Solid-phase synthesis of bleomycin group antibiotics. Construction of a 108-member deglycobleomycin library

Article abstract of DOI:10.1021/ja021388w

The bleomycins (BLMs) are structurally related glycopeptide antibiotics isolated from Streptomyces verticillus that mediate the sequence-selective oxidative damage of DNA and RNA. Deglycobleomycin, which lacks the carbohydrate moiety, cleaves DNA analogously to bleomycin itself, albeit less potently, and has been used successfully for analyzing the functional domains of bleomycin. Although structural modifications to bleomycin and deglycobleomycin have been reported, no bleomycin or deglycobleomycin analogue having enhanced DNA cleavage activity has yet been described. The successful synthesis of a deglycobleomycin on a solid support has permitted the facile solid-phase synthesis of 108 unique deglycobleomycin analogues through parallel solid-phase synthesis. Each of the deglycobleomycin analogues was synthesized efficiently; the purity of each crude product was greater than 60%, as determined by HPLC integration. The solid-phase synthesis of the deglycobleomycin library provided near-milligram to milligram quantities of each deglycobleomycin, thereby permitting characterization by ¹H NMR and high-resolution mass spectrometry. Each analogue demonstrated supercoiled plasmid DNA relaxation above background cleavage; the library included two analogues that mediated plasmid relaxation to a greater extent than the parent deglycobleomycin molecule.

Full text of DOI:10.1021/ja021388w

Published on Web 06/13/2003
Solid-Phase Synthesis of Bleomycin Group Antibiotics.
Construction of a 108-Member Deglycobleomycin Library
Christopher J. Leitheiser, Kenneth L. Smith, Michael J. Rishel, Shigeki Hashimoto,
Kazuhide Konishi, Craig J. Thomas, Chunhong Li, Michael M. McCormick, and
Sidney M. Hecht*
Contribution from the Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22904
Received November 27, 2002; E-mail: sidhecht@virginia.edu
Abstract: The bleomycins (BLMs) are structurally related glycopeptide antibiotics isolated from Streptomyces
verticillus that mediate the sequence-selective oxidative damage of DNA and RNA. Deglycobleomycin,
which lacks the carbohydrate moiety, cleaves DNA analogously to bleomycin itself, albeit less potently,
and has been used successfully for analyzing the functional domains of bleomycin. Although structural
modifications to bleomycin and deglycobleomycin have been reported, no bleomycin or deglycobleomycin
analogue having enhanced DNA cleavage activity has yet been described. The successful synthesis of a
deglycobleomycin on a solid support has permitted the facile solid-phase synthesis of 108 unique
deglycobleomycin analogues through parallel solid-phase synthesis. Each of the deglycobleomycin
analogues was synthesized efficiently; the purity of each crude product was greater than 60%, as determined
by HPLC integration. The solid-phase synthesis of the deglycobleomycin library provided near-milligram to
1
milligram quantities of each deglycobleomycin, thereby permitting characterization by H NMR and high-
resolution mass spectrometry. Each analogue demonstrated supercoiled plasmid DNA relaxation above
background cleavage; the library included two analogues that mediated plasmid relaxation to a greater
extent than the parent deglycobleomycin molecule.
Introduction
Despite a lesser cleavage efficiency and a decreased double- to
single-strand cleavage ratio for deglycobleomycin, the aglycon
The bleomycins (BLMs) are a family of glycopeptide-derived
antitumor antibiotics that were originally isolated from a culture
broth of Streptomyces Verticillus as copper chelates.1 A mixture
of bleomycins, consisting primarily of bleomycin A2 (∼60%)
and bleomycin B2 (∼30%) (Figure 1),2 are used for the treatment
of cancers.3 The therapeutic effects of bleomycin are believed
to derive in part from damage to chromosomal DNA in the
presence of a metal ion cofactor.4 Bleomycin can also degrade
some RNAs at micromolar concentrations,5 in both the presence5a,c
and absence of Fe2+ 5b
therapeutic locus.4e,5a,d
Deglycobleomycin (Figure 2) cleaves DNA in a sequence-
selective manner quite similar to that of bleomycin itself.6
is more readily accessible synthetically and has been used
successfully to define the essential structural elements necessary
to perform sequence-selective oxidative DNA degradation.7
Over the past two decades, numerous analogues of bleomycin
and deglycobleomycin have been synthesized to better under-
stand the mechanism of DNA cleavage by bleomycin.7 Most
of these have differed from the parent (deglyco)bleomycin only
within a single amino acid constituent.
Recently, the solid-phase synthesis of deglycobleomycin A5
has been reported.8 This has also permitted the solid-phase
syntheses of deglycobleomycins having varied C-termini9 and
,
and RNA may represent another
(6) (a) Oppenheimer, N. J.; Chang, C.; Ehrenfeld, G.; Rodriguez, L. O.; Hecht,
S. M. J. Biol. Chem. 1982, 257, 1606. (b) Sugiyama, H.; Ehrenfeld, G.
M.; Shipley, J. B.; Kilkuskie, R. E.; Chang, L.-H.; Hecht, S. M. J. Nat.
