Sequence and structure dependence of the hybridization-triggered reaction of oligonucleotides bearing conjugated cyclopropapyrroloindole

Article abstract of DOI:10.1021/ja9703133

Oligodeoxyribonucleotides (ODNs) with conjugated reactive groups are potential sequence-specific gene inactivating agents. The antitumor antibiotic CC-1065 binds preferably in the minor groove of A-T-rich sites of double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adenines at their N3 position. Pure enantiomeric (+)- and -)- CPI and its N5-methyl homologue (MCPI) were synthesized and conjugated to an ODN. These conjugates were evaluated for their ability to alkylate a target containing a duplex region immediately adjacent to a single-stranded complementary binding region for the ODN conjugate. The conjugates demonstrated excellent stability in physiologic conditions and stereospecific, hybridization-triggered alkylation of the synthetic ODN targets. The dependence of the reaction rates on sequence of the duplex target region was in accord with the predicted minor groove binding of the conjugated CPI. The reactivity was highly dependent on the structure of the cross-linking group. Natural (+)-enantiomers alkylate 10-20 times faster than the corresponding (-)-enantiomers. Regiospecificity of the alkylation reaction is conferred by the length of the spacer arm. N5Methylation of the CPI moiety suppresses the reactivity by a factor of 3-5. Addition of a 1,2- dihydro-3H-pyrrolo[3,2-ε]indole-7-carboxylate (DPI) binding subunit of CC- 1065 between CPI or MCPI residues and an ODN results in a significant enhancement of the reactivity which is especially pronounced for (-)- enantiomers. The main products of sequence-specific alkylation were determined for complexes with the most efficient reactions.

Full text of DOI:10.1021/ja9703133

6214
J. Am. Chem. Soc. 1997, 119, 6214-6225
Articles
Sequence and Structure Dependence of the
Hybridization-Triggered Reaction of Oligonucleotides Bearing
Conjugated Cyclopropapyrroloindole
Eugeny A. Lukhtanov,* Igor V. Kutyavin, Vladimir V. Gorn, Michael W. Reed,
A. David Adams, Deborah D. Lucas, and Rich B. Meyer, Jr.
Contribution from Epoch Pharmaceuticals, Inc., 1725 220th Street Southeast,
#104, Bothell, Washington 98021
ReceiVed January 29, 1997X
Abstract: Oligodeoxyribonucleotides (ODNs) with conjugated reactive groups are potential sequence-specific gene
inactivating agents. The antitumor antibiotic CC-1065 binds preferably in the minor groove of A-T-rich sites of
double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adenines at their N3
position. Pure enantiomeric (+)- and (-)-CPI and its N5-methyl homologue (MCPI) were synthesized and conjugated
to an ODN. These conjugates were evaluated for their ability to alkylate a target containing a duplex region
immediately adjacent to a single-stranded complementary binding region for the ODN conjugate. The conjugates
demonstrated excellent stability in physiologic conditions and stereospecific, hybridization-triggered alkylation of
the synthetic ODN targets. The dependence of the reaction rates on sequence of the duplex target region was in
accord with the predicted minor groove binding of the conjugated CPI. The reactivity was highly dependent on the
structure of the cross-linking group. Natural (+)-enantiomers alkylate 10-20 times faster than the corresponding
(-)-enantiomers. Regiospecificity of the alkylation reaction is conferred by the length of the spacer arm. N5-
Methylation of the CPI moiety suppresses the reactivity by a factor of 3-5. Addition of a 1,2-dihydro-3H-pyrrolo-
[3,2-e]indole-7-carboxylate (DPI) binding subunit of CC-1065 between CPI or MCPI residues and an ODN results
in a significant enhancement of the reactivity which is especially pronounced for (-)-enantiomers. The main products
of sequence-specific alkylation were determined for complexes with the most efficient reactions.
Introduction
tized with (haloacetamido)alkyl groups3,4 or aromatic 2-chlo-
roethylamines (mustards) such as p-(N-(2-chloroethyl)-N-methyl)-
benzyl5 and chlorambucil6,7 have been shown to alkylate
complementary DNA targets with high efficiency and sequence
specificity. ODNs bearing haloacetamides have limited ap-
plication in cellular systems due to their inherent reactivity, both
with themselves and with nontarget cellular nucleophiles prior
to nucleic acid binding. The mustards offer advantages in
reactivity due to their spontaneous activation,8 but may still react
at nontarget sites.
Direct and specific modification of gene function could
provide a general approach to treatment of many diseases.
Efficiency and selectivity of action can be achieved by synthetic
oligodeoxynucleotides (ODNs) which are able to form comple-
mentary complexes with preselected sequences of double-
stranded DNA (the antigene approach) or mRNA (the antisense
approach). Although development of antisense ODNs is well
into the clinical stage,1 ODNs that target DNA have barriers
that have hindered their development. Lacking the ability to
catalyze the degradation of the target, as most antisense ODNs
do via RNAse H action, DNA-directed ODNs must either bind
very tightly or carry a DNA modifying group to attain efficacy
of action.
We have been concentrating on achieving modification of
gene function with use of ODNs bearing an alkylating group.
While the therapeutic utility of these agents shares some of the
hurdles facing antisense ODNs, such as cell uptake and
sensitivity of the phosphodiester backbone to nucleases, the
properties of the reactive group add unique considerations. A
large number of studies have been directed toward enhancing
stabilization of the backbone,2 but the stability of the reactive
group remains an important issue. For example, ODNs deriva-
The most attractive way to address the selectivity problem is
to use hybridization-triggered agents, which are chemically
stable when conjugated to a single-stranded ODN, but highly
activated after hybridization of the ODN to DNA target.
Matteucci and co-workers investigated N4-ethanocytosine-
containing ODNs as hybridization triggered electrophiles,9-11
(3) Tabone, J. C.; Stamm, M. R.; Gamper, H. B.; Meyer, R. B.
Biochemistry 1994, 33, 375-383.
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Chem. Soc. 1993, 115, 9303-9304.
* Corresponding author. Telephone: 206-485-8566. Facsimile: 206-486-
8336. E-mail: elukhtan@epochpharm.com.
(7) Podyminogin, M. A.; Meyer, R. B.; Gamper, H. B. Biochemistry 1995,
34, 13098-13108.
X Abstract published in AdVance ACS Abstracts, July 1, 1997.
(1) Crooke, S. T.; Bennett, C. F. Annu. ReV. Pharmacol. Toxicol. 1996,
36, 107-129.
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S0002-7863(97)00313-2 CCC: $14.00 © 1997 American Chemical Society

