Solid-phase synthesis of bleomycin A5 and three monosaccharide analogues: Exploring the role of the carbohydrate moiety in RNA cleavage

Article abstract of DOI:10.1021/ja0208916

The solid-phase synthesis of bleomycin A₅ (BLM A₅) and three monosaccharide analogues is presented. The monosaccharide analogues incorporated α-D-mannose, α-L-gulose, and α-L-rhamnose moieties in lieu of the disaccharide normally pre

Full text of DOI:10.1021/ja0208916

Published on Web 10/15/2002
Solid-Phase Synthesis of Bleomycin A5 and Three Monosaccharide
Analogues: Exploring the Role of the Carbohydrate Moiety in RNA Cleavage
Craig J. Thomas, Alexander O. Chizhov, Christopher J. Leitheiser, Michael J. Rishel,
Kazuhide Konishi, Zhi-Fu Tao, and Sidney M. Hecht*
Departments of Chemistry and Biology, UniVersity of Virginia, CharlottesVille, Virginia 22901
Received June 28, 2002
The bleomycins (BLMs), exemplified by BLM A5 (1) (Figure
1), are anticancer antibiotics isolated from Streptomyces Verticillus.1
They effect single- and double-strand DNA cleavage,2 as well as
RNA cleavage3 in the presence of a metal cofactor and oxygen.4
Further, cleavage has been shown to be sequence- and shape-
selective for both DNA and RNA.2,3
The role of the carbohydrate moiety is not well understood. It
has been implicated in metal binding5 and cellular uptake and likely
participates in defining the nature of BLM binding to DNA, but
has minimal effect on the sequence selectivity or potency of DNA
cleavage.6,7 While deglycoBLM (2) analogues have provided
insights into the function of the amino acid constituents of BLM,8
the sugar moiety has not been modified systematically. Boger et
al. prepared BLM A2 derivatives containing the monosaccharides
R-D-mannose and 2-O-methyl-R-L-gulose.9 These retained the DNA
cleavage selectivity of BLM; however, only the 2-O-methyl-R-L-
gulose BLM A2 derivative cleaved DNA efficiently.
Figure 1. Structures of bleomycin A5 (1), deglycobleomycin A5 (2), and
BLM A5 analogues containing R-D-mannose (3), R-L-gulose (4), and R-L-
rhamnose (5).
Scheme 1
The accumulating evidence that RNA may be a plausible
therapeutic locus for BLM has focused interest on the exploration
of BLM analogues that target RNA specifically. While the
carbohydrate moiety apprently has little influence on the sequence
selectivity of DNA cleavage by BLM,7 no study has defined the
effect of the carbohydrate moiety on RNA cleavage. Presently, we
describe the solid-phase synthesis of BLM A5 and monosaccharide
analogues of BLM A5 containing R-D-mannose (3), R-L-gulose (4),
and R-L-rhamnose (5). Also described is the ability of 3-5 to cleave
DNA and RNA.
The solid-phase synthesis of BLM A5 and the monosaccharide
derivatives 3-5 was carried out in analogy with the synthesis of
deglycoBLM.10 The syntheses (Scheme 1) utilized Boc (6)- or NBS
(2-nitrobenzenesulfonyl) (7)-protected spermidine resins. Addition
of bithiazole intermediate (8)10 to the free amine of 6 or 7 was
accomplished utilizing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (HBTU) and Hunig’s base in DMF
over a 30-min period.11 Fmoc group removal was accomplished
using 20% piperidine in DMF. NR-Fmoc-(S)-threonine (9) was then
attached using HBTU, hydroxybenzotriazole (HOBt), and Hunig’s
base in DMF.12 Following Fmoc deprotection, NR-Fmoc-(2S,3S,4R)-
4-amino-3-hydroxy-2-methylvalerate (10)10 was conjugated to the
resin in the same fashion.12
Fmoc- and trityl-protected histidine analogues 11-14 containing
the native disaccharide and each of the three monosaccharide
derivatives, respectively, were prepared using NR-Fmoc-Nim-trityl-
(S)-erythro-â-hydroxyhistidine.13 Coupling of 11-14 to resin-bound
tetrapeptide was accomplished using a combination of benzotriazol-
1-yloxy-tris(dimethyamino)phosphonium hexafluorophosphate (BOP)
and Hunig’s base in DMF at 0 °C over a 12-h period.12 Coupling
of the Boc pyrimidoblamic acid moiety (15)14 to the resin-bound
pentapeptide was effected using BOP reagent and Hunig’s base in
DMF at 0 °C for 12 h. The resin-bound, fully functionalized BLM
A5 and each of the monosaccharide analogues was then depro-
tected15 and cleaved from the resin, affording BLM A5 (1) and BLM
derivatives 3, 4, and 5.12
The DNA cleavage efficiency, selectivity, and the ratio of double-
strand to single-strand DNA cleavage was determined for each of
the monosaccharide analogues. The R-L-gulosyl BLM A5 analogue
4 effected single- and double-strand DNA cleavage to nearly the
same extent as BLM A5 itself (not shown). The R-D-mannosyl
analogue of BLM A5 (3) and the R-L-rhamnosyl analogue (5) of
BLM A5 had diminished DNA cleavage efficiencies comparable
to that of deglycoBLM A5. Monosacharide analogues 3-5 each
had the same sequence selectivity of DNA cleavage as BLM A5.
