DNA-tethered Hoechst groove-binding agents: Duplex stabilization and fluorescence characteristics

Article abstract of DOI:10.1021/ja960948m

Fluorescent Hoechst 33258 analogues have been synthesized in which the terminal phenol moiety is employed as a site for the introduction of a linker to permit covalent attachment of the fluorophores to oligo(deoxynucleotides). Hybridization by the DNA-Hoechst conjugates to target sequences generates the DNA minor groove structure and triggers a binding event by the tethered Hoechst agent. The attendant fluorescence signal reports upon this hybridization event. Conjugation of the Hoechst derivatives to the DNA sequences employs a cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation maximizes the versatility of linker placement and minimizes the associated chemistry required to introduce the linker. Two related Hoechst derivatives have been synthesized; both contain a bromoacetamido linker for conjugation to the oligonucleotides. With each Hoechst derivative, two pairs of diastereomeric (R(p) and S(p)) oligo(deoxynucleotide) conjugates were prepared to provide the tethered Hoechst groove binders with two different orientations within the dA-dT rich minor groove. T(m) measurements suggest that while all of the conjugates provide some increased duplex stability, the diastereomeric conjugates tentatively assigned the R(p) configuration exhibit nearly 20°C increases in T(m) values for the double-stranded dodecameric complexes, while those tentatively assigned as the S(p) diastereomers exhibit only moderate 4-8°C increases in T(m) values. The fluorescence characteristics of the conjugates are more variable, with one complex exhibiting a 23-fold enhancement in quantum yield effects, very similar to that observed for a free untethered Hoechst 33258 fluorophore bound to duplex DNA.

Full text of DOI:10.1021/ja960948m

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J. Am. Chem. Soc. 1996, 118, 7055-7062
7055
DNA-Tethered Hoechst Groove-Binding Agents: Duplex
Stabilization and Fluorescence Characteristics
Kristin Wiederholt, Sharanabasava B. Rajur, John Giuliano, Jr.,
Maryanne J. O’Donnell, and Larry W. McLaughlin*
Contribution from the Department of Chemistry, Merkert Chemistry Center, Boston College,
Chestnut Hill, Massachusetts 02167
ReceiVed March 22, 1996X
Abstract: Fluorescent Hoechst 33258 analogues have been synthesized in which the terminal phenol moiety is
employed as a site for the introduction of a linker to permit covalent attachment of the fluorophores to oligo-
(deoxynucleotides). Hybridization by the DNA-Hoechst conjugates to target sequences generates the DNA minor
groove structure and triggers a binding event by the tethered Hoechst agent. The attendant fluorescence signal
reports upon this hybridization event. Conjugation of the Hoechst derivatives to the DNA sequences employs a
cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation maximizes the versatility
of linker placement and minimizes the associated chemistry required to introduce the linker. Two related Hoechst
derivatives have been synthesized; both contain a bromoacetamido linker for conjugation to the oligonucleotides.
With each Hoechst derivative, two pairs of diastereomeric (Rp and Sp) oligo(deoxynucleotide) conjugates were prepared
to provide the tethered Hoechst groove binders with two different orientations within the dA-dT rich minor groove.
Tm measurements suggest that while all of the conjugates provide some increased duplex stability, the diastereomeric
conjugates tentatively assigned the Rp configuration exhibit nearly 20 °C increases in Tm values for the double-
stranded dodecameric complexes, while those tentatively assigned as the Sp diastereomers exhibit only moderate
4-8 °C increases in Tm values. The fluorescence characteristics of the conjugates are more variable, with one
complex exhibiting a 23-fold enhancement in quantum yield effects, very similar to that observed for a free untethered
Hoechst 33258 fluorophore bound to duplex DNA.
