Alteration of DNA primary structure by DNA topoisomerase I. Isolation of the covalent topoisomerase I-DNA binary complex in enzymatically competent form

Article abstract of DOI:10.1021/ja961788h

DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in uncoupling of the cleavage and ligation reac

Full text of DOI:10.1021/ja961788h

J. Am. Chem. Soc. 1996, 118, 11701-11714
11701
Alteration of DNA Primary Structure by DNA Topoisomerase I.
Isolation of the Covalent Topoisomerase I-DNA Binary
Complex in Enzymatically Competent Form
Kristine A. Henningfeld, Tuncer Arslan, and Sidney M. Hecht*
Contribution from the Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22901
ReceiVed May 28, 1996X
Abstract: DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing
a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in
uncoupling of the cleavage and ligation reactions of the enzyme, thereby trapping the covalent enzyme-DNA
intermediate. DNA cleavage could be reversed by the addition of acceptor oligonucleotides containing a free 5′-OH
group and capable of hybridizing to the noncleaved strand of the “suicide substrates”. Utilizing acceptors with
partial complementarity, modification of nucleic acid structure has been obtained. Modifications included the formation
of DNA insertions, deletions, and mismatches. To further evaluate the potential of topoisomerase I to mediate
structural transformations of DNA, acceptor oligonucleotides containing nucleophiles other than OH groups at the
5′-end were studied as substrates for the topoisomerase I-mediated ligation reaction. Toward this end, oligonucleotides
containing 5′-thio, amino, and hydroxymethylene moieties were synthesized. Initial investigations utilizing a coupled
cleavage-ligation assay suggested that only the modified acceptor containing an additional methylene group underwent
efficient enzyme-mediated ligation. However, as linear DNA is not a preferred substrate for topoisomerase I, the
enzyme-DNA intermediate was purified to homogeneity, thereby allowing investigation of the ligation reaction
independent of the forward reaction that formed the covalent binary complex. The isolated complex consisted of
equimolar enzyme and DNA, with topoisomerase I covalently bound to a specific site on the DNA duplex in an
enzymatically competent form. Displacement of the enzyme-linked tyrosine moiety of the enzyme-DNA binary
complex was effected by all the modified acceptor oligonucleotides, affording unnatural internucleosidic linkages at
a specific site. Characterization of the formed linkages was effected both by enzymatic and chemical degradation
studies. Comparative analysis revealed overall differences in the efficiency and rate of the topoisomerase I-mediated
ligation of the modified acceptors. Moreover, the facility of ligation of the amino acceptor was significantly enhanced
at increasing pH values. In addition, the method utilized to obtain the topoisomerase I-DNA intermediate is capable
of affording large quantities required for further mechanistic and physicochemical characterization of the formed
binary complex.
The DNA topoisomerases control DNA topology through the
introduction of transient breaks in the phosphodiester backbone.1
Owing to their unique function, these enzymes are central to
the essential cellular processes of replication and transcription,2
as well as recombination.3 DNA topoisomerases are classified
into two groups based on the mode of DNA strand scission:
the type I enzymes mediate the transient single-strand breakage
of duplex DNA, while the type II topoisomerases break both
strands of the duplex.
Mechanistically, the transient strand breaks mediated by
eukaryotic DNA topoisomerase I involve reversible formation
of an intermediate in which the active site of topoisomerase I
is linked to the DNA substrate covalently via a 3′-O-phospho-
rotyrosine bond, with concomitant production of a free 5′-OH
group on the DNA at the site of the break.4 DNA ligation is
coordinated with cleavage and restores continuity to the DNA
duplex. Under normal circumstances the cleavage and ligation
reactions of the enzyme are tightly coupled, with a low steady-
state concentration of the covalent intermediate.5 However, the
two half-reactions of topoisomerase I can be separated in Vitro
by site specific cleavage of partial duplex substrates containing
a high affinity cleavage site toward the 3′-end of the scissile
strand (Figure 1A).6 Cleavage of the suicide substrate occurs
without sequential religation due the instability of the truncated
strand downstream from the site of cleavage; loss of this short
oligonucleotide traps the topoisomerase I-DNA covalent
intermediate. The covalently bound enzyme is catalytically
competent; admixture of an acceptor oligonucleotide comple-
mentary to the single-stranded region of the covalent binary
complex results in ligation to reform a duplex.6 The ability of
topoisomerase I to facilitate the formation of phosphodiester
bonds other than those broken in the original transesterification
reaction suggests that the enzyme can catalyze covalent
alterations in DNA connectivity.7 In fact, through the use of
acceptor strands of varying length and sequence, topoisomerase
X Abstract published in AdVance ACS Abstracts, November 1, 1996.
(1) (a) Chen, A. Y.; Liu, L. F. Annu. ReV. Pharmacol. Toxicol. 1994,
34, 191. (b). Gupta, M.; Fujimori, A.; Pommier, Y. Biochim. Biophys. Acta
1995, 1262, 1.
(2) (a) Wang, J. C. HarVey Lectures 1987, 81, 93. (b) Osheroff, N.
Pharmacol. Ther. 1989, 41, 223.
(3) (a) Champoux, J. J.; Bullock, P. A. In Genetic Recombination;
Kucherlapati, R.; Smith, G. R., Eds.; American Society for Microbiology,
Washington, D. C., 1988, pp 655-666. (b) Ikeda, H. In DNA Topology
and its Biological Effects; Cozzarelli, N. R.; Wang, J. C., Eds.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1990, pp 341-359. (c)
Zhu, J.; Schiestl, R. H. Mol. Cell. Biol. 1996, 16, 1805.
(5) Champoux, J. J. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3488.
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Westergaard, O. J. Mol. Biol. 1991, 222, 669.
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78, 2883. (b) Halligan, B. D.; Davis, J. L.; Edwards, K. A.; Liu, L. F. J.
Biol. Chem. 1982, 257, 3995. (c) McCoubrey, W. K., Jr.; Champoux, J. J.
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S0002-7863(96)01788-X CCC: $12.00 © 1996 American Chemical Society

11702 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
Figure 1. (A) DNA substrate utilized to uncouple the cleavage and ligation reactions of topoisomerase I, thereby allowing DNA strand exchange.
(B) Topoisomerase I-mediated ligation reaction.
I-mediated DNA insertions, deletions and mismatches have been
the covalently bound topoisomerase I with concomitant alter-
ation in the internucleosidic bond and release of free topo-
isomerase I. These results further expand the repertoire of
topoisomerase I-catalyzed nucleic acid rearrangements.
shown to obtain in Vitro with quite reasonable efficiency.8,9
To further define the capacity of topoisomerase I to catalyze
structural transformations of DNA, modified DNA oligonucleo-
tides containing nucleophiles other than the 5′-OH group of
deoxyribose were studied as substrates for the ligation reaction.
Oligonucleotides containing 5′-OH, -SH, -NH2 or -CH2OH
functionalities at their 5′-termini were prepared and the ability
of these acceptors to undergo topoisomerase I-mediated ligation
was investigated both in a coupled cleavage-ligation assay, and
also employing the purified topoisomerase I-DNA covalent
binary complex (Figure 1B). Presently, we describe the
purification of the topoisomerase I-DNA covalent binary
complex and the chemical synthesis of the nucleoside analogues
which permitted the study of the reverse reaction in the presence
of several different nucleophilic functionalities. All modified
oligonucleotide acceptors studied were capable of displacing
Results
Synthesis of 5′-Homo-2′-deoxyadenosine. Although several
methods have been described for the preparation of homoad-
enosine10 and the corresponding thymidine analogue,11 the
application of these procedures for preparative purposes in this
study proved to be problematic owing to poor yields and side
reactions. The route employed for the synthesis of the 5′-
homologue of deoxyadenosine is outlined in Scheme 1. Ben-
zylation of R-D-allofuranose 5 gave benzyl ether 6 in quantitative
(10) (a) Ryan, K. J.; Arzoumanian, H.; Acton, E. M.; Goodman, L. J.
Am. Chem. Soc. 1964, 86, 2503. (b) Pfitzner, K. E.; Moffatt, J. G. J. Am.
Chem. Soc. 1965, 87, 5661. (c) Kappler, F.; Hampton, A. In Nucleic Acid
Chemistry, Part 4; Townsend, L. B., Tipson, R. S. Eds.; Wiley Interscience,
New York, 1991, p 240.
(11) (a) Lebreton, J.; De Mesmaeker, A.; Waldner, A.; Valerie, F.; Wolf,
R. M.; Freier, S. M. Tetrahedron Lett. 1993, 34, 6383. (b) Wendeborn, S.;
Wolf, R. M.; De Mesmaeker, A. Tetrahedron Lett. 1995, 36, 6879.
(8) Henningfeld, K. A.; Hecht, S. M. Biochemistry 1995, 34, 6120.
(9) (a) Shuman, S. J. Biol. Chem. 1992, 267, 8620. (b) Christiansen, K.;
Westergaard, O. In DNA Repair Mechanisms, Alfred Benzon Symposium
35 ; Bohr, V. A., Wassermann, K., Kraemer, K. H., Eds; Munksgaard,
Copenhagen, 1992, pp 361-371.

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11703
Scheme 1a
a (a) BnBr, NaH, DMF, quantitative; (b) Ac2O, AcOH, TsOH, 97%; (c) BisTMS-ABz, TMSOTf, CH3CN, 71%; (d) 2 N NaOH, EtOH-pyridine,
86%; (e) TBDPSCl, imidazole, CH2Cl2, 97%; (f) Im2CS, THF, 65 °C, 90%; (g) Bu3SnH, AIBN, toluene, 75 °C, 80%; (h) TBAF, THF, 76%; (i)
Pd black, EtOH, 45 °C, 9 h, 83%; (j) DMTrCl, pyridine, 81%.
Scheme 2a
yield. The diacetonide (6) was then hydrolyzed and protected
as the tetra-O-acetate by treatment with acetic anhydride in the
presence of a catalytic amount of p-toluenesulfonic acid in
AcOH, which afforded 7 in 97% yield as a colorless solid. A
diastereomeric ratio of 9:1 was obtained, with the â-anomer
presumably being the major isomer.12,13 Initial attempts to
introduce a bis-TMS-N6-benzoyladenine moiety into tetraacetate
a (a) NaIO4, THF-H2O, NaBH4, MeOH; (b) Pd black, EtOH, 45 °C,
9 h.
7 under standard Vorbru¨ggen conditions14 at room temperature
using 1 equivalent of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) as a Lewis acid were unsuccessful. However, a 71%
yield of nucleoside 8 was obtained if the condensation was
performed at 55 °C in the presence of 0.28 equivalent of
TMSOTf. Selective removal of the acetyl groups of 8 was
effected by treatment with 2 N NaOH in ethanol-pyridine to
afford triol 9 in 86% yield.15 Protection of the primary hydroxyl
group of 9 as a silyl ether gave 10 in 97% yield. Subsequent
thioacylation of the free alcohols with thiocarbonyldiimidazole
in THF afforded 11 as a colorless powder in 90% yield.16
Deoxygenation of nucleoside 11 via the Barton procedure17
using tributyltin hydride and azo-bis-isobutyronitrile (AIBN)
furnished 12 in 80% yield. The silyl group was removed
utilizing TBAF in THF to give 13 in 76% yield. Debenzylation
with Pd black in ethanol18 afforded the corresponding 5′-homo-
2′-deoxyadenosine (14) as a colorless powder in 83% yield. The
primary hydroxyl group of 14 was protected with dimethoxy-
trityl chloride in pyridine to furnish the 5′-DMTr-homodeoxy-
adenosine 2a in 81% yield.
Verification of the anomeric configuration of this nucleoside
analogue was obtained by the conversion of triol 9 to N6-
benzoyladenosine as outlined in Scheme 2. Oxidative cleavage
of the diol moiety of 9 with sodium periodate19 and subsequent
reduction of the derived aldehyde with NaBH4 (to prevent
epimerization at C4′)20 afforded adenosine derivative 15 in 83%
overall yield. Debenzylation of 15 was effected utilizing
palladium black to furnish N6-benzoyladenosine (16) in 68%
yield. The 1H NMR spectrum and the optical rotation of
synthetic nucleoside 16 were identical to those of an authentic
sample of 16.
Synthesis of 5′-Thio-2′,5′-dideoxyriboadenosine. The syn-
thesis of the 5′-thio-2′-deoxyadenosine derivative (3a) is outlined
in Scheme 3. The 5′-hydroxyl group of 2′-deoxyadenosine (17)
was converted to the thioacetate via the Mitsunobu reaction21
using PPh3, DEAD and thioacetic acid in THF to furnish 18 in
88% yield.22 Treatment of 18 with benzoyl chloride gave the
(12) Huang, Z.; Schneider, K. C.; Benner, S. A. J. Org. Chem. 1991,
56, 3869.
(13) Under the same reaction conditions the 3-O-TBDPS-protected
diacetonide gave the corresponding tetraacetate as a 1:1 diastereomeric
mixture.
(14) (a) Johnson, T. B.; Hilbert, G. E. Science 1929, 69, 579. (b) Hilbert,
G. E.; Johnson, T. B. J. Am. Chem. Soc. 1930, 52, 4489. (c) Niedballa, U.;
Vorbru¨ggen, H. J. Org. Chem. 1974, 39, 3654. (d) Niedballa, U.;
Vorbru¨ggen, H. J. Org. Chem. 1974, 39, 3660. (e) Niedballa, U.;
Vorbru¨ggen, H. J. Org. Chem. 1976, 41, 2084. (f) Vorbru¨ggen, H.; Hofle,
G. Chem. Ber. 1981, 114, 1256.
(15) Neilson, T.; Werstiuk, E. S. Can. J. Chem. 1971, 49, 493.
(16) (a) Rasmussen, J. R.; Slinger, C. J.; Kordish, R. J.; Newman-Evans,
D. D. J. Org. Chem. 1981, 46, 4843. (b) Robins, M. J.; Wilson, J. S. J. Am.
Chem. Soc. 1981, 103, 932. (c) Lessor, R. A.; Leonard, N. J. J. Org. Chem.
1981, 46, 4300.
1
N6-bis benzoyl protected nucleoside 19 in 92% yield. The H
NMR ratio of ortho proton signals of the benzoyl groups of the
amide relative to the ortho proton signals of the benzoyl ester,
as well as the absence of an amide proton signal, provided
structural evidence for nucleoside 19. Thio ester 19 was treated
with 2 N NaOH to afford the desired modified nucleoside 20
1
in 90% yield. The amide proton signal at 8.92 ppm in the H
(19) (a) Lai, C. K.; Gut, M. J. Org. Chem. 1987, 52, 685. (b) Svansson,
L.; Kvarnstro¨m, I.; Classon, B.; Samuelsson, B. J. Org. Chem. 1991, 56,
2993.
(17) (a) Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans.
1, 1975, 1574. (b) Barton, D. H. R.; Motherwell, W. B. Pure Appl. Chem.
1981, 53, 15.
(18) Felix, A. M.; Jimehez, H. M.; Meienhofer, J. Org. Syn. 1979, 59,
159.
(20) Ranganathan, R. S.; Jones, G. H.; Moffatt, J. G. J. Org. Chem. 1974,
39, 290.
(21) (a) Mitsunobu, O. Synthesis 1981, 1.
(22) (a) Hugles, D. L. Org. React. 1992, 42, 335. (b) Dodge, J. A.;
Trujillo, J. I.; Presnell, M. J. Org. Chem. 1994, 59, 234.