Prod. 1985, 48, 869. (c) Shipley, J. B.; Hecht, S. M. Chem. Res. Toxicol.
1988, 1, 25.
(1) Umezawa, H.; Suhara, Y.; Takita, T.; Maeda, K. J. Antibiot. 1966, 19A,
210.
(2) Anticancer Drugs: ReactiVe Metabolism and Drug Interactions; Powis,
G., Ed.; Pergamon Press: New York, 1994.
(7) (a) Takita, T.; Umezawa, Y.; Saito, S.; Morishima, H.; Naganawa, H.;
Umezawa, H.; Tsuchiya, T.; Miyake, T.; Kageyama, S.; Umezawa, S.;
Muraoka, Y.; Suzuki, M.; Otsuka, M.; Narita, M.; Kobayashi, S.; Ohno,
M. Tetrahedron Lett. 1982, 23, 521. (b) Aoyagi, Y.; Murugesan, N.;
Ehrenfeld, G. M.; Chang, L.-H.; Ohgi, T.; Shekhani, M. S.; Kirkup, M. P.;
Hecht, S. M. J. Am. Chem. Soc. 1982, 104, 5237. (c) Aoyagi, Y.; Katano,
K.; Suguna, H.; Primeau, J.; Chang, L.-H.; Hecht, S. M. J. Am. Chem.
Soc. 1982, 104, 5537. (d) Sugiura, Y.; Suzuki, T.; Otsuka, M.; Kobayashi,
S.; Ohno, M.; Takita, T.; Umezawa, H. J. Biol. Chem. 1983, 258, 1328.
(e) Umezawa, H.; Takita, T.; Sugiura, Y.; Otsuka, M.; Kobayashi, S.; Ohno,
M. Tetrahedron 1984, 40, 501. (f) Boger, D. L.; Honda, T.; Menezes, R.
F.; Colletti, S. L. J. Am. Chem. Soc. 1994, 116, 5631. (g) Boger, D. L.;
Honda, T. J. Am. Chem. Soc. 1994, 116, 5647. (h) Boger, D. L.; Cai, H.
Angew. Chem., Int. Ed. 1999, 38, 448. (i) Katano, K.; An, H.; Aoyagi, Y.;
Overhand, M.; Sucheck, S. J.; Stevens, W. C., Jr.; Hess, C. D.; Zhou, X.,
Hecht, S. M. J. Am. Chem. Soc. 1998, 120, 11285.
(3) Bleomycin Chemotherapy; Sikic, B. I., Rozencweig, M., Carter, S. K., Eds.;
Academic Press: Orlando, FL, 1985.
(4) (a) Hecht, S. M. Acc. Chem. Res. 1986, 19, 383. (b) Stubbe, J.; Kozarich,
J. W. Chem. ReV. 1987, 87, 1107. (c) Natrajan, A.; Hecht, S. M. In
Molecular Aspects of Anticancer Drug-DNA Interactions; Neidle, S.,
Waring, M. J., Eds.; Macmillan: London, 1994; pp 197-242. (c) Kane,
S. A.; Hecht, S. M. Prog. Nucleic Acid Res. Mol. Biol. 1994, 49, 313. (d)
Aso, M.; Kondo, M.; Suemune, H.; Hecht, S. M. J. Am. Chem. Soc. 1999,
121, 9023. (e) Hecht, S. M. J. Nat. Prod. 2000, 63, 158.
(5) (a) Hecht, S. M. Bioconjugate Chem. 1994, 5, 513. (b) Keck, M. V.; Hecht,
S. M. Biochemistry 1995, 34, 12029. (c) Holmes, C. E.; Duff, R. J.; van
der Marel, G. A.; van Boom, J.; Hecht, S. M. Bioorg. Med. Chem. 1997,
5, 1235. (d) Hecht, S. M. RNA as a Therapeutic Target for Bleomycin. In
The Many Faces of RNA; Eggleston, D. S., Prescott, C. D., Pearson, N.
D., Eds; Academic Press Ltd.: London, 1998; pp 3-17.
9
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Solid-Phase Synthesis of Bleomycin Group Antibiotics
A R T I C L E S
Figure 1. Structures of bleomycin A2, B2, and A5.
for BLM catabolism in a therapeutic setting. In an effort to
achieve these changes, the initial deglycobleomycin library
included analogues of the bithiazole moiety, the threonine/
methylvalerate linker structure, and the â-hydroxyhistidine
moiety (Figure 1). The individual building blocks for the
modified deglycobleomycins were either commercially available
or readily accessible through existing synthetic methods. To
prepare 108 analogues, three bithiazole, three threonine, three
methylvalerate, and four â-hydroxyhistidine substitutions were
made (Figure 3). The constituents present in deglycobleomycin
were also included in this library, so that the effects of the
modifications could be analyzed systematically. The selection
of the amino acid fragments was based on previously reported
observations as discussed in the following.
Selection of Fragment Analogues for Library Synthesis.
