Total synthesis of bleomycin group antibiotics. Total syntheses of bleomycin demethyl A2, bleomycin A2, and decarbamoyl bleomycin demethyl A2

Article abstract of DOI:10.1021/ja9819458

The total syntheses of bleomycin A₂ (1) by two routes are described. The final step in the synthesis of bleomycin A₂ involves methylation of bleomycin demethyl A₂ (2). This bleomycin derivative is of interest mechanistical

Full text of DOI:10.1021/ja9819458

J. Am. Chem. Soc. 1998, 120, 11285-11296
11285
Total Synthesis of Bleomycin Group Antibiotics. Total Syntheses of
Bleomycin Demethyl A2, Bleomycin A2, and Decarbamoyl Bleomycin
Demethyl A2
Kiyoaki Katano, Haoyun An, Yoshiaki Aoyagi, Mark Overhand, Steven J. Sucheck,
William C. Stevens, Jr., Cynthia D. Hess, Xiang Zhou, and Sidney M. Hecht*
Contribution from the Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22901
ReceiVed June 3, 1998
Abstract: The total syntheses of bleomycin A2 (1) by two routes are described. The final step in the synthesis
of bleomycin A2 involves methylation of bleomycin demethyl A2 (2). This bleomycin derivative is of interest
mechanistically, and can also provide access to other bleomycins via its known chemical conversion to
bleomycinic acid. Accordingly, the synthetic strategy presented represents a particularly versatile approach
for the elaboration of a wide variety of BLM congeners. Bleomycin was constructed from five key intermediates,
the syntheses of which are described. 1,6-Di-O-acetyl-3,4-di-O-benzyl-2-O-[2,4,6-tri-O-acetyl-3-O-(N-
acetylcarbamoyl)-R-D-mannopyranosyl]-â-L-gulopyranose (3) was converted quantitatively to its disaccharide
chloride (4), the latter of which was condensed with NR,Nim-bis(t-Boc)-(S)-erythro-â-hydroxyhistidine (7) to
provide R-O-glycosidated product 16. The subsequent couplings with benzyl valerate 8, threonylbithiazole 9,
and NR-t-Boc-pyrimidoblamic acid (10) afforded access to bleomycin demethyl A2 (2) and decarbamoyl
bleomycin demethyl A2 (26). Although it was obtained as a byproduct of the synthesis of BLM, the synthesis
of decarbamoyl bleomycin demethyl A2 nonetheless constitutes the first report of synthetic access to this BLM
congener that is of particular importance from a mechanistic perspective. This BLM can be used to resolve
the issue of the participation of the carbamoyl group as a metal ligand in metallobleomycins. Also reported
is a new route to bleomycin demethyl A2 that employed an unprotected carbamoyl group throughout the
synthesis. In conjunction with the use of the uncharged methylthiopropylamide C-substituent, the latter strategy
permitted improved functional group manipulation and thereby afforded intermediates that could be purified
with greater facility. Because the synthesis of bleomycin is limited by the efficiency of elaboration of the
carbohydrate moiety, a study was carried out to define improved methods for the preparation of this constituent
of BLM. Three new routes were explored, and a route involving the intermediacy of disaccharide activated
as a glycosyl bromide was found to be particularly efficient and convenient. Also of importance was the
finding that activation of carbohydrate intermediates as their glycosyl trichloroacetimidates permitted the requisite
couplings to be carried out conveniently and in good yields. Synthetic BLM demethyl A2 was shown to have
the same potency as a naturally derived sample in a plasmid DNA relaxation assay. The sequence selectivity
of DNA cleavage by synthetic and authentic Fe(II)‚BLMs was also shown to be the same. Also established
for the first time for any synthetic bleomycin was the actual chemistry of DNA degradation, which is the same
as that for naturally derived bleomycins.
The bleomycins (BLMs) are a family of structurally related
glycopeptide-derived antibiotics with significant antitumor activ-
ity.1 They were originally isolated from fermentation broths
of Streptomyces Verticillus by Umezawa and co-workers.2
Blenoxane, the clinically used mixture of bleomycins, contains
mainly bleomycin A2 and bleomycin B2, in addition to smaller
amounts of other bleomycins.3 The bleomycins were initially
thought to be â-lactam-type antibiotics.4 However, this structure
was later revised,1,5 and the revised structure was confirmed
by total synthesis in 1982 by the Umezawa6 and Hecht7
laboratories. Subsequently, Umezawa and co-workers reported
improvements in their synthesis.8 More recently, Boger and
co-workers have also reported a total synthesis of BLM A2.9
The therapeutic effects of bleomycins are believed to derive
from their ability to mediate the degradation of DNA10-12 and
possibly also RNA.12,13 Polynucleotide degradation mediated
by BLM is metal ion and oxygen dependent. Of particular
interest has been the sequence selectivity of DNA cleavage,
* To whom correspondence should be addressed at the Department of
Chemistry.
(3) Hecht, S. M. In Cancer Chemotherapeutic Agents; Foye, W. O., Ed.;
American Chemical Society: Washington, DC, 1995; pp 369-388.
(4) Takita, T.; Muraoka, Y.; Yoshioka, T.; Fujii, A.; Maeda, K.;
Umezawa, H. J. Antibiot. 1972, 25, 755.
(5) Takita, T.; Muraoka, Y.; Nakatani, T.; Fujii, A.; Umezawa, Y.;
Naganawa, H.; Umezawa, H. J. Antibiot. 1978, 31, 801.
(6) 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.
(7) Aoyagi, Y.; Katano, K.; Suguna, H.; Primeau, J.; Chang, L.-H.; Hecht,
S. M. J. Am. Chem. Soc. 1982, 104, 5537.
(1) (a) Umezawa, H. In Bleomycin: Current Status and New DeVelop-
ments; Carter, S. K., Crooke, S. T., Umezawa, H., Eds.; Academic Press:
New York, 1978. (b) Hecht, S. M. In Bleomycin: Chemical, Biochemical
and Biological Aspects; Hecht, S. M., Ed.; Springer-Verlag: New York,
1979. (c) Sugiura, Y.; Takita, T.; Umezawa, H. Met. Ions Biol. Syst. 1985,
19, 81.
(2) (a) Umezawa, H.; Maeda, K.; Takeuchi, T.; Okami, Y. J. Antibiot.
Ser. A 1966, 19, 200. (b) Umezawa, H.; Suhara, Y.; Takita, T.; Maeda, K.
J. Antibiot. Ser. A 1966, 19, 210. (c) Umezawa, H. Lloydia 1977, 40, 67.
(d) Umezawa, H. Prog. Biochem. Pharmacol. 1976, 11, 18.
10.1021/ja9819458 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/22/1998

11286 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
which involves predominantly 5′-GC-3′ and 5′-GT-3′ se-
quences.10,11 BLM has also been shown to recognize nucleic
acid tertiary structure, as exemplified by the cleavage of a
duplex-triplex junction in DNA,14 bulges in DNA15 and
RNA,13c and the putative junctions between single- and double-
stranded regions in RNA.13
As a consequence of their clinical utility, as well as their
interesting structures and mechanism of action, the BLMs have
been the focus of considerable attention. In addition to the
bleomycin analogues and fragments accessible by degradation
or modification of the natural product,16-18 ongoing synthetic
efforts have facilitated the preparation of synthetic19-23 and
semisynthetic24 BLMs as potential therapeutic agents and as
tools for dissecting the mechanism(s) of bleomycin action.
Studies of the mechanism of action, in particular, have benefited
from access to BLM subunits and analogues.19-25
Presently, we report the full details of two total syntheses of
bleomycin A2, using bleomycin demethyl A2 as a key interme-
diate. One of these syntheses employed carbohydrate interme-
diates containing a protected mannose carbamoyl group; the
other synthesis was achieved without protection of the carbam-
oyl group. Also obtained as a byproduct from one synthetic
route was decarbamoyl bleomycin demethyl A2, a species of
special interest for mechanistic studies of BLM metal ion
Figure 1. Structures of bleomycin A2 (1) and bleomycin demethyl A2
(2).
binding and activation. In addition, we report three new routes
for the elaboration of the carbohydrate moiety of bleomycin,
two of which gave particularly favorable results from the
perspectives of efficiency and convenience for the elaboration
of the carbohydrate moiety.
Results and Discussion
From the perspective of total synthesis, bleomycin A2 (1) and
bleomycin demethyl A2 (2) (Figure 1) may be envisioned to
consist of five key structural elements, the coupling of which
could provide synthetic access to the natural products. These
constituents, shown as intermediates protected in a fashion
suitable for the elaboration of bleomycin (Figure 2), include
the disaccharide moiety (3-6), (S)-erythro-â-hydroxyhistidine
(7) (cf. Scheme 1), (2S,3S,4R)-4-amino-3-hydroxy-2-methylva-
leric acid (8), the threonylbithiazole moiety (9) (cf. Scheme 2),
and pyrimidoblamic acid (10). Synthetic studies in three
laboratories have demonstrated amply that the success of
strategies for the synthesis of bleomycin depends critically on
the choice of protecting groups and the order in which the
coupling of key intermediates is effected. The modified
approach described by Umezawa and co-workers8 includes the
O-glycosidation of erythro-NR-t-Boc-Nim-tos-(S)-â-hydroxyhis-
tidine methyl ester with the glycosyl bromide analogous to
disaccharide derivative 6, subsequent coupling to tetrapeptide
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Umezawa, H. In Peptide Chemistry 1982; Sakakibara, S., Ed.; Protein
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Total Synthesis of Bleomycin
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11287
Scheme 1
Scheme 2
Figure 2. Key intermediates employed for the synthesis of bleomycin.
nose (3) and 1,6-di-O-acetyl-3,4-di-O-benzyl-2-O-(2,4,6-tri-O-acet-
yl-3-O-carbamoyl-R-D-mannopyranosyl)-â-L-gulopyranose (5),
were synthesized according to our previously reported proce-
dures.29 NR,Nim-bis(tert-butoxycarbonyl)-(S)-erythro-â-hydrox-
yhistidine (11) (Scheme 1) was prepared from (S)-erythro-â-
hydroxyhistidine.30 Benzylation of 11 with phenyldiazomethane,
prepared in situ from benzaldehyde tosylhydrazone, provided
the corresponding benzyl ester 7 in 88% yield.
