A systematic evaluation of the bleomycin A2 L-threonine side chain: Its role in preorganization of a compact conformation implicated in sequence- selective DNA cleavage

Article abstract of DOI:10.1021/ja9816638

The preparation and examination of 3-7 are detailed and constitute analogues of deglycobleomycin A₂ (2) containing systematic modifications in the L-threonine side chain. The studies revealed a substantial impact of the substituent on the DNA cleavage efficiency and ratio of double strand/single strand (ds/ss) cleavage without affecting the characteristic 5'-GPy selectivity. The results of the comparisons proved consistent with conformational effects of the substituent within the linker domain which restrict the number of accessible conformations (Φ ? - 120°, Ψ = 60- 180°) and favor adoption of a compact conformation (Φ ? -120°, Ψ ? 180°) implicated in sequence-selective DNA cleavage. The studies also identify one potential site that may adopt two nearly equivalent turn conformations. This site may constitute one swivel point in the structure that permits access to a class of related bound structures (Φ = 60-180°) adaptable to variable conformational characteristics required of multiple cleavage sites, including access to both strands of duplex DNA from a single intercalation site important for both primary and secondary DNA cleavage.

Full text of DOI:10.1021/ja9816638

J. Am. Chem. Soc. 1998, 120, 9139-9148
9139
A Systematic Evaluation of the Bleomycin A2 L-Threonine Side
Chain: Its Role in Preorganization of a Compact Conformation
Implicated in Sequence-Selective DNA Cleavage
Dale L. Boger,*,† Timothy M. Ramsey,† Hui Cai,† Silvia T. Hoehn,‡ and JoAnne Stubbe‡
Contribution from the Department of Chemistry and the Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, and
Departments of Chemistry and Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
ReceiVed May 13, 1998
Abstract: The preparation and examination of 3-7 are detailed and constitute analogues of deglycobleomycin
A2 (2) containing systematic modifications in the L-threonine side chain. The studies revealed a substantial
impact of the substituent on the DNA cleavage efficiency and ratio of double strand/single strand (ds/ss)
cleavage without affecting the characteristic 5′-GPy selectivity. The results of the comparisons proved consistent
with conformational effects of the substituent within the linker domain which restrict the number of accessible
conformations (Φ = -120°, Ψ ) 60-180°) and favor adoption of a compact conformation (Φ = -120°, Ψ
= 180°) implicated in sequence-selective DNA cleavage. The studies also identify one potential site that may
adopt two nearly equivalent turn conformations. This site may constitute one swivel point in the structure
that permits access to a class of related bound structures (Ψ ) 60-180°) adaptable to variable conformational
characteristics required of multiple cleavage sites, including access to both strands of duplex DNA from a
single intercalation site important for both primary and secondary DNA cleavage.
Bleomycin A2 (1, Figure 1),1-13 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 metal ion- and
† The Scripps Research Institute.
‡ Massachusetts Institute of Technology.
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S0002-7863(98)01663-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/28/1998

9140 J. Am. Chem. Soc., Vol. 120, No. 36, 1998
Boger et al.
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Herein we report the synthesis and evaluation of 3-7 in which
the L-threonine side chain of 2 has been systematically modified
in efforts to dissect the subtle contributions of this subunit
(Figure 2). In an initial comparison involving substantial
modifications in the linker region of deglycobleomycin A2 (2),
the replacement of the L-threonine subunit with glycine (6) was
found to have no impact on the characteristic 5′-GT, 5′-GC
cleavage selectivity but did have a substantial impact on the
cleavage efficiency of the resulting agent, placing it among the
worst analogues examined to date containing a single substituent
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of the adoption of a compact, bent conformation productive for
DNA cleavage, enlisting a turn at the threonine/valerate junction
rather than adoption of an extended conformation characteristic
of glycine. These studies complemented an earlier study of
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for the L-threonine subunit caused no significant change in the
cleavage selectivity although varying efficiencies of cleavage
were noted.50 Similarly, we have shown that N-methylation of
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less efficient (0.1-0.08×) at cleaving DNA and that its
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Bleomycin A2 L-Threonine Side Chain
J. Am. Chem. Soc., Vol. 120, No. 36, 1998 9141
characteristic 5′-GC, 5′-GT cleavage selectivity was diminished,
while the corresponding D-allo N-methyl threonine analogue
failed to cleave DNA in a sequence specific manner.38 Neither
the glycine substitution nor the N-methylation of the L-threonine
subunit substantially altered the metal chelation, H2O2 activation,
catalytic efficiency, or inherent oxidation capabilities of the
analogues. Rather, the studies suggest that the diminished DNA
cleavage efficiency and selectivity may stem from a disruption
of the deglycobleomycin A2 interaction with duplex DNA and/
or its reduced ability to adopt a productive bound conformation
capable of C4′-H abstraction resulting in reduced DNA cleavage.
The conformation of the linker of the metal-ligated bleomycin
is especially interesting to probe, in light of the recent NMR
structural models of Co(III)OOH and Zn bleomycin A2.51-53
Both studies highlight a turn in the acyclic peptide of the DNA-
bound agent within the L-threonine subunit, implying that it may
be induced by the L-threonine substitute. In addition, the NMR
studies of the model Co(III)OOH complex of free and DNA-
bound bleomycin revealed that the observed conformation may
additionally be facilitated by a H-bond from the threonine NH
to the proximal oxygen of the metal-bound hydroperoxide.51,52
This H-bond could stabilize the productive bound conformation
of the activated agent or fix the position or alignment of the
reacting Fe-oxo intermediate for C4′ hydrogen abstraction.
Additionally, it could stabilize the metal-bound hydroperoxide54
or contribute to catalysis by assisting the chemistry of oxygen-
oxygen bond cleavage in a fashion not yet understood. Finally,
the threonine carbonyl of the DNA-bound conformation, but
not of the free solution conformation, of Co(III)OOH bleomycin
is positioned to accept a H-bond from the terminal oxygen of
the hydroperoxide ligand. These related studies all highlight a
potential important role for the L-threonine subunit of 1-2, and
the examination of 3-7 was anticipated to provide a detailed
accounting of the role of the side chain substituents.