11704 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
Scheme 3a
a (a) AcSH, PPh3, DEAD, THF, 88%; (b) BzCl, pyridine, 92%; (c) 2 N NaOH, EtOH-pyridine, 90%; (d) DMTrCl, pyridine, 91%.
Scheme 4a
a (a) TBDPSCl, imidazole, CH2Cl2, 78%; (b) AcOH, 77%; (c) TsCl, pyridine, 85%; (d) NaN3, DMF, 95%; (e) H2, Pd on C, EtOH, 80%; (f)
MTrCl, pyridine, 91%; (g) TBAF, THF, 81%.
Scheme 5
NMR spectrum, as well as the the triplet signal of the SH group
at 1.60 ppm and the ratio of protons, indicated that the disulfide
had not formed during the reaction and that one of the amide
benzoyl groups was hydrolyzed under the reaction conditions.
Loss of the benzoyl group was also confirmed by mass
spectrometry. Selective protection of the 5′-thiol moiety of 20
with DMTrCl afforded the thio ether 3a as a colorless foam in
91% yield.
Synthesis of 5′-Amino-2′,5′-dideoxyadenosine. The syn-
thesis of the 5′-amino nucleoside 4a was carried out in analogy
with a previously reported synthesis.23 The 5′ protecting group
was changed to a lipophilic, acid-labile group, thereby allowing
oligonucleotides were purified and the trityl group was then
reverse phase purification of the modified oligonucleotide
cleaved with trifluoroacetic acid using Nensorb Prep chroma-
prepared using monomer 4b, following removal of the base and
tography to isolate II and IV. During oligonucleotide synthesis
phosphorous protecting groups. The preparation of 5′-amino-
the thiol-linked DMTr group was removed, presumably as a
consequence of iodine treatment, resulting in the formation of
the disulfide-containing oligonucleotide. This dimeric oligo-
nucleotide was purified on a polyacrylamide gel. Subsequent
reduction with dithiothreitol (DTT) afforded the desired 5′-thiol-
containing product (III). Loss of the DMTr moiety most likely
occurred during the oxidation step of DNA synthesis as S-trityl
2′, 5′-dideoxyadenosine is illustrated in Scheme 4. 5′-O-DMTr-
N6-benzoyl-2′-deoxyadenosine (21) was protected as the silyl
ether by treatment with TBDPSCl, affording 22 in 78% yield.
Following detritylation to afford 23 in 77% yield, the 5′-OH
group was activated with tosyl chloride24 in pyridine to furnish
nucleoside 24 in 85% yield. Displacement of the tosylate was
effected via the agency of NaN3 in DMF to give the 5′-azido
nucleoside 25 in 95% yield. The 5′-azido group was reduced
over 10% palladium-on-carbon by hydrogenation in ethanol,
cleavage by iodine has previously been shown to occur via
intermolecular disulfide bond formation.26 Interestingly, another
report describes the preparation of an oligonucleotide containing
affording the 5′-amino compound 26 in 80% yield. The 5′-
a modified S-trityl moiety at the 5′-terminus that was stable
amino group of the nucleoside was protected with monometh-
toward iodine oxidation during oligonucleotide synthesis.27
oxytrityl chloride in pyridine to furnish 27 in 91% yield.
Topoisomerase I Coupled Cleavage-Ligation. The 5′-32P
end labeled partial duplex shown in Figure 1A was treated with
Treatment with TBAF effected removal of the tert-butyldiphen-
ylsilyl group to give 4a as a colorless foam in 81% yield.
calf thymus DNA topoisomerase I in the presence of an excess
Oligonucleotide Synthesis. Phosphitylation of the nucleoside
of the acceptor oligonucleotide at 37 °C for 60 min. Following
monomers using 2-cyanoethyl N,N-diisopropylchlorophosphor-
proteolysis to remove the covalently bound enzyme, the
amidite and diispropylethylamine in CHCl2 afforded the 3′-O-
reactions were analyzed on a 20% denaturing polyacrylamide
phosphoramidite derivatives 2b-4b (Scheme 5). The monomers
gel. The acceptor oligonucleotide containing a 5′-OH group
were incorporated at the 5′-termini of the acceptor oligonu-
(I) afforded a single ligated product (supporting information,
cleotides using standard solid phase phosphoramidite chemis-
Figure 1). DNA sequence analysis of the isolated product
try.25 Cleavage from the solid support and removal of the base
verified that the ligation product resulted from cleavage of the
and phosphate protecting groups was effected with concentrated
partial duplex at site 1, with concomitant loss of the penta-
NH4OH for 12 h at 55 °C. The DMTr and MTr-containing
nucleotide 5′-AGAGA-3′, and ligation of the acceptor strand
to the truncated DNA (supporting information, Figure 2). As
(23) Sproat, S. B.; Beijer, B.; Rider, P. Nucleic Acids Res. 1987, 15,
6181.
(24) Reist, E. J.; Benitez, A.; Goodman, L. J. Org. Chem. 1964, 29,
554.
(25) Caruthers, M. H. Science 1985, 230, 281.
(26) (a) Kamber, B.; Rittel, W. HelV. Chim. Acta 1968, 51, 2061. (b)
Kamber, B. HelV. Chim. Acta 1971, 54, 398.
(27) Connolly, B. A. Nucleic Acids Res. 1985, 13, 4485.

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11705
described previously, the presence of the acceptor strand
complementary to the single-stranded region downstream from
the intended site of cleavage promoted cleavage at an additional
site two nucleotides upstream from the intended site of cleavage,
i.e. site 2 (cf. Figure 1A).8 When topoisomerase I-mediated
cleavage and ligation were performed in the presence of the
modified acceptor oligonucleotide II, significantly lower levels
of ligation were observed compared with acceptor I (supporting
information, Figure 1, cf lanes 2 and 4). However, the
topoisomerase I-covalent intermediate was formed at both sites,
as demonstrated by the cleavage products observed.
While the modified acceptor oligonucleotide underwent
ligation to a significant extent, complete release of the topo-
isomerase covalently bound at site 1 was not observed.
Attempts to increase the ligation yields of duplex in the presence
of acceptor II employed agents that facilitate steric exclusion
or DNA aggregation, such as polyethylene glycol, spermidine
or hexammine cobalt chloride; however, these increased the
ligation yields only marginally (data not shown). Moreover, a
temperature profile of the course of the ligation reaction simply
paralleled the thermostability of the enzyme suggesting that the
native conformation of the enzyme is uniquely required for
catalysis (data not shown). As the ligation product incorporating
acceptor oligonucleotide I was formed only to the extent of 34%
in the coupled cleavage-ligation assay, less efficient transfor-
mations leading to structurally altered duplexes would not have
been detected readily utilizing this assay system. Moreover,
the presence of an acceptor strand has been shown to influence
both the efficiency and the specificity of initial topoisomerase
I-mediated cleavage.8 Thus, to investigate the ligation reaction
independent of the forward reaction, the topoisomerase I-DNA
covalent intermediate was purified.
Purification of the Topoisomerase I-DNA Covalent
Intermediate. The topoisomerase I-DNA covalent intermedi-
ate was formed by site-specific cleavage of the nicked duplex
shown in Figure 2A. The addition of a 12-nt oligomer (5′-
pAATTTGGCGCGG-3′) complementary to the single-stranded
region of the partial duplex was introduced to increase the
efficiency and specificity of cleavage by topoisomerase I.8 The
5′-termini of all the oligomers used to assemble the substrate
were phosphorylated to prevent ligation with the formed
enzyme-DNA binary complex.
Figure 2. (A) DNA substrate utilized to obtain the covalent topo-
isomerase I-DNA intermediate. The oligonucleotide which completes
the “duplex” is indicated in bold italics. (B) Elution profile showing
products formed by treatment of the 5′-32P end labeled partial duplex
with topoisomerase I, followed by chromatography on a Mono Q
column.
The substrate partial duplex, 5′-32P end labeled on the scissile
strand, was treated with topoisomerase I for 60 min at 37 °C.
Separation of the covalent topoisomerase I-DNA intermediate
from both the unreacted DNA substrate and topoisomerase I
was achieved utilizing anion exchange chromatography on a
Mono Q column. The reaction mixture was applied to a Mono
Q column then washed with a linear gradient of aqueous NaCl.
As shown in Figure 2B, the DNA substrate is strongly retained
on this column; preincubation with topoisomerase I resulted in
the formation of a new, early eluting peak. Control experiments
demonstrated that topoisomerase I alone did not bind to this
matrix.
To determine if the peak formed in the presence of topo-
isomerase I actually contained topoisomerase I covalently linked
to the DNA, the isolated complex was analyzed by 20%
denaturing polyacrylamide gel electrophoresis (PAGE). It has
been reported previously that topoisomerase I retains DNA to
which it is linked covalently in the wells of denaturing
polyacrylamide gels.28 The putative enzyme-DNA complex
enters the gel only upon incubation with proteinase K, demon-
strating that all of the radiolabeled DNA is covalently bound
Figure 3. Autoradiogram of a 20% denaturing polyacrylamide gel
illustrating the enzymatic activity of the purified topoisomerase I-DNA
intermediate: lane 1, DNA alone; lane 2, DNA + proteinase K; lane
3, DNA + I; lane 4, putative topoisomerase I-DNA complex alone;
lane 5, putative topoisomerase I-DNA complex + proteinase K; lane
6, putative topoisomerase I-DNA complex + I.
to protein (Figure 3, cf lanes 4 and 5). Treatment with the
protease afforded a DNA fragment with increased electro-
phoretic mobility compared with the original uncleaved DNA
substrate (Figure 3, cf lanes 1 and 5). These results are
consistent with topoisomerase I cleavage of the DNA substrate,
concomitant formation of the covalent intermediate and subse-
quent loss of the pentanucleotide downstream from the cleavage
site. As expected, the electrophoretic mobility of the DNA
substrate itself was not altered by treatment with proteinase K
(cf. lanes 1 and 2).
(28) Christiansen, K.; Bonven, B. J.; Westergaard, O. J. Mol. Biol. 1987,
193, 517.