In addition to bithiazole itself, a chlorobithiazole analogue was
selected, since previous reports of deglycobleomycins containing
halobithiazoles demonstrated DNA cleavage in the presence of
light.12 A trithiazole-containing deglycobleomycin analogue
recently synthesized demonstrated diminished 5′-GC-3′ cleavage
patterns when incorporated into deglycobleomycin, indicating
a preference for 5′-GT-3′ cleavage.13 In addition, the analogue
containing the trithiazole demonstrated enhanced RNA cleavage.
It was hoped that incorporating these two analogues into the
bleomycin library would provide biochemically interesting new
analogues.
Figure 2. Structures of deglycobleomycin A2, A5, and A6.
the synthesis of bleomycin A5 itself.10 As a result of these efforts,
the preparation of a combinatorial library of deglycobleomycins
seemed plausible and could further reveal the roles of individual
bleomycin constituents. Presently, we report the combinatorial
synthesis, purification, and structural characterization of 108
deglycobleomycin analogues.
Results
The role of the threonine moiety of bleomycin has been
studied in two laboratories.14 The data suggested that simple
It was hoped that the incorporation of multiple substitutions
simultaneously within the bleomycin core structure would afford
analogues having improved biochemical properties. These might
include enhanced potency of DNA cleavage, enhanced double-
strand DNA cleavage, preferential RNA cleavage, or resistance
to the action of bleomycin hydrolase,11 an enzyme responsible
(11) (a) Umezawa, H. Pure Appl. Chem. 1971, 28, 665. (b) Lazo, J. S.; Sebti,
S. M.; Filderman, A. E. In Metabolism and Mechanism of Action of Anti-
Cancer Drugs; Powis, G., Prough, R. A., Eds.; Taylor and Francis, Ltd:
London, 1987; p 194ff. (c) Bennett, J. M.; Reich, S. D. Ann. Intern. Med.
1979, 90, 945. (d) Sebti, S. M.; Lazo, J. S. Pharmacol. Ther. 1988, 38,
321. (e) Lazo, J. S.; Sebti, S. M. In Anticancer Drug Resistance; Kessel,
D., Ed.; CRC Press: Boca Raton, FL, 1989; p 267 ff. (f) Umezawa, H.;
Hori, S.; Sawa, T.; Yoshioka, T.; Takeuchi, T. J. Antibiot. 1974, 27, 419.
(12) (a) Quada, J. C., Jr.; Levy, M. J.; Hecht, S. M. J. Am. Chem. Soc. 1993,
115, 12171. (b) Quada, J. C., Jr.; Zuber, G. F.; Hecht, S. M. Pure Appl.
Chem. 1998, 70, 307. (c) Zuber, G.; Quada, J. C., Jr.; Hecht, S. M. J. Am.
Chem. Soc. 1998, 120, 9368. (d) Quada, J. C., Jr.; Boturyn, D.; Hecht, S.
M. Bioorg. Med. Chem. 2001, 9, 2303.
(8) Leitheiser, C. J.; Rishel, M. J.; Wu, X.; Hecht, S. M. Org. Lett. 2000, 2,
3397.
(9) (a) Smith, K. L.; Tao, Z.-F.; Hashimoto, S.; Leitheiser, C. J.; Wu, X.; Hecht,
S. M. Org. Lett. 2002, 4, 1079. (b) Tao, Z.-F.; Leitheiser, C. J.; Smith, K.
L.; Hashimoto, S.; Hecht, S. M. Bioconjugate Chem. 2002, 13, 426.
(10) Thomas, C. J.; Chizhov, A. C.; Leitheiser, C. J.; Rishel, M. J.; Konishi,
K.; Tao, Z.-F.; Hecht, S. M. J. Am. Chem. Soc. 2002, 124, 12926.
(13) Thomas, C. J.; McCormick, M. M.; Vialas, C.; Tao, Z.-F.; Leitheiser, C.
J.; Rishel, M. J.; Wu, X.; Hecht, S. M. J. Am. Chem. Soc. 2002, 124, 3875.
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Leitheiser et al.
Figure 3. Deglycobleomycin fragment analogues used for combinatorial library synthesis.
modifications of threonine do not affect sequence selectivity
but do affect the efficiency of strand scission. It was also
concluded that conformational effects imposed by this constitu-
ent can play an important role in the efficiency of DNA
cleavage. Since previous studies did not address the range of
structural parameters that can affect BLM function, additional
structural factors were examined in this study in an attempt to
define the effects of hydrogen bonding or π-stacking interactions
among those that are commercially available. Accordingly,
tryptophan, methionine, thienylalanine, and histidine were used.
Each of these amino acids has a nitrogen or sulfur atom in a
position roughly equivalent to those of the imidazole nitrogens
of â-hydroxyhistidine. While previous studies have defined the
importance of the τ-nitrogen for sequence selectivity,17 it was
believed that the altered heterocyclic and nonheterocyclic
substitutions might demonstrate alternative RNA binding motifs,
a property not explored previously. Using these criteria for
selection of the amino acid fragments, the synthesis of the
required amino acid fragments was performed.