S and NR-t-Boc-pyrimidoblamic acid, followed by deprotection
to give bleomycin A2. Boger and co-workers,9 in their initial
efforts, found that the O-glycosidation of NR-t-Boc-Nim-trityl-
(S)-â-hydroxyhistidine methyl ester with the â-glycosyl diphenyl
phosphate provided a mixture of at least three components with
the NR-Boc deprotection product. However, this reaction was
conducted in good yield by using an NR-CBz protecting group
on (S)-â-hydroxyhistidine instead of the NR-Boc group to avoid
the competitive NR-Boc deprotection. Subsequent coupling with
tetrapeptide S and NR-t-Boc-pyrimidoblamic acid similarly
afforded bleomycin A2.9 Our strategy included successful
O-glycosylation of NR,Nim-di-t-Boc-(S)-erythro-â-hydroxyhis-
tidine benzyl ester (7) with disaccharide chlorides 4 and 6
(Schemes 3 and 7) and subsequent, sequential coupling with
benzyl valerate 8, tripeptide S derivative 9, and NR-t-Boc-
pyrimidoblamic acid (10) (Schemes 4-6, 8, and 9). This
synthetic strategy has also proven useful for the synthesis of
four other BLM congeners.
A unique feature of the synthetic scheme employed here
involves the intermediacy of bleomycin demethyl A2. In
addition to providing uncharged synthetic intermediates, ame-
nable to purification with greater facility, bleomycin demethyl
A2 is of great interest intrinsically for its DNA binding and
cleaving properties26 and has also been used as a convenient
starting material to obtain BLMs modified within the C-
substituent via chemical transformation to bleomycinic acid.3,27,28
Synthesis of Key Intermediates. The requisite disacchar-
ides, 1,6-di-O-acetyl-3,4-di-O-benzyl-2-O-[2,4,6-tri-O-acetyl-3-
O-(N-acetylcarbamoyl)-R-D-mannopyranosyl]-â-L-gulopyra-
(2S,3S,4R)-4-Amino-3-hydroxy-2-methylvaleric acid (12)
([R]25 ) +10.5° (c 1.0, H2O), lit.31 [R]23 ) +10.7° (c 7.25,
D
D
H2O), lit.32 [R]25 ) +12.1° (c 1.03, H2O)) was synthesized
D
according to the reported procedures.31,33 Benzylation of this
acid with benzyl alcohol in the presence of anhydrous hydrogen
chloride afforded the corresponding benzyl ester (8) in 89%
yield.21c Threonylbithiazole derivative 9 was synthesized using
reported procedures19a,21a,34,35 (Scheme 2). Specifically, 2′-(2-
aminoethyl)-2,4′-bithiazole-4-[(3-methylthio)propyl]carboxam-
ide (14) was prepared by the reported method from the
corresponding carboxylic acid.19a,34,35 The coupling of 14 with
commercially available (S)-NR-t-Boc-threonine (13) via the
agency of DCC-HOBt afforded the coupled product NR-t-Boc-
threonylbithiazole 1535 in 68% yield. Deprotection of 15 with
trifluoroacetic acid in the presence of dimethyl sulfide gave
threonylbithiazole derivative 9 in 93% yield. NR-t-Boc-pyrimi-
doblamic acid (10) was prepared by our reported procedure;36
(29) (a) Pozsgay, V.; Ohgi. T.; Hecht, S. M. J. Org. Chem. 1981, 46,
3761. (b) Katano, K.; Chang, P.-I.,; Millar, A.; Pozsgay, V.; Minster, D.
K.; Ohgi, T.; Hecht, S. M. J. Org. Chem. 1985, 50, 5807. (c) Millar, A.;
Kim, K. H.; Minster, D. K.; Ohgi, T.; Hecht, S. M. J. Org. Chem. 1986,
51, 189. (d) Katano, K.; Millar, A.; Pozsgay, V.; Primeau, J. L.; Hecht, S.
M. J. Org. Chem. 1986, 51, 2927.
(30) Hecht, S. M.; Rupprecht, K. M.; Jacobs, P. M. J. Am. Chem. Soc.
1979, 101, 3982.
(31) Takita, T.; Muraoka, Y.; Maeda, K.; Umezawa, H. J. Antibiot. 1986,
19, 79.
(32) Ohgi. T.; Hecht, S. M. J. Org. Chem. 1981, 46, 1233.
(33) Narita, M.; Otsuka, M.; Kobayashi, S.; Ohno, M.; Umezawa, Y.;
Morishima, H.; Saito, S. Tetrahedron Lett. 1982, 23, 525.
(34) Levin, M. D.; Subrahamanian, K.; Katz, H.; Smith, M. B.; Burlett,
D. J.; Hecht, S. M. J. Am. Chem. Soc. 1980, 102, 1452.
(35) (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.
(26) (a) Takeda, K.; Kato, F.; Kawai, S.; Konno, K. J. Antibiot. 1979,
32, 43. (b) Berry, D. E.; Chang, L.-H.; Hecht, S. M. Biochemistry 1985,
24, 3207. (c) Levy, M. J.; Hecht, S. M. Biochemistry 1988, 27, 2647.
(27) (a) Umezawa, H. Heterocycles 1979, 13, 23. (b) Tanaka, W.; Takita,
T. Heterocycles 1979, 13, 469. (b) Takita, T.; Fujii, A.; Fukuoka, T.;
Umezawa, H. J. Antibiot. 1973, 26, 252.
(28) (a) DeRiemer, L. H.; Meares, C. F.; Goodwin, D. A.; Diamanti, C.
I. J. Med. Chem. 1979, 22, 1019. (b) Vloon, W. J.; Kruk, C.; Pandit, U. K.;
Hofs, H. P.; McVie, J. G. Heterocycles 1984, 22, 779. (c) Vloon, W. J.;
Kruk, C.; Pandit, U. K.; Hofs, H. P.; McVie, J. G. J. Med. Chem. 1987, 30,
20.

11288 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
Scheme 3
Scheme 4
this species is also accessible using other synthetic strategies.37
The key intermediates were all characterized thoroughly,
including NMR spectroscopic and mass spectral analyses.
Elaboration of BLM from a Disaccharide Having an
N-Protected Carbamoyl Group. Disaccharide 3 was con-
verted to the corresponding gulopyranosyl chloride (4) by using
hydrogen chloride gas in dichloromethane (Scheme 3). Gu-
lopyranosyl chloride 4 was isolated as a colorless foam in
quantitative yield after workup, and was used directly in the
next step. The condensation of 4 with benzyl NR,Nim-di-t-
butoxycarbonyl-(S)-erythro-â-hydroxyhistidine (7) (CF3SO3Ag,
(CH3)2NCON(CH3)2) in anhydrous 1,2-dichloroethane at 45 °C
for 24 h afforded the coupling product 16 in 21% yield.
Treatment of 16 with di-tert-butyl dicarbonate in dry pyridine
at 25 °C for 2 h provided tri-t-Boc product 17 in which the
NR,Nim and carbamoyl nitrogens were all protected by Boc
groups. Compound 17 underwent hydrogenolysis selectively
over 5% palladium-on-carbon in ethyl acetate to afford the
corresponding free carboxylic acid 18 in 68% yield without the
loss of the Boc protecting groups.
Scheme 5
The tri-Boc derivative 18 was condensed with benzyl (2S,3S,
4R)-4-amino-3-hydroxy-2-methylvalerate (8) using DCC-HOBt
in the presence of N,N-diisopropylethylamine at 25 °C for 3 h
(Scheme 4). Dipeptide disaccharide 19 was obtained as a
Scheme 6
colorless foam in 77% yield ([R]24 -12.2° (c 2.3, EtOH)).
D
Benzyl ester 19 was deesterified by hydrogenolysis over
commercially available palladium black at 50-53 °C for 20 h.
The Nim-Boc protecting group was also solvolyzed under these
conditions, affording compound 20. An attempt to purify this
compound by simple extractive procedures under neutral
conditions resulted in conversion to compound 21, in which
the acetyl group of the O-carbamoyl substituent had been lost,
as judged by NMR spectroscopy. Also formed to some extent
was dipeptide disaccharide derivative 22, in which the O-
carbamoyl group itself underwent solvolysis. The mixture of
N-Boc carbamoyl protected 21 and 22 (the latter of which is a
precursor of decarbamoyl BLM) was used for the next coupling
step without further attempts at purification. As shown in
Scheme 5, condensation of 21 and threonylbithiazole 9 was
carried out at 25 °C for 20 h in dry DMF in the presence of
DCC-HOBt. Pentapeptide disaccharide 23 was obtained as a
Final coupling of pentapeptide disaccharide 24 with NR-t-
Boc-pyrimidoblamic acid (10)36 was conducted at 25 °C for 2
days using DCC-HOBt in anhydrous DMF (Scheme 6). The
resulting product (25) was deprotected with a 1:1 mixture of 2
N aqueous NH4OH-methanol at 25 °C for 4.5 h (to remove
the acetyl groups), followed by treatment with trifluoroacetic
acid-dimethyl sulfide at 0 °C for 75 min (to effect removal of
the N-t-Boc protecting group).
1
colorless glass in 68% yield and characterized by H NMR
spectroscopy. The Boc protecting group was removed by
treatment with trifluoroacetic acid in the presence of dimethyl
sulfide, affording 24 in 90% yield as a glass.
Crude bleomycin demethyl A2 (2), putatively containing
decarbamoyl bleomycin demethyl A2 as a consequence of
adventitious solvolysis of the carbamoyl group (vide supra), was
initially purified by chromatography on an Amberlite XAD-2
column, followed by chromatographic fractionation of the Cu-
(36) Aoyagi, Y.; Chorghade, M. S.; Padmapriya, A. A.; Suguna, H.;
Hecht, S. M. J. Org. Chem. 1990, 55, 6291.
(37) (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) Boger, D. L.; Honda, T.; Dang, Q. J. Am. Chem. Soc.
1994, 116, 5619.

Total Synthesis of Bleomycin
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11289
(II) complex on a CM Sephadex C-25 column. The first peak
to elute was treated with EDTA disodium salt to remove Cu-
(II), and then purified on an Amberlite XAD-2 column, affording
pure bleomycin demethyl A2 (2) as a colorless powder. The
purified product 2 was found to have chromatographic properties
identical with those of authentic bleomycin demethyl A2 on CM-
Sephadex C-25 and silica gel TLC in several solvent systems.
This compound also had the same retention time as authentic
BLM demethyl A2 on C18 reversed phase HPLC. The structure
of synthetic BLM demethyl A2 was also confirmed by high-
resolution FAB mass spectrometry and by its 1H NMR spectrum,
which was identical with that of an authentic sample.
