Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene

Article abstract of DOI:10.1021/ja802118p

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the

Full text of DOI:10.1021/ja802118p

Published on Web 06/28/2008
Synthesis of an Oligodeoxyribonucleotide Adduct of
Mitomycin C by the Postoligomerization Method via a
Triamino Mitosene
Elise Champeil,*,‡ Manuel M. Paz,§ Sweta Ladwa, Cristina C. Clement,¶
Andrzej Zatorski,0 and Maria Tomasz*,†
Department of Chemistry, Hunter College, City UniVersity of New York,
New York, New York 10021, Department of Science, John Jay College, City UniVersity of New
York, New York 10019, and Facultade de Ciencias, Departamente Quimica Organica,
UniVersidade de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain
Received March 26, 2008; E-mail: mtomasz@hunter.cuny.edu; echampeil@jjay.cuny.edu
Abstract: The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA mono-
functionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known
structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was
accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific
placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the
synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected
triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by
reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14
or 24 in several steps. Both were successfully coupled to the 2-fluoro-O6-(2-trimethylsilylethyl)deoxyinosine
residue of the 12-mer oligonucleotide. The N2-phenylacetyl protecting group of 14 after its coupling to the
12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc
protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2
to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the
nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included
HPLC coelution and identical spectral properties with a natural sample, and 1H NMR. Structure proof of
the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct
8, as well as MS and MS/MS analysis.
such adduct of a natural antibiotic5 (Chart 1). The same six
DNA adducts were shown to form in tumor cells treated with
Introduction
Mitomycin C (1; MC1), an antitumor antibiotic, is used in
clinical cancer chemotherapy.2 Its cytotoxic and antitumor
activity is attributed to its ability to alkylate DNA monofunc-
tionally and bifunctionally, the latter mode resulting in DNA
interstrand and intrastrand cross-links.3 Six major DNA adducts
have been isolated from in Vitro systems, formed under
biomimetic conditions, and their structures have been eluci-
dated,4 including the DNA interstrand cross-link (ICL), the first
MC.6 The ICL was also isolated from rat liver DNA of animals
injected with the drug.5 The structures of the six MC-DNA
adducts illustrate that the exclusive target of alkylation of DNA
by MC is the guanine base. Individual structure-activity
relationships of multiple DNA adducts generated by a single
agent have been investigated mostly in the case of organic
mutagens and carcinogens.7 In general, such studies utilize
synthetic oligonucleotides bearing a specific adduct at a unique
position of their base sequences. The MC adducts present an
opportunity for similar studies, enabling in this case direct
comparisons of biological effects of the various DNA adducts
of a cancer chemotherapeutic agent. Using this approach,
comparison of the effects of monoadducts and interstrand cross-
links of MC, its interstrand and intrastrand cross-links, DNA
adducts of MC and DNA adducts of the MC metabolite 2,7-
† Hunter College, City University of New York.
‡ John Jay College, City University of New York.
§ Universidade de Santiago de Compostela.
Current address: A2SP Ltd., 117 Pagitt Street, Chatham, Kent, ME4
6RD, United Kingdom.
¶ Current address: Pathology Department, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
0 Current address: Weill Medical College, Citigroup Biomedical Imaging
Center, 516 East 72nd Street, New York, NY 10021.
(1) Abbreviations: MC, mitomycin C; DAM, diaminomitosene;8 ICL,
interstrand cross-link; TAM, triaminomitosene;8 TMSE, 2-trimethyl-
silylethyl.
(4) Palom, Y.; Belcourt, M. F.; Musser, S. M.; Sartorelli, A. C.; Rockwell,
S.; Tomasz, M. Chem. Res. Toxicol. 2000, 13, 479–488.
(5) Tomasz, M.; Lipman, R.; Chowdary, P.; Pawlak, J.; Verdine, G. L.;
Nakanishi, K. Science 1987, 235, 1204–1208.
(2) Verveij, J. D. H.; Pinedo, H. M. In Cancer Chemotherapy, Chabner,
B. A., Collins, J. M., Eds; Lippincott; Philadelphia, PA, 1990; pp 382-
396.
(6) Bizanek, R.; Chowdary, D.; Arai, H.; Kasai, M.; Hughes, C. S.;
Sartorelli, A. C.; Rockwell, S.; Tomasz, M. Cancer Res. 1993, 53,
5127–5134.
(3) Tomasz, M. Chem. Biol. 1995, 2, 575–579.
(7) Basu, A. K.; Essigmann, J. M. Mutat. Res. 1990, 233, 189–201.
9
9556 J. AM. CHEM. SOC. 2008, 130, 9556–9565
10.1021/ja802118p CCC: $40.75
2008 American Chemical Society

Synthesis of Oligodeoxyribonucleotide Adduct of Mitomycin C
A R T I C L E S
Chart 1. Six Major DNA Adducts of Mitomycin C
diaminomitosene8 (2; 2,7-DAM) formed in situ in MC-treated
cells,4 for example, would be possible. To date, we carried out
a comparison between the mutagenic and cytotoxic effects of
MC-N2-guanine monoadduct 39a and compared these properties
with those of the 2,7-DAM-N7-guanine adduct 7.9b
Synthesis of most of the six MC adducts (Chart l) incorpo-
rated in oligonucleotides has been accomplished by the biomi-
metic route, consisting of the alkylation reaction of MC with a
short DNA duplex of appropriate sequence in the presence of
a reductive MC-activating agent. Varying the activation condi-
tions leads to different adducts.10 This approach has been
successfully applied in the case of adducts 3,11 4,11 5,12 and
7.13 However, the method is usually inefficient, due to the
difficulty of purification of the product to homogeneity.
Furthermore, adducts 6 and 8 are formed in very low yield by
the biomimetic approach to be practical.
postoligomerization/convertible nucleoside approach14a–c in
which the normal nucleophile-electrophile relationship of the
DNA nucleoside and the drug is reversed by using an amine
derivative of the latter and 2-fluorodeoxyinosine as the DNA
target site. Specifically, we describe a synthesis of monoadduct
8 on both the nucleoside and oligonucleotide levels. In view of
the highly sensitive functional groups of the MC molecule its
selective conversion to the requisite amine derivatives 14 and
24 took six steps to be accomplished. The adduct described
herein is, to the best of our knowledge, the most complex DNA
adduct synthesized by the postoligomerization strategy to date.
This strategy was necessitated by the fact that, although adduct
8 is a major adduct in MC-treated tumor cells, it is formed only
in trace quantities in Vitro by biomimetic reactions. Assignment
of its structure was previously based only on absorption
spectrophotometric and mass spectroscopic data, mostly on
1
We report here the first alternative access to one of the adducts
of MC, based on organic synthetic methods, featuring the
material obtained from cells.4 We present now definitive H
NMR characterization of adduct 8.
Adduct 8, along with adduct 7, is the product of reductive
alkylation of DNA by 2,7-DAM (2), the reduced metabolite of
MC. 2,7-DAM is the major product of the reductive activation
of MC in ViVo,15a as well as the enzymatic or chemical reduction
of MC in Vitro (Scheme 1).15b Its 10-carbamoyloxy function is
activated for displacement by a DNA nucleophile after a new
reduction of the quinone function in situ.13 DNA from 2,7-
(8) For the original definition of the trivial name “mitosene” see: Webb,
J. S.; Cosulich, D. B.; Mowat, J. H.; Patrick, J. B.; Broschard, W. E.;
Meyer, R. P.; Williams, R. W.; Wolf, C. F.; Fulmore, W.; Pidacks,
C.; Lancaster, J. E. J. Am. Chem. Soc. 1962, 84, 3185–3186.