These results agree with those of Boger et al.8,9 and support the
* Address correspondence to this author. E-mail: sidhecht@virginia.edu.
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J. AM. CHEM. SOC. 2002, 124, 12926-12927
10.1021/ja0208916 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Supporting Information Available: Experimental procedures and
spectral data for BLM and its synthetic intermediates as well as
experimental details of the cleavage of the 53-nt 5′-32P end-labeled
RNA (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) (a) Umezawa, H.; Maeda, K.; Takeuchi, T.; Okami, Y. J. Antibiot. 1966,
19, 200. (b) Umezawa, H.; Suhara, Y.; Takita, T.; Maeda, K. J. Antibiot.
1966, 19, 210.
(2) (a) Stubbe, J.; Kozarich, J. W. Chem. ReV. 1987, 87, 1107. (b) Natrajan,
A.; Hecht, S. M. In Molecular Aspects of Anticancer Drug-DNA
Interactions; Neidle, S., Waring, M. J., Eds.; Macmillan Press: London,
1994; pp 197-242. (c) Kane, S. A.; Hecht, S. M. Prog. Nucleic Acid
Res. Mol. Biol. 1994, 49, 313.
(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, 87, 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.
(4) (a) Sausville, E. A.; Peisach, J.; Horwitz, S. B. Biochemistry 1978, 17,
2740. (b) Sausville, E. A.; Stein, R. W.; Peisach, J.; Horwitz, S. B.
Biochemistry 1978, 17, 2746. (c) Burger, R. M.; Peisach, J.; Horwitz, S.
B. J. Biol. Chem. 1981, 256, 11636. (d) Kuramochi, H.; Takahashi, K.;
Takita, T.; Umezawa, H. J. Antibiot. 1981, 34, 576.
(5) (a) Oppenheimer, N. J.; Rodriguez, L. O.; Hecht, S. M. Proc. Natl. Acad.
Sci. U.S.A. 1979, 76, 5616. (b) Akkerman, M. A. J.; Neijman, E. W. J.
F.; Wijmenga, S. S.; Hilbers, C. W.; Bermel, W. J. Am. Chem. Soc. 1990,
112, 7462.
(6) Choudhury, A. K.; Tao, Z.-F.; Hecht, S. M. Org. Lett. 2001, 3, 1291.
(7) Sugiyama, H.; Ehrenfeld, G. M.; Shipley, J. B.; Kilkuskie, R. E.; Chang,
Figure 2. Cleavage of a 53-nucleotide RNA related to B. subtilis tRNAHis
precursor by Fe(II)‚BLM derivatives. Lane 1, RNA alone; lane 2, 100 µM
Fe2+; lane 3, 20 µM 3; lane 4, 1 µM 3 + 1 µM Fe2+; lane 5, 10 µM 3 +
10 µM Fe2+; lane 6, 20 µM 3 + 100 µM Fe2+; lane 7, 20 µM 4; lane 8, 1
µM 4 + 1 µM Fe2+; lane 9, 10 µM 4 + 10 µM Fe2+; lane 10, 20 µM 4 +
100 µM Fe2+; lane 11, 20 µM 5; lane 12, 1 µM 5 + 1 µM Fe2+; lane 13,
10 µM 5 + 10 µM Fe2+; lane 14, 20 µM 5 + 100 µM Fe2+; lane 15, 20
L.-H.; Hecht, S. M. J. Nat. Prod. 1985, 48, 869.
(8) Boger, D. L.; Cai, H. Angew Chem., Int. Ed. 1999, 38, 448.
(9) Boger, D. L.; Teramoto, S.; Zhou, J. J. Am. Chem. Soc. 1995, 117, 7344.
(10) (a) Leitheiser, C. J.; Rishel, M. J.; Wu, X.; Hecht, S. M. Org. Lett. 2000,
2, 3397. (b) Tao, Z.-F.; Leitheiser, C. J.; Smith, K. L.; Hashimoto, S.;
Hecht, S. M. Bioconjugate Chem. 2002, 13, 426. (c) Smith, K. L.; Tao,
Z.-F.; Hashimoto, S.; Leitheiser, C. J.; Wu, X.; Hecht, S. M. Org. Lett.