Scheme 7
The second peak to elute from the CM Sephadex C-25 was
also treated with EDTA to remove Cu(II) and then purified on
an XAD-2 column to afford decarbamoyl bleomycin demethyl
A2 (26). Decarbamoyl BLM demethyl A2 was obtained as a
as a metal ligand since it can be compared functionally with
the corresponding BLM derivative itself. In fact, Sugiyama et
al.18c demonstrated that in the absence of the carbamoyl group
there was a dramatic change in the strand selectivity of DNA
oligonucleotide degradation by Fe(II)‚BLM. More recently, it
has been shown that the absence of the carbamoyl group
produced fundamental changes in the spectral properties of Fe-
(II)‚BLM, again suggesting an important role for the carbamoyl
moiety in metal binding by BLM.44
Despite the synthesis of numerous analogues of BLM, to date
there has been no report on the synthesis of decarbamoyl BLM.
This species has been accessible in low yield exclusively by
treatment of BLM under basic aqueous conditions over a period
of several months.45 The sample of decarbamoyl BLM dem-
ethyl A2 prepared in this study was shown to be identical in
structure and behavior with a sample derived from BLM
demethyl A2 by base treatment, and also with an authentic
synthetic sample prepared recently by an unambiguous total
synthesis.38
Synthesis of Bleomycin without Protection of the Car-
bamoyl Group. Because the N-protected O-carbamoyl group
employed in our original synthesis of bleomycin proved to be
unstable hydrolytically during routine manipulations, we ex-
plored another route for the synthesis of bleomycin. This route,
which is outlined in Schemes 7-9, incorporated a few important
modifications, including (i) the use of an unprotected carbamoyl
group throughout the entire synthesis, (ii) successful retention
of the Nim-t-Boc protecting group until its intentional removal
prior to the last peptide coupling, and (iii) the use of freshly
prepared palladium black which permitted the clean removal
of all benzyl groups in a single step. As a consequence of these
changes, the intermediates obtained could be purified readily
and exhibited excellent spectral characteristics.
colorless glass. Although this BLM derivative has not been
reported previously, it was shown to be identical with an
authentic sample38 on silica gel TLC using several solvent
systems.
Synthetic BLM demethyl A2 (2) was converted to BLM A2
(1) by methylation of its Cu(II) chelate, as described.7,39 The
sample of 1 so obtained was identical in all respects with
naturally derived bleomycin A2.
Synthesis of Decarbamoyl BLM Demethyl A2. Although
obtained here as a byproduct during our initial synthesis of BLM
demethyl A2, the synthesis of decarbamoyl BLM demethyl A2
represents a particularly important achievement. The carbamoyl
group has been postulated to function as a metal ligand for at
least three different metal ions that bind to BLM. This includes
the Fe(II)‚CO complex of BLM,40 for which two groups have
assigned the role of ligand to this group. It has also been
suggested that Zn(II)‚BLM41 and Co(II)‚BLM42 complexes
employ the carbamoyl moiety as metal ligands, although
alternative ligands have been suggested as well.43
Decarbamoyl BLM represents a particularly valuable species
for determining the possible participation of the carbamoyl group
(38) An, H.-Y.; Katano, K.; Padmapriya, A. A.; Hess, C. D.; Hecht, S.
M. Manuscript in preparation.
(39) Roy, S. N.; Orr, G. A.; Brewer, C. F.; Horwitz, S. B. Cancer Res.
1981, 41, 4471.
(40) (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.
(41) (a) Akkerman, M. A. J.; Haasnoot, C. A. G.; Hilbers, C. W. Eur. J.
Biochem. 1988, 173, 21l. (b) Manderville, R. A.; Ellena, J. F., Hecht, S.
M. J. Am. Chem. Soc. 1994, 116, 10851. (c) Manderville, R. A.; Ellena, J.
F.; Hecht, S. M. J. Am. Chem. Soc. 1995, 117, 7891.
(42) Cortes, J. C.; Sugiyama, H.; Ikudome, K.; Saito, I.; Wang, A. H.-J.
Eur. J. Biochem. 1997, 224, 818.
(43) See, e.g. (a) Stubbe, J.; Kozarich, J. W.; Wu, W.; Vanderwall, D.
E. Acc. Chem. Res. 1996, 29, 322 and references therein. (b) Calafat, A.
M.; Won, H.; Marzilli, L. G. J. Am. Chem. Soc. 1997, 119, 3656.
Initially, disaccharide 5, having an unprotected O-carbamoyl
group, was converted to the respective gulopyranosyl chloride
6 in quantitative yield by the use of gaseous hydrogen chloride
in CH2Cl2 (Scheme 7). Derived chloride 6 was condensed with
benzyl NR,Nim-di-t-Boc-(S)-erythro-â-hydroxyhistidine (7) at 48
(44) Loeb, K. E.; Zaleski, J. E.; Hess, C. D.; Hecht, S. M.; Solomon, E.
I. J. Am. Chem. Soc. 1998, 120, 1249.
(45) Naganawa, H.; Muraoka, Y.; Takita, T.; Umezawa, H. J. Antibiot.
1977, 30, 388.

11290 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
Scheme 8
Scheme 9
confirmed by 13C NMR spectroscopy, and by low- and high-
resolution FAB mass spectrometry. Dipeptide disaccharide 31
was coupled with threonylbithiazole 9 at 25 °C for 24 h using
DCC-HOBt in the presence of N,N-diisopropylethylamine in
anhydrous DMF. Di-Boc pentapeptide 32 was obtained as a
colorless foam in 59% yield and was characterized fully. The
Boc protecting groups on 32 were removed using trifluoroacetic
acid at 0 °C for 3 h to provide pentapeptide disaccharide 24 as
the free amine in 87% yield after chromatographic purification
on an Amberlite XAD-2 column. This intermediate was also
°C for 22 h by using silver trifluoromethanesulfonate as the
catalyst. Chromatographic separation of the reaction mixture
on a silica gel flash column gave the desired di-Boc disaccharide
27 in 12% yield, as well as mono-Boc disaccharide 28 (11%
yield). The conversion of 28 to the corresponding di-Boc
derivative 27 was effected by treatment of 28 with di-tert-butyl
dicarbonate at 25 °C for 1 h; this limited Boc substitution
exclusively to the Nim nitrogen, instead of both Nim and
carbamoyl nitrogen atoms (cf. 16 f 17, Scheme 3). Compound
27 obtained in this fashion had exactly the same spectroscopic
properties as compound 27 isolated directly from the coupling
of 6 and 7. Compounds 27 and 28 were characterized by their
1H NMR and high-resolution FAB mass spectra. Compound
27 was then debenzylated selectively by hydrogenation over
10% palladium-on-carbon at 25 °C for 20 h. The pure
carboxylic acid 29 was obtained as a colorless foam character-
ized by its 1H and 13C NMR spectra as well as a molecular ion
in the high-resolution FAB mass spectrum.
1
characterized by H NMR spectroscopy and high-resolution
FAB mass spectrometry. The final coupling of 24 with NR-t-
Boc-pyrimidoblamic acid (10)36 was effected by the use of
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluo-
rophosphate48 (Scheme 9). The reaction was conducted in
anhydrous DMF in the presence of N,N-diisopropylethylamine
at 25 °C for 28 h. The condensation product 25 so obtained
was the same as compound 25 obtained by the use of
N-protected carbamoyl intermediates (cf. Schemes 6 and 9).
However, the material obtained by the latter route was a single
compound. After extractive workup, crude 25 was deacetylated
directly with a 1:1 mixture of 2 N aqueous NH4OH-methanol
at 25 °C for 3.5 h. The Boc-protected intermediate was treated
further with trifluoroacetic acid in dimethyl sulfide at 0 °C for
1.5 h to remove the Boc protecting group. Crude bleomycin
demethyl A2 (2) was converted to its Cu(II) complex, which
was purified by chromatography on a CM Sephadex C-25
column. The purified Cu(II) complex of bleomycin demethyl
A2 (2) was decomplexed with Na EDTA and purified by flash
chromatography on a reversed-phase (C18) column. Compound
The condensation of carboxylic acid 29 with the hydrochlo-
ride of benzyl (2S,3S,4R)-4-amino-3-hydroxy-2-methylvalerate
(8) was effected by the use of DCC-HOBt in the presence of
N,N-diisopropylethylamine at 25 °C for 5 h (Scheme 8). The
corresponding benzyl ester 30 was obtained as a colorless foam
1
in 70% yield and was characterized by its H and 13C NMR
spectra as well as its high-resolution FAB mass spectrum. To
debenzylate 30 effectively, the palladium black catalyst was
freshly made according to the reported procedure46 with some
improvement. In particular, freshly prepared palladium black
had to be washed thoroughly with water and then anhydrous
methanol to preclude the solvolysis of the Nim-Boc protecting
group.47 Benzyl ester 30 was hydrogenated over the freshly
prepared palladium black in absolute ethanol under 1 atm of
hydrogen at 25 °C for 40 h. The completely debenzylated
product (31) was obtained as a colorless foam in 52% yield.
1H NMR spectroscopy confirmed that the two benzyl groups
on gulose had been removed, and that no solvolysis of either
Boc group had occurred. The structure of 31 was also
2, [R]25 ) 17.1° (c 0.14, H2O), was obtained as a colorless
D
foam in 23% overall yield from 24. The purified product had
chromatographic properties identical with those of an authentic,
naturally derived sample, as well as with those of BLM demethyl
A2 (2) obtained by the protected carbamoyl route. The structure
of bleomycin demethyl A2 was further confirmed by its 1H NMR
spectrum, which was identical with that of an authentic sample,
and by its high-resolution FAB mass spectrum. Intermediates
27-32 and 25 were all obtained in a good state of purity after
chromatographic separation, as judged by HPLC analysis.
1
These compounds were characterized by their H NMR and
high-resolution FAB mass spectral analyses. Compounds 29
and 30 were also characterized by their 13C NMR spectra. The
(46) Felix, A. M.; Jimenez, M. H.; Meienhofer, J.; Revesz, L.; Bu¨chi,
G. Org. Synth. 1979, 59, 159.
(47) The catalyst should be saturated with solvent at all times; the dry
catalyst is pyrophoric.
(48) (a) Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron Lett.
1975, 1219. (b) Castro, B.; Dormoy, J. R.; Dourtoglou, B.; Evin, G.; Selve,
C.; Ziegler, J. C. Synthesis 1976, 751. (c) Castro, B.; Evin, G.; Selve, C.;
Seyer, R. Synthesis 1977, 413.