11706 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
Table 1.a Quantification of Full Length Ligation Products
Resulting from Treatment of the Purified Topoisomerase I-DNA
Complex with Acceptor Oligonucleotides I-IV
acceptor
% ligation
acceptor
% ligation
I
II
100
29
III
IV
13
4.3
a The calculated values were normalized to the ligation obtained with
acceptor oligonucleotide I.
Figure 4. Autoradiogram of a 20% denaturing polyacrylamide gel
illustrating the ligation of the modified acceptor oligonucleotides to
the 5′-32P end labeled topoisomerase I-DNA intermediate: lane 1,
topoisomerase I-DNA complex alone; lane 2, topoisomerase I-DNA
complex + proteinase K; lane 3, topoisomerase I-DNA complex + I;
lane 4, topoisomerase I-DNA complex + II; lane 5, topoisomerase
I-DNA complex + III; lane 6, topoisomerase I-DNA complex +
IV. Following incubation at 37 °C for 60 min, the reactions in lanes
2-6 were treated with proteinase K prior to analysis. The arrow denotes
the position of the ligation products. Although not visible in the gel
photograph, the formation of product in the presence of acceptor
oligonucleotide IV was readily apparent by phosphorimager analysis
(cf. Table 1 and supporting information, Figure 3).
Having demonstrated that topoisomerase I was covalently
bound to the truncated DNA, the enzymatic competence of the
complex was next examined by assaying for strand transfer
activity. Incubation of the purified complex with an excess of
acceptor oligonucleotide I for 60 min at 37 °C effected efficient
conversion to a single ligated product (94% yield), demonstrat-
ing the enzymatic competence of the covalently bound topo-
isomerase I (Figure 3, lane 6).
Figure 5. Autoradiogram of a 20% denaturing polyacrylamide gel
illustrating the ligation of IV as a function of pH: lane 1, pH 7.5; lane
2, pH 8.0; lane 3, pH 8.5; lane 4, pH 9.0. The putative hydrolysis
product is indicated by the asterisk.
when Mg2+ or Mn2+ was employed as the divalent cation
(supporting information, Figure 4). The relative efficiency of
ligation of the oligonucleotide having a 5′-NH2 group was only
about 4%.
Verification that the duplex structure was maintained during
the isolation was accomplished by 5′-32P end labeling the
individual strands of the substrate. The purified topoisomerase
I-DNA complex was treated with proteinase K and applied to
a 20% denaturing polyacrylamide gel confirming the presence
of the truncated scissile strand, the 12-mer (5′-AATTTG-
GCGCGG-3′) and the noncleaved strand (data not shown).
Topoisomerase I-Mediated Ligation of Modified Accep-
tors. The topoisomerase I-DNA covalent intermediate, 5′-32P
end labeled on the scissile strand, was purified by Mono Q
chromatography and then a portion was treated with an acceptor
oligonucleotide containing a 5′-OH (I), -CH2OH (II), -SH (III),
or -NH2 (IV) functional group. After 60 min at 37 °C, each
reaction was quenched by the addition of 1% sodium dodecyl
sulfate (SDS), and the covalently bound enzyme was removed
by proteolysis with proteinase K prior to analysis by 20%
denaturing polyacrylamide gel electrophoresis. As shown in
Figure 4, each of the modified acceptor oligonucleotides (II-
IV) afforded a ligation product that had the same electrophoretic
mobility as that obtained with the unmodified acceptor oligo-
nucleotide (I). However, the ability of the individual acceptor
oligonucleotides to serve as substrates for topoisomerase
I-mediated ligation varied. The percent ligation obtained with
each acceptor oligonucleotide, relative to unmodified oligo-
nucleotide I, was determined by phosphorimager analysis. The
results are shown in Table 1. The actual extent of ligation
obtained using the unmodified oligonucleotide (I) varied from
64-94% in individual experiments. The most effective of the
modified oligonucleotides was II, containing an additional
methylene group at the 5′-end. This species afforded almost
30% ligation product, relative to the unmodified acceptor
oligonucleotide. The acceptor oligonucleotide containing an SH
group at the 5′-terminus (III) gave about 13% ligation product
relative to the unmodified acceptor; no difference was observed
The ability of the individual acceptor oligonucleotides to dis-
place the covalently bound topoisomerase I was also investigated
over a range of pH values. Shown in Figure 5 is the topo-
isomerase I-mediated ligation of acceptor oligonucleotide IV
(containing a 5′-NH2 group) in the presence of Tris-HCl at
several pH values (7.5, 8.0, 8.5 and 9.0) for 1 h at 37 °C.
Phosphorimager quantification (supporting information, Figure
5) clearly demonstrates that as pH value is raised the efficiency
of ligation is increased, with more than a 5-fold enhancement
of ligation obtained by increasing the pH from 7.5 to 9. In
contrast, the ligation of the other acceptors did not show a
significant pH dependence (supporting information, Table 1).
Additionally, another product having increased electrophoretic
mobility compared with the cleavage product, became readily
apparent at high pH (indicated by the asterisk in Figure 5). This
product most likely resulted from the displacement of the bound
enzyme by water, affording the oligonucleotide GGCGCG-
GAGACTTp, which has a 3′-phosphate group.29 That this
product actually contained a 3′-phosphate group was demon-
strated by the decrease in electrophoretic mobility upon treat-

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11707
ment with T4 polynucleotide kinase,30 which is known to have
an associated 3′-phosphatase activity (data not shown).
Further characterization of differences in the ligation of the
modified acceptors to the topoisomerase-DNA complex involved
determination of the time course of the ligation reaction. The
5′-32P end labeled complex was incubated with an excess of
the individual acceptor oligonucleotides for various times,
quenched by the addition of SDS, treated with proteinase K
and analyzed by 20% denaturing PAGE. As shown in Figure
6, the modified acceptor oligonucleotides (II-IV) exhibited a
significantly slower rate of ligation compared with the unmodi-
fied acceptor strand (I). Moreover, the observed efficiency of
ligation at 60 min paralleled the rates of ligation of the individual
acceptor oligonucleotides. It is interesting that the ligation
reactions at 60 min in this experiment appear incomplete and
the yields of ligation products were lower than those reflected
in Table 1. This may well reflect the different amounts of
acceptor oligonucleotides employed.
To establish the chemical nature of the newly formed bonds
in the ligation products, these products were isolated from
polyacrylamide gels and subjected to chemical and enzymatic
degradation analysis. To determine the nature of the linkage
formed by oligonucleotide II, an authentic synthetic product
having the sequence shown in Figure 1B was synthesized by
incorporation of the phosphoramidite monomer 2b at cleavage
site 1 (Figure 1). A synthetic oligonucleotide was also pre-
pared containing the unmodified linkage at this site. The
synthetic oligonucleotides were 5′-32P end labeled and annealed
to the noncleaved strand. The synthetic and enzymatically
derived products were then incubated with exonuclease III, a
nuclease that catalyzes the sequential removal of nucleotides
from the ends of double-stranded DNA proceeding in a 3′ f 5′
direction. After incubation for 20 min at 37 °C, the reactions
were quenched by the addition of EDTA and then analyzed on
a 20% denaturing polyacrylamide gel. The synthetic and
enzymatic ligation products containing the unmodified linkage
at site 1 were completely degraded by exonuclease III (sup-
porting information, Figure 6, lanes 3 and 6). In contrast,
exonuclease III digestion of the ligation product putatively con-
taining nucleoside 2a at site 1, as well as the authentic synthetic
standard containing 2a, “stalled” at a specific site (arrow, lanes
4 and 5). Comparison of the degradation products with the
Maxam-Gilbert G and G + A base-specific reactions revealed
that exonuclease III digestion was strongly inhibited prior to
Figure 6. Time courses for the topoisomerase I-mediated ligation of
the acceptor oligonucleotides (open circle, I; filled circle, II; filled
square, III, filled triangle, IV).
the modified linkage (5′-32pGGCGCGGAGACTTCH AGA-3′).
2
Consistent with the formation of a phosphorous-sulfur linkage,
the ligation product afforded by the acceptor oligonucleotide
III underwent oxidative cleavage when treated with 50 mM
iodine in pyridine for 2 h at room temperature (Figure 7, cf
lanes 3 and 4).31 In contrast, the unmodified ligation product
derived from I was refractory to cleavage with I2 (Figure 7, cf.
lanes 1 and 2).
Figure 7. Autoradiogram of a 20% denaturing polyacrylamide gel
demonstrating the topoisomerase I-mediated formation of the bridging
phosphorothioate linkage. Lane 1, I2 treatment of the product resulting
from ligation of I with the topoisomerase I-DNA covalent complex;
lane 2, product resulting from ligation of I with the topoisomerase
I-DNA covalent complex; lane 3, I2 treatment of the product resulting
from ligation of III with the topoisomerase I-DNA covalent complex;
lane 4, product resulting from ligation of III with the topoisomerase
I-DNA covalent complex.
The extreme lability of internucleosidic phosphoramidate
linkages in dilute acid, relative to the phosphodiester linkage,
provided a facile method to authenticate the linkage at site 1 of
the ligation product formed by IV.32 The putative phosphor-
amidate and phosphodiester-containing ligation products, formed
by incubation of IV and I, respectively, were treated with 15%
acetic acid for 12 h at room temperature. As shown in Figure
8, the product afforded by enzymatic ligation of IV underwent
site-specific cleavage, yielding a single cleavage product (cf.
lanes 2 and 3). The hydrolytic product had slightly increased
mobility compared with synthetic 13-mer cleavage product (5′-
32pGGCGCGGAGACTT-3′) owing to the presence of a 3′-
(29) Christiansen, K.; Knudsen, B. R.; Westergaard, O. J. Biol. Chem.
1994, 269, 11367.
(30) Cameron, V.; Uhlenbeck, O. C. Biochemistry 1977, 16, 5120.
(31) Cosstick, R.; Vyle, J. S. Nucleic Acids Res. 1990, 18, 829.
(32) Letsinger, R. L.; Wilkes, J. S.; Dumas, L. B. Biochemistry 1976,
15, 2810.

11708 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
oligonucleotide. Enzyme-mediated ligation of the modified
acceptors would afford a site-specific alteration of the ligated
oligonucleotide at the site of ligation (Figure 1B). Acceptor
oligonucleotides were synthesized containing 5′-thio and amino
moieties; also prepared was an acceptor oligonucleotide with
an additional methylene group at the 5′-terminus to permit
investigation of spatial constraints for ligation.
Initial attempts utilizing a coupled cleavage-ligation assay
suggested that topoisomerase I could mediate the joining of
modified acceptor oligonucleotides, but product formation was
limited by the availability of the enzyme-DNA intermediate.
Therefore, anion exchange chromatography was utilized to
obtain homogenous preparations of the covalent intermediate.
This species was shown to be catalytically competent (Figure
3), thereby allowing investigation of the ligation reaction
independent of the cleavage reaction. This approach has also
been utilized by other workers to study DNA modifying
enzymes; for example, Chen et al.35 have isolated a stable
covalent complex formed between DNA and the DNA (cytosine-
5)-methyltransferase. For topoisomerase I, this method should
provide access to large quantities of the enzyme-DNA inter-
mediate required for physicochemical characterization of the
formed binary complex. The results obtained here further
suggest the general utility of this method for the isolation of
protein-DNA complexes, e.g. for the site-specific prokaryotic
integrases which have biochemical properties similar to topo-
isomerase I.36,37
Figure 8. Autoradiogram of 20% denaturing polyacrylamide gel
demonstrating the topoisomerase I-mediated formation of the phos-
phoramidate linkage. Lane 1, synthetic 13-mer cleavage product
5′32pGGCGCGGAGACTT; lane 2, ligation product derived from IV;
lane 3, ligation product derived from IV + acetic acid; lane 4, ligation
product derived from I; lane 5, ligation product derived from I + acetic
acid.
phosphate (cf. lanes 1 and 3). The cleavage product observed
supports the presence of a phosphoramidate linkage at site 1.
Discussion
The catalytic cycle mediated by DNA topoisomerase I
involves cleavage and ligation of the phosphodiester backbone
of DNA via a transient DNA-enzyme covalent intermediate
containing a phosphorotyrosine linkage. The equilibrium for
the cleavage and ligation reactions that comprise the catalytic
cycle lies strongly in the direction of the ligation reaction,
affording a low steady-state concentration of covalent binary
complexes.5 The cleavage-ligation equilibrium can be altered
by the camptothecins, which stabilize the covalent intermediate
by inhibiting the ligation reaction.33 In ViVo, increasing the
concentration of the topoisomerase I-DNA covalent complex
facilitates chromosomal aberrations, sister chromatid exchange
and illegitimate recombination.3,34 This process can be modeled
in Vitro though the use of DNA duplexes containing a single
discontinuity on the scissile strand in proximity to the preferred
site of cleavage by topoisomerase I.8,9 Site specific cleavage
by the enzyme results in uncoupling of the cleavage and ligation
reactions due to the instability of the short DNA duplex structure
downstream from the site of cleavage (Figure 1A). The
topoisomerase I trapped using such “suicide substrates” can
undergo ligation with exogenously added DNA acceptors,
affording structural transformations of the DNA; these have
included insertions, deletions and mismatches.8,9 We were,
therefore, interested in determining whether topoisomerase I
could catalyze additional alterations of DNA structure.
Reaction of the purified DNA-topoisomerase I intermediate
with acceptor oligonucleotides containing free 5′-CH2OH, -SH,
and -NH2 moieties afforded DNA products containing modified
internucleosidic bonds. The enzyme-DNA complex exhibited
the greatest tolerance (29% yield) for acceptor oligonucleotide
II, containing an additional methylene group at the 5′-terminus.
This is consistent with the ability of topoisomerase I to facilitate
the formation of insertions.8,9 The acceptor oligonucleotides
incorporating 5′-SH and -NH2 groups both afforded significantly
lower levels of ligation (13% and 4% conversion, respectively).
It was possible to increase the ligation yields obtained with
acceptor oligonucleotide IV by increasing the pH of the
incubation mixture. At pH 7.5, only 2.6% ligation was obtained,
while at pH 9.0 12% of the substrate oligonucleotide was
converted to full length product. As the pKa of alkylamines is
approximately 10, the pH effect most likely resulted from
deprotonation of the amino moiety, thus increasing the nucleo-
philicity of this acceptor. Consistent with this interpretation,
the incorporation of the other acceptors did not follow this trend
as a function of pH. The latter observation is entirely consistent
with the previous finding that the topoisomerase I-mediated
ligation reaction of acceptor oligonucleotides containing a free
5′-OH group was independent of pH over the range 5.1-9.5.29,38
As noted above, Westergaard and coworkers have reported
that the covalently bound topoisomerase I in the enzyme-DNA
complex can be displaced by nucleophiles present in the reaction
mixtures at high concentrations, including water and a variety
of compounds with free hydroxyl groups, such as glycerol. The
pH optima observed for these reactions were between 8.5 and
9.5.29 Interestingly, mononucleosides and mononucleotides
Christiansen et al.29 have noted that nucleophilic species such
as H2O and glycerol, present at high concentrations in incubation
mixtures containing topoisomerase I and DNA, could react with
the electrophilic linkage formed between the active site tyrosine
moiety of the enzyme and the phosphate ester of DNA.
Accordingly, a nicked DNA substrate was utilized to uncouple
the cleavage and ligation reactions of topoisomerase I, thereby
allowing investigation of the nature of the nucleophiles capable
of displacing the enzyme-linked tyrosine moiety from the DNA
when the nucleophiles were present as part of the acceptor
(35) Chen, L.; MacMillan, A. M.; Chang, W.; Ezaz-Nikpay; K.; Lane,
W. S.; Verdine, G. L. Biochemistry 1991, 30, 11018.
(36) Sadowski, P. D. FASEB J. 1993, 7, 760.
(37) While no definitive studies have been carried out to define the
stability of the isolated covalent binary complex, experiments carried out
during this study suggest that it can be maintained at low temperature at
least for several hours. Significant loss of enzymatic competence was
observed in one experiment after storage of the complex for 19 h at 4 °C.
(38) Stivers, J. T.; Shuman, S.; Mildvan, A. S. Biochemistry 1994, 33,
15449.
(33) Hsiang, Y.-H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem.
1985, 260, 14873.
(34) (a) Liu, L. F. Annu. ReV. Biochem. 1989, 58, 351. (b) Froelich-
Ammon, S. J.; Osheroff, N. J. Biol. Chem. 1995, 270, 21429.

