Alteration of the selectivity of DNA cleavage by a deglycobleomycin analogue containing a trithiazole moiety

Article abstract of DOI:10.1021/ja011820u

The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both involved in the binding of BLM to DNA. The metal-binding

Full text of DOI:10.1021/ja011820u

Published on Web 03/22/2002
Alteration of the Selectivity of DNA Cleavage by a
Deglycobleomycin Analogue Containing a Trithiazole Moiety
Craig J. Thomas, Michael M. McCormick, Corine Vialas, Zhi-Fu Tao,
Christopher J. Leitheiser, Michael J. Rishel, Xihan Wu, and Sidney M. Hecht*
Contribution from the Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22901
Received July 26, 2001
Abstract: The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective
manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both
involved in the binding of BLM to DNA. The metal-binding domain is normally the predominant structural
element in determining the sequence selectivity of DNA binding, but it has been shown that replacement
of the bithiazole moiety with a strong DNA binder can alter the sequence selectivity of DNA binding and
cleavage. To further explore the mechanism by which BLM and DNA interact, a trithiazole-containing
deglycoBLM analogue was synthesized and tested for its ability to relax supercoiled DNA and cleave linear
duplex DNA in a sequence-selective fashion. Also studied was cleavage of a novel RNA substrate. Solid-
phase synthesis of the trithiazole deglycoBLM A5 analogue was achieved using a TentaGel resin containing
a Dde linker and elaborated from five key intermediates. The ability of the resulting BLM analogue to relax
supercoiled DNA was largely unaffected by introduction of the additional thiazole moiety. Remarkably,
while no new sites of DNA cleavage were observed for this analogue, there was a strong preference for
cleavage at two 5′-GT-3′ sites when a 5′-32P end-labeled DNA duplex was used as a substrate. The alteration
of sequence selectivity of cleavage was accompanied by some decrease in the potency of DNA cleavage,
albeit without a dramatic diminution. In common with BLM, the trithiazole analogue of deglycoBLM A5 effected
both hydrolytic cleavage of RNA in the absence of added metal ion and oxidative cleavage in the presence
of Fe2+ and O2. In comparison with BLM A5, the relative efficiencies of hydrolytic cleavage at individual
sites were altered.
in DNA binding as well as chelation to the metal ion cofactor.5
The carbohydrate moiety may participate in metal ion binding6
Introduction
The bleomycin (BLM) family of antitumor antibiotics pos-
sesses clinical utility in the treatment of squamous cell
carcinomas, lymphomas, and testicular cancer.1 It is believed
that the observed therapeutic activity of BLM results from the
metal-dependent oxidative degradation of DNA2 and possibly
RNA.3 DNA cleavage is known to be sequence-selective,
preferentially occurring at 5′-GpC-3′ and 5′-GpT-3′ sequences.4
and possibly also in cell surface recognition and uptake.7 The
C-terminus consists of the bithiazole moiety and a positively
charged alkyl substituent, both of which take part in DNA
binding and possibly sequence recognition.2
Numerous structure-activity and molecular modeling studies
have contributed to the current view of the molecular mechanism
of action of BLM.8 Key unresolved issues include a complete
understanding of the binding interaction of the bithiazole moiety
The structure of BLM, exemplified by one member of this
family of antitumor agents (BLM A5 (1)), can be subdivided
into three domains (Figure 1). The metal-binding domain
contains the pyrimidine moiety, which is known to participate
(3) (a) Carter, B. J.; deVroom, E.; Long, E. C.; van der Marel, G. A.; van
Boom, J. H.; Hecht, S. M. Proc. Natl. Acad. Sci. U.S.A. 1990, 97, 9373.
(b) Holmes, C. E.; Carter, B. J.; Hecht, S. M. Biochemistry 1993, 32, 4293.
(c) Hecht, S. M. Bioconjugate Chem. 1994, 5, 513. (d) Hecht, S. M. In
The Many Faces of RNA.; Eggleston, D. S., Prescott, C. D., Pearson, N.
D, Eds.; Academic Press: San Diego, 1998; pp 3-17.
(1) (a) Bleomycin: Current Status and New DeVelopments; Carter, S. K.,
Crooke, S. T., Umezawa, H., Eds.; Academic Press: New York, 1978. (b)
Bleomycin Chemotherapy; Sikic, B. I., Rozencweig, M., Carter, S. K., Eds.;
Academic Press: Orlando, FL, 1985. (c) Lazo, J. S. In Cancer Chemo-
therapy and Biological Response Modifiers Annual 18; Pinedo, H. M.,
Longo, D. L., Chabner, B. A., Eds.; Elsevier: New York, 1999.
(2) (a) Sausville, E. A.; Peisach, J.; Horwitz, S. B. Biochemistry 1978, 17,
2740. (b) Sausville, E. A.; Stein, R. W. P.; Horwitz, S. B. Biochemistry
1978, 17, 2746. (c) Burger, R. M.; Peisach, J.; Horwitz, S. B. J. Biol. Chem.
1981, 256, 11636. (d) Hecht, S. M. Fed. Proc., Fed. Am. Soc. Exp. Biol.
1986, 45, 2784. (e) Hecht, S. M. Acc. Chem. Res. 1986, 19, 383. (f) Stubbe,
J.; Kozarich, J. W. Chem. ReV. 1987, 87, 1107. (g) Natrajan, A.; Hecht, S.
M. In Molecular Aspects of Anticancer Drug-DNA Interactions; Neidle,
S., Waring, M., Eds.; Macmillan: London, 1993; p 197ff. (h) Kane, S. A.;
Hecht, S. M. Prog. Nucleic Acid Chem. Mol. Biol. 1994, 49, 313. (i) Burger,
R. M. Chem. ReV. 1998, 98, 1153.
(4) (a) D’Andrea, A. D.; Haseltine, W. A. Proc. Natl. Acad. Sci. U.S.A. 1978,
75, 3608. (b) Takeshita, M.; Grollman, A. P.; Ohtsubo, E.; Ohtsubo, H.
Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5983. (c) Mirabelli, C. K.; Ting,
A.; Huang, C. H.; Mong, S.; Crooke, S. T. Cancer Res. 1982, 42, 2779.
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M.; van der Marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1986, 108,
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Brown, S. J.; Mascharak, P. K. J. Am. Chem. Soc. 1993, 115, 7971.
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J. AM. CHEM. SOC. 2002, 124, 3875-3884
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A R T I C L E S
Thomas et al.
Figure 2. Structures of bithiazole (4) and trithiazole (5) analogues that
mediate sequence-selective DNA cleavage.
unfused aromatic ring systems could alter the sequence selectiv-
ity of BLM, as already demonstrated in one case by Ohno and
co-workers.11
Kuroda and co-workers have prepared a number of model
compounds containing one or more thiazoles and incorporating
a p-nitrobenzoyl group capable of mediating DNA cleavage.12
Interestingly, a model compound containing a bithiazole moiety
(4) (Figure 2) cleaved DNA substrates at 5′-AAATN (N not
equal to G) sequences, while a structurally related trithiazole
(5) exhibited selectivity for 5′-GG-3′ and 5′-GA-3′ sequences.
Presently, we describe the synthesis of an analogue of
deglycoBLM A5 containing a trithiazole moiety (3). Also
described is the ability of 3 to effect the relaxation of supercoiled
plasmid DNA in comparison with deglycoBLM A5 (2) and the
effect of the additional thiazole moiety on the sequence
selectivity of DNA cleavage. The hydrolytic and oxidative
cleavage of an RNA substrate is discussed as well.
Figure 1. Structures of bleomycin A5 (1), deglycoBLM A5 (2), and a
trithiazole analogue of deglycoBLM A5 (3).