Total Synthesis of Bleomycin
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11291
Scheme 11
Scheme 10
reprotection with N-(9-fluorenylmethoxycarbonyl)succinimide51
(67% overall yield) (Scheme 10).
absence of racemization during the peptide couplings was
verified both by detailed NMR spectral analysis and by
hydrolysis of key intermediates following peptide coupling and
analysis of the recovered fragments for loss of optical integrity.
In addition to the syntheses of BLM demethyl A2, BLM A2,
and decarbamoyl BLM demethyl A2 reported here, the methods
outlined here have been applied successfully for the synthesis
of a number of novel BLM analogues.21,38,49
Optimization of the Synthesis of the Carbohydrate Moiety
of Bleomycin. While the route employed for the synthesis of
bleomycin without protection of the carbamoyl group is entirely
workable, facets of the overall scheme that may be considered
less than optimal include the low yield associated with the
introduction of â-hydroxyhistidine (i.e., 6 + 7 f 27, Scheme
7) and the partial loss of the imidazole protecting group in the
same transformation, as well as the occasional difficulty in
effecting the debenzylation of intermediate 30 (Scheme 8).
Further, although the glycosylated intermediates isolated were
single isomers having the anomeric configurations indicated in
each case, it was not possible to analyze the anomeric composi-
tion of the products in most cases. Accordingly, we have
investigated alternative synthetic approaches.
As outlined in Scheme 10, the difficulty in removing the
benzyl protecting groups from advanced synthetic intermediates
can be obviated by replacing the benzyl groups prior to
introduction of the â-hydroxyhistidine moiety. Because the
additional acetyl groups introduced into the disaccharide
deactivate the anomeric center, the anomeric center must be
converted to an intermediate more reactive than the species
accessible by treatment of a glycosyl chloride with AgOTf.
Accordingly, dibenzyl disaccharide 5 was converted to per-
acetylated disaccharide 33 in essentially quantitative yield by
debenzylation over 20% palladium-on-carbon followed by
acetylation.9 Removal of the anomeric acetate (33 f 34)
followed by conversion to the diphenyl phosphate derivative
(34 f 35) also followed literature precedent9 and proceeded in
80% overall yield. As an alternative, the anomeric center in
33 was also converted directly to the bromide (33 f 36) in
93% yield via the agency of 30% HBr in HOAc.
Coupling of glycosyl disaccharide 35 with di-Fmoc â-hy-
droxyhistidine derivative 37 in 2:1 ether-CH2Cl2 in the presence
of TMSOTf gave the desired product 38 after 20 min at 0 °C.
Compound 38 was obtained as an 8:1 mixture of R and â
anomers from which the desired R anomer was isolated in 58%
yield by preparative silica gel TLC (Scheme 11). Compound
38 was deblocked with piperidine at 25 °C and then converted
to the di-Boc derivative by treatment with di-tert-butyl dicar-
bonate.
Compound 39 was isolated as a foam in 70% yield and
converted promptly to the corresponding carboxylic acid 40 by
hydrogenolysis over 10% palladium-on-carbon (99% yield).
Compound 40 was also accessible in a more direct fashion
via the coupling of glycosyl bromide 36 with di-Boc â-hy-
droxyhistidine derivative 7 in the presence of tetramethylurea
(28% conversion; 54% yield based on consumed 7). Because
this transformation was carried out at a lower temperature over
a shorter period of time (40 °C for 14 h) than the analogous
coupling of 6 and 7 (48 °C for 22 h) (cf. Schemes 7 and 11),
loss of the Nim protecting group was not an important side
reaction. While the overall yields of 40 accessible from 35 and
36 are comparable, the route involving glycosyl bromide 36 is
shorter and more convenient operationally and has been used
successfully to prepare quantities of intermediates for the
elaboration of bleomycin congeners.
Also explored with quite favorable results was application
of the carbohydrate trichloroacetimidate intermediates pioneered
by Schmidt52 for the elaboration of the carbohydrate moiety of
bleomycin (Scheme 12). Accordingly, key saccharide inter-
mediates 1,2,4,6-tetra-O-acetyl-3-O-(p-nitrophenoxycarbonyl)-
R-D-mannopyranose (41)29c and 3,4-di-O-benzyl-1,6-O-diacetyl-
â-L-gulopyranose (44)29b were prepared as described previously.
Treatment of saccharide 41 with anhydrous ammonia afforded
the 3-O-carbamoylated product after 1 h; deacetylation of the
anomeric position occurred after extended reaction to afford
2,4,6-tri-O-acetyl-3-O-carbamoyl-R-D-mannopyranose (42) cleanly
in 74% yield. The Schmidt trichloroacetimidate 43 was
prepared essentially quantitatively from saccharide 42 using
trichloroacetonitrile in the presence of anhydrous potassium
carbonate; the product was predominantly the â-imidate.52
Treatment of glycosyl acceptor 4429b with imidate 43 in the
presence of 5 mole percent of BF3‚OEt2 provided the 1,2-trans-
linked disaccharide 5 in 86% yield as a >10:1 mixture of R
To circumvent the difficulty encountered with loss of the Nim
protecting group during introduction of the â-OH histidine
moiety, the latter was protected as the di-Fmoc derivative 37.
The Fmoc group is generally easily removed with amine bases,
but is stable under acidic conditions.50 Compound 37 was
readily accessible by deprotection of 7 (CF3COOH) and
(50) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1980, 45, 72.
(51) Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis
1983, 671.
(52) Schmidt, R. R. Angew Chem., Int. Ed. Engl. 1986, 25, 212.
(53) Sakai, T. T.; Riordan, J. M.; Glickson, J. D. Biochim. Biophys. Acta
1983, 758, 176.
(49) (a) Carter, B. J.; Murty, V. S.; Reddy, K. S.; Wang, S.-N.; Hecht,
S. M. J. Biol. Chem. 1990, 265, 4193. (b) Kane, S. A.; Sasaki, H.; Hecht,
S. M. J. Am. Chem. Soc. 1995, 117, 9107. (c) Zuber, G.; Quada, Jr., J. C.;
Hecht, S. M. J. Am. Chem. Soc. 1998, 120, 9368.

11292 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
Scheme 12
to essentially the same extent when incubated in the presence
of equimolar Fe2+. As reported previously,26a,b both samples
of 2 were less potent than Fe(II)‚BLM A2 in mediating DNA
cleavage. Also studied was the sequence selectivity of DNA
cleavage by synthetic bleomycin demethyl A2. As shown in
Figure 1 (Supporting Information), incubation of a 158-base
pair 3′-32P-end-labeled DNA duplex with Fe(II)‚BLM demethyl
A2 afforded the same pattern of cleavage as that obtained with
Fe(II)‚BLM A2 itself. The essentially identical patterns of
cleavage, as well as the lesser potency of BLM demethyl A2,
were both as anticipated on the basis of literature precedent.26a,b,53
Also apparent from the better resolved bands on the polyacry-
lamide gel was the appearance of products having both
3′-phosphate and 3′-phosphoroglycolate termini.10,11 This find-
ing confirms that synthetic BLM demethyl A2 also mediates
the same chemical transformations of DNA noted for naturally
derived bleomycins. Although the syntheses of naturally
occurring BLMs have been reported previously, this represents
the first confirmation of the nature of the actual chemistry of
DNA degradation mediated by a synthetic BLM.
Experimental Section
General Methods. NMR chemical shifts are referenced to CHCl3
at 7.24 ppm, CH3OH at 3.30 ppm, or HOD at 4.80 ppm. The following
abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad; q, quartet; br s, broad singlet; br d, broad doublet; dd, doublet
of doublets; dq, doublet of quartets. Optical rotation data are reported
as [R]Dtemp (concentration in g/100 mL of solvent). Chemical ionization
(CI) mass spectra were recorded using isobutane. Some of the high-
resolution data were obtained with the assistance of the Michigan State
University Mass Spectrometry Facility (supported by NIH/NCRR Grant
RR0480) or the Washington University Mass Spectrometry Resource,
supported by the NIH/NCRR (Grant RR0954). All organic chemicals,
as well as palladium black, were purchased from Aldrich Chemical
Co., as were all deuterated NMR solvents. Pyridine was heated at reflux
for 3 h over KOH and then distilled prior to use.
Ferrous ammonium sulfate [FeII(NH4)2(SO4)2] was purchased from
EM Science. Agarose was obtained from Bethesda Resarch Labora-
tories. Acrylamide, N,N′-methylenebisacrylamide, ethidium bromide,
piperidine, cacodylic acid, Trizma base, bromophenol blue, and herring
sperm DNA (carrier for Maxam-Gilbert sequencing reactions) were
purchased from Sigma Chemicals. The disodium salt of (ethylene-
dinitrilo)tetraacetic acid (EDTA) was purchased from J. T. Baker, and
xylene cyanol was from Bio-rad Laboratories. Endonuclease HindIII
was purchased from Promega; EcoRV was from New England BioLabs.
Alkaline phosphatase, Sephadex G-50 spin columns, and plasmid
pBR322 were obtained from Boehringer Mannheim Biochemicals. T4
Polynucleotide kinase was purchased from United States Biochemicals.
[γ-32P]ATP (7000 Ci/mmol) was from ICN Radiochemicals.
Coupling of Pentapeptide Disaccharide 24 with N-t-Boc-pyrimi-
doblamic Acid (10). Deprotection and Purification of Bleomycin
Demethyl A2 (2) and Decarbamoyl Bleomycin Demethyl A2 (26).
To a solution containing 29 mg (23 µmol) of N-t-Boc-pyrimidoblamic
acid (10)36,37 in 0.3 mL of anhydrous DMF were added successively 5
mg (39 µmol) of N,N-diisopropylethylamine, 3.5 mg (26 µmol) of
1-hydroxybenzotriazole, and 6 mg (26 µmol) of N,N′-dicyclohexyl-
carbodiimide (DCC). The reaction mixture was stirred at 25 °C for 2
days. The solvent was concentrated under diminished pressure, and
the residue was partitioned between water and ethyl acetate. The
aqueous phase was extracted with ethyl acetate, and the combined
organic phase was back-extracted with water. The combined aqueous
phase was concentrated to dryness under diminished pressure to afford
the crude coupled product (25).