(9) (a) Ramos, L. A.; Lipman, R.; Tomasz, M.; Basu, A. K. Chem. Res.
Toxicol. 1998, 11, 64–69. (b) Utzat, D. C.; Clement, C. C.; Ramos,
L. A.; Das, A.; Tomasz, M.; Basu, A. K. Chem. Res. Toxicol. 2005,
18, 213–223.
(10) Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73–87.
(11) Kumar, S.; Lipman, R.; Tomasz, M. Biochemistry 1992, 31, 1399–
1407.
(14) (a) Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T. M. J. Am. Chem.
Soc. 1991, 113, 4328–4329. (b) Erlanson, D. A.; Chen, L.; Verdine,
G. L. J. Am. Chem. Soc. 1993, 115, 12583–12584. (c) DeCorte, B. L.;
Tsarouhtsis, D.; Kuchimanchi, S.; Cooper, M. D.; Horton, P.; Harris,
C. M.; Harris, T. M. Chem. Res. Toxicol. 1996, 9, 630–637. (d) Cao,
H.; Jiang, Y.; Wang, Y. J. Am. Chem. Soc. 2007, 129, 12123–1230.
(12) Borowy-Borowsky, H.; Lipman, R.; Tomasz, M. Biochemistry 1990,
29, 2999–3006.
(13) Suresh Kumar, G.; Musser, S. M.; Cummings, J.; Tomasz, M. J. Am.
Chem. Soc. 1996, 118, 9209–9217.
9
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A R T I C L E S
Champeil et al.
Scheme 1. Reductive Conversion of Mitomycin C to
2,7-Diaminomitosene
Coupling of 14 to the 12-mer Oligonucleotide 5′-CTAGT-
GXTATCC(19a;X)2-fluoro-O6-(2-trimethylsilylethyl)deoxy-
inosine;20 Scheme 4) was accomplished using DMSO as
solvent and diisopropyl ethylamine as a base, with incubation
at 42 °C, for 72 h. The crude mixture was hydrolyzed by mild
acid to remove the TMSE protecting group (20a).14c,d The
mixture was then neutralized to pH 7, and the N2-phenylacetyl-
protected mitosene-oligonucleotide adduct 20a was purified
by HPLC.
Attempts to remove the phenylacetyl protecting group from
the mitosene residue in oligonucleotide 20a by penicillin
amidase have failed repeatedly. In order to verify that this failure
was independent of the sequence we used, we also coupled 14
to a different 12-mer oligonucleotide 5′-GCTAGCXAGTCC-
3′ (19b).20 Deprotection of the analogous alkylated oligo (20b)
also failed consistently. These failures necessitated the synthesis
of a triaminomitosene with a more suitable 2-amino protecting
group that would be removable from the oligonucleotide adduct.
This was accomplished as follows:
Synthesis of a New N2-Protected Triaminomitosene 24
(Scheme 5). Mitosene 21 was hydrolyzed by penicillin amidase
to 22. The free 2-amino group of 22 was then outfitted with the
new protecting group trimethylsilylethoxycarbonyl (Teoc)21 to
give 23. Reduction of the 10-azido group of 23 to the 10-amine
gave the desired protected triaminomitosene derivative 24,
intended for the coupling to oligonucleotides.
DAM-treated tumor cells contains adducts 7 and 8, formed at
relatively high frequency.4 Yet, surprisingly, 2,7-DAM is
essentially noncytotoxic to the same cells, in contrast to the
parent MC.16 Consistent with this finding, in a structure-activity
study, adduct 7 was found to be noncytotoxic and nonmutagenic
in transfection experiments and could be bypassed by high-
fidelity DNA polymerases in Vitro.9b The present synthetic
approach to the previously unavailable sister adduct 8 will
provide a substrate to examine the biological and structural
properties of 8 in parallel with its major groove adduct
counterpart 7. It will also be interesting to compare properties
of 8 with those of the other minor groove guanine-N2 monoad-
ducts of MC, 3 and 4.
Results
Synthesis of the 2-Amino-Protected 2,7,10-Triamino-10-
decarbamoylmitosene 14 from MC (Scheme 2). MC was
converted in two steps to 2,7-diamino-10-decarbamoylmitosene
1015b,c followed by four additional steps to obtain 14 protected
at its 2-amino group by a phenylacetyl group. Conversion of
the 10-hydroxyl function to the 10-amine (12 f 13 f 14)
proceeded smoothly by the Mitsunobu reaction.17 The phenyl-
acetyl protecting group has been shown previously to be
removable from oligonucleotide bases enzymatically by penicil-
lin amidase.18 Deprotection of 14 to the free amine 15 was
accomplished accordingly, but since the necessary intermediate
in the desired transformations was 14, the free mitosene 15 was
not rigorously characterized. Alternatively, access to compound
14 was also possible Via the conversion of 12 to the 10-azide
21 followed by a Staudinger reaction to give 14. Both synthetic
routes were explored and were found to give similar overall
yields (40%).
Synthesis of the Adducted Nucleoside 8 (Scheme 3) was
accomplished by reaction of 14 with 2-fluoro-O6-(2-p-nitro-
phenylethyl)deoxyinosine 1619 to give 17 followed by two
consecutive deprotection steps: first, removal of the 2-p-
nitrophenylethyl group by DBU from the nucleobase,19 then
hydrolysis of the phenylacetamide 18 by penicillin amidase to
give the free amine 8. This substance was shown to be identical
with adduct 8 isolated from MC-treated tumor cells6 and from
a biomimetic reaction in Vitro,4 with respect to their coelution
by HPLC, and its mass, UV, and CD spectra (Figure 1). Its 1H
NMR and COSY spectra (Figure 2 and S1 respectively) support
the previously derived structure as 8.4
Coupling of the New Triaminomitosene 24 and the 12-mer
Oligonucleotide 5′-CTAGTGXTATCC (19a; X ) 2-fluoro-
O6-(2-trimethylsilylethyl)deoxyinosine)20 and Successful Depro-
tection to the Adducted Oligonucleotide 26 (Scheme 6). Com-
pounds 24 and 19a were incubated in DMSO in the presence
of diethylamine for 72 h at RT. HPLC indicated that the
coupling yielded 80% of a product, presumably 25, as it showed
absorbance at 315 nm. Oligonucleotide-type materials were
isolated through ethanol precipitation. Deprotections at the
guanine-O6 and mitosene-N2 positions were performed in one
step using ZnBr2. The final product 26 was isolated by Sephadex
G-25 gel filtration followed by HPLC purification in 57% overall
yield.
Verification of the Structure and Purity of the Adducted
Oligonucleotide 26. The oligonucleotide product 26 was isolated
by HPLC, and a mass spectrum was obtained, confirming the
identity of the correct site-specific-modified oligonucleotide.
Negative ESI-MS results showed that the deconvoluted mass
of 26 was 243 Da higher than the calculated mass of the
unmodified 5′-CTAGTGGTATCC which is consistent with the
presence of adduct 8 in the substrate. Moreover, high-resolution
negative ESI-MS results revealed that the deconvoluted mass
of the molecular ion, 3877.74444, differed from the calculated
m/z of 3877.74104 for the corresponding peak by only 0.8 ppm
(Figure S2). We next characterized the above oligonucleotide
by MS/MS (Figure 3) as was described previously in an
analogous case.14b The product ion spectrum of the [M - 3H]3-
ion (m/z 1292) of the oligonucleotide 26 showed the formation
2-
of wn ions, that is, w1-, w3-, w52-, w62-, w8 and [an-base]
(15) (a) Chirrey, L.; Cummings, J.; Halbert, G. W.; Smyth, J. F. Cancer
Chemother. Pharmacol. 1995, 35, 318–3226. (b) Tomasz, M.; Lipman,
R. Biochemistry 1981, 20, 5065–5071. (c) Kinoshita, S.; Uzu, K.;
Nakano, K.; Takahashi, T. J. Med. Chem. 1971, 109–110.