Scheme 1
Synthesis of 3-7. We prepared the agents 3-7, employing
a recent modification49 of our original synthesis of 244 that was
also utilized in our prior preparation of 6.37 The approach entails
the synthesis of the pentapeptide S analogues 18a-e and a final
amide coupling at the erythro-â-hydroxy-L-histidine and py-
rimidoblamic acid juncture rather than tetrapeptide S analogue
(15a-e) coupling with the linked pyrimidoblamic acid/erythro-
â-hydroxy-L-histidine. The latter coupling was found to suffer
occasional dehydration with elimination of the â-hydroxy group
of the L-histidine subunit upon carboxylate activation for amide
bond coupling when conducted on relative large scales. This
was not observed upon carboxylate activation of the N-BOC
derivative of erythro-â-hydroxy-L-histidine presumably due to
the lower acidity of its R-proton (N-BOC versus N-acyl
derivative). Notably, this was accomplished with the sulfonium
(51) (a) 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. (b)
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.
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Tang, X.-J.; Turner, C. J.; Boger, D. L.; Kozarich, J. W.; Stubbe, J. J. Am.
Chem. Soc. 1998, 120, 2239.
(53) 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.
salt installed, on substrates incorporating only one protecting
group, without deliberate protection of the imidazole, and
without competitive imidazole acylation, provided that the
coupling was effected by diphenylphosphoryl azide (DPPA).
Coupling (2 equiv EDCI,55 1 equiv HOBt, DMF, 25 °C, 3 h,
72-80%) of L-Ser-OCH3, methyl (S)-2-aminobutyrate, L-Ala-
OCH3, Gly-OCH3, and L-Val-OCH3 (9a-e) with (2S,3S,4R)-
N-BOC-4-amino-3-hydroxy-2-methylpentanoic acid (8)42 pro-
vided the dipeptides 10a-e (Scheme 1). Sequential methyl ester
(54) Fulmer, P.; Zhao, C.; Li, W.; DeRose, E.; Antholine, W. E.; Petering,
D. H. Biochemistry 1997, 36, 4367. Mao, Q.; Fulmer, P.; Li, W.; DeBose,
E. F.; Petering, D. H. J. Biol. Chem. 1996, 271, 6185. Xu, R. X.; Nettesheim,
D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907.
(55) EDCI ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; BOP reagent ) benzotriazol-1-yloxytris(dimethylamino)phos-
phonium hexafluorophosphate; DCC ) 1,3-dicyclohexylcarbodiimide.

9142 J. Am. Chem. Soc., Vol. 120, No. 36, 1998
Boger et al.
Table 1. Summary of ΦX174 and w794 DNA Cleavage Properties
relative efficiency of DNA cleavage
agent
ΦX174a
w794b
ratio of double to single strand cleavagec
DNA cleavage selectivityb
1, bleomycin A2
2, deglycobleomycin A2
3, R ) CH2OH
4, R ) CH2CH3
5, R ) CH3
2-5
1.0
5.8
1.0
1.0
0.45
0.40
0.25
0.20
0.03
1:6
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
5′-GC, 5′-GT > 5′-GA
none
1:12
1:17
1:21
1:24
1:31d
1:25
1:98
1-0.9
0.50
0.45
0.20d
0.17
0.04
6, R ) H
7, R ) CH(CH3)2
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 [32P]-5′-end-labeled w794, Fe(III)-H2O2. The results are the average of five experiments. c Ratio of double to single stranded
cleavage of supercoiled ΦX174 DNA calculated as FIII ) n2 exp(-n2), FI ) exp[-(n1 + n2)]. d Previously reported as 0.20 and 1:29, ref 37.
Figure 4. Representative kinetics of supercoiled ΦX174 DNA cleavage
by Fe(II)-7 (12 µM) in buffer solution containing 2-mercaptoethanol.
The DNA cleavage reactions were run at 25 °C for various lengths of
time, and electrophoresis was conducted on a 1% agarose gel. Direct
fluorescence quantitation of the percentage of forms I-III DNA present
at each time point was conducted using a Millipore BioImage 60S RFLP
system visualized on a UV (312 nm) transilluminator in the presence
of 0.1 µg/mL ethidium bromide taking into account the relative
fluorescence intensities of forms I-III ΦX174 DNA (forms II and III
have fluorescence intensities that are 0.7 times that of form I).
Figure 3. Agarose gel illustrating the cleavage reactions of supercoiled
ΦX174 DNA by Fe(II)-agents at 25 °C for 1 h in buffer solutions
containing 2-mercaptoethanol. After electrophoresis 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.
the L-threonine side chain had little or no impact on the
properties of 2. The cleavage efficiencies of 4 and 5 were
comparable although 4 was perceptibly better and both were
less effective than 2 or 3 (0.45-0.5×). Thus, removal of the
hydroxyl group of the L-threonine side chain reduced the
cleavage efficiency 2-fold. Removal of the side chain altogether
with 6 further and substantially reduced the cleavage efficiency
(0.2× relative to 2). Interestingly, replacement of the L-
threonine subunit with valine in which the hydroxyl group is
replaced with a methyl group led to an even greater 6-fold
reduction in the efficiency of DNA cleavage.
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. We assumed a Poisson distribution for the formation of
ss and ds breaks to calculate the average number of double and
single strand cleavages per DNA molecule using the Freifelder-
Trumbo equation.56 The ratio of ds to ss cleavages observed
with the Fe(II) complexes is illustrated in Figure 4 for 7, and
the full set of results is summarized in Table 1. The ratio of
double to single strand DNA cleavage for 3 and 4 was
established to be 1:17 and 1:21, that of 5 and 7 was 1:25, and
that of 6 was 1:31. Consistent with the relative efficiencies of
DNA cleavage, this was lower than bleomycin A2 (1:6) or
deglycobleomycin A2 (1:12) but well above the ratio derived
from uncomplexed Fe(II) cleavage (1:98).57 With the significant
exception of that for 6 (Gly), this diminishing trend in the ds/
ss cleavage ratio parallels the trends observed in DNA-cleavage
efficiency. For 6, the ratio was 2-3× lower than that of 2
hydrolysis (2 equiv LiOH, t-BuOH/H2O, 0 °C, 1 h, 89-94%),
subsequent coupling of the resulting carboxylic acids 11a-e
with the bithiazole amine 1242 (2 equiv EDCI, 1 equiv HOBt,
DMF, 25 °C, 5 h, 74-79%), and S-methylation (50 equiv CH3I,
CH3OH, 25 °C, 24 h, 97-99%) of 13a-e cleanly provided the
N-BOC-tetrapeptide S analogues 14a-e. Acid-catalyzed N-
BOC deprotection (3.4 N HCl-EtOAc, 25 °C, 1.5 h) with
liberation of the free amines 15a-e (NH4OH-CH3OH, 25 °C,
1 h, 91-96%), subsequent coupling (1.5 equiv BOP reagent,55
2 equiv i-Pr2NEt, DMF, 0-25 °C, 8 h, 72-78%) with
NR-BOC,Nim-CPh3-erythro-â-hydroxy-L-histidine (16)42 to pro-
vide 17a-e, and acid-catalyzed deprotection (20% TFA-CH2Cl2,
0 °C, 2 h) with liberation of the free amines (NH4OH-CH3OH,
25 °C, 1 h) provided the pentapeptide S analogues 18a-e in
excellent conversions (82-86%). Their final couplings with
NR-BOC-pyrimidoblamic acid (19)43 proceeded smoothly when
effected by diphenylphosphoryl azide (DPPA, 1.5 equiv, 2 equiv
i-Pr2NEt, DMF, 0 °C, 10 h, 67-71%), and final acid-catalyzed
deprotection of 20a-e (20% TFA-CH2Cl2, 0 °C, 1.5 h) provided
3-7 (83-86%). When this last coupling reaction with 18d was
conducted with DCC55 (2 equiv, 1 equiv HOBt, DMF, 0-25
°C, 10 h), the conversion to 20d was much lower (29%).
