Assessment of the role of the bleomycin A2 pyrimidoblamic acid C4 amino group

Article abstract of DOI:10.1021/ja971889v

The preparation and examination of 3 and 4 and their unnatural epimers 6 and 7 by following a new and alternative synthesis of 2 was conducted to assess the role of the pyrimidine C4 amine of bleomycin A₂ (1) and deglycobleomycin A₂ (2). The agent 3 bearing a pyrimidine C4 dimethylamino substituent exhibited a substantially diminished DNA cleavage efficiency (10-15x) relative to 2 and the loss of the characteristic 5'-GC/5'-GT cleavage selectivity. The agent 4 in which the pyrimidine C4 amino group was removed exhibited an even greater diminished DNA cleavage efficiency (30x) relative to that for 2. For this agent, the characteristic cleavage selectivity is either slightly or significantly reduced depending on the assay conditions. Even in the instances where it was not substantially altered, the ability to detect it required a temperature of 4 versus 25-37 °C. This information and temperature dependence suggest a reduced binding interaction and are consistent with the participation of the pyrimidine C4 amine in one of two critical hydrogen bonds of a minor groove triplex-like recognition between the metal binding domain of 1 and 2 and guanine at the 5'-GC/5'-GT cleavage sites responsible for the characteristic cleavage selectivity. These observations have further implications on the potential origin of inherent 5'-GPy > 5'-APy cleavage selectivity of bleomycin A₂ itself. This cleavage preference of 5'-GPy > 5'-APy may be analogously attributed to a reduced binding affinity at the 5'-APy sites resulting from one versus two triplex-like hydrogen bonds to adenine.

Full text of DOI:10.1021/ja971889v

J. Am. Chem. Soc. 1998, 120, 53-65
53
Assessment of the Role of the Bleomycin A2 Pyrimidoblamic Acid
C4 Amino Group
Dale L. Boger,*,† Timothy M. Ramsey,† Hui Cai,† Silvia T. Hoehn,‡
John W. Kozarich,§ and JoAnne Stubbe‡
Contribution from the Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, Departments of Chemistry and Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, and Merck Research Laboratory, P.O. Box 2000,
Rahway, New Jersey 07065
ReceiVed June 9, 1997
Abstract: The preparation and examination of 3 and 4 and their unnatural epimers 6 and 7 by following a
new and alternative synthesis of 2 was conducted to assess the role of the pyrimidine C4 amine of bleomycin
A2 (1) and deglycobleomycin A2 (2). The agent 3 bearing a pyrimidine C4 dimethylamino substituent exhibited
a substantially diminished DNA cleavage efficiency (10-15×) relative to 2 and the loss of the characteristic
5′-GC/5′-GT cleavage selectivity. The agent 4 in which the pyrimidine C4 amino group was removed exhibited
an even greater diminished DNA cleavage efficiency (30×) relative to that for 2. For this agent, the characteristic
cleavage selectivity is either slightly or significantly reduced depending on the assay conditions. Even in the
instances where it was not substantially altered, the ability to detect it required a temperature of 4 versus
25-37 °C. This information and temperature dependence suggest a reduced binding interaction and are
consistent with the participation of the pyrimidine C4 amine in one of two critical hydrogen bonds of a minor
groove triplex-like recognition between the metal binding domain of 1 and 2 and guanine at the 5′-GC/5′-GT
cleavage sites responsible for the characteristic cleavage selectivity. These observations have further implications
on the potential origin of inherent 5′-GPy > 5′-APy cleavage selectivity of bleomycin A2 itself. This cleavage
preference of 5′-GPy > 5′-APy may be analogously attributed to a reduced binding affinity at the 5′-APy sites
resulting from one versus two triplex-like hydrogen bonds to adenine.
Bleomycin A2 (1, Figure 1),1-11 the major constituent of the
clinical antitumor drug blenoxane, is thought to derive its
therapeutic effects from the ability to mediate the oxidative
cleavage of double-stranded DNA or RNA by a process that is
metal ion and oxygen dependent.12-18 Extensive studies
employing derivatives of the natural product,19,20 its degradation
products or semisynthetic analogues,21-30 and closely related
or substantially simplified analogues31-34 have contributed to
(13) Sausville, E. A.; Stein, R. W.; Peisach, J.; Horwitz, S. B.
Biochemistry 1978, 17, 2746.
(14) D’Andrea, A. D.; Haseltine, W. A. Proc. Natl. Acad. Sci. U.S.A.
1978, 75, 3608.
(15) Takeshita, M.; Grollman, A. P.; Ohtsubo, E.; Ohtsubo, H. Proc.
Natl. Acad. Sci. U.S.A. 1978, 75, 5983.
† The Scripps Research Institute.
‡ Massachusetts Institue of Technology.
§ Merck Research Laboratory.
(1) Natrajan, A.; Hecht, S. M. In Molecular Aspects of Anticancer Drug-
DNA Interactions; Neidle, S., Waring, M. J., Eds.; CRC: Boca Raton FL,
1994; Vol. 2, p 197. Kane, S. A.; Hecht, S. M. In Progress in Nucleic
Acids Research Molecular Biology; Cohn, W. E., Moldave, K., Eds.;
Academic: San Diego, CA, 1994; Vol. 49, p 313.
(2) Ohno, M.; Otsuka, M. In Recent Progress in the Chemical Synthesis
of Antibiotics; Lukacs, G., Ohno, M., Eds.; Springer-Verlag: New York,
1990; p 387.
(3) Dedon, P. C.; Goldberg, I. H. Chem. Res. Toxicol. 1992, 5, 311.
(4) Petering, D. H.; Byrnes, R. W.; Antholine, W. E. Chem.-Biol. Interact.
1990, 73, 133.
(5) Stubbe, J.; Kozarich, J. W. Chem. ReV. 1987, 87, 1107. Stubbe, J.;
Kozarich, J. W.; Wu, W.; Vanderwall, D. E. Acc. Chem. Res 1996, 29,
322.
(6) Hecht, S. M. Acc. Chem. Res. 1986, 19, 383.
(7) Sugiura, Y.; Takita, T.; Umezawa, H. Metal Ions Biol. Syst. 1985,
19, 81.
(8) Twentyman, P. R. Pharmacol. Ther. 1984, 23, 417.
(9) Povirk, L. F. In Molecular Aspects of Anti-Cancer Drug Action;
Neidle, S., Waring, M. J., Eds.; MacMillian: London, 1983.
(10) Bleomycin: Chemical, Biochemical and Biological Aspects; Hecht,
S. M., Ed.; Springer-Verlag: New York, 1979.
(16) Magliozzo, R. S.; Peisach, J.; Cirolo, M. R. Mol. Pharmacol. 1989,
35, 428.
(17) Carter, B. J.; de Vroom, 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.
Hecht, S. M. Bioconjugate Chem. 1994, 5, 513.
(18) Carter, B. J.; Reddy, K. S.; Hecht, S. M. Tetrahedron 1991, 47,
2463. Holmes, C. E.; Carter, B. J.; Hecht, S. M. Biochemistry 1993, 32,
4293.
(19) Takita, T.; Muraoka, Y.; Nakatani, T.; Fujii, A.; Umezawa, Y.;
Naganawa, H.; Umezawa, H. J. Antibiot. 1978, 31, 801.
(20) Umezawa, H. Pure Appl. Chem. 1971, 28, 665.
(21) Deglycobleomycin A2: Oppenheimer, N. J.; Chang, C.; Chang, L.-
H.; Ehrenfeld, G.; Rodriguez, L. O.; Hecht, S. M. J. Biol. Chem. 1982,
257, 1606. Sugiura, Y.; Suzuki, T.; Otsuka, M.; Kobayashi, S.; Ohno, M.;
Takita, T.; Umezawa, H. J. Biol. Chem. 1983, 258, 1328. Sugiura, Y.;
Kuwahara, J.; Suzuki, T. FEBS Lett. 1985, 182, 39. Kenani, A.; Lamblin,
G.; Henichart, J.-P. Carbohydr. Res. 1988, 177, 81. Boger, D. L.; Menezes,
R. F.; Yang, W. Bioorg. Med. Chem. Lett. 1992, 2, 959.
(22) iso-Bleomycin A2: Nakayama, Y.; Kunishima, M.; Omoto, S.;
Takita, T.; Umezawa, H. J. Antibiot. 1973, 26, 400.
(23) epi-Bleomycin A2: Muraoka, Y.; Kobayashi, H.; Fujii, A.; Kun-
ishima, M.; Fujii, T.; Nakayama, Y.; Takita, T.; Umezawa, H. J. Antibiot.
1976, 29, 853.
(24) Deamidobleomycin A2: Umezawa, H.; Hori, S.; Sawa, T.; Yoshioka,
T.; Takeuchi, T. J. Antibiot. 1974, 27, 419.
(11) Umezawa, H. In Bleomycin: Current Status and New DeVelopments;
Carter, S. K., Crooke, S. T., Umezawa, H., Eds.; Academic: New York,
1978.
(12) Ishida, R.; Takahashi, T. Biochem. Biophys. Res. Commun. 1975,
66, 1432.
(25) Deamidobleomycin A2 and depyruvamidobleomycin A2: Sugiura,
Y. J. Am. Chem. Soc. 1980, 102, 5208.
S0002-7863(97)01889-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/14/1998

