A novel DNA hairpin substrate for bleomycin

Article abstract of DOI:10.1021/ol800445x

(Chemical Equation Presented) A 16-nucleotide DNA hairpin containing 4-aminobenzo[g]quinazoline-2-one 2′-deoxyribose at position 15 has been prepared and found to lack significant fluorescence. When treated with Fe(II)·BLM, the hairpin was found to undergo oxidative transformation selectively at position 15. The predominant fluorescent product was characterized and quantified. The pro-fluorescent DNA hairpin was used as a substrate for 15 bleomycin congeners, and the results were compared with those obtained following cleavage of a radiolabeled DNA duplex and PAGE analysis.

Full text of DOI:10.1021/ol800445x

ORGANIC
LETTERS
2008
Vol. 10, No. 11
2127-2130
A Novel DNA Hairpin Substrate for
Bleomycin
Yoshitsugu Akiyama,† Qian Ma,† Erin Edgar,‡ Andrei Laikhter,‡ and
Sidney M. Hecht*,†
Departments of Chemistry and Biology, UniVersity of Virginia, CharlottesVille,
Virginia 22904, and Integrated DNA Technologies, 1710 Commercial Park,
CoralVille, Iowa 52241
sidhecht@Virginia.edu
Received March 3, 2008
ABSTRACT
A 16-nucleotide DNA hairpin containing 4-aminobenzo[g]quinazoline-2-one 2′-deoxyribose at position 15 has been prepared and found to lack
significant fluorescence. When treated with Fe(II)·BLM, the hairpin was found to undergo oxidative transformation selectively at position 15.
The predominant fluorescent product was characterized and quantified. The pro-fluorescent DNA hairpin was used as a substrate for 15
bleomycin congeners, and the results were compared with those obtained following cleavage of a radiolabeled DNA duplex and PAGE analysis.
The bleomycins (BLMs) are glycopeptide-derived antitumor
antibiotics.1 BLM exhibits sequence selective DNA strand
scission, predominantly at 5′-GC-3′ and 5′-GT-3′ sequences
in the presence of O2 and metal ions such as Fe or Cu,2 and
also shape-selective cleavage of some RNAs.3 tRNA3Lys has
been shown to be especially susceptible to cleavage by
Fe(II)•BLM and may plausibly represent a therapeutically
relevant locus for the drug.4 DNA and RNA degradation by
BLM depends critically on several facets of its structure,
which is organized into discrete structural domains, including
DNA binding, metal binding and linker domains, and a
disaccharide moiety and C-terminal substituent (Figure 1).
The individual structural elements in BLM control metal
binding, O2 binding and activation, selective DNA or RNA
† University of Virginia.
‡ Integrated DNA Technologies.
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Figure 1
. Structure of bleomycin A5 (BLM A5) including the
nitrogen atoms (red) believed to coordinate the metal ion cofactor.
10.1021/ol800445x CCC: $40.75
Published on Web 04/30/2008
2008 American Chemical Society