Synthesis of the Fmoc Amino Acid Fragments. The Fmoc-
bithiazole moiety was synthesized by treating 2′-aminoethyl-
2,4′-bithiazole-4-carboxylic acid hydrochloride (1) with 9-flu-
orenylmethyl-succinimidyl carbonate in a 1:1 aqueous 5%
K2CO3-dioxane solution.18 The desired product 2 was obtained
in 72% yield. The synthesis of the Fmoc-chlorobithiazole
moiety was accomplished by adding an additional step to the
reported procedure12d for the preparation of Boc-chlorobithiazole
methyl ester. Fully protected methyl ester 3 was saponified (3:1
methanol-NaOH (1 N aq)). The Boc protecting group was then
with DNA and RNA. As a result, tyrosine and methionine were
selected for use in the library.
A systematic study of the methylvalerate moiety revealed that,
in addition to the optimized four-carbon chain length, substit-
uents at the 2- and 4-positions were important for efficient DNA
strand scission.15 The modifications provided important insights
into the preferred conformation of the methylvalerate moiety.15
On this basis, it was felt that increased steric bulk at the C-4
position might provide a better preferential fit to DNA compared
to the natural methylvalerate moiety. As a result, the isopro-
pylmethylvalerate and phenylmethylvalerate moieties were
utilized for analogue synthesis.
The histidine moiety provides two potential metal coordina-
tion sites for metal-dependent DNA strand scission.16 Replace-
ments for this amino acid should logically contain analogous
potential metal coordination sites. To expedite deglycobleomycin
library synthesis, these amino acids were also chosen from
(14) (a) Carter, B. J.; Murty, V. S.; Reddy, K. S.; Wang, S.-N.; Hecht, S. M. J.
Biol. Chem. 1990, 265, 4193. (b) Carter, B. J.; Reddy, K. S.; Hecht, S. M.
Tetrahedron 1991, 47, 2463. (c) Boger, D. L.; Ramsey, T. M.; Cai, H.;
Hoehn, S. T.; Stubbe, J. J. Am. Chem. Soc. 1998, 120, 9139.
(15) Boger, D. L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.; Stubbe, J. J. Am.
Chem. Soc. 1998, 120, 9149.
(17) Boger, D. L.; Ramsey, T. M.; Cai, H. Bioorg. Med. Chem. 1996, 4, 195.
(16) Iitaka, Y.; Nakamura, H.; Nakatani, T.; Muraoka, A.; Fujii, A.; Takita, T.;
Umezawa, H. J. Antibiot. 1978, 31, 1070.
(18) Lapatsanis, L.; Milias, G.; Froussions, K.; Kolovos, M. Synthesis 1983,
671.
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Solid-Phase Synthesis of Bleomycin Group Antibiotics
A R T I C L E S
Scheme 2
removed with TFA in the presence of dimethyl sulfide, and the
resulting free amino acid was treated with N-(9-fluorenyl-
methoxycarbonyloxy)-succinimide in alkaline aqueous dioxane.
After precipitation by addition of TFA, desired Fmoc-protected
chlorobithiazole analogue 4 was obtained in 49% yield.
Likewise, 7b and 7c were obtained in 40 and 27% yields,
respectively. The chiral auxiliary was removed with lithium
peroxide in 3:1 THF-H2O at 0 °C and gave amino acid
derivative 8a in 88% yield (84 and 87% yields for 8b and 8c,
respectively). The Boc group was then removed with TFA in
the presence of dimethyl sulfide and treated with N-(9-
fluorenylmethoxycarbonyloxy)succinimide in alkaline aqueous
dioxane, which yielded the requisite Fmoc-protected amino acid
9a in 63% yield. Amino acid derivatives 9b and 9c were
obtained analogously in yields of 63 and 37%, respectively. This
method proved to be extremely versatile and readily applicable
to the synthesis of additional methylvalerate analogues, including
conformationally constrained valerates.23 It may be noted that
further modifications were made in the synthesis of the
methylvalerate analogues following Evans’ groundbreaking
work in the use of chiral enolates to mediate highly stereocon-
trolled aldol reactions.21 Most notable was the stereoselective
generation of (2S,3S,4R)-4-amino-3-hydroxy-2-methylvalerate
in 4 steps and 46% overall yield from Boc-R-alanine anhy-
dride.24 It was demonstrated that the outcome of the crucial
Zn(BH4)2 mediated reduction of the 3-keto intermediate leading
Scheme 1
The synthesis of the Fmoc-methylvalerate moiety was
accomplished by adding an additional step to the previously
reported route for Boc-methylvalerate synthesis.19,20 The Z-boron
enolate of the acylated chiral auxiliary 521 was condensed with
Boc-D-alanal22 (6a) (Scheme 1). The stereoselective aldol
condensation gave the desired product 7a in 71% yield.
(20) (a) Boger, D. L.; Menezes R. F J. Org. Chem. 1992, 57, 4331. (b) Boger,
D. L.; Colletti, S. L.; Honda, T.; Menezes, R. F. J. Am. Chem. Soc. 1994,
116, 5607.