54 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
A2 (2) bind to an oligonucleotide at a cleavage site in essentially
identical manners albeit with 2 exhibiting a lower affinity
(30×).35-37 Both 1 and 2 exhibit comparable DNA cleavage
selectivities but with 2 exhibiting lower efficiencies (2-6×).
This comparable behavior suggests that the disaccharide con-
tributes to the cleavage efficiency of 1 but not its DNA cleavage
selectivity and that it may do so by increasing DNA binding
affinity or altering the kinetic parameters for activation.
Regardless of the role, these and related studies illustrate that
deglycobleomycin A2 analogues may provide important and
relevant information on the nature of the interaction of 1 with
duplex DNA. In our own efforts,38-48 this has entailed single
point changes in the structure of deglycobleomycin A2 conducted
with the intention of defining the role of each subunit, functional
group, or substituent. These studies, carried out in conjunction
with structural studies, have begun to unravel many of the subtle
structural features contributing to the properties of the natural
product which suggest that they are greater than the sum of its
parts.
(32) Kilkuskie, R. E.; Suguna, H.; Yellin, B.; Murugesan, N.; Hecht, S.
M. J. Am. Chem. Soc. 1985, 107, 260. Shipley, J. B.; Hecht, S. M. Chem.
Res. Toxicol. 1988, 1, 25. Carter, B. J.; Murty, V. S.; Reddy, K. S.; Wang,
S.-N.; Hecht, S. M. J. Biol. Chem. 1990, 265, 4193. Hamamichi, N.;
Natrajan, A.; Hecht, S. M. J. Am. Chem. Soc. 1992, 114, 6278. Kane, S.
A.; Natrajan, A.; Hecht, S. M. J. Biol. Chem. 1994, 269, 10899. Quada, J.
C.; Levy, M. J.; Hecht, S. M. J. Am. Chem. Soc. 1993, 115, 12171.
(33) Guajardo, R. J.; Tan, J. D.; Mascharak, P. K. Inorg. Chem. 1994,
33, 2838. Guajardo, R. J.; Chavez, F.; Farinas, E. T.; Mascharak, P. K. J.
Am. Chem. Soc. 1995, 117, 3883. Loeb, K. E.; Zaleski, J. M.; Westre, T.
E.; Guajardo, R. J.; Mascharak, P. K.; Hedmen, B.; Hodgson, K. O.;
Solomon, E. I. J. Am. Chem. Soc. 1995, 117, 4545. Guajardo, R. J.; Hudson,
S. E.; Brown, S. J.; Mascharak, P. K. J. Am. Chem. Soc. 1993, 115, 7971.
Tan, J. D.; Hudson, S. E.; Brown, S. J.; Olmstead, M. M.; Mascharak, P.
K. J. Am. Chem. Soc. 1992, 114, 3841.
(34) Kenani, A.; Lohez, M.; Houssin, R.; Helbecque, N.; Bernier, J. L.;
Lemay, P.; Henichart, J. P. Anti-Cancer Drug Des. 1987, 2, 47. Kenani,
A.; Bailly, C.; Helbecque, N.; Houssin, R.; Bernier, J.-L.; Henichart, J.-P.
Eur. J. Med. Chem. 1989, 24, 371.
(35) Wu, W.; Vanderwall, D. E.; Lui, S. M.; Tang, X.-J.; Turner, C. J.;
Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1268. Wu, W.;
Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem.
Soc. 1996, 118, 1281. Wu, W.; Vanderwall, D. E.; Stubbe, J.; Kozarich, J.
W.; Turner, C. J. J. Am. Chem. Soc. 1994, 116, 10843. See also:
Vanderwall, D. E.; Lui, S. M.; Wu, W.; Turner, C. J.; Kozarich, J. W.;
Stubbe, J. Chem. Biol. 1997, 4, 373. Lui, S. M.; Vanderwall, D. E.; Wu,
W.; Tang, X.-J.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem.
Soc. 1997, 119, 9603.
(36) Wu, W.; Lui, S. M.; Hoehn, S.; Turner, C. J.; Stubbe, J.; Teramoto,
S.; Boger, D. L.; Vanderwall, D. E.; Kozarich, J. W.; Tang, X.-J. J. Am.
Chem. Soc., in press.
(37) Manderville, R. A.; Ellena, J. F.; Hecht, S. M. J. Am. Chem. Soc.
1994, 116, 10851. Manderville, R. A.; Ellena, J. F.; Hecht, S. M. J. Am.
Chem. Soc. 1995, 117, 7891.
(38) Boger, D. L.; Teramoto, S.; Cai, H. Bioorg. Med. Chem. 1996, 4,
179.
(39) Boger, D. L.; Teramoto, S.; Honda, T.; Zhou, J. J. Am. Chem. Soc.
1995, 117, 7338.
(40) Boger, D. L.; Menezes, R. F.; Dang, Q.; Yang, W. Bioorg. Med.
Chem. Lett. 1992, 2, 261. Boger, D. L.; Menezes, R. F.; Dang, Q. J. Org.
Chem. 1992, 57, 4333.
(41) Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L.; Dang, Q.;
Yang, W. J. Am. Chem. Soc. 1994, 116, 82. Boger, D. L.; Yang, W. Bioorg.
Med. Chem. Lett. 1992, 2, 1649.
Figure 1.
an emerging model of the structural features responsible for
the sequence-selective cleavage of duplex DNA. Recent
structural studies have demonstrated that the Co(III) hydro-
peroxide complexes of bleomycin A2 (1) and deglycobleomycin
(26) Decarbamoylbleomycin A2: Sugiyama, H.; Ehrenfeld, G. M.;
Shipley, J. B.; Kilkuskie, R. E.; Chang, L.-H.; Hecht, S. M. J. Nat. Prod.
1985, 48, 869.
(27) PEMH and iso-bithiazole bleomycin A2: Morii, T.; Matsuura, T.;
Saito, I.; Suzuki, T.; Kuwahara, J.; Sugiura, Y. J. Am. Chem. Soc. 1986,
108, 7089. Morii, T.; Saito, I.; Matsuura, T.; Kuwahara, J.; Sugiura, Y. J.
Am. Chem. Soc. 1987, 109, 938.
(28) Peplomycin and liblomycin: Umezawa, H.; Takita, T.; Saito, S.;
Muraoka, Y.; Takahashi, K.; Ekimoto, H.; Minamide, S.; Nishikawa, K.;
Fukuoka, T.; Nakatani, T.; Fujii, A.; Matsuda, A. In Bleomycin Chemo-
therapy; Sikic, B. I., Rozenweig, M., Carter, S. K., Eds.; Academic:
Orlando, FL, 1985; p 289.
(29) Takita, T.; Maeda, K. J. Heterocycl. Chem. 1980, 17, 1799.
(30) Vloon, W. J.; Kruk, C.; Pandit, U. K.; Hofs, H. P.; McVie, J. G. J.
Med. Chem. 1987, 30, 20.
(42) Boger, D. L.; Colletti, S. L.; Honda, T.; Menezes, R. F. J. Am. Chem.
Soc. 1994, 116, 5607.
(43) Boger, D. L.; Honda, T.; Dang, Q. J. Am. Chem. Soc. 1994, 116,
5619.
(44) Boger, D. L.; Honda, T.; Menezes; R. F.; Colletti, S. L. J. Am. Chem.
Soc. 1994, 116, 5631. Boger, D. L.; Menezes, R. F.; Honda, T. Angew.
Chem., Int. Ed. Engl. 1993, 32, 273.
(45) Boger, D. L.; Honda, T. J. Am. Chem. Soc. 1994, 116, 5647.
(46) Boger, D. L.; Teramoto, S.; Zhou, J. J. Am. Chem. Soc. 1995, 117,
7344.
(47) Boger, D. L.; Colletti, S. L.; Teramoto, S.; Ramsey, T. R.; Zhou, J.
Bioorg. Med. Chem. 1995, 3, 1281.
(48) Boger, D. L.; Ramsey, T. M.; Cai, H. Bioorg. Med. Chem. 1996, 4,
195. Boger, D. L.; Teramoto, S.; Cai, H. Bioorg. Med. Chem. 1997, 5,
1577.
(31) Kittaka, A.; Sugano, Y.; Otsuka, M.; Ohno, M. Tetrahedron 1988,
44, 2811, 2821. Suga, A.; Sugiyama, T.; Otsuka, M.; Ohno, M.; Sugiura,
Y.; Maeda, K. Tetrahedron 1991, 47, 1191. 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. Otsuka, M.;
Masuda, T.; Haupt, A.; Ohno, M.; Shiraki, T.; Sugiura, Y.; Maeda, K. J.
Am. Chem. Soc. 1990, 112, 838. Otsuka, M.; Kittaka, A.; Ohno, M.; Suzuki,
T.; Kuwahara, J.; Sugiura, Y.; Umezawa, H. Tetrahedron Lett. 1986, 27,
3639. Sugiyama, T.; Ohno, M.; Shibasaki, M.; Otsuka, M.; Sugiura, Y.;
Kobayashi, S.; Maeda, K. Heterocycles 1994, 37, 275. Otsuka, M.; Satake,
H.; Sugiura, Y.; Murakami, S.; Shibasaki, M.; Kobayashi, S. Tetrahedron
Lett. 1993, 34, 8497.

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 55
characteristic DNA cleavage selectivity, while its removal with
4 and 7 maintained most of the characteristic 5′-GC/5′-GT
cleavage selectivity but did substantially diminish the ability
to detect it (4 versus 25 or 37 °C) for one set of assays whereas
very little cleavage specificity could be detected in another set
of assays even at 4 °C. These results suggest that the basis for
sequence selectivity defined by the structural models obtained
with the cobalt bleomycins is indicative of the interaction of
the physiologically important iron bleomycins with DNA.
Alternative Synthesis of Deglycobleomycin A2 (2). In our
preceding efforts culminating in the total synthesis of degly-
cobleomycin A2 and bleomycin A2, the final coupling to link
the full agent was conducted at the tetrapeptide S and â-hydroxy-
L-histidine juncture.44,45 This convergent assembly proved
advantageous for the synthesis of the natural product and was
adopted for the preparation of a range of analogues. For the
efforts herein, a more direct preparation would entail a final
coupling of NR-BOC-pyrimidoblamic acid (BOC ) tert-
butoxycarbonyl) or its analogues to pentapeptide S. In an effort
to establish the viability of this approach and the level of
required protecting groups, an alternative synthesis of degly-
cobleomycin A2 (2) was developed (Scheme 1). Coupling of
tetrapeptide S (8)42 and NR-BOC,Nim-CPh3-â-hydroxy-L-histidine
(9)42 effected by (benzotriazol-1-yloxy)tris(dimethylamino)-
phosphonium hexafluorophosphate (BOP reagent, 1.5 equiv, 2
equiv of i-Pr2NEt, DMF, 25 °C, 8 h, 75-88%) cleanly provided
10. Acid-catalyzed deprotection of 10 (20% TFA-CH2Cl2, 0
°C, 2.5 h) followed by liberation of the amine free base provided
pentapeptide S (11, 88%)42 resulting from both BOC and trityl
removal. Subsequent coupling of 11 with NR-BOC-pyrimido-
blamic acid (12)43 proceeded smoothly when effected by
diphenylphosphoryl azide (DPPA) treatment (1.5 equiv, 3 equiv
of i-Pr2NEt, DMF, 25 °C, 10 h, 72%) providing NR-BOC-
deglycobleomycin A2 (13) incorporating only one protecting
group with the sulfonium salt installed. This was accomplished
without deliberate protection or competitive acylation of the
imidazole. In preceding studies with bleomycin A2,45 we
reported that a related coupling effected by activation with
dicyclohexylcarbodiimide (DCC-HOBt, DMF, 25 °C, 36 h)
provided predominately imidazole versus primary amine cou-
pling. Whether these observations are unique to bleomycin A2
and result from the attempted reaction of a more hindered
primary amine or whether they are related to the activation
conditions (DPPA versus DCC-HOBt) has not been established.
However, the same coupling of 11 with 12 conducted with
DCC-HOBt (DMF, 25 °C, 10 h) provided 13 in much lower
conversions (25-29%). It is even plausible that the acylated
imidazole serves as an intermediate but is effectively converted
to 13 under the DPPA reaction conditions (3 equiv of i-Pr2-
NEt). Acid-catalyzed deprotection of 13 (20% TFA-CH2Cl2,
0 °C, 2 h, 86-89%) provided deglycobleomycin A2 (2) identical
in all respects with authentic material. The success of this
approach provided the basis for our synthesis of 3 and 4.
Synthesis of C4 (Dimethylamino)pyrimidoblamic Acid and
Its Epimer and Their Incorporation into 3 and 6. Displace-
ment of the 4-chloro substituent of 1552 by treatment with
dimethylamine (2 M in THF, 25 °C, 1.5 h, 97%) cleanly
provided 16, and subsequent acetal hydrolysis afforded the
aldehyde 17 and our key intermediate for diastereoselective
introduction of the C2 side chain (Scheme 2). Following
protocols developed in our synthesis of 1 and 2,43,53 condensation
of 17 with NR-BOC-â-amino-L-alanineamide (18)41 followed by
Figure 2.
Herein we report the synthesis and evaluation of 3 and 4 as
well as their epimers 6 and 7 in which the pyrimidoblamic acid
C4 primary amine of 2 has been replaced with a tertiary
dimethylamine or removed altogether. In addition to the impact
this may have on the metal chelation and oxygen activation
properties of 1 and 2, their examination allows the functional
assessment of the consequences of preventing formation of a
key hydrogen bond from this primary amine to guanine N3
implicated in recent structural studies.35,36 These studies
suggested two previously unrecognized and critical hydrogen
bonds between the pyrimidine of the metal binding domain of
bleomycin A2 and guanine of the cleavage sites 5′-GC/5′-GT.
The pyrimidoblamic acid N3 was found to be hydrogen bonded
to the non-base-pairing hydrogen of the guanine C2 amine, and
one of the pyrimidoblamic acid C4 amine hydrogens was
hydrogen bonded to guanine N3, providing a triplex-like
recognition interaction in the minor groove (Figure 2). These
observations provide a structural basis for the metal binding
domain control of the cleavage sequence selectivity which had
been implicated in preceding studies.32
This key interaction potentially provides the basis for the
sequence-selective cleavage of DNA by 1 and 2 and explains
the requirement for the guanine C2 amine for 5′-GC/5′-GT
cleavage.49-51 However, this interaction differs from the earlier
Sugiura and Dickerson models that enlist bithiazole minor
groove binding and its hydrogen bonding to the cleavage site
guanine C2 amine.50,51 Rather, it defined a previously unrec-
ognized role of the bleomycin A2 pyrimidine C4 amine which
the analogues 3 and 4 and their epimers 6 and 7 may address
directly. As detailed herein, both changes dramatically reduce
the DNA cleavage efficiency (10-30×). The C4 dimethyl-
amino substitution with 3 and 6 results in the loss of the
(49) Bailly, C.; Waring, M. J. J. Am. Chem. Soc. 1995, 117, 7311. Bailly,
C.; Kenani, A.; Waring, M. J. FEBS Lett. 1995, 372, 144. Suh, D.; Povirk,
L. F. Biochemistry 1997, 36, 4248.
(50) Kuwahara, J.; Sugiura, Y. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
2459. Sugiura, Y.; Suzuki, T. J. Biol. Chem. 1982, 257, 10544.
(51) Dickerson, R. E. In Mechanisms of DNA Damage and Repair:
Implications for Carcinogenesis and Risk Assessment in Basic Life Sciences;
Sini, M. G., Grossman, L., Eds.; Plenum: New York, 1986; p 245.
(52) Otsuka, M.; Kobayashi, S.; Ohno, M.; Umezawa, Y.; Morishima,
H.; Umezawa, H. Chem. Pharm. Bull. 1985, 33, 515.
(53) Boger, D. L.; Honda, T. Tetrahedron Lett. 1993, 34, 1567.