Results
with DNA and its possible role in sequence selectivity of DNA
cleavage. The interaction between the bithiazole moiety and
DNA may entail intercalation, minor-groove binding, or partial
intercalation. Evidence has been presented to support each of
these possibilities, including numerous molecular models and
NMR-based experiments.9 While there is compelling evidence
that the metal-binding domain is primarily responsible for the
sequence selectivity of DNA cleavage by BLM, studies with a
series of chlorinated bithiazoles and related BLM analogues have
clearly established that the bithiazole is also capable of binding
to DNA with its own preferred sequence selectivity.10 We
reasoned that replacement of the bithiazole moiety with related
Synthesis of Trithiazole Intermediate 11. The elaboration
of key Fmoc-protected trithiazole intermediate 11 (Scheme 1)
was accomplished by analogy with the work of Kobayashi et
al.13 N-Boc bithiazole methyl ester 7 was synthesized from the
previously reported carboxylic acid 614 by acid-catalyzed
esterification. Protection of the free amine with di-tert-butyl
dicarbonate was accomplished in 91% yield. Treatment of N-Boc
bithiazole methyl ester 7 with ammonium hydroxide in methanol
afforded the corresponding amide 8 in 77% yield. The conver-
sion of 8 to the respective thioamide 9 was then effected in
60% yield using Lawesson’s reagent.15 Formation of the third
thiazole ring was realized via Hantzsch cyclization16 with ethyl
(8) (a) Calafat, A. M.; Marzilli, L. G. Comments Inorg. Chem. 1998, 20, 121.
(b) Boger, D. L.; Cai, H. Angew. Chem., Int. Ed. Engl. 1999, 38, 448. (c)
Hecht, S. M. J. Nat. Prod. 2000, 63, 158.
(11) Owa, T.; Haupt, A.; Otsuka, M.; Kobayashi, S.; Tomioka, N.; Itai, A.;
Ohno, M.; Shiraki, T.; Uesugi, M.; Sugiura, Y.; Maeda, K. Tetrahedron
1992, 48, 1193.
(9) (a) Lin, S. Y.; Grollman, A. P. Biochemistry 1981, 20, 7589. (b)
Manderville, R. A.; Ellena, J. F.; Hecht, S. M. J. Am. Chem. Soc. 1994,
116, 10851. (c) Wu, W.; Vanderwall, D. E.; Stubbe, J.; Kozarich, J. W.;
Turner, C. J. Am. Chem. Soc. 1994, 116, 10843. (d) Manderville, R. A.;
Ellena, J. F.; Hecht, S. M. J. Am. Chem. Soc. 1995, 117, 7891. (e) Wu,
W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am.
Chem. Soc. 1996, 118, 1281. (f) Cortes, J. C.; Sugiyama, H.; Ikudome, K.;
Saito, I.; Wang, A. H.-J. Biochemistry 1997, 36, 9995. (g) Lui, S. M.;
Vanderwall, D. E.; Wu, W.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J.
Am. Chem. Soc. 1997, 119, 9603. (h) Wu, W.; Vanderwall, D. E.; Teramoto,
S.; Lui, S. M.; Hoehn, S. T.; Tang, X.-J.; Turner, C. J.; Boger, D. L.;
Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1998, 120, 2239. (i) Sucheck,
S. J.; Ellena, J. F.; Hecht, S. M. J. Am. Chem. Soc. 1998, 120, 7450.
(10) (a) Quada, J. C., Jr.; Levy, M. J.; Hecht, S. M. J. Am. Chem. Soc. 1993,
115, 12171. (b) Zuber, G.; Quada, J. C., Jr; Hecht, S. M. J. Am. Chem.
Soc. 1998, 120, 9368. (c) Quada, J. C., Jr.; Zuber, G. F.; Hecht, S. M.
Pure Appl. Chem. 1998, 70, 307. (d) Quada, J. C., Jr.; Boturyn, D.; Hecht,
S. M. Bioorg. Med. Chem. 2001, 9, 2303.
(12) (a) Kuroda, R.; Satoh, H.; Shinomiya, M.; Watanabe, T.; Otsuka, M. Nucleic
Acid Res. 1995, 1524. (b) Ninomiya, K.; Satoh, H.; Sugiyama, T.;
Shinomiyz, M.; Kuroda, R. J. Chem. Soc., Chem. Commun. 1996, 1825.
(13) Kobayashi, S.; Kuroda, R.; Watanabe, T.; Otsuka, M. Synlett. 1992, 59.
(14) (a) Zee-Cheng, K. Y.; Cheng, C. C. J. Heterocycl. Chem. 1970, 7, 1439.
(b) Minster, D. K.; Jordis, U.; Evans, D. L.; Hecht, S. M. J. Org. Chem.
1978, 43, 1624. (c) Levin, M. D.; Subrahamanian, K.; Katz, H.; Smith, M.
B.; Burlett, D. J.; Hecht, S. M. J. Am. Chem. Soc. 1980, 102, 1452. (d)
Takita, T.; Umezawa, Y.; Saito, S.; Morishima, H.; Umezawa, H.; Muraoka,
Y.; Suzuki, M.; Otsuka, M.; Kobayashi, S.; Ohno, M. Tetrahedron Lett.
1981, 22, 671. (e) Boger, D. L.; Menezes, R. F. J. Org. Chem. 1992, 57,
4331.
(15) (a) Cherkasov, R. A.; Kutyrev, G. A.; Pudovik, A. N. Tetrahedron 1985,
41, 2567. (b) Cava, M. P.; Lewinson, M. I. Tetrahedron 1985, 41, 5061.
(16) Metzger, J. V. In ComprehensiVe Heterocyclic Chemistry; Katritzky, A.
R., Ed.; Pergamon Press: Oxford, 1984; p 235.
9
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Alteration of the Selectivity of DNA Cleavage
A R T I C L E S
Scheme 1
Scheme 2
bromopyruvate in toluene, providing Boc trithiazole ethyl ester
10 in 43% yield. Saponification of 10 with aqueous sodium
hydroxide was followed by CF3COOH-catalyzed removal of
the Boc protecting group. Fmoc protection was then carried out
by a standard procedure that employed Fmoc succinimide,17
affording Fmoc trithiazole 11 as a colorless solid in 79% yield
for the final three steps.
Solid-Phase Synthesis of Trithiazole DeglycoBLM Ana-
logue 3. The synthesis of deglycobleomycin 3 was carried out
by solid-phase synthesis using a TentaGel resin (preloading of
0.45 mmol/g) and utilizing the versatile Dde linker developed
by Bycroft et al.18 The elaboration of a support containing the
spermidine moiety of BLM (13) was accomplished as shown
in Scheme 2. The TentaGel resin containing the Dde linker was
treated with 1,4-diaminobutane to effect transamination, after
which that terminal amine was protected with 2-nitrobenzene-
sulfonyl chloride. Nucleophilic displacement of N-Boc-3-
aminopropanol under Mitsunobu conditions19 produced the
resin-bound intermediate (12) containing the protected spermi-
dine moiety. Removal of the Boc group was carried out
immediately prior to the synthesis of the BLM analogue by
treatment with CF3COOH in the presence of triisopropylsilane;
subsequent washing with 20% Hunig’s base in DMF afforded
the resin-bound spermidine (13) used for the synthesis of
deglycoBLM analogue 3.
The synthesis of resin-bound tetrapeptide 18 was accom-
plished using resin 13, as outlined in Scheme 3. Initially, resin
13 was condensed with Fmoc trithiazole 11 using O-benzo-
triazol-1-yl- N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HBTU) in the presence of Hunig’s base in DMF over a period
of 30 min. The addition of the Fmoc-protected trithiazole
afforded the first opportunity for quantitative measurement of
loading. Fmoc cleavage analysis of an aliquot of dry resin
indicated that the loading of resin 14 was 0.226 mmol/g. This
corresponded to a 75% yield for the production of resin-bound
dipeptide 14 over the first six steps. Preparative removal of the
Fmoc group from 14 was accomplished using a 20% piperidine
solution in DMF. Condensation with commercially available
NR-Fmoc-(S)-threonine (15) was accomplished via the agency
HBTU, HOBt, and Hunig’s base in DMF, providing resin-bound
tripeptide 16 in 90% yield, as judged by subsequent Fmoc
analysis. Using conditions as described above, Fmoc removal
and coupling with NR-Fmoc-(2S,3S,4R)-4-amino-3-hydroxy-2-
methylvalerate (17)20 resulted in the synthesis of resin-bound
(17) Lapatsamis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis 1983, 671.
(18) (a) Bycroft, B. W.; Chan, W. C.; Hone, N. D.; Millington, S.; Nash, I. A.
J. Am. Chem. Soc. 1994, 116, 7415. (b) Chhabra, S. R.; Khan, A. N.;
Bycroft, B. W. Tetrahedron Lett. 1998, 39, 3585.
(19) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett. 2000, 41,
3585.