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11709
present at the same molar concentrations were unable to serve
as substrates for ligation.39
also operate during topoisomerase I-mediated nucleotide deletion
and insertion reactions.8
Finally, definition of the ease with which specific nucleophiles
can react with the electrophilic topoisomerase I-DNA binary
complex should facilitate the exploration of novel strategies for
intercepting the activated enzyme-DNA binary complex.
Recently, the effect of a modified DNA linkage on the
topoisomerase I-mediated cleavage reaction has been studied
utilizing a DNA substrate containing a bridging phosphorothio-
ate moiety.40 Topoisomerase I was capable of cleaving the
modified linkage to afford an oligonucleotide having a free 5′-
SH moiety; the latter was not observed to undergo the reverse
(i.e., ligation) reaction and was concluded to be incapable of
doing so. This conclusion was based on the observed enhance-
ment of cleavage of the substrate containing the phosphorothio-
ate linkage, relative to the cleavage of the analogous substrate
containing a phosphodiester linkage. The observed topo-
isomerase I cleavage represents the steady state population of
covalent binary complexes. For the unmodified substrate, the
covalent binary complex is present at low levels reflecting an
equilibrium that favors the ligation reaction. For the substrate
containing the phosphorothioate linkage, the increase in covalent
binary complex formation was interpreted as the inability of
the derived sulfhydryl-containing oligonucleotide to serve as a
substrate for the reverse (i.e., ligation) reaction. It should be
noted, however, that if the ligation reaction actually were
infeasible, all of the phosphorothioate-containing substrate
should eventually undergo cleavage. The fact that only partial
conversion to cleaved product was actually observed implies
either that insufficient time was provided to permit complete
substrate cleavage, or else that there actually was an equilibrium
between cleavage and ligation. In fact, the present results
demonstrate that the 5′-SH functionality can displace the
covalently bound enzyme to effect ligation, but at a decreased
rate compared with the 5′-OH group normally present. This
could well alter the cleavage-ligation equilibrium, consistent
with the previously reported results.40 Caution should be
exercised when utilizing phosphorothioate-containing oligo-
nucleotides as suicide substrates, if one is to conclude that the
reaction is irreversible.
We have demonstrated previously that DNA topoisomerase
I can promote the rearrangment of DNA structure; catalysis of
nucleotide insertions and deletions was documented for a few
types of systems.8 In the present study, we have extended these
findings by exploring the range of nucleophiles capable of
reacting with the activated enzyme-DNA complex. In par-
ticular, it is known that phenoxides undergo phosphoryl transfer
reactions with facility,41 a type of transformation that nature
apparently exploits in the use of tyrosine as the active site
nucleophile for reaction with the phosphate ester backbone of
DNA. The ability of S and N nucleophiles to promote the loss
of this tyrosine phenoxide moiety is of fundamental interest from
a chemical perspective, as it reflects the extent to which a
biochemical transformation can be influenced by simple alter-
ation of key chemical parameters. In fact, the pH-dependent
extent of ligation obtained using acceptor oligonucleotide IV
argues that the facility of the ligation reaction is explicable in
terms of simple chemical principles.
Experimental Section
General Methods. Elemental analyses were carried out by Atlantic
Microlab, Inc., Norcross, GA. Melting points were taken on a Thomas
Hoover apparatus and are not corrected. Optical rotations were
determined on a Perkin-Elmer Model 141 polarimeter. 1H NMR and
13C NMR spectra were recorded on a General Electric QE-300
spectrophotometer. Chemical shifts values are expressed relative to
added tetramethylsilane. Experiments requiring anhydrous conditions
were performed under an argon atmosphere. Solvents were J. T. Baker
p. a. and were used without further purification unless noted. THF
and diethyl ether were distilled from potassium-benzophenone. Thin
layer chromatography (TLC) was carried out on Merck silica gel F245
pre-coated plates; spots were visualized by dipping the plates in a Ce-
Mo staining reagent. Column chromatography employed Fluka silica
gel 60, mesh size 230-400.
T4 polynucleotide kinase and proteinase K were purchased from
United States Biochemical; exonuclease III was from Gibco BRL.
â-Cyanoethylphosphoramidites, activator solution and solid support
were obtained from Cruachem Inc. Nensorb prep nucleic acid
purification cartridges were from DuPont-New England Nuclear; [γ-32P]-
ATP (7000 Ci/mmol) was obtained from ICN Biochemicals. Scintil-
lation counting was performed on a Beckman LS-100C instrument using
Beckman Ready Safe scintillation fluid. The Mono Q (HR 5/5) column
was from Pharmacia and FPLC was performed on a Pharmacia system.
Polyacrylamide gel electrophoresis was carried out on 20% gels [19%
(w/v) acrylamide, 1% (w/v) N,N-methylenebisacrylamide, 8 M urea]
in 90 mM Tris-borate buffer, pH 8.3, containing 5 mM EDTA.
Polyacrylamide gel loading solution contained 10 M urea, 1.5 mM
EDTA, 0.05% (w/v) xylene cyanol and 0.05% (w/v) bromphenol blue.
Gels were visualized by autoradiography at -80 °C with Kodak XAR-2
film and quantified utilizing the Molecular Dynamics 400E Phospho-
rimager using ImageQuant version 3.2 software. Sequencing analysis
was performed using a modification42 of the traditional Maxam-Gilbert
method43 for short, single-stranded deoxyoligonucleotides. Distilled,
deionized water from a Milli-Q system was used for all aqueous
manipulations.
Synthesis of Nucleosides. 3-O-Benzyl-1,2:5,6-di-O-isopropyli-
dene-r-D-allofuranose (6). To a stirred solution containing 3.0 g (11.5
mmol) of diacetone allofuranose (5) and 660 mg (16.5 mmol) of sodium
hydride (60% dispersion in mineral oil) in 50 mL of DMF was added
1.40 mL (11.7 mmol) of benzyl bromide. The combined solution was
stirred at room temperature for 1 h, then cooled to 0 °C. The reaction
mixture was partitioned between 50 mL of saturated NaHCO3 and 200
mL of ethyl acetate. The organic phase was washed with 50 mL of
brine and dried over MgSO4. The solution was concentrated under
diminished pressure to yield a crude product which was purified by
flash chromatography on a silica gel column (25 cm × 3 cm). Elution
with 3:7 ethyl acetate-hexanes afforded 6 as a viscous, colorless oil
that solidified at room temperature: yield 4.03 g (quantitative); mp 64
°C; silica gel TLC Rf 0.61 (3:7 ethyl acetate-hexanes); 1H NMR
(CDCl3) δ 1.35 (s, 3 H), 1.36 (s, 3 H), 1.38 (s, 3 H), 1.58 (s, 3 H), 3.87
(dd, 1 H, J ) 4.5, 8.5 Hz), 3.93-4.04 (m, 2 H), 4.14 (dd, 1 H, J ) 3,
8.5 Hz), 4.35 (ddd, 1 H, J ) 3, 7, 10 Hz), 4.57 (d, 1 H, J ) 8 Hz),
4.59 (d, 1 H, J ) 11.5 Hz), 4.77 (d, 1 H, J ) 11.5 Hz), 5.74 (d, 1 H,
J ) 3.5 Hz) and 7.30-7.41 (m, 5 H); 13C NMR (CDCl3) δ 25.5, 26.6,
27.0, 27.3, 65.6, 72.6, 75.2, 77.9, 78.2, 78.5, 104.3, 110.0, 113.3, 128.4,
128.6, 128.8 and 137.9. Anal. Calcd for C19H26O6: C, 65.12; H, 7.48.
Found: C, 65.42; H, 7.56.
In similar fashion, the lack of effect of Mg2+ or Mn2+ on the
ligation reaction carried out in the presence of acceptor
oligonucleotide III has important implications for the role of
metal ions in DNA strand scission and ligation by topoisomerase
I. The ability of oligonucleotide II to undergo ligation with
quite reasonable efficiency reflects considerable flexibility in
the spatial requirements for religation, a parameter that may
3-O-Benzyl-1,2:5,6-tetra-O-acetyl-â-D-allofuranose (7). To a stirred
solution containing 4.03 g (11.5 mmol) of diacetonide 6 in 120 mL of
acetic acid was added 4.8 mL (69 mmol) of acetic anhydride, followed
(39) Christiansen, K.; Westergaard, O. J. Biol. Chem. 1994, 269, 721.
(40) Burgin Jr., A. B.; Huizenga, B. N.; Nash, H. A. Nucleic Acids Res.
1995, 23, 2973.
(41) Blackburn, G. M., Gait, M. J., Eds. Nucleic Acids in Chemistry and
Biology, 2nd ed.; Oxford: New York, 1996, pp 100-107.
(42) (a) Jay, E.; Seth, A. K.; Rommens, J.; Sood, A.; Jay, G. Nucleic
Acids Res. 1982, 10, 6319. (b) Banaszuk, A. M.; Deugau, K. V.; Sherwood,
J.; Michalak, M.; Glick, B. R. Anal. Biochem. 1983, 128, 281.