Figure 3. Relaxation of pBR322 Form I DNA by BLM congeners:
lane 1, DNA alone; lane 2, 1 µM Fe(II)‚BLM A2; lane 3, 5 µM Fe-
(II)‚BLM A2; lane 4, 1 µM Fe(II)‚BLM demethyl A2; lane 5, 5 µM
Fe(II)‚BLM demethyl A2; lane 6, 1 µM synthetic Fe(II)‚BLM demethyl
A2; lane 7, 5 µM synthetic Fe(II)‚BLM demethyl A2; lane 8, 5 µM
BLM A2; lane 9, 5 µM BLM demethyl A2; lane 10, 5 µM Fe2+
.
and â anomers, respectively. Disaccharide 5 was then deacy-
lated at the anomeric position (C6H5CH2NH2, 25 °C, 72%) and
activated as trichloroacetimidate 46 in 97% yield.52
To prepare glycosylated NR,Nim-bis(t-Boc)-(S)-erythro-â-
hydroxyhistidine benzyl ester (27), a key intermediate in the
synthesis of BLM A2 (cf. Scheme 7), imidate 46 was used to
glycosylate NR,Nim-bis(Fmoc)-(S)-erythro-â-hydroxyhistidine
benzyl ester (37). The R glycoside was formed using TMSOTf
under thermodynamic conditions.52 Glycosylated NR,Nim-bis-
(Fmoc)-(S)-erythro-â-hydroxyhistidine benzyl ester (47), ob-
tained in 44% yield as a colorless glass, was deprotected with
piperidine and refunctionalized using di-tert-butyl dicarbonate
to provide key intermediate 27.
Biochemical Characterization of Synthetic BLM Demethyl
A2. Synthetic BLM demethyl A2 was also characterized for its
ability to mediate the cleavage of DNA. As shown in Figure
3, synthetic and naturally derived samples of bleomycin
demethyl A2 effected the relaxation of supercoiled plasmid DNA
Crude 25 was treated with 2 mL of 2 N ammonium hydroxide and
2 mL of methanol at 25 °C for 4.5 h to remove the acetyl groups.
After concentration of the solution, the residue was treated with 1 mL
of 5:3 trifluoroacetic acid-dimethyl sulfide at 0 °C for 75 min to
remove the Boc protecting group. The solvent was concentrated, and

Total Synthesis of Bleomycin
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11293
the residue was dissolved in 5 mL of water. The aqueous solution
was neutralized (to pH 7.3) with aqueous sodium bicarbonate solution
and applied to an Amberlite XAD-2 column (5 × 1.4 cm). The column
was washed with 30 mL of water and then with 100 mL of methanol.
The methanol eluate was concentrated under diminished pressure to
afford crude bleomycin demethyl A2.
The synthesized Cu(II)‚BLM A2 was dissolved in 15% aqueous
EDTA solution and stirred at 25 °C for 12 h. The mixture was applied
to a C18 open column (7 × 1 cm) and washed with 250 mL of deionized
water. Then BLM A2 was eluted from the column with 4:1 MeOH-2
mM HCl. After concentration of the eluate, BLM A2 was purified
further by reversed-phase HPLC on an Alltima C18 column (250 × 4.6
mm) using a linear gradient of 0.1 M NH4OAc, pH 4.5, containing
increasing amounts of CH3CN (0 f 30 min, linear gradient from 5 to
12% CH3CN in NH4OAc) at a flow rate of 1.5 mL/min. The eluate
was monitored by 290 nm; the product eluted at 28.8 min. Concentra-
tion of the eluate under diminished pressure to a small volume, followed
by lyophilization, afforded BLM A2 as a colorless solid: yield 0.38
mg (61% from Cu(II)‚BLM A2); 1H NMR (D2O) δ 1.00-1.10 (m, 9H),
1.92 (s, 3H), 2.10 (m, 1H), 2.40 (m, 1H), 2.50-2.70 (m, 4H), 2.83 (s,
6H), 2.91 (m, 1H), 3.18 (m, 1H), 3.25-4.04 (m, 23H), 4.12 (d, 1H, J
) 5 Hz) 4.65 (m, 1H), 4.85 (br s, 1H), 5.00 (d, 1H, J ) 2 Hz), 5.20
(m, 2H), 7.20 (s, 1H), 7.78 (s, 1H), 7.95 (s, 1H), and 8.15 (s, 1H);
mass spectrum (FAB), m/z 1414.5 (M+); mass spectrum (FAB), m/z
1414.523 (M+) (C55H84N17O4S3 requires 1414.519) and 1415.530 (M
+ H)+ (C55H85N17O4S3 requires 1414.527).
Coupling of Disaccharide Chloride 6 with Benzyl Nr,Nim-Bis-
(tert-butoxycarbonyl)-(S)-erythro-â-hydroxyhistidine (7). â-Hydrox-
yhistidine benzyl ester derivative 7 (1.0 g, 2.16 mmol) and 855 mg
(3.32 mmol) of silver trifluoromethanesulfonate were dried overnight
under vacuum and dissolved in 3 mL of anhydrous 1,2-dichloroethane
and 0.5 mL (0.48 g, 4.1 mmol) of 1,1,3,3-tetramethylurea. A solution
of 2.27 g (3.02 mmol) of disaccharide chloride 6 in 10 mL of anhydrous
1,2-dichloroethane was added dropwise to this solution at 0 °C. The
reaction mixture was stirred at 48 °C for 22 h. To the cooled reaction
mixture was added 100 mL of brine. The well-shaken mixture was
diluted with 400 mL of ethyl acetate, filtered through a bed of Celite,
and washed with ethyl acetate. The organic phase was washed with
saturated sodium bicarbonate and water. The dried (MgSO4) solution
was filtered, and the filtrate was concentrated. The residue was purified
by flash chromatography on a silica gel column (30 × 2 cm). Elution
with 1:2 toluene-ethyl acetate afforded the di-Boc-protected disac-
The crude product and 4 mg of CuSO4 were dissolved in 1 mL of
0.05 M ammonium formate. The dark blue solution was applied to a
CM-Sephadex C-25 column (75 × 1 cm, buffered with 0.05 M
ammonium formate). After washing with 35 mL of 0.05 M ammonium
formate, the column was washed with a linear gradient of 0.05-0.5 M
ammonium formate. The fractions were monitored by UV absorbance
at 292 nm. The first product peak was collected, treated with 350 mg
of EDTA disodium salt for 30 min, and then applied to an Amberlite
XAD-2 column (5 × 1.4 cm). After washing successively with 20
mL of water, 30 mL of 50% sodium chloride, and 30 mL of water, the
product was eluted with 50 mL of 4:1 methanol-0.002 N hydrochloric
acid. The solvent was concentrated, and the residue was dissolved in
a small amount of water. The frozen aqueous solution was lyophilized
to give bleomycin demethyl A2 (2) as a colorless powder: yield 1.1
mg (3.4% from 24); silica gel TLC Rf 0.63 (1:2 10% NH4OAc-EtOH);
Rf 0.47 (1:3 10% NH4OAc-EtOH); 1H NMR (D2O) δ 1.06 (d, 3 H, J
) 6 Hz), 1.10 (d, 3 H, J ) 7 Hz), 1.13 (d, 3 H, J ) 7 Hz), 1.87-1.94
(m, 2 H), 1.97 (s, 3 H), 2.07 (s, 3 H), 2.53-2.60 (m, 4 H), 2.67 (s, 1
H), 2.72-2.86 (m, 2 H), 3.15-3.30 (m, 4 H), 3.44-3.52 (m, 2 H),
3.54-3.63 (m 3 H), 3.65-3.83 (m, 6 H), 3.87-3.92 (m, 2 H), 3.97-
4.06 (m, 4 H), 4.11-4.15 (m, 1 H), 4.17-4.24 (m, 2 H), 4.99 (s, 1 H),
5.06 (d, 1 H, J ) 6.5 Hz), 5.22 (d, 1 H, J ) 3.5 Hz), 5.45 (d, 1 H, J
) 6.5 Hz), 7.57 (s, 1 H), 8.00 (s, 1 H), 8.14 (s, 1 H), and 8.76 (s, 1 H);
mass spectrum (FAB), m/z 1400 (M + H)+; mass spectrum (FAB),
m/z 1400.497 (M + H)+ (C54H82N17O21S3 requires 1400.503).
A second peak from the Sephadex column was collected and treated
with 400 mg of EDTA disodium salt. This solution was also purified
by Amberlite XAD-2 column chromatography as described above to
give decarbamoyl bleomycin demethyl A2 (26) as a colorless powder:
yield 2.7 mg (8.6%); silica gel TLC Rf 0.33 (1:2 10% NH4OAc-EtOH);
Rf 0.26 (1:3 10% NH4OAc-EtOH); 1H NMR (D2O) δ 0.95 (d, 3 H, J
) 7 Hz), 1.03 (d, 3 H, J ) 7 Hz), 1.07 (d, 3 H, J ) 7 Hz), 1.92 (s, 3
H), 2.08 (m, 2 H), 2.67 (q, 1 H, J ) 7 Hz), 2.70 (s, 3 H), 2.67-2.92
(m, 4 H), 3.15 (m, 3 H), 3.19 (s, 3 H), 3.25 (m, 1 H), 3.35-3.58 (m,
6 H), 3.60 (t, 2 H, J ) 6 Hz), 3.75 (m, 2 H), 3.75 (s, 1 H), 3.85 (m,
2 H), 3.80-4.00 (m, 4 H), 4.10 (d, 1 H, J ) 6 Hz), 4.18 (m, 2 H), 4.97
(s, 1 H), 5.09 (d, 1 H, J ) 9 Hz), 5.18 (d, 1 H, J ) 2 Hz), 5.29 (d, 1
H, J ) 9 Hz), 7.60 (s, 1 H), 8.05 (s, 1 H), 8.18 (s, 1 H), and 8.77 (s,
1 H). This material was identical in all respects with an authentic
sample of decarbamoyl bleomycin demethyl A2.38
Cu(II)‚BLM Demethyl A2. A solution of 1.0 mg (7.1 µmol) of
BLM demethyl A2 in 120 µL of 50 mM citrate buffer, pH 4.7, was
treated with 1.2 mg (7.3 µmol, 1.1 equiv) of CuSO4. The solution
was maintained at 0-4 °C for 30 min, and the resulting blue solution
was purified by reversed-phase HPLC on an Alltima C18 column (250
× 4.6 mm) using a linear gradient of 0.1 M NH4OAc, pH 5.5, containing
increasing amounts of CH3CN (0 f 25 min, linear gradient from 0 to
88% CH3CN in aqueous NH4OAc) at a flow rate of 1.0 mL/min. The
eluate was monitored at 290 nm; the product eluted at 13.3 min.
Concentration of the eluate under diminished pressure provided Cu-
(II)‚BLM demethyl A2 as a blue powder: yield 0.75 mg (73%).