9
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A R T I C L E S
Thomas et al.
Scheme 3
tetrapeptide 18 in 93% yield, as judged by subsequent Fmoc
analysis.
Completion of the synthesis of deglycoBLM analogue 3 was
accomplished as shown in Scheme 4. Resin-bound tetrapeptide
18 was treated with 20% piperidine in DMF to effect removal
of the Fmoc group, followed by coupling with NR-Fmoc-Nim-
trityl-(S)-erythro-â-hydroxyhistidine (19)21 using a combination
of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole
(HOAt), and Hunig’s base in DMF. Fmoc analysis indicated a
81% yield for the synthesis of resin-bound pentapeptide 20. Final
Fmoc removal with 20% piperidine in DMF was followed by
coupling with NR-Boc-pyrimidoblamic acid (21)23 by employing
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) reagent and Hunig’s base in DMF at 0
°C. This coupling was allowed to proceed over a 24 h period
to afford protected, resin-bound deglycoBLM 22.
Figure 3. Cleavage of supercoiled pBR322 DNA by a trithiazole-containing
bleomycin analogue: lane 1, DNA alone; lane 2, 1.5 µM Fe2+; lane 3, 5
µM deglycoBLM 3; lane 4, 1 µM deglycoBLM 3 + 1.5 µM Fe2+; lane 5,
3 µM deglycoBLM 3 + 1.5 µM Fe2+; lane 6, 5 µM deglycoBLM 3 + 1.5
µM Fe2+; lane 7, 5 µM deglycoBLM A5 (2); lane 8, 1 µM deglycoBLM
A5 + 1.5 µM Fe2+; lane 9, 3 µM deglycoBLM A5 + 1.5 µM Fe2+; lane 10,
5 µM deglycoBLM A5 + 1.5 µM Fe2+
.
Deprotection of the trityl and Boc groups was accomplished
using CF3COOH in the presence of dimethyl sulfide, triisopro-
pylsilane, and water. Removal of the NBS group was carried
out using repetitive treatments with the sodium salt of thiophenol
in DMF. Cleavage of the product from the resin was achieved
using a 2% solution of hydrazine in DMF. The ultraviolet
spectrum of the crude product indicated that the final coupling
had taken place in high yield. Following two successive
purifications of the product by C18 reversed-phase HPLC, the
overall yield of the final coupling, deprotection, and cleavage
steps was 42%. Lyophilization provided deglycoBLM analogue
3 as a colorless solid.
(20) Synthesized by acid-catalyzed deprotection and Fmoc-succinimide-mediated
reprotection of the previously reported Boc-protected derivative. See:
Boger, D. L.; Colletti, S. L.; Honda, T.; Menezes, T. F. J. Am. Chem. Soc.
1994, 116, 5607.
(21) Prepared from (2′S,3′R,4R)-3-[2′-azido-3′-hydroxy-3′-(N-(triphenylmethyl)-
imidazol-4′′-yl) propanoyl]-4-isopropyl-2-oxazolidinone22 as described in
the Experimental Section.
(22) See, e.g. Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L. J. Am.
Chem. Soc. 1994, 116, 5631.
(23) (a) Umezawa, Y.; Morishima, H.; Saito, S.; Takita, T.; Umezawa, H.;
Kobayashi, S.; Otsuka, M.; Narita, M.; Ohno, M. J. Am. Chem. Soc. 1980,
102, 6630. (b) Aoyagi, Y.; Chorghade, M. S.; Padmapriya, A. A.; Suguna,
H.; Hecht, S. M. J. Org. Chem. 1990, 55, 6291. (c) Boger, D. L.; Honda,
T.; Dang, Q. J. Am. Chem. Soc. 1994, 116, 5619.
Characterization of DNA Degradation by DeglycoBLM
3. As shown in Figure 3, Fe(II)‚deglycoBLM analogue 3 was
9
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Alteration of the Selectivity of DNA Cleavage
A R T I C L E S
Scheme 4
quite effective in mediating the relaxation of supercoiled
pBR322 plasmid DNA. In the presence of 3 µM deglycoBLM
3 + 1.5 µM Fe2+, about 50% of the 300 ng of supercoiled DNA
utilized was converted to relaxed (form II) DNA. Moreover, 5
µM deglycoBLM 3 + 1.5 µM Fe2+ produced a significant
amount of form III (linear duplex DNA). Densitiometric analysis
of the form III bands for deglycoBLM 3 (lane 6) and deglyco-
BLM A5 (lane 10) revealed that 3 retained 80% of the ability
of deglycoBLM A5 to produce double-stranded DNA cleavage.
Also investigated was the ability of deglycoBLM 3 to effect
the sequence-selective cleavage of DNA. This was studied using
a 5′-32P end-labeled 158-base pair DNA restriction fragment as
a substrate. As shown in Figure 4, Fe(II)‚deglycoBLM A5 (2)
effected sequence-selective cleavage of the DNA substrate at
several sites. Five strong cleavage sites were identified by
sequence; these included two 5′-GC-3′ sites and three 5′-GT-3′
sites. The strongest cleavage was at 5′-G33C34-3′. The same five
sites were also cleaved to some extent by Fe(II)‚BLM A5
analogue 3. However, three of these sites, including 5′-G33C34-
3′, were cleaved very weakly by this BLM analogue. The ratio
of cleavage at 5′-G33C34-3′ relative to that at 5′-G54T55-3′ was
5:1 for deglycoBLM A5, as determined by phosphorimager
analysis. In comparison, the cleavage ratio at the same two sites
was 1:6 for deglycoBLM analogue 3. Both of the strong
cleavage sites for 3 involved 5′-GT-3′ sequences. Additionally,
the overall extent of DNA cleavage mediated by analogue 3
was significantly less than that obtained in the presence of 2
(cf. lanes 4-6 and 8-10). The slight decrease in band intensities
Figure 4. Cleavage of 5′-32P end-labeled 158-base pair DNA duplex by
deglycoBLM 3: lane 1, DNA alone; lane 2, 10 µM Fe2+; lane 3, 10 µM
deglycoBLM 3; lane 4, 1 µM deglycoBLM 3 + 1 µM Fe2+; lane 5, 5 µM
deglycoBLM 3 + 5 µM Fe2+; lane 6, 10 µM deglycoBLM 3 + 10 µM
Fe2+; lane 7, 10 µM deglycoBLM A5 (2); lane 8, 1 µM deglycoBLM A5 +
1 µM Fe2+; lane 9, 5 µM deglycoBLM A5 + 5 µM Fe2+; lane 10, 10 µM
deglycoBLM A5 + 10 µM Fe2+. The bands of intermediate mobility in
lanes 1, 2, 3, and 7 were due to nondenatured DNA duplex.
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A R T I C L E S
Thomas et al.
Figure 5. Structures of B. subtilis RNAHis precursor (left) and a 53-nucleotide RNA (right) that serve as substrates for cleavage by Fe(II)‚BLM.
greatly diminished by the addition of Fe2+, as already established
for BLM itself.24 Direct comparison with hydrolytic cleavage
mediated by 10 µM Fe(II)‚BLM A5 is shown in lane 9. As can
be seen, most of the sites of cleavage were the same, although
3 produced cleavage at one site (upper arrow) not readily
apparent when BLM A5 was employed.
Oxidative cleavage by 1 and 3 were also compared (lanes
4-7 and 11, respectively). The predominant cleavage site
observed for Fe(II)‚BLM A5 was the same as that produced by
deglycoBLM 3, although the latter cleaved the RNA with lesser
efficiency.