11710 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
by 200 mg (1.15 mmol) of p-toluenesulfonic acid monohydrate. The
reaction mixture was heated at reflux for 1 h and then the solvent was
coevaporated with two 100-mL portions of toluene under diminished
pressure. The residue was dissolved in 250 mL of ethyl acetate, washed
with 100 mL of saturated NaHCO3, and then dried over MgSO4. The
solution was concentrated under diminished pressure to afford a residue,
which was purified by flash chromatography on a silica gel column
(20 cm × 4 cm). Elution with 3:7 ethyl acetate-hexanes afforded 7
as a colorless solid: yield 4.90 g (97%) as a 9:1 mixture of anomers.
The diastereomeric ratio was determined according to the signals of
anomeric protons in the crude product. The major anomer was isolated
as colorless microcrystals from ethyl acetate-hexanes; mp 86 °C; silica
gel TLC Rf 0.50 (3:7 ethyl acetate-hexanes); 1H NMR (CDCl3) δ 2.03
(s, 3 H), 2.04 (s, 3 H), 2.09 (s, 3 H), 2.12 (s, 3 H), 4.05 (dd, 1 H, J )
6.5, 12 Hz), 4.19 (dd ) t, 1 H, J ) 7.5 Hz), 4.30 (dd, 1 H, J ) 5, 8
Hz), 4.36 (dd, 1 H, J ) 3, 9.5 Hz), 4.43 (d, 1 H, J ) 11 Hz), 4.58 (d,
1 H, J ) 11 Hz), 5.23-5.28 (m, 1 H), 5.30 (d, 1 H, J ) 4 Hz), 6.14
(s, 1 H) and 7.28-7.35 (m, 5 H); 13C NMR (CDCl3) δ 21.10, 21.12,
21.3, 21.4, 63.0, 71.8, 73.8, 78.7, 80.3, 98.8, 128.0, 128.5, 128.6, 128.9,
137.2, 169.2, 170.1, 170.4 and 170.9. Anal. Calcd for C21H26O10: C,
57.53; H, 5.98. Found: C, 57.47; H, 5.85.
133.7, 138.3, 143.9, 150.2, 151.7, 152.0 and 166.7; mass spectrum
(FABMS) m/z 492.1891 (M + H)+ (C25H26N5O6 requires 492.188).
N6-Benzoyl-9-[(2R,3R,4R,5R)-4-(benzoxy)-5-[(1R)-2-(tert-butyl-
diphenylsilyloxy)-1-(hydroxy)ethyl]-3-(hydroxy)tetrahydrofuran-2-
yl] adenine (10). tert-Butyldiphenylsilyl chloride (790 µL, 3.05 mmol)
was added dropwise to a stirred solution of 1.50 g (3.05 mmol) of triol
9 and 829 mg (12.2 mmol) of imidazole in 80 mL of CH2Cl2. The
combined solution was stirred at room temperature for 1 h, then diluted
with 150 mL of ethyl acetate. The reaction mixture was washed
successively with 50 mL of saturated NaHCO3 and 50 mL of brine,
then dried over MgSO4. The solution was concentrated under
diminished pressure to afford a residue which was purified by flash
chromatography on a silica gel column (20 cm × 3 cm). Elution with
9:1 CH2Cl2-methanol afforded 10 as a colorless foam: yield 2.17 g
(97%); mp 112 °C; silica gel TLC Rf 0.70 (9:1 CH2Cl2-methanol); 1H
NMR (CD3OD) δ 0.99 (s, 9 H), 3.59 (dd, 1 H, J ) 7, 10.5 Hz), 3.70
(dd, 1 H, J ) 5, 10 Hz), 4.01-4.06 (m, 1 H), 4.21 (d, 1 H, J ) 5 Hz),
4.50 (s, 1 H), 4.62 (d, 1 H, J ) 12 Hz), 4.67 (d, 1 H, J ) 12 Hz), 4.92
(t, 1 H, J ) 6 Hz), 6.09 (d, 1 H, J ) 7 Hz) 7.12-7.71 (m, 18 H), 7.98
(d, 2 H, J ) 7.5 Hz), 8.42 (s, 1 H) and 8.51 (s, 1 H); 13C NMR (CD3-
OD) δ 19.1, 26.6, 64.9, 72.1, 72.2, 74.4, 77.0, 85.5, 89.6, 124.3, 127.6,
127.9, 127.99, 128.5, 128.7, 129.5, 130.0, 132.2, 132.9, 133.3, 133.4,
133.8, 135.0, 135.7, 138.2, 143.9, 150.2, 151.7, 151.9 and 166.7; mass
spectrum (FABMS) m/z 730.308 (M + H)+ (C41H44N5O6Si requires
730.306).
N6-Benzoyl-9-[(2R,3R,4R,5R)-4-(benzoxy)-5-[(1R)-2-[(tert-butyl-
diphenylsilyl)oxy]-1-[(imidazolethiocarbonyl)oxy]ethyl]-3-[(imida-
zolethiocarbonyl)oxy]tetrahydrofuran-2-yl]adenine (11). Thiocar-
bonyldiimidazole (2.0 g, 10.0 mmol) was added quickly to a stirred
solution containing 2.17 g (2.97 mmol) of 10 in 75 mL of THF. The
reaction mixture was stirred at 65 °C for 12 h in the dark, then the
solution was concentrated under diminished pressure. The resulting
residue was dissolved in 200 mL of 5:1 ethyl acetate-CH2Cl2. The
solution was washed successively with cold 1 N HCl (50 mL), saturated
NaHCO3 (75 mL) and brine (75 mL), then dried over MgSO4. The
solution was concentrated under diminished pressure to afford a residue
which was purified by flash chromatography on a silica gel column
(25 cm × 3 cm). Elution with ethyl acetate followed by 9:1 CH2Cl2-
methanol afforded 11 as a colorless powder: yield 2.52 g (90%); silica
gel TLC Rf 0.60 (9:1 CH2Cl2-methanol); 1H NMR (CDCl3) δ 1.04 (s,
9 H), 3.94 (dd, 1 H, J ) 5.5, 10.5 Hz), 4.00 (dd, 1 H, J ) 5.5, 10.5
Hz), 4.50 (d, 1 H, J ) 11.5 Hz), 4.56 (d, 1 H, J ) 11.5 Hz), 4.74 (t,
1 H, J ) 5.5 Hz), 5.21 (t, 1 H, J ) 6 Hz), 6.21 (d, 1 H, J ) 4 Hz),
6.24 (t, 1 H, J ) 6 Hz), 6.45 (dd, 1 H, J ) 4, 5 Hz), 7.02-7.63 (m, 22
H), 7.98 (s, 1 H), 8.01 (m, 2 H), 8.26 (s, 1 H), 8.33 (s, 1 H), 8.50 (s,
1 H) and 8.80 (s, 1 H); 13C NMR (CDCl3) δ 18.6, 26.3, 61.0, 72.9,
75.0, 79.9, 80.1, 81.0, 86.0, 117.4, 123.9, 127.3, 127.7, 128.0, 128.1,
129.5, 130.3, 130.7, 131.9, 132.1, 133.1, 134.9, 135.1, 136.6, 136.8,
142.6, 149.9, 150.8, 151.9, 165.0, 182.0 and 182.7. Anal. Calcd for
C49H47O9N9SiS2: C, 61.93; H, 4.99. Found: C, 61.71; H, 5.18.
N6-Benzoyl-9-[(2R,4S,5R)-4-(benzoxy)-5-[(tert-butyldiphenylsi-
lyloxy)ethyl]tetrahydrofuran-2-yl]adenine (12). To a stirred solution
containing 804 µL (2.98 mmol) of Bu3SnH and 25 mg (0.15 mmol) of
azobisisobutyronitrile (AIBN) in 50 mL of toluene at 75 °C was added
dropwise a solution of 951 mg (1.00 mmol) of 11 in 30 mL of toluene.
The combined solution was stirred at 75 °C for 90 min and then
concentrated under diminished pressure to afford a residue, which was
purified by flash chromatography on a silica gel column (20 cm × 2
cm). Elution with ethyl acetate afforded 12 as a colorless foam: yield
560 mg (80%); mp 58-61 °C; silica gel TLC Rf 0.40 (3:1 ethyl acetate-
hexanes); 1H NMR (CDCl3) δ 1.05 (s, 9 H), 1.91-1.97 (m, 2 H), 2.63
(ddd, 1 H, J ) 3, 6, 9 Hz), 2.85 (dt, 1 H, J ) 6, 13.5 Hz), 3.78 (t, 2
H, J ) 6 Hz), 4.21-4.25 (m, 1 H), 4.46 (m, 1 H), 4.57 (d, 1 H, J )
10.5 Hz), 4.68 (d, 1 H, J ) 10.5 Hz), 6.33 (t, 1 H, J ) 6.5 Hz), 7.50
(m, 18 H), 7.99 (m, 2 H), 8.03 (s, 1 H), 8.77 (s, 1 H) and 8.91 (s, 1 H);
13C NMR (CDCl3) δ 19.6, 27.4, 37.5, 61.0, 66.2, 71.8, 82.2, 82.5, 85.4,
124.8, 127.1, 128.1, 128.2, 128.3, 128.5, 128.9, 130.2, 132.9, 134.1,
135.9, 138.1, 142.4, 150.4, 152.1, 152.6 and 165.9; mass spectrum
(FABMS) m/z 698.315 (M + H)+ (C41H44N5O4Si requires 698.316).
N6-Benzoyl-9-[(2R,4S,5R)-4-(benzoxy)-5-(2-hydroxyethyl)tetrahy-
drofuran-2-yl]adenine (13). To a solution containing 512 mg (0.73
mmol) of 12 in 25 mL of THF was added 800 µL (0.80 mmol) of a
N6-Benzoyl-9-[(2R,3R,4R,5R)-3-(acetoxy)-4-(benzoxy)-5-[(1R)-1,2-
(diacetoxy)ethyl]tetrahydrofuran-2-yl]adenine (8). Trimethylsilyl
trifluoromethanesulfonate (540 µL, 2.70 mmol) was added to a stirred
solution containing 4.90 g (11.2 mmol) of tetraacetate 7 and 4.30 g
(11.2 mmol) of bis-TMS-N6-benzoyladenine in 200 mL of CH3CN at
room temperature. The combined solution was heated at 55 °C for 12
h. After cooling to 0 °C, the reaction was quenched by the addition of
50 mL of saturated NaHCO3 followed by the addition of 250 mL of
ethyl acetate. The organic layer was separated, washed successively
with two 100-mL portions of saturated NaHCO3 and two 100-mL
portions of brine, then dried over MgSO4. The solution was concen-
trated under diminished pressure to afford a residue which was purified
by flash chromatography on a silica gel column (25 cm × 4 cm). Elution
with 9:1 CH2Cl2-methanol afforded nucleoside 8 as a colorless foam:
yield 4.91 g (71%); mp 69 °C; silica gel TLC Rf 0.70 (9:1 CH2Cl2-
methanol); 1H NMR (CDCl3) δ 1.97 (s, 3 H), 2.04 (s, 3 H), 2.11 (s, 3
H), 4.05 (dd, 1 H, J ) 6, 12 Hz), 4.32 (t, 1 H, J ) 6 Hz), 4.38 (dd, 1
H, J ) 3.5, 12 Hz), 4.62 (d, 1 H, J ) 12.5 Hz), 4.65 (d, 1 H, J ) 12.5
Hz), 4.82 (t, 1 H, J ) 5.5 Hz), 5.43-5.48 (m, 1 H), 5.95 (t, 1 H, J )
4 Hz), 6.09 (d, 1 H, J ) 4 Hz), 7.30-7.40 (m, 5 H), 7.50-7.65 (m, 3
H), 8.01-8.04 (m, 3 H), 8.02 (s, 1 H) and 8.91 (s, 1 H); 13C NMR
(CDCl3) δ 21.0, 21.1, 21.2, 62.5, 70.6, 73.6, 76.7, 81.2, 88.1, 124.4,
128.3, 128.6, 128.7, 128.9, 129.2, 133.2, 133.9, 137.2, 142.8, 150.3,
151.8, 153.1, 165.2, 170.1, 170.2 and 170.8; mass spectrum (FABMS)
m/z 618.218 (M + H)+ (C31H32N5O9 requires 618.220).
N6-Benzoyl-9-[(2R,3R,4R,5R)-4-(benzoxy)-5-[(1R)-1,2-(dihy-
droxy)ethyl]-3-(hydroxy)tetrahydrofuran-2-yl]adenine (9). A solu-
tion of 4.91 g (7.96 mmol) of nucleoside analogue 8 in 18 mL of 2:1
ethanol-pyridine was treated with 6 mL of 2 N NaOH in 6 mL of
ethanol at room temperature. The combined solution was stirred at
room temperature for 10 min, then cooled to 0 °C. The solution was
adjusted to pH 7 with 1 N HCl, then extracted with 250 mL of ethyl
acetate. The organic layer was washed successively with 100 mL of
saturated NaHCO3 and 100 mL of brine, then dried over MgSO4. The
solution was concentrated under diminished pressure to afford a residue,
which was purified by flash chromatography on a silica gel column
(20 cm × 3 cm). Elution with 9:1 CH2Cl2-methanol afforded
nucleoside 9 as a white foam: yield 3.36 g (86%); mp 110 °C; silica
1
gel TLC Rf 0.50 (9:1 CH2Cl2-methanol); H NMR (CD3OD) δ 2.54
(d, 2 H, J ) 6 Hz), 3.92-3.97 (m, 1 H), 4.23 (d, 1 H, J ) 5 Hz), 4.30
(s, 1 H), 4.63 (d, 1 H, J ) 7.5 Hz), 4.80 (d, 1 H, J ) 7.5 Hz), 4.91 (d,
1 H, J ) 6 Hz), 6.07 (d, 1 H, J ) 7 Hz), 7.33 (m, 8 H), 7.97 (d, 2 H,
J ) 7 Hz), 8.43 (s, 1 H) and 8.54 (s, 1 H); 13C NMR (CD3OD) δ 62.9,
72.2, 72.4, 74.2, 77.3, 85.5, 89.6, 124.2, 127.7, 128.5, 128.7, 132.9,
(43) Maxam, A. M., Gilbert, W. Methods Enzymol. 1980, 65, 499.
(44) Wang, L.-K.; Johnson, R. K.; Hecht, S. M. Chem. Res. Toxicol.
1993, 6, 813.
(45) Schmitt, B.; Buhre, U.; Vosberg, H.-P. Eur. J. Biochem. 1984, 144,
127.
(46) Sambrook, J.; Fritsh, E.; Maniatis, T. In Molecular Cloning: A
Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, 1989, pp 11.31-11.32.