DNA Cleavage Properties. Four assays were used to
examine the DNA cleavage properties of 3-7. The initial study
of the relative efficiency of DNA cleavage was conducted with
the Fe(II) complexes and supercoiled ΦX174 DNA in the
presence of O2 and 2-mercaptoethanol. Like Fe(II)-bleomycin
A2 and deglycobleomycin A2, the Fe(II) complexes of all five
agents produced single and double strand cleavage to afford
relaxed (Form II) and linear (Form III) DNA, respectively
(Figure 3 and Table 1). The replacement of threonine with
serine gave 3 which was essentially indistinguishable from
deglycobleomycin A2. Thus, removal of the methyl group of
(56) Freifelder, D.; Trumbo, B. Biopolymers 1969, 7, 681.
(57) A theoretical ratio of approximately 1:100 is required for the linear
DNA to be the result of the random accumulation of single strand breaks
within the 5386 base-pair size of ΦX174 DNA, assuming that sequential
cleavage on the complementary strands within 15 base-pairs is required
for formation of linear DNA.

Bleomycin A2 L-Threonine Side Chain
J. Am. Chem. Soc., Vol. 120, No. 36, 1998 9143
Figure 5. Cleavage of double-stranded DNA by Fe(III)-agent (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 30 min at 37 °C, and electrophoresis was run on an 8% denaturing
PAGE and visualized by autoradiography.
while the remaining agents were <2× less effective, suggesting
that 6 is uniquely less prone to promote ds cleavage as suggested
in prior studies.37
Figure 6. Internally labeled hairpin oligonucleotide containing GTAC
The assessment of the selectivity of DNA cleavage along with
hot spot for ds cleavage. 32P-labeling site is marked by /, ss cleavage
an additional assessment of the relative efficiency of DNA
sites are underlined, and ds cleavage site is in bold.
cleavage were carried out with duplex w794 DNA58 by
monitoring strand cleavage of singly [32P]-5′-end-labeled double-
assay. With this 5-fold compression of the dynamic range
stranded DNA by the Fe(III)-complexes upon activation with
(167×/33× ) 5), the 4-fold reduction in cleavage efficiency
59
H2O2 in 10 mM phosphate buffer (pH 7.0). This protocol
observed herein with 6 would appear to be consistent with our
has proven more sensitive to the distinctions in the relative
efficiency of DNA cleavage than the ΦX174 supercoiled DNA
cleavage assay, but both have always provided the same trends
in our hands. Thus, incubation of the labeled duplex DNA with
the agents in the presence of equimolar FeCl3 and excess H2O2
led to DNA cleavage. Because of the poor efficiency of DNA
prior observations (25×/4× ) 6.25).37
Typical comparisons of 3-7 are illustrated in Figure 5.
Under all conditions examined, 3-7 were found to cleave w794
double-stranded DNA with the characteristic 5′-GT, 5′-GC >
5′-GA sequence selectivity of bleomycin A2 and deglycobleo-
mycin A2 (2). Consistent with the ΦX174 DNA studies, 3
proved to be the most efficient analogue and was essentially
indistinguishable from deglycobleomycin A2. The analogues
4 and 5 were progressively less efficient, and 6 and 7 were even
cleavage exhibited by 6 in our earlier study (6 ) 0.04×, Fe(III)
) 0.006×), the assay was modified by reducing the amount of
end-labeled DNA employed. The net result was that the agent
concentration range over which cleavage occurred was smaller
less efficient (Table 1). Thus, removal of the L-threonine
and the absolute concentration required for DNA cleavage was
hydroxyl group or its replacement with a methyl group
reduced but the cleavage selectivity was more readily distin-
significantly reduced the cleavage efficiency, while the removal
guishable. Thus, the magnitude of the relative efficiency
of the methyl group had no impact. A further significant
reduction in cleavage efficiency was observed when the side
chain was removed altogether.
differences was reduced, but the relative trend was not altered.
For example, 6 was found to be 25× less effective than 2 but
7× more effective than Fe(III) itself in our earlier study,37 while
In a fourth assessment of the DNA cleavage properties, 6
in the present assay it was 4× less effective than 2 and 8×
was examined in a hairpin cleavage assay that enlists an
more effective than Fe(III). Similarly, deglycobleomycin A2
internally labeled site to establish the relative ss and ds cleavage
was 167× more effective than Fe(III) in our prior studies but
(Figure 6).60 For bleomycin A2, the ds to ss cleavage ratio at
33× more effective under the conditions employed in the present
the T13 + T38 ds cleavage site is 1:3 under single turnover
(58) 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.
(59) 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.
Hamamichi, N.; Natrajan, A.; Hecht, S. M. J. Am. Chem. Soc. 1992, 114,
6278.
(60) (a) Absalon, M. J.; Stubbe, J.; Kozarich, J. W. Biochemistry 1995,
34, 2065. (b) The larger increases in the magnitude of differences observed
with the hairpin assay and, to a lesser extent, with the w794 DNA versus
the ΦX174 DNA can be attributed in part to the corresponding amount of
unlabeled carrier DNA which competes for consumption of agent and its
oxidizing capability. Additionally, the hairpin assay is run under single
turnover conditions which may affect the magnitude of the differences.