Discussion
Experiments from a few laboratories have demonstrated
convincingly that in addition to its role in binding metal ions
and dioxygen and in mediating the chemical transformations
catalyzed by BLM, the metal-binding domain of BLM (Figure
1) is the primary determinant of the sequence selectivity of DNA
cleavage by BLM.5 There is general agreement that the metal-
binding domain must associate with DNA in the minor groove,
since C4′-H of deoxyribose must be abstracted by an activated
metallobleomycin in order to initiate the chemical transforma-
tions that are actually observed.2
In contrast, the specific molecular role(s) played by the
Figure 6. Cleavage of a 53-nucleotide RNA by Fe(II)‚BLM: lane 1, RNA
bithiazole moiety in supporting BLM-mediated DNA degrada-
alone; lane 2, 100 µM Fe2+; lane 3, 100 µM deglycoBLM 3; lane 4, 1 µM
tion is uncertain. While not the primary mediator of sequence-
Fe(II)‚deglycoBLM 3; lane 5, 5 µM Fe(II)‚deglycoBLM 3; lane 6, 10 µM
selective DNA cleavage by BLM, studies involving photo-
induced DNA cleavage by chlorobithiazoles and structurally
related deglycoBLMs incorporating chlorinated bithiazole moi-
eties have made it clear that the bithiazole moiety of BLM may
also exhibit sequence-selective DNA binding;10 it seems likely
that strongly preferred sites of DNA binding and cleavage by
BLM may include those that accommodate the DNA binding
preferences of both the metal binding and C-terminal domains
of BLM.
Fe(II)‚deglycoBLM 3; lane 7, 50 µM Fe(II)‚deglycoBLM 3; lane 8, 100
µM Fe(II)‚deglycoBLM 3; lane 9, 10 µM BLM A5; lane 10, 10 µM Fe2+
;
lane 11, 10 µM Fe(II)‚BLM A5. The upper arrow illustrates a site of
hydrolytic cleavage mediated exclusively by deglycoBLM 3. The lower
arrow illustrates the site of oxidative cleavage mediated both by Fe(II)‚
deglycoBLM 3 (lanes 4-7) and Fe(II)‚BLM A5 (lane 11) (1).
at higher deglycoBLM concentrations was due to multiple
cleavage events.
Characterization of RNA Degradation by DeglycoBLM
3. To assess the ability of deglycoBLM 3 to effect RNA
degradation, we utilized a synthetic 53-nucleotide RNA that has
the same primary sequence as the “core” of Bacillus subtilis
tRNAHis precusor (Figure 5), the latter of which has been shown
to be an excellent substrate for oxidative cleavage by Fe(II)‚
BLM.3
As shown in Figure 6 (lane 3), 100 µM deglycoBLM 3
effected cleavage of the RNA substrate at several sites in the
absence of added metal ion. Further, these sites of cleavage were
Although the metal-binding domain of BLM is the primary
determinant of sequence-selective DNA cleavage, it is clear that
replacement of the bithiazole moiety with an unfused aromatic
ring system that binds strongly to DNA can alter the importance
of the structural elements responsible for determining sequence
selectivity. Ohno and co-workers demonstrated that the intro-
duction of an oligopyrrole moiety in lieu of the bithiazole altered
(24) Keck, M. V.; Hecht, S. M. Biochemistry 1995, 34, 12029.
9
3880 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002

Alteration of the Selectivity of DNA Cleavage
A R T I C L E S
the sequence selectivity of DNA cleavage by BLM to AT-rich
regions, although the efficiency of DNA cleavage was dimin-
ished significantly.11
cleaved to a significant extent by 10 µM Fe(II)‚BLM A5 (cf.
lanes 3 and 9, upper arrow). Thus, the presence of the trithiazole
moiety also effected some changes in the sequence selectivity
of RNA cleavage by (deglyco)BLM.
To permit better definition of the way in which the C-terminal
domain of BLM contributes to the sequence selectivity of DNA
binding and cleavage, we have employed solid-phase synthesis25
to prepare a deglycoBLM A5 analogue (3) in which the
bithiazole moiety has been replaced by a trithiazole. In a
previous study of oligothiazoles incorporating a nitrobenzoyl
group as a DNA cleaving agent, the number of thiazoles had a
significant effect on the sequence selectivity of DNA cleavage.12
Specifically, the presence of a third thiazole ring (cf. compounds
4 and 5, Figure 2) altered the sequence selectivity of DNA
cleavage for these compounds from 5′-AAATN-3′ (N not equal
to G) regions to 5′-GG-3′ and 5′-GA-3′ regions. It was of interest
to determine whether the introduction of a third thiazole ring
into a BLM analogue would have an analogous effect on the
sequence selectivity of DNA cleavage.
As shown in Figure 3, the introduction of a third thiazole
moiety into deglycoBLM A5 afforded a species capable of
relaxing supercoiled plasmid DNA and producing linear duplex
(form III) DNA. The proportions of forms II and III DNA
produced were not much different than that resulting from
cleavage by deglycoBLM A5 (2). The latter is quite unusual;
most modifications of BLM structure, including removal of the
disaccharide moiety, have resulted in significantly less efficient
DNA cleavage and a smaller proportion of form III DNA.22
No less striking were the results obtained when a linear DNA
duplex was employed as a substrate for cleavage by BLM
analogues 2 and 3. As shown in Figure 4, trithiazole analogue
3 cleaved DNA efficiently at only two of the five sites cleaved
efficiently by deglycoBLM analogue 2. Although analogous
effects had previously been noted for several bithiazole-
containing BLM congeners,26 and also for Fe(II)‚BLM vs Cu-
(I)‚BLM,27 the differences in the present case are much more
pronounced. This finding argues strongly that while not the
primary mediator of sequence-selective cleavage by BLM, the
bithiazole + C-substituent does contribute to the observed
efficiency of DNA cleavage at certain sites.
As we have noted previously, it seems likely that the 5′-G‚
pyr-3′ specificity noted for cleavage of B-form DNA by
bleomycin reflects the fact that these sequences correspond to
the widest, shallowest part of the DNA minor groove.28
Physicochemical studies of the binding of metalloBLMs to the
minor groove suggest that the metal-binding domain of BLM
is actually somewhat larger than the minor groove;9 thus, binding
to 5′-G‚pyr-3′ sequences would best accommodate this ligand.
The differences noted for BLMs 2 and 3 in the specific 5′-G‚
pyr-3′ sequences targeted the most efficiently obviously must
reflect the behavior of the trithiazole moiety in 3 vs the
bithiazole moiety in 2. That the differences are not dramatic
argues that the fundamental binding mode for the bithiazole
moiety in 2 and trithiazole moiety in 3 are the same. In this
context, it may be noted that a minor groove binding mode could
readily be envisioned for these two moieties, while one might
anticipate that threading intercalation seems unlikely to be
optimal for both the bi- and trithiazole moieties.
Experimental Section
General Methods. NMR chemical shifts are referenced to (CD3)2-
SO at 2.50 ppm or CDCl3 at 7.26 ppm or D2O at 4.79 ppm. Mass
determination was accomplished by electrospray ionization on a
Finnigan 3200 quadrupole mass spectrometer. Elemental analyses were
performed by Atlantic Microlab, Inc. HPLC purifications were per-
formed on a Varian Associates HPLC using an Alltech Alltima C18
reversed-phase column (250 × 10 mm, 5 µm).
TentaGel resin, HBTU, HOBt, BOP, Fmoc-threonine, and Fmoc-
succinimide were purchased from Novabiochem. DMF was purchased
from Acros Organics. All other chemicals were purchased from Aldrich
Chemical Co. Deuterated NMR solvents were purchased from Cam-
bridge Isotope. Agarose was obtained from Bethesda Research Labo-
ratories. Acrylamide, N,N′-methylenebisacrylamide and ethidium bro-
mide were purchased from Sigma Chemicals. All solvents were of
analytical grade. Tetrahydrofuran was distilled from potassium metal
and benzophenone ketyl prior to use. Methanol was dried over 3 Å
sieves for 48 h prior to use. All synthetic transformations were carried
out under dry argon or nitrogen. Dry resin was swollen in CH2Cl2 and
the appropriate reaction solvent prior to usage.
In comparison with other BLM analogues that exhibited
significant alterations in the patterns of DNA cleavage, degly-
coBLM 3 retained much of the potency associated with the
parent deglycoBLM (2) to which it was related structurally. The
proportion of single- vs double-strand DNA cleavage was also
largely unaffected.
Yield determinations for reactions performed on the solid support
were accomplished following Fmoc cleavage by UV measurement of
the dibenzylfulvene-piperidine adduct formed upon treatment of the
resin with piperidine.29 The molar absorptivities of 5540 M-1 at 290
nm and 7300 M-1 at 300 nm were used to calculate the loading from
a known weight of dry resin.