(21) (a) Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103,
2127. (b) Evans, D. A.; Nelson, J. V.; Vogel, E.; Taber, T. R. J. Am. Chem.
Soc. 1981, 103, 3099.
(19) 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.
(22) Fehrentz, J.-A.; Castro, B. Synthesis 1983, 676.
(23) Rishel, M. J.; Hecht, S. M. Org. Lett. 2001, 3, 2867.
(24) Dipardo, R. M.; Bock, M. G. Tetrahedron Lett. 1983, 24, 4805.
9
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Leitheiser et al.
Figure 4. Representative deglycobleomycin analogues 10-17 characterized and listed in the Experimental Section in the Supporting Information.
to 7a was directed almost exclusively by the presence and
absolute stereochemistry of the group at C2 and not by the
chirality at C4 or that present in the chiral auxiliary. Ohno19
and Boger20 further refined this method for the synthesis
(2S,3S,4R)-4-amino-3-hydroxy-2-methylvalerate as they dem-
onstrated that the synthesis could be accomplished from
Boc-R-alanal22 in three steps and 61% overall yield.
Synthesis of a 108-Member Deglycobleomycin Library.
The synthesis of the deglycobleomycin A6 library was carried
out as outlined in Scheme 2 for deglycobleomycin A6 itself.
The linker-bound resin25 was treated with di-Boc spermine26 in
the presence of Hunig’s base in DMF for 24 h.25b This provided
the spermine-functionalized resin. The bithiazole moiety of
deglycobleomycin was immediately added to the solid support
via HBTU-mediated coupling in DMF. After washing the resin
with DMF and CH2Cl2, we assayed a small sample using the
Kaiser test27 and the bromophenol blue test.28 The coupling was
quantified by Fmoc cleavage and gave a loading of 0.17 mmol/
g, representing a transamination and coupling efficiency of
40%.29 Since neither test indicated the presence of any free
amine, the resin was treated with a solution of 20% piperidine
in DMF, and the threonine moiety was coupled to the resin by
the use of HBTU, HOBt, and Hunig’s base, providing the
tripepide with a coupling efficiency of 0.17 mmol/g (>98%).
The methylvalerate moiety was then added by the same
sequence of treatments; the coupling efficiency was 0.15 mmol/g
(>98%). Following an additional treatment with piperidine, the
histidine moiety was coupled to the resin using HATU, HOAt,
(25) (a) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett. 1998,
39, 3585. (b) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron
Lett. 2000, 41, 1099.
(26) (a) O’Sullivan, M. C.; Zhou, Q.; Li, Z.; Durham, T. B.; Rattendi, D.; Lane,
S.; Bacchi, C. J. Bioorg. Med. Chem. 1997, 5, 2145. (b) Carrington, S.;
Fairlamb, A. H.; Blagbrough, I. S. J. Chem. Soc., Chem. Commun. 1998,
21, 2335. (c) Ronsin, G.; Perrin, C.; Guedat, P.; Kremer, A.; Camilleri, P.;
Kirby, A. J. J. Chem. Soc., Chem. Commun. 2001, 21, 2234.
(27) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. J. Anal. Biochem.
1970, 34, 595.
(28) Krchnak, V.; Vagner, J.; Safar, P.; Lebl, M. Collect. Czech. Chem. Commun.
1988, 53, 2542.
(29) Fmoc cleavage was based on the dibenzylfulvene-piperidine adduct formed
upon treatment of the resin with piperidine. The optical densities 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.
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Solid-Phase Synthesis of Bleomycin Group Antibiotics
A R T I C L E S
Figure 5. Representative deglycobleomycin analogues 18-23 characterized and listed in the Experimental Section in the Supporting Information.
and Hunig’s base which yielded resin-bound pentapeptide resin
with a final loading of 0.13 mmol/g (>95%).
parallel, that is, spatially separated, library was constructed in
order to test the robustness of the synthetic methodology. The
spatially separated library was based on 36 bithiazole-containing
deglycobleomycins, 36 chlorobithiazole-containing deglycob-
leomycins, and 36 trithiazole-containing deglycobleomycins.
The resin was initially functionalized with the protected
spermine and the bithiazole moieties. Each resin was then
divided into three separate vessels, and the appropriate threonine
analogue was then added. The splitting process was repeated
with the addition of the methylvalerate and histidine moieties.
This continuous division technique minimized the number of
reactions required and also provided flexibility in the amounts
of resin employed for specific analogues, thus affording a larger
quantity of final products compared to other combinatorial
techniques. The final deglycobleomycin analogues were readily
deprotected under acidic conditions and cleaved from the resin
using 2% hydrazine in DMF.
Initial attempts to couple Boc pyrimidoblamic acid at room
temperature demonstrated less than quantitative coupling ef-
ficiencies, typically less than 50%. Additional attempts to
improve the coupling efficiency with more active coupling
reagents or heat were not successful, and in fact, the attempted
coupling of Boc pyrimidoblamic acid via the agency of HATU
eliminated coupling altogether. However, when the final cou-
pling of Boc pyrimidoblamic acid was performed at 0 °C in
the presence of BOP and Hunig’s base, nearly quantitative
coupling was observed, as determined by reversed phase HPLC.