56 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
Scheme 1
13 (21 + 22:23) corresponds exactly to that observed in our
efforts on the synthesis of pyrimidoblamic acid (87:13)43 where
the relative and absolute configurations were unambiguously
established for all three diastereomers by independent synthesis.
Thus, they would appear to be safely assigned. Nonetheless,
to ensure that such an ambiguity might not inadvertently alter
the interpretation of the properties, both diastereomers 24 and
27 were carried forward to provide the final agents 3 and 6.
Aminolysis of the acyloxazolidinone (16% NH3-EtOH, 0 °C,
1 h, 65-70%) followed by ethyl ester hydrolysis of 25 and 28
provided 26 and 29, respectively.
Coupling of both 26 and 29 with pentapeptide S (11, 1.5 equiv
of DPPA, 3 equiv, i-Pr2NEt, DMF, 0 °C, 12 h, 69-72%) and
Scheme 2
addition of 19 to the stannous (Z)-enolate 20, generated by
treatment of the corresponding oxazolidinone with i-Pr2NEt (2.2
equiv) in the presence of Sn(OTf)2 (2.0 equiv), provided a
separable mixture of 21-23 (75%, 5.5:1:1 ratio). Both 21 and
22 provided 24 upon reductive removal of the thiomethyl group
(Bu3SnH, C6H6, 80 °C, 1 h, 90-92%) and thus possess the same
configuration at the newly introduced amine center. In contrast,
23 provided the diastereomer 27 upon reductive removal of the
thiomethyl group and the unnatural (R)-configuration at the
newly introduced amine center. Analogous to observations
made in our synthesis of pyrimidoblamic acid,43 1H NMR
analysis (J, C4-H/C5-H) of the cyclic carbamate 30 (J ) 1.6
Hz, eq 1) established that the major product 21 is the anti aldol
imine addition product (J ) 1.5 Hz typically), while 22
constitutes the minor syn addition product (J ) 5.6 Hz typically).
The remaining minor diastereomer 23, which possessed the
unnatural (R)-configuration at the newly introduced amine
center, was assigned the anti aldol imine addition product
stereochemistry by analogy to our prior work.43 Although we
did not unambiguously establish the absolute configuration at
the newly introduced amine center, the diastereoselection of 87:

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 57
Scheme 3
Scheme 5
Scheme 4
for 1 and 243 failed to provide clean C2 ethyl ester reduction
and typically provided both C2 and C6 reduction (1:1-4,
respectively) under a range of reaction conditions. Subsequent
oxidations of the corresponding alcohols provided 17 (pyr-SO3,
Et3N, DMSO-CH2Cl2, 0 °C, 1 h, 70%) and 36 (BaMnO4, CH2-
Cl2, 20-25 °C, 12 h, 80%).
Synthesis of C4 Desaminopyrimidoblamic Acid and Its
Epimer and Their Incorporation into 4 and 7. Following
unsuccessful efforts at reductive deamination of a pyrimido-
blamic acid precursor, the synthesis of 46 and its epimer 49
was accomplished as shown in Scheme 5. Reductive dechlo-
rination of 15 (H2, Pd-C, 2 N aqueous KOH-Et2O 1:1, 25
°C, 1 h, 88%) under conditions that resulted in ethyl ester
hydrolysis cleanly provided 37. Methyl ester formation (CH3I,
K2CO3, DMF, 25 °C, 10 h, 74%) followed by acetal hydrolysis
(91%) of 38 provided the key aldehyde 39 (Scheme 5).
Condensation of 39 with 1841 followed by addition of 40 to the
stannous (Z)-enolate 20 provided a separable 7:1:1 mixture of
41-43 (73%). Both 41 and 42 provided 44 upon reductive
removal of the thiomethyl group, indicating that they both
subsequent acid-catalyzed deprotection of the NR-BOC group
(20% TFA-CH2Cl2, 0 °C, 1.5 h, 86-88%) provided 3 and 6,
respectively (Scheme 3).
An alternative and less satisfactory preparation of 17 was
also accomplished from the key intermediate 33 derived from
the [4 + 2] cycloaddition of 1-(dimethylamino)propyne with
2,4,6-tris(ethoxycarbonyl)-1,3,5-triazine (Scheme 4).43,54,55 At-
tempted selective reduction of 33 (1 equiv of NaBH4, EtOH,
20-25 °C, 5 h) analogous to the successful efforts employed
(54) Boger, D. L.; Dang, Q. Tetrahedron 1988, 44, 3379.
(55) Ott, E. Chem. Ber. 1919, 52, 656.

58 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
Scheme 6
Figure 3. Agarose gel illustrating the cleavage reactions of supercoiled
ΦX174 DNA by Fe(II)-3 and 6 (top) or 4 and 7 (bottom) at 25 °C for
1 h in buffer solutions containing 2-mercaptoethanol. After electro-
phoresis on a 1% agarose gel, the gel was stained with 0.1 µg/mL
ethidium bromide and visualized on a UV transilluminator and
quantified on a Millipore BioImage 60S RFLP system. The results are
tabulated in Table 1.
at cleaving ΦX174 RFI DNA than deglycobleomycin A2 and
only 1.5-2× more effective than uncomplexed Fe(II) itself.
Their epimers 6 and 7 were even less effective and were nearly
indistinguishable from Fe(II) itself.
The relative extent of double-strand (ds) to single-strand (ss)
DNA cleavage was established in a study of the kinetics of
supercoiled ΦX174 DNA cleavage to produce linear and circular
DNA. The reactions exhibit initial fast kinetics in the first 1-5
min, and the subsequent decreasing rate may reflect conversion
to a less active or inactive agent or the kinetics of metal complex
reactivation. We assumed a Poisson distribution for the
formation of ss and ds breaks to calculate the average number
of ds and ss cuts per DNA molecule using the Freifelder-
Trumbo equation.56 The ratios of ds to ss cleavages observed
with the Fe(II) complexes are summarized in Table 1. The ratios
for 3 and 4 were established to be 1:53 and 1:45, and those of
their epimers 6 and 7 were 1:61 and 1:48, respectively.
Consistent with the relative efficiencies of DNA cleavage, this
was substantially lower than that of bleomycin A2 (1:6) or
deglycobleomycin A2 (1:12) and is nearly indistinguishable from
the ratio derived from uncomplexed Fe(II) cleavage (1:98). A
theoretical ratio of approximately 1:100 is required for the linear
DNA to be the result of the random accumulation of ss breaks
within the 5386 base-pair size of ΦX174 RFI DNA assuming
that sequential cleavage on the complementary strands within
15 base pairs is required to permit formation of linear DNA.
Thus, all four agents have effectively lost their ability to promote
ds cleavage of DNA. The significance of these observations is
not yet completely understood. It is not yet clear whether the
second cleavage adheres to a special sequence selectivity. Early
studies examining a limited number of ds cleavage sites implied
no special selectivity,57 while more recent studies have clearly
defined hot spots for ds cleavage that include 5′-GT cleavage
on both strands, i.e. 5′-GTAC.58 It could be argued that the
ds:ss cleavage ratio with 3 and 4 and their epimers should not
diminish with the decreased cleavage efficiency if the former
is accurate while the later would be substantially effected if its
basis for selectivity also involved the triplex-like hydrogen bond
model. While the results suggest that the latter may prove
accurate, typically, the ratio of ds:ss cleavage diminishes as the
cleavage efficiency decreases, and the results with 3 and 4 and
possess the same natural (S)-configuration at the newly intro-
duced amine center while 43 provided the diastereomer 47.
Although we did not unambiguously establish the absolute or
relative stereochemistry for the imine aldol addition products,
the diastereoselection of 89:11 (41 + 42:43) and relative
amounts of each of the three products parallel those observed
in our preceding efforts on pyrimidoblamic acid (87:13)43 where
they were unambiguously established and are analogous to those
defined in Scheme 2. However, to ensure that an inadvertent
misassignment might not alter the interpretation of the proper-
ties, both diastereomers were carried forward to provide 4 and
7. Following reductive desulfurization (3 equiv of Bu3SnH,
C6H6, 80 °C, 1 h, 89-91%), aminolysis of the acyloxazolidi-
nones 44 and 47 (16% NH3-EtOH, 0 °C, 52-58%) and final
methyl ester hydrolysis of 45 and 48 (LiOH, t-BuOH-H2O (2:
1), 0 °C, 1 h, 91-92%) afforded 46 and 49, respectively.
Coupling of both 46 and 49 with pentapeptide S (11, 1.5 equiv
of DPPA, 3 equiv of i-Pr2NEt, DMF, 0 °C, 12 h, 68-71%) and
subsequent acid-catalyzed deprotection of the NR-BOC group
(20% TFA-CH2Cl2, 0 °C, 1.5 h, 86-88%) cleanly provided 4
and 7, respectively (Scheme 6).
DNA Cleavage Properties. Four assays were used to
examine the DNA cleavage properties of 3 and 4 and their
epimers. The initial study of the relative efficiency of DNA
cleavage was conducted with the Fe(II) complexes and super-
coiled ΦX174 DNA in the presence of O2 and 2-mercaptoet-
hanol. Like Fe(II)-bleomycin A2 and deglycobleomycin A2,
the Fe(II) complexes of all four agents produced single- and
double-strand cleavage to afford relaxed (form II) and linear
(form III) DNA, respectively (Figure 3, Table 1). Both 3 and
4 were found to be substantially less effective (12× and 17×)
(56) Freifelder, D.; Trumbo, B. Biopolymers 1969, 7, 681.
(57) Povirk, L. F.; Han, Y.-H.; Steighner, R. J. Biochemistry 1989, 28,
5808. Steighner, R. J.; Povirk, L. F. Proc. Natl. Acad. Sci. U.S.A. 1990,
87, 8350.