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11711
1M solution of tetrabutylammonium fluoride in THF. The reaction
mixture was stirred at room temperature for 5 h. The solution was
concentrated under diminished pressure to afford a residue, which was
purified by flash chromatography on a silica gel column (25 cm × 3
cm). Elution with ethyl acetate, followed by 9:1 CH2Cl2-methanol,
afforded nucleoside analogue 13 as a colorless foam: yield 254 mg
(76%); mp 115-117 °C; silica gel TLC Rf 0.70 (9:1 CH2Cl2-methanol);
1H NMR (CDCl3) δ 1.96-2.03 (m, 2 H), 2.42 (s, 1 H), 2.59 (ddd, 1 H,
J ) 3.5, 6.5, 13.5 Hz), 2.91-2.99 (m, 1 H), 3.72-3.83 (m, 2 H), 4.29-
4.37 (m, 2 H), 4.58 (d, 1 H, J ) 11.5 Hz), 4.62 (d, 1 H, J ) 11.5 Hz),
6.36 (t, 1 H, J ) 6.5 Hz), 7.29-7.37 (m, 5 H), 7.51-7.63 (m, 3 H),
8.01 (d, 2 H, J ) 7 Hz), 8.10 (s, 1 H), 8.77 (s, 1 H) and 8.99 (s, 1 H);
13C NMR (CDCl3) δ 36.7, 37.4, 59.5, 72.0, 82.1, 83.2, 85.3, 124.1,
128.1, 128.2, 128.3, 128.4, 128.9, 129.0, 133.0, 134.0, 137.8, 142.3,
150.1, 151.7, 152.7 and 165.7; mass spectrum (FABMS) m/z 458.183
(M + H)+ (C25H26N5O4 requires 458.183).
N6-Benzoyl-9-[(2R,4S,5R)-4-(hydroxy)-5-(2-hydroxyethyl)tetrahy-
drofuran-2-yl]adenine (14). A solution containing 80 mg (0.17 mmol)
of nucleoside 13 and freshly prepared palladium black (prepared18 from
420 mg of PdCl2) in 17 mL of dry ethanol was stirred under 1 atm of
H2 at 45 °C for 9 h. The solution was decanted and the catalyst was
washed with 20 mL of ethanol. The combined solution was concen-
trated under diminished pressure to afford a residue that was purified
by flash chromatography on a silica gel column (15 cm × 3 cm). Elution
with 9:1 CH2Cl2-methanol afforded 14 as a colorless powder: yield
62 mg (83%); mp 142-145 °C; silica gel TLC Rf 0.40 (9:1 CH2Cl2-
methanol); 1H NMR (CD3OD) δ 1.91 (m, 2 H), 2.48 (ddd, 1 H, J ) 5,
6.5, 11.5 Hz), 2.94 (d, t, 1 H, J ) 6.5, 13 Hz), 3.61-3.70 (m, 2 H),
4.05 (m, 1 H), 4.47 (m, 1 H), 6.48 (t, 1 H, J ) 6.5 Hz), 7.51-7.65 (m,
3 H), 8.06 (d, 2 H, J ) 7 Hz), 8.50 (s, 1 H) and 8.69 (s, 1 H); 13C
NMR (CDCl3) δ 36.5, 39.2, 58.8, 74.5, 84.7, 124.4, 128.4, 128.5, 128.7,
132.8, 133.9, 143.4, 150.0, 152.1 and 167.1; mass spectrum (FABMS)
m/z 370.149 (M + H)+ (C18H20N5O4 requires 370.151).
(m, 1 H), 6.37 (t, 1 H, J ) 6.5 Hz), 6.79 (dd, 4 H, J ) 3, 9 Hz),
7.16-7.64 (m, 12 H), 7.97-8.03 (m, 3 H), 8.77 (s, 0.5 H), 8.79 (s, 0.5
H) and 8.96 (s, 1 H); mass spectrum (FABMS) m/z 872.390 (M + H)+
(C48H55N7O7P requires 872.390).
N6-Benzoyl-3′-O-benzyladenosine (15). Sodium periodate (120 mg,
0.56 mmol) was added to a stirred solution containing 200 mg (0.41
mmol) of nucleoside analogue 9 in 10 mL of 9:1 THF-H2O. The
suspension was stirred vigorously at room temperature for 2 h, then
concentrated under diminished pressure. The residue was dissolved
in 25 mL of methanol and filtered. To the filtrate was added 200 mg
(5.28 mmol) of NaBH4 at 0 °C. The solution was stirred at room
temperature for 30 min and then cooled to 0 °C. The solution was
adjusted to pH 7 with 1 N HCl, then extracted with 150 mL of ethyl
acetate. The organic layer was separated, washed successively with
50 mL of saturated NaHCO3 and 50 mL brine, then dried over MgSO4.
The dried solution was concentrated under diminished pressure to afford
a residue which was purified by flash chromatography on a silica gel
column (15 cm × 3 cm). Elution with 9:1 CH2Cl2-methanol afforded
diol 15 as a colorless foam: yield 146 mg (83%); mp 200 °C; silica
1
gel TLC Rf 0.60 (9:1 CH2Cl2-methanol); H NMR (CD3OD) δ 3.58
(dd, 1 H, J ) 1.5, 13 Hz), 3.88 (dd, 1 H, J ) 1.5, 13 Hz), 4.22 (dd, 1
H, J ) 1.5, 5 Hz), 4.30 (d, 1 H, J ) 1.5 Hz), 4.65 (d, 1 H, J ) 11.5
Hz), 4.71 (d, 1 H, J ) 11.5 Hz), 4.83 (dd, 1 H, J ) 6, 7 Hz), 5.88 (d,
1 H, J ) 7 Hz), 7.30 (m, 5 H), 7.43-7.57 (m, 3 H), 8.00 (m, 2 H),
8.15 (s, 1 H) and 8.67 (s, 1 H). Anal. Calcd for C24H23O5N5: C, 62.46;
H, 5.02. Found: C, 62.09; H, 4.98.
N6-Benzoyladenosine (16). A suspension of palladium black
(freshly prepared18 from 500 mg of PdCl2) and 110 mg (0.24 mmol)
of nucleoside 15 in 10 mL of ethanol was stirred under 1 atm of H2 at
45 °C for 9 h. The solution was removed by decatenation and the
catalyst was washed with 20 mL of ethanol. The combined solution
was concentrated under diminished pressure to afford a residue which
was purified by flash chromatography on a silica gel column (20 cm
× 3 cm). Elution with 9:1 CH2Cl2-methanol afforded N6-benzoylad-
enosine (16) as a colorless powder: yield 70 mg (68%); silica gel TLC
Rf 0.50 (9:1 CH2Cl2-methanol); [R]D -48.0° (c 0.5, MeOH) (authentic
sample [R]D -47.8° (c 0.5, MeOH)); 1H NMR (CD3OD) δ 3.75 (dd,1
H, J ) 3, 12 Hz), 3.88 (dd, 1 H, J ) 3, 13 Hz), 4.15-4.17 (m, 1 H),
4.34 (dd, 1 H, J ) 3, 3.5 Hz), 4.73 (t, 1 H, J ) 5.5 Hz), 6.12 (d, 1 H,
J ) 6 Hz), 7.52-7.65 (m, 3 H), 8.05-8.07 (m, 2 H), 8.65 (s, 1 H) and
8.69 (s, 1 H).
N6-Benzoyl-9-[(2R,4S,5R)-5-[2-(4,4′-dimethoxytriphenylmethoxy)-
ethyl]-4-(hydroxy)tetrahydrofuran-2-yl]adenine (2a). To a solution
containing 35 mg (0.095 mmol) of diol 14 in 1 mL of pyridine at 0 °C
was added slowly 35.8 mg (0.10 mmol) of dimethoxytrityl chloride in
0.5 mL of pyridine. The combined solution was stirred at 0 °C for 1
h, then extracted with a mixture of saturated NaHCO3 solution (25 mL)
and ethyl acetate (100 mL). The organic layer was washed with 25
mL of brine, then dried over MgSO4. The dried solution was
concentrated under diminished pressure to afford a residue which was
purified by flash chromatography on a silica gel column (20 cm × 3
cm). Elution with 9:1 CH2Cl2-methanol afforded 2a as a colorless
powder: yield 52 mg (81%); mp 113-115 °C; silica gel TLC Rf 0.50
(9:1 CH2Cl2-methanol); 1H NMR (CDCl3) δ 1.97-2.08 (m, 2 H), 2.58
(dt, 1 H, J ) 7, 13 Hz), 2.89 (ddd, 1 H, J ) 4, 7, 11.5 Hz), 3.15 (dd,
1 H, J ) 4, 9 Hz), 3.21 (d, 1 H, J ) 2.5 Hz), 3.41-3.47 (m, 1 H), 3.78
(s, 3 H), 3.79 (s, 3 H), 3.91-3.97 (m, 1 H), 4.54-4.60 (m, 1 H), 6.48
(dd, 1 H, J ) 4, 7 Hz), 6.82-6.86 (m, 4 H), 7.19-7.64 (m, 12 H),
5′-[(Acetyl)thio]-2′,5′-dideoxyadenosine (18). A solution contain-
ing 1.05 g (3.89 mmol) of 2′-deoxyadenosine (17) and 1.3 g (4.95
mmol) of Ph3P in 45 mL of THF was treated successively with 779
µL (4.05 mmol) of diethyl azodicarboxylate and 353 µL (4.95 mmol)
of thiolacetic acid. The reaction mixture was stirred at room temper-
ature for 1 h and then concentrated under diminished pressure to afford
a residue. The residue was purified by flash chromatography on a silica
gel column (25 cm × 3 cm). Elution with 9:1 CH2Cl2-methanol
afforded 18 as a colorless foam: yield 1.06 g (88%); mp 74-75 °C;
8.01-8.04 (m, 2 H), 8.10 (s, 1 H), 8.80 (s, 1 H) and 8.94 (s, 1 H); 13
C
1
silica gel TLC Rf 0.30 (9:1 CH2Cl2-methanol); H NMR (CD3OD) δ
NMR (CDCl3) δ 32.0, 34.4, 40.1, 55.6, 61.1, 74.7, 84.5, 86.2, 87.4,
113.7, 124.0, 127.3, 128.3, 128.4, 129.0, 130.2, 130.3, 133.1, 136.0,
136.3, 141.7, 144.9, 153.0, 158.0, and 166.2; mass spectrum (FABMS)
m/z 672.284 (M + H)+ (C39H38N5O6 requires 672.282).
2.38 (s, 3 H), 2.50-2.60 (m, 1 H), 2.88-2.96 (m, 1 H), 3.27 (dd, 1 H,
J ) 6, 12.5 Hz), 3.38 (dd, 1 H, J ) 6, 12.5 Hz), 4.10-4.18 (m, 1 H),
4.45-4.55 (m, 1 H), 5.80 (br s, 2 H), 6.40 (t, 1 H, J ) 6 Hz), 8.02 (s,
1 H) and 8.35 (s, 1 H); 13C NMR (CD3OD) δ 29.4, 31.4, 39.0, 73.6,
84.8, 86.1, 119.6, 140.1, 149.4, 152.8, 156.3 and 195.6; mass spectrum
(FABMS) m/z 310.098 (M + H)+ (C12H16N5O3S requires 310.098).
5′-[(Acetyl)thio]-2′,5′-dideoxy-3′-O,N6,N6-tribenzoyl)adenosine(19).
To a stirred solution containing 800 mg (2.58 mmol) of thioester 18 in
15 mL of pyridine was added 1.19 mL (10.2 mmol) of benzoyl chloride
at 0 °C. The combined solution was stirred at room temperature for
12 h, then diluted with 200 mL of ethyl acetate. The reaction mixture
was washed successively with 100 mL of saturated NaHCO3 and 100
mL of brine, then dried over MgSO4. The dried solution was
concentrated under diminished pressure to afford a residue which was
purified by flash chromatography on a silica gel column (20 cm × 4
cm). Elution with 2:1 ethyl acetate-hexanes afforded 19 as a colorless
foam: yield 1.48 g (92%); mp 97-101 °C; silica gel TLC Rf 0.65 (2:1
ethyl acetate-hexanes); 1H NMR (CDCl3) δ 2.36 (s, 3 H), 2.74 (dd, 1
H, J ) 2, 6 Hz), 3.14-3.24 (m, 1 H), 3.39 (dd, 1 H, J ) 6, 12 Hz),
3.47 (dd, 1 H, J ) 6, 14 Hz), 4.45-4.50 (m, 1 H), 5.55-5.58 (m, 1
H), 6.51 (dd, 1 H, J ) 6, 8 Hz), 7.34-7.64 (m, 9 H), 7.86 (d, 4 H, J
N6-Benzoyl-9-[(2R,4S,5S)-4-[(2-cyanoethyl N,N-diisopropylphos-
phoramidityl)oxy]-5-[2-(4,4′-dimethoxytriphenylmethoxy)ethyl]-
tetrahydrofuran-2-yl]adenine (2b). To a stirred solution containing
60 mg (0.089 mmol) of 2a in 4 mL of CH2Cl2 was added successively
at room temperature 40 µL (0.23 mmol) of N,N-diisopropylethylamine
and 19.8 µL (0.089 mmol) of 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite. The reaction mixture was stirred at room temperature
for 1 h, then diluted with 50 mL of ethyl acetate. The solution was
washed with 15 mL of saturated NaHCO3, dried over MgSO4, and then
concentrated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (15 cm × 3 cm). Elution
with 45:45:10 CH2Cl2-ethyl acetate-Et3N afforded monomer 2b as a
colorless foam: yield 61 mg (77%) of a 1:1 mixture of diastereomers;
silica gel TLC Rf 0.70 (45:45:10 CH2Cl2-ethyl acetate-Et3N); 1H NMR
(CDCl3) δ 1.21-1.25 (m, 12 H), 1.87-2.11 (m, 2 H), 2.53-2.70 (m,
3 H), 2.85-2.93 (m, 1 H), 3.18-3.24 (m, 2 H), 3.52-3.71 (m, 3.5 H),
3.77 (s, 6 H), 3.80-3.88 (m, 0.5 H), 4.31-4.37 (m, 1 H), 4.59-4.62