Oligonucleotide-Cyclopropapyrroloindole Conjugates
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6215
In our initial approach13 to design a sequence-specific,
hybridization-triggered ODN reagent, we conjugated racemic
CPI to a 3′-thiophosphate tailed ODN using CPI reagent 2
(Figure 1). The sequence of the ODN reagent and comple-
mentary single-stranded target was designed in such a way that,
after hybridization, the CPI moiety would be directed to adenines
of the target. Indeed, 3′-CPI-ODN conjugate was found to
alkylate the complement, which contained an A6 run im-
mediately next to its 5′ end, very rapidly and efficiently. In
the present study, we have further investigated (i) the effect of
sequence of the target site on the efficiency of alkylation, (ii)
specificity and efficiency of alkylation by purified conjugated
CPI enantiomers, (iii) the effect of the linker connecting an ODN
and the CPI moiety, and (iv) whether efficient alkylation could
be maintained with reduced propensity for side reactions. A
new type of CPI reagent, N5-methyl CPI (MCPI), was devel-
oped to modulate the powerful reactivity of CPI. The structure-
activity studies reported here address these questions and
establish the effect of N5-methylation on CPI reactivity.
Figure 1. Structures of (+)-CC-1065 (1) and (N6-(bromoacetyl)amino)-
hexanoyl CPI (2).
but they are slow in the cross-linking reaction, with a half-life
of approximately 30 h. The alkylated base was a mismatched
cytosine. Rokita et al. used a stable silylated phenolic quinone
methide precursor,12 which upon hybridization, lost the silyl
group to generate the reactive species. Alkylation of the
complementary strand occurred with moderate efficiency over
several hours.
Results and Discussion
Synthesis of MCPI and CPI Derivatives. The synthetic
approach to the preparation of the chloromethyl compound (7a),
the key intermediate of new MCPI analogues, is shown in
Scheme 1. Oxindole 3a was exhaustively methylated, affording
bis-methylated oxindole 3b. Borane-methyl sulfide reduction16
gave racemic MCPI precursor 4a in 80% yield from 3a.
Esterification of the primary alcohol 4a with N-BOC-L-
tryptophan25 afforded a mixture of diastereomeric esters 4b,c.
The (S,S) diastereomer 4b was selectively crystallized from
hexane/ethyl acetate in 50-60% yield. (R,S)-Diastereomer 4c
was isolated from the mother liquor by preparative chromatog-
raphy on silica. Methanolysis of 4b afforded 99% pure alcohol
(1S)-4a with 1S absolute configuration (natural configuration
of (+)-CC-1065). This assignment was later unequivocally
proved by the difference observed in the DNA alkylating
properties of MCPI agents with opposite absolute configuration.
All of the following steps were performed analogously for each
enantiomer. Red-Al removal of the methanesulfonyl group gave
unstable dihydrobenzodipyrrole 5a, which was not purified but
reacted directly with di-tert-butyl dicarbonate to provide the
protected N3-BOC dihydrobenzodipyrrole 5b. A two-step
reaction,25 involving the transient formation of mesylate 6a
followed by chloride substitution, provided the 1-chloromethyl
intermediate 6b. Finally, debenzylation by phase transfer
catalysis gave the desired intermediate 7a. The analogous CPI
intermediate (15a) (Scheme 3) was synthesized as previously
described.16,24,26
To prepare CPI derivatives suitable for further conjugation
with an ODN, we employed two general synthetic routes. In
the first case (method A), reactive haloacetamide groups, suitable
for coupling with a thiophosphate on the ODN, were incorpo-
rated into the CPI residue directly at the N2 position or via
variable in length linkers as shown on Scheme 2. The synthesis
of haloacetamido derivatives 11b, 12b, and 14 proceeded from
unstable indoline hydrochloride 7b, prepared by treatment of
7a with saturated HCl in ethyl acetate. Coupling of 7b and
N-BOC-aminohexanoic acid or N-BOC-aminopropanoic acid in
the presence of N-(3-(dimethylamino)propyl)-N′-ethylcarbodi-
We have recently developed13 a new class of hybridization-
triggered cross-linking ODNs using a conjugated cyclopropa-
pyrroloindole (CPI) subunit derived from the potent antitumor
antibiotic (+)-CC-106514 (1) (Figure 1). This reactive CPI
subunit is responsible for the N3 alkylation of adenine.15 CC-
1065 is very stable in neutral aqueous solution16,17 but becomes
much more reactive when bound to the minor groove of double-
stranded DNA. The precise mechanism of activation of the
cyclopropane function is not clear. Catalytic effects such as
DNA-mediated general acid catalysis (protonation of C4 car-
bonyl)18,19 or alkylation site catalysis20 have been proposed, as
have the conformational effects of DNA-induced bond strain
on the CPI moiety.21,22 The activation involves a high-affinity
minor groove binding interaction, which is strengthened by the
middle and right-hand subunits. The DNA alkylation occurs
with sequence dependence, with a preference to 5′-PuNTTA*
and 5′-AAAAA*, where * marks the preferred alkylation
site.23,24
(10) Webb, T. R.; Matteucci, M. D. Nucleic Acids Res. 1986, 14, 7661-
7674.
(11) Shaw, J.-P.; Milligan, J. F.; Krawczyk, S. H.; Matteucci, M. J. Am.
Chem. Soc. 1991, 113, 7765-7766.
(12) Li, T.; Zeng, Q.; Rokita, S. E. Bioconjugate Chem. 1994, 5, 497-
500.
(13) Lukhtanov, E. A.; Podyminogin, M. A.; Kutyavin, I. V.; Meyer, R.
B.; Gamper, H. B. Nucleic Acids Res. 1996, 24, 683-687.
(14) Reynolds; V. L.; McGovren, J. P.; Hurley, L. H. J. Antibiotics
(Tokyo) 1986, 39, 319-334.
(15) Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold, G. L.;
Scahill, T. Science 1984, 226, 843-844.
(16) Warpehoski, M. A.; Gebhard, I.; Kelly, R. C.; Krueger, W. C.; Li,
L. H., McGovren, J. P.; Prairie, M. D.; Wicnienski, N.; Wierenga, W. J.
Med. Chem. 1988, 31, 590-603.
(17) Warpehoski, M. A.; Harper, D. E. J. Am. Chem. Soc. 1994, 116,
7573-7580.
(18) Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988, 1,
315-333.
(19) Lin, C. H.; Beale, J. M.; Hurley, L. H. Biochemistry 1991, 30, 3597-
3602.
(20) Warpehoski, M. A., Harper, D. E. J. Am. Chem. Soc. 1995, 117,
2951-2952.
(24) Hurley, L. H.; Warpehoski, M. A.; Lee, C. -S.; McGovren, J. P.;
Scahill, T. A.; Kelly, R. C.; Mitchell, M. A.; Wicnienski, N. A.; Gebhard,
I.; Johnson, P. D.; Bradford, V. S. J. Am. Chem. Soc. 1990, 112, 4633-
4649.
(25) Warpehoski, M. A. Tetrahedron Lett. 1986, 27, 4103-4106.
(26) Kelly, R. C.; Gebhard, I.; Wicnienski, N.; Aristoff, P. A.; Johnson,
P. D.; Martin, D. G. J. Am. Chem. Soc. 1987, 109, 6837-6838.
(21) Boger, D. L.; Boyce, C. W.; Johnson, D. S. Bioorg. Med. Chem.
Lett. 1997, 7, 233-238.
(22) Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263-
276.
(23) Reynolds, V. L.; Molineux, I. J.; Kaplan, D. J.; Swenson, D. H.;
Hurley, L. H. Biochemistry 1985, 24, 6628-6237.

6216 J. Am. Chem. Soc., Vol. 119, No. 27, 1997
Scheme 1. Synthesis of MCPI Precursorsa
LukhtanoV et al.
Scheme 2. Synthesis of Haloacetamide Intermediatesa
a Reagents: (a) HCl/EtOAc; (b) (N-BOC-amino)hexanoic acid or
(N-BOC-amino)propanoic acid, EDC, DMA; (c) Et3N, CH3CN, H2O;
(d) bromoacetic acid N-hydroxysuccinimide, DMF; (e) bromoacetic
acid, EDC; (f) Cl-.
2,3,5,6-tetrafluorophenyl (TFP) trifluoroacetate27 gave MCPI-
TFP ester (18b) (37% overall yield from 7a) or CPI-TFP ester
(19b) (44% overall yield from 15a). MCPI-DPI (DPI ) 1,2-
dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate) (22b) and CPI-
DPI (23b) TFP esters were prepared in a manner identical with
that described for 18b and 19b, except that addition of the DPI
fragment was accomplished using EDC-mediated coupling of
7b (or 15b) with BOC-DPI.28 UV characteristics of the new
derivatives are summarized in Table 1.
Solvolytic Reactivity of MCPI. An important characteristic
of CC-1065 alkylating subunit and related analogues is the pH-
rate profile for solvolysis. These reagents undergo acid-
catalyzed ring opening with nucleophilic addition to the least
substituted cyclopropane carbon.17,29 Comparison of a series
of agents possessing a modified CC-1065 alkylating subunit has
shown that the rate of acid-catalyzed hydrolysis directly
correlates with biological potency.29,30 The more potent of these
agents are also the most stable and take best advantage of the
selectivity achieved by DNA binding-induced reactivity en-
hancement.31
a Reagents: (a) CH3I, Na2CO3, acetone, DMF; (b) BH3‚(CH3)2S,
THF; (c) N-BOC-L-tryptophan, DCC, N-methylimidazole, 1,4-dioxane;
(d) NaOCH3, MeOH; (e) Red-Al, THF, toluene; (f) di-tert-butyl
dicarbonate, THF; (g) CH3SO2Cl, pyridine, CH2Cl2; (h) LiCl, DMF;
(i) 10% Pd/C, NH4COOH, THF, MeOH; (j) HCl/EtOAc; (k) Et3N,
CH3CN, H2O.
imide hydrochloride (EDC) (Scheme 2) afforded compound 9a
or 10a, respectively. The hydrochlorides 9b and 10b obtained
from 9a and 10a were treated with 1:1:3 Et3N/H2O/CH3CN to
effect Ar-3′ spirocyclization26 (11a and 12a). Final coupling
with N-hydroxysuccinimidyl bromoacetate afforded the desired
MCPI-bromoacetamido derivatives 11b and 12b in 50% overall
yield from 7b. When 7b was coupled with bromoacetic acid
in presence of EDC (Scheme 2), the bromoacetamide 13a was
not obtained. Chloroacetamide 13b was isolated instead from
the reaction mixture as a result of halogen exchange driven by
the excess chloride ion (EDC counterion). Although the reaction
scheme could be modified to avoid using the excess chloride
ion, chloroacetamido MCPI 14, prepared from 13b using the
standard cyclyzation conditions, proved to be reactive enough
in the following conjugation reaction with the nucleophilic ODN.
We determined the solvolytic reactivity of the cyclopropyl
function in our compounds. Model N-BOC protected analogue
8 (N-BOC-MCPI), whose reactivity could be directly compared
(27) Gamper, H. B.; Reed, M. W.; Cox, T.; Virosco, J. S.; Adams, A.
D.; Gall, A. A.; Scholler, J. K.; Meyer, R. B., Jr. Nucleic Acids Res. 1993,
21, 145-150.
(28) Boger, D. L.; Coleman, R. S.; Invergo, B. J. J. Org. Chem. 1987,
52, 1521-1530.
(29) Boger, D. L.; Machiya, K.; Hertzog, D. L.; Kitos, P. A.; Holmes,
D. J. Am. Chem. Soc. 1993, 115, 9025-9036.
(30) Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. Engl. 1996,
35, 1439-1474.
(31) Boger, D. L.; Johnson, D. S. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 3642-3649.
An alternative conjugation method (method B) is to use CPI
and MCPI derivatives bearing a linker with activated carboxy
group to be coupled to an amine tailed ODN. For use in this
method (Scheme 3), 7b (or 15b)24 was selectively derivatized
with succinic anhydride, providing succinate 16 (or 17) followed
by Ar-3′ spirocyclization as described above to afford succinate
18a (or 19a). Activation of the terminal carboxy group using