(16) Palom, Y.; Belcourt, M. F.; Suresh Kumar, G.; Arai, H.; Kasai, M.;
Sartorelli, A. C.; Rockwell, S.; Tomasz, M. Oncol. Res. 1998, 10,
509–521.
ions, that is, [a2-T]- [a3-A]- [a4-G]- [a6-G]- [a7-8]2- [a11-T]6-
ions (nomenclature for fragment ions follows that reported by
(20) Oligonucleotides 19a and 19b were a kind gift of Dr. Carmelo Rizzo,
Vanderbilt University, synthesized by the general method described
in ref 14a.
(21) Ferreira, F.; Morvan, F. Nucleosides, Nucleotides, Nucleic Acids 2005,
24, 1009–1013.
(17) Hughes, D. L. Org. React. 1992, 42, 335–656.
(18) Waldman, H.; Heuser, A.; Reidel, A. Synlett 1994, 65–67.
(19) Lee, H.; Hinz, M.; Stezowsky, J. J.; Harvey, R. G. Tetrahedron Lett.
1990, 31, 6773–6780.
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Synthesis of Oligodeoxyribonucleotide Adduct of Mitomycin C
A R T I C L E S
Scheme 2. Synthesis of 2,7,10-Triamino-10-decarbamoylmitosene 15a
a Reagents: (a) CH3ONa, toluene, 80%; (b) 5% Pd/C, MeOH, 75%; (c) phenylacetic anhydride, pyridine, 70%; (d) NH3, MeOH, 90%; (e) phtalimide,
DIAD, PPh3, DMF, 50%; (f) hydrazine, EtOH, 80%; (g) methanesulfonyl chloride, pyridine followed by NaN3, DMSO, 48%; (h) PPh3, NH4OH, THF/H2O,
96%;(i) penicillin amidase, potassium phosphate buffer, pH 8.5, aq MeOH, 90%.
McLuckey et al.22). The measured masses for the w1, w3, and
w5 ions were the same as the calculated masses for the
corresponding ions of the unmodified oligonucleotide, whereas
the w6 and w8 ions exhibited 243 Da higher in mass than the
calculated corresponding fragments formed from the unmodified
d(CTAGTGGTATCC). These results are consistent with the
presence of adduct 8 at the seventh position in the oligonucle-
otide. The above conclusion is further substantiated by the
observed masses for the [an-base] ions. In this respect, the
measured masses for the [a11-T]6- ion is 243 Da higher, whereas
the measured masses of the [a2-T]- [a3-A]- [a4-G]- [a6-G]-
[a7-8]2- are the same as the calculated ones of the corresponding
fragment ions for the unmodified d(CTAGTGGTATCC). This
technique confirmed the site of adduct 8 incorporation.
19 from 2,7-diamino-10-decarbamoylmitosene 10 required two
main synthetic imperatives: (i) conversion of the 10-hydroxyl
group to a 10-amino group and (ii) suitable protection for the
2-amino group, so that the coupling would occur solely at the
10-position. The protecting group also had to be removable
under mild conditions so as not to damage the mitosene moiety
and be compatible with the presence of amines, i.e., base
resistant.
Both tasks demanded that we explore the chemical reactivity
of 2,7-diamino-10-decarbamoylmitosene 10. This was a difficult
undertaking as MC derivatives have several highly reactive
functional groups; therefore, it is hard to find suitably selective
protection and deprotection methods applicable to these mol-
ecules in general.
In addition, oligonucleotide 26 was submitted to enzymatic
digestion to nucleosides followed by HPLC analysis. The
chromatogram of the digest established the presence of the
mitosene-adducted nucleoside 8. This was further validated by
the coelution of an authentic sample of 8 with this digestion
product (Figure 4b). Furthermore, we integrated the peak areas
for adduct 8, dC dG, and dT in the HPLC traces of the digest
of oligonucleotide 26 (Figure 4a) and estimated the molar ratios
We first chose the phenylacetyl group as the protecting group
for the 2-amino position and synthesized the amino mitosene
14 from MC. The phenylacetyl group has been previously used
by other research groups to protect peptides and oligonucle-
otides.23 This group was utilized for the protection and depro-
tection of amino functions of nucleobases since, being removable
by enzymatic hydrolysis around neutral pH, it offered an
alternative route to the acidic or basic conditions required for
the removal of the established blocking groups. 2,7-Diamino-
10-decarbamoylmitosene 10 was submitted to phenylacetylation
using phenylacetic anhydride. The anhydride was found to be
highly reactive and led to extremely short reaction times. Both
the hydroxyl and amino groups were acylated in less than 5
min; after longer times a new yellow compound was formed,
probably resulting from hyperacylation of the mitosene moiety.
Selective removal of the phenylacetyl group from the 10-
hydroxyl group was executed using ammonia in methanol.
Two routes for the conversion of the 10-hydroxyl function
of 12 to the 10-amine of 14 were explored and were found to
give similar yields. One proceeded smoothly by the Mitsunobu
reaction. Formation of the N7-iminophosphorane was observed
with the consideration of the molar extinction coefficients (E260
)
of the three nucleosides. In the case of adduct 8, a correction
for E260 was done by adding 5189 L mol-1 cm-1 (E260 of 2,7-
DAM) to that of dG.9b It turned out that the calculated molar
ratios of 8:dC:dG:dT were 1:2.8:2:4.3 which are consistent with
the presence of one adduct 8, three dC, two dG, and four dT
residues in oligonucleotide 26. Because of the partial deami-
nation of dA to dI during enzymatic digestion, the peak area
for dA was not determined.
Discussion
The synthesis of a suitable aminomitosene for the post-
oligomerization coupling with nucleoside 16 and oligonucleotide
(22) McLuckey, S. A.; Vankerbel, G. J.; Glish, G. L. J. Am. Soc. Mass.
Spectrom. 1992, 3, 60–70.
(23) (a) Waldman, H.; Reidel, A. Angew. Chem., Int. Ed. Engl. 1997, 36,
647–649. (b) Waldman, H. Tetrahedron Lett. 1988, 29, 1131–1134.
9
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A R T I C L E S
Champeil et al.
Scheme 3. Synthesis of the Nucleoside Adduct 8: Coupling of the
N2-Protected Triaminomitosene 14 to the
a
2-Fluoro-O6-(2-p-nitrophenylethyl)deoxyinosine 16
Figure 1. Ultraviolet spectrum of adduct 8 (a) and circular dichroism
spectrum of adduct 8 (b).
The condensation reactions of 14 with oligonucleotides 19a and
19b occurred in more dilute solutions than the reaction with
nucleoside 16 ([amine] ) 0.98 M for the nucleoside versus [amine]
) 0.3 M for the oligonucleotides). As a result, the rates were
significantly lower,and maximal couplings were achieved after 72 h.