1H NMR spectroscopy was used to determine the overall yield
for deglycobleomycin A6 by careful integration of the exocyclic
C5-methyl substituent on pyrimidoblamic acid compared to a
known amount of added tert-butyl alcohol. The overall yield
of the desired deglycobleomycin A6 analogue was calculated
to be 66%, which corresponded to a stepwise coupling yield of
92% from the attached bithiazole moiety. This demonstrated
that the coupling efficiency of pyrimidoblamic had improved
greatly compared to that of the previous reported procedure.8
The fully protected deglycobleomycin A6 was then treated with
a 90:5:5 TFA-Me2S-triisopropylsilane30 solution and gave the
fully functionalized resin-bound deglycobleomycin A6. The
resin-bound deglycobleomycin was then liberated from the resin
with a solution of 2% hydrazine in DMF.
Each of the deglycobleomycin analogues was purified by
reversed phase HPLC. In every case, the major peak was
subsequently identified as the desired product. The purity of
the crude products, as determined by HPLC peak integration,
was >60% and in some cases >70%. All of the purified
deglycobleomycin analogues were obtained in near-milligram
or milligram quantities after reversed phase HPLC purification.
The deglycobleomycin analogues were characterized by 1H
NMR spectroscopy, electrospray mass spectrometry, and, in
some cases, high-resolution mass spectrometry. Representative
deglycobleomycins are shown in Figures 4 and 5, and charac-
terization data for these compounds can be found in the
Experimental Section in the Supporting Information. Electro-
The remaining deglycobleomycins in the library were syn-
thesized in analogy with deglycobleomycin A6 (Scheme 2). A
(30) Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A. Tetrahedron
Lett. 1989, 30, 2739.
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Figure 6. Electrospray mass spectrum of crude deglycobleomycin analogue 11.
spray mass spectra, HPLC chromatograms, and 1H NMR spectra
are shown in Figures 6 and 7 for deglycobleomycin analogue
11 and Figures S1 and S2 (Supporting Information) for
deglycobleomycin analogue 23. Electrospray mass spectrometric
analysis of the crude sample was used in order to determine
whether the desired deglycobleomycin analogue had been
synthesized. Clearly present in Figures 6 and S1 are the desired
molecular ions for deglycobleomycin analogues 11 and 23,
respectively. In addition to the presence of the molecular ion,
the di- and tripositive ions are also present, which was expected
as a result of the multiple positions on deglycobleomycin that
could be protonated. The absence of any undesired peptides
resulting from incomplete coupling reactions was also verified,
suggesting that the coupling reactions were nearly quantitative.
Figures 7 and S2 show the reversed phase HPLC chromatograms
shifts characteristic of the amino acid fragments present within
the deglycobleomycin analogue. Thus, we verified that this
methodology was robust and could be employed for the
synthesis of a large number of deglycobleomycin analogues.
The derived deglycobleomycin analogues could be readily
identified through classical chemical characterization techniques.
Methionine-Containing Deglycobleomycin Analogues. The
incorporation of methionine into the deglycobleomycin library
was accompanied by an additional transformation. Methionine
has been reported to oxidize to the sulfoxide during the protocol
used for acidic cleavage.31 All deglycobleomycin congeners
containing a methionine moiety were isolated as the respective
methionine oxide species, as exemplified by deglycobleomycin
analogue 23 (Figure S1). Analysis of these products by reversed
phase HPLC and electrospray mass spectrometry indicated that
1
and H NMR spectra for deglycobleomycin analogues 11 and
(31) (a) Hofmann, K.; Haas, W.; Smithers, M. J.; Wells, R. D.; Wolman, Y.;
Yanaihara, N.; Zanetti, G. J. Am. Chem. Soc. 1965, 87, 620. (b) Joppich-
Kuhn, R.; Corkill, J. A.; Giese, R. W. Anal. Biochem. 1982, 119, 73. (c)
Hammer, R. P.; Alberico, F.; Gera, L.; Barany, G. Int. J. Pept. Protein
Res. 1990, 36, 31.
23. The major peak isolated from each reversed phase HPLC
1
purification was analyzed by H NMR spectroscopy. Clearly
1
present in these H NMR spectra are protons having chemical
9
8224 J. AM. CHEM. SOC. VOL. 125, NO. 27, 2003

Solid-Phase Synthesis of Bleomycin Group Antibiotics
A R T I C L E S
Figure 7. HPLC chromatogram and 1H NMR spectrum of 11. The major peak at 17.4 min was found to be the desired product: HPLC analysis was carried
out on a C18 reversed phase HPLC column (250 × 10 mm) using a linear gradient of 14 f 20% CH3CN in 0.1% aq TFA at a flow rate of 4 mL/min over
a period of 30 min.
the oxidized species were, in fact, the major products, analogous
to 23 (Figure S2). In most cases, the methionine-containing
species was not present at all. Fortunately, methionine-oxide-
containing peptides can be reduced in high yields with excess
ammonium iodide in the presence of methyl sulfide in neat
TFA.32 This reduction procedure could be used for the facile
conversion of methionine oxide to methionine in our degly-
cobleomycin analogues. For example, the methionine-oxide-
containing deglycobleomycin 24 shown in Figure 8 was purified
by HPLC and reduced quantitatively to 24a using the afore-
mentioned protocol, as demonstrated quantitatively by reversed
phase HPLC (Figure S3) and high-resolution mass spectrometry.