Introduction
“walls” of the minor groove. The extended heterocycles are
capable of adopting a crescent shape that permits them to follow
the convex curvature of the minor groove. They penetrate
deeply into the minor groove and become “isohelical” with the
B-form duplex.10 Functional groups that protrude into the minor
grove, such as the 2-amino group of dG-dC base pairs,6a,11 or
that of related base analogues such as 2-aminopurine,12 interfere
with optimal binding and give rise to the noted sequence
preferences. Tight binding within the groove by such agents
typically results in increased duplex stability.13,14
A variety of extended heterocycles including Netropsin,1
Distamycin,2 DAPI,3 and the Hoechst derivatives4 bind tightly
to duplex DNA.5 On the basis of early chemical protection
studies,6 and more recent crystal structures,1-4 binding by such
agents occurs primarily in the minor groove of B-form duplexes
at sequences containing from three to six dA-dT base pairs.7
Binding appears to be mediated by the formation of specific
hydrogen bonds to the O2 oxygens of the dT residues and to
the N3 nitrogens of the dA residues in the target sites,4 but
electrostatic effects8 or van der Waals contacts9 may also be
prominent between the binding agent and the “floor” or the
Ligands such as DAPI and the Hoechst derivatives are
moderately fluorescent in aqueous solutions, but upon binding
to double-stranded DNA exhibit a dramatically enhanced
quantum yield,15 some 20-fold greater than that observed for
the free dyes.16 The fluorescent properties of these agents have
been employed to visualize chromosomes,17,18 and to quantitate
DNA concentrations.19 The enhanced quantum yield effects are
likely related to the tight binding by the ligand deep within the
minor groove such that solvent access is restricted and non-
* To whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, July 15, 1996.
(1) (a) Kopka, M.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
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Pjura, P.; Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376-
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radiative decay processes for the excited state are reduced.15
A
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S0002-7863(96)00948-1 CCC: $12 00 © 1996 American Chemical Society

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7056 J. Am. Chem. Soc., Vol. 118, No. 30, 1996
Wiederholt et al.
variety of Hoechst analogues have been prepared to date.20 In
most of these studies, the interest in the analogues has been to
alter base specificity by altering functional group interactions
between the dye and the minor groove. In some cases, the
modified Hoechst derivatives have been shown to bind to altered
sequences, most notably to those containing a dG-dC base pair
as part of a dA-dT binding site,20a,g or even to a dG-dC rich
binding site.21 Most of the analogues described in such studies
still exhibit enhanced quantum yield effects upon binding to
double-stranded DNA. Those studies suggest the possibility
of functionalizing the Hoechst dye with a linker in order to
provide a site for covalent attachment of the fluorophore to a
DNA probe sequence without deleterious effects on the attendant
fluorescent characteristics.
generates the minor-groove-binding site and triggers a binding
event by the tethered agent. Formation of a complex between
the groove-binding agent and the hybridization product should
result in enhanced thermal stability, and should generate a
spectroscopic signal to report upon the occurrence of the
hybridization event. A conjugate of this type could function
as a valuable solution-based diagnostic or in situ hybridization
probe. In this study we describe the synthesis of Hoechst-33258
derivatives appropriately modified with linkers that allow
covalent attachment to DNA sequences. We report upon the
covalent conjugation of such agents to DNA as well as the helix-
stabilizing and fluorescent properties resulting from hybridiza-
tion of these DNA-Hoechst conjugates to target sequences.
Experimental Section
Covalent tethering of fluorophores and other agents to DNA
has been accomplished by a number of protocols.22 Virtually
any functional group present in the DNA sequence, whether it
is located on a base residue, carbohydrate, or internucleotide
phosphorus, can be exploited for postsynthetic conjugation
reactions.23 Most commonly a short tether is introduced into
the sequence to provide a terminal aliphatic amino or thiol group
as the site for conjugation. Such tethers have been commonly
employed at the 5′-terminus of the DNA sequence,24 or
internally using the C-5 position of dT,25 one of the base amino
groups,26 the 2′-hydroxyl of a single ribonucleotide,27 or one
of the prochiral oxygens of an internucleotide phosphodi-
ester.28,29 A variety of agents have been tethered to DNA
sequences to improve antisense or antigene activities by
stabilizing duplex and triplex structures; such agents have
included intercalators30 and polypeptides31 or peptide-like
agents.32
Materials. Oligo(deoxynucleotides) were synthesized using 2′-
deoxynucleoside phosphoramidites on an Applied Biosystems 381A
DNA synthesizer. The four common 2′-deoxynucleoside phosphor-
amidites containing aryl- or isobutyrylamides were purchased from
Cruachem through Fisher Chemical Co. H-phosphonate derivatives
were obtained from Glen Research (Sterling, VA). The controlled pore
glass support containing the 3′-terminal nucleoside was a product of
CPG Inc. (Fairfield, NJ). Cystamine dihydrochloride, adamantanecar-
bonyl chloride, anhydrous solvents, and ammonium hydroxide were
all obtained from the Aldrich Chemical Co. (Milwaukee, WI).
