6.1 Hz, 1H), 4.8 (d, J=6.1 Hz, 1H), 4.46 (d,
J=1.6 Hz, 1H), 4.51 (m, 1H), 3.70 (m, 2H), 3.60 (t, J=9.6 Hz, 2H), 3.46 (t, J=3.0 Hz, 2H), 3.40 (t, J=3.6 Hz,

9498
2H), 3.20 (m, 2H), 3.02 (t, J=5.8 Hz, 2H), 2.95 (s, 6H), 2.63 (m, 1H), 2.27 (s, 3H), 2.16 (t, J=3.6 Hz, 2H), 1.22
(d, J=3.4 Hz, 3H), 1.21 (d, J=3.4 Hz, 3H), 1.15 (d, J=3.3 Hz, 3H); IR (neat) wmax 3346, 2997, 1646, 1551, 1426,
1259, 1065 cm−1; HRMALDI FTMS (DHB) m/z 1048.3869 (M+, C42H62N15O11S3 requires 1048.3915).
+
23. After electrophoresis on a 1% agarose gel, the gel was stained with 0.1 mg/mL ethidium bromide and visualized
on a UV transilluminator. The image has been inverted for clarity.
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25. Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L. J. Am. Chem. Soc. 1994, 116, 5631. Boger, D. L.;
Menezes, R. F.; Honda, T. Angew. Chem., Int. Ed. Engl. 1993, 32, 273.
26. Natrajan, A.; Hecht, S. M.; van der Marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1990, 112, 3997. Natrajan,
A.; Hecht, S. M.; van der Marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1990, 112, 4532.
27. The DNA cleavage reactions were run for 30 min at 37°C and electrophoresis was run on an 8% denaturing
PAGE and visualized by autoradiography.
.