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 59
Table 1. Summary of ΦX174 and w794 DNA Cleavage Properties
relative efficiency of DNA cleavagea
ratio of double to
agent
ΦX174a
w794b
single strand cleavagec
DNA cleavage selectivityb
1, bleomycin A2
2, deglycobleomycin A2
3
2-5
1.0
5.8
1.0
1:6
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
disrupted
weak 5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
disrupted
1:12
1:53
1:45
1:29
1:61
1:48
1:98
0.08
0.06
0.25
0.04
0.03
0.04
0.09
0.03
0.04
0.10
0.03
0.03
4
544
6
7
weak 5′-GC, 5′-GT > 5′-GA
none
Fea,b
a Relative efficiency of supercoiled ΦX174 DNA cleavage, Fe(II)-O2, 2-mercaptoethanol. The results are the average of six experiments.
b Examined within 5′ 32P-end-labeled w794, Fe(III)-H2O2. The results are the average of four experiments. c Ratio of double- to single-stranded
cleavage of supercoiled ΦX174 DNA calculated as FIII ) n2 exp(-n2), FI ) exp[-(n1 + n2)].
their epimers also suggest that they may simply conform to this
established trend.40-48
Consistent with past observations,44 the natural epimers 3 and
4 were slightly more effective (2×) than the unnatural epimers
6 and 7. In past studies where this has been examined (e.g., 5
versus 2),44 the unnatural epimers proved to be 2-4× less
effective than the natural epimers and indistinguishable from
the agents that lack the acetamido side chain altogether. The
comparisons of 3 and 4 with 6 and 7 conform nicely to these
past trends, further establishing the stereochemical assignments.
The prior studies suggested that the acetamido side chain natural
configuration may contribute to adoption of a metal chelate or
DNA-bound conformation productive for cleavage while the
unnatural epimers lack this enhancement but are not adversely
affected by the presence of the acetamido side chain. Finally,
in both the assays with ΦX174 supercoiled DNA, 3 and 4 and
their epimers 6 and 7 are among the worst analogues of
deglycobleomycin A2 examined to date that contain a single
site structural change.
Most revealing was the comparison of the DNA cleavage
selectivity of 3 and 4 determined using end-labeled duplex
DNA59 and a hairpin oligonucleotide previously used to study
ds cleavage.58 The selectivity of DNA cleavage and an
additional assessment of the relative efficiency of DNA cleavage
were examined with duplex w794 DNA59 by monitoring strand
cleavage of singly 32P 5′-end-labeled double-stranded DNA by
60
the Fe(III) complexes upon activation with H2O2 in 10 mM
phosphate buffer (pH 7.0). This protocol has proven to be more
sensitive to the distinctions in the relative efficiency of DNA
cleavage than the ΦX174 supercoiled DNA cleavage assays,
Figure 4. Cleavage of double-stranded DNA by Fe(III)-3 and 6 (SV40
DNA fragment, 144 base pairs, nucleotide no. 5238-138, clone w794)
in phosphate/KCl buffer containing H2O2. The DNA cleavage reactions
were run for 90 min at 4 °C, and electrophoresis was run on an 8%
but both have always provided the same trends in our hands.
denaturing PAGE and visualized by autoradiography.
Thus, incubation of the labeled duplex DNA with the agents in
Fe(III) complexes of both agents were 3× more effective than
the presence of equimolar FeCl3 and excess H2O2 led to DNA
Fe(III) itself but 10× less effective than deglycobleomycin A2.
cleavage. Following a quench of the reaction with the addition
Under a range of experimental conditions, the DNA cleavage
of glycerol, removal of the agent by EtOH precipitation of the
selectivity characteristic of bleomycin A2 and deglycobleomycin
DNA, resuspension of the treated DNA in aqueous buffer, and
A2 was essentially lost. Only when the assay was conducted
at 4 versus 25 or 37 °C were the remnants of the characteristic
cleavage detectable with 3, and even then, the cleavage pattern
is substantially altered (Figure 4). Within w794 DNA small
high-resolution polyacrylamide gel electrophoresis (PAGE) of
the resultant DNA under denaturing conditions adjacent to
Sanger sequencing standards permitted the identification of the
sites of DNA cleavage. Typical comparisons for agent 3 and
enhancements above nonselective cleavage were observed at
its epimer 6 are illustrated in Figure 4.
sites characteristic of 2 but typically appeared as doubled bands
Under all conditions examined, 3 and 6 were found to cleave
indicating proximal or adjacent cleavage sites. The clear, crisp
DNA only slightly above background Fe(III) (Table 1). The
pattern of 2 is not observed, and this slight cleavage preference
(58) Absalon, M. J.; Stubbe, J.; Kozarich, J. W. Biochemistry 1995, 34,
barely rises above nonselective cleavage. At 25 or 37 °C, this
2065. Absalon, M. J.; Wu, W.; Stubbe, J.; Kozarich, J. W. Biochemistry
slight preference disappears. The epimer 6 was even less
1995, 34, 2076.
selective and cleaves DNA with essentially no selectivity. We
have interpreted these observations to indicate that the charac-
teristic sequence-selective cleavage of 1 and 2 is disrupted by
the C4 dimethylamino substitution of the pyrimidoblamic acid
subunit.
(59) Ambrose, C.; Rajadhyaksha, A.; Lowman, H.; Bina, M. J. Mol. Biol.
1989, 209, 255. Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J.; Bina, M. Tetrahedron 1991, 47, 2661.
(60) Natrajan, A.; Hecht, S. M.; van der Marel, G. A.; van Boom, J. H.
J. Am. Chem. Soc. 1990, 112, 3997. Natrajan, A.; Hecht, S. M.; van der
Marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1990, 112, 4532.

60 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
Figure 6. Hairpin oligonucleotide containing GTAC hot spot for
double-strand cleavage. 32P labeling site is marked by . Good ss
*
cleavage sites are underlined; the ds cleavage site is in bold.
DNA cleavage but does so with largely the same sequence and
chemical specificity as the Fe bleomycins.61 Thus, it is not
surprising that at 25 °C the Co(III) hydroperoxide complexes
of 3, 4, 6, and 7 exhibited analogous decreases in cleavage
efficiency and all four resulted in a loss of the cleavage
selectivity relative to 2 (data not shown).61
Recently, a method for quantitatively evaluating the ratio of
ss versus ds cleavage has been developed using a hairpin
(51mer) oligonucleotide in which a hot spot for ds cleavage, a
GTAC box, is built into the center of the duplex region and is
flanked by good ss cleavage sites at C10 and C43 (Figure 6).58
Initially, efforts focused on the use of Fe(II) and O2 to generate
the activated bleomycin analogues. Since essentially no cleav-
age had been observed under these conditions even in the
absence of carrier DNA for ineffective analogues, compounds
3 and 4 were activated by an alternative protocol using Fe(III)
and H2O2. The cleavage efficiency with this construct was
estimated to be at least 50 times less than that of bleomycin.
Furthermore, even at 4 °C, the cleavage pattern resembled that
of hydroxide radical with essentially no specificity for cleavage
at GT or GC’s in this DNA shown to be excellent sites for ss
cleavage (Figure 7). In addition no apparent ds cleavage band
was observed.
The lack of significant sequence specificity for 4 seems to
differ somewhat from the studies with w794 DNA. A careful
examination of the w794 sequence reveals a few GC and GT
sites cleaved by 2, but not by 4 or 7 (see Figure 5). Around
these sites nonselective chemistry appears to occur. Even with
the selective w794 cleavage conducted at 4 °C, the selectivity
is weakened and suffers from competitive nonselective strand
breakage. Since there are only two GC and GT cleavage sites
in the hairpin oligonucleotide, these results suggest that different
sequence contexts may have different affinities for the analogues
which could lead to different cleavage efficiencies. Moreover,
since the method of activation required to detect cleavage is
effecting multiple turnovers, the results suggest that 3 and 4
can shift between selective iron-oxo chemistry and nonselective
iron-oxo or hydroxide radical chemistry and that, in the absence
of an effectively bound conformation, the latter may dominate.
Importantly, these results provide additional support that the
putative H-bonding interaction between the 4-amino group of
the pyrimidine plays a key role in the specificity and efficiency
of both ss and ds cleavage.
Figure 5. Cleavage of double-stranded DNA by Fe(III)-4 and 7 (SV40
DNA fragment, 144 base pairs, nucleotide no. 5238-138, clone w794)
in phosphate/KCl buffer containing H2O2. The DNA cleavage reactions
were run for 90 min at 4 °C, and electrophoresis was run on an 8%
denaturing PAGE and visualized by autoradiography.
A comparison of the DNA cleavage sequence selectivity for
the desamino agent 4 and its epimer 7 proved even more
interesting, and a representative examination is illustrated in
Figure 5 while the results are summarized in Table 1. Both
agents were found to cleave w794 double-stranded DNA
substantially less effectively (30×) than deglycobleomycin A2
or even 3 and 6. Unlike 3 and 6, the DNA cleavage sequence
selectivity characteristic of 1 and 2 remained when the amino
group on the pyrimidine ring was removed. However, the ability
to detect the cleavage selectivity was most pronounced at 4 °C
and, unlike 1 and 2, diminished as the assay temperature was
raised to 25 or 37 °C. Even at 4 °C, the cleavage selectivity
was not nearly as clear or crisp as that of 1 or 2, and suffers
from competitive nonselective cleavage. Although similar, the
cleavage selectivity of both 4 and 7 is distinguishable from that
of 1 and 2. For example, the clean and pronounced 5′-GC
cleavage of deglycobleomycin A2 at G113 and the weak 5′-GT
cleavage at G61 are not observed with either 4 or 7. The
significance of this subtle alteration is not yet known. In
addition, the relative efficiency of cleavage at the minor 5′-AT
sites was diminished and essentially disappeared. Our inter-
pretation of these observations is that the removal of the
pyrimidoblamic acid C4 amine removes the key guanine N3
hydrogen bond to the C4 amine but does not preclude formation
of the second hydrogen bond from the guanine C2 NH2 to the
pyrimidine N3 of 4. Thus, the effectiveness of the interaction
is diminished by the loss of one hydrogen bond but not
completely lost. Both epimers 4 and 7 were nearly indistin-
guishable in their efficiency, and both were comparable to, or
perhaps even slightly less effective than, uncomplexed Fe(III).
The Co(III) hydroperoxide complex of bleomycin A2 stoi-
chiometrically versus catalytically cleaves DNA only in the
presence of light by a mechanism that precludes double-stranded
Oxidation Capabilities of 2-4 and 6 and 7. In final efforts
to characterize the properties of the agents, the abilities of their
(61) Chang, C.-H.; Meares, C. F. Biochemistry 1982, 21, 6332. Chang,
C.-H.; Meares, C. F. Biochemistry 1984, 23, 2268. Suzuki, T.; Kuwahara,
J.; Goto, M.; Sugiura, Y. Biochim. Biophys. Acta 1985, 824, 330. Saito, I.;
Morii, T.; Sugiyama, H.; Matsura, T.; Meares, C. F.; Hecht, S. M. J. Am.
Chem. Soc. 1989, 111, 2307. Worth, Jr., L.; Frank, B. L.; Christner, D. F.;
Absalon, M. J.; Stubbe, J.; Kozarich, J. W. Biochemistry 1993, 32, 2601.
The cleavage studies were conducted by formation of the Co complexes
(equimolar CoCl2, 2 h, 25 °C, open to air, 0.5-128 µM), addition of w794
end-labeled DNA in pH 7 phosphate buffer, and illumination of the mixture
with a 312-nm UV illuminator (20 min, 25 °C). Workup and PAGE of the
cleavage reactions was conducted as described for the Fe(III) complex
cleavages of DNA. Appropriate controls of CoCl2 alone, agents in the
absence of metal, and reactions conducted in the dark or under room light
ensured that the DNA cleavage reactions were due to the Co(III)-agent
complexes initiated by 312-nm irradiation. Under these conditions 0.5-2
µM deglycobleomycin A2 selectively cleaved the w794 DNA.