Nuclease P1 and snake venom phosphodiesterase were products of
Boehringer Mannheim (Indianapolis, IN). Acrylamide and bisacryl-
amide were obtained from ICN Biomedicals (Cleveland, OH). Thin
layer chromatography was performed on aluminum-backed precoated
silica gel 60 F254 plates purchased from EM Science (Gibbstown, NJ).
NMR spectra were obtained at 300 MHz on a Varian XL-300
multinuclear NMR spectrometer (residual solvent and water peaks
present in the spectra have not been reported). UV measurements for
thermal denaturation studies employed an AVIV 14DS spectropho-
tometer equipped with digital temperature control. Fluorescence
emission spectra were collected on a Shimadzu RF5000U fluorescence
spectrophotometer containing a Shimadzu DR-15 microprocessor and
graphics display terminal. Mass spectra were obtained using FAB
ionization from the Mass Spectrometry Laboratory, School of Chemical
Sciences, University of Illinois, Urbana, IL.
Methods. 2-[2-(4-Nitrophenyl)-6-benzimidazolyl]-6-(1-methyl-4-
piperazinyl)benzimidazole (3). To 0.35 g (1.08 mmol) of 2-(3,4-
diaminophenyl)-6-(1-methyl-4-piperazinyl)benzimidazole (1)33 dis-
solved in anhydrous methanol was added 0.46 g (2.17 mmol) of
4-nitrobenzimidic acid methyl ester (2)34 followed by 0.13 mL (2.17
mmol) of glacial acetic acid. The mixture was refluxed for 2 h. At
this point TLC analysis (dichloromethane/methanol, 1:1) indicated the
complete absence of 1. The solution was basified with ammonium
hydroxide and the resulting precipitate collected. This material was
purified by flash chromatography to yield 0.28 g (0.622 mmol) of 3
(57% yield). Rf (dichloromethane/methanol, 1:1): 0.56. 1H-NMR
(DMSO-d6): δ ) 2.00 (s, 3H, NCH3), 2.80 (s, 4H, -CH2-), 3.20 (s,
4H, -CH2-), 6.6-8.0 (m, 6H, Ar H), 8.20 (q, 4H, Ar H) ppm. UV
(H2O): λmax ) 265, 342 λmin ) 304 nm. IR (KBr): 3114, 2931, 2848,
2804, 1632, 1604, 1521, 1449, 1340, 1290, 1180, 1144, 857, 812, 708
cm-1. Mass spectrum: calcd for C25H24N7O2 (M + H+), 454.199 91,
found 454.199 50.
Tethering an appropriate groove-binding fluorophore to a
short DNA sequence should permit the development of a probe,
which upon hybridization to a complementary target sequence
(20) (a) Latt, S. A.; Stetten, G. J. Histochem. Cytochem. 1976, 24, 24-
33. (b) Bathini, Y.; Rao, K. E.; Shea, R. G.; Lown, J. W. Chem. Res. Toxicol.
1990, 3, 268-280. (c) Gupta, R.; Wang, H. Y.; Huang, L. R.; Lown, J. W.
Anti-Cancer Drug Des. 1995, 10, 25-41. (d) Gravatt, G. L.; Baguley, B.