binding, H atom abstraction and subsequent oxidative
transformation of the nucleic acid substrates. Accordingly,
the design of bleomycin congeners with altered and improved
properties requires a systematic approach and significant
effort. Several groups have worked to elucidate the relation-
ship between chemical structure and functions of BLM by
the use of (semi)synthetic approaches.5 An important mile-
stone in efforts to prepare BLMs with altered properties in
nucleic acid binding and degradation was realized with the
solid-phase synthesis of BLM congeners.6 In addition to
greatly facilitating the preparation of single BLMs, the
analogues could be prepared on solid supports compatible
with on-bead assay of the fully deprotected BLMs.6 Solid-
phase synthesis has also permitted the elaboration of a
combinatorial library of 108 BLM analogues,7 the properties
of which have recently been described.8 The library contained
potent (deglyco)BLM analogues that cleave DNA to a greater
extent than the corresponding parent (deglyco)BLM. In
addition, some BLM analogues in the library exhibited an
altered selectivity of DNA binding and cleavage.8 It is
anticipated that larger libraries may afford access to bleo-
mycin congeners exhibiting potentially useful properties, e.g.,
the ability to cleave individual cellular RNAs specifically.
While the synthesis of BLM libraries on solid supports is
readily accomplished and the elaborated BLMs can be
assayed for DNA cleavage while still attached to the
synthesis beads, there is a key unresolved issue for the
evaluation of larger BLM libraries. The library whose
synthesis and evaluation has been described7,8 was a parallel
library, i.e., containing compounds in individual containers.
While this strategy has important advantages, it also requires
that each of the several assays employed for characterization
of the new analogues be carried out individually on each
member of the library.
To facilitate the evaluation of much larger numbers of
BLM analogues, it is anticipated that future studies may
involve mix-and-split libraries.9 This would result in a large
number of beads, each containing multiple copies of a single
BLM congener. Such libraries can be assayed en masse, e.g.,
by the use of appropriate molecular beacons, potentially
permitting visual identification of beads containing BLM
analogues that are particularly proficient in a specific assay.
We previously described a DNA molecular beacon that can
be used for this purpose.10
The molecular beacon initially described employed fluo-
rescence resonance energy transfer (FRET). Kinetics10 and
visualization6b,c of BLM-mediated DNA cleavage were
achieved using a 16-nucleotide (nt) hairpin DNA (5′-
CGCT3A7GCG-3′) having a fluorescence-quencher pair at
the 5′- and 3′-ends.10 While this oligonucleotide permitted
the characterization of individual BLMs, it was not cleaved
with an efficiency comparable to unmodified DNA,10 owing
presumably to the interaction of the attached fluorescein or
dabcyl moieties with the sites on the DNA-substrate cleaved
by BLM. Thus, the characterization of libraries using this
beacon may be idiosyncratic, rather than reflecting the
intrinsic properties of individual BLM analogues.
Presently, we report the site-specific incorporation of a
fluorescent nucleobase (Nf) into 16-nt hairpin DNAs at BLM
cleavage sites (hairpin DNA-Nf) and the strong enhancement
in fluorescence caused by BLM-mediated DNA strand
scission. Several types of Nf have been incorporated into
DNA as probes of nucleic acid structure, dynamics, and
interactions.11,12 To detect cleavage of DNA by BLM based
on the use of a DNA containing a fluorescent nucleobase,
we focused on the position of insertion and selection of Nf.
In our previous study on the cleavage of a 16-nt hairpin
DNA, it was found that cleavage occurred at cytidine15 for
Fe(II)·BLM.10 As regards the choice of Nf, we sought a
nucleobase having a compact ring structure because a larger
fluorophore-nucleobase13 or fluorophore-linked base14 seemed
more likely to affect the sequence recognition of BLM due
to steric effects. Certain benzo[g]quinazoline heterocycles
have exhibited strong fluorescence emission with favorable
quantum yields in buffered aqueous solution at neutral pH.15
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Org. Lett., Vol. 10, No. 11, 2008