To prevent reoxidation upon storage, the methionine-contain-
ing deglycobleomycin analogues were characterized and assayed
initially as the oxidized species. Those species of special interest
in any given assay can be reduced to the respective methionine-
Figure 8. Oxidation and reduction of methionine-containing deglycobleo-
mycins.
(32) Nicola´s, E.; Vilaseca, M.; Giralt, E. Tetrahedron 1995, 51, 5701.
9
J. AM. CHEM. SOC. VOL. 125, NO. 27, 2003 8225

A R T I C L E S
Leitheiser et al.
Figure 9. Effect of single and multiple substitutions in deglycobleomycin A6 on the efficiency of relaxation of a supercoiled DNA plasmid. Combinations
of + and ++ constituents yielded deglycoBLMs that produced Form III supercoiled plasmid breaks. Combinations of ++ and +++ constituents yielded
deglycoBLMs that produced Form III supercoiled plasmid breaks with good and sometimes enhanced potency. Any analogue containing one or more -
constituents yielded deglycoBLMs that did not produce Form III DNA but did produce Form II DNA.
containing analogues. While the presence of methionine oxide
in the analogues initially isolated was not planned, the where-
withal to convert these species readily to those containing
methionine does provide facile access to an additional 34
deglycobleomycin analogues.
DNA Cleavage Mediated by Members of the Deglycob-
leomycin A6 Library. The deglycobleomycin library was
utilized in the supercoiled DNA plasmid relaxation assay to
identify compounds having altered DNA cleavage activity. In
the presence of equimolar Fe2+, all compounds synthesized as
part of the library mediated plasmid relaxation to some extent.
Two analogues (11 and 15) demonstrated supercoiled DNA
plasmid relaxation to an extent greater than deglycobleomycin
A6 itself.
important observations relate to coupling efficiencies and the
facility of analogue preparation. As judged by the Fmoc cleavage
assay, all couplings except those involving the Boc pyrimido-
blamic acid moiety were calculated to be greater than 90%.
Since the major product isolated by reversed phase HPLC was
the desired deglycobleomycin conjugate, it was apparent that
coupling of the pyrimidoblamic acid moiety was also quite
efficient, when the low temperature coupling protocol was
employed. While the basis for the success of the low temperature
protocol is uncertain, it may relate to the stability of the activated
intermediate. In any case, the procedure was generally applicable
for those species studied. This suggests that the solid-phase
synthetic methodology is robust and that additional (deglyco)-
bleomycins can be synthesized without undue concern over
incomplete coupling reactions. The individual deglycobleomy-
cins were obtained in near-milligram and milligram quantities
after purification, which is altogether sufficient for chemical
and biochemical evaluation. Also notable was the facility with
which individual analogues can be prepared. Once the protected
amino acid constituents and functionalized resin are obtained,
synthesis and purification of a deglycobleomycin analogue can
be accomplished routinely in 2-3 days.
Discussion
Synthesis and Characterization of Analogues. In recent
years, the properties of a number of DNA binding agents have
been explored by the preparation of libraries of compounds.33
The ability to synthesize (deglyco)bleomycin on a solid support
has permitted the preparation of a significant number of
analogues for evaluation. From a synthetic perspective, the most
All deglycobleomycin analogues were purified readily by
reversed phase HPLC and characterized by 1H NMR spectros-
copy and low-resolution (electrospray) or high-resolution (FAB)
mass spectrometry. Each compound was identified readily by
mass spectrometry, as a consequence of the presence of a
(33) (a) Sagnou, M. J.; Howard, P. W.; Gregson, S. J.; Eno-Amooquaye, E.;
Burke, P. J.; Thurston, D. E. Bioorg. Med. Chem. Lett. 2000, 10, 2083. (b)
Baraldi, P. G.; Balboni, G.; Pavani, M. G.; Spalluto, G.; Tabrizi, M. A.;
De Clercq, E.; Balzarini, J.; Bando, T.; Sugiyama, H.; Romagnoli, R. J.
Med. Chem. 2001, 44, 2536. (c) Kers, I.; Dervan, P. B. Bioorg. Med. Chem.
2002, 10, 3339.
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Solid-Phase Synthesis of Bleomycin Group Antibiotics
A R T I C L E S
summarized in Figure 9. As shown, analogues prepared from
constituents having scores of + or ++ produced deglycobleo-
mycin analogues of reasonable potency that generally mediated
double-strand DNA cleavage. Deglycobleomycin analogues
prepared from constituents having scores of ++ and +++
exhibited a potency of cleavage of supercoiled plasmid DNA
comparable to or greater than that of deglycobleomycin itself.