Oligonucleotide-Cyclopropapyrroloindole Conjugates
Scheme 3. Synthesis of TFP Intermediatesa
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6217
tive 11b, 12b, or 14. The reactions were carried out in 50%
aqueous DMF using 2-3-fold excess of the reagents. As we
had noticed before,13 this reaction produced a minor side
product, identified as the conjugate lacking the characteristic
long-wavelength absorbance (max1 in Table 1). Most likely
this product resulted from competitive reaction of the cyclo-
propyl function with the terminal phosphorothioate residue. This
was supported by the fact that no ODN-related side products
were observed in the reaction using TFP ester type reagents
and amino tailed ODNs (method B).
In method B, the alkylating residues were linked to 5′-
alkylamine-modified ODNs via amide bonds. The amine-
modified ODNs were first converted to the triethylammonium
(TEA) salts by reverse phase HPLC. The TEA salts of ODNs
as well as TFP esters are soluble in DMSO and allow the
conjugation chemistry to be carried out under anhydrous
conditions. After treatment with 5-20 equiv of the appropriate
TFP ester (18b, 19b, 22b, or 23b), the crude CPI-ODN
conjugates were precipitated and purified by reverse phase
HPLC to give the desired reagents in 35-78% yield.
The conjugates were routinely analyzed by nuclease digestion
assay to monitor the nucleoside content as well as the presence
of alkylating group. These preparation and analysis steps are
shown in Figure 2 for a typical MCPI-ODN conjugate.
Stability of CPI-ODN Conjugates. We have previously
shown that a conjugated CPI moiety is somewhat more labile
than free CPI in mild acidic conditions. A conjugate which
had been prepared by reaction of bromoacetamido CPI (2) with
an ODN-bearing terminal thiophosphate had a half-life of about
3 h at pH 4.5 as measured by UV spectroscopy at 360 nm.13
The conjugates synthesized in this work had similar stability
(Table 2). Half-lives of tens of minutes were observed at a pH
of 4 (22 °C) for the tethered CPI function. This is in contrast
to the unconjugated 8 and 22c (prepared by reacting 22b with
3-aminopropanol), which were significantly more stable under
these conditions. We assume that this enhanced lability (50-
100 times) is due to an effect of the ODN. Conjugation of the
CPI moiety to the ODN either might allow a proximity effect
(proximity to nucleophilic DNA centers) or might involve acid
catalysis by phosphate-coordinated Mg+2 cations. At pH 4,
phosphate-mediated acid catalysis may also be a factor.
Whatever the mechanism is, CPI hydrolysis and/or alkylation
of adjacent bases or phosphates (self-alkylation) are potential
results of the activation.
a Reagents: (a) HCl/EtOAc; (b) succinic anhydride, Et3N, (MeOH,
CH2Cl2) or DMA; (c) Et3N, CH3CN, H2O; (d) 2,3,5,6-tetrafluorophenyl
trifluoroacetate, Et3N, CH2Cl2, or DMA; (e) 3-(tert-butyloxycarbonyl)-
1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid, EDC, DMA; (f)
3-aminopropanol, DMF.
The acid lability of ODN-conjugated CPI (MCPI) was not a
problem at pH 7.2, where most of the conjugates used in this
work did not show any significant decomposition over hours
of incubation even at elevated temperature (37 °C). The rate
of decomposition of alkylating function for the CPI- or MCPI-
ODN conjugates is independent of the absolute configuration
of the reactive center at either pH 4 or 7.2, indicating that
hydrolysis and not ODN interaction is the main degradation
pathway. This was supported by the fact that one major product
was observed by HPLC. The CPI-DPI-ODN and MCPI-DPI-
ODN conjugates, however, when incubated under the same
conditions, gave multiple unidentified products. This observa-
tion supports a self-alkylation pathway for degradation of MCPI-
DPI or CPI-DPI conjugates. Natural (+)-enantiomers were
considerably more reactive than the unnatural ones. For
example, the ODN-bearing (+)-CPI-DPI residue (also the most
reactive DNA alkylating conjugate used in this work) was the
least stable (half-life about 10 h at 37 °C in a neutral solution),
while (-)-MCPI-DPI-ODN was the most stable with a half-
life of more than 100 h. Double-stranded DNA structures could
be involved in these cases. Although the oligonucleotide
with other N-BOC-protected CPI analogues,29 was synthesized
as shown in Scheme 1. Compound 8 was found to have a half-
life of 35.8 h (k ) 5.38 × 10-6 s-1) at pH 4 (22 °C) and was
about as stable as the natural analogue N-BOC-CPI (t1/2 ) 36.7
h29). This shows that N5-methylation has negligible effect on
the electronic properties of the aromatic system and on the pKa
of the C4 oxygen protonated in the acid-catalyzed reaction. This
is also supported by the fact that both N-methylated (18b) and
natural (19b) CPI analogues have remarkably similar UV spectra
(Table 1). This observation is interesting in a context of the
comparative reactivities of corresponding CPI and MCPI-
carrying ODN conjugates in reaction with complementary
targets discussed below.
Conjugation Chemistry. Two types of conjugation chem-
istries were used to couple an ODN to a cross-linking agent. In
method A, ODNs containing a highly nucleophilic terminal
thiophosphate residue were reacted with haloacetamido deriva-

6218 J. Am. Chem. Soc., Vol. 119, No. 27, 1997
LukhtanoV et al.
Table 1. UV Properties of Some MCPI and CPI Compounds
λ max1
ꢀ max1
λ max2
ꢀ max2
λ max3
ꢀ max3
λ min2
ꢀ min2
λ min1
ꢀ min1
compd
solvent
EtOH
MeOH
MeOH
MeOH/H2O
MeOH
MeOH
MeCN
(nm)
(M-1 cm-1
)
(nm)
(M-1 cm-1
)
(nm)
(M-1 cm-1
)
(nm)
(M-1 cm-1
)
(nm)
(M-1 cm-1
)
8
14
342
344
344
351
344
347
350
349
13800
14200
13300
13600
14000
14000
26000
26600
279
283
284
289
283
283
312
312
17400
14700
15500
14700
14800
17400
22000
24200
248
251
251
253
250
248
291
290
1600
4000
2500
3200
2800
2700
18000
20700
302
302
303
305
302
312
325
325
8000
8500
7600
7400
8000
8000
18700
19500
12b
11b
18b
19b
22b
23b
262
262
35100
37300
MeCN
Table 2. Stability of ODN Conjugates
pH 4.0b
pH 7.2c
conjugatea or
compd
kobs × 104
t1/2
(min)
kobs × 106
t1/2
(h)
(s-1
)
(s-1
)
(+)-CPI-DPI-ODN
(-)-CPI-DPI-ODN
8.5 ( 0.5
7.2 ( 0.1
14 1.9 ( 0.3
16 3.0 ( 0.2
20 4.0 ( 0.3
10
63
48
(+)-MCPI-DPI-ODN 5.9 ( 0.1
(-)-MCPI-DPI-ODN 4.4 ( 0.2
26 1.7 ( 0.4 >115
(+)-CPI-ODN
(-)-CPI-ODN
(+)-MCPI-ODN
(-)-MCPI-ODN
8
2.6 ( 0.1
3.1 ( 0.1
1.9 ( 0.1
2.0 ( 0.1
44 4.4 ( 0.3
37 5.2 ( 0.2
60 3.4 ( 0.3
58 3.6 ( 0.3
44
37
56
53
0.054 ( 0.001 2148
0.091 ( 0.003 1275
d
d
22c
a The conjugates were prepared by reaction of TFP esters of
corresponding alkylating agents with 5′-hexylamino tailed ODN
(prepared using method B). b Determination of rates of acid catalyzed
hydrolysis was carried out at 22 °C in an acidic buffer (pH 4). The
buffer contained 8 mM citric acid and 4 mM Na2HPO4 in 50% aqueous
methanol. c The experiments were carried out in a buffer containing
140 mM KCl, 10 mM MgCl2, and 20 mM HEPES (pH 7.2) at 37 °C.
d Reaction was too slow to measure accurately.
by three types of consensus sequences: PyNTTA*, AAAAA*,
and T/AT/AA*.23,24 The effects of target site sequence on
reaction rates, however, are less investigated.20,35 The system
described here offers an opportunity to evaluate the reactivity
of CPI with any possible duplex site via direct positioning of
the ODN-conjugated CPI into the target site.
Figure 3 shows the design of the ODN hairpin target we used
to investigate the target sequence preference and effect of the
tether to the ODN. The targets were designed with an invariable
5′ single-stranded region, which was the complementary binding
site for (+)- and (-)-CPI conjugates of the test dodecanucleotide
ODN, and an adjacent variable, double-stranded hairpin segment
for binding and reaction of the alkylating group. The sequences
are listed in Table 3. Since the conjugated alkylating residue
can, in principle, interact with adjacent hairpin duplex as well
as fold back into the duplex formed by the ODN, the ODN
sequence was made G-C rich, which is unfavorable for binding
and alkylation by CPI. In addition, molecular modeling
indicated that the fifth and sixth positions of the double-stranded
region would be alkylated.
The alkylation rate was determined by monitoring the
decrease in the characteristic long-wavelength band shown in
Figure 4. A 2-fold excess of the target hairpin was used to
insure complete duplexing of the reactive ODN. For determi-
nation of efficiency and site of alkylation, 32P-labeled targets
were treated with reactive ODN conjugate and incubated
overnight (15 h) at 37 °C. The yield of alkylation was
determined by denaturing polyacrylamide gel electrophoresis
(PAGE) of the reaction; products of alkylation reactions have
different mobilities from starting oligonucleotides. The sites
of alkylation were determined by denaturing PAGE analyses
of heat/piperidine-treated reactions.
Figure 2. Analysis of a MCPI-ODN conjugate: (A) crude reaction
mixture; (B) purified conjugate; (C) nuclease digest. The analytical
reverse phase HPLC was performed on a Rainin C-18 column (4.5 ×
150 mm) in a gradient of acetonitrile (0-60%) in 0.1 M triethylam-
monium acetate buffer (pH 7.5). Flow rate was 1 mL/min. Dual
wavelength detection was used: fine line, 260 nm; bold line, 350 nm.
sequence used in this study does not have any obvious self-
complementarity, some nonperfect duplexes could be formed
due to the increased ability of the dimeric agents to bind to and
stabilize such duplexes.32-34 This could lead to more rapid
reaction for the one isomer and stabilization for the other.
Reactivity and Site Specificity. The site specificity of
alkylation for CC-1065 and its analogues can be summarized
(32) Kim, D.-Y.; Swenson, D. H.; Cho, D.-Y.; Taylor, H. W.; Shih, D.
S. Antisense Res. DeV. 1995, 5, 149-154.
(33) Kim, D.-Y.; Shih, D. S.; Cho, D.-Y.; Swenson, D. H. Antisense
Res. DeV. 1995, 5, 49-57.
(34) Lukhtanov, E. A.; Kutyavin, I. V.; Gamper, H. B.; Meyer, R. B.,
Jr. Bioconjugate Chem. 1995, 6, 418-426.
(35) Boger, D. L.; Jonson, D. S.; Yun, W. J. Am. Chem. Soc. 1994, 116,
1635-1656.