Effective quenching of intrinsic fluorescence by duplex
formation containing 4-aminobenzo[g]quinazoline-2-one 2′-
O-CH3 riboside was also observed.15b Consequently, 4-ami-
nobenzo[g]quinazoline-2-one 2′-deoxyribonucleoside (Cf)
was chosen for insertion into a 16-nt hairpin DNA at C15
(hairpin DNA-Cf15) in lieu of cytidine (Figure 2).
according to synthesis procedures for 4-triazolothymidine or
uridine derivatives.18 This mode of conversion into the Cf
derivative via the triazolide phosphoramidite obviated the
need for the additional transformations that had been
employed for the 2′-O-CH3 ribonucleoside of 4-aminoben-
zo[g]quinazoline-2-one.15b The conversion of 1 into triazolide
phosphoramidite 2 was accomplished in 92% yield.19 Hairpin
DNA-Cf15 was synthesized by standard automated DNA
synthesis protocols (1 µmol scale). The overall and average
stepwise coupling yields were determined to be 91.4% and
99.4%, respectively. Deprotected and purified hairpin DNA-
Cf15 was obtained as a colorless powder in 13% yield (1 µmol
scale). Hairpin DNA-Cf3 was prepared analogously.
The fluorescence emission of hairpin DNA-Cf15 following
treatment with Fe(II)·BLM was monitored by fluorescence
spectroscopy. Figure 3 shows the emission spectrum of 0.72
Figure 2. Chemical structures of hairpin DNA-Cf15 and hairpin
DNA-Cf3 having the 2′-deoxyriboside of 4-aminobenzo[g]quinazo-
line-2-one (Cf) at positions 15 and 3, respectively.
Since DNA binding and cleavage mediated by BLM is
achieved from the minor groove of DNA,16 projection of
the benzo[g]quinazoline heterocycle in the DNA duplex into
the major groove was anticipated not to affect DNA binding
and cleavage by Fe(II)·BLM. Accordingly, it was hoped that
hairpin DNA-Cf15 would exhibit efficient quenching of
benzo[g]quinazoline fluorescence due to formation of the
base-paired duplex, while strong fluorescence emission would
result from release of Cf derivatives following treatment with
Fe(II)·BLM analogues. Because we wish to identify BLM
analogues having altered DNA cleavage properties, and have
noted previously that some BLM congeners exhibit altered
strand selectivity of DNA cleavage,17 a second hairpin DNA
containing the fluorescent nucleobase in lieu of cytidine3
(hairpin DNA-Cf3) was also prepared (Figure 2).
Figure 3. Emission spectra of hairpin DNA-Cf15 (0.72 µM) treated
with 2.5 µM Fe(II)·BLM A5 (curve 1) and 2.5 µM BLM A5 (curve
4) after 30 min (λex 310 nm) at 25 °C in 10 mM Na cacodylate
buffer, pH 7.0, with 100 mM NaCl. The emission spectra of hairpin
DNA-Cf3 treated with Fe(II)·BLM A5 (curve 2) and BLM A5 (curve
3) under the same conditions are also shown.
To incorporate Cf into the 16-nt hairpin DNAs, triazolide
phosphoramidite 2 was prepared from benzo[g]quinazoline-
2,4-(3H)-dione phosphoramidite 115a (Scheme 1). We found
µM hairpin DNA-Cf15 following treatment with 2.5 µM
Fe(II)·BLM A5. The spectrum exhibited emission enhance-
ment (maximum λem 455 nm) when excited at 310 nm. In
addition, an 8-fold enhancement of fluorescence emission
was observed relative to the hairpin DNA-Cf15 treated with
BLM A5 in the absence of Fe2+ (as a control); this was
similar to the fluorescence enhancement observed with our
previous DNA beacon.10 This indicates that BLM can
recognize and cleave a DNA containing the non-natural
fluorescent nucleobase Cf, releasing Cf derivatives. As
expected, hairpin DNA-Cf15 itself exhibited little fluorescence
when excited at 310 nm. As anticipated based on earlier
studies with DNA duplexes of similar sequence,17,20 analo-
gous treatment of hairpin DNA-Cf3 with Fe(II)·BLM A5
produced less fluorescence enhancement (Figure 3).
Scheme 1. Synthesis of Hairpin DNA-Cf15
that the triazolide moiety could be introduced into the uridine
analogues at the phosphoramidite level and converted to the
requisite cytidine derivative concomitant with the deblocking
of the formed oligonucleotide by treatment with NH4OH,
To characterize the formed products, HPLC analysis of
(18) (a) Webb, T. R.; Matteucci, M. K. Nucleic Acids Res. 1986, 14,
7661. (b) Cowart, M.; Gibson, K. J.; Allen, D. J.; Benkovic, S. J.
Biochemistry 1989, 28, 1975. (c) Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J.
Org. Chem. 1992, 57, 3839. (d) Chirakul, P.; Sigurdsson, S. T. Org. Lett.
2003, 5, 917. (e) Jin, S.; Miduturu, C. V.; McKinney, D. C.; Silverman,
S. K. J. Org. Chem. 2005, 70, 4284.
(16) (a) Kuwahara, J.; Sugiura, Y. Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 2459. (b) Bailly, C.; Waring, M. J. J. Am. Chem. Soc. 1995, 117, 7311.
(17) Sugiyama, H.; Kilkuskie, R. E.; Chang, L.-H.; Ma, L.-T.; Hecht,
S. M.; van der Marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1986,
108, 3852.
(19) The conversion of 1 to 2 was 98% based on the peak areas of the
signals from the H-5 protons of 1 (8.73 ppm) and 2 (8.31 ppm).
Org. Lett., Vol. 10, No. 11, 2008
2129