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 61
examination of 3, 4, 6, and 7 revealed that the same products
and same product distribution were observed with all four
analogues and 2. Thus, like 2, 3, 4, 6, and 7 provide competent
Fe-oxo intermediates distinguishable from hydroxide radical
oxidations. The relative efficiencies of the two dimethylamino
analogues (3 and 6) were nearly indistinguishable from that of
2 itself, while the efficiencies of the two desamino analogues
(4 and 7) were somewhat lower. This is consistent with the
expectation that a pyrimidine C4 electron-donating substituent
would improve the oxygen activation properties of the metal
complexes.2,31 However, both 4 and 7 were still quite effective,
providing roughly four oxidations per Fe(III) complex, and this
subtle distinction from 2 is insufficient to account for the
distinctions in the DNA cleavage efficiencies of the agents. This
is especially true of 3 and 6, which possess the oxidation
capabilities of 2 but which failed to produce the characteristic
DNA cleavage selectivity.
Discussion. The synthesis and examination of 3 and 4 and
their unnatural epimers 6 and 7 by following an alternative
approach to the preparation of 2 provide direct evidence for
the critical role that the pyrimidoblamic acid C4 amine plays
in the polynucleotide recognition of bleomycin A2. These
results support the proposal that CoBLM A2 is a good mimic
of “activated iron BLM” and that the NMR spectrum of the
CoBLM species bound site specifically to DNA provides a
useful model for the physiologically important FeBLM A2.35
Substitution of 2 with a pyrimidine C4 dimethylamino group
did not significantly alter the inherent metal chelation, oxygen
activation, or oxidation capabilities of the resulting agent 3 but
did substantially diminish the DNA cleaving efficiency of the
agent (10-15×) and resulted in the loss of the characteristic
5′-GC/5′-GT cleavage selectivity of the parent agent 2. Simi-
larly, the removal of the C4 pyrimidine amino group of 2 only
subtly reduced the oxidation capabilities of the resulting agent
4 (1.6×) but had a much more substantial diminishing effect
on the DNA cleavage efficiency of the agent (30×). The
characteristic 5′-GC/5′-GT cleavage selectivity of the agent 4
was substantially diminished, and the ability to detect the
selectivity above nonselective cleavage was greatly reduced,
requiring assay conditions of 4 versus 25-37 °C. Both of these
observations are consistent with the involvement of the pyri-
midine C4 amine in one of a pair of hydrogen bonds in a triplex-
like recognition of the cleavage site guanine (Figure 2).
Substitution with the C4 dimethylamino group not only pre-
cludes the formation of a pyrimidine C4 amine/guanine N3
hydrogen bond but also sterically prevents formation of the
remaining guanine C2 amine/pyrimidine N3 hydrogen bond,
reducing the cleavage efficiency and destroying the cleavage
selectivity of the agent. In contrast, removal of the C4 amino
only precludes formation of the pyrimidine C4 amine/guanine
N3 hydrogen bond but does not prevent formation of the
remaining guanine C2 amine/pyrimidine N3 hydrogen bond. The
resulting interaction involving only one but not both of the key
hydrogen bonds is weaker. Consequently, the DNA cleavage
efficiency is substantially reduced (30× relative to 2) and the
cleavage selectivity more difficult to detect (4 versus 25-37
°C).
Figure 7. Cleavage of internally radiolabeled hairpin oligonucleotide
by Fe(III)-3 and 4 in HEPES/NaCl buffer containing H2O2: Lane 1,
G+A Maxam-Gilbert standard (5′-radiolabeled hairpin oligonucle-
otide); lane 2, 1 µM BLM; lane 3, 400 µM 3; lane 4, 200 µM 3; lane
5, 400 µM 4; lane 6, 200 µM 4. Lanes 1 and 2 were run on a separate
gel under identical conditions and lined up according to an internal
standard.
Table 2. Styrene Oxidationa
styrene
oxide phenylacetaldehyde
total
product
rel
agent
(mM)
(mM)
ratio (mM) efficiency
Fe(III)-2 1.80
Fe(III)-3 1.67
Fe(III)-4 1.11
Fe(III)-6 1.55
Fe(III)-7 1.07
1.32
1.21
0.88
1.00
0.79
0
1.36
1.38
1.26
1.55
1.35
3.12
2.88
1.99
2.55
1.86
0
1.0
0.92
0.64
0.82
0.59
0
Fe(III)b
0
0
Fe(III)-2
(no H2O2)
0
0
0
a 500 µM Fe(III)-agent, 50 mM styrene, 30 mM H2O2, 0 °C, 1.5 h,
80% CH3OH-H2O. Results reported are the average of two runs. b 500
µM Fe(III) under identical conditions with H2O2 present.
Fe(III) complexes to mediate the oxidation of styrene were
investigated.62 The oxidation of styrene by deglycobleomycin
A2 produces both styrene epoxide and phenylacetaldehyde. A
solution of 500 µM Fe(III)-2, 50 mM styrene, and 30 mM H2O2
(0 °C, 1.5 h) produced 1.80 mM styrene epoxide and 1.32 mM
phenylacetaldehyde constituting slightly over six oxidations for
each Fe(III)-2 utilized (Table 2).
Although the pyrimidine C4 amine of bleomycin A2 is not
directly engaged in the metal chelation, the electronic character
of C4 substituents has been shown to affect the oxygen
activation properties of the agents.31,63 Electron-donating sub-
stituents have been found to increase the O2 activation properties
of a series of related synthetic metal chelation subunits.2
Notably, a C4 dimethylamino substituent incorporated into a
pyridine analogue (PYML-8) of the pyrimidoblamic acid subunit
has been reported to be superior at activating O2.2,31 The
These observations have further implications on the inherent
DNA cleavage selectivity of bleomycin A2 itself. Bleomycin
A2 not only cleaves essentially all 5′-GT and 5′-GC sites in
duplex DNA but also cleaves 5′-AT and 5′-AC sites albeit less
effectively (25-40% versus 100% of available sites) and
typically does so with a weaker relative efficiency of cleavage
(Table 3). Although many of the minor or unusual cleavage
(62) Hamamichi, N.; Natrajan, A.; Hecht, S. M. J. Am. Chem. Soc. 1992,
114, 6278.
(63) Loeb, K. E.; Zaleski, J. M.; Westre, T. E.; Guajardo, R. J.;
Mascharak, P. K.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am.
Chem. Soc. 1995, 117, 4545.
(64) Full characterization of intermediates are provided in the Supporting
Information.

62 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
Table 3. Summary of DNA Cleavage Sites for Fe(III)-Bleomycin
13C NMR (CD3OD, 100 MHz) δ 168.0, 161.4, 156.9, 154.9, 148.6,
146.7, 140.9, 128.3, 126.7, 122.7, 116.2, 73.6, 66.9, 65.8, 57.1, 41.6,
39.9, 37.4; IR (neat) νmax 3338, 2927, 1651, 1549, 1497, 1446, 1364,
1251, 1148 cm-1; FABHRMS (NBA-CsI) m/z 1082.4361 (M+,
C54H68N9O9S3 requires 1082.4302).
a
A2
no. of
cleavage no. of
total
no. of
cleavage no. of
total
%
%
site
sites
sites cleaved site
sites
sites cleaved
(3-(2′-(2-((2(S)-(N-(4(R)-(N-(2(S)-Amino-3(R)-hydroxy-3-imida-
zol-4-ylpropanoyl)amino)-3(S)-hydroxy-2(S)-methylpentanoyl)amino)-
3(R)-hydroxybutanoyl)amino)ethyl)-2,4′-bithiazole-4-carboxamido)-
5′-GC
5′-GT
5′-GA