11712 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
) 7 Hz), 8.07 (d, 2 H, J ) 7 Hz), 8.29 (s, 1 H) and 8.66 (s, 1 H); 13
C
was concentrated under diminished pressure to afford a residue which
was purified by flash chromatography on a silica gel column (20 cm
× 3 cm). Elution with 3:1 ethyl acetate-hexanes afforded 22 as a
white powder: yield 1.09 g (78%); mp 102-105 °C; silica gel TLC Rf
NMR (CDCl3) δ 30.9, 31.8, 76.9, 84.0, 85.3, 128.4, 129.0, 129.1, 129.4,
129.8, 130.1, 133.4, 134.0, 144.0, 152.0, 152.4, 153.0, 166.0, 172.7
and 194.9; Anal. Calcd for C33H27O6N5S: C, 63.75; H, 4.38. Found:
C, 63.61; H, 4.48.
1
0.60 (3:1 ethyl acetate-hexanes); H NMR (CDCl3) δ 1.13 (s, 9 H),
N6-Benzoyl-2′,5′-dideoxy-5′-thioadenosine (20). A solution con-
taining 900 mg (1.45 mmol) of nucleoside 19 in 10 mL of 2:1 EtOH-
pyridine was treated under argon with 2.17 mL of 2 N NaOH and 2
mL of EtOH. The solution was degassed by stirring under vacuum
for 2 min, then stirred for an additional 5 min at room temperature
under argon. The solution was adjusted to pH 7 with 1 N HCl and
extracted with a mixture of saturated NaHCO3 solution (50 mL) and
ethyl acetate (250 mL). The organic layer was dried over MgSO4.
The solvent was concentrated under diminished pressure to afford a
residue, which was purified by flash chromatography on a silica gel
column (25 cm × 3 cm). Elution with 9:1 CH2Cl2-methanol afforded
20 as a colorless foam: yield 484 mg (90%); mp 87-90 °C; silica gel
2.41-2.50 (m, 1 H), 2.52-2.65 (m, 1 H), 3.00 (dd, 1 H, J ) 4.5, 10.5
Hz), 3.07 (dd, 1 H, J ) 4.5, 10.5 Hz), 3.72 (s, 6 H), 4.22-4.28 (m, 1
H), 4.60-4.66 (m, 1 H), 6.52 (t, 1 H, J ) 6 Hz), 6.68 (d, 4 H, J ) 9
Hz), 7.08-7.74 (m, 22 H), 7.98 (d, 2 H, J ) 10 Hz), 8.02 (s, 1 H),
8.70 (s, 1 H) and 8.82 (s, 1 H); mass spectrum (FABMS) m/z 896.381
(M + H)+ (C54H54N5O6Si requires 896.385).
N6-Benzoyl-3′-O-(tert-butyldiphenylsilyl)-2′-deoxyadenosine (23).
A solution containing 1.06 g (1.18 mmol) of nucleoside 22 in 30 mL
of 80% acetic acid was stirred at room temperature for 15 min. The
solution was adjusted to pH 7 with saturated Na2CO3 solution, then
diluted with 250 mL of ethyl acetate. The reaction mixture was washed
successively with 50 mL of saturated NaHCO3 solution and 50 mL of
brine and was then dried over MgSO4. The dried solution was
concentrated under diminished pressure to afford a residue which was
purified by flash chromatography on a silica gel column (20 cm × 3
cm). Elution with 3:1 ethyl acetate-hexanes afforded 23 as a colorless
foam: yield 538 mg (77%); mp 98-100 °C; silica gel TLC Rf 0.30
1
TLC Rf 0.40 (9:1 CH2Cl2-methanol); H NMR (CDCl3) δ 1.60 (t, 1
H, J ) 7.5 Hz), 2.40 (br s, 1 H), 2.52-2.62 (m, 1 H), 2.82 (m, 2 H),
3.03-3.12 (m, 1 H), 4.08-4.14 (m, 1 H), 4.73 (m, 1 H), 6.45 (t, 1 H,
J ) 6 Hz), 7.48-7.62 (m, 3 H), 7.98-8.04 (m, 2 H), 8.20 (s, 1 H),
8.80 (s, 1 H) and 8.92 (s, br, 1 H); 13C NMR (CDCl3) δ 27.3, 40.1,
73.4, 85.0, 87.9, 123.9, 128.3, 129.2, 133.3, 133.9, 149.8, 151.8, 152.9
and 165.5; mass spectrum (FABMS) m/z 372.113 (M + H)+
(C17H18N5O3S requires 372.113).
1
(3:1 ethyl acetate-hexanes); H NMR (CDCl3) δ 1.15 (s, 9 H), 2.31
(dd, 1 H, J ) 5, 13 Hz), 2.89-2.99 (m, 1 H), 3.71-3.75 (m, 2 H),
4.16 (s, 1 H), 4.53 (d, 1 H, J ) 5 Hz), 6.42 (dd, 1 H, J ) 5.5, 11.5
Hz), 7.37-7.73 (m, 13 H), 7.99-8.02 (m, 2 H), 8.08 (s, 1 H), 8.72 (s,
1 H) and 8.93 (s, 1 H); 13C NMR (CDCl3) δ 19.5, 27.4, 41.5, 63.2,
75.2, 88.0, 90.4, 124.9, 128.3, 129.1, 130.5, 133.1, 133.5, 133.6, 133.9,
136.1, 142.9, 150.6, 151.2, 152.3 and 165.3; mass spectrum (FABMS)
m/z 594.255 (M + H)+ (C33H35N5O4Si requires 594.254).
N6-Benzoyl-2′,5′-dideoxy-5′-[(4,4′-dimethoxytriphenylmethyl)thio]-
adenosine (3a). To a solution containing 300 mg (0.80 mmol) of 20
in 5 mL of pyridine was added 272 mg (0.80 mmol) of dimethoxytrityl
chloride in 2 mL of pyridine at 0 °C. The combined solution was
stirred for 1 h and was then concentrated under diminished pressure to
afford a residue which was purified by flash chromatography on a silica
gel column (20 cm × 3 cm). Elution with 95:5 CH2Cl2-methanol
afforded 3a as a colorless foam: yield 490 mg (91%); mp 120-125
°C; silica gel TLC Rf 0.50 (95:5 CH2Cl2-methanol); 1H NMR (CDCl3)
δ 1.77 (br s, 1 H), 2.38-2.50 (m, 1 H), 2.58 (dd, 1 H, J ) 6, 12 Hz),
2.69 (dd, 1 H, J ) 6, 12 Hz), 2.78-2.86 (m, 1 H), 3.74 (s, 6 H), 3.86
(m, 1 H), 4.33-4.40 (m, 1 H), 6.43 (t, 1 H, J ) 7 Hz), 6.78 (d, 4 H,
J ) 10.5 Hz), 7.18-7.40 (m, 9 H), 7.48-7.63 (m, 3 H), 8.00 (d, 2 H,
J ) 9 Hz), 8.09 (s, 1 H), 8.72 (s, 1 H) and 8.85 (s, 1 H); 13C NMR
(CDCl3) δ 35.1, 39.7, 55.6, 66.6, 74.1, 85.2, 86.2, 113.6, 123.9, 127.2,
128.3, 129.2, 129.7, 131.0, 133.2, 134.0, 137.1, 142.2, 145.4, 152.9,
158.6 and 165.1; mass spectrum (FABMS) m/z 674.244 (M + H)+
(C38H36N5O5S requires 674.244).
N6-Benzoyl-3′-O-(tert-butyldiphenylsilyl)-2′-deoxy-5′-O-(p-tolu-
enesulfonyl)adenosine (24). To a solution containing 180 mg (0.30
mmol) of nucleoside 23 in 2 mL of pyridine was added 68.4 mg (0.36
mmol) of p-toluensulfonyl chloride in 0.50 mL of pyridine at 0 °C.
The solution was stirred at room temperature for 3 h, then diluted with
250 mL of ethyl acetate. The reaction mixture was washed successively
with 50 mL of saturated NaHCO3 and 50 mL of brine, then dried over
MgSO4. The dried solution was concentrated under diminished pressure
to afford a residue, which was purified by flash chromatography on a
silica gel column (25 cm × 3 cm). Elution with 95:5 CH2Cl2-methanol
afforded tosylate 24 as a colorless foam: yield 190 mg (85%); mp
83-85 °C; silica gel TLC Rf 0.50 (95:5 CH2Cl2-methanol); 1H NMR
(CDCl3) δ 1.09 (s, 9 H), 2.37 (s, 3 H), 2.41-2.47 (m, 1 H), 2.56-2.58
(m, 1 H), 3.68 (dd, 1 H, J ) 4, 12 Hz), 3.90 (dd, 1 H, J ) 4, 12.5 Hz),
4.16 (s, 1 H), 4.52-4.60 (m, 1 H), 6.48 (dd, 1 H, J ) 1.5, 6.5 Hz),
7.19 (d, 2 H, J ) 9 Hz), 7.33-7.62 (m, 15 H), 7.72-7.86 (m, 2 H),
8.09 (s, 1 H) and 8.50 (s, 1 H); 13C NMR (CDCl3) δ 19.4, 22.0, 27.3,
40.5, 69.1, 74.1, 85.2, 85.5, 123.8, 128.2, 128.4, 128.5, 129.3, 130.2,
130.6, 130.7, 132.7, 133.1, 133.2, 134.0, 136.0, 136.1, 141.9, 145.4,
149.9, 152.9 and 164.9; mass spectrum (FABMS) m/z 748.265 (M +
H)+ (C40H41N5O6SiS requires 748.262).
5′-Azido-N6-benzoyl-3′-O-(tert-butyldiphenylsilyl)-2′,5′-dideoxy-
adenosine (25). A solution containing 170 mg (0.22 mmol) of tosylate
24 and 52 mg (0.80 mmol) of NaN3 in 5 mL of DMF was stirred at
100 °C for 15 min. The solution was cooled to room temperature and
diluted with 200 mL of ethyl acetate. The reaction mixture was washed
with 50 mL of saturated NaHCO3 solution and then dried over MgSO4.
The solution was concentrated under diminished pressure and the
residue was coevaporated with portions of methanol. Precipitation of
the product from ethyl acetate-hexanes afforded 25 as a white foam:
yield 130 mg (95%); mp 112-115 °C; silica gel TLC Rf 0.70 (3:1
ethyl acetate-hexanes); 1H NMR (CDCl3) δ 1.13 (s, 9 H), 2.55-2.63
(m, 1 H), 2.69-2.81 (m, 1 H), 3.10 (dd, 1 H, J ) 3, 13 Hz), 3.33 (dd,
1 H, J ) 5, 13 Hz), 4.18 (s, 1 H), 4.61 (s, 1 H), 6.55 (t, 1 H, J ) 6
Hz), 7.40-7.78 (m, 13 H), 8.03-8.12 (m, 2 H) 8.22 (s, 1 H), 8.80 (s,
1 H) and 9.50 (s, 1 H); 13C NMR (CDCl3) δ 19.4, 27.3, 40.6, 52.2,
74.0, 85.0, 86.3, 124.1, 128.3, 128.4, 129.1, 130.6, 130.7, 133.0, 133.2,
133.3, 134.0, 136.1, 142.1, 150.1, 151.9, 152.9 and 165.3; mass
spectrum FABMS m/z 619.257 (M + H)+ (C33H35N8O3Si requires
619.260).
N6-Benzoyl-2′,5′-dideoxy-5′-[(4,4′-dimethoxytriphenylmethyl)thio]-
adenosine 3′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (3b).
To a stirred solution containing 60 mg (0.089 mmol) of 3a in 1 mL of
CH2Cl2 was added successively at room temperature 45 µL (0.23 mmol)
of N,N-diisopropylethylamine and 19.8 µL (0.089 mmol) of 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite. The reaction mixture was
stirred at room temperature for 1 h and then diluted with 100 mL of
ethyl acetate. The solution was washed with 10 mL of saturated
NaHCO3 solution and dried over MgSO4. The dried solution was
concentrated under diminished pressure to afford a residue which was
purified by flash chromatography on a silica gel column (15 cm × 2
cm). Elution with 45:45:10 CH2Cl2-ethyl acetate-Et3N afforded
monomer 3b as a colorless foam: yield 68 mg (86%) of a 1:1 mixture
of diastereomers; silica gel TLC Rf 0.70 (45:45:10 CH2Cl2-ethyl
1
acetate-Et3N); H NMR (CDCl3) δ 1.14-1.19 (m, 12 H), 2.48-2.67
(m, 5 H), 2.82-2.91 (m, 1 H), 3.52-3.74 (m, 4 H), 3.77 (s, 3 H), 3.78
(s, 3 H), 4.10-4.18 (m, 1 H), 4.49-4.56 (m, 1 H), 6.31-6.40 (m, 1
H), 6.78 (d, 2 H, J ) 4 Hz), 6.79 (d, 2 H, J ) 4 Hz), 7.16-7.19 (m,
9 H), 7.48-7.63 (m, 3 H), 8.02 (d, 2 H, J ) 7 Hz), 8.16 (s, 1 H), 8.72
(s, 1 H) and 8.87 (s, 1 H); mass spectrum (FABMS) m/z 874.351 (M
+ H)+ (C47H53N7O6PS requires 874.352).
N6-Benzoyl-3′-O-(tert-butyldiphenylsilyl)-5′-O-(4,4′-dimethoxy-
triphenylmethyl)-2′-deoxyadenosine (22). To a solution containing
1.0 g (1.52 mmol) of nucleoside 21 and 155 mg (2.28 mmol) of
imidazole in 15 mL of CH2Cl2 was added 433 µL (1.66 mmol) of tert-
butyldiphenylsilyl chloride. The solution was stirred at room temper-
ature for 12 h and then diluted with 250 mL of ethyl acetate. The
reaction mixture was washed successively with 50 mL of saturated
NaHCO3 and 50 mL of brine, then dried (MgSO4). The dried solution
5′-Amino-N6-benzoyl-3′-O-(tert-butyldiphenylsilyl)-2′,5′-dideoxy-
adenosine (26). A solution containing 130 mg (0.21 mmol) of