9144 J. Am. Chem. Soc., Vol. 120, No. 36, 1998
Table 2. Styrene Oxidationa
Boger et al.
agent
Styrene epoxide (mM)
phenylacetaldehyde (mM)
ratio
total product (mM)
rel efficiency
Fe(III)-2
Fe(III)-6
Fe(III)
1.79
0.87
0
0.73
0.40
0
2.45
2.18
0
2.52
1.27
0
1.0
0.50
0
H2O2
0
0
0
0
0
Fe(III)-H2O2
2
0
0
0
0
0
0
0
0
0
0
2-H2O2
Fe(III)-2b
0
0
0
0
0
0
0
0
0
0
a 500 µM (Fe(III)-agent, 30 mM H2O2, 50 mM styrene, 0 °C, 1.5 h, 80% CH3OH-H2O. Results reported are the average of two runs. b 500
µM Fe(III)-2 under identical conditions without H2O2.
Figure 7. Cleavage of hairpin oligonucleotide by 6 activated with
Fe(II)-O2. Lane 1, hairpin only; lane 2, hairpin + Fe(II); lane 3,
hairpin + 6, no Fe(II); lane 4, 20 µM bleomycin A2; lane 5, 10 s
digestion with 300 µM 6; lane 6, 30 s digestion with 300 µM 6; lane
Figure 8. Kinetics of styrene (50 mM, 30 mM H2O2) oxidation
7, 60 s digestion with 300 µM 6; lane 8, 120 s digestion with 300 µM
catalyzed by the Fe(III) complex of agent 2 and 6 (500 µM). The
6.
ratio of total concentration of products (styrene epoxide and phenyl-
acetaldehyde) versus the initial concentration of Fe(III)-agent complex
(turnover) is plotted against time.
conditions.60 In these studies, 6 was activated with either
Fe(III)/H2O2 or with Fe(II)/O2 in the absence of external
reducing equivalents. The latter activation gives a good
the first 5 min were not distinguishable (Figure 8). A full
indication of the ds versus ss cleavage ratio since it ensures
oxidation turnover occurred in less than 30 s, the first time point
single turnover conditions. As can be seen in Figure 7, the ds
in the kinetic measurements, and at that point, the two agents
cleavage band is significantly less pronounced for 6 than it is
both had oxidized 1.74-1.80 equiv of styrene. Over the next
for 1 with a ratio of ds to ss cleavage at the T13 + T38 site of
approximately 1:20. Thus, the ability of 6 to promote ds
cleavage is greatly impaired but not completely suppressed. Its
DNA cleavage selectivity (5′-GC/5′-GT) as well as its reduced
efficiency (0.03-0.04× deglycobleomycin A2) in the hairpin
assay were in accord with the results obtained in the ΦX174
and w794 DNA-cleavage assays.60b
5 min, the two agents exhibited essentially indistinguishable
kinetics of oxidation, most likely reflecting the reactivation
versus oxidation kinetics. After 5-10 min, the rates of
oxidation rates diverged, slowed, and the rate of 6 decreased
more rapidly than that of 2. Ultimately the oxidation halted at
15 min for 6 and 45 min for 2, constituting 2.52 and 5.04
oxidations for 6 and 2, respectively, indicating the exhaustion
of the Fe(III)-agents. Although the genesis of the initial
oxidation burst and the initial oxidation kinetics of 2 and 6 were
not determined, the studies did allow us to establish that
differences in the styrene oxidation efficiencies of 2 and 6 may
be attributed to differences in total turnover and not to the
relative rates of oxidation. Since these conditions parallel those
enlisted in the w794 DNA cleavage studies, the results imply
that part of the efficiency differences observed with 6 and 2
may be attributed to their relative stabilities and number of
catalyst turnovers and is unlikely due to differences in steady-
state oxidation kinetics. However, the effect is modest and
insufficient to account for the extent of the differences in 2 and
6.
Oxidation of Styrene. Relative Kinetics versus Efficiencies
of Oxidation. In our final efforts to characterize the differences
in the properties of the agents, the ability of the Fe(III)
complexes of 6 and deglycobleomycin A2 (2) to mediate the
oxidation of styrene were compared.59 The oxidation produces
both styrene epoxide and phenylacetaldehyde and a solution of
500 µM Fe(III)-2, 50 mM styrene (excess), and 30 mM H2O2
(0 °C, 1.5 h) produced 1.79 mM styrene epoxide and 0.73
mM phenylacetaldehyde constituting five oxidations for each
Fe(III)-2 (Table 2). Under identical conditions, the glycine-
containing agent produced 0.87 mM styrene epoxide and 0.40
mM phenylacetaldehyde constituting 2.5 oxidations for each
Fe(III)-6. Thus, the same products and product distribution
were observed, but 6 proved to be only half as efficient as 2.
The kinetics of the styrene oxidation were examined in efforts
to establish whether this distinction was derived from different
rates of oxidation or different catalytic efficiencies of the two
agents. Under the pseudo-first-order oxidation conditions of
excess styrene and H2O2, the oxidation rates of 2 and 6 within
A similar set of observations were made in a study of the
N-methyl threonine analogue of deglycobleomycin A2.38 The
DNA cleavage efficiency of the Fe(III) complex was substan-
tially diminished (0.08-0.1×) but the inherent styrene oxidation
capability of the agent was relatively unaffected (0.52×) and
similar to that observed with 6. These results and those of 6

Bleomycin A2 L-Threonine Side Chain
J. Am. Chem. Soc., Vol. 120, No. 36, 1998 9145
observed in the refined solution conformation of Co(III)OOH
bleomycin A2 (Φ ) -121.5°, Figure 10a)51 and its DNA-bound
conformation (Φ ) -145.5°, Figure 10b)51 as well as in the
DNA-bound conformation of Zn bleomycin A253 and constitutes
one site at which a bend in the structure is found (Φ dihedral
angle ca. -120°). This facilitates the adoption of a compact
conformation implicated in earlier studies.37,44 Removal of the
L-threonine side chain (R ) H) provides two equivalent and
equally accessible H-eclipsed conformations (Φ ) 120° and
-120°), of which only one would be productive for DNA
binding and cleavage, and one fully extended conformation (Φ
) 180°) of an equal energy (Figure 9). It also reduces the
energy difference between these three conformations and the
remaining two bisected conformations (Φ ) 60° and -60°) to
ca. 0.6 kcal/mol. The consequences are that the adoption of
the single conformation implicated in DNA cleavage (Φ =
-120°) out of these accessible five is considerably less favorable
when R ) H. This is consistent with the observation that
removal of the L-threonine side chain has a substantial effect
on the cleavage efficiency (w794, 0.2-0.04×).