Oligonucleotide-Cyclopropapyrroloindole Conjugates
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6219
Figure 4. Kinetic determination from loss of CPI chromophore. (A)
UV-visible spectra of 8 µM MCPI-ODN (1) and 8 µM MCPI-DPI-
ODN (2) conjugates in 4 mM HEPES (pH 7.2). (B) Time course for
the cross-linking reaction of the (+)-MCPI-ODN conjugate with the
target ODN (I). The spectra were taken in a buffer containing 20 mM
HEPES (pH 7.2) (37 °C) in 10 min intervals. (C) Time course for the
cross-linking reaction of the (+)-MCPI-DPI-ODN conjugate with the
target ODN (I). The spectra were taken in a buffer containing 20 mM
HEPES (pH 7.2) (37 °C) in 2 min intervals.
this case supports the case for the minor groove-specific,
hybridization-triggered nature of the reactions with a duplex
target.
Figure 3. Diagram of target hairpin ODNs with bound MCPI-DPI-
ODN conjugate. The structure of the linker for the MCPI-DPI-ODN
conjugate is illustrated. Structures of the alkylation sites and the results
of cross-linking reactions are shown in Table 3.
The importance of correct placement of the CPI in the targeted
site can be seen when the 5′-TTTTAC sequence is placed at
varying distances from the ODN-CPI conjugate. This sequence
provides a good binding site for the (+)-MCPI-DPI and contains
a single A for reaction. It is found in the four target ODNs
V-VIII. The optimum reaction was observed when the
alkylated adenine was in the sixth position from 5′-end of the
target (VI). Shift of the binding site one base in the 5′-direction
(V, closer to the bound ODN) results in slightly less reactivity.
Shift of the target site away from the ODN binding site either
significantly reduces (target VII) or completely diminishes the
reaction (target VIII). Sequencing of the alkylation reactions
confirms this stereospecificity: the most reactive targets (I, III,
VI, and XII) all have an adenine in the sixth residue from the
5′-end of the hairpin, and all show reaction at that site.
The natural (+)-MCPI-DPI enantiomer is at least 10 times
more reactive than the (-)-isomer, reflecting their similarity
with CPI enantiomers. As suggested by Boger and co-
workers,31,35,36 the origin for the difference in efficiency of DNA
alkylation arises from steric interaction between the C7 methyl
group and the base adjacent to the alkylated adenine. This steric
Table 3 summarizes the results for an ODN carrying natural
(+)-MCPI-DPI, (+)-MCPI, and unnatural (-)-MCPI-DPI enan-
tiomers. Even with this limited set of DNA targets, some
general trends can be observed. The tethered (+)-MCPI-DPI
can alkylate any accessible adenine residue, with the reaction
rates varying with the target sequence and linker. When the
linker allows movement of the alkylating group, conjugation
does not change the known sequence preference characteristic
for free CPI-DPI. For example, oligoadenylate sequence I
proved to be a very good target, predominantly alkylated at the
A6 position, consistent with previous observation.13 Other A-T-
rich sequences such as 5′TTTAA (III), 5′TTTTA (V, VI),
5′TAAAA (XI), and 5′GTAAA (XII) also demonstrate good
reactivity, in accord with previously established rules for free
CPI.23,24 An alternating AT sequence (XVIII) is less suitable
as a target. Not surprisingly, VIII and XVI, with no adenine
residues in the target site, show no reactivity.
It is important to note that no reaction was detected for target
XX. In contrast to all targets tested, this 21 nucleotide ODN
has a single-stranded A6 target site. The lack of reactivity in
(36) Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. Bioorg. Med.
Chem. 1994, 2, 115-135.

6220 J. Am. Chem. Soc., Vol. 119, No. 27, 1997
LukhtanoV et al.
Table 3. Efficiency and Position of Alkylation by Tethered (+)-MCPI-DPI, (-)-MCPI-DPI, and (+)-MCPI at Various DNA Sites
b
a See Figure 3 for structure and numbering of hairpin targets. Kobs was determined from loss of CPI chromophore (330-450 nm) as shown in
Figure 4. c Half-lives of reactions with (+)-MCPI conjugate were determined as times required for absorbance at 355 nm to decrease by one-half.
(Many of the reactions did not go to completion.) d Extent of alkylation by (+)-MCPI-ODN was determined after 15 h incubation by two different
assays: by UV spectrometry as illustrated in Figure 4 (open numbers) or by gel-electrophoretic analysis of radiolabeled cross-link products (numbers
f
in parentheses). e No change in the UV spectrum of the reaction mixture was observed after 3 h. No reaction was observed by analysis of radiolabeled
cross-link products after 15 h (37 °C).
interaction is more pronounced for the unnatural (-)-enantiomer.
Introduction of the N5-methyl group, which protrudes away
from the minor groove, does not affect the general distinction
between (+)- and (-)-enantiomers but, as will be pointed out
later, significantly decreases rate of DNA alkylation for both
enantiomers. In addition, the observed difference (∼10×) is
substantially smaller than was reported for free (+)- and (-)-
CPI-DPI (100-500×).29,36
s-1). For example, a (+)-MCPI-DPI-ODN derivative slowly
but quantitatively reacts with target XV (t1/2 of 27.5 min). The
corresponding (-)-MCPI-DPI-ODN conjugate showed just two
times slower reaction with another target IV (t1/2 of 64 min),
but overnight incubation of this complex resulted in 75% yield
of the cross-linking reaction. Having almost identical reactivity
toward targets III and IV (t1/2 of 61 and 64 min, respectively),
a (-)-MCPI-DPI-carrying ODN showed lower efficiency of
alkylation (95 and 75%). The difference in the alkylation
efficiencies could be explained by reversibility of the N3-
adduct,37 which could be more pronounced for the unnatural
enantiomer.36
Extended incubation of (+)-MCPI-DPI-ODN with targets
usually resulted either in a quantitative reaction or in no reaction
at all, depending on the sequence of the targets (Table 3). In
contrast, (-)-MCPI-DPI-ODN frequently showed intermediate
efficiency in the overnight reaction (20-95%), even when there
was a reasonably high rate of alkylation (k ) 1-3 × 10-4
(37) Warpehoski, M. A.; Harper, D. E.; Mitchell, M. A.; Monroe, T. J.
Biochemistry 1992, 31, 2502-2508.