Because of the ease with which the enzymatic deprotection
occurred at the nucleoside level as well as of the reported enzymatic
a Reagents: (a) Et3N, DMF, 30%; (b) DBU, DMF/acetonitrile, 80%; (c)
penicillin amidase, potassium phosphate buffer, pH 8.5, aq MeOH, 85%.
deprotection of phenylacetylated bases in oligonucleotides,18,23
a
similar outcome was anticipated for the oligonucleotide coupling
product, perhaps at a slower rate. However, when oligonucleotide
20a was submitted to penicillin amidase treatment, the deprotection
failed repeatedly. We varied the reaction conditions (i.e., temper-
ature, enzyme concentration, oligonucleotide concentration) and
the sequence of the substrate (20a vs 20b) to no avail. Although
some removal of the phenylacetyl group was detected, this was
accompanied by degradation, as HPLC of the reaction mixture
revealed the appearance of several new uncharacterized alkylated
oligonucleotides. Evidently, the efficacy of the enzymatic trans-
formation was subtly and strongly dependent on the type of
substrate. We concluded that the enzymatic deprotection was not
appropriate at the oligonucleotide level, and this approach was
abandoned.
This repeated failure prompted us to look for new protecting
groups. A proper protecting group had to be removable under
nonbasic (since the coupling occurs in the presence of base)
and nonacidic conditions (to prevent depurination). Our focus
shifted to the Teoc group which can be removed with ZnBr2
under mild conditions. The Teoc group has been recently used
to protect nucleosides in oligonucleotide synthesis.21 The
synthesis of mitosene 24 was performed according to Scheme
5 as attempts to selectively protect the 2-amino group of
in some cases due to the presence of excess triphenylphosphine.
However, acidic hydrolysis efficiently converted the imino-
phosphorane back to the free 7-amino group. The second route
we investigated was the conversion of the 10-hydroxyl group
to an azido group. The hydroxyl group was first mesylated. The
nucleophilic substitution of the mesylate by the azido group
proceeded smoothly in DMSO to yield 21. Reduction of the
azido group to the amino group was achieved Via a Staudinger
reaction in ammonium hydroxide to give 14. The Staudinger
reaction did not proceed without the presence of ammonia in
the reaction mixture.
The reactivity of the aminomitosene 14 was sufficient for
the synthesis of adduct 17 which was then deprotected enzy-
matically to adduct 8 using penicillin amidase. Previous
elucidation of the structure of adduct 8 isolated from sources
of biomimetic material was based primarily on the difference
UV spectral method,24 MS, and chemical tests.4 However, due
to the scarcity of material, no NMR data of the adduct has been
obtained, and definite proof of its structure was still lacking.
The present organic synthesis of adduct 8 enabled the unam-
biguous characterization of this adduct.
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Synthesis of Oligodeoxyribonucleotide Adduct of Mitomycin C
A R T I C L E S
Figure 2. 1H NMR spectrum of 8 in pyridine-d5/D2O. H8(g), 8-proton of guanine.
Scheme 4. Coupling of 14 to the 12-mer Oligonucleotide
Scheme 5. Synthesis of N2-Protected 2,7,10-Triaminomitosene
(24)
5′-CTAGTGXTATCC-3′ (19a)a
a Coupling of 14 to 5′-GCT-AGC-XAG-TCC (19b) resulted in the
corresponding product 20b which, similarly to 20a, was resistant to penicillin
amidase.
number of transformations performed on oligonucleotide 25,
hence increasing the overall yield (Scheme 6).
compound 10 with the Teoc group in one step was unsuccessful.
The protected mitosene 24 was coupled to oligonucleotide 19a.
The coupling occurred readily as monitored by HPLC. The
product appeared as a new peak with an absorbance at 320 nm,
serving as evidence of the incorporation of the mitosene moiety
into the oligonucleotide. The crude modified oligonucleotide
25 was subsequently subjected to deprotection. ZnBr2 was found
to be the best reagent leading to complete and rapid deprotection
of the Teoc group at the nucleoside level.21 These conditions
also removed the O6-TMSE group, offering the advantage of
complete deprotection of oligonucleotide 25 at the mitosene-
N2 and guanine-O6 positions in a single step. This limited the
Conclusion and Significance
The aim of the present work was to prepare a mitomycin C
(1) derivative suitable for incorporation of the known MC-
deoxyguanosine adduct 8 in oligonucleotides, using the posto-
ligomerization adduct synthesis method.14 This was accom-
plished by conversion of MC (1) to the N2-protected triamino-
10-decarbamoyl-mitosene 24 in six steps, followed by coupling
of this mitosene to the O6-protected 2-fluorodeoxyinosine
residue of oligonucleotide 19a. Chemical removal of both
protecting groups of the product 25 in one step yielded the final
product 26. This is the first report, to our knowledge, of an
application of postoligomerization synthesis to a DNA adduct
of an antitumor antibiotic natural product. The mitosene
(24) Verdine, G. L.; Nakanishi, K. J. Am. Chem. Soc. 1985, 107, 6118–
6120.
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A R T I C L E S
Champeil et al.
Scheme 6. Postoligomerization Synthesis of the Adduct
8-Substituted Oligonucleotide (26)
derivative 14 was utilized for an authentic synthesis of deoxy-
guanosine adduct 8, which was previously not characterized
rigorously.4
This work extends the preparative availability of known MC
adducts on the oligonucleotide level, enabling further studies
of adduct structure and adduct structure-activity relationships.
Our results raise the possibility that the last oligonucleotide-
level MC adduct, intrastrand cross-link 6 (Chart 1) that is still
Figure 4. (a) HPLC chromatogram of the digest of oligonucleotide 26.
(b) HPLC chromatogram of the coelution of the digest with an authentic
sample of 8 (s 260 nm, --- 320 nm).
unavailable for such studies, may be synthesized by a similar
postoligomerization approach.
Experimental Section
Materials. Mitomycin C was obtained from Bristol-Myers
Squibb, Wallingford, CT, and from Kyowa Hakko Kogyo Co., Ltd.,
Japan. Synthetic oligonucleotides containing O6-protected 2-fluo-
rodeoxyinosine were a kind gift of Dr. Carmelo Rizzo from
Vanderbilt University and were further purified by us as described
below. Reagents were obtained from commercial sources and were
used without further purification. Thin layer chromatographic
analyses were carried out on 250 µm silica gel plates containing a
fluorescent indicator, and if necessary, spots were visualized with
I2. Column chromatographic purifications were performed using
200-300 mesh silica gel. All reactions were carried out under an
1
atmosphere of Ar unless otherwise noted. H NMR spectra were
obtained at 500 MHz and are referenced to residual protonated
solvent. Chemical shifts are reported in parts per million and
coupling constants are in hertz (Hz). Carbon-13 data was recorded
at 125 MHz. Mass spectra of modified oligonucleotides and HRMS
of all compounds were recorded using Agilent Technologies 6210
TOF (HPLC: Agilent 1200), except for oligonucleotide 26 the mass
spectrum of which was recorded using Agilent Technologies 6520
Figure 3. MS-MS characterization of oligonucleotide 26. Product ion
spectrum of the [M - 3H]3- ion (m/z ) 1292.24). For clarity purposes,
w1- (306.04), w82- (1360.80), [a2-T]- (401.01), and [a6-G]- (1636.18) are
not represented.
9
9562 J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008

Synthesis of Oligodeoxyribonucleotide Adduct of Mitomycin C
A R T I C L E S
Q-TOF (HPLC: Agilent 1200), at Hunter College Mass Spectrom-
etry Facility. Synthetic oligonucleotides were purified by C-18
reverse phase HPLC in NH4HCOO (0.1 M in water)/acetonitrile
with a diode array detector monitoring at 260 and 320 nm. The
following gradient was used: initial condition 99% NH4COO (0.1
M in water)-1% acetonitrile, then 60 min linear gradient to 80%
acetonitrile, 5 min at 80% acetonitrile followed by 5 min linear
gradient to initial conditions. The conventional numbering system
is used for the MC and mitosene8 moieties, and the purine carbons
are numbered 1-9 also as per convention.