The substituent has a less pronounced and more subtle effect
on the conformation within the L-threonine subunit itself (Ψ
dihedral angle). When substituted, two accessible carbonyl-
eclipsed conformations are of comparable energy (Figure 9, Ψ
) 180° and 60°). The former is observed in the DNA-bound
conformation of Co(III)OOH bleomycin A2 (Ψ ) 173.9°) while
an average intermediate bisected conformation is observed free
in solution (Ψ ) 120.4°).51 As the size of this substituent
increases, the productive NHCO eclipsed conformation (Ψ )
180°) would be expected to become increasingly disfavored. It
may be that the diminished DNA cleavage efficiency within
the series (R ) CH3 = Et > i-Pr) reflects this increasing
difficulty in accessing the conformation (Ψ ) 180°), observed
with the DNA-bound agent. The exceptions to this generaliza-
tion, R ) CH(OH)CH3 and CH2OH, can enlist the side-chain
hydroxyls to adopt intramolecular backbone carbonyl H-bonded
conformations to restabilize the extended conformation (Figure
11).64 This is especially true of the six-membered ring H-bond
to the threonine carbonyl which is observed in the free solution
conformation (Figure 11). However, close examination of the
model Co(III)OOH bleomycin A2-bound conformation suggests
an additional and provocative explanation for the behavior of
the agents containing a side-chain hydroxyl group. Although
neither the model of DNA-bound Co(III)OOH BLM A2 nor the
Hecht model of DNA-bound Zn BLM A2 reveals a potential
interaction of the hydroxyl group with DNA itself, the primary
amide carbonyl of the pyrimidoblamic acid C2 acetamido side
chain lies within H-bonding distance (1.70 Å, O‚‚H-O angle
) 147°) of the threonine side-chain hydroxyl group in the
Stubbe model (Figure 10b). The adoption of this H-bond
releases the H-bond to the threonine carbonyl allowing the
adoption of the extended (Ψ ) 180°) conformation and aligns
the threonine carbonyl to accept a H-bond from the peroxide
ligand of Co(III)OOH bleomycin A2 (see Figure 10, 2.24 f
1.87 Å, O-H- - -O angle 130.7 f 162.7°). This provides a
tightly organized H-bonded metal complex that also includes
the additional threonine NH H-bond to the proximal oxygen of
Figure 9. Analysis of the Φ and Ψ angles of the threonine subunit.62
detailed herein suggest that any H-bonding interaction of the
threonine subunit with a metal-bound hydroperoxide or subse-
quent activated species does not appear to be contributing to
the catalysis of the oxidation reaction but rather may be
contributing to the inherent stability of the complex, providing
an improvement in the number of oxidation turnovers.
Correlation with Conformational Effects of the Substitu-
tions. The results of the DNA cleavage comparisons proved
consistent with the conformational effects of the substituent
within the linker region. The most dominant effect of the
substitutions resides with the amide bond linking the valerate
and threonine subunits. When the L-threonine subunit bears a
substituent (Figure 9, R * H), the H-eclipsed conformation (Φ
) -120°) can be expected to dominate, on the basis of the
well-established conformational preference for amides and esters
of cyclohexylamines or alcohols.61 This conformation is
(62) H/H, H/R, and R/R 1,2-eclipsing interactions were estimated using
H/H (1.0 kcal/mol), H/CH3 (1.3 kcal/mol), and CH3/CH3 (3.7 kcal/mol).
1
Gauche interactions were estimated, enlisting /2 the known A-values.
(63) Boger, D. L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.; Stubbe, J. J.
Am. Chem. Soc. 1998, 120, 9149.
(61) Hoffman, R. W. Chem. ReV. 1989, 89, 1841.
(64) Ser H-bonding: Siemion, I. Z.; Picur, B. Biophys. Chem. 1988, 31,
71. Lipkind, G. M.; Arkhipova, S. F.; Popov, E. M. Int. J. Pept. Res. 1973,
5, 381. Marcus, E.; Keller, D. A.; Shibota, M.; Ornstein, R. L.; Rein, R.
Chem. Phys. 1996, 204, 157.

9146 J. Am. Chem. Soc., Vol. 120, No. 36, 1998
Boger et al.
Figure 10. Views of the (a) free and (b) DNA-bound conformation51a of Co(III)OOH bleomycin A2, highlighting the compact conformations, the
L-threonine subunit conformations, and the entire rigid, compact linker domain.
Figure 11.
the peroxide ligand. It is plausible that the acetamido side chain
CH2CONH2 (4×)44 and serine/threonine OH (2×) contribution
to the DNA cleavage efficiency rests with these intramolecular
H-bonding interactions that could stabilize or induce the
adoption of the compact conformation implicated in productive
DNA cleavage.
High level ab initio computational studies of NAc-Gly-
Figure 12. Ab initio calculations.65
NHMe, NAc-Ala-NHMe, NAc-Val-NHMe, and NAc-Ser-
NHMe (Figure 12)65 support the conclusions drawn from the
qualitative analysis illustrated in Figure 9. NAc-Gly-NHMe
may adopt two equivalent low energy perpendicular conforma-
tions (Φ ) -85.4°, +84.5°) which represent opposite turns in
the peptide, a readily accessible extended conformation (Φ )
180°, ∆E ) 0.8 kcal/mol), and two Φ ) -162° conformations
(∆E ) 1.5 kcal/mol). In contrast, Ala and Val are restricted to
a single NHCO perpendicular conformation (Φ ) -84.6, ∆E
for 74.6° ) 2.6 kcal/mol for Ala) and a related Φ ) -157.3°
conformation (∆E ) 0.4 kcal/mol for Ala). Both induce a
common turn direction (Φ ) -85° to -157°), possess
comparable energies (a near flat barrier to interconversion), and
approximate the conformations found for free (Φ ) -121°)
and DNA-bound Co(III)OOH bleomycin A2 (Φ ) -145°).51
Moreover, the Ψ = 180° conformational isomer implicated in
DNA cleavage becomes increasingly disfavored as the size of
the substituent increases.