5′-GG
5′-AT
5′-AC
5′-AA
5′-AG
29
5
11
0
7
2
29
5
14
28
18
7
100
100
79
0
39
28
13
0
5′-TT
5′-TA
5′-TC
5′-TG
5′-CT
5′-CC
5′-CA
5′-CG
1
1
0
0
1
0
0
0
13
15
19
10
20
38
18
17
8
7
0
0
5
0
0
0
propyl)dimethylsulfonium Chloride (11, Pentapeptide S).
A
suspension of 10 (20 mg, 0.018 mmol) in CH2Cl2 (200 µL) at 0 °C
was treated with 20% TFA-CH2Cl2 (200 µL) and the mixture stirred
at 0 °C for 2.5 h before the solvent was evaporated in vacuo under a
stream of N2. The residue was dissolved in CH3OH, and the solution
was treated with aqueous NH4OH (28%, 20 µL) and stirred at 23 °C
(1 h). The solvent was removed in vacuo and the mixture purified by
reverse-phase chromatography (C-18, 1.0 × 4 cm, 0-60% CH3OH-
H2O), affording 11 (12.3 mg, 14.0 mg theoretical, 88%) as a white
film: Rf ) 0.25 (SiO2, 10:9:1 CH3OH-10% aqueous CH3CO2NH4-
10% aqueous NH4OH); [R]25D +6.3 (c 0.20, CH3OH); 1H NMR (CD3-
OD, 400 MHz) δ 8.21 (s, 1H), 8.13 (s, 1H), 7.79 (br s, 1H), 7.15 (br
s, 1H), 5.04 (d, J ) 5.5 Hz, 1H), 4.29 (d, J ) 4.0 Hz, 1H), 4.14 (d, J
) 5.5 Hz, 1H), 4.09 (dd, J ) 6.0, 6.0 Hz, 1H), 3.87 (m, 1H), 3.67 (m,
4H), 3.59 (dd, J ) 7.0, 7.0 Hz, 2H), 3.37 (dd, J ) 7.5, 7.5 Hz, 2H),
3.28 (dd, J ) 6.5, 6.5 Hz, 2H), 2.93 (s, 6H), 2.52 (dq, J ) 7.0, 7.0 Hz,
1H), 2.14 (tt, J ) 7.0, 7.0 Hz, 2H), 1.18 (d, J ) 6.5 Hz, 3H), 1.12 (d,
J ) 6.5 Hz, 3H), 1.05 (d, J ) 6.5 Hz, 3H); 13C NMR (CD3OD, 100
MHz) δ 177.8, 172.7, 170.8, 166.2, 164.1, 161.2, 152.4, 149.5, 136.7,
132.3, 125.4, 118.6, 117.8, 75.5, 68.5, 66.2, 59.7, 58.4, 44.8, 42.6, 40.1,
38.6, 33.6, 25.6, 25.3, 20.1, 15.6, 14.3; IR (neat) νmax 3266, 2937, 1662,
1549, 1431, 1199, 1127 cm-1; FABHRMS (NBA-CsI) m/z 740.2662
(M+, C30H46N9O7S3 requires 740.2682).
3
0
24
22
a Within w794 and w836 DNA, ref 44.
Figure 8.
sites could result from ds cleavage originating from primary
sites on the complementary strand, the more general cleavage
preference of 5′-GPy versus 5′-APy may be analogously
attributed to reduced binding affinity at the 5′-APy sites resulting
from one versus two triplex-like hydrogen bonds to adenine
(Figure 8). Thus, the behavior of 4 has shed further light on
the potential origin of the inherent 5′-GPy > 5′-APy cleavage
selectivity of bleomycin A2. These results complement the
studies where Waring and co-workers have shown that the
cleavage is greatly diminished but not lost when guanine is
replaced with inosine and that when adenine is replaced with
2-aminoadenine the cleavage at pyrimidines 3′ to this site is
greatly enhanced.49 Both of these complementary single site
modifications in DNA also support the basis of the hydrogen-
bonding specificity proposed.
Deglycobleomycin A2 (2). A solution of 12 (3.6 mg, 8 µmol, 1.2
equiv) and 11 (5 mg, 7 µmol) in DMF (100 µL) at 0 °C under Ar was
treated with DPPA (2.2 µL, 10 µmol, 1.5 equiv) followed by i-Pr2NEt
(3.8 µL, 14 µmol, 3 equiv). The reaction mixture was stirred at 0 °C
for 10 h in the dark before the solvent was removed in vacuo.
Chromatography (C-18, 1.2 × 4 cm, 0-70% CH3OH-H2O gradient
elution) afforded 13 (6 mg, 8.1 mg theoretical, 72%) as a white solid.
A solution of 13 (6 mg, 5 µmol) in CH2Cl2 (100 µL) at 0 °C was
treated with 20% TFA-CH2Cl2 (100 µL), and the reaction mixture
was stirred at 0 °C for 2 h. The solvent was removed in vacuo.
Chromatography (C-18, 1.2 × 4 cm, 0-90% CH3OH-H2O gradient
elution) afforded 2 (4.6 mg, 5.5 mg theoretical, 86%) as a white solid
identical in all respects with authentic material.21,44
Ethyl 4-Chloro-2-(diethoxymethyl)-5-methylpyrimidine-6-car-
boxylate (15). A solution of 1452 (2.2 g, 7.64 mmol) in DMF (32
mL) at 23 °C under Ar was treated with freshly distilled SOCl2 (distilled
over collidine, 1.1 mL, 15.1 mmol, 2 equiv), and the mixture was stirred
(2 h). The mixture was treated with K2CO3 (0.5 g) and concentrated
to a red oil. Chromatography (SiO2, 40% EtOAc-hexane) afforded
15 (2.0 g, 2.3 g theoretical, 82%) as a pale yellow oil: Rf ) 0.5 (SiO2,
50% Et2O-hexane); 1H NMR (CDCl3, 400 MHz) δ 5.51 (s, 1H), 4.45
(q, J ) 7.0 Hz, 2H), 3.79 (dq, J ) 9.5, 7.0 Hz, 2H), 3.66 (dq, J ) 9.5,
7.0 Hz, 2H), 2.47 (s, 3H), 1.40 (t, J ) 7.0 Hz, 3H), 1.24 (t, J ) 7.0
Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 164.5, 163.8, 163.7, 157.5,
128.0, 101.4, 63.0, 62.6, 15.14, 15.08, 14.0; IR (neat) νmax 2978, 2931,
1732, 1558, 1532, 1381, 1334, 1225, 1115, 1060 cm-1; FABHRMS
(NBA-CsI) m/z 303.1122 (M + H+, C13H19N2ClO4 requires 303.1111).
Ethyl 2-(Diethoxymethyl)-4-(dimethylamino)-5-methylpyrimidine-
6-carboxylate (16). A solution of 15 (2.0 g, 6.50 mmol) in THF (5
mL) at 23 °C was treated with HN(CH3)2 (2 M in THF, 6.8 mL, 2.1
equiv), and the mixture was stirred (1.5 h). The reaction mixture was
diluted with H2O (10 mL) and extracted with EtOAc (5 × 10 mL),
dried (Na2SO4), and concentrated to a pale yellow oil affording 16 (1.98
g, 2.0 g theoretical, 97%) which required no further purification: Rf )
Experimental Section
(3-(2′-(2-((2(S)-(N-(4(R)-(N-(2(S)-((tert-Butyloxycarbonyl)amino)-
3(R)-hydroxy-3-(N-(triphenylmethyl)imidazol-4-yl)propanoyl)amino)-
3(S)-hydroxy-2(S)-methylpentanoyl)amino)-3(R)-hydroxybutanoyl)-
amino)ethyl)-2,4′-bithiazole-4-carboxamido)propyl)dimethyl-
sulfonium Chloride (10). A solution of tetrapeptide S42 (8, 26.4 mg,
0.042 mmol), 942 (25 mg, 0.049 mmol, 1.15 equiv), and BOP reagent
(27.8 mg, 0.063 mmol, 1.5 equiv) in DMF (450 µL) at 0 °C was treated
with i-Pr2NEt (14.6 µL, 0.084 mmol, 2 equiv) under Ar and stirred for
8 h. The DMF was removed in vacuo and the resulting oil triturated
with H2O (2 mL) and filtered through Celite. The resulting solid was
dissolved and eluted from the Celite with CH3OH and concentrated to
provide a white solid. Chromatography (C-18, 1.0 × 4.0 cm, 0-90%
CH3OH-H2O gradient elution) afforded 10 (35.2 mg, 46.9 mg
theoretical, 75%; 75-88%) as a white solid: Rf ) 0.5 (SiO2, 10:9:1
CH3OH-10% aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25
D
+21.5 (c 0.06, CH3OH); 1H NMR (CD3OD, 400 MHz) δ 8.18 (s, 1H),
8.12 (s, 1H), 7.42 (br s, 1H), 7.35 (m, 6H), 7.11 (m, 4H), 6.91 (br s,
1H), 4.81 (d, J ) 6.5 Hz, 1H), 4.36 (d, J ) 6.5 Hz, 1H), 4.32 (d, J )
4.0 Hz, 1H), 3.91 (m, 1H), 3.66 (m, 4H), 3.58 (dd, J ) 7.0, 7.0 Hz,
2H), 3.36 (dd, J ) 7.0, 7.0 Hz, 2H), 3.27 (dd, J ) 6.5, 6.5 Hz, 2H),
2.92 (s, 6H), 2.60 (dq, J ) 7.0, 7.0 Hz, 1H), 2.13 (tt, J ) 7.0, 7.0 Hz,
2H), 1.40 (s, 9H), 1.13 (d, J ) 6.5 Hz, 3H), 1.06 (d, J ) 6.5 Hz, 3H);
1
0.45 (SiO2, 50% Et2O-hexane); H NMR (CDCl3, 400 MHz) δ 5.48
(s, 1H), 4.39 (q, J ) 7.0 Hz, 2H), 3.76 (dq, J ) 9.5, 7.0 Hz, 2H), 3.63
(dq, J ) 9.5, 7.0 Hz, 2H), 3.13 (s, 6H), 2.28 (s, 3H), 1.38 (t, J ) 7.0
Hz, 3H), 1.21 (t, J ) 7.0 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ
166.7, 166.6, 161.7, 156.6, 113.7, 102.4, 62.6, 61.8, 40.6, 16.2, 15.2,
14.1; IR (neat) νmax 2975, 2931, 1735, 1565, 1535, 1400, 1379, 1215,