2′-[2-(tert-Butoxycarbonyl)amino]ethyl-2,4′-bithiazole-4-carboxy-
late Methyl Ester (7). To a solution of 10.0 g (34.3 mmol) of 2′-(2-
amino)ethyl-2,4′-bithiazole-4-carboxylate (6) in 100 mL of methanol
was added 2 mL of concentrated sulfuric acid. The mixture was heated
to reflux for 3 h, and the cooled reaction mixture was neutralized with
18 mL of triethylamine and concentrated under diminished pressure.
To the resulting mixture was added 75 mL of water, 75 mL of THF,
and 11.2 g (51.3 mmol) of di-tert-butyl dicarbonate. The reaction
mixture was then stirred for 72 h and concentrated under diminished
pressure. The resulting aqueous phase was extracted with ethyl acetate.
The pattern of RNA cleavage produced by deglycoBLM 3 is
also worthy of note. Analogue 3 mediated both hydrolytic24 and
oxidative cleavage of a 53-nucleotide RNA identical with the
core region of B. subtilis tRNAHis. The latter undergoes oxidative
cleavage at a single site by Fe(II)‚BLM (U35; see arrow in Figure
5) and the 53-nucleotide RNA substrate is apparently cleaved
at the analogous (i.e., U6) site by Fe(II)‚BLM A5. Fe(II)‚
deglycoBLM 3 also cleaved the 5′-32P end-labeled substrate at
this site, although only weakly. More interesting was the
hydrolytic cleavage mediated by 3 in the absence of added Fe2+
.
Several sites were cleaved efficiently, including one site not
(28) Dickerson, R. E. In Structure and Methods, vol. 3; DNA and RNA.
Proceedings of the Sixth ConVersation in Biomolecular Stereodynamics;
Sarma, R. H.; Sarma, M. H., Eds.; Academic Press: Schenectady, NY,
1990; pp 1-38.
(29) (a) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161. (b)
Gordeev, M. F.; Luehr, G. W.; Hui, H. C.; Gordon, E. M.; Patel, D. V.
Tetrahedron 1998, 54, 15879.
(25) Leitheiser, C. J.; Rishel, M. J.; Wu, X.; Hecht, S. M. Org. Lett. 2000, 2,
3397.
(26) Shipley, J. B.; Hecht, S. M. Chem. Res. Toxicol. 1988, 1, 25.
(27) Ehrenfeld, G. M.; Shipley, J. B.; Heimbrook, D. C.; Sugiyama, H.; Long,
E. C.; van Boom, J. H.; van der Marel, G. A.; Oppenheimer, N. J.; Hecht,
S. M. Biochemistry 1987, 26, 931.
9
J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002 3881

A R T I C L E S
Thomas et al.
The combined organic extract was dried (MgSO4), filtered, and
concentrated under diminished pressure to give bithiazole 7 as a
colorless solid: yield 11.5 g (91%); silica gel TLC Rf 0.35 (1:1
hexanes-EtOAc); 1H NMR (CDCl3) δ 1.43 (s, 9H), 3.22 (t, 2H), 3.61
(q, 2H), 3.97 (s, 3H), 5.04 (s, 1H), 8.05 (s, 1H), and 8.20 (s, 1H).
Anal. Calcd for C15H19N3O4S2: C, 48.76; H, 5.18; N, 11.37.
Found: C, 48.81; H, 5.17; N, 11.37.
50 mL of 10% aqueous K2CO3 and 2 mL of dioxane. To this mixture
was added a solution of 1.84 g (5.45 mmol) of Fmoc succinimide in
50 mL of dioxane. The reaction mixture was stirred for 3 h and then
concentrated under diminished pressure. The resulting precipitate was
washed with acetone and water and then dried, providing Fmoc
trithiazole 11 as an off-white solid: yield 2.43 g (76%); silica gel TLC
1
Rf 0.72 (1:1 EtOAc-MeOH); H NMR ((CD3)2SO) δ 3.14 (m, 2H),
3.38 (m, 2H), 4.18 (m, 1H), 4.26 (m, 2H), 7.22-7.32 (m, 4H), 7.38
(m, 1H), 7.61 (d, 2H, J ) 8 Hz), 7.82 (d, 2H, J ) 8 Hz), 8.22 (s, 1H),
8.30 (s, 1H), and 8.46 (s, 1H); 13C ((CD3)2SO) δ 32.89, 46.77, 65.39,
118.27, 118.71, 120.18, 125.17, 127.05, 127.58, 127.65, 129.29, 140.79,
143.90, 146.94, 148.18, 148.58, 162.06, 163.12, 166.99, and 169.45;
mass spectrum (FAB), m/z 561.0727 (C27H21N4O4S3 requires 561.0725).
Spermidine Resin 12. To a suspension of 1.01 g (0.40 mmol/g) of
swollen Dde-modified TentaGel resin in 2 mL of DMF was added 2.00
g (22.7 mmol) of 1,4-diaminobutane. The mixture was shaken for 12
h, then the solvent was removed by filtration, and the resin was washed
with three 30-mL portions of DMF, three 30-mL portions of CH2Cl2,
and three 30-mL portions of DMF. The treatment with 1,4-diaminobu-
tane and subsequent washing was repeated a second time and an aliquot
was removed; a qualitative Kaiser free-amine test30 showed the presence
of free amine. The resin was washed with three 30-mL portions of
THF and three 30-mL portions of CH2Cl2. To a suspension of resin in
3 mL of CH2Cl2 was added 516 µL (383 mg, 3.0 mmol) of Hunig’s
base. The resin was shaken for 30 s and 440 mg (2.0 mmol) of
2-nitrobenzenesulfonyl chloride was added. The reaction mixture was
shaken for 12 h and the solvent was removed by filtration. The resin
was washed with three 30-mL portions of CH2Cl2, three 30-mL portions
of THF, and three 30-mL portions of CH2Cl2. The treatment with
2-nitrobenzenesulfonyl chloride and subsequent washing procedure was
repeated a second time and an aliquot (<1 mg) was removed; a
qualitative Kaiser free-amine test showed the absence of free amine.
The resin was suspended in a solution containing 315 mg (1.80 mmol)
of Boc-protected 3-aminopropanol and 474 mg (1.80 mmol) of
triphenylphosphine in 3 mL of THF. The mixture was shaken for 30 s,
and 283 µL (1.80 mmol) of diethyl azodicarboxylate was added. The
reaction mixture was shaken for 12 h in the absence of light, and then
solvent was removed by filtration. The treatment with Boc-protected
3-aminopropanol, triphenylphosphine, and diethyl azodicarboxylate was
repeated a second time. Resin 12 was washed with three 30-mL portions
of THF, three 30-mL portions of CH2Cl2, and another three 30-mL
portions of THF. This washing procedure was repeated a second time
and an aliquot was removed; a qualitative Kaiser free-amine test
indicated the absence of free amine. The resin was dried under
diminished pressure.
Resin-Bound Dipeptide 14. To 300 mg of swollen spermidine resin
12 was added a 10% solution of triisopropylsilane in 2 mL of CH2Cl2
and 1 mL of CF3COOH. The reaction mixture was allowed to stir for
1 h, then the solvent was removed by filtration, and the (iPr)3SiH/TFA
treatment was repeated. The resin was washed with 20 mL of a 10%
solution of isopropylsilane in CH2Cl2, and then the solvent was removed
by filtration and the resin was washed successively with three 30-mL
portions of CH2Cl2, three 30-mL portions of DMF, and three 30-mL
portions of a 10% solution of Hunig’s base in DMF. An aliquot was
removed; a qualitative Kaiser free-amine test showed the presence of
free amine. The resin 13 was suspended in a solution of 175 mg (0.31
mmol) of Fmoc-protected trithiazole 11, 119 mg (0.31 mmol) of HBTU,
and 110 µL (0.63 mmol) of Hunig’s base in 2 mL of DMF. The
resulting mixture was stirred for 30 min, then the solvent was removed
by filtration, and the resin was washed successively with three 30-mL
portions of DMF, three 30-mL portions of CH2Cl2, and again with three
30-mL portions of DMF. An aliquot was removed; a qualitative Kaiser
free-amine test showed the absence of free amine. An aliquot of the
putative “dipeptide” was cleaved from the resin with a 1-mL solution
containing 2% hydrazine in DMF. After concentration, the residue was
N-Boc Bithiazole Carboxamide 8. To a solution of 8.04 g (21.8
mmol) of bithiazole methyl ester 7 in 200 mL of methanol was added
100 mL (14.8 N, 1.48 mol) of concentrated ammonium hydroxide. The
reaction was stirred at room temperature for 12 h and then concentrated
under diminished pressure. The resulting solid was purified by
crystallization from ethyl acetate and hexanes, affording bithiazole 8
1
as a colorless solid: yield 5.96 g (77%); H NMR ((CD3)2SO) δ 1.30
(s, 9H), 3.10 (t, 2H, J ) 6 Hz), 3.26 (t, 2H, J ) 6 Hz), 6.98 (m, 1H),
7.61 (s, 1H), 7.74 (s, 1H), 8.12 (s, 1H), and 8.21 (s, 1H); 13C NMR
((CD3)2SO) δ 28.27, 33.05, 77.86, 117.65, 124.36, 147.40, 151.12,
155.55, 161.95, 162.26, and 169.42; MS (FAB), m/z 355.0890
(C14H19N4O3S2 requires 355.0899).