3.15, 5.5 Hz, 2H) and 7.87 (dd, J ) 3.1, 5.5 Hz, 2H). HRMS m/z
calcd for C29H25N4O5 [M + H]+ 509.1825, found 509.1810.
10-Azido-2,7-diamino-N2-phenylacetyl-10-decarbamoylmito-
sene (21). Compound 12 (46 mg, 0.121 mmol) was diluted in 1
mL of dry pyridine and cooled to 0°. Methanesulfonyl chloride
(60 µL, 88.44 mg, 0.772 mmol) was added dropwise and the
reaction mixture was warmed to room temperature and stirred for
2 h. TLC indicated the disappearance of all starting material.
Solvents were evaporated and the residue was dried in a desiccator.
The crude material was dissolved in dry DMSO and dry sodium
azide (100 mg, 1.53 mmol) was added. The reaction mixture was
heated at 100° for 2.5 h. The resulting red residue was purified by
flash chromatography (SiO2:2% to 10% MeOH in CH2Cl2). (23.5
mg, 48% yield). 1H NMR (methanol-d4) δ 1.79 (s, CH3, 3H), 2.79
(dd, H1, J ) 4.3, 16.5 Hz, 1H), 3.24 (dd, H1, J ) 7.5, 16.5 Hz,
1H), 3.49 (s, CH2, 2H), 4.08 (dd, H3, J ) 4.2, 13.1 Hz, 1H), 4.44
(dd, H3, J ) 7.1, 13.1 Hz, 1H), 4.45 (s, CH2, 2H), 4.96 (m, H2,
1H), 7.25 (m, Har, 5 H). 13C NMR (methanol-d4) δ 6.8 (1C, CH3),
29.5 (1C), 39.0 (1C), 42.2 (1C), 44.7 (1C), 52.5 (1C), 104.8 (1C,
CdC), 110.3 (1C, CdC), 121.9 (1C, CdC), 126.5 (1C, Ph), 128.4
(2C, Ph), 128.7 (2C, Ph), 135.5 (1C, Ph), 139.5 (1C, CdC), 147.5
(1C, CdC), 172.6 (1C, CO), 177.5 (1C, CO), 178.6 (1C, CO).
HRMS m/z calcd for C21H19N6O3 [M - H]+ 403.1519, found
403.1524.
2,7-Diamino-10-decarbamoylmitosene8 (10). Palladium over
charcoal (71.3 mg) was added to a solution of 10-decarbamoyl
mitomycin C15c (170.7 mg, 0.587 mmol) in deaerated methanol
(18 mL). H2 gas was bubbled through the solution while acetic
acid (54.8 µL, 57.5 mg, 0.957 mmol) was added by a syringe. H2
gas was bubbled gently for a additional 10 min. TLC indicated the
disappearance of all starting material. The reaction mixture was
filtered through Celite and concentrated in Vacuo. The product (136
mg, 90% yield) was isolated by silica gel chromatography (SiO2:
10% to 15% MeOH in CH2Cl2).1H NMR (methanol-d4) δ 1.82 (s,
CH3, 3H), 2.73 (dd, H1, J ) 4.5, 16.5 Hz, 1H), 3.23 (dd, H1, J )
3.8, 16.5 Hz, 1H), 3.99 (dd, H3, J ) 4.3, 12.9 Hz, 1H), 4.24 (m,
H2, 1H), 4.41 (dd, H3, J ) 6.7, 12.9 Hz, 1H), 4.60 (s, CH2, 2H).
13C NMR (methanol-d4) δ 6.8 (1C, CH3), 31.7 (1C, CH2), 53.9
(1C), 57.9 (1C), 58.7 (1C), 104.6 (1C, CdC), 112.1 (1C, CdC),
121.8 (1C, CdC), 128.6 (1C, CdC), 139.7 (1C, CdC), 147.5 (1C,
CdC), 177.8 (1C, CO), 178.5 (1C, CO). HRMS m/z calcd for
C13H15N3O3Na [M + Na]+ 284.1005, found 284.1026.
2,7-Diamino-N2-phenylacetyl-10-decarbamoylmitosene (12).
2,7-Diamino-10-decarbamoylmitosene (10 mg, 0.039 mmol) was
dissolved in 1 mL of dry pyridine, and the reaction vessel was
sonicated. Phenylacetic anhydride (43.8 mg, 0.42 mmol) was added.
TLC indicated the disappearance of all starting material after 10
min. The reaction mixture was diluted with ethyl acetate and washed
with water, dilute HCl, and NaHCO3. Drying (MgSO4) and
evaporation of solvents gave a red residue which was dried in a
desiccator under vacuum. The residue was redissolved in 2.5 mL
of 7 N ammonia in methanol. The mixture was stirred overnight at
room temperature. The resulting product was diluted with ethyl
acetate, washed with water and satd. NaHCO3, and once more with
water. The organic phase was dried (MgSO4). Evaporation of
solvents gave a red residue. The final product was purified by
chromatography (SiO2:10% MeOH in CH2Cl2) (13 mg, 89%
yield).1H NMR (methanol-d4) δ 1.81 (s, CH3, 3H), 2.81 (dd, H1, J
) 4.5, 16.5 Hz, 1H), 3.25 (dd, H1, J ) 7.5, 16.5 Hz, 1H), 3.52 (s,
CH2, 2H), 4.07 (dd, H3, J ) 3.5, 12.5 Hz, 1H), 4.44 (dd, H3, J )
7.5, 12.5 Hz, 1H), 4.69 (s, CH2, 2H), 4.96 (m, H2, 1H), 7.30 (m,
Har, 5 H). 13C NMR (methanol-d4) δ 6.8 (1C, CH3), 29.4 (1C, CH2),
42.2 (1C), 52.3 (1C), 52.6 (1C), 55.5 (1C), 104.7 (1C, CdC), 116.8
(1C, CdC), 121.8 (1C, CdC), 126.5 (1C, Ph), 128.2 (2C, Ph), 128.7
(2C, Ph), 135.4 (1C, Ph), 138.3 (1C, CdC), 147.5 (1C, CdC), 172.6
(1C, CO), 177.8 (1C, CO), 178.9 (1C, CO). HRMS m/z calcd for
C21H22N3O4 [M + H]+ 380.1610, found 380.1614.
2,7,10-Triamino-N2-phenylacetyl-10-decarbamoylmitosene (14).
Synthesis from 21: Compound 21 (20 mg, 0.0494 mmol) was
dissolved in 1.2 mL of pyridine at room temperature. Ammonium
hydroxide (0.6 mL of a 30% solution) and triphenylphosphine (45
mg, 0.171 mmol) were added. The mixture was stirred at room
temperature for 36 h. The resulting purple product was isolated by
chromatography (SiO2:7.5% MeOH, 2% NEt3, 81.5% CH2Cl2) (18
mg, 96% yield). Synthesis from 13: Compound 13 (15 mg, 0.0295
mmol) was dissolved in 20 mL of 80% hydrazine in ethanol. The
mixture was stirred overnight at room temperature. Solvents were
evaporated and the purple residue was purified by chromatography
(SiO2:7.5% MeOH, 2% NEt3, 81.5% CH2Cl2). (10 mg, 89%
yield).1H NMR (methanol-d4) δ 1.83 (s, CH3, 3H), 2.93 (dd, H1, J
) 4.8, 16.3 Hz, 1H), 3.29 (dd, H1, J ) 7.5, 16.3 Hz, 1H), 3.54 (s,
CH2, 2H), 4.12 and 4.16 (AB quartet, H10, J ) 13.9 Hz, 2H), 4.12
(dd, H3, J ) 3.5, 12.5 Hz, 1H), 4.51 (dd, H3, J ) 7.5, 12.9 Hz,
1H), 4.96 (m, H2, 1H), 7.30 (m, Har, 5 H). 13C NMR (CDCl3/
methanol-d4) δ 7.9 (1C, CH3), 29.4 (1C), 35.7 (1C), 42.9 (1C),
52.6 (1C), 53.5 (1C), 107.1 (1C, CdC), 113.2 (1C, CdC), 122.4
(1C, CdC), 126.9 (1C, Ph), 128.6 (2C, Ph), 129.1 (2C, Ph), 129.4
(C, CdC), 134.8 (1C, Ph), 139.1 (1C, CdC), 145.6 (1C, CdC),
171.7 (1C, CO), 177.5 (1C, CO), 179.6 (1C, CO). HRMS m/z calcd
for C21H23N4O3 [M + H]+: 379.1770, found: 379.1751.