Bleomycin A2 (1). A solution of 0.75 mg (5.1 µmol) of Cu‚BLM
demethyl A2 in 200 µL of dry MeOH under Ar was treated with 30 µL
(510 µmol) of CH3I. The reaction mixture was stirred in the dark at
room temperature. The reaction was monitored by reversed-phase
HPLC on an Alltima C18 column (250 × 4.6 mm) using a linear gradient
of 0.1 M NH4OAc, pH 5.5, containing increasing amounts of CH3CN
(0 f 25 min, linear gradient from 0 to 88% CH3CN in NH4OAc) at a
flow rate of 1.0 mL/min. The eluate was monitored at 290 nm. After
6 days, the reaction was complete. After the reaction mixture was
concentrated under diminished pressure, crude Cu(II)‚BLM A2 was
isolated by preparative reversed-phase HPLC using the same mobile
phase described above. The product eluted at 11.1 min. Concentration
of the eluate afforded Cu(II)‚BLM A2 as a blue powder: yield 0.55
mg (76%).
charide 27 as a colorless foam: yield 308 mg (12%); silica gel TLC Rf
1
0.33 (10:1 chloroform-methanol); [R]25 -19.0° (c 0.8, CHCl3); H
D
NMR (CDCl3) δ 1.39 (s, 9 H), 1.60 (s, 9 H), 1.84 (s, 3 H), 2.00 (s, 3
H), 2.02 (s, 3 H), 2.10 (s, 3 H), 3.67 (m, 1 H), 3.86 (m, 1 H), 3.99 (m,
1 H), 4.04 (m, 1 H), 4.10 (m, 1 H), 4.30 (m, 2 H), 4.41 (m, 1 H), 4.53
(m, 1 H), 4.61 (m, 1 H), 4.66 (m, 2 H), 4.81 (m, 1 H), 4.90 (m, 1 H),
4.99 (m, 1 H), 5.07 (m, 1 H), 5.16 (m, 1 H), 5.21 (m, 1 H), 5.25 (m,
1 H) 5.39 (m, 1 H), 6.42 (br d, 1 H), 7.12-7.40 (m, 16 H), and 7.92
(s, 1 H); mass spectrum (FAB), m/z 1177 (M + H)+; mass spectrum
(FAB), m/z 1177.467 (M)+ (C58H73N4O22 requires 1177.471).
The flash silica gel column was further eluted with 20:1 chloroform-
methanol, affording mono-Boc-protected disaccharide 28 as a pale
yellow foam: yield 252 mg (11%); silica gel TLC Rf 0.20 (10:1 CHCl3-
1
CH3OH); H NMR (CDCl3) δ 1.37 (s, 9 H), 1.86 (s, 3 H), 2.00 (s, 3
H), 2.04 (s, 3 H), 2.10 (s, 3 H), 3.67 (m, 1 H), 3.86 (m, 1 H), 3.99 (m,
1 H), 4.04 (m, 1 H), 4.10 (m, 1 H), 4.30 (m, 2 H), 4.41 (m, 1 H), 4.53
(m, 1 H), 4.61 (m, 1 H), 4.66 (m, 2 H), 4.81 (m, 1 H), 4.90 (m, 1 H),
4.99 (m, 1 H), 5.07 (m, 1 H), 5.16 (m, 1 H), 5.21 (m, 1 H), 5.25 (m,
1 H), 5.39 (m, 1 H), 6.71 (s, 1 H), 7.10-7.40 (m, 16 H), and 7.92 (s,
1 H); mass spectrum (FAB), m/z 1077.418 (M + H)+ (C53H65N4O20
requires 1077.419).
Reprotection of Mono-Boc-Protected Disaccharide 28. Com-
pound 28 (252 mg, 0.234 mmol) in 2 mL of dry pyridine was treated
with 140 mg (0.64 mmol) of di-tert-butyl dicarbonate at 25 °C for 1 h.
The solution was concentrated under diminished pressure with co-
evaporation of portions of toluene; the foamy residue was purified by
flash chromatography on a silica gel column (20 × 2 cm). Elution
with 1:2 toluene-ethyl acetate gave compound 27 as a white foam:
yield 177 mg (64%). The di-Boc product obtained in this fashion was
identical to the compound described above, as judged by silica gel TLC
1
and H NMR spectroscopy.

11294 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
Selective Debenzylation of Disaccharide Benzyl Ester 27.
A
chloroform-methanol); [R]
+11.8° (c 0.5, methanol); 1H NMR
25
D
mixture of 480 mg (0.408 mmol) of disaccharide benzyl ester 27 and
0.11 g of 10% palladium-on-carbon in 10 mL of absolute ethanol was
stirred at 25 °C under 1 atm of hydrogen for 20 h. The catalyst was
filtered through Celite and washed with ethanol. The filtrate was
concentrated under diminished pressure, and the residue was purified
by flash chromatography on a silica gel column (20 × 2 cm). Elution
with 1:2 toluene-ethyl acetate, and then 15:1 and finally 10:1
chloroform-methanol, afforded disaccharide derivative 29 as a colorless
foam: yield 241 mg (54%); silica gel TLC Rf 0.41 (5:1 chloroform-
methanol); [R] D25 +0.33° (c 1.2, CHCl3); 1H NMR (CDCl3) δ 1.39 (s,
9 H), 1.59 (s, 9 H), 1.87 (s, 3 H), 2.03 (s, 3 H), 2.07 (s, 3 H), 2.13 (s,
3 H), 3.40 (br s, 1 H), 3.65 (s, 1 H), 3.77-4.18 (m, 6 H), 4.20-4.60
(m, 5 H), 4.65-4.84 (m, 2 H), 5.15-5.37 (m, 4 H), 5.47-5.57 (m, 1
H), 5.85 (br s, 1 H), 7.14 (br s, 2 H), 7.22-7.40 (m, 10 H), 7.42 (s, 1
H), and 8.04 (s, 1 H); 13C NMR (CDCl3) δ 21.08, 21.33, 28.24, 28.73,
62.68, 62.84, 62.97, 65.33, 66.12, 69.67, 69.72, 70.37, 70.78, 73.16,
73.26, 74.14, 80.11, 86.37, 98.70, 115.33, 128.54, 128.91, 129.05,
129.42, 137.12, 137.67, 137.85, 138.11, 142.32, 147.24, 155.99, 170.48,
170.56, 170.68, 171.03, 171.24, 171.68, and 172.40; mass spectrum
(FAB), m/z 1125 (M + Na)+; mass spectrum (FAB), m/z 1125.380 (M
+ Na)+ (C51H66N4O22Na requires 1125.380).
Coupling of Disaccharide 29 with Benzyl (2S,3S,4R)-4-Amino-
3-hydroxy-2-methylvalerate (8). A mixture of 72 mg (66 µmol) of
disaccharide 29 and 27 mg (98 µmol) of benzyl valerate hydrochloride
8 was coevaporated with portions of toluene, dried under vacuum
overnight, and then dissolved in 2 mL of anhydrous CH2Cl2. To the
stirred solution were added successively 27 µL (20 mg, 150 µmol) of
N,N-diisopropylethylamine, 15 mg (110 µmol) of 1-hydroxybenzot-
riazole, and 25 mg (120 µmol) of N,N′-dicyclohexylcarbodiimide. The
reaction mixture was stirred at 25 °C under Ar for 5 h and then diluted
with 80 mL of ethyl acetate. The insoluble material was removed by
filtration through Celite and washed with 20 mL of ethyl acetate. The
filtrate was washed successively with 30 mL of 0.5 M aqueous citric
acid solution, 30 mL of saturated sodium bicarbonate, 30 mL of water,
and 30 mL of brine. The combined organic phase was dried (Na2SO4)
and concentrated. The residue was purified by flash chromatography
on a silica gel column (15 × 2 cm). Elution with 80:1 and then 40:1
chloroform-methanol afforded the desired product 30 as a colorless
foam: yield 60 mg (70%); silica gel TLC Rf 0.41 (10:1 chloroform-
methanol); [R]25D -30° (c 1.0, CHCl3); 1H NMR (CDCl3) δ 1.10 (d, 3
H, J ) 6 Hz), 1.19 (d, 2 H, J ) 6.5 Hz), 1.41 (s, 9 H), 1.58 (s, 9 H),
1.94 (s, 3 H), 1.98 (s, 3 H), 2.07 (s, 3 H), 2.16 (s, 3 H), 2.40-2.51 (m,
1 H), 3.50-3.65 (m, 1 H), 3.78-3.96 (m, 3 H), 4.00-4.15 (m, 4 H),
4.20-4.50 (m, 6 H), 4.54-4.70 (m, 4 H), 4.76-4.90 (m, 2 H), 5.05-
5.18 (m, 4 H), 5.24-5.44 (m, 3 H), 6.45 (m, 1 H), 7.17 (br s, 2 H),
7.22-7.40 (m, 15 H), 7.44 (s, 1 H), and 7.99 (s, 1 H); 13C NMR (CDCl3)
δ 12.66, 16.08, 21.35, 21.17, 28.22, 28.69, 42.48, 48.16, 62.84, 66.59,
66.71, 67.75, 69.59, 70.22, 70.42, 70.57, 70.76, 72.54, 73.31, 73.44,
73.54, 74.09, 74.61, 75.30, 75.63, 80.46, 86.66, 97.66, 116.78, 128.57,
128.94, 136.40, 136.52, 137.53, 137.82, 137.95, 138.27, 138.68, 140.43,
146.97, 155.82, 168.10, 168.77, 170.39, 170.49, 170.68, 171.11, and
175.66; mass spectrum (FAB), m/z 1328 (M + Na)+; mass spectrum
(FAB), m/z 1328.526 (M + Na)+ (C64H83N5O24Na requires 1328.532).
(CD3OD) δ 1.11 (d, 3 H, J ) 6.5 Hz), 1.27 (d, 3 H, J ) 5 Hz), 1.40
(s, 9 H), 1.60 (s, 9 H), 1.99 (s, 3 H), 2.04 (s, 6 H), 2.11 (s, 3 H),
2.40-2.50 (m, 1 H), 3.20-3.32 (m, 2 H), 3.60-3.70 (m, 1 H), 3.70-
3.80 (m, 1 H), 3.90-4.18 (m, 8 H), 4.50-4.65 (m, 1 H), 5.00-5.15
(m, 4 H), 5.20-5.35 (m, 3 H), 5.38-5.50 (m, 2 H), 7.58 (s, 1 H), and
8.15 (s, 1 H); mass spectrum (FAB), m/z 1036 (M + H)+; mass
spectrum (FAB), m/z 1036.407 (M + H)+ (C43H66N5O24 requires
1036.409).