N-Boc Bithiazole Thiocarboxamide 9. To a suspension of 5.00 g
(14.1 mmol) of bithiazole carboxamide 8 in 50 mL of toluene was
added 4.42 g (10.9 mmol) of Lawesson’s reagent. The resulting mixture
was heated to reflux for 3 h and concentrated under diminished pressure.
The residue was partitioned between 200 mL of water and 200 mL of
CH2Cl2. The aqueous phase was extracted twice with 200-mL portions
of CH2Cl2. The combined organic fraction was dried (MgSO4), filtered,
and concentrated under diminished pressure. The residue was purified
by flash chromatography on a silica gel column. Elution with 1:1
hexanes-ethyl acetate afforded bithiazole 9 as a colorless solid: yield
3.16 g (60%); silica gel TLC Rf 0.41 (1:1 hexanes-EtOAc); 1H NMR
((CD3)2SO) δ 1.31 (s, 9H), 3.10 (t, 2H, J ) 6 Hz), 3.27 (t, 2H, J ) 6
Hz), 6.98 (m, 1H), 8.16 (s, 1H), 8.40 (s, 1H), 9.57 (s, 1H), and 10.05
(s, 1H); 13C NMR ((CD3)2SO) δ 28.25, 33.08, 77.86, 117.98, 127.39,
147.35, 154.31, 155.59, 161.43, 169.51, and 189.03; MS (FAB), m/z
371.0672 (C14H19N4O2S3 requires 371.0670).
Boc Trithiazole Ethyl Ester 10. To a suspension of 5.16 g (13.9
mmol) of bithiazole thiocarboxamide 9 in 100 mL of toluene was added
2.50 mL (3.89 g, 17.9 mmol) of ethyl bromopyruvate. The reaction
mixture was stirred at ambient temperature for 8 h. A second portion
of 2.50 mL (17.9 mmol) of ethyl bromopyruvate was added, the reaction
mixture was heated to reflux for 2 h, and the cooled reaction mixture
was then concentrated under diminished pressure. The resulting residue
was dissolved in 150 mL of THF and 150 mL of water. To this solution
was added 3.05 g (13.9 mmol) of Boc anhydride and 4 mL (2.90 g,
28.7 mmol) of technical grade triethylamine to ensure that the primary
amine was protected. The reaction mixture was stirred at ambient
temperature for 48 h and then concentrated to a small volume under
diminished pressure. The remaining aqueous phase was extracted with
three portions of CH2Cl2. The combined organic fraction was dried
(MgSO4), filtered, concentrated under diminished pressure and then
washed with methanol to afford trithiazole 10 as an off-white solid:
1
yield 2.78 g (43%); silica gel TLC Rf 0.38 (1:1 hexanes-EtOAc); H
NMR ((CD3)2SO) δ 1.26 (t, 3H, J ) 7 Hz), 1.28 (m, 9H), 3.10 (t, 2H,
J ) 6 Hz), 3.26 (t, 2H, J ) 6 Hz), 4.26 (q, 2H, J ) 7 Hz), 7.01 (m,
1H), 8.22 (s, 1H), 8.35 (s, 1H), and 8.54 (s, 1H); 13C NMR ((CD3)2-
SO) δ 14.28, 28.27, 33.03, 60.98, 77.87, 118.31, 118.93, 129.76, 146.87,
147.07, 148.40, 148.42, 160.73, 163.19, 166.99, and 169.51.
Anal. Calcd for C19H22N4O4S3: C, 48.91; H, 4.75. Found: C48.97;
H, 4.74.
N-Fmoc Trithiazole Carboxylate 11. To a suspension of 2.68 g
(5.74 mmol) of trithiazole ethyl ester 10 in 50 mL of methanol was
added 4.60 mL (23.0 mmol) of aqueous 5 M NaOH. The reaction
mixture was stirred for 3 h and then concentrated under diminished
pressure. To this residue was added 30 mL of dimethyl sulfide and 10
mL of trifluoroacetic acid. The reaction mixture was stirred at room
temperature for 12 h and then concentrated under diminished pressure
and coevaporated with portions of chloroform. To the residue was added
(30) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595.
9
3882 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002

Alteration of the Selectivity of DNA Cleavage
A R T I C L E S
dissolved in 1 mL of CF3COOH and the solution was treated with 10
mL of ether to effect precipitation of the cleaved product. ESI mass
spectroscopy showed a molecular ion at m/z 651.3, consistent with the
presence of the desired product. An aliquot was used for Fmoc cleavage
analysis and revealed a loading of 0.226 mmol/g (corresponding to a
75% overall yield for the first six steps). The resin was dried under
diminished pressure.
EtOAc and two 30-mL portions of CHCl3. The combined organic
extract was washed with two 25-mL portions of brine, and the excess
solvent was removed under diminished pressure to give a colorless
foam. The residue was dissolved in 50 mL of a 9:1 THF-H2O mixture,
to which was carefully added 45 mg of 10% Pd on carbon, 0.56 g (4.1
mmol) of K2CO3, and 1.36 g (4.05 mmol) of FmocOSu. The reaction
mixture was carefully vacuum purged five times with H2 and then stirred
under an H2 atmosphere for 16 h. The suspension was filtered through
a pad of Celite to remove the catalyst, and then the Celite pad was
rinsed thoroughly with 100 mL of a 90:8:2 CHCl3-MeOH-AcOH
solution, followed by 25 mL of toluene. The excess solvent was
carefully removed under diminished pressure and the remaining residue
was coevaporated with two additional 25-mL portions of toluene. The
crude product was an off-white foam. The crude product was purified
by flash chromatography on a silica gel column (35 × 5 cm). Elution
with 90:8:2 CHCl3-MeOH-AcOH gave the product in a semipure
form; this was purified further by flash chromatography on a silica gel
column (15 × 2 cm). Elution with 80:20:0.1 EtOAc-hexanes-acetic
acid provided the product as a colorless oil. The product was precipitated
from EtOAc and petroleum ether to give a colorless powder: yield
0.56 g (63%); mp 133 °C (dec); silica gel TLC Rf 0.24 (90:8:2 CHCl3-
Resin-Bound Tripeptide 16. To 290 mg (0.226 mmol/g) of swollen
dipeptide resin 14 was added 3 mL of a 20% solution of piperidine in
DMF. The reaction mixture was shaken for 10 min, then the solvent
was removed by filtration, and the resin was washed with 10 mL of a
20% solution of piperidine in DMF. The piperidine deblocking and
subsequent wash procedure was repeated three times and resin washed
successively with three 30-mL portions of DMF, three 30-mL portions
of CH2Cl2, and another three 30-mL portions of DMF. An aliquot was
removed; a qualitative Kaiser free-amine test showed the presence of
free amine. The resin was suspended in a solution containing 68 mg
(0.20 mmol) of Fmoc-protected threonine (15), 103 mg (0.20 mmol)
of HBTU, 26 mg (0.19 mmol) of HOBt, and 70 µL (51.9 mg, 0.40
mmol) of Hunig’s base in 2 mL of DMF. The reaction mixture was
allowed to stir for 30 min, then the solvent was removed by filtration,
and the resin was washed with three 30-mL portions of DMF, three
30-mL portions of CH2Cl2, and three 30-mL portions of methanol. An
aliquot was removed; a qualitative Kaiser free-amine test showed the
absence of free amine. An aliquot was cleaved from the resin with a
solution of 2% hydrazine in DMF. After concentration, the residue was
dissolved in CF3COOH and the solution was treated with ether to effect
precipitation of the cleaved product. ESI mass spectrometry gave a
molecular ion at m/z 752.3, consistent with the presence of desired
“tripeptide” 16. An aliquot was removed for Fmoc cleavage analysis
and revealed a loading of 0.197 mmol/g (corresponding to a 90% yield
from resin-bound dipeptide 14, calculated on the basis of a theoretical
loading of 0.219 mmol/g). The resin was dried under vacuum.