Conformational energy calculations of NAc-X-NHMe (X )
amino acid) and calculated Boltzmann probabilities (300 K,
Table 3) for the 20 naturally occurring amino acids and
2-aminobutyric acid have been disclosed.66 In addition to
defining the characteristic Φ = -120°, Ψ ) 60-180° confor-
mational space for the amino acids discussed, the DNA cleavage
efficiencies detailed herein correlate remarkably well with the
probability for adoption of the solution phase conformation of
Co(III)OOH bleomycin A2 (Ψ ) 120.4°), do not appear to
correlate with the probability of adoption of the DNA-bound
conformation (Ψ ) 173.9°), and with the exception of Val,
correlate with the total distribution within these two related
conformations (Table 3). The simplest interpretation of this
correlation is that the L-threonine substituents facilitate preor-
ganization of bleomycin A2 into a conformation that ultimately
leads to productive DNA cleavage.
(65) NAc-Gly-NHMe: Klimkowski, V. J.; Schafer, L.; Momany, F. A.;
Alsenoy, C. V. J. Mol. Struct. (THEOCHEM) 1985, 124, 143. Schafer, L.;
Alsenoy, C. V.; Scarsdale, J. N. J. Chem. Phys. 1982, 76, 1439. Scarsdale,
J. N.; Alsenoy, C. V.; Klimkowski, V. J.; Momany, F. A. J. Am. Chem.
Soc. 1983, 105, 3438. NAc-Ala-NHMe: Schafer, L.; Klimkowski, V. J.;
Momany, F. A.; Chuman, H.; Alsenoy, C. V. Biopolymers 1984, 23, 2335.
Schafer, L.; Bin Drees, I. S.; Frey, R. F.; Alsenoy, C. V.; Ewbank, J. D. J.
Mol. Struct. (THEOCHEM) 1995, 338, 71. Mohle, K. R.; Hofmann, H.-J.
J. Mol. Struct. (THEOCHEM) 1993, 285, 211. NAc-Ser-NHMe: Siam, K.;
Kimkowski, V. J.; Alsenoy, C. V.; Ewbank, J. D.; Schafer, L. J. Mol. Struct.
(THEOCHEM) 1987, 152, 261. Siam, K.; Kulp, S. Q.; Ewbank, J. D.;
Schafer, L. J. Mol. Struct. (THEOCHEM) 1989, 184, 143. For NAc-Val-
NHMe: Paterson, Y.; Leach, S. J. Macromolecules 1978, 11, 409. See
also: Kollman, P. A.; Gould, I. R. J. Phys. Chem. 1992, 96, 9255. Gould,
I. R.; Cornell, W. D.; Hillier, I. H. J. Am. Chem. Soc. 1994, 116, 9250.
In our own modeling studies (OPLS Amber, H2O), we have
also observed a subtle interplay between the Ψ conformational
isomers (Ψ ) ca. 60° or 180°) which influences the Φ dihedral
(66) Vasquez, M.; Nemethy, G.; Scheraga, H. Macromolecules 1983,
16, 1043. Zimmerman, S. S.; Pottle, M. S.; Nemethy, G.; Scheraga, H. A.
Macromolecules 1977, 10, 1.

Bleomycin A2 L-Threonine Side Chain
J. Am. Chem. Soc., Vol. 120, No. 36, 1998 9147
Table 3.
rel cleavage
efficiency (ΦX174)
Boltzmann prob.a
Ψ ) 20 to 130°
Boltzmann prob.
Ψ ) 130 to -140°
total
Boltzmann prob.
agent
Thr, R ) CH(OH)CH3
Ser, R ) CH2OH
Abu, R ) Et
1.0
0.798
0.776
0.605
0.589
0.425
0.390
0.153
0.087
0.191
0.138
0.049
0.335
0.951
0.863
0.796
0.727
0.474
0.725
1-0.9
0.50
0.45
0.20
0.17
Ala, R ) CH3
Gly, R ) H
Val, R ) i-Pr
a Taken from ref 66, for AcN-X-NHMe. Free Co(III)OOH BLM A2 (Φ ) -121.5°, Ψ ) 120.4°). DNA-bound Co(III)OOH BLM A2 (Φ )
-145.5°, Ψ ) 173.9°).
angle. As the R substituent increases in size, the Ψ ca. ) 180°
conformation becomes increasingly disfavored, diminishing the
capabilities for adoption of the conformation implicated in DNA
cleavage. The exceptions to this are serine and threonine which
provide compensating stabilization of the conformation through
H-bonding. The difference in energy is small for the two
accessible Ψ conformational isomers, and both are only slightly
more stable than a third, bisected conformation (Ψ ) 120°)
that lies between the two. In fact, the structure for both free
Co(III)OOH bleomycin A2 as well as the DNA-bound confor-
conformation observed in free Co(III)OOH bleomycin A2 and
both models of DNA-bound bleomycin A2.51-53 Removal of
this substituent (R ) H) results in five accessible conformations,
at least three of which may be nonproductive for DNA cleavage.
Clearly, from the relative behavior of 6, the extended conforma-
tion characteristic of Gly is not optimal for DNA cleavage. In
addition, as the size of this substituent increases, the DNA-
cleavage efficiency decreases (R ) Me = Et > i-Pr), consistent
with the observation that the conformations nonproductive for
primary strand cleavage becomes increasingly favored. Both
of these observations are consistent with the premise that linker
region substituents preorganize the agent, facilitating adoption
of the productive DNA-bound conformation that enhances the
cleavage selectivity over nonproductive background cleavage
and increases the overall cleavage efficiency.
53
mation of Zn bleomycin A2 are represented as this bisected
conformation (Ψ ) 120°). Thus, the local threonine conforma-
tion is characterized by a Φ ) -120, Ψ ) 60-180° accessible
conformational space. It is thus tempting to suggest that this
site also constitutes one of the critical swivel points for the agent
that permits the adoption of a class of related bound conforma-
tions (Ψ ) 60-180°) adaptable to variable conformational
characteristics required of the multiple cleavage sites, including
both primary cleavage and subsequent ds cleavage on the
complementary strand from a common intercalation site.