Coupling of Compound 31 with Threonylbithiazole 9. A mixture
of 20 mg (19 µmol) of disaccharide dipeptide acid 31, 20 mg (45 µmol)
of threonylbithiazole 9, and 6 mg (44 µmol) of 1-hydroxybenzotriazole
was coevaporated with portions of toluene, dried overnight under
vacuum in the presence of P2O5, and then dissolved in 1 mL of
anhydrous DMF. To this reaction mixture were added 10 mg (48 µmol)
of N,N′-dicyclohexylcarbodiimide and 10 µL (7.3 mg, 5.6 µmol) of
N,N-diisopropylethylamine. The reaction mixture was stirred at 25 °C
under Ar for 24 h. The solvent was concentrated, and the residue was
coevaporated with portions of toluene under diminished pressure. The
residue was purified by flash chromatography on a silica gel column
(17 × 2 cm). Elution with 20:1 and then 15:1 chloroform-methanol
afforded Boc-pentapeptide disaccharide 32 as a colorless foam: yield
16 mg (59%); silica gel TLC Rf 0.45 (5:1 chloroform-methanol); [R]25
+18.8° (c 1.0, methanol); H NMR (CDCl3) δ1.18 (d, 9 H, J ) 6.5
D
1
Hz), 1.42 (s, 9 H), 1.59 (s, 9 H), 1.90 (m, 2 H), 2.00 (s, 3 H), 2.06 (s,
6 H), 2.10 (s, 3 H), 2.13 (s, 3 H), 2.60 (t, 2 H, J ) 6.5 Hz), 3.20-3.44
(m, 4H), 3.44-3.53 (m, 2 H), 3.54-3.63 (m, 2 H), 3.65-3.83 (m, 6
H), 3.87-3.92 (m, 2 H), 3.97-4.06 (m, 4 H), 4.11-4.15 (m, 1 H),
4.17-4.24 (m, 2 H), 4.99 (m, 1 H), 5.06 (m, 1 H), 5.22 (m, 1 H), 5.45
(m, 1 H), 7.18-7.30 (m, 3 H), 7.49-7.58 (m, 2 H), 7.67-7.75 (m, 1
H), 7.75 (s, 1 H), 8.02 (s, 1 H), and 8.65 (s, 2 H); mass spectrum
(FAB), m/z 1462 (M + H)+; mass spectrum (FAB), m/z 1461.509 (M
+ H)+ (C60H89N10O26S3 requires 1461.510).
Partial Deprotection of Pentapeptide Disaccharide 32. Boc-
pentapeptide disaccharide 32 (16 mg, 10.9 µmol) was treated with 0.4
mL of dimethyl sulfide and 0.8 mL of trifluoroacetic acid at 0 °C under
Ar for 3 h. The reaction mixture was concentrated under diminished
pressure, and the residue was coevaporated with portions of toluene.
The crude product was dissolved in 1.0 mL of water and loaded onto
an Amberlite XAD-2 column (20 × 1 cm). The column was washed
with 80 mL of water, and the product was eluted from the column by
washing with 100 mL of methanol. The combined methanol eluate
was concentrated under diminished pressure, and the residue was
coevaporated with portions of toluene to afford the pentapeptide
1
disaccharide 24 as a pale yellow foam: yield 12 mg (87%); H NMR
(CD3OD) δ 1.07-1.37 (m, 9 H), 1.97 (m, 3 H), 2.17 (s, 9 H), 2.24 (s,
3 H), 2.60 (m, 2 H), 3.20-3.45 (m, 4 H), 3.44-3.52 (m, 2 H), 3.54-
3.63 (m, 2 H), 3.65-3.83 (m, 6 H), 3.87-3.92 (m, 2 H), 3.97-4.06
(m, 4 H), 4.11-4.15 (m, 1 H), 4.17-4.24 (m, 2 H), 4.99 (m, 1 H),
5.06 (m, 1 H), 5.22 (m, 1 H), 5.45 (m, 1 H), 7.67 (s, 1 H), 8.05 (s, 1
H), 8.15 (s, 1 H), and 8.94 (s, 1 H); mass spectrum (FAB), m/z 1261
(M + H)+; mass spectrum (FAB), m/z 1261.407 (M + H)+
(C50H73N10O22S3 requires 1261.406).
This product was employed in the next transformation without further
purification.
Debenzylation of Disaccharide Dipeptide Benzyl Ester 30. To a
suspension of 80 mg (0.45 mmol) of PdCl2 in 40 µL of 2 N HCl were
added 4 mL of boiling water, 19.2 µL (23 mg, 0.51 mmol) of formic
acid, and 1.25 mL of 10% potassium hydroxide solution. The pH of
the mixture was adjusted to ∼7-8 with formic acid, and the mixture
was boiled for 7-9 min to form the palladium black as big chunks.
The liquid phase was removed, and the catalyst was washed succes-
sively with 30 mL of water and 50 mL of methanol. The disaccharide
dipeptide benzyl ester 30 (29 mg, 22 µmol) was dissolved in 3 mL of
absolute ethanol and added to the flask containing the freshly prepared
palladium black catalyst. The reaction mixture was stirred at 25 °C
under 1 atm of hydrogen for 40 h. The catalyst was filtered through
Celite and washed with methanol. The filtrate was concentrated under
diminished pressure, and the residue was purified by flash chromatog-
raphy on a silica gel column (12 × 2 cm). Elution with 5:1 and then
1:1 chloroform-methanol afforded the debenzylated product 31 as a
colorless foam: yield 12 mg (52%); silica gel TLC Rf 0.35 (3:1
Coupling of Pentapeptide Disaccharide 24 with N-t-Boc-pyrimi-
doblamic Acid (10). Deprotection and Purification of Bleomycin
Demethyl A2 (2). A mixture of 12 mg (9.5 µmol) of pentapeptide
disaccharide 24 and 8 mg (18 µmol) of N-t-Boc-pyrimidoblamic acid
(10)36 was coevaporated with portions of toluene and then dried
overnight under vacuum in the presence of P2O5. To this mixture was
added 14 mg (32 µmol) of benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate. The reaction mixture was dissolved
in 1 mL of anydrous DMF and cooled to 0 °C. N,N-Diisopropylethy-
lamine (33 µL, 24.5 mg, 0.18 mmol) was added, and the reaction
mixture was stirred at 25 °C under Ar for 28 h. The solution was
concentrated under diminished pressure. The residue was coevaporated
with portions of toluene and then treated successively with 30 mL of
water and 30 mL of ethyl acetate. The aqueous phase was extracted
with ethyl acetate. The combined organic phase was washed with three

Total Synthesis of Bleomycin
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11295
10-mL portions of water. The combined aqueous phase was concen-
trated and coevaporated with toluene to give crude product 25.
were added 236 mg (1.64 mmol) of trichloroacetonitrile and 122 mg
(0.884 mmol) of anhydrous potassium carbonate. The solution was
stirred vigorously for 6 h and then filtered. The filtered solid was
washed with 10 mL of CH2Cl2. The organic filtrate was partitioned
against 5 mL of water. The water layer was back-extracted with 5 mL
of CH2Cl2. The combined organic phase was then concentrated under
diminished pressure to afford trichloroacetimidate 46 as a colorless
Crude 25 was dissolved in 2 mL of methanol and treated with 2 mL
of 2 N NH4OH at 25 °C for 3.5 h to remove the acetyl groups. The
solvent was concentrated and coevaporated with portions of toluene to
give a colorless glass. This glass was treated with 1 mL of dimethyl
sulfide and 2 mL of trifluoroacetic acid at 0 °C for 1.5 h. The solvent
was concentrated, and the residue was treated with 20 mL of ethyl
acetate and 20 mL of water. The organic phase was washed with three
8-mL portions of water. The combined aqueous phase was concentrated
to afford crude bleomycin demethyl A2 (2).
foam: yield 140 mg (97%); silica gel TLC Rf 0.42 (1:1 hexanes-
1
acetone); [R]20 26.1° (c 0.20, CHCl3); H NMR (CDCl3) δ 1.99 (s,
D
3H), 2.05 (s, 3H), 2.05 (s, 3H), 2.20 (s, 3H), 3.55 (m, 1H), 4.01 (m,
1H), 4.08-4.13 (m, 3H), 4.16-4.22 (m, 3H), 4.31 (m, 1H), 4.34 (d,
1H, J ) 12.5 Hz), 4.37 (d, 1 H, J ) 11.5 Hz), 4.50 (d, 1H, J ) 12.5
Hz), 4.57 (br s, 2H), 4.60 (m, 1H), 4.68 (d, 1H, J ) 11 Hz), 4.87 (m,
1H), 5.21-5.31 (m, 2 H) 6.10 (d, 1H, J ) 8 Hz), 7.20-7.38 (m, 10H),
and 8.67 (s, 1H); 13C NMR (CDCl3) δ 20.52, 20.61, 20.72, 20.88, 61.81,
62.56, 65.98, 68.44, 69.49, 70.18, 72.12, 72.83, 73.83, 73.34, 73.72,
93.10, 94.45, 95.13, 128.14, 128.23, 128.53, 137.32, 137.09, 155.11,
160.74, 169.79, 169.95, 170.45, and 170.53; mass spectrum (FAB),
m/z 899.157 (M + Na)+ (C37H43N2O16Cl3Na requires 899.157).
Crude BLM 2 and 8 mg of CuSO4‚5H2O were dissolved in 1 mL of
50 mM sodium citrate buffer, pH 4.5, and applied to a CM Sephadex
C-25 column (24 × 1.5 cm). The column was washed with a linear
gradient of NaCl (0 f 2 N) in 50 mM sodium citrate buffer, pH 4.5.
The column was monitored at 292 nm, and the appropriate fractions
were pooled and concentrated. This Cu(II)‚BLM complex and 0.2 g
of Na2EDTA salt in 7 mL of water were stirred at 25 °C for 1 h. The
solution was purified by chromatography on a C18 reversed-phase
column (9.5 × 1 cm). The column was first washed with 70 mL of
water and then with 60 mL of 4:1 MeOH-0.002 N HCl. The
methanol-0.002 N HCl eluate was pooled and concentrated. The
residue was dissolved in 4 mL of water, frozen, and lyophilized to
give bleomycin demethyl A2 (2) as a colorless powder: yield 3 mg
(23% from 24); silica gel TLC Rf 0.47 (1:3 10% NH4OAc-EtOH);
Benzyl erythro-Nr,Nim-Bis(9-fluorenylmethoxycarbonyl)-â-[6-O-
acetyl-3,4-di-O-benzyl-2-O-(2,4,6-tri-O-acetyl-3-O-carbamoyl-r-D-
mannopyranosyl)-r-L-gulopyranosyl]-S-histidine (47). A sample of
59.2 mg (67.1 µmol) of disaccharide imidate 46 and 15 mg (21.3 µmol)
of â-hydroxyhistidine derivative 37 were combined and dried under
vacuum. Dichloromethane (0.5 mL) was added to the mixture, and
the solution was treated with 13.3 µL (16.9 mg, 73.8 µmol) of TMSOTf.