MeOH-AcOH); [R]22 +35.7° ( 1.2° (c 1.03, CHCl3); 1H NMR
D
((CD3)2SO) δ 4.19 (m, 2H), 4.33 (m, 1H), 4.57 (d, 1H, J ) 5.2 Hz),
5.30 (s, 1H), 6.30 (s, 1H), 6.96 (s, 1H), 7.08 (m, 6H), 7.31 (m, 14H),
7.58 (d, 2H, J ) 7.0 Hz), and 7.75 (d, 2H, J ) 7.8 Hz); 13C NMR
(CDCl3) δ 46.67, 60.38, 67.04, 67.17, 119.56, 127.71, 124.91, 125.01,
126.78, 127.35, 128.08, 128.29, 129.28, 136.75, 137.59, 140.56, 140.84,
143.32, 143.51, 156.68, and 171.31; MS (FAB), m/z 636.2474 (M +
H)+ (C40H34N3O5 requires 636.2498).
Resin-Bound Pentapeptide 20. To 260 mg (0.176 mmol/g) of
swollen tetrapeptide resin 18 was added 3 mL of a 20% solution of
piperidine in DMF. The reaction mixture was shaken for 10 min, then
the solvent was removed by filtration, and the resin was washed with
10 mL of a 20% piperidine solution. The piperidine treatment and
subsequent washing procedure was repeated three times, and the resin
was washed with three 30-mL portions of DMF, three 30-mL portions
of CH2Cl2, and three additional 30-mL portions of DMF. An aliquot
was removed; a qualitative Kaiser free-amine test showed the presence
of free amine. The resin was suspended in a solution containing 87
mg (0.14 mmol) of Fmoc-protected â-hydroxyhistidine (19), 52 mg
(0.14 mmol) of HATU, 20 mg (0.15 mmol) of HOAt, and 48 µL (36
mg, 0.28 mmol) of Hunig’s base in 2 mL of DMF. The reaction mixture
was stirred for 30 min, then the solvent was removed, and the resin
was washed successively with three 30-mL portions of DMF, three
30-mL portions of CH2Cl2, and three 30-mL portions of methanol. An
aliquot was removed; a qualitative Kaiser free-amine test showed the
absence of free amine. An aliquot was cleaved from the resin with a
solution containing 2% hydrazine in DMF. After concentration, the
residue was dissolved in CF3COOH and the solution was treated with
ether to effect precipitation of the cleaved, detritylated product. ESI
mass spectrometry showed the presence of a molecular ion at m/z
1034.5, consistent with the presence of “pentapeptide” 20. An aliquot
was removed for Fmoc cleavage analysis and revealed a loading of
0.127 mmol/g (corresponding to a 81% yield from the tetrapeptide 18,
calculated on the basis of a theoretical loading of 0.156 mmol/g). The
resin was dried under vacuum.
Resin-Bound Tetrapeptide 18. To 280 mg (0.197 mmol/g) of
swollen tripeptide resin 16 was added 2 mL of a 20% solution of
piperidine in DMF. The reaction mixture was shaken for 10 min, then
the solvent was removed by filtration, and the resin was washed with
10 mL of a 20% piperidine solution in DMF. The piperidine treatment
and subsequent washing procedure was repeated three times, and resin
was washed successively with three 30-mL portions of DMF, three
30-mL portions of CH2Cl2, and again with three 30-mL portions of
DMF. An aliquot was removed; a qualitative Kaiser free-amine test
showed the presence of free amine. The resin was suspended in a
solution containing 61 mg (0.17 mmol) of Fmoc-protected valerate 17,20
64 mg (0.17 mmol) of HBTU, 22 mg (0.16 mmol) of HOBt, and 58
µL (43 mg, 0.33 mmol) of Hunig’s base in 2 mL of DMF. The reaction
mixture was stirred for 30 min, then the solvent was removed, and the
resin was washed successively with three 30-mL portions of DMF,
three 30-mL portions of CH2Cl2, and three 30-mL portions of methanol.
An aliquot was removed; a qualitative Kaiser free-amine test showed
the absence of free amine. An aliquot was cleaved from the resin with
a solution containing 2% hydrazine in DMF. After concentration, the
residue was dissolved in CF3COOH and the solution was treated with
ether to effect precipitation of the cleaved product. ESI mass spec-
trometry indicated a molecular ion at m/z 881.21, consistent with the
presence of “tetrapeptide” 18. An aliquot was removed for Fmoc
cleavage analysis and revealed a loading of 0.176 mmol/g (correspond-
ing to a 93% yield from the resin-bound tripeptide 16, calculated on
the basis of a theoretical loading of 0.189 mmol/g). The resin was dried
under vacuum.
Trithiazole DeglycoBLM 3. To 100 mg (0.127 mmol/g) of swollen
pentapeptide resin 20 was added 3 mL of a 20% solution of piperidine
in DMF. The reaction mixture was shaken for 10 min, then the solvent
was removed, and the resin was washed with 10 mL of 20% aqueous
piperidine in DMF. The piperidine treatment and subsequent washing
procedure was repeated three times and the resin was washed with three
30-mL portions of DMF, three 30-mL portions of CH2Cl2, and again
with three 30-mL portions of DMF. An aliquot was removed; a
qualitative Kaiser free-amine test showed the presence of free amine.
The resin was suspended in solution of 8 mg (0.019 mmol) of Boc
pyrimidoblamic acid (21),23 22 mg (0.05 mmol) of BOP reagent, and
Nr-Fmoc-Nim-trityl-(S)-erythro-â-hydroxyhistidine (19). To a
solution of 0.75 g (1.4 mmol) of (2′S,3′R,4R)-3-[2′-azido-3′-hydroxy-
3′-(N-(triphenylmethyl)imidazol-4′′-yl)propanoyl]-4-isopropyl-2-oxazo-
lidinone25 in 60 mL of a 4:1 THF-H2O mixture at 25 °C was added
0.28 g (6.8 mmol) of LiOH‚H2O. The reaction mixture was stirred for
40 min and then quenched by the addition of 15 mL of 1 N HCl to pH
≈2.5. The acidic mixture was extracted with three 25-mL portions of
9
J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002 3883

A R T I C L E S
Thomas et al.
13 µL (10 mg, 0.07 mmol) of Hunig’s base in 1 mL of DMF at 0 °C.
The reaction mixture was stirred for 12 h, then the solvent was removed
by filtration, and the resin was washed successively with three 30-mL
portions of DMF, three 30-mL portions of CH2Cl2, and three 30-mL
portions of methanol. An aliquot was removed; a qualitative Kaiser
free-amine test showed the absence of free amine. An aliquot was
cleaved from the resin with a solution containing 2% hydrazine in DMF.