Experimental Section67
Nâ-[1-Amino-3(S)-[4-amino-6-[[[1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-
dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithiazol-2-yl)-1-
ethyl)amino)carbonyl)-2-hydroxy-1-ethyl)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 (3).67 A solution of 20a67 (5 mg, 0.005
mmol) in CH2Cl2 (100 µL) at 0 °C was treated with 20% TFA-CH2Cl2
(200 µL) and the mixture was stirred at 0 °C (1.5 h) before the solvent
was evaporated in vacuo. Reverse phase chromatography (C-18, 1.2
× 2.0 cm, 0-60% CH3OH-H2O gradient elution) afforded 3 (3.8 mg,
4.6 mg theoretical, 85%) as a white film: Rf ) 0.2 (SiO2, 10:9:1 CH3-
OH-10% aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25D -8.0
Conclusions
Variations in the L-threonine subunit substituents had no
impact on the characteristic 5′-GC, 5′-GT cleavage selectivity
of deglycobleomycin but did have a large effect on the cleavage
efficiency. The studies define a clear potentiating role for the
L-threonine hydroxyl group, highlight the additional importance
of the presence of a substituent on the L-threonine subunit (R
* H), and suggest the effect is unusually sensitive to the size
of the substituent (R ) CH3 = Et > i-Pr). A good correlation
was observed between the cleavage efficiencies and the
calculated Boltzmann probabilities66 of adopting the L-threonine
subunit local conformation observed with free Co(III)OOH
bleomycin A2 and, with the exception of Val, with the total
distribution within the free and DNA-bound conformations of
Co(III)OOH bleomycin A2. This suggests that the L-threonine
side chain facilitates preorganization of bleomycin A2 into a
conformation that leads to productive DNA cleavage. The
important role of the L-threonine hydroxyl group may be
attributed to either intramolecular H-bonding to the threonine
carbonyl or, more provocatively, to the distal pyrimidoblamic
acid C2 acetamido side-chain carbonyl and the metal-coordi-
nated, â-amino group of the C2 side chain, stabilizing a
preorganized conformation of activated bleomycin productive
for DNA cleavage. However, we cannot rule out an intermo-
lecular H-bond with duplex DNA itself although structural
models to date have not provided evidence for such an
interaction. The additional, simpler effect of the substituent
presence is consistent with a subtle but important role in
restricting the available conformations accessible to the agent.
The presence of a substituent (R * H) restricts the valerate-
threonine amide to a single orientation (Φ = -120°), inducing
a turn in the linker domain characteristic of the compact bound
1
(c 0.10, CH3OH); H NMR (CD3OD, 400 MHz) δ 8.19 (s, 1H), 8.11
(s, 1H), 7.68 (br s, 1H), 7.10 (br s, 1H), 5.17 (d, J ) 6.5 Hz, 1H), 4.79
(d, J ) 6.5 Hz, 1H), 4.41 (dd, J ) 5.5, 5.5 Hz, 1H), 3.97 (dd, J ) 5.5,
9.0 Hz, 1H), 3.91 (dd, J ) 5.5, 5.5 Hz, 1H), 3.78 (dd, J ) 6.0, 6.0 Hz,
1H), 3.74 (dd, J ) 5.5, 5.5 Hz, 2H), 3.65 (m, 4H), 3.57 (dd, J ) 6.0,
6.0 Hz, 2H), 3.35 (dd, J ) 6.5, 6.5 Hz, 2H), 3.26 (dd, J ) 7.0, 7.0 Hz,
2H), 2.91 (s, 6H), 2.86 (m, 2H), 2.71 (dd, J ) 4.5, 15.0 Hz, 1H), 2.53
(dd, J ) 9.0, 15.0 Hz, 1H), 2.41 (dq, J ) 7.0, 7.0 Hz, 1H), 2.14 (tt, J
) 7.0, 7.0 Hz, 2H), 1.11 (d, J ) 6.5 Hz, 3H), 1.08 (d, J ) 6.5 Hz,
3H); IR (neat) νmax 3282, 2912, 1656, 1641, 1589, 1548, 1487, 1364,
1241, 1205, 1092 cm-1; FABHRMS (NBA-CsI) m/z 1033.3975 (M+,
C41H61N16O10S3 requires 1033.3919).
Nâ-[1-Amino-3(S)-[4-amino-6-[[[1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-
dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithiazol-2-yl)-1-
ethyl)amino)carbonyl)-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 (4):67 (86%) Rf ) 0.2 (SiO2, 10:9:1 CH3OH-
10% aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25 -6.0 (c
D
1
0.05, CH3OH); H NMR (CD3OD, 400 MHz) δ 8.19 (s, 1H), 8.10 (s,
1H), 7.67 (br s, 1H), 7.11 (br s, 1H), 5.13 (d, J ) 6.5 Hz, 1H), 4.76 (d,
J ) 6.5 Hz, 1H), 4.23 (dd, J ) 6.0, 8.0 Hz, 1H), 3.96 (dd, J ) 4.5, 9.5
Hz, 1H), 3.91 (dd, J ) 5.0, 6.5 Hz, 1H), 3.76 (dd, J ) 6.0, 6.0 Hz,
1H), 3.69 (dd, J ) 7.0, 7.0 Hz, 1H), 3.61 (m, 3H), 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,
(67) Full characterization of intermediates 10a-e, 11a-e, 13a-e, 14a-
e, 15a-e, 17a-e, 18a-e, and 20a-e is provided in Supporting Information.

9148 J. Am. Chem. Soc., Vol. 120, No. 36, 1998
Boger et al.
2H), 2.92 (s, 6H), 2.85 (d, J ) 9.0 Hz, 2H), 2.71 (dd, J ) 4.5, 15.0
Hz, 1H), 2.52 (dd, J ) 9.0, 15.0 Hz, 1H), 2.44 (dq, J ) 7.0, 7.0 Hz,
1H), 2.27 (s, 3H), 2.14 (tt, J ) 7.0, 7.0 Hz, 2H), 1.75 (m, 1H), 1.61
(m, 1H), 1.12 (d, J ) 7.0 Hz, 3H), 1.07 (d, J ) 7.0 Hz, 3H), 0.88 (dd,
J ) 7.5, 7.5 Hz, 3H); IR (neat) νmax 3292, 2933, 1656, 1641, 1589,
1482, 1241, 1205, 1092 cm-1; FABHRMS (NBA-CsI) m/z 1031.4182
(M+, C42H63N16O9S3 requires 1031.4126).
7.99 (s, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.17 (s, 1H), 5.10 (m, 1H),
4.21 (m, 1H), 3.93 (m, 3H), 3.64 (m, 3H), 3.41 (m, 4H), 3.31 (m, 2H),
3.19 (t, J ) 7.5 Hz, 2H), 3.07 (m, 2H), 2.71 (s, 6H), 2.53 (m, 2H),
2.37 (m, 1H), 2.24 (s, 3H), 1.97 (m, 2H), 1.01 (d, J ) 6.5 Hz, 3H),
0.90 (d, J ) 7.0 Hz, 3H); IR (neat) νmax 3451, 1643, 1256, 1094, 1021
cm-1; FABMS (NBA) m/z 1003 (M+, C40H59N16O9S3).