C.; Wilson, W. R.; Denny, W. A. J. Med. Chem. 1994, 37, 4338-4345. (e)
Czarny, A.; Boykin, D. W.; Wood, A. A.; Nunn, C. M.; Neidle, S.; Zhao,
M.; Wilson, W. D. J. Am. Chem. Soc. 1995, 117, 4716-4717. (f) Beerman,
T. A.; McHugh, M. M.; Sigmund, R.; Lown, J. W.; Rao, K. E.; Bathini, Y.
Biochim. Biophys. Acta 1992, 1131, 53-61. (g) Parkinson, J.; Sadat-
Ebrahimi, S.; Wilton, A.; McKie, J. H.; Andrews, J.; Douglas, K. T.
Biochemistry 1995, 34, 16240-16244.
(21) Singh, M. P.; Joseph, T.; Kumar, S.; Bathini, Y.; Lown, J. W. Chem.
Res. Toxicol. 1992, 5, 597-607.
(22) Goodchild, J. Bioconjugate Chem. 1990, 1, 165-187.
(23) O’Donnell, M.; McLaughlin, L. W. In Bioorganic Chemistry:
Nucleic Acids; Hecht, S., Ed.; Oxford University Press: Oxford, 1996; pp
216-243.
(24) (a) Agrawal, S.; Christodoulou, C.; Gait, M. J. Nucleic Acids Res.
1986, 14, 6227-6245. (b) Connolly, B. A. Nucleic Acids Res. 1987, 15,
3131-3139 (c) Coull, J. M.; Weith, H. L.; Bischoff, R. Tetrahedron Lett.
1986, 27, 3991-3994.
(25) Bergstrom, D. E.; Ruth, J. L. J. Am. Chem. Soc. 1976, 98, 1587-
1589.
(26) MacMillan, A. M.; Verdine, G. L. J. Org. Chem. 1991, 55, 5931-
5933.
(27) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron Lett. 1991,
32, 7171-7174.
(28) (a) Fidanza, J. A.; McLaughlin, L. W. J. Am. Chem. Soc. 1989,
111, 9117-9120. (b) Fidanza, J.; McLaughlin, L. W. J. Org. Chem. 1992,
57, 2340-2346. (c) Fidanza, J. A.; Ozaki, H.; McLaughlin, L. W. J. Am.
Chem. Soc. 1992, 114, 5509-5517.
(29) Ja¨ger, A.; Levy, M. H.; Hecht, S. M. Biochemistry 1988, 27, 7237-
7246.
(30) Thuong, N. T.; Asseline, U.; Moneney-Garestier, T. In Oligo-
deoxynucleotides. Antisense Inhibitors of Gene Expression; Cohen, J., Ed.;
CRC Press, Inc.: Boca Raton, FL, 1989; pp 25-48.
(31) Lemaitre, M.; Bayard, B.; Lebleu, B. Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 648-652.
(32) Sinyakov, A. N.; Lokhov, S. G.; Kutyavin, I. V.; Gamper, H. B.;
Meyer, R. B. J. Am. Chem. Soc. 1995, 117, 4995-4996.
2-[2-(4-Aminophenyl)-6-benzimidazolyl]-6-(1-methyl-4-piper-
azinyl)benzimidazole (4). To 0.27 g (0.60 mmol) of 2-[2-(4-nitro-
phenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazinyl)benzimidazole (3)
in anhydrous methanol (15 mL) was added 0.13 g of Pd/C. The reaction
mixture was stirred under a hydrogen atmosphere (1 atm) for 2 h. TLC
analysis (dichloromethane/methanol, 1:1) after this time indicated the
absence of starting material and the presence of a new product. The
reaction mixture was filtered through Celite, and the resulting filtrate
was evaporated to dryness. The product was purified by flash
chromatography to yield 0.21 g (0.50 mmol) of 4 as a yellow solid
(33) Loewe, H.; Urbanietz, J. Arzneim.-Forsch. 1974, 27, 1927-1933.