Alteration of DNA Primary Structure by DNA Topoisomerase I
J. Am. Chem. Soc., Vol. 118, No. 47, 1996 11713
nucleoside 25 and 45 mg of 5% palladium-on-carbon in 10 mL of
ethanol was stirred under 1 atm H2 at room temperature for 8 h. The
catalyst was removed by filtration and the filtrate was concentrated
under diminished pressure to afford a residue which was purified by
flash chromatography on a silica gel column (15 cm × 3 cm). Elution
with 9:1 CH2Cl2-methanol afforded 26 as a colorless powder: yield
100 mg (80%); mp 81-83 °C; silica gel TLC Rf 0.30 (9:1 CH2Cl2-
3.765 (s, 1.5 H), 4.38-4.47 (m, 1 H), 4.92-5.06 (m, 1 H), 6.31-6.36
(m, 1 H), 6.72-6.74 (m, 2 H), 7.14-7.63 (m, 15 H), 7.98-8.05 (m, 3
H), 8.15 (s, 0.5 H), 8.23 (s, 0.5 H) and 8.93 (s, 1 H); mass spectrum
(FABMS) m/z 827.380 (M + H)+ (C46H52N8O5Si requires 827.380).
Enzyme Purification. Calf thymus DNA topoisomerase I was
isolated and purified by slight modification of a published procedure.44
The isolated protein exhibited two major bands (Mr ≈ 96 000 and
82 000) when analyzed by SDS-polyacrylamide gel electrophoresis and
visualization of the protein by silver staining. The heterogeneity of
the isolated topoisomerase I can be attributed to proteolysis during the
isolation procedures.45 The purified protein had a specific activity of
1.4 × 107 units/mg protein.
1
methanol); H NMR (CDCl3) δ 1.13 (s, 9 H), 2.30 (dd, 1 H, J ) 6,
11.5 Hz), 2.90-3.03 (m, 1 H), 3.22 (d, 1 H, J ) 12 Hz), 3.75 (dd, 1
H, J ) 12 Hz), 4.08 (s, 1 H), 4.76 (d, 1 H, J ) 6 Hz), 5.60 (d, 2 H, J
) 12 Hz), 6.44 (dd, 1 H, J ) 6, 12 Hz), 7.35-7.75 (m, 13 H), 8.00 (d,
2 H, J ) 9 Hz), 8.10 (s, 1 H), 8.72 (s, 1 H) and 8.93 (s, 1 H); 13C
NMR (CD3OD) δ 18.9, 26.5, 38.7, 41.8, 75.2, 84.6, 86.2, 123.4, 128.2,
128.26, 128.4, 128.8, 130.4, 130.5, 133.0, 133.1, 133.9, 135.9, 144.0,
149.5, 151.5, 154.0 and 166.9; mass spectrum (FABMS) m/z 593.272
(M + H)+ (C33H37N6O3Si requires 593.270).
Oligonucleotide Substrates. Synthetic oligonucleotides were pur-
chased from Cruachem Inc. or synthesized on a Biosearch 8600 series
DNA synthesizer using standard phosphoramidite chemistry.25 The
synthesized oligonucleotides were deblocked and cleaved from the solid
support by treatment with concentrated NH4OH at 55 °C for 12 h. The
5′-CH2OH and -NH2 acceptor oligomers were detritylated and purified
by Nensorb chromatography according to the manufacturer’s protocol.
Removal of the MTr group was effected by treatment with 2%
trifluoroacetic acid and the DMTr group with 0.5% trifluoroacetic acid.
All oligonucleotides were purified on a preparative 20% denaturing
polyacrylamide gel, and the DNA recovered by crush and soak, then
by precipitation. The DNA was 5′-32P end labeled with T4 polynucleo-
tide kinase + [γ-32P]ATP.46
Hybridization of Substrates. Oligonucleotides were hybridized in
a solution (100 µL total volume) containing 10 mM Tris-HCl, pH 7.5,
40 mM NaCl, 5 mM MgCl2 and 5 mM CaCl2. The solution was heated
to 80 °C for 5 min and cooled slowly to room temperature under
ambient conditions over a period of ∼3 h. Due to the low DNA strand
concentrations, hybridization mixtures contained 0.13 pmol of the
labeled strand and a 100-fold excess of the unlabeled strands to ensure
complete hybridization of the labeled DNA.
N6-Benzoyl-3′-O-(tert-butyldiphenylsilyl)-2′,5′-dideoxy-5′-[(4-meth-
oxytriphenylmethyl)amino]adenosine (27). To a solution containing
70 mg (0.11 mmol) of nucleoside 26 in 2 mL of pyridine was added
slowly 43.6 mg (0.14 mmol) of methoxytrityl chloride in 0.5 mL of
pyridine at 0 °C. The combined solution was stirred at room
temperature for 1 h and then partitioned between 25 mL of saturated
NaHCO3 solution and 100 mL of ethyl acetate. The organic layer was
separated, washed with 25 mL of brine and then dried over MgSO4.
The dried solution was concentrated under diminished pressure to afford
a residue which was purified by flash chromatography on a silica gel
column (20 cm × 3 cm). Elution with 95:5 CH2Cl2-methanol afforded
27 as a colorless powder: yield 87 mg (91%); mp 112-115 °C; silica
1
gel TLC Rf 0.70 (95:5 CH2Cl2-methanol); H NMR (CDCl3) δ 1.11
(s, 9 H), 2.12-2.25 (m, 2 H), 2.40 (ddd, 1 H, J ) 1.5, 5.5, 13 Hz),
2.65 (s, 1 H), 2.91-3.00 (m, 1 H), 3.75 (s, 3 H), 4.25-4.30 (m, 1 H),
4.74-4.78 (m, 1 H), 6.39 (dd, 1 H, J ) 5.5, 8 Hz), 6.69 (d, 2 H, J )
9 Hz), 7.13-7.68 (m, 25 H), 7.88 (s, 1 H), 7.99 (d, 2 H, J ) 7 Hz),
8.17 (s, 1 H) and 8.84 (s, 1 H); 13C NMR (CDCl3) δ 19.5, 27.4, 40.7,
46.1, 55.6, 70.6, 75.2, 85.9, 88.7, 113.5, 124.3, 126.6, 128.2, 128.3,
128.9, 129.0, 129.2, 130.2, 130.5, 133.1, 133.5, 133.7, 134.1, 136.1,
138.2, 142.5, 146.4, 146.5, 150.0, 151.8, 152.9, 158.2 and 165.0; mass
spectrum (FABMS) m/z 865.387 (M + H)+ (C53H53N6O4Si requires
865.389).
Formation of the Topoisomerase I-DNA Covalent Complex.
The labeled duplex (6.5 fmol, 100,000 dpm) was incubated with
topoisomerase I (8.8 ng) in reaction mixture (20 µL total volume)
containing 10 mM Tris-HCl, pH 7.5, 40 mM NaCl, 5 mM MgCl2,
5
mM CaCl2, 0.5 mM EDTA and 0.5 mM DTT for 60 min at 37 °C
(13:1 enzyme-duplex DNA).
N6-Benzoyl-2′,5′-dideoxy-5′-[(4-methoxytriphenylmethyl)amino]-
adenosine (4a). To a solution containing 83 mg (0.096 mmol) of
nucleoside 27 in 1 mL of THF was added 105 µL (0.10 mmol) of
TBAF as a 1 M solution in THF. The reaction mixture was stirred for
5 h at room temperature, then the solution was concentrated under
diminished pressure to afford a residue which was purified by flash
chromatography on a silica gel column (15 cm × 3 cm). Elution with
9:1 CH2Cl2-methanol afforded nucleoside analogue 4a as a colorless
foam: yield 48 mg (81%); mp 105-107 °C; silica gel TLC Rf 0.70
(9:1 CH2Cl2-methanol); 1H NMR (CDCl3) δ 2.41 (ddd, 1 H, J ) 3.5,
6, 9 Hz), 2.48 (d, 2 H, J ) 4 Hz), 2.75 (s, 1 H), 3.05 (2 t, 1 H, J ) 6.5
Hz), 3.54 (s, 1 H), 3.73 (s, 3 H), 4.16 (d, 1 H, J ) 3 Hz), 4.80-4.90
(m, 1 H), 6.31 (t, 1 H, J ) 6 Hz), 6.75 (d, 2 H, J ) 9 Hz), 7.11-7.59
(m, 15 H), 7.91 (s, 1 H), 7.98 (d, 2 H, J ) 7.5 Hz), 8.28 (s, 1 H) and
9.23 (s, 1 H); 13C NMR (CDCl3) δ 40.5, 46.2, 55.6, 70.7, 73.4, 85.7,
87.9, 113.6, 124.0, 126.7, 128.2, 129.0, 129.2, 130.2, 133.2, 134.0,
138.2, 142.4, 146.3, 146.4, 149.9, 151.7, 153.0, 158.3 and 165.2.
N6-Benzoyl-2′,5′-dideoxy-5′-[(4-methoxytriphenylmethyl)amino]-
adenosine 3′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (4b).
To a solution containing 43 mg (0.069 mmol) of nucleoside 4a in 2
mL of CH2Cl2 was added successively at room temperature 32.8 µL
(0.19 mmol) of N,N-diisopropylethylamine and 15.3 µL (0.069 mmol)
of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. The solution
was stirred for 1 h and then diluted with 50 mL of ethyl acetate. The
reaction mixture was washed with 15 mL of saturated NaHCO3 and
then dried over MgSO4. The dried solution was concentrated under
diminished pressure to afford a residue which was purified by flash
chromatography on a silica gel column (15 cm × 3 cm). Elution with
45:45:10 CH2Cl2-ethyl acetate-Et3N afforded monomer 4b as a
colorless foam: yield 52 mg (91%) as a 1:1 mixture of diastereomers;
mp 96-98 °C; silica gel TLC Rf 0.50 (5:45:10 CH2Cl2-ethyl acetate-
Et3N); 1H NMR (CDCl3) δ 1.14-1.29 (m, 12 H), 2.42-2.70 (m, 5 H),
2.95 (m, 1 H), 3.30 (m, 1 H), 3.60-3.95 (m, 4 H), 3.763 (s, 1.5 H),
Purification of the Topoisomerase I-DNA Covalent Intermedi-
ate. The topoisomerase I cleavage reaction mixture was applied to an
FPLC Mono Q HR 5/5 column equilibrated in 20 mM Tris-HCl, pH
7.5, containing 0.5 mM EDTA, followed by a 30-mL wash with the
same buffer. Elution was effected with a 30-mL linear gradient of
NaCl (0 f 1 M) in the same buffer at a flow rate of 1 mL/min. One-
mL fractions were collected and an aliquot of each fraction (100 µL)
was utilized for scintillation counting. Typically, 20 individual
topoisomerase I cleavage reactions were combined and applied to the
column.
Proteolysis and Strand Transfer of the Topoisomerase I-DNA
Complex. The fractions containing 5′-32P end labeled covalent complex
(2.9 × 10-16 mol, 4400 dpm) were incubated with 1.25 × 10-11 mol
of the indicated acceptor oligonucleotides promptly after separation
by FPLC in a 110 µL (total volume) reaction mixture containing 20
mM Tris-HCl, pH 7.5, 400 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2
and 1 mM DTT. The thiol acceptor oligonucleotide was reduced
immediately prior to use with 10 mM DTT. The reactions were
incubated for 60 min at 37 °C followed by proteolysis with 1 mg/mL
proteinase K (1 h; 37 °C) and lyophilization. The samples were
reconstituted in 30 µL of 20 mM Tris-HCl, pH 7.5 and 15 µL of loading
solution. The reactions were heat-denatured at 90 °C for 5 min and
quick chilled on ice; 15 µL was applied to a 20% denaturing PAGE.
Effect of pH on Topoisomerase I-Mediated Ligation. The 5′-32P
end labeled covalent complex (2.0 × 10-16 mol, 3000 dpm) in 58 µL
of 20 mM Tris-HCl, pH 7.5, containing 400 mM NaCl and 0.5 mM
EDTA was diluted with an equal volume of 100 mM Tris-HCl at pH
7.5, 8.0, 8.5 or 9.0. MgCl2 and DTT were added to a final
concentrations of 5 mM, and 1 mM, respectively, and the acceptor
oligonucleotide (6.25 × 10-11 mol) was added to initiate the reaction.
The reactions were incubated for 60 min at 37 °C followed by
proteolysis with 1 mg/mL proteinase K (1 h; 37 °C) and lyophilization.
The samples were reconstituted in 30 µL of 20 mM Tris-HCl, pH 7.5

11714 J. Am. Chem. Soc., Vol. 118, No. 47, 1996
Henningfeld et al.
and 15 µL of loading solution. The reactions were heat-denatured at
90 °C for 5 min and quick chilled on ice; 15 µL was applied to a 20%
denaturing PAGE.
iodine in pyridine. After 2 h at room temperature the reactions were
dried under diminished pressure, then redissolved in 5 µL of water
and 3 µL of loading solution. The reactions were heat denatured at 90
°C for 5 min and quick chilled on ice; 5 µL of each was applied to a
20% denaturing PAGE.
Time Course of Topoisomerase I-Mediated Ligation. The 5′-32P
end labeled covalent complex (3.25 × 10-16 mol, 5000 dpm) was
incubated with 6.25 × 10-12 mol of the indicated acceptor oligonucleo-
tide in a reaction mixture (70 µL total volume) containing 20 mM Tris-
HCl, pH 7.5, 400 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2 and 1 mM
DTT. The reactions were incubated for 5, 30 or 60 min at 37 °C,
quenched by the addition of 1% SDS and proteolyzed with 1 mg/mL
proteinase K (1 h; 37 °C). The reactions were lyophilized and then
dissolved in 30 µL of 20 mM Tris-HCl, pH 7.5 and 15 µL of loading
solution. The reactions were heat denatured at 90 °C for 5 min and
quick chilled on ice; 15 µL of each was applied to a 20% denaturing
PAGE.
DNA Strand Scission by Acetic Acid. The 5′-32P end labeled DNA
(1500 dpm) was treated with 15% acetic acid for 15 h at room
temperature. The reactions were dried under diminished pressure, then
redissolved in 5 µL of water and 3 µL of loading solution. The
reactions were heat denatured at 90 °C for 5 min and quick chilled on
ice; 5 µL of each was applied to a 20% denaturing PAGE.
Acknowledgment. This work was supported by Research
Grant CA-53913, awarded by the National Cancer Institute.
Supporting Information Available: Data illustrating to-
poisomerase I-mediated ligation of I and II, DNA sequencing
analysis of topoisomerase I-mediated ligation of I, the effect
of Mg2+ and Mn2+ on the ligation of I and III, ligation of the
modified acceptors as a function of pH, and the results of
exonuclease III digestion of products formed in the presence
of oligonucleotides I and II (9 pages). See any current masthead
page for ordering and Internet access instructions.
Exonuclease III Digestion. Reaction mixtures consisted of 10 µL
(total volume) of 50 mM Tris-HCl, pH 8.0, containing 5 mM MgCl2,
1 mM DTT, 6000 dpm of 5′-32P end labeled duplex DNA and 2 µg of
calf thymus DNA. The reactions were initiated by the addition of 64
units of exonuclease III. After 20 min, the reactions were terminated
by the addition of 2 µL of 200 mM EDTA and 6 µL of loading solution.
The reactions were heat denatured at 90 °C for 5 min and quick chilled
on ice; 5 µL of each was applied to a 20% denaturing PAGE.
DNA Strand Scission by Iodine. To a 10 µL aqueous solution of
5′-32P end labeled DNA (5000 dpm) was added 10 µL of 100 mM
JA961788H