2,7,10-Triamino-10-decarbamoylmitosene (15). Synthesis from
14: A solution of 14 (20 mg, 53 mmol) in MeOH (50 mL) and
potassium phosphate buffer (0.05M, pH 8.5, 100 mL) was treated
with penicillin amidase (0.540 mL, 500 units). The resulting solution
was gently stirred for 18 h at room temperature, when TLC analysis
(5% MeOH in CH2Cl2) showed the disappearance of 14. The
mixture was partially concentrated to remove MeOH. The resulting
aqueous solution was acidified with 1 M HCl and extracted with
EtOAc-hexane (1:1, 2 × 40 mL) to remove traces of 14. The
aqueous phase was treated with saturated aqueous Na2CO3 until
pH 10-11, then extracted with CH2Cl2 (3 × 40 mL). The combined
extracts were dried and concentrated, to give 15 as a purple solid
(12 mg, mmol, 87%). Synthesis from (22): Triphenylphosphine
(45 mg, 0.17 mmol) was added to a solution of 22 (10 mg, 0.035
mmol) in pyridine (1.2 mL) and concentrated ammonia (0.6 mL).
The reaction mixture was stirred for 36 h under Ar at room
temperature. TLC analysis (1% concentrated NH4OH, 7% MeOH,
92% CH2Cl2) showed the disappearance of 22, and the formation
of a single new compound. The reaction mixture was concentrated
in vacuum, and the resulting residue was redissolved in water. The
aqueous phase was washed with hexane, EtOAc, and CH2Cl2 (2 ×
40 mL each). The aqueous phase was reduced to 3 mL and eluted
Synthesis of 13. A mixture of 12 (20 mg, 0.0527 mmol),
triphenyl phosphine (40.16 mg, 0.158 mmol), and phtalimide (62.1
mg, 0.422 mmol) was dissolved in dry DMF. tert-Butyl-azodicar-
boxylate was added (36.46 mg, 0.158 mmol). The solution was
stirred at room temperature for 2 h. TLC analysis showed
disappearance of the starting material. The reaction mixture was
diluted with ethyl acetate and washed three times with water. Drying
(Na2SO4) and evaporation of solvents gave a crude purple oil. The
final product was isolated by chromatography (SiO2:20% CH2Cl2
80% ethyl acetate) to yield 15 mg of product (56% yield).1H NMR
(CDCl3) δ 1.79 (s, CH3, 3H), 2.59 (dd, H1, J ) 4.1, 16.5 Hz, 1H),
3.11 (dd, H1, J ) 7.6, 16.5 Hz, 1H), 3.57 (s, CH2, 2H), 3.99 (dd,
H3, J ) 4, 13.3 Hz, 1H), 4.40 (dd, H3, J ) 6.9, 13.4 Hz, 1H), 4.88
(d, J ) 9.4 Hz, 1H), 4.88 and 5.01 (AB quartet, CH2, J ) 15.5 Hz,
2H), 5.07 (m, H2, 1H), 7.24-7.35 (m, Har, 5 H), 7.75 (dd, J )
9
J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008 9563

A R T I C L E S
Champeil et al.
through a Sep-Pak cartridge with water using a gradient of 0% to
40% methanol in water. The eluting purple solution was collected
and lyophilized to give 15 (7 mg, 0.027 mmol, 77%) as a purple
solid. 1H NMR (methanol-d4) δ 1.83 (s, CH3, 3H), 2.63 (dd, H1, J
) 4.5, 16.3 Hz, 1H), 3.13 (dd, H1, J ) 7.5, 16.3 Hz, 1H), 3.95 (dd,
H3, J ) 3.5, 12.5 Hz, 1H), 3.97 (s, H10, 2H), 4.20 (m, H2, 1H),
4.39 (dd, H3, J ) 6.5, 12.9 Hz, 1H). HRMS m/z calcd for
C13H17N4O2 [M + H]+: 261.1346, found: 261.1348.
2,7-Diamino-10-decarbamoyl-10-azido-mitosene (22). A solu-
tion of 21 (30 mg, 0.074 mmol) in MeOH (50 mL) and 0.05 M
potassium phosphate buffer pH 8.5 (100 mL) was treated with
penicillin amidase (0.540 mL, 500 units). The resulting solution
was gently stirred for 18 h at room temperature, when TLC analysis
(5% MeOH in CH2Cl2) showed the disappearance of 21 and the
formation of a single new compound. The mixture was partially
concentrated to remove MeOH. The resulting aqueous solution was
acidified with 1 M HCl and extracted with EtOAc-hexane (1:1, 2
× 40 mL) to remove traces of 21. The aqueous phase was treated
with saturated aqueous Na2CO3 to pH 10-11, then extracted with
CH2Cl2 (3 × 40 mL). The combined extracts were dried and
concentrated, to give 22 as a purple solid (20 mg, 0.0699 mmol,
94%). TLC and NMR analysis showed that the product was of high
purity, and was used in the next step without further purification.1H
NMR (methanol-d4): δ 1.82 (s, CH3, 3H), 2.62 (dd, H1, J ) 4.5,
16.1 Hz, 1H), 3.21 (dd, H1, J ) 6.9, 16.1 Hz, 1H), 3.96 (dd, H3, J
) 4.4, 12.9 Hz, 1H), 4.26 (m, H2, 1H); 4.43 (dd, H3, J ) 6.5, 12.9
Hz, 1H), 4.45 and 4.50 (AB quartet, H10, J ) 14.0 Hz, 2H), 4.87
(br s, NH2, 2H). 13C NMR (methanol-d4) δ 8.1 (1C, CH3), 29.7
(1C), 33.7 (2C), 45.6 (1C), 55.8 (1C), 107.3 (1C, Ph), 110.7 (1C,
Ph), 122.1 (1C, Ph), 128.7 (1C, Ph), 139.8 (1C, Ph), 145.5 (1C,
Ph), 177.7 (1C, CO), 178.6 (1C, CO). HRMS m/z calcd for
C13H15N6O2 [M + H]+: 287.1251, found: 287.1250.