(34) Prepared by dissolving 4-nitrobenzonitrile in methanol and bubbling
anhydrous HCl through the solution. The precipitate formed (5) was
collected, dried, and used without further purification.

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DNA-Tethered Hoechst GrooVe-Binding Agents
J. Am. Chem. Soc., Vol. 118, No. 30, 1996 7057
(83% yield). Rf (dichloromethane/methanol, 7:3, + trace of tri-n-
butylamine): 0.36. 1H-NMR (DMSO-d6): δ ) 2.10 (s, 3H, NCH3),
3.00 (s, 4H, -CH2-), 3.10 (s, 4H, -CH2-), 5.60 (s, 2H, -NH2), 6.4-
8.2 (m, 10 H, Ar H). UV (H2O): λmax 272, 342, λmin 254, 298 nm. IR
(KBr): 2933, 2808, 1608, 1439, 1491, 1280, 1234, 1178, 1140, 1005,
963, 832 cm-1. HRMS: calcd for C25H26N7 (M + H+), 424.224 97,
found 424.224 50.
330 nm, λmin 296 nm. HRMS: calcd for C29H29N7O2F3 (M + H+),
564.233 48, found 564.232 30.
2-[4-(2-Aminoethoxy)phenyl]-6-benzimidazolyl]-6-(1-methyl-4-
piperazinyl)benzimidazole (8). The trifluoroacetamide 7 (140 mg,
0.248 mmol) was dissolved in 10 mL of potassium carbonate in
methanol and water (5:2, v/v). The turbid solution was heated gently
until a clear solution resulted and then allowed to stir overnight at
ambient temperature. TLC analysis at this point indicated that the
starting material was absent and a new fluorescent spot was present.
The solvents were removed, and the residue 8 was purified by flash
chromatography on silica gel (dichloromethane with a gradient of
methanol containing trace amounts of tri-n-butylamine to yield 110
mg of 8 (0.235 mmol) 94%. Rf (dichloromethane/methanol, 1:1,
containing a trace of tri-n-butylamine): 0.18. 1H-NMR (DMSO-d6):
δ ) 2.2 (s, 3H, -CH3), 2.4-2.8 (m, 8H, -CH2-), 3.0 (t, 2H,
-CH2-), 4.0 (t, 2H, -CH2-), 5.4 (s, 2H, -NH2), 6.9 (d, 2H, Ar H),
7.1 (d, 2H, Ar H), 7.4 (d, 1H, Ar H), 7.6 (d, 2H, Ar H), 7.9 (d, 1H, Ar
H), 8.2 (d, 2H, Ar H), 8.24 (s, 1H, Ar H) ppm. UV (methanol): λmax
254, 330 nm, λmin 296 nm. HRMS: calcd for C27H30N7O (M + H+),
468.251 18, found 468.251 30.
2-[2-[4-[2-(Bromoacetamido)ethoxy]phenyl]-6-benzimidazolyl]-
6-(1-methyl-4-piperazinyl)benzimidazole (9). To 50 mg (0.107
mmol) of 8 dissolved in 3 mL of anhydrous DMF was added ∼1 mg
of 4-(N,N-dimethylamino)pyridine, and the solution was cooled to -78
°C. To the cooled solution was added 56 mg (0.214 mmol) of
bromoacetic anhydride, and the reaction mixture was stirred for 3 h at
-78 °C. TLC analysis after 3 h indicated the absence of starting
material and the presence of a new compound. The reaction was
quenched with methanol, and the mixture was reduced in volume. Ether
was added, and the resulting precipitate was collected and purified by
column chromatography on silica gel using a gradient of dichloro-
methane/methanol containing a trace amount of tri-n-butylamine to yield
35 mg (0.059 mmol, 56%) of 9. Rf (dichloromethane/methanol, 7:3,
containing a trace of tri-n-butylamine): 0.52. 1H-NMR (DMSO-d6):
δ ) 2.2 (s, 3H, -CH3), 3.2-3.6 (m, 8H, -CH2-), 3.8 (t, 2H,
-CH2-), 4.2 (t, 2H, -CH2-), 4.4 (s, 2H, -CH2-), 7.0-8.4 (m, 10H,
Ar H), 9.0 (s, 1H, NH) ppm. UV (methanol): λmax 255, 335 nm, λmin
295 nm. HRMS: calcd for C29H31N7O2Br (M + H+), 588.172 26,
found 588.171 40.