Oligonucleotide-Cyclopropapyrroloindole Conjugates
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6221
The rate of alkylation by tethered (+)-MCPI was substantially
less than both (+)-MCPI-DPI and (-)-MCPI-DPI. Except for
the super reactive site I, alkylation by the monomeric agent
was about 100 times slower than by (+)-MCPI-DPI or ∼10
times slower than by (-)-MCPI-DPI (Table 3). Alkylation at
the site I appears to be favored by the unusual bent structure of
long A tracts,38 thus diminishing the difference between the
monomeric and dimeric agents.
No alkylation was observed when targeted adenines were
placed six bases or farther into the adjacent duplex (sequences
VI, VII, and VIII). These sequences were not used for
comparison of reactivity since their adenines, apparently, are
out of reach for the tethered (+)-MCPI residue.
The reversibility of alkylation by the monomeric (+)-MCPI
was even more pronounced than for (-)-MCPI-DPI. After 15
h incubation (at 37 °C), most of the reactions involving the
monomeric agent did not go to completion and reached plateaus
(as followed by UV spectroscopy). Moreover, the efficiency
of alkylation as measured by gel electrophoresis of the radio-
labeled cross-link products was significantly lower than that
measured by the UV method (Table 3). Decomposition of the
N3-adenine adduct during electrophoresis, which was observed
as a smear below the cross-link bands, may account for the
difference. This is in accord with the known retroalkylation of
the N3-adenine adduct,22 which is highly dependent on the
extent of noncovalent binding.37
Non-Oligonucleotide-Specific (“Anchor”) Effects. Since
these ODNs are conjugated to strong minor groove binding
agents such as simplified analogues34,35 of CC-1065,32,33 it is
important to show that the observed alkylation is completely
directed by ODN binding and not controlled by the minor groove
binder (“anchor” effect). A noncomplementary ODN, CpCpC-
pApTpGpTpTpGpTpGpC, carrying a (+)-MCPI-DPI residue
conjugated to its 3′-end was prepared and reacted with a target
sequence (I). A small nonspecific reaction due to the anchor
effect, with a half-life of approximately 6 h (37 °C), was seen.
The same concentration (2 µM) of free (+)-MCPI-DPI (22c)
alkylated the same target with a half-life of 7 min. The
complementary ODN bearing the (+)-MCPI-DPI residue (Table
3) alkylated the target with a half-life of 2 min. Similar results
were obtained with the other hairpin targets. These data
demonstrate that the observed reactions (Table 3) are ODN-
sequence dependent and not a result of an anchor effect, and
that conjugation of the minor groove binding agent to the ODN
significantly masks its inherent propensity to bind to A-T-rich
sites in the minor groove. This is likely due to repulsion
between the negatively charged backbones of target DNA and
reactive ODN. The anchor effect was found to be negligible
for (-)-MCPI-DPI, (+)- or (-)-MCPI, and (+)- or (-)-CPI
conjugates, a reflection of their reduced minor groove binding
affinity (data not shown).
Figure 5. Possible cross-linking modes for the tethered (+)-CPI-DPI
group. ∆ indicates positioning of the cyclopropane ring.
can slide along the minor groove or flip around to achieve
alkylation of the opposite strand in the same DNA site, the
conjugated CPI moiety is structurally constrained by tethering.
This difference is very effectively demonstrated when alkylation
of A6/T6 DNA site is compared for free MCPI-DPI (23c) and
the MCPI-DPI-ODN conjugates. This same sequence is
presented in two target ODNs (sequences I and XIX, Table 3)
with opposite positioning. Only target I was efficiently alky-
lated by the (+)-MCPI-DPI-ODN, while no reaction was
observed for target XIX since it would require unfavorable mode
3 for alkylation (Figure 5). Unconjugated 23c, on the other
hand, reacted with both targets with approximately equal
efficiency (data not shown). Similarly, tethered (-)-MCPI-DPI
reacted efficiently with target XIX but not with target I in
accordance with the opposite alkylation mode. However, in
contrast to (+)-MCPI-DPI, a very slow contra strand alkylation
(t1/2 > 600 min) was observed for (-)-enantiomer (target
sequences I and XI) despite the unfavorable orientation of the
adenine stretch. This difference may lie in the strength of
noncovalent minor groove binding. While (+)-dimer preferably
binds very strongly to target XIX in mode 1, the (-)-enantiomer
(being directed to target I) may bind more weakly and also
accept an inverted binding mode (mode 3).
Another important feature of conjugated CPI is that it tends
to occupy the most distal position to avoid looping out the spacer
arm. The overall length of the conjugated CPI residue (includ-
ing the spacer arm) determines the reachable range. For
example, MCPI-DPI residue with the standard C6 linker has
the highest alkylation rate when the target adenine in the
TTTTAC site is in the sixth position relative to the attachment
point (sequence VI, Table 3). Shifting the site further out results
in almost complete lost of reactivity (sequence VII), while closer
placement (sequence V) gives a significant drop in the reaction
rate. Adjustment of the spacer arm length can fine tune the
reactivity for target V. For instance, replacement of the C6
linker with a C4 linker provides a slight increase (∼20%) in
the reaction rate (data not shown).
Binding Mode and Effect of Spacer Arm Structure. An
interesting feature of CPI compounds is that their orientation
in the minor groove dictates which strand can be alkylated. Thus
(+)-CC-1065 covers a four-base region within the minor groove
in 5′-direction from the covalently modified adenosine. Con-
versely, the (-)-enantiomer of CC-1065 binds in the reverse
orientation relative to the modified adenine. We found that the
same rules apply to the tethered alkylators (Figure 5). If (+)-
CPI is conjugated to the 5′ end of the ODN, it would be expected
to alkylate an adenine either in the complementary strand of
the adjacent duplex region (mode 1) or in the carrier ODN if it
folds back on itself (mode 2). However, unlike free CPI, which
Figure 6 shows the effect of the spacer arm structure on
reactivity of the MCPI-ODN conjugates with target I. Gener-
ally, conjugates which have linker arms of 10-12 atoms in
length (counting between the 5′ terminal phosphorus atom and
N2 nitrogen) have similar rates of reaction with the target. A
reasonable degree of tether flexibility is apparently necessary
for conjugated CPI to bind in the minor groove and attain the
required conformation for alkylation. This can explain the low
reactivity for two conjugates with short and rigid spacer arms.
Structural features of the spacer arm such as hydrophobicity
and hydrogen bonding amide groups are factors which could
(38) Crothers, D. M.; Haran, T. E.; Nadeau, J. G. J. Biol. Chem. 1990,
265, 7093-7096.

6222 J. Am. Chem. Soc., Vol. 119, No. 27, 1997
LukhtanoV et al.
addition of the DPI subunit results in 10-15-fold increase in
reactivity for (+)-enantiomers (this difference becomes as large
as 100× for sequences other than I and XIX (Table 3)) and
∼50-fold for (-)-enantiomers, (ii) all (+)-enantiomers react
faster than the corresponding (-)-enantiomers by a factor of
10-20, and (iii) addition of the N5-methyl group to CPI
decreases reactivity by a factor of 3-5. The reactivity of the
tested conjugates was therefore (+)-CPI-DPI > (+)-MCPI-DPI
> (-)-CPI-DPI > (+)-CPI > (-)-MCPI-DPI > (+)-MCPI .
(-)-CPI > (-)-MCPI.
The rate of N3-adenine alkylation by CPI and analogues is
affected by the noncovalent minor groove binding interaction
and by the inherent electrophilic reactivity of the cyclopropyl
function.31 The electrophilic reactivity can be estimated for
different analogues by the rates of acid-catalyzed solvolysis.29
In addition, structural elements, such as a C7 substituent, may
affect the DNA alkylating reactivity.39 We were surprised,
therefore, to find that the rate of DNA alkylation by CPI
analogues was 3-5 times higher than for the corresponding
methylated MCPI compounds. Both have similar acid hydroly-
sis rates (half-lives of 35.8 and 36.7 h, respectively, at pH 4)
and the same C7 substituent. Computer modeling indicates that
the N5-methyl group is situated on the outer periphery of the
agent and should not interfere with the interaction with minor
groove binding. The difference may lie in hindrance to catalysis
by phosphate-bound magnesium, as discussed above, or in the
manner in which the agent’s conformation is affected by DNA
binding.
Figure 6. Effect of spacer arm structure on reactivity of (+)-MCPI-
ODN conjugates. The 2 µM conjugates were reacted with target ODN
I (4 µM) at 37 °C in a buffer containing 140 mM KCl, 10 mM MgCl2,
and 20 mM HEPES (pH 7.2). Kinetics were determined from loss of
CPI chromophore.
This new class of cross-linking oligonucleotides has promise
in the research and diagnostic areas as a tool for DNA site
specific modification and cleavage and in the therapeutic area
as inhibitors of gene expression. The remarkable stability of
the CPI-alkylating function in physiological buffer allows the
reactive ODN to reach a DNA target while avoiding side
reactions with cellular nucleophiles, after which the unique
hybridization-triggered mechanism of activation provides a rapid
and efficient cross-link with that target. The target site of the
CPI moiety can be specified by the oligonucleotide, not by the
much more restricted binding mode of CC-1065. The efficiency
and rate of the reaction will depend somewhat on favorable
binding sites for the tethered CPI, but the number of DNA sites
suitable for conjugated CPI residue are significantly increased
over CC-1065. The general requirement is that the site has to
be three-to-four A-T base pairs long. The linker to the
oligonucleotide limits freedom of alkylating CPI such that the
targeted adenine has to be in the fifth or sixth position from
the end of the ODN carrier. The reaction preference differences
between the natural (+) and the unnatural (-)-enantiomers
offers the advantage of targeting one DNA strand or the other.
Structural modifications such as the introduction of the DPI
binding subunit or N5-methylation allows for modulation of the
reactivity. These same conjugates might also alkylate double-
stranded DNA, either as part of a classical triple-stranded
complex40 or, as we have recently shown, in a recombinase-
stabilized synaptic joint.41
Figure 7. Relative reactivity of cross-linking ODNs. The conjugates
(2 µM) were reacted either with target ODN I (4 µM) for (+)-
enantiomers or with target XIX for (-)-enantiomers at 37 °C in a buffer
containing 140 mM KCl, 10 mM MgCl2, and 20 mM HEPES (pH 7.2).
Kinetics were determined from loss of CPI chromophore.
additionally contribute to the interaction with the minor groove
and subsequently affect reactivity. The hydrophilic 2-(2-
aminoethoxy)ethyl linker, for instance, gives a (+)-MCPI-ODN
conjugate that reacted almost three times slower than the equal
length but more hydrophobic aminopentyl linker.
Relative Reactivity of CPI Reagents and Analogues.
Figure 7 shows a comparison of the reactivity of various CPI
reagents using an A6/T6 motif in the target. Among the tested
sequences, this motif was shown to be the best target site for
all CPI reagents. To allow each enantiomer to bind in an
orientation preferred for reaction, sequence I was used for (+)-
enantiomers and sequence XIX for (-)-enantiomers. The rate
of reaction within the site should not be affected significantly
by the length of the linker because any adenine residue could
act as a target. Three factors affected the rate of reaction: (i)
Experimental Section
(1S)-3-(6-(N-(tert-Butyloxycarbonyl)amino)hexanoyl)-1-
(chloromethyl)-6,8-dimethyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-5-
ol ((1S)-9a). To a solution of freshly prepared (1S)-7b (0.28 mmol)
in 3.5 mL of dry DMA were added 6-(N-BOC-amino)hexanoic acid
(39) Asai, A.; Nagamura, S.; Saito, H. J. Am. Chem. Soc. 1994, 116,
4171-4177.
(40) Helene, C. Anti-Cancer Drug Des. 1991, 6, 569-584.
(41) Podyminogin, M. A.; Meyer, R. B.; Gamper, H. B. Biochemistry
1995, 34, 13098-13108.