While all of the deglycobloemycin analogues prepared were
capable of relaxing supercoiled plasmid DNA to some extent,
the presence of an amino acid having a score of - resulted in
a substantial diminution of DNA relaxation potency, and the
presence of two such constituents afforded weakly active
deglycobleomycins that produced minimal amounts of Form II
DNA and no Form III DNA. Significantly, when an amino acid
having a score of - was present, no combination of other
constituents could restore the potency of DNA relaxation.
In terms of the effects of specific substitutions, the replace-
ment of threonine with tyrosine or methionine afforded degly-
cobleomycin analogues that mediated plasmid relaxation with
minimal efficiency; those deglycoBLMs tested for cleavage of
a linear DNA duplex gave only sequence neutral cleavage. Thus,
while the structure of the threonine moiety is not unique in its
ability to afford deglycoBLM analogues capable of sequence-
selective DNA cleavage,14 the range of amino acid structures
that can be employed in this position are clearly somewhat
limited and require more precise definition. The same conclusion
was reached for the â-hydroxyhistidine moiety in which
replacement of the imidazole moiety of histidine with an indole,
thiophene, or dialkylsulfoxide moiety greatly diminished the
supercoiled DNA relaxation potential.
Deglycobleomycin analogues 11 and 15 exhibited potencies
of supercoiled plasmid DNA relaxation greater than those of
deglycobleomycin A6 itself. Further, each of these analogues
produced Form III DNA with enhanced efficiency. This is
illustrated in Figure 10 for deglycoBLM derivative 11. These
represent the first examples of the preparation of (deglyco)-
bleomycin analogues having potencies greater than those of the
parent molecule from which they were derived. It seems
reasonable to assert that the use of solid-phase synthesis of
additional bleomycin libraries, employing the principles defined
in the present study, may afford bleomycins having improved
properties in the several types of assays used to characterize
bleomycin function.
Figure 10. Relaxation of supercoiled pBR322 plasmid DNA by Fe-
(II)•deglycoBLM 11. Lane 1, DNA + 10 µM deglycoBLM; lane 2, 1 µM
deglycoBLM; lane 3, 5 µM deglycoBLM; lane 4, 10 µM deglycoBLM;
lane 5, 10 µM deglycoBLM 11; lane 6, 1 µM deglycoBLM 11; lane 7, 5
µM deglycoBLM 11; lane 8, 10 µM deglycoBLM 11. Lanes 2-4 and 6-8
also contained 10 µM Fe2+; essentially no cleavage was observed in the
presence of Fe2+ alone.
molecular ion, in addition to the presence of the di- and
tripositive molecular ions. Characterization by 1H NMR afforded
definitive evidence for the structures of individual deglycob-
leomycins synthesized. Chemical shifts corresponding to the
individual amino acid fragments were easily identifiable through-
out the entire range of analogues synthesized, providing more
conclusive evidence for the actual structures of individual
analogues.
As noted previously, methionine-containing deglycobleomy-
cins were isolated as the respective methionine S-oxides. A
protocol for the reduction of these species to deglycobleomycins
containing methionine has been demonstrated utilizing degly-
cobleomycin 24. The facile reduction of methionine-oxide-
containing deglycobleomycins will clearly permit the introduc-
tion of methionine into additional deglycobleomycin libraries.
Electrospray mass spectrometry was helpful in characterizing
both the oxidized and unoxidized methionine-containing species.
Purification of the oxidized and unoxidized methionine com-
pounds was accomplished readily by reversed phase HPLC, and
the individual species were easily identifiable by a dramatic
shift in retention time. As anticipated, the less polar unoxidized
methionine-containing analogues were retained several minutes
longer than the respective oxidized methionine-containing
species under identical gradient conditions. HPLC analysis of
the reduced species also indicated quantitative reduction,
providing an exceptionally clean product.
Preliminary Biochemical Evaluation. Each member of the
deglycobleomycin library was assayed and found to mediate
concentration-dependent plasmid DNA relaxation to some
extent. The efficiency of cleavage by individual analogues was
strongly dependent upon the specific substitutions made to the
parent molecule. To permit a semiquantitative evaluation of the
effects of individual amino acid substitutions, the individual
amino acid constituents in deglycobleomycin A6 were each
assigned a score of ++. Each of the modified amino acid
constituents employed for analogue synthesis was then assigned
a score (-, +, ++, or +++) by evaluating the potency of
cleavage of the deglycobleomycin resulting from the single
substitution of that amino acid constituent in place of the one
normally present in deglycobleomycin A6. The results of the
plasmid relaxation assays utilizing all of the analogues are
Acknowledgment. This paper is dedicated to the memory of
Professor Claude He´le`ne. This work was supported by NIH
Research Grants CA76297 and CA77284 awarded by the
National Cancer Institute.
Supporting Information Available: Experimental procedures
1
for the synthesis and bioassay of deglycoBLM derivatives. H
NMR and mass spectral data for members of the combinatorial
library. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA021388W
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