Synthesis of Oligonucleotides Tethering a Masked Thiol Group
(10). DNA sequences were initially assembled using phosphoramidite
protocols.37 At the site of the tether, 25 µmol of the appropriate fully
protected nucleoside H-phosphonate was dissolved in 600 µL of 1:1
anhydrous acetonitrile/pyridine and activated by the addition of 25 µmol
of adamantanecarbonyl chloride.38,39 The column was removed from
the synthesizer and the activated monomer was added using a syringe.
The column was then rinsed with 1:1 anhydrous pyridine/acetonitrile,
followed by acetonitrile, and was then dried in a desiccator under high
vacuum. The N-(triphenylacetyl)cystamine linker was then added as
described.40 The remaining monomers were added using standard
phosphoramidite protocols, but the capping steps were deleted.40 After
removal of the terminal DMT group, the sequences were deprotected
(concentrated ammonia for 6 h at 50 °C), and the diastereomeric
sequences were separated and isolated using a 9.4 × 250 mm column
of ODS-Hypersil at a flow rate of 3.0 mL/min in 20 mM potassium
phosphate (pH 5.5) with a gradient of methanol (0-100% over 90 min),
desalted (Sephadex G-10), and lyophylized to dryness.
Oligonucleotide Conjugation. To 1 A260 unit of a single diastereo-
mer of the desired dodecamer in 100 mM Tris‚HCl, pH 8, was added
DTT to a concentration of 10 mM, and the mixture was incubated at
50 °C for 1 h. HPLC analysis [50 mM triethylammonium acetate, pH
7.0, with a linear gradient of acetonitrile (7-28% over 30 min)] after
this time period indicated the complete absence of the (triphenylmethyl)-
acetyl-protected sequence (retention time ∼28 min) and the presence
2-[2-[4-(Bromoacetamido)phenyl]-6-benzimidazolyl]-6-(1-methyl-
4-piperazinyl)benzimidazole (5). To 0.1 g (0.236 mmol) of 2-[2-(4-
aminophenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazinyl)benz-
imidazole (4) dissolved in a mixture of DMF/dichloromethane (1:1,
v/v) (4 mL) cooled to -78 °C was added 0.26 mL (0.472 mmol) of
bromoacetic anhydride followed by 0.028 g (0.023 mmol) of 4-(N,N-
dimethylamino)pyridine. The reaction mixture was stirred at -78 °C
for 3 h. TLC analysis (dichloromethane/methanol, 7:3, + trace tri-n-
butylamine) indicated the completion of the reaction. Methanol (1 mL)
and then water (1 mL) were added at -78 °C to destroy any unreacted
bromoacetic anhydride. The solvents were evaporated, and the residue
was dissolved in the minimum amount of methanol and added to ether.
The resulting yellow precipitate was filtered and washed thoroughly
with ether. The resulting yellow solid was purified on a small column
of silica gel using a gradient of dichloromethane and methanol. The
pooled fractions were evaporated to yield 0.098 g (0.234 mmol) of 5
(97% yield). Rf (dichloromethane/methanol, 7:3, + trace of tri-n-
butylamine): 0.52. 1H-NMR (DMSO-d6): δ ) 2.25 (s, 3 H, NCH3),
2.50 (m, 8 H, -CH2-), 4.25 (s, 2 H, bromoacetyl H), 6.95 (d, 1 H, Ar
H), 7.05 (s, 1 H, Ar H), 7.45 (d, 1 H, Ar H), 7.71 (d, 1 H, Ar H), 7.82
(d, 2 H, Ar H), 8.04 (d, 1 H, Ar H), 8.19 (d, 2 H, Ar H), 8.38 (s, 1 H,
Ar H), 10.43 (s, 1 H, amido H) ppm. The imidazole NH residues could
not be located under these solvent conditions. The amide resonance
at 10.43 ppm was exchangeable. UV (H2O): λmax 268, 345 nm, λmin
245, 296 nm. HRMS: calcd for C27H27N7BrO (M + H+), 544.146 04,
found 544.144 00.