Oligonucleotide-Cyclopropapyrroloindole Conjugates
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6223
(110 mg, 0.48 mmol) and EDC (300 mg, 1.56 mmol). After being
stirred for 30 min, the mixture was cooled in ice and water (15 mL)
was added. The resulting solid was collected by filtration, washed with
water, and dried in Vacuo. The crude product was purified by flash
chromatography CH2Cl2-MeOH (20:1) to give (1S)-9a as an off-white
solid after evaporation of the solvent (95 mg, 73%): 1H NMR (CDCl3)
δ 8.96 (br s, 1H), 8.04 (s, 1H), 6.73 (s, 1H), 4.54 (br s, 1H), 4.28 (d,
1H, J ) 11 Hz), 4.13 (t, 1H, J ) 8 Hz), 4.02 (s, 3H), 3.93 (m, 1H),
3.84 (m, 1H), 3.31 (t, 1H, J ) 11 Hz), 3.15 (m, 2H), 2.7-2.4 (m, 2H),
2.37 (s, 3H), 1.83 (m, 2H), 1.7-1.4 (m, 4H overlapping with H2O and
BOC), 1.43 (s, 9H). Anal. Calcd for C24H34N3O4Cl‚0.3H2O: C, 61.41;
H, 7.43; N, 8.95. Found: C, 61.66; H, 7.30; N, 8.61.
(1S)-3-(3-(N-(tert-Butyloxycarbonyl)amino)propanoyl)-1-(chlo-
romethyl)-6,8-dimethyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-5-ol ((1S)-
10a). Compound (1S)-10a was synthesized by analogy with (1S)-9a
using 3-(N-BOC-amino)propanoic acid in 83% yield: 1H NMR (CDCl3)
δ 8.69 (br s, 1H), 7.99 (s, 1H), 6.74 (s, 1H), 5.53 (br s, 1H), 4.25 (d,
1H, J ) 11 Hz), 4.11 (t, 1H, J ) 8 Hz), 4.02 (s, 3H), 3.93 (m, 1H),
3.85 (m, 1H), 3.60 (m, 2H), 3.33 (t, 1H, J ) 11 Hz), 3.0-2.6 (m, 2H),
2.37 (s, 3H), 1.42 (s, 9H). Anal. Calcd for C21H28N3O4Cl: C, 59.78;
H, 6.69; N, 9.96. Found: C, 59.88; H, 6.59; N, 9.56.
(200 mg, 2 mmol) and triethylamine (0.5 mL, 3.65 mmol), and the
resulting solution was stirred for 3 h. The solvents were removed by
evaporation, and the crude product was purified by flash chromatog-
raphy (CH2Cl2-MeOH-triethylamine, 90:10:2) to give the desired
succinate (1S)-16 as a tan solid foam (200 mg, 50%). It was dissolved
in a mixture of acetonitrile (10 mL), triethylamine (3 mL), and water
(3 mL). After being stirred for 45 min, the mixture was concentrated
in Vacuo. Residual water was removed by coevaporation with
acetonitrile (3 × 10 mL). The resulting cyclic product (8aS)-18a (tan
oil) was taken to the next step without additional purification. To a
solution of the crude succinate ((8aS)-18a) in 5 mL of dry CH2Cl2 were
added dropwise triethylamine (200 mL) and 2,3,5,6-tetrafluorophenyl
trifluoroacetate27 (200 mL, 1.14 mmol). After being stirred for 1 h,
the solution was concentrated in Vacuo. Flash chromatography (3.5 ×
15 cm, 40% ethyl acetate-hexane) afforded (8aS)-18b (180 mg) as a
white crystalline solid (150 mg, 74% from (8aS)-18a): mp 158-160
°C (hexane/ethyl acetate): 1H NMR (CDCl3) δ 7.00 (m, 1H), 6.58 (s,
1H), 4.4-4.1 (m, 1H), 3.99 (s with overlapping m, 4H), 3.3-2.7 (m,
5H), 1.98 (s, 3H), 1.92 (m, 1H), 1.22 (t, 1H, J ) 4.5 Hz). Anal. Calcd
for C23H18N2O4F4: C, 59.74; H, 3.92; N, 6.06. Found: C, 59.51; H,
3.72; N, 5.92. [R]22
: +112° (c 0.005, CDCl3). (8aR)-18b was
D
prepared analogously from (1R)-7b: [R]22D: -115° (c 0.004, CDCl3).
(8aS)-2,3,5,6-Tetrafluorophenyl 1,2,8,8a-tetrahydro-7-methyl-4-
oxo-cyclopropa[c]pyrrolo[3,2-e]indole-2-succinate ((8aS)-19b). It
was prepared by analogy with (8aS)-18b by starting from (1S)-3-(tert-
butyloxycarbonyl)-1-(chloromethyl)-5-hydroxy-8-methyl-1,2-dihydro-
3H-pyrrolo[3,2-e]indole ((1S)-15a)16,24,26 in 44% yield: 1H NMR
(CDCl3) δ 9.61 (br s, 1H), 7.00 (m, 1H), 6.85 (s, 1H), 4.4-3.9 (m,
2H), 3.3-2.8 (m, 5H), 2.03 (s, 3H), 1.99 (m, 1H), 1.27 (m, 1H). Anal.
Calcd for C22H16N2O4F4: C, 58.93; H, 3.60; N, 6.25. Found: C, 58.61;
H, 3.78; N, 5.98. (8aR)-19b was prepared analogously from (1R)-
15a.
(1S)-20a. To a solution of freshly prepared (1S)-7b (0.33 mmol) in
5 mL of dry DMA were added 3-(tert-butyloxycarbonyl)-1,2-dihydro-
3H-pyrrolo[3,2-e]indole-7-carboxylic acid28 (100 mg, 0.33 mmol) and
EDC (120 mg, 0.63 mmol). After being stirred for 5 h, the mixture
was cooled in ice and water (15 mL) was added. The resulting solid
was collected by filtration, washed with 1 M potassium phosphate and
water, and dried in Vacuo affording (1S)-20a as a green-yellow solid
(161 mg, 91%). This was 95% pure by reverse phase HPLC. An
analytical sample was purified by flash chromatography (5% MeOH
in CH2Cl2): 1H NMR (DMSO-d6) δ 11.62 (s, 1H), 9.79 (s, 1H), 7.8 (br
s, 1H), 7.61 (br s, 1H), 7.31 (d, 1H, J ) 9 Hz), 6.96 (br s, 2H), 4.63
(t, 1H, J ) 10 Hz), 4.50 (d, 1H, J ) 11 Hz), 4.1-3.8 (m with
overlapping singlet (CH3) at 3.92 ppm, 6H), 3.55 (t, 1H, 10 Hz), 3.4-
3.1 (m, 3H overlapping with the water peak), 2.31 (s, 3H), 1.51 (s,
9H). Anal. Calcd for C29H31N4O4Cl: C, 65.10; H, 5.84; N, 10.47.
Found: C, 64.86; H, 5.94; N, 10.26. (1R)-20a was prepared analo-
gously from (1R)-7b.
(1S)-21a. It was prepared by analogy with (1S)-20a starting from
(1S)-3-(tert-butyloxycarbonyl)-1-(chloromethyl)-5-hydroxy-8-methyl-
1,2-dihydro-3H-pyrrolo[3,2-e]indole ((1S)-15a) and 3-(tert-butyloxy-
carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid. Crude
(1S)-21a was purified by flash chromatography in ethyl acetate to give
the title compound as a yellow-green solid (70% yield): 1H NMR
(DMSO-d6) δ 11.63 (s, 1H), 10.72 (s, 1H), 9.77 (s, 1H), 7.82 (m, 1H),
7.61 (m, 1H), 7.31 (d, 1H, J ) 9 Hz), 7.04 (s, 1H), 6.96 (s, 1H), 4.65
(t, 1H, J ) 10 Hz), 4.52 (d, 1H, J ) 11 Hz), 4.02 (t, 2H, J ) 8.5 Hz),
3.91 (d, 1H, J ) 8.5 Hz), 3.58 (t, 1H, 9 Hz), 3.4-3.1 (m, 3H
overlapping with the water peak), 2.35 (s, 3H), 1.52 (s, 9H). Anal.
Calcd for C28H29N4O4Cl: C, 64.55; H, 5.61; N, 10.75. Found: C,
64.33; H, 5.73; N, 10.46. (1R)-21a was prepared analogously from
(1R)-15a and 3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]-
indole-7-carboxylic acid.
(8aS)-22b. (1S)-20a (150 mg, 0.28 mmol) was suspended in 5 mL
of ethyl acetate saturated with HCl. The suspension was stirred for 40
min and concentrated in Vacuo. Residual HCl was removed by
coevaporation with CH2Cl2 (2 × 10 mL). The resulting orange solid
((1S)-20b) was dissolved in 1 mL of dry DMA. Succinic anhydride
(30 mg, 0.3 mmol) and triethylamine (50 mL, 0.36 mmol) were added
to produce succinate (1S)-20c. After being stirred for 3 h, the reaction
mixture was diluted with acetonitrile (10 mL) followed by the addition
(8aS)-2-6-((N-(Bromoacetyl)amino)hexanoyl)-1,2,8,8a-tetrahydro-
5,7-dimethylcyclopropa[c]pyrrolo[3,2-e]indol-4-one ((8aS)-11b). (1S)-
9a (80 mg, 0.17 mmol) was suspended in 3 mL of ethyl acetate saturated
with HCl. The suspension was stirred for 30 min and concentrated in
Vacuo. Residual HCl was removed by coevaporation with CH2Cl2 (2
× 10 mL). The resulting hydrochloride (1S)-9b was dissolved in a
mixture of acetonitrile (10 mL), triethylamine (3 mL), and water (3
mL). After 40 min, the solution was concentrated in Vacuo to give an
oil. This was dried by coevaporation with acetonitrile (3 × 10 mL) to
give (8aS)-11a as a tan solid (95% pure by analytical reverse phase
HPLC). To a suspension of the amine (8aS)-11a in 2 mL of DMF
were added triethylamine (50 mL) and bromoacetic acid N-hydroxy-
succinimide ester (50 mg, 0.21 mmol). After 30 min, the reaction
mixture was partitioned between water and CH2Cl2. The water layer
was separated and reextracted. The combined organic layer was washed
with 5% NaHCO3, dried over Na2SO4, and concentrated in Vacuo.
Chromatography on silica gel, eluting with 5% MeOH-CH2Cl2,
afforded 52 mg (68%) of (8aS)-11b as an off-white crystalline solid:
1H NMR (CDCl3) δ 6.66 (br s 1H), 6.57 (s, 1H), 4.3-3.8 (m, 7H),
3.35 (m, 2H), 2.85 (m, 1H), 2.52 (m, 2H), 1.98 (s, 3H), 1.92 (m, 1H),
1.73 (m, 2H), 1.59 (m, 2H), 1.43 (m, 2H), 1.16 (t, 1H, J ) 5 Hz).
Anal. Calcd for C21H26N3O3Br: C, 56.26; H, 5.85; N, 9.37. Found:
C, 56.55; H, 6.02; N, 9.45.
(8aS)-2-3-((N-(Bromoacetyl)amino)propanoyl)-1,2,8,8a-tetrahy-
dro-5,7-dimethylcyclopropa[c]pyrrolo[3,2-e]indol-4-one ((8aS)-12b).
(8aS)-12b was synthesized by analogy with (8aS)-11b in 62% yield:
1H NMR (CDCl3) δ 7.2 (br s 1H), 6.58 (s, 1H), 4.3-3.8 (m, 7H), 3.65
(m, 2H), 2.88 (m, 1H), 2.72 (m, 2H), 1.98 (s, 3H), 1.92 (m, 1H), 1.18
(m, 1H). Anal. Calcd for C18H20N3O3Br: C, 53.21; H, 4.96; N, 10.34.
Found: C, 53.41; H, 5.01; N, 10.57.
(8aS)-2-(Chloroacetyl)-1,2,8,8a-tetrahydro-5,7-dimethylcyclopro-
pa[c]pyrrolo[3,2-e]indol-4-one ((8aS)-14). To a solution of freshly
prepared (1S)-7b (0.28 mmol) in 3.5 mL of dry DMA were added
bromoacetic acid (100 mg, 0.72 mmol) and EDC (300 mg, 1.56 mmol).
After being stirred for 30 min, the solution was diluted with 10 mL of
acetonitrile. Triethylamine (3 mL) and water (3 mL) were added to
convert chloromethyl intermediate (1S)-13b into cyclic compound (8aS)-
14. After 1 h, acetonitrile was removed on a rotary evaporator. The
residual solution (∼6 mL) was diluted with 15 mL of brine and
extracted with dichloromethane (2 × 15 mL). The organic phase was
washed with 5% NaHCO3 (15 mL) and dried over Na2SO4. Purification
by flash chromatography (CH2Cl2-MeOH, 20:1) gave (8aS)-14 as an
off-white solid (21 mg, 22%): 1H NMR (CDCl3) δ 6.59 (s, 1H), 4.4-
4.0 (m, 4H), 3.98 (s, 3H), 2.87 (m, 1H), 1.98 (s, 3H), 1.95 (m, 1H,
partially overlapping with CH3 singlet), 1.23 (t, 1H, J ) 5 Hz). Anal.
Calcd for C15H15N2O2Cl: C, 61.97; H, 5.20; N, 9.64. Found: C, 61.67;
H, 5.14; N, 9.46.
(8aS)-2,3,5,6-Tetrafluorophenyl 1,2,8,8a-tetrahydro-5,7-dimethyl-
4-oxo-cyclopropa[c]pyrrolo[3,2-e]indole-2-succinate ((8aS)-18b). To
a suspension of freshly prepared (1S)-7b (0.9 mmol) in a mixture of
dry CH2Cl2 (7 mL) and MeOH (7 mL) were added succinic anhydride