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 63
1116, 1059 cm-1; FABHRMS (NBA-CsI) m/z 444.0898 (M + Cs+,
C15H25N3O4 requires 444.0899).
6H), 2.91 (m, 2H), 2.30 (s, 3H), 2.04 (s, 3H), 1.45 (s, 9H), 1.38 (t, J
) 7.0 Hz, 3H), 0.81 (d, J ) 6.5 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
δ 173.9, 171.1, 166.8, 166.4, 165.7, 156.6, 155.8, 133.2, 128.8, 125.6,
112.4, 78.9, 78.8, 64.1, 61.9, 60.5, 54.8, 53.5, 49.9, 41.5, 40.6, 28.3;
4-(Dimethylamino)-6-(ethoxycarbonyl)-5-methylpyrimidine-2-
carboxaldehyde (17). A solution of 16 (1.97 g, 6.25 mmol) in
acetone-H2O (2:1, 25 mL) was treated with TsOH (108 mg, 0.62 mmol,
0.10 equiv), and the mixture was warmed at 80 °C (8 h). The mixture
was neutralized with the addition of 10% aqueous NaHCO3 and
extracted with EtOAc (5 × 20 mL), dried (Na2SO4), and concentrated
to an oil. Chromatography (SiO2, 30% EtOAc-hexane) afforded 17
(1.42 g, 1.60 g theoretical, 89%): Rf ) 0.35 (SiO2, 50% Et2O-hexane);
1H NMR (CDCl3, 400 MHz) δ 9.93 (s, 1H), 4.99 (q, J ) 7.0 Hz, 2H),
3.16 (s, 6H), 2.38 (s, 3H), 1.41 (t, J ) 7.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz) δ 191.4, 166.5, 165.8, 156.7, 155.7, 117.0, 62.5, 40.6, 16.7,
IR (neat) νmax 2919, 1772, 1681, 1558, 1365, 1193, 1054 cm-1
;
FABHRMS (NBA-CsI) m/z 820.2122 (M + Cs+, C32H45N7O8S
requires 820.2105).
Ethyl 2(S)-(1-((2(S)-((tert-Butoxycarbonyl)amino)-2-carbamoyl-
ethyl)amino)-2-(((4S,5R)-4-methyl-5-phenyl-2-oxazolidinon-3-yl)car-
bonyl)-2(S)-(methylthio)ethyl)-4-(dimethylamino)-5-methylpyrimidine-
6-carboxylate (23): Rf ) 0.33 (SiO2, 15% CH3OH-CHCl3); [R]25
D
1
+21.1 (c 0.085, CHCl3); H NMR (CD3OD, 400 MHz) δ 7.38 (m,
5H), 5.74 (d, J ) 8.5 Hz, 1H), 5.12 (d, J ) 11.0 Hz, 1H), 4.42 (q, J
) 7.0 Hz, 2H), 4.23 (m, 2H), 4.15 (d, J ) 11.0 Hz, 1H), 4.09 (m, 1H),
3.15 (s, 6H), 2.89 (m, 2H), 2.31 (s, 3H), 2.04 (s, 3H), 1.42 (s, 9H),
1.39 (t, J ) 7.0 Hz, 3H), 0.91 (d, J ) 6.5 Hz, 3H); 13C NMR (CD3OD,
100 MHz) δ 176.5, 173.5, 171.0, 168.0, 167.6, 166.5, 157.0, 154.5,
135.3, 128.8, 126.5, 125.6, 113.2, 80.3, 64.6, 63.0, 56.0, 55.7, 50.9,
41.0, 30.7, 28.7, 28.3, 16.6, 14.5; IR (neat) νmax 3337, 2929, 1778,
1687, 1563, 1359, 1193, 1059 cm-1; FABHRMS (NBA-CsI) m/z
820.2120 (M + Cs+, C32H45N2O8S requires 820.2105).
14.1; IR (neat) νmax 2938, 1728, 1568, 1403, 1380, 1203, 1061 cm-1
;
FABHRMS (NBA-NaI) m/z 260.1017 (M + Na+, C11H15N3O3 requires
260.1011).
NR-((tert-Butyloxy)carbonyl)-Nâ-(((4-(dimethylamino)-6-(ethoxy-
carbonyl)-5-methylpyrimidin-2-yl)methylene)amino)-(S)-â-aminoala-
nineamide (19). A solution of 17 (35 mg, 0.15 mmol) and 1841,43 (33.5
mg, 0.17 mmol, 1.1 equiv) in anhydrous CH3CN (1.5 mL) was stirred
at 23 °C (1 h). The reaction mixture was concentrated in vacuo and
pumped dry, affording 19 (63.4 mg, 63.4 mg theoretical, 100%) as a
Ethyl 2(S)-(1-((2(S)-((tert-Butoxycarbonyl)amino)-2-carbamoyl-
ethyl)amino)-2-(((4S,5R)-4-methyl-5-phenyl-2-oxazolidinon-3-yl)car-
bonyl)ethyl)-4-(dimethylamino)-5-methylpyrimidine-6-carboxy-
late (24). A solution of 21 (50 mg, 0.72 mm) in C6H6 (0.5 mL) was
treated with Bu3SnH (63 µL, 0.23 mmol, 3 equiv) and AIBN (2.5 mg,
0.015 mmol, 2.0 equiv), and the reaction mixture was warmed at 80
°C (1 h) under Ar. The mixture was cooled to 23 °C and the solvent
evaporated in vacuo. Chromatography (SiO2, 2 × 5 cm, 5% CH3OH-
CHCl3) afforded 24 (45 mg, 50 mg theoretical, 90%) as a white solid:
Rf ) 0.30 (5% CH3OH-CHCl3); [R]25D -38 (c 0.15, CHCl3); 1H NMR
(CD3OD, 400 MHz) δ 7.41 (m, 5H), 5.74 (d, J ) 7.0 Hz, 1H), 4.77
(dq, J ) 6.5, 7.0 Hz, 1H), 4.39 (q, J ) 7.0 Hz, 2H), 4.16 (dd, J ) 6.0,
8.0 Hz, 1H), 4.04 (dd, J ) 6.0, 6.0 Hz, 1H), 3.54 (dd, J ) 8.0, 16.0
Hz, 1H), 3.25 (dd, J ) 6.0, 16.0 Hz, 1H), 3.14 (s, 6H), 2.84 (dd, J )
6.5, 12.0 Hz, 1H), 2.76 (dd, J ) 5.5, 12.0 Hz, 1H), 2.28 (s, 3H), 1.41
(s, 9H), 1.37 (t, J ) 7.0 Hz, 3H), 0.86 (d, J ) 6.5 Hz, 3H); 13C NMR
(CD3OD, 100 MHz) δ 173.6, 170.3, 168.0, 167.6, 166.7, 164.4, 162.3,
157.5, 136.9, 129.5, 129.4, 127.1, 126.6, 113.5, 82.4, 63.0, 61.5, 55.5,
54.8, 53.5, 50.2, 40.9, 28.7, 17.5, 16.4, 14.4; IR (neat) νmax 3333, 2974,
1733, 1687, 1564, 1405, 1379, 1225, 1164 cm-1; FABHRMS (NBA-
CsI) m/z 774.2217 (M + Cs+, C31H43N7O8 requires 774.2227).
Similar treatment of 22 provided 24, identical in all respects.
NR-(tert-Butoxycarbonyl)-Nâ-(1-amino-3(S)-(4-(dimethylamino)-
6-(ethoxycarbonyl)-5-methylpyrimidin-2-yl)propion-3-yl)-(S)-â-ami-
noalanineamide (25). Solid 24 (24.7 mg, 0.036 mmol) was treated
with 15% NH3-EtOH (4.5 mL), and the solution was stirred for 1.0 h
at 0 °C. The solvent was evaporated in vacuo. Chromatography (SiO2,
2 × 5 cm, 10% CH3OH-CHCl3) afforded 25 (12.5 mg, 17.3 mg
theoretical, 72%) as a white solid: Rf ) 0.25 (10% CH3OH-CHCl3);
[R]25D -28 (c 0.28, CH3OH); 1H NMR (CD3OD, 400 MHz) δ 4.39 (q,
J ) 7.0 Hz, 2H), 4.12 (m, 1H), 4.01 (dd, J ) 5.0, 8.5 Hz, 1H), 2.82
(dd, J ) 12.0, 6.0 Hz, 1H), 2.78 (dd, J ) 12.0, 6.0 Hz, 1H), 2.62 (dd,
J ) 5.5, 15.0 Hz, 1H), 2.54 (dd, J ) 8.5, 15.0 Hz, 1H), 2.28 (s, 3H),
1.43 (s, 9H), 1.38 (t, J ) 7.0 Hz, 3H); 13C NMR (CD3OD, 100 MHz)
δ 176.5, 176.4, 168.0, 167.7, 167.0, 157.5, 113.5, 102.5, 80.7, 63.1,
61.9, 55.6, 41.9, 41.0, 28.7, 16.4, 14.4; IR (neat) νmax 3323, 2927, 1666,
1531, 1400, 1255, 1164 cm-1; FABHRMS (NBA-CsI) m/z 482.2715
(M + H+, C21H35N7O6 requires 482.2727).
1
white foam: [R]25 +38 (c 0.85, CH3OH); H NMR (CD3OD, 400
D
MHz) δ 8.18 (s, 1H), 4.40 (q, J ) 7.0 Hz, 2H), 4.14 (m, 1H), 4.03
(dd, J ) 5.0, 7.5 Hz, 1H), 3.87 (dd, J ) 5.0, 12.0 Hz, 1H), 3.15 (s,
6H), 2.28 (s, 3H), 1.37 (t, J ) 7.0 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 191.7, 172.9, 169.6, 166.6, 166.3, 163.6, 160.6, 157.3, 156.6,
155.5, 115.2, 70.1, 62.3, 61.5, 54.3, 40.6, 16.5, 14.1; IR (neat) νmax
3333, 2977, 1682, 1564, 1510, 1403, 1366, 1249, 1167, 1062 cm-1
;
FABHRMS (NBA-CsI) m/z 423.2369 (M + H+, C19H30N6O5 requires
423.2356).
Diastereoselective Reaction of the Stannous (Z)-Enolate of
(4S,5R)-3-((Methylthio)acetyl)-4-methyl-5-phenyl-2-oxazolidinone
with 19. A solution of Sn(OTf)2 (245 mg, 0.58 mmol, 4 equiv)
dissolved in anhydrous THF (1.0 mL) under Ar was cooled to -78 °C
and treated sequentially with (4S,5R)-3-((methylthio)acetyl)-4-methyl-
5-phenyl-2-oxazolidinone (78 mg, 0.29 mmol, 2 equiv) in anhydrous
THF (1 mL) and i-Pr2NEt (115 µL, 0.66 mmol, 4.5 equiv). The mixture
was stirred for 1 h at -20 °C for complete enolate formation, and the
reaction mixture was recooled to -78 °C. A solution of 19 (62 mg,
0.14 mmol, 1 equiv) in anhydrous THF (1.5 mL) was slowly added,
and the reaction mixture allowed to warm to 0 °C where it was stirred
for 7.5 h. The reaction mixture was poured into a two-layer solution
of CHCl3 (15 mL) and saturated aqueous NaHCO3 (7 mL) with vigorous
stirring. The organic layer was separated off, dried (MgSO4), and
concentrated in vacuo. Chromatography (SiO2, 3 × 6 cm, 5% CH3-
OH-CHCl3) gave 70.2 mg (96.2 mg theoretical, 75%) of a 5.5:1:1
mixture of the diastereomers. Further chromatography (PCTLC, 4 mm
SiO2, 4% CH3OH-CHCl3) afforded 21 (51 mg), 22 (10 mg), and 23
(9 mg) as white solids.
Ethyl 2(R)-(1-((2(S)-((tert-Butoxycarbonyl)amino)-2-carbamoyl-
ethyl)amino)-2-(((4S,5R)-4-methyl-5-phenyl-2-oxazolidinon-3-yl)car-
bonyl)-2(R)-(methylthio)ethyl)-4-(dimethylamino)-5-methylpyrimidine-
6-carboxylate (21): Rf ) 0.35 (SiO2, 15% CH3OH-CHCl3); [R]25
D
-26.3 (c 0.315, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.42 (m, 5H),
5.82 (d, J ) 7.5 Hz, 1H), 5.22 (d, J ) 11 Hz, 1H), 4.85 (m, 1H), 4.41
(q, J ) 7.0 Hz, 2H), 4.10 (m, 1H), 4.03 (dd, J ) 5.0, 5.0 Hz, 1H), 3.16
(s, 6H), 2.71 (dd, J ) 5.0, 5.0 Hz, 2H), 2.31 (s, 3H), 2.05 (s, 3H), 1.39
(t, J ) 7.0 Hz, 3H), 1.37 (s, 9H), 0.97 (d, J ) 6.5 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) δ 173.8, 173.7, 170.4, 166.8, 166.4, 156.1, 155.9,
152.7, 133.2, 129.0, 128.6, 125.7, 113.1, 79.8, 78.7, 64.1, 61.7, 54.9,
53.4, 49.1, 46.5, 40.6, 28.3, 16.3, 14.7, 14.2, 11.9; IR (neat) νmax 3347,
2078, 1777, 1690, 1566, 1365, 1196, 1061 cm-1; FABHRMS (NBA-
CsI) m/z 820.2118 (M + Cs+, C32H45N7O8S requires 820.2105).
Ethyl 2(R)-(1-((2(S)-((tert-Butoxycarbonyl)amino)-2-carbamoyl-
ethyl)amino)-2-(((4S,5R)-4-methyl-5-phenyl-2-oxazolidinon-3-yl)car-
bonyl)-2(S)-(methylthio)ethyl)-4-(dimethylamino)-5-methylpyrimidine-
NR-(tert-Butoxy)carbonyl)-Nâ-(1-amino-3(S)-(6-carboxy-4-(di-
methylamino)-5-methylpyrimidin-2-yl)propion-3-yl)-(S)-â-aminoala-
nineamide (26). A solution of 25 (10.6 mg, 0.002 mmol) in t-BuOH-
H2O (2:1, 0.30 mL) at 0 °C was treated with aqueous 1 N LiOH (49
µL, 0.044 mmol, 2 equiv), and the mixture was stirred for 1 h. The
solution was acidified to pH 4-5 with the addition of aqueous 1 N
HCl, and the solvent was evaporated in vacuo. Chromatography (C-
18, 1.2 × 5 cm, 0-40% CH3OH-H2O) afforded 26 (9.5 mg, 10 mg
6-carboxylate (22): Rf ) 0.34 (SiO2, 15% CH3OH-CHCl3); [R]25
theoretical, 95%) as a white solid: Rf ) 0.75 (SiO2, 4:1:1 i-PrOH-
D
1
1
-9.3 (c 0.82, CHCl3); H NMR (CD3OD, 400 MHz) δ 7.37 (m, 5H),
H2O-HOAc); [R]25 -31 (c 0.90, CH3OH); H NMR (CD3OD, 400
D
5.72 (d, J ) 7.5 Hz, 1H), 5.47 (d, J ) 11 Hz, 1H), 4.35 (m, 1H), 4.40
(q, J ) 7.0 Hz, 2H), 4.25 (m, 1H), 4.15 (d, J ) 11.0 Hz, 1H), 3.16 (s,
MHz) δ 4.18 (m, 2H), 3.16 (s, 6H), 3.14 (m, 1H), 2.94 (dd, J ) 4.0,
16.0 Hz, 1H), 2.84 (dd, J ) 8.0, 16.0 Hz, 1H), 2.33 (s, 3H), 1.45 (s,