After concentration, the residue was dissolved in CF3COOH and the
solution was treated with ether to effect precipitation of the cleaved
product lacking trityl and Boc protecting groups. ESI mass spectrometry
indicated a molecular ion having m/z 1341.5, consistent with the
presence of trithiazole deglycoBLM 22. To 80.0 mg of swollen resin
was added 0.1 mL of triisopropylsilane, 0.1 mL of dimethyl sulfide,
and 0.1 mL of water. The reaction mixture was shaken for 30 s and 1
mL of CF3COOH was added. The reaction mixture was shaken for 2
h, then the solvent was removed by filtration, and the resin was washed
successively with three 30-mL portions of DMF, three 30-mL portions
of CH2Cl2, and three additional 30-mL portions of DMF. The resin
was suspended in 1.5 mL of a 1 M solution of sodium benzenethiolate
in DMF. The solution was shaken for 30 min and the solvent was then
removed by filtration The treatment with sodium benzenethiolate was
repeated, then the solvent was filtered, and the resin was washed
successively with three 30-mL portions of DMF, three 30-mL portions
of CH2Cl2, and another three 30-mL portions of DMF. The product
was cleaved from resin by treatment with a 2% hydrazine solution in
DMF; the crude ultraviolet spectrum had an absorption at 300 nm; the
ratio of A240/A300 indicated that the final coupling had proceeded in
good yield. 1H NMR of the crude sample containing a known
concentration of tert-butyl alcohol as an internal standard indicated that
the coupling of Boc pyrimidoblamic and proceeded in 70% yield (as
judged by integration of the methyl resonance in pyrimidoblamic acid
vs those in tert-butyl alcohol). The sample was lyophilized, yielding a
colorless, solid product. Purification was achieved by C18 reversed-
phase HPLC using a linear gradient of 0.1% CF3COOH containing
increasing amounts of CH3CN (0 f 25 min, linear gradient from 13%
f 22% CH3CN at a flow rate of 4 mL/min: tR 14.8 min); this gave
trithiazole deglycoBLM analogue 3 as a colorless solid following
lyophilization (yield 4.9 mg, 41.7% overall for the pyrimidoblamic acid
coupling, deprotection and cleavage from the resin): 1H NMR (D2O)
δ 0.99-1.04 (m, 6H), 1.07 (d, 3H, J ) 6.8 Hz), 1.61-1.71 (m, 4H),
1.86 (s, 3H), 1.95 (m, 2H), 2.52 (m, 1H), 2.64-2.66 (m, 2H), 2.93-
3.09 (m, 8H), 3.17-3.25 (m, 2H), 3.41-3.47 (m, 2H), 3.52-3.59 (m,
2H), 3.65-3.68 (m, 1H), 3.78-3.81 (m, 1H), 3.96-4.15 (m, 4H), 4.76
(d, 1H, J ) 7.9 Hz), 5.13 (d, 1H, J ) 7.5 Hz), 7.37 (s, 1H), 8.01 (s,
1H), 8.10 (s, 1H), 8.14 (s, 1H) and 8.63 (s, 1H); MS (electrospray),
m/z 1156.5 (M+) (C47H69N19O10S3 requires 1156.4).
mixture was incubated at 37 °C for 1 h. The enzyme was inactivated
by heating at 75 °C for 10 min in 5 mM EDTA. The DNA was then
recovered by ethanol precipitation following phenol extraction. The
dephosphorylated DNA was 5′-32P end-labeled by incubation with 20
units of T4 polynucleotide kinase in a reaction mixture (50 µL total
volume) containing 7 mM Tris-HCl, pH 7.6, 1 mM MgCl2, 0.5 mM
DTT, and 0.5 mCi of [γ-32P]ATP. The reaction mixture was incubated
at 37 °C for 1 h. The enzyme was inactivated by heating at 65 °C for
20 min. Unreacted [γ-32P]ATP was removed by the use of a microspin
G-25 column, and the DNA was recovered by ethanol precipitation.
The 5′-32P end-labeled DNA was digested with 100 units of restriction
endonuclease EcoRV in a reaction mixture (150 µL total volume)
containing 10 mM NaCl, 5 mM Tris-HCl, pH 7.9, 1 mM MgCl2, and
0.1 mM DTT. The reaction medium was incubated at 37 °C for 3 h,
and the enzyme was then inactivated by heating at 80 °C for 20 min.
The sample was desalted using a microspin G-25 column. The sample
was applied to an 8% native polyacrylamide gel after addition of 50
µL of loading dye [50% glycerol (w/v), 25 mM EDTA, 0.25%
bromophenol blue]. Electrophoresis was carried out at 10 W for 3.5 h.
The DNA was visualized by autoradiography and the band of interest
was excised from the gel. The 158-base pair 5′-32P end-labeled DNA
duplex was eluted into 2 M LiClO4 at 37 °C for 12 h and finally
recovered by ethanol precipitation.
Cleavage of 5′-32P End-Labeled DNA Duplex by a Trithiazole-
Containing Bleomycin Analogue. Reactions were carried out in 20
µL (total volume) of 10 mM sodium cacodylate, pH 7.0, containing
32P end-labeled DNA (∼3 × 104 cpm, 0.1 nM duplex concentration)
and the appropriate concentrations of BLM and Fe2+. Each reaction
was initiated by the simultaneous addition of solutions of BLM and
Fe2+ to the buffered solution containing DNA. The reaction mixture
was incubated at 4 °C for 30 min and lyophilized. The samples were
dissolved in 5 µL of loading dye [80% formamide, 2 mM EDTA, 1%
(w/v) xylene cyanol and 1% (w/v) bromophenol blue], heated at 90 °C
for 10 min, and then chilled on ice. The solutions were finally applied
to a 10% denaturing polyacrylamide gel (31 cm × 38.5 cm × 0.4 mm,
containing 7 M urea). Electrophoresis was carried out at 50 W for 2 h.
The gel was analyzed using a phosphoimager (Molecular Dynamics).
The bands were correlated with those produced according to a Maxam-
Gilbert A+G sequencing protocol.31
Cleavage of a 53-Nucleotide 5′-32P End-Labeled RNA by a
Trithiazole-Containing Bleomycin Analogue. The 53-nucleotide RNA
was 5′-32P end-labeled essentially as described.32 RNA cleavage
reactions were carried out in 10 µL (total volume) of 10 mM sodium
phosphate, pH 7.0, containing 5′-32P end-labeled RNA (5 × 104 cpm,
0.32 nM concentration) and the appropriate concentrations of reagents
as indicated in the legend to Figure 6. Each reaction was initiated by
the simultaneous addition of solutions of BLM and Fe2+ to the buffered
solution containing RNA. Each reaction mixture was incubated at 23
°C for 30 min, and then the reaction mixture was frozen in dry ice,
lyophilized, and dissolved in a gel loading solution [80% formamide,
2 mM EDTA, 0.05% (w/v) xylene cyanol, and 0.05% (w/v) bromo-
phenol blue]. The resulting solution was heated at 70 °C for 3 min,
chilled on ice, and then applied to a 20% denaturing polyacrylamide
gel (31 cm × 38.5 cm × 0.4 mm). Electrophoresis was carried out at
45 W for 2 h. The gel was analyzed by the use of a Molecular Dynamics
Phosphorimager.
Relaxation of Supercoiled DNA by a Trithiazole-Containing
Deglycobleomycin Analogue. Reactions were carried out in 25 µL
(total volume) of 10 mM sodium cacodylate buffer, pH 7.0, containing
300 ng (4.32 nM plasmid concentration, 18.8 µM base pair concentra-
tion) of pBR322 plasmid DNA and the appropriate concentrations of
BLM and Fe2+. Reaction mixtures were incubated at 37 °C for 30 min.
The reactions were quenched by the addition of 5 µL of loading dye
[30% glycerol containing 0.125% (w/v) bromophenol blue] and applied
to a 1% agarose gel (15 × 9 × 0.5 cm) containing 0.5 µg/mL ethidium
bromide. Horizontal gel electrophoresis was carried out in 9 mM Tris-
borate buffer, pH 8.3, containing 320 µM disodium EDTA at 156 V
for 2 h. The DNA bands were visualized under UV light.
Preparation of a 5′-32P End-Labeled DNA Restriction Fragment.
Plasmid pBR322 (25 µg) was incubated with 100 units of restriction
endonuclease HindIII in a 100 µL (total volume) reaction mixture
containing 5 mM NaCl, 1 mM Tris-HCl, pH 7.9, 1 mM MgCl2, and
0.1 mM DTT. The digestion reaction was carried out at 37 °C for 3 h.
The DNA was recovered by ethanol precipitation. The linearized DNA
was dephosphorylated with 1 unit of calf intestinal alkaline phosphatase
in a reaction mixture (200 µL total volume) containing 10 mM NaCl,
5 mM Tris-HCl, pH 7.9, 1 mM MgCl2, and 0.1 mM DTT. The reaction
Acknowledgment. We thank Dr. James Quada, Jr. for
assistance with the characterization of compound 7. This work
was supported by NIH Research Grants CA76297 and CA77284
awarded by the National Cancer Institute.
JA011820U
(31) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499.
(32) Holmes, C. E.; Duff, R. J.; van der Marel, G. A.; van Boom, J.; Hecht, S.
M. Bioorg. Med. Chem. 1997, 5, 1235.
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3884 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002