2002, 4, 1079.
(11) Quantification of coupling efficiency employed Fmoc analysis involving
piperidine treatment of a known weight of dry resin and subsequent
spectrophotometric measurement of the resulting dibenzylfulvene-piperi-
dine adduct.10,12 The resin-bound dipeptide derived from 6 (used for the
synthesis of 1) was produced in 43% yield from the initial resin; the resin-
bound dipeptide derived from 7 (employed for the syntheses of 3-5) was
obtained in yields >75%.
(12) Threonine was attached in >85% yield, as judged by Fmoc analysis. The
valerate was attached in >90% yield. Yields for the coupling of each of
the monosaccharide-containing histidine intermediates and the native
disaccharide were consistently >85%. Boc pyrimidoblamic acid coupling
to the pentapeptide containing 11 gave 1 in 15% overall yield following
deprotection and removal from the resin. The respective yields for 3, 4,
and 5 were 22, 29, and 17%.
(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.
(14) (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.
(15) Removal of the NBS group from resin 7 and its coupling products required
repeated exposure to the sodium salt of thiophenol. This treatment removed
the carbamoyl group from the disaccharide of 1, prompting the use of
resin-bound Boc-protected spermidine 6 for the synthesis of 1. Depro-
tection of both Boc and trityl groups from resin-bound 1 was accomplished
by treatment with TFA, isopropylsilane, and dimethyl sulfide. Following
deprotection, fully functionalized BLM A5 (1) was removed from the resin
using 20% hydrazine in DMF, which also effected disaccharide moiety
deacetylation. The monosaccharide derivatives, lacking the carbamoyl
group, were prepared using the NBS-protected spermidine resin 7.
(16) This RNA, identical in structure with the core of B. subtilis tRNAHis
precursor transcript,3a has been shown to undergo oxidative cleavage
predominantly at a single position13 that is presumably analogous to U35
in B. subtilis tRNAHis precursor transcript.
(17) (a) Sugiura, Y.; Suzuki, T.; Otsuka, M.; Kobayashi, S.; Ohno, M.; Takita,
T.; Umezawa, H. J. Biol. Chem. 1983, 258, 1328. (b) Kittaka, A.; Sugano,
Y.; Otsuka, M.; Ohno, M. Tetrahedron 1988, 44, 2821. (c) Kenani, A.;
Bailly, C.; Helbecque, N.; Catteau, J.-P.; Houssin, R.; Bernier, J.-L.;
Henichart, J.-P. Biochem. J. 1988, 253, 497. (d) Kenani, A.; Bailly, C.;
Houssin, R.; Henichart, J. P. Anticancer Drugs 1994, 5, 199.
µM 1; lane 16, 1 µM 1 + 1 µM Fe2+; lane 17, 10 µM 1 + 10 µM Fe2+
;
lane 18, 20 µM 1 + 100 µM Fe2+
.
suggestion that the nature of the first carbohydrate moiety is
important to BLM function.
The cleavage of a 53-nt RNA16 by each of the monosaccharide
analogues (Figure 2) showed that each retained a selectivity pattern
for hydrolytic (lanes 3, 7, 11, and 15) and oxidative RNA cleavage
(lanes 4-6, 8-10, 12-14, and 16-18) most similar to that of BLM
A5 itself. Again, only the R-L-gulosyl analogue (4) exhibited a
relatively high level of oxidative RNA cleavage. Quantification of
the major oxidative cleavage site (arrow) revealed that 4 cleaved
the RNA substrate 47% as efficiently as BLM A5, while 3 and 5
effected 16 and 19% cleavage, respectively. Interestingly, the
hydrolytic RNA cleavage efficiency of analogue 4 was superior to
that of BLM A5.
The role of the carbohydrate moiety during DNA and RNA
cleavage has been the subject of much speculation. It has been
postulated that the carbohydrate moiety forms part of a protective
pocket for the reactive metal-oxygen intermediates that are directly
responsible for both DNA and RNA strand scission.17 It is clear
from the present experiments that the carbohydrate moiety of BLM
plays a significant role in defining DNA and RNA cleavage
competence. Further, it is apparent that the sugar can be of critical
importance to the efficiency of oxidative DNA and RNA cleavage.
This work establishes the utility of our solid-phase synthesis
strategy for the facile production of BLM analogues containing one
or more sugar moieties. The elaboration of BLM libraries that
include modified carbohydrate moieties is now technically feasible.
Acknowledgment. This work was supported by NIH Research
Grants CA76297 and CA77284 awarded by the National Cancer
Institute.
JA0208916
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