hairpin DNA-Cf15 was carried out after treatment with
Fe(II)·BLM A5. It is known that cleavage of DNA by BLM
is achieved both through C4′-OH and C4′-OOH
intermediates,1b,c which can be quantified by C18 reversed-
phase HPLC using oligonucleotides having sequences similar
to those employed here.17,20 As shown in Scheme 2, cleavage
observed for the unmodified duplexes. In both these regards,
hairpin DNA-Cf15 is clearly better than the DNA beacon
reported initially.10 Interestingly, the ratio of products formed
using the two pathways shown in Scheme 2 was substantially
different at the two sites of cleavage (i.e., C3 vs C15). This
type of difference in degradation chemistry at two sites in
one DNA-substrate has been documented previously.22 The
hairpin DNA-Cf3 was degraded with even greater efficiency
by Fe(II)·BLM A5 (Table S1, Supporting Information).23
As the new hairpin DNA was prepared to facilitate the
rapid evaluation of BLM combinatorial libraries, it seemed
important to demonstrate that the results obtained were not
dissimilar to those obtained using more laborious techniques.
Accordingly, DNA cleavage efficiency by fluorescence
emission and its correlation with results obtained by gel
electrophoresis was determined for 15 BLM congeners
(Figure S1, Supporting Information) known to exhibit quite
different DNA degradation efficiencies. The cleavage of
hairpin DNA-Cf15 by the BLM analogues confirmed that
small differences in structure between the analogues could
strongly affect the resulting fluorescence intensity (Figure
S2, Supporting Information). BLM A5 exhibited the strongest
fluorescent enhancement of the analogues studied. With the
exception of isoBLM A2, the analogues of BLM A5 differing
in the nature of the carbohydrates such as tallysomycin A,
decarbamoyl BLM A2, and L-gulose BLM A5 afforded
diminished fluorescence intensity compared to that of BLM
A5.24 As is clear from Figure S2 (Supporting Information),
several of the BLM analogues tested (e.g., phleomyin, BLM
BAPP, decarbamoyl BLM A5, decarbamoyl BLM A2, and
iso BLM A2) produced significant enhancement of fluores-
cence from hairpin DNA-Cf15. In comparison, other BLMs
produced minimal DNA cleavage under the conditions
employed. To evaluate whether or not the fluorescence
intensities produced from hairpin DNA-Cf15 correlated with
those that would have been obtained by routinely used DNA
cleavage assays, a 5′-32P end labeled 16-nucleotide hairpin
DNA lacking Cf was treated with the same 15 Fe(II)·BLMs.
Fe(II)·BLM A5 was also the most potent in this assay (Figure
S3, Supporting Information). Critically, the results of poly-
acrylamide gel analysis generally showed quite similar trends
in efficiencies of cleavage activity for most BLMs as that
determined by fluorescence emission, indicating that hairpin
DNA-Cf15 can likely be used to characterize BLMs produced
as members of BLM combinatorial libraries. Any BLM
congener producing cleavage at C15, or a significant alteration
of hairpin DNA ternary structure as a result of cleavage
elsewhere, should be detectable using this new substrate.
Scheme 2
.
Cleavage Products Resulting from Treatment of
Hairpin DNA-Cf15 with Fe(II)·BLM
of hairpin DNA-Cf15 at cytidine15 would result in the release
of fluorescent cytosine (fCytosine, 3) and fluorescent cytosine
propenal (fCytosine propenal, 4). The formation of 3 and 4
would be readily distinguished from the products released
upon cleavage at cytidine3 (i.e., cytosine and cytosine
propenal) and consistent with cleavage of the ribose moiety
at C15 via C4′-OH and C4′-OOH intermediates, respectively.
In fact, degradation of hairpin DNA-Cf15 by Fe(II)·BLM A5
was accompanied by the release of two fluorescent com-
pounds when monitored chromatographically. By comparison
with authentic synthetic fCytosine (3) (Scheme 1, Supporting
Information), it was found that one of the fluorescent
products comigrated with authentic synthetic fCytosine.21
Quantification of the cleavage of hairpin DNA-Cf15 by
Fe(II)·BLM indicated that the majority of cleavage (78%)
occurred at C15, with the remainder at C3. This was in quite
good agreement with earlier studies that employed DNA
duplexes of similar sequence.17,20 The total of cleavage
events at C3 and C15 was also not significantly less than that
(20) (a) Sugiyama, H.; Kilkuskie, R. E.; Hecht, S. M.; van der Marel,
G. A.; van Boom, J. H. J. Am. Chem. Soc. 1985, 107, 7765. (b) Sugiyama,
H.; Xu, C.; Murugesan, N.; Hecht, S. M. J. Am. Chem. Soc. 1985, 107,
4104. (c) Sugiyama, H.; Xu, C.; Murugesan, N.; Hecht, S. M.; van der
Marel, G. A.; van Boom, J. H. Biochemistry 1988, 27, 58
.
(21) Moreover, mass spectrometric analysis of 3 collected by C18
reversed-phase HPLC had m/z 212.2, in good agreement with that
determined for authentic synthetic fCytosine (m/z 211.6).
(22) Long, E. C.; Hecht, S. M.; van der Marel, G. A.; van Boom, J. H.
J. Am. Chem. Soc. 1990, 112, 5272.
(23) While the ratio of cleavage events at C3 vs C15 was essentially
identical with that observed for hairpin DNA-Cf15, the actual chemistry of
degradation at the two sites was actually reversed from that found for hairpin
DNA-Cf15. Thus the presence of Cf within the duplex structure influences
the specific chemistry of degradation at a given site, but not the total
degradation events (which is the process intended to be monitored by the
use of this sensor).
Acknowledgment. This work was supported by NIH
Research Grant No. CA76297, awarded by the National
Cancer Institute.
Supporting Information Available: Experimental pro-
cedures for the synthesis and characterization of Cf phos-
phoramidite (2), fCytosine (3), and hairpin DNA-Cf15 as well
as experimental details of the BLM-mediated DNA cleavage.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(24) Although the exact function(s) of the carbohydrate moiety in BLM
are incompletely understood, this result supports earlier observations
concerning the importance of the sugars in BLM.6d,25
(25) (a) Sugiyama, H.; Ehrenfeld, G. M.; Shipley, J. B.; Kilkuskie, R. E.;
Chang, L.-H.; Hecht, S. M. J. Nat. Prod. 1985, 48, 869. (b) Boger, D. L.;
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