64 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Boger et al.
9H); IR (neat) νmax 3328, 3201, 2975, 1668, 1580, 1448, 1367, 1252,
1160 cm-1; FABHRMS (NBA-NaI) m/z 476.2247 (M + Na+,
C19H31N7O6 requires 476.2234).
NR-(tert-Butoxy)carbonyl)-Nâ-(1-amino-3(S)-(4-(((1(S)-(((4(S)-
(((1(S)-(((2-(4′-((((3-dimethylsulfonio)-1-propyl)amino)carbonyl)-2′4-
bithiazol-2-yl)-1-ethyl)amino)carbonyl)-2(R)-hydroxy-1-propyl)-
amino)carbonyl)-3(S)-hydroxy-2(R)-pentyl)amino)carbonyl)-2(R)-
hydroxy-2-(imidazol-4-yl)-1-ethyl)amino)carbonyl)-5-methylpyrimidin-
2-yl)propion-3-yl)-(S)-â-aminoalanineamide (50). A solution of 4664
(4.0 mg, 0.01 mmol, 1.15 equiv) and pentapeptide S (11, 7.0 mg, 0.009
mmol, 1.0 equiv) in DMF (100 µL) at 0 °C under Ar was treated with
DPPA (3.2 µL, 0.015 mmol, 1.4 equiv) followed by i-Pr2NEt (5.2 µL,
0.03 mmol, 2 equiv). The reaction mixture was stirred for 10 h at 0
°C in the dark before the solvent was removed in vacuo. Chromatog-
raphy (C-18, 1.2 × 4.5 cm, 0-70% CH3OH-H2O gradient elution)
afforded 50 (8.0 mg, 11.3 mg theoretical, 71%): Rf ) 0.50 (SiO2, 10:
9:1 CH3OH-10% aqueous CH3CO2NH4-10% aqueous NH4OH);
[R]25D +29 (c 0.15, CH3OH); 1H NMR (CD3OD, 400 MHz) δ 8.70 (s,
1H), 8.19 (s, 1H), 8.11 (s, 1H), 8.04 (br s, 1H), 7.14 (br s, 1H), 4.98
(d, J ) 7.0 Hz, 1H), 4.59 (d, J ) 7.0 Hz, 1H), 4.43 (m, 1H), 4.31 (d,
J ) 4.0 Hz, 1H), 4.27 (m, 1H), 4.12 (dd, J ) 6.5, 4.0 Hz, 1H), 3.92
(dd, J ) 6.5, 6.5 Hz, 1H), 3.66 (m, 3H), 3.57 (dd, J ) 6.5, 6.5 Hz,
2H), 3.34 (dd, J ) 7.5, 7.5 Hz, 2H), 3.27 (dd, J ) 7.0, 7.0 Hz, 2H),
3.10 (m, 1H), 2.97 (m, 1H), 2.90 (s, 6H), 2.55 (s, 3H), 2.49 (dq, J )
7.0, 7.0 Hz, 1H), 2.11 (tt, J ) 7.0, 7.0 Hz, 2H), 1.42 (s, 9H), 1.16 (d,
J ) 6.5 Hz, 3H), 1.13 (d, J ) 6.5 Hz, 3H), 1.09 (d, J ) 6.5 Hz, 3H);
IR (neat) νmax 3313, 2892, 1650, 1587, 1542, 1485, 1241, 1201, 1086
cm-1; FABHRMS (NBA-CsI) m/z 1132.4471 (M+, C47H70N15O12S3
requires 1132.4491).
Nâ-(1-Amino-3(S)-(4-(((1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-dimethyl-
sulfonio)-1-propyl)amino)carbonyl)-2,4′-bithiazol-2-yl)-1-ethyl)ami-
no)carbonyl)-2(R)-hydroxy-1-propyl)amino)carbonyl)-3(S)-hydroxy-
2(R)-pentyl)amino)carbonyl)-2(R)-hydroxy-2-(imidazol-4-yl)-1-
ethyl)amino)carbonyl)-5-methylpyrimidin-2-yl)propion-3-yl]-(S)-â-
aminoalanineamide (4). A solution of 50 (5 mg, 0.005 mmol) in CH2-
Cl2 (100 µL) at 0 °C was treated with 20% TFA-CH2Cl2 (200 µL)
and the mixture stirred at 0 °C (1.5 h). The solvent was evaporated in
vacuo. Chromatography (C-18, 1.2 × 2.0 cm, 0-60% CH3OH-H2O
gradient elution) afforded 4 (4.4 mg, 5.2 mg theoretical, 88%) as a
white film: Rf ) 0.20 (SiO2, 10:9:1 CH3OH-10% aqueous CH3CO2-
NH4-10% aqueous NH4OH); [R]25D -16 (c 0.011, CH3OH); 1H NMR
(CD3OD, 400 MHz) δ 8.70 (s, 1H), 8.20 (s, 1H), 8.11 (s, 1H), 7.64 (s,
1H), 7.01 (s, 1H), 4.97 (d, J ) 7.0 Hz, 1H), 4.62 (d, J ) 7.0 Hz, 1H),
4.31 (d, J ) 4.0 Hz, 1H), 4.23 (dd, J ) 6.5, 6.5 Hz, 1H), 4.11 (m, 1H),
4.01 (dd, J ) 9.0, 4.0 Hz, 2H), 3.93 (dd, J ) 7.0, 7.0 Hz, 1H), 3.69
(m, 1H), 3.63 (m, 4H), 3.59 (dd, J ) 6.0, 6.0 Hz, 2H), 3.36 (dd, J )
8.0, 7.0 Hz, 2H), 3.27 (m, 2H), 2.92 (s, 6H), 2.68 (dd, J ) 13.0, 5.0
Hz, 1H), 2.63 (m, 1H), 2.55 (s, 3H), 2.49 (dq, J ) 7.0, 7.0 Hz, 1H),
2.13 (tt, J ) 7.0, 7.0 Hz, 2H), 1.17 (d, J ) 6.5 Hz, 3H), 1.13 (d, J )
6.5 Hz, 3H), 1.06 (d, J ) 6.5 Hz, 3H); IR (neat) νmax 3304, 1652,
1590, 1549, 1488, 1242, 1206, 1088, 913 cm-1; FABHRMS (NBA-
CsI) m/z 1165.3011 (M+ + Cs, C42H62N15O10S3 requires 1165.3021).
NR-(tert-Butoxycarbonyl)-Nâ-(1-amino-3(S)-(4-(dimethylamino)-
6-(((1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-dimethylsulfonio)-1-propyl)ami-
no)carbonyl)-2′,4-bithiazol-2-yl)-1-ethyl)amino)carbonyl)-2(R)-hy-
droxy-1-propyl)amino)carbonyl)-3(S)-hydroxy-2(R)-pent-
yl)amino)carbonyl)-2(R)-hydroxy-2-(imidazol-4-yl)-1-ethyl)amino)-
carbonyl)-5-methylpyrimidin-2-yl)propion-3-yl)-(S)-â-aminoala-
nineamide (31). A solution of 26 (7.5 mg, 0.016 mmol, 1.15 equiv)
and pentapeptide S (11, 11.0 mg, 0.014 mmol, 1.0 equiv) in DMF (200
µL) at 0 °C under Ar was treated with DPPA (4.5 µL, 0.022 mmol,
1.4 equiv) followed by i-Pr2NEt (7.5 µL, 0.043 mmol, 2 equiv). The
reaction mixture was stirred for 10 h at 0 °C in the dark before the
solvent was removed in vacuo. Chromatography (C-18, 1.2 × 4.5 cm,
0-70% CH3OH-H2O gradient elution) afforded 31 (11.5 mg, 16.4
mg theoretical, 72%): Rf ) 0.45 (SiO2, 10:9:1 CH3OH-10% aqueous
CH3CO2NH4-10% aqueous NH4OH); [R]25 -5.0 (c 0.05, CH3OH);
D
1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 8.11 (s, 1H), 7.93 (br s,
1H), 7.17 (br s, 1H), 5.17 (d, J ) 7.0 Hz, 1H), 4.76 (d, J ) 7.0 Hz,
1H), 4.40 (m, 1H), 4.29 (dd, J ) 4.0, 5.0 Hz, 1H), 4.11 (m, 1H), 3.97
(dd, J ) 12.5, 6.5 Hz, 1H), 3.71 (dd, J ) 6.5, 6.5 Hz, 2H), 3.64 (m,
4H), 3.56 (dd, J ) 6.0, 6.0 Hz, 2H), 3.35 (dd, J ) 7.0, 7.0 Hz, 2H),
3.27 (dd, J ) 7.0, 7.0 Hz, 2H), 3.08 (s, 6H), 2.91 (s, 6H), 2.52 (dq, J
) 7.0, 7.0 Hz, 1H), 2.31 (s, 3H), 2.12 (tt, J ) 7.0, 7.0 Hz, 2H), 1.43
(s, 9H), 1.14 (d, J ) 6.5 Hz, 3H), 1.12 (d, J ) 6.5 Hz, 3H), 1.09 (d,
J ) 6.5 Hz, 3H); IR (neat) νmax 3291, 1659, 1590, 1487, 1401, 1246,
1206, 1092, 914 cm-1; FABHRMS (NBA-CsI) m/z 1175.4966 (M+,
C49H75N16O12S3 requires 1175.4912).
Nâ-(1-Amino-3(S)-(4-(dimethylamino)-6-(((1(S)-(((4(S)-(((1(S)-
(((2-(4′-((((3-dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithia-
zol-2-yl)-1-ethyl)amino)carbonyl)-2(R)-hydroxy-1-propyl)amino)-
carbonyl)-3(S)-hydroxy-2(R)-pentyl)amino)carbonyl)-2(R)-hydroxy-
2-(imidazol-4-yl)-1-ethyl]amino]carbonyl)-5-methylpyrimidin-2-
yl)propion-3-yl)-(S)-â-aminoalanineamide (3). A solution of 31 (5
mg, 0.005 mmol) in CH2Cl2 (100 µL) at 0 °C was treated with 20%
TFA-CH2Cl2 (200 µL) and the mixture stirred at 0 °C (1.5 h). The
solvent was evaporated in vacuo. Chromatography (C-18, 1.2 × 2.0
cm, 0-60% CH3OH-H2O gradient elution) afforded 3 (4.7 mg, 5.4
mg theoretical, 88%): Rf ) 0.20 (SiO2, 10:9:1 CH3OH-10% aqueous
CH3CO2NH4-10% aqueous NH4OH); [R]25 -18 (c 0.078, CH3OH);
D
1H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H), 8.11 (s, 1H), 7.68 (br s,
1H), 7.10 (br s, 1H), 5.11 (d, J ) 7.0 Hz, 1H), 4.78 (d, J ) 7.0 Hz,
1H), 4.11 (dd, J ) 6.0, 4.0 Hz, 1H), 4.06 (dd, J ) 9.0, 4.0 Hz, 1H),
3.93 (dd, J ) 6.0, 6.0 Hz, 1H), 3.83 (dd, J ) 6.0, 6.0 Hz, 1H), 3.69
(m, 1H), 3.65 (m, 4H), 3.57 (dd, J ) 6.0, 6.0 Hz, 2H), 3.35 (dd, J )
6.5, 6.5 Hz, 2H), 3.27 (dd, J ) 7.0, 7.0 Hz, 2H), 3.07 (s, 6H), 2.92 (s,
6H), 2.76 (dd, J ) 11.0, 5.0 Hz, 1H), 2.56 (dd, J ) 15.0, 9.5 Hz, 1H),
2.48 (dq, J ) 7.0, 7.0 Hz, 1H), 2.30 (s, 3H), 2.16 (tt, J ) 7.0, 7.0 Hz,
2H), 1.14 (d, J ) 6.5 Hz, 3H), 1.12 (d, J ) 6.5 Hz, 3H), 1.08 (d, J )
6.5 Hz, 3H); IR (neat) νmax 3352, 1647, 1588, 1481, 1255, 1200, 1066,
900 cm-1; FABHRMS (NBA-CsI) m/z 1075.4366 (M+, C44H67N16O10S3
requires 1075.4388).
Nâ-(1-Amino-3(R)-(4-(((1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-dimeth-
ylsulfonio)-1-propyl)amino)carbonyl)-2,4′-bithiazol-2-yl)-1-ethyl)-
amino)carbonyl)-2(R)-hydroxy-1-propyl)amino)carbonyl)-3(S)-hy-
droxy-2(R)-pentyl)amino)carbonyl)-2(R)-hydroxy-2-(imidazol-4-yl)-
1-ethyl)amino)carbonyl)-5-methylpyrimidin-2-yl)propion-3-yl)-(S)-
â-aminoalanineamide (7):64 (86%); Rf ) 0.20 (SiO2, 10:9:1 CH3OH-
10% aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25D -11 (c 0.15,
Nâ-(1-Amino-3(R)-(4-(dimethylamino)-6-(((1(S)-(((4(S)-(((1(S)-
(((2-(4′-((((3-dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithia-
zol-2-yl)-1-ethyl)amino)carbonyl)-2(R)-hydroxy-1-propyl)amino)-
carbonyl)-3(S)-hydroxy-2(R)-pentyl)amino)carbonyl)-2(R)-hydroxy-
2-(imidazol-4-yl)-1-ethyl)amino)carbonyl)-5-methylpyrimidin-2-
yl)propion-3-yl)-(S)-â-aminoalanine Amide (6):64 (86%); Rf ) 0.20
(SiO2, 10:9:1 CH3OH-10% aqueous CH3CO2NH4-10% aqueous NH4-
OH); [R]25D -9.0 (c 0.090, CH3OH); 1H NMR (CD3OD, 400 MHz) δ
8.20 (s, 1H), 8.11 (s, 1H), 7.67 (br s, 1H), 7.10 (br s, 1H), 5.11 (d, J
) 7.0 Hz, 1H), 4.77 (d, J ) 7.0 Hz, 1H), 4.10 (m, 1H), 4.07 (dd, J )
9.0, 4.0 Hz, 1H), 3.92 (dd, J ) 6.0, 6.0 Hz, 1H), 3.66 (m, 2H), 3.58
(dd, J ) 6.0, 6.0 Hz, 2H), 3.35 (dd, J ) 6.5, 6.5 Hz, 2H), 3.27 (dd, J
) 7.0, 7.0 Hz, 2H), 3.06 (s, 6H), 2.92 (s, 6H), 2.76 (dd, J ) 11.0, 5.0
Hz, 1H), 2.55 (dd, J ) 15.0, 9.0 Hz, 1H), 2.48 (dq, J ) 7.0, 7.0 Hz,
1H), 2.30 (s, 3H), 2.13 (tt, J ) 7.0, 7.0 Hz, 2H), 1.14 (d, J ) 6.5 Hz,
3H), 1.12 (d, J ) 6.5 Hz, 3H), 1.10 (d, J ) 6.5 Hz, 3H); IR (neat) νmax
3280, 1641, 1587, 1550, 1487, 1366, 1241, 1066 cm-1; FABHRMS
(NBA-CsI) m/z 1075.4333 (M+, C44H67N16O10S3 requires 1075.4388).
1
CH3OH); H NMR (CD3OD, 400 MHz) δ 8.70 (s, 1H), 8.20 (s, 1H),
8.11 (s, 1H), 7.64 (s, 1H), 7.01 (s, 1H), 4.95 (d, J ) 7.0 Hz, 1H), 4.61
(d, J ) 7.0 Hz, 1H), 4.30 (d, J ) 4.0 Hz, 1H), 4.23 (dd, J ) 6.5, 6.5
Hz, 1H), 4.11 (m, 1H), 4.00 (m, 2H), 3.92 (dd, J ) 7.0, 7.0 Hz, 2H),
3.66 (m, 5H), 3.61 (dd, J ) 6.5, 6.5 Hz, 1H), 3.57 (dd, J ) 6.0, 6.0
Hz, 2H), 3.36 (dd, J ) 8.0, 7.5 Hz, 2H), 2.93 (s, 6H), 2.71 (dd, J )
11.5, 7.0 Hz, 1H), 2.64 (m, 1H), 2.55 (s, 3H), 2.48 (dq, J ) 7.0, 7.0
Hz, 1H), 2.15 (tt, J ) 7.0, 7.0 Hz, 2H), 1.17 (d, J ) 6.5 Hz, 3H), 1.14
(d, J ) 6.5 Hz, 3H), 1.06 (d, J ) 6.5 Hz, 3H); IR (neat) νmax 3304,
2925, 1647, 1590, 1544, 1488, 1236, 1201, 1093, 913 cm-1; FABHRMS
(NBA-CsI) m/z 1032.3960 (M+, C42H62N15O10S3 requires 1032.3973).
DNA Cleavage and Oxidation Properties. Experimental details
for the DNA cleavage studies and the oxidation of styrene may be found
in the Supporting Information.

Role of C4 Amino Group of Bleomycin A
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 65
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA42056, D.L.B.;
GM34454, J.S.) and the Skaggs Institute of Chemical Biology
(D.L.B.).
30 and the intermediates 27-29, 32, 47-49, and 51 leading to
the epimeric series 6 and 7, and experimental details for the
DNA cleavage and styrene oxidation studies (8 pages). See
any current masthead page for ordering and Internet access
instructions.
Supporting Information Available: Full experimental
details for the preparation of 2 and 37-46, characterization of
JA971889V