6224 J. Am. Chem. Soc., Vol. 119, No. 27, 1997
LukhtanoV et al.
of triethylamine (3 mL) and water (3 mL) to effect cyclization. After
1 h, the volatiles were removed in Vacuo, and to the residue (DMA
∼1 mL) was added ether (20 mL) to precipitate the product (8aS)-22a.
It was collected by filtration and dried in Vacuo affording the succinate
as a grey-yellow solid. To a suspension of (8aS)-22a in 3 mL of DMA
were added triethylamine (200 µL, 1.44 mmol) and 2,3,5,6-tetrafluo-
rophenyl trifluoroacetate (140 µL, 0.8 mmol). After being stirred for
1 h, the reaction mixture was added dropwise to 10 mL of 0.3 M
potassium phosphate buffer (pH 7.5), the resulting precipitate was
collected by filtration and washed with water (15 mL) and ether (5
mL). Drying in Vacuo afforded (8aS)-22b as a yellow-green solid (138
mg, 72%): 1H NMR (DMSO-d6) δ 11.81 (s, 1H), 8.21 (d, 1H, J ) 9
Hz), 7.94 (m, 1H), 7.31 (d, 1H, J ) 9 Hz), 7.08 (s, 1H), 6.88 (s, 1H),
6.65 (s, 1H), 4.6-4.4 (m, 2H), 4.24 (t, 2H, J ) 8 Hz), 3.87 (s, 3H),
3.3 (m, 2H, overlapping with water), 3.18 (m, 1H), 3.06 (m, 2H), 2.91
(m, 2H), 1.97 (s, 3H), 1.92 (m, 1H, overlapping with CH3), 1.38 (m,
1H). Anal. Calcd for C34H26N4O5F4‚2H2O: C, 59.82; H, 4.43; N, 8.21.
Found: C, 59.80; H, 4.04; N, 8.07. (8aR)-22b was prepared analo-
gously from (1R)-20a.
by reverse phase HPLC using standard procedures.44 After detritylation,
the dried ODN was taken up in 0.5 mL of water and repurified on a
Hamilton PRP-1 column (305 × 7.0 mm) using a linear gradient of
0-40% acetonitrile in 0.1 M triethylammonium bicarbonate (pH 7)
over 20 min (flow rate ) 3 mL/min). The desired fraction was taken
to dryness on a Savant SpeedVac and reconstituted in 0.5 mL of water.
The concentration was determined from the UV absorbance at 260 nm.
An aliquot of this stock solution containing ∼0.25 mg of the ODN
was dried in a 1.7 mL Eppendorf tube (Savant SpeedVac), then
dissolved in 100 µL of dry DMSO (Aldrich). Ethyldiisopropylamine
(5 µL) and 50 µL (0.5 mg, 1.1 µmol) of a 10 mg/mL solution of 18b,
19b, 22b, or 23b in DMSO were added, and the solution was kept at
ambient temperature for 16 h. The crude ODN product was precipitated
by adding the reaction mix to 2 mL of 2% sodium perchlorate in
acetone. After centrifugation, the pellet was dried, dissolved in 0.5
mL of water, and purified by HPLC as described in method A. Yields
varied from 35 to 78% (from starting ODN). Analytical preparation
is shown in Figure 2. The conjugates were at least 90% pure by C18
analytical HPLC.
Nuclease Digestion Analysis. To a solution of CPI-ODN (∼15 µg)
in 50 µL of a buffer containing 50 mM Tris-HCl (pH 8.5) and 10 mM
MgCl2 were added RQ1 DNase (5 µL, 1000 U/mL), phosphodiesterase
I (5 µL, 100 U/mL), and calf intestine alkaline phosphatase (1 µL, 650
U/mL). The solution was incubated at 37 °C for 30 h. Aliquots (10
µL) were taken during this period to monitor the progress of the reaction
by reversed phase HPLC in a gradient of acetonitrile 0-60% (50 mM
triethylammonium acetate, pH 7.5) using a Rainin C18 (4.5 × 150
mm) column. Waters 994 photodiode array detector was used to
identify products of digestion by their UV spectra.
Determination of Stability of ODN Conjugates in Physiological
Buffer. A 100 µM solution of ODN conjugated with one of the
alkylating agents was incubated at 37 °C in a buffer containing 140
mM KCl, 20 mM HEPES (pH 7.2), and 10 mM MgCl2. As an internal
unreactive standard, 100 µM of a modified scrambled 12-mer ODN
was added. The ODN had a CDPI2 (3-carbamoyl-3H-pyrrolo[3,2-e]-
indole-7-carboxamide dimer) residue attached at the 3′-end as previously
described.34 To monitor the progress of the reaction, 10 µL aliquots
were taken every 15-24 h and analyzed by reverse phase HPLC in a
gradient of acetonitrile 0-60% (50 mM triethylammonium acetate, pH
7.5) in 20 min using a Rainin C18 (4.6 × 150 mm) column with a
flow rate of 1 mL/min. The extent of degradation was measured as
loss of the starting CPI-ODNs relative to the internal unreactive
standard. This internal standard had a retention time longer than CPI-
ODNs and did not interfere with measurement of starting material or
degradation products. Pseudo-first-order rate constants were calculated
from linear plots of log(C/C0) versus time. At least five time points
were used for the calculation during at least 2 half-lives. The
experiments were done in duplicate.
Determination of Reaction Rates of DNA Alkylation and Acid-
Catalyzed Solvolysis. Determination of the reaction rate of cross-
linking between the CPI-ODN conjugate and hairpin target was based
on monitoring of long wavelength band as illustrated in Figure 4. The
experiments were carried out in a buffer containing 140 mM KCl, 10
mM MgCl2, and 20 mM HEPES (pH 7.2) at 37 °C using a Lambda 2
(Perkin-Elmer) spectrophotometer equipped with a PT-6 automatic
temperature controller. Preheated at 37 °C, solutions of a CPI-ODN
conjugate and a hairpin target were mixed in a UV cuvette to final
concentration of 2 and 4 µM, respectively. UV spectra (340-450 nm)
were recorded periodically until no further change was detected in the
spectra. Residual absorbance at the long-wavelength region (355 nm
for MCPI-ODN and 375 nm for MCPI-DPI-ODN conjugates) was
subtracted from the measured absorbances. Pseudo-first-order rate
constants were calculated from linear plots of log(A/A0) versus time
using at least eight time points spanning more than 2 half-lives. The
error ranges (Table 3) represent maximal deviations from the calculated
rate constants. Some of the experiments were performed in triplicate
in order to ensure reproducibility. Variation did not exceed 5%.
Determination of rates of acid-catalyzed hydrolysis was carried out
analogously at 22 °C in an acidic buffer (pH 4). The buffer contained
8 mM citric acid, and 4 mM Na2HPO4 in 50% aqueous methanol.
(Note: It was recently specified17 that this mixture has a pH of 4 not
3 as earlier reported.16) The UV spectra were recorded every 5 min
(8aS)-23b. It was prepared by analogy with (8aS)-22b starting from
(1S)-21a in 70% yield: 1H NMR (DMSO-d6) δ 11.83 (s, 1H), 11.55 (s,
1H), 8.22 (d, 1H, J ) 9 Hz), 7.94 (m, 1H), 7.31 (d, 1H, J ) 9 Hz),
7.09 (s, 1H), 6.89 (s, 1H), 6.69 (s, 1H), 4.7-4.3 (m, 2H), 4.25 (t, 2H,
J ) 8 Hz), 3.3 (m, 2H, overlapping with water), 3.18 (m, 1H), 3.06
(m, 2H), 2.91 (m, 2H), 2.00 (s, 3H), 1.97 (m, 1H, overlapping with
CH3), 1.40 (m, 1H). Anal. Calcd for C33H24N4O5F4‚1.5H2O: C, 60.09;
H, 4.13; N, 8.49. Found: C, 60.29; H, 4.07; N, 8.29. (8aR)-23b was
prepared analogously from (1R)-21a.
Oligonucleotide Synthesis. Oligonucleotides were synthesized on
an Applied Biosystems Model 394 DNA synthesizer utilizing the 1
µmol coupling program supplied by the manufacturer. 5′-Aminolinkers
were introduced into ODNs on the DNA synthesizer using the
appropriate phosphoramidite reagent. Phosphoramidites for introduction
of 6-aminohexyl or 2-(2-aminoethoxy)ethyl residues were purchased
from Glen Research (Sterling, VA). 5-(N-(Monomethoxytrityl)amino)-
pentyl and 4-(N-(monomethoxytrityl)amino)butyl (2-cyanoethyl)diiso-
propyl phosphoramidites were prepared as described by Connolly42 for
similar 3-aminopropyl compound. Oligonucleotides with terminal 3′-
or 5′-thiophosphate group were synthesized using Chemical Phospho-
rylation Reagent from Glen Research (Sterling, VA) and 3H-1,2-
benzodithiol-3-one 1,1-dioxide as the sulfurizing agent.43 After
ammonia deprotection, the ODNs were HPLC purified, detritylated,
and precipitated from butanol as described previously.44
Conjugation Method A. CPI-ODN Conjugates with a Thio-
phosphate Linkage. To a solution of an ODN with a terminal
thiophosphate (∼0.25 µmol) in 20 µL of water were added triethylamine
(0.5 µL) and haloacetamido reagent (11b, 12b or 14) (20 µL of a 33
mM solution in DMF, 0.66 µmol). The progress of the reaction was
monitored by analytical reverse phase HPLC on a Rainin C-18 column
(4.5 × 150 mm) in a gradient of acetonitrile (0-60%) in 0.1 M
triethylammonium acetate buffer (pH 7.5). After 2 h, the solution was
diluted with 0.8 mL of water and loaded onto a semipreparative reverse
phase HPLC column (Polymer Labs, PLRP-S, 250 × 4.6 mm). The
conjugates were resolved as cleanly separated peaks using a linear
gradient of 0-60% acetonitrile in 0.1 M sodium perchlorate (pH 7)
over 20 min (flow rate ) 2 mL/min). The desired fraction was taken
to dryness on a Savant SpeedVac and reconstituted in 50 µL of water.
Acetone (1.5 mL) was added to precipitate the product while leaving
the excess sodium perchlorate in solution. After centrifugation, the
pellet was additionally washed with acetone and dried in Vacuo. After
reconstitution in ∼0.1 mL of 10 mM HEPES (pH 7.2), the concentration
was determined from the UV absorbance using 1 A260 ) 30 µg/mL.45
Typical yield was 60-70% (from starting ODN).
Conjugation Method B. ODN-CPI Conjugates with an Amide
Linkage. 5′-Alkylamine-modified ODN was synthesized and purified
(42) Connolly, B. A. Nucleic Acids Res. 1987, 15, 3131-3139.
(43) Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Am. Chem.
Soc. 1990, 112, 1253-1254.
(44) Reed, M. W.; Adams, A. D.; Nelson, J. S.; Meyer, R. B., Jr.
Bioconjugate Chem. 1991, 2, 217-225.
(45) Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers 1970, 9,
1059-1077.