Synthesis of Protected Adduct 17. 2-Fluoro-O6-(2-p-nitrophen-
ylethyl)deoxyinosine19 (16) (18.8 mg, 0.0448 mmol) was dissolved
in dry DMF (40 µL). Compound 14 (17 mg, 0.0449 mmol) and
diisopropylethylamine (10 µL, 7.42 mg, 0.958 mmol) were added
to the reaction mixture which was incubated at 45° for 18 h. The
resulting crude material was diluted with water (100 µL) and
lyophilized. The desired product was isolated by preparative thin
layer chromatography (SiO2:40% acetone:60% EtOAc) to give 15
1
mg (43% yield) of 17. H NMR (methanol-d4): δ 1.80 (s, CH3,
3H), 2.34 (ddd, H2′, J ) 3, 6.2, 13.4 Hz, 1H), 2.64 (dd, H1, J )
4.6, 16 Hz, 1H), 2.82 (ddd, H2′′, J ) 2, 6.2, 13.4 Hz, 1H), 3.13
(dd, H1, J ) 7.1, 15.5 Hz, 1H), 3.19 (t, CH2, J ) 4.6 Hz, 2H), 3.42
and 3.43 (AB quartet, CH2, J ) 14 Hz, 2H), 3.67 (bd, H5′, J )
12.2 Hz, 1H) and 3.77 (bd, H5′′, J ) 8.5 Hz, 1H), 3.99 (d, H3, J )
4.6 Hz, 1H), 4.02 (d, H4′, J ) 4.4 Hz, 1H), 4.39 (dd, H3, J ) 7.1,
12.9 Hz, 1H), 4.52 (m, H3′, 1H), 4.67 (s, CH2, 2H), 4.75 (t, CH2,
J ) 6.6 Hz, 2H), 4.87 (m, H2, 1H), 6.35 (t, H1′, J ) 3.3 Hz, 1H),
7.20-7.27 (m, Har, 5 H), 7.52 (d, Har, J ) 8.5 Hz, 2H), 8.13 (d,
Har, J ) 8.5 Hz, 2H), 8.25 (bs, 1H). HRMS m/z calcd for
C39H40N9O9 [M + H]+: 778.2949, found: 778.2922.
2,7-Diamino-N2-(2-trimethylsilyl)ethoxycarbonyl-10-decar-
bamoyl-10-azido-mitosene (23). 4-Nitrophenyl-2-(trimethylsilyl)-
ethyl carbonate (29 mg, 0.10 mmol) and i-Pr2NEt (0.021 mL, 16
mg, 0.10 mmol) were added to a solution of 22 (16 mg, 0.056
mmol) in i-PrOH (0.70 mL). The reaction mixture was stirred for
3 h at room temperature. TLC analysis (5% MeOH in CH2Cl2)
showed the disappearance of 23, and the formation of a single new
compound. The reaction was quenched by addition of 0.050 mL
of concentrated aqueous ammonia. The resulting mixture was
concentrated in vacuum, and the residue was dissolved in EtOAc
(25 mL) and washed with saturated Na2CO3 (5 × 10 mL) to remove
4-nitrophenol. The organic phase was dried and concentrated in
vacuum. The residue was purified by flash chromatography using
a gradient of 10% to 0% hexane in CH2Cl2 to give 23 (22 mg,
Synthesis of 18. The N2- and O6-protected mitosene-nucleoside
adduct 17 (6 mg, 0.00771 mmol) was dissolved in acetonitrile/
DMF (100 µL/200 µL) and was treated with DBU (30 µL, 30.54
mg, 0.201 mmol). TLC showed the disapearance of all starting
material after 1.5 h. Water was added to the sample (1 mL), and
the solution was lyophilized. The desired product was isolated by
preparative thin layer chromatography (SiO2:3% triethylamine:15%
MeOH:82% CH2Cl2) to give 4 mg (82% yield) of 18 as a red solid.
1H NMR (methanol-d4): δ ) 1.81 (s, CH3, 3H), 2.40 (ddd, H2′, J
) 3.5, 6.2, 13.5 Hz, 1H), 2.69 (m, H2′′, 1H), 2.83 (dd, H1, J ) 4.5,
16 Hz, 1H), 3.19 (m, H1, 1H), 3.67 (dd, H5′, J ) 4.8, 12.8 Hz, 1H)
and 3.75 (dd, H5′′, J ) 3.9, 8.1 Hz, 1H), 3.99 (m, H4′, 1H), 4.02
(dd, H3, J ) 4.8, 13.2 Hz, 1H), 4.45 (dd, H3, J ) 7.5, 13.2 Hz,
1H), 4.51 (bm, H3′, 1H), 4.58 and 4.64 (AB quartet, CH2, J ) 15
Hz, 2H), 4.96 (m, H2, 1H), 6.35 (t, H1′, J ) 7.1 Hz, 1H), 7.20-7.29
(m, Har, 5 H), 8.00 (s, H8, 1H), 8.11 (s, H8, 1H). HRMS m/z calcd
for C31H33N8O7 [M + H]+: 629.2472, found: 629.2485.
1
0.051 mmol, 91%) as a purple solid. H NMR (methanol-d4): δ
0.07 (s, TMS, 9H), 1.02 (t, CH2Si, J ) 8.2 Hz, 2H), 1.79 (s, 3H,
CH3), 2.80 (dd, H1, J ) 5.2, 16.3 Hz, 1H), 3.24 (dd, H1, J ) 7.8,
16.3 Hz, 1H), 4.04 (dd, H3, J ) 5.2, 12.9 Hz, 1H), 4.18 (t, CH2O,
J ) 8.1 Hz, 2H), 4.45 (s, H10, 2H), 4.47 (dd, H3, J ) 7.2, 12.9 Hz,
1H), 4.80 (m, H2, 1H). 13C NMR (methanol-d4) δ-1.3 (3C, TMS),
8.1 (1C, CH3), 17.8 (1C), 31.1 (1C), 45.6 (1C), 55.3 (1C), 54.1
(1C), 63.6 (1C), 107.3 (1C, Ph), 110.9 (1C, Ph), 122.2 (1C, Ph),
128.8 (1C, Ph), 136.7 (1C, Ph), 145.5 (1C, Ph), 155.9 (1C, CO),
174.4 (1C, CO), 178.6 (1C, CO). HRMS m/z calcd for
C19H26N6O4NaSi [M + Na]+: 453.1677, found: 453.1681.
Synthesis of 8. The N2-protected mitosene-nucleoside adduct
18 (4 mg, 0.00636 mmol) was dissolved in a mixture of methanol
(0.6 mL) and a potassium phosphate buffer (1.8 mL, pH ) 8.5, M
) 0.05, KH2PO4). Penicillin amidase (37 units, 40 µL) was added,
and the reaction was incubated overnight at room temperature. The
crude mixture was diluted with methanol (15 mL), and a white
precipitate appeared which was discarded after centrifugation. The
solution was evaporated, and the resulting brown residue was
redissolved in methanol. A second white precipitate appeared and
was discarded as above. The methanol solution was then evapo-
rated to dryness. The black residue was taken up in water and
washed three times with chloroform. The aqueous layer was
collected and lyophilized to give 8 (3 mg, 92% yield) as a brown
solid. The compound was found to be pure by HPLC and NMR
analysis. 1H NMR (pyridine-d5-D2O): δ ) 1.53 (s, CH3, 3H), 2.19
(ddd, H2′, J ) 3.5, 6.2, 13.3 Hz, 1H), 2.38 (app quint, H2′′, J ) 6.9
Hz, 1H), 2.65 (dd, H1, J ) 4, 16 Hz, 1H), 2.77 (dd, H1, J ) 7.1,
16 Hz, 1H), 3.52 (dd, H5′, J ) 4.5, 12 Hz, 1H) and 3.59 (dd, H5′′,
J ) 3.9, 11.8 Hz, 1H), 3.68 (dd, H3, J ) 4.7, 12.5 Hz, 1H), 3.82
(m, H2, 1H), 3.96 (dd, H3, J ) 6.8, 12.8 Hz, 1H), 4.02 (app quartet,
H4′, J ) 5.7 Hz, 1H), 4.13 and 4.15 (AB quartet, CH2, J ) 15 Hz,
2H), 4.50 (app quint, H3′, J ) 3.4 Hz, 1H), 6.23 (t, H1′, J ) 6.8
Hz, 1H), 7.85 (s, H8, 1H). HRMS m/z calcd for C23H27N8O6 [M +
H]+: 511.2054, found: 511.2049.