Nâ-[1-Amino-3(S)-[4-amino-6-[[[1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-
dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithiazol-2-yl)-1-
ethyl)amino)carbonyl)-2-methyl-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 (7):67 (83%) Rf ) 0.2 (SiO2, 10:9:1
Nâ-[1-Amino-3(S)-[4-amino-6-[[[1(S)-(((4(S)-(((1(S)-(((2-(4′-((((3-
dimethylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithiazol-2-yl)-1-
ethyl)amino)carbonyl)-1-ethyl)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-ami-
noalanine amide (5):67 (85%) Rf ) 0.2 (SiO2, 10:9:1 CH3OH-10%
CH3OH-10% aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25
D
aqueous CH3CO2NH4-10% aqueous NH4OH); [R]25 -7.5 (c 0.07,
1
-4.0 (c 0.05, CH3OH); H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H),
D
1
CH3OH); H NMR (CD3OD, 400 MHz) δ 8.19 (s, 1H), 8.11 (s, 1H),
8.10 (s, 1H), 7.68 (br s, 1H), 7.12 (br s, 1H), 5.12 (d, J ) 6.5 Hz, 1H),
4.75 (d, J ) 6.5 Hz, 1H), 4.14 (d, J ) 7.5 Hz, 1H), 4.08 (m, 1H), 3.95
(dd, J ) 5.5, 9.0 Hz, 1H), 3.91 (dd, J ) 5.5, 5.5 Hz, 1H), 3.73 (m,
1H), 3.67 (m, 4H), 3.58 (dd, J ) 6.0, 6.0 Hz, 2H), 3.36 (dd, J ) 6.5,
6.5 Hz, 2H), 3.12 (m, 1H), 2.92 (s, 6H), 2.85 (m, 2H), 2.69 (dd, J )
4.5, 15.0 Hz, 1H), 2.51 (dq, J ) 7.0, 7.0 Hz, 1H), 2.28 (s, 3H), 2.14
(tt, J ) 7.0, 7.0 Hz, 2H), 2.00 (m, 1H), 1.13 (d, J ) 7.0 Hz, 3H), 1.06
(d, J ) 7.0 Hz, 3H), 0.89 (d, J ) 4.0 Hz, 3H), 0.87 (d, J ) 4.0 Hz,
3H); IR (neat) νmax 3220, 2923, 1656, 1641, 1589, 1548, 1492, 1446,
1246, 1092 cm-1; FABHRMS (NBA-CsI) m/z 1045.4215 (M+,
C43H65N16O9S3 requires 1045.4282).
7.67 (br s, 1H), 7.11 (br s, 1H), 5.11 (d, J ) 6.5 Hz, 1H), 4.76 (d, J )
6.5 Hz, 1H), 4.33 (dd, J ) 7.0, 7.0 Hz, 1H), 3.96 (dd, J ) 5.5, 9.0 Hz,
1H), 3.91 (dd, J ) 12.0, 5.5 Hz, 1H), 3.75 (dd, J ) 6.0, 6.0 Hz, 1H),
3.66 (dd, J ) 7.0, 7.0 Hz, 1H), 3.61 (m, 4H), 3.58 (dd, J ) 6.0, 6.0
Hz, 2H), 3.37 (dd, J ) 6.5, 6.5 Hz, 2H), 3.27 (dd, J ) 7.0, 7.0 Hz,
2H), 2.92 (s, 6H), 2.85 (m, 2H), 2.70 (dd, J ) 4.5, 15.0 Hz, 1H), 2.52
(dd, J ) 9.0, 15.0 Hz, 1H), 2.39 (dq, J ) 7.0, 7.0 Hz, 1H), 2.27 (s,
3H), 2.15 (tt, J ) 2.0, 7.0 Hz, 2H), 1.30 (d, J ) 7.0 Hz, 3H), 1.10 (d,
J ) 7.0 Hz, 3H), 1.08 (d, J ) 7.0 Hz, 3H); IR (neat) νmax 3333, 2923,
1656, 1635, 1589, 1482, 1241, 1205, 1087 cm-1; FABHRMS (NBA-
CsI) m/z 1017.3607 (M+, C41H61N16O9S3 requires 1017.3969).
Nâ-[1-Amino-3(S)-[4-amino-6-[[[1(S)-(((4(S)-(((1-(((2-(4′-((((3-di-
methylsulfonio)-1-propyl)amino)carbonyl)-2′,4-bithiazol-2-yl)-1-
ethyl)amino)carbonyl)methyl)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-ami-
noalanine (6):67 (83%) Rf ) 0.10 (SiO2, 10:9:1 CH3OH-10% aqueous
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.).
CH3CO2NH4-10% aqueous NH4OH); [R]23 +14 (c 0.04, 0.1 N
Supporting Information Available: Experimental proce-
dures and characterization of 10a-e, 11a-e, 13a-e, 14a-e,
15a-e, 17a-e, 18a-e, and 20a-e, experimental details of the
DNA-cleavage studies, and the styrene oxidation studies are
provided (14 pages, print/PDF). See any current masthead page
for ordering information and Web access instructions.
D
1
aqueous HCl); H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H), 8.11 (s,
1H), 7.77 (s, 1H), 7.13 (s, 1H), 5.11 (d, J ) 6.6 Hz, 1H), 4.77 (d, J )
6.6 Hz, 1H), 4.02 (m, 2H), 3.93 (m, 2H), 3.64 (m, 4H), 3.60 (m, 3H),
3.37 (dd, J ) 7.5, 7.5 Hz, 2H), 3.26 (dd, J ) 6.6, 6.6 Hz, 2H), 3.00
(m, 2H), 2.93 (s, 6H), 2.75 (m, 1H), 2.53 (m, 1H), 2.38 (dd, J ) 6.8,
6.8 Hz, 1H), 2.26 (s, 3H), 2.15 (tt, J ) 6.8, 6.8 Hz, 2H), 1.12 (d, J )
1
6.8 Hz, 3H), 1.11 (d, J ) 6.7 Hz, 3H); H NMR (D2O, 400 MHz) δ
JA9816638