Oligonucleotide-Cyclopropapyrroloindole Conjugates
J. Am. Chem. Soc., Vol. 119, No. 27, 1997 6225
for ODN conjugates or every 3 h for compound 8. Analyses of the
data were performed as described above.
were incubated in boiling water for 30 min followed by treatment with
10% piperidine for 15 min at the same temperature. After the high-
temperature treatment, reaction mixtures were dried using a SpeedVac,
dissolved in 25-50 µL of loading solution and analyzed by denaturing
PAGE (55-60 °C). Statistical cleavage of each individual 32P-labeled
ODN hairpin at the purine bases (A + G reaction; 60% formic acid,
37 °C, 12 min) was used as a control lane to assign detected bands of
cleavage pattern to the bases of the targeted ODN hairpins which
underwent alkylation.
Determination of Sequence Specificity and Efficiency of DNA
Alkylation by ODN Conjugates. To determine efficiency and
specificity of reaction between targeted hairpins and CPI- or MCPI-
ODN conjugates, the hairpins were 5′-end-labeled with 32P using
polynucleotide kinase and [γ-32P]ATP. The 32P-labeled hairpin targets
(2 × 10-7 M) were mixed with reactive ODN derivatives (4 × 10-7
M) in 140 mM KCl, 10 mM MgCl2, and 20 mM HEPES (pH 7.2).
The reactions (25 µL) were incubated at 37 °C. After 15 h, samples
(1-2 µL) were mixed with 5-10 µL of loading solution (8 M urea,
0.1% Bromophenol Blue, 0.1% Xylene Cyanole FF) and analyzed by
electrophoresis on a denaturing polyacrylamide gel (∼40 °C). Gels
were transferred on a Whatman 3MM Chr paper (Whatman Interna-
tional Ltd, England) and dried in Vacuo. The dried gels were visualized
by autoradiography, and bands were quantified using a GS-250
Molecular Imager (Bio-Rad Laboratories, Inc., U.S.). Products of the
cross-linking reactions were identified as slower migrating bands
relatively to unmodified hairpin ODN. The yield of cross-linking
reaction (%) or efficiency of alkylation was determined as the ratio
between counts assigned to the products and total number of counts in
the lane. To determine sequence specificity of alkylation, reactions
Acknowledgment. We thank Dr. Alexander Gall for helpful
discussions and computer modeling. A portion of this work
was funded by Grant GM52774 from the National Institutes of
Health, USPHS.
Supporting Information Available: Synthesis and charac-
terization of 3-8. Separation and characterization of diaster-
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