2-[2-[4-[2-(Trifluoroacetamido)ethoxy]phenyl]-6-benzimidazolyl]-
6-(1-methyl-4-piperazinyl)benzimidazole (7). 4-[2-(Trifluoroacet-
amido)ethoxy]benzonitrile was prepared by the condensation of (tert-
butoxycarbonyl)ethanolamine with 4-cyanophenol using a Mitsonobu
protocol.35 The tert-butoxycarbonyl group was removed by passing
HCl(g) through the crude product dissolved in dichloromethane, and
the resulting amine was protected with trifluoroacetic anhydride in
quantitative yield using a standard literature procedure.36
To prepare the necessary imino methyl ester 6, 1.2 g (4.65 mmol)
of 4-[2-(trifluoroacetamido)ethoxy]benzonitrile was dissolved in 15 mL
of dichloromethane, 0.267 g (8.37 mmol) of anhydrous methanol was
added, and the reaction was cooled to 0 °C. The reaction mixture was
saturated with HCl(g) and maintained at 0 °C overnight. The resulting
white precipitate was collected, washed thoroughly with diethyl ether,
and dried to yield 1.3 g (4.40 mmol, 96%) of a white solid which was
used in the following step without purification.
To 100 mg (0.310 mmol) of 2-(3,4-diaminophenyl)-6-(1-methyl-4-
piperazinyl)benzimidazole (1)33 dissolved in 3 mL of anhydrous
methanol was added 134 mg (0.465 mmol) of 4-[2-(trifluoroacetamido)-
ethoxy]benzimidic acid methyl ester followed by 37 µL (∼0.6 mmol)
of glacial acetic acid. The mixture was stirred at 65-70 °C for 3 h.
At this time, TLC analysis (dichloromethane/methanol, 7:3, containing
a trace of tri-n-butylamine) indicated the absence of starting material
and the presence of a new compound that was fluorescent under long-
wavelength UV excitation (365 nm). Solvents were removed by
evaporation, the residue was dissolved in a minimum amount of
methanol, and the solution was neutralized with ammonium hydroxide.
The resulting brown precipitate was collected and purified by silica
gel column chromatography (dichloromethane and a gradient of
methanol, containing a trace amount of tri-n-butylamine) to yield 80
mg (0.138 mmol, 45%) of 7. Rf (dichloromethane/methanol, 7:3,
containing a trace of tri-n-butylamine): 0.38. 1H-NMR (DMSO-d6):
δ ) 2.2 (s, 3H, CH3), 3.0-3.2 (m, 8H, -CH2-), , 3.6 (t, 2H,
-CH2-), 4.2 (t, 2H, -CH2-), 6.9 (d, 2H, Ar H), 7.1 (d, 2H, Ar H),
7.4 (s, 1H, Ar H), 7.6 (s, 1H, Ar H), 7.9 (d, 1H, Ar H), 8.2 (d, 2H, Ar
H) 8.3 (s, 1H, Ar H), 9.8 (s, 1H, NH) ppm. UV (methanol): λmax 254,
(37) Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. Soc. 1981, 103,
3185-3191.
(38) Froehler, B. C.; Ng, P. G.; Matteucci, M. D. Nucleic Acids Res.
1986, 14, 5399-5407.
(39) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1986, 27, 469-
472.
(35) Mitsunobu, O. Synthesis 1981, 1-28.
(36) Pyne, S. G. Tetrahedron Lett. 1987, 28, 4737-4741.
(40) O’Donnell, M. J.; Hebert, N.; McLaughlin, L. W. Biol. Med. Chem.
Lett. 1994, 4, 1001-1004.

+
+
7058 J. Am. Chem. Soc., Vol. 118, No. 30