2,7,10-Triamino-N2-(2-trimethylsilyl)ethoxycarbonyl-10-de-
carbamoyl-mitosene (24). Triphenylphosphine (35 mg, 0.13 mmol)
was added to a solution of 23 (8 mg, 0.018 mmol) in THF/H2O (1
mL; 1:1) and concentrated aqueous ammonia (0.5 mL). The reaction
mixture was stirred for 18 h under Ar at room temperature. TLC
analysis (1% concentrated NH4OH, 7% MeOH, 92% CH2Cl2)
showed the disappearance of 23, and the formation of a single new
compound. The reaction mixture was concentrated in vacuum, and
the resulting residue was purified by flash chromatography using a
gradient of 0 to 2% NH4OH in MeOH/CH2Cl2 (7:100) to give 24
1
(7 mg, 0.017 mmol, 93%) as a purple solid. H NMR (methanol-
d4): δ 0.07 (s, TMS, 9H), 1.07 (t, CH2Si, J ) 8.2 Hz, 2H), 1.80 (s,
CH3, 3H), 2.76 (dd, H1, J ) 5.1, 16.1 Hz, 1H), 3.21 (dd, H1, J )
7.6, 16.1 Hz, 1H), 3.79 (s, H10, 2H), 4.04 (dd, H3, J ) 5.0, 12.9
Hz, 1H), 4.18 (t, CH2O, J ) 8.1 Hz, 2H), 4.44 (dd, H3, J ) 7.0,
12.9 Hz, 1H), 4.78 (m, H2, 1H). 13C NMR (methanol-d4) δ-2.9
(3C, TMS), 6.8 (1C, CH3), 17.2 (1C), 29.1 (1C), 35.3 (1C), 55.4
(1C), 53.7 (1C), 62.7 (1C), 104.8 (1C, Ph), 114.6 (1C, Ph), 122.1
(1C, Ph), 129.2 (1C, Ph), 138.8 (1C, Ph), 147.4 (1C, Ph), 157.2
9
9564 J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008

Synthesis of Oligodeoxyribonucleotide Adduct of Mitomycin C
A R T I C L E S
(1C, CO), 177.5 (1C, CO), 179.2 (1C, CO). HRMS m/z calcd for
C19H29N4O4Si [M + H]+: 405.1952, found: 405.1950.
containing the desired oligonucleotide 26 was lyophilized and
purified by HPLC (17 A260 units, 57% overall yield). It was
characterized by nuclease digestion and HRMS (negative electro-
spray; deconvolution); m/z calcd for [M]: 3877.7411, found:
3877.7318.
Enzymatic Digestion of 26 to Nucleosides. The oligonucleotide
adduct 26 (1.3 A260 units) was dissolved in the following mixture:
700 µL of water, 200 µL of 0.5 M Tris-HCl pH 8.6,and 350 µL of
0.025 M MgCl2 and was incubated with snake venom phosphodi-
esterase (4 units) and alkaline phosphatase (4 units) at room
temperature for 8 h. The mixture was analyzed by reverse phase
HPLC using the following gradient: 5-60% buffer B in buffer A
in 75 min. (Buffer A: 0.3 M potassium phosphate, pH 5.8; buffer
B: 70% buffer A, 30% acetonitrile). The modified nucleoside 8
was identified by comparison with an authentic sample based on
retention time, coinjection, and UV spectrum.
Synthesis of 5′-d(CTAGTG(18)TATCC) (20a). The oligo-
nucleotide 5′-d(CTAGTG(X)TATCC) (19a; X ) 2-fluoro-O6-(2-
trimethylsilylethyl)deoxyinosine; 10 A260 units) was mixed in a vial
with diisopropylethylamine (25 µL), DMSO (60 µL), and 14 (1
mg, 0.0026 mmol). The mixture was stirred at 42 °C for 72 h,
adding additional portions of 14 (0.5 mg) after 24 and 48 h. The
reaction was stopped, and the mixture was diluted with water (0.5
mL) and then lyophilized. One milliliter of 5% acetic acid was
added, and the solution was stirred at room temperature for 3 h,
followed by neutralization with 0.1 M NaOH to pH 7. The modified
oligonucleotide (20a) was isolated by HPLC (6 A260 units, 60%)
and characterized by MS (negative electrospray; deconvolution);
m/z calcd for [M]: 3997.57, found: 3997.77.
Synthesis of 5′-d(GCTAGC(18)AGTCC) (20b). Following the
procedure described for 20a, 20b was prepared from oligonucleotide
19b in 65% yield. It was characterized by MS (negative electro-
spray; deconvolution); m/z calcd for [M]: 4007.57, found: 4007.66.
Synthesis of 5′-d(CTAGTG(8)TATCC) (26). The oligonucle-
otide 5′-d(CTAGTG(X)TATCC) (19a; X ) 2-fluoro-O6-(2-tri-
methylsilylethyl)-deoxyinosine; 30 A260 units) was mixed in a vial
with diethylamine (20 µL), DMSO (150 µL), and 24 (6 mg, 0.015
mmol). The mixture was stirred at room temperature. The reaction
was monitored by HPLC after 24, 48, and 72 h. No starting mate-
rial was observed after 72 h, and the reaction was stopped. The
mixture was diluted with 750 µL of water and 100 µL of 3 M
NaOAc, pH ) 5. Ethanol (3 mL) was added, and the mixture was
left at -20 °C for 20 min. The suspension was centrifuged (13000
rpm for 15 min), and the supernatant was discarded. The brown
residue was washed twice more with ethanol (750 µL). The crude
modified oligonucleotide 25 (24 A260 units, 80%) was dried in air
and dissolved in a ZnBr2 solution (300 µL, made as follow: 250
mg of ZnBr2, 150 µL of nitromethane, 150 µL of isopropanol).
The mixture was incubated at room temperature for 48 h and
monitored by HPLC. The reaction was quenched with EDTA (12
mL of a 0.5 M solution), and the volume was reduced to half. A
Sephadex G-25 gel filtration was performed. The void fraction
Acknowledgment. This work was supported by NIH Grant
5R01 CA28681 to M.T., by a PSC-CUNY research award 60035-
37 38 to E.C., and a Research Centers for Minority Institutions
Award RR03037, supporting the infrastructure at Hunter College
from the National Center for Research Resources (NCRR), a
component of NIH. We thank Dr. Dinesh M. Vyas of Bristol-Myers
Squibb Co., Ltd., and Dr. Hitoshi Arai of Kyowa Hakko Kogyo
Co., Ltd., respectively, for generously providing mitomycin C. We
are most grateful to Dr. Carmelo J. Rizzo and Albena Kozekova
of the Department of Chemistry and Center for Molecular Toxicol-
ogy, Vanderbilt University, for the synthesis of 2-fluorodeoxyi-
nosine-containing oligonucleotides. We also thank Dr. Cliff Soll
for performing the mass spectroscopy measurements.
Supporting Information Available: Figure S1: COSY NMR
spectrum of synthetic adduct 8; Figure S2: ion spectrum of
oligonucleotide 26 [M - 3H]3-. This material is available free
of charge via the Internet at http://pubs.acs.org.
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J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008 9565