The reaction mixture was stirred at 25 °C for 1.5 h and then quenched
with saturated NaHCO3 solution. The reaction mixture was partitioned
against 5 mL of CH2Cl2, and the aqueous layer was extracted with
three 10-mL portions of ethyl acetate. The combined organic fraction
was dried (MgSO4). The solvent was concentrated under diminished
pressure, and the product was purified by preparative silica gel TLC;
development was with 3:1 ethyl acetate-hexanes. The product was
eluted from the silica gel with ethyl acetate to afford the Fmoc-protected
histidinyldisaccharide 47 as a colorless glass: yield 12.5 mg (44%);
λ
max 292 nm (ꢀ 14 000); [R]25D -17.1° (c 0.14, H2O); 1H NMR (D2O)
δ 1.06 (d, 3 H, J ) 6 Hz), 1.10 (d, 3 H, J ) 7 Hz), 1.13 (d, 3 H, J )
7 Hz), 1.87-1.94 (m, 2 H), 1.97 (s, 3 H), 2.07 (s, 3 H), 2.53-2.60 (m,
4 H), 2.67 (s, 1 H), 2.72-2.86 (m, 2 H), 3.15-3.30 (m, 4 H), 3.44-
3.52 (m, 2 H), 3.54-3.63 (m, 3 H), 3.65-3.83 (m, 6 H), 3.87-3.92
(m, 2 H), 3.97-4.06 (m, 4 H), 4.11-4.15 (m, 1 H), 4.17-4.24 (m, 2
H), 4.99 (s, 1 H), 5.06 (d, 1 H, J ) 6.5 Hz), 5.22 (d, 1 H, J ) 3.5 Hz),
5.45 (d, 1 H, J ) 6.5 Hz), 7.57 (s, 1 H), 8.00 (s, 1 H), 8.14 (s, 1 H),
and 8.76 (s, 1 H); mass spectrum (FAB), m/z 1400 (M + H)+; mass
spectrum (FAB), m/z 1400.500 (M + H)+ (C54H82N17O21S3 requires
1400.503).
silica gel TLC Rf 0.38 (3:1 ethyl acetate-hexanes); [R]23 -6.4° (c
D
Benzyl erythro-Nr,Nim-Bis(9-fluorenylmethoxycarbonyl)-â-[3,4,6-
tri-O-acetyl-2-O-(2,4,6-tri-O-acetyl-3-O-carbamoyl-r-D-mannopyra-
nosyl)-r-L-gulopyranosyl]-S-histidine (38). Compound 35 (11.5 mg,
13 µmol) was coevaporated with three 1-mL portions of toluene. To
the residue was added 300 µL of CH2Cl2 containing 11.7 mg (16 µmol)
of di-Fmoc â-hydroxyhistidine 37, which had been coevaporated with
three 1-mL portions of toluene. The stirred reaction mixture was diluted
with 600 µL of ether, cooled (0 °C), and treated with 10.3 µL (12.2
mg, 53 µmol) of trimethysilyl trifluoromethanesulfonate. Stirring was
continued for 20 min at 0 °C, and then the reaction mixture was poured
into 8 mL of a 2:1 ethyl acetate-aqueous NaHCO3 mixture with
vigorous stirring. The organic phase was washed twice with 5 mL of
brine, dried (MgSO4), and concentrated under diminished pressure. The
residue was purified using preparative silica gel TLC (0.5-mm plate
thickness). Elution with 8:2 ethyl acetate-hexanes gave compound
38 as a colorless foam: yield 10.2 mg (58%); silica gel TLC Rf 0.38
(4:1 ethyl acetate-hexanes); 1H NMR (CDCl3) δ 1.77, 1.87, 2.02, 2.07,
2.08, and 2.10 (each s, 3H), 3.9-4.4 (m, 10H), 4.5 (br s, 2H), 4.70-
4.75 (m, 3H), 4.77 (d, 1H, J ) 12 Hz), 4.97 (d, 1H, J ) 12 Hz), 5.0-
5.35 (m, 8H), 5.4 (t, 1H, J ) 2 Hz), 5.68 (m, 1H), 6.33 (d, 1H, J ) 8.5
Hz), 7.2-7.5 (m, 14H), 7.5-7.7 (m, 4H), 7.77 (dd, 4H, J ) 13, 7.5
Hz), and 7.95 (s, 1H); 13C NMR (CDCl3) δ 20.91, 21.00, 21.11, 47.02,
47.54, 57.05, 60.80, 62.01, 62.58, 62.91, 65.28, 65.82, 66.63, 67.64,
67.85, 68.86, 69.65, 69.99, 70.27, 71.20, 73.35, 96.35, 96.94, 116.10,
120.35, 125.15, 125.64, 125.70, 127.44, 127.58, 127.82, 128.08, 128.58,
128.72, 128.79, 135.71, 137.35, 141.35, 141.52, 141.70, 141.79, 143.05,
144.26, 144.57, 148.54, 155.61, 156.63, 169.54, 169.78, 169.89, 170.44,
170.49, 170.79, 170.91, and 171.03; mass spectrum (FAB), m/z
1325.426 (M + H)+ (C68H69N4O24 requires 1325.430).
1
0.14, CHCl3); H NMR (CDCl3) δ 1.82 (s, 3H), 1.87 (s, 3H), 1.99 (s,
3H), 2.13 (s, 3H), 3.60 (m, 1H), 3.90-4.12, 4.09 (m, 6H), 4.22 (m,
3H), 4.28-4.42 (m, 5H), 4.50-4.66 (m, 4H), 4.75-4.87 (m, 1H), 5.11
(m, 3H), 5.19-5.32 (m, 6H), 5.39 (m, 1H), 6.63 (d, 1H, J ) 8.5 Hz),
7.20-7.81 (m, 32H), and 7.93 (s, 1H); mass spectrum (FAB), m/z
1443.487 (M + Na)+ (C78H76N4O22Na requires 1443.483).
Relaxation of Supercoiled DNA by BLM. DNA relaxation was
measured using 200 ng of supercoiled pBR322 plasmid DNA in 5 µL
of 10 mM sodium cacodylate buffer, pH 7.2. The reactions contained
equimolar BLM and FeII(NH4)2(SO4)2 at the concentration indicated
in the figure legend. The reactions were initiated by the addition of
FeII(NH4)2(SO4)2 from a freshly prepared solution, and incubated at 23
°C for 10 min. The reactions were quenched by the addition of 10 µL
of a 1:1 mixture of 100 mM EDTA, pH 7.4, and 40% sucrose loading
solution containing 0.1% (w/v) bromophenol blue. A portion of each
reaction (7 µL) was analyzed by horizontal electrophoresis on a 1%
agarose gel containing 1 µg/mL ethidium bromide (30 v, 2 h; then 40
v, 12 h). The bands were visualized by UV transillumination.
Preparation of a 5′-32P-End-Labeled DNA Restriction Fragment.
Plasmid pBR322 (50 µg) was incubated with 100 units of restriction
endonuclease HindIII in 10 mM Tris HCl, pH 8.0, containing 5 mM
MgCl2, 100 mM NaCl, and 1 mM â-mercaptoethanol. The digestion
reaction was carried out at 37 °C for 1 h, and then the DNA was
dephosphorylated with 5 units of alkaline phosphatase (37 °C). The
DNA was recovered by ethanol precipitation following phenol extrac-
tion. The linearized DNA (0.5 A260 unit; 10 pmol) was 5′-32P-end-
labeled by incubation with 320 µCi of [γ-32P]ATP and 6 units of T4
polynucleotide kinase at 37 °C for 30 min. The DNA was purified on
a Sephadex G-50 column and then digested with 300 units of restriction
enzyme EcoRV in 350 µL (total volume) of 10 mM Tris-HCl, pH
8.0, containing 5 mM MgCl2, 100 mM NaCl, and 1 mM â-mercapto-
ethanol at 37 °C for 1 h. The 158-base pair 5′-32P-end-labeled DNA
duplex was isolated from an 8% nondenaturing polyacrylamide gel.
6-O-Acetyl-3,4-di-O-benzyl-2-O-(2,4,6-tri-O-acetyl-3-O-carbam-
oyl-r-D-mannopyranosyl)-â-L-gulopyranosyl Trichloroacetimidate
(46). A sample of 120 mg (0.164 mmol) of 6-O-acetyl-3,4-di-O-benzyl-
2-O-(2,4,6-tri-O-acetyl-3-O-carbamoyl-R-D-mannopyranosyl)-R/â-gu-
lopyranoside (45) was dissolved in 5 mL of CH2Cl2. To this solution

11296 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
Katano et al.
Cleavage of 32P-End Labeled Duplex DNA by BLM. Reactions
contained ∼30 000 cpm of 158-base pair 5′-32P-end-labeled DNA
duplex in 5 µL of 10 mM sodium cacodylate, pH 7.4. The reactions
containing BLM at the concentrations indicated in the figure legend
were initiated by the addition of equimolar Fe2+ from a freshly prepared
solution of FeII(NH4)2(SO4)2. The reactions were incubated at 23 °C
for 20 min, then quenched by the addition of 5 µL of loading buffer (7
M urea, 1 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene
cyanol), and applied to a 14% polyacrylamide gel containing 8 M urea.
Electrophoresis was carried out at 10 W for 1 h and then at 50 W for
2 h. The gel was analyzed using a phosphorimager; autoradiography
(Kodak XAR-2 film) was carried out at -80 °C.
Acknowledgment. We thank Michiel Lodder, Edward
Locke, and Dr. Tuncer Arslan for assistance with some of the
1H NMR spectra. This work was supported by Research Grants
CA27603, CA53913, and CA77284, awarded by the National
Cancer Institute, National Institutes of Health.
Supporting Information Available: Polyacrylamide gel
showing the DNA cleavage activities of synthetic bleomycin
congeners and synthetic methods for compounds 4-7, 9, 15-
24, 27, 36, 37, 39, 40, 42, 43, and 45 (17 pages, print/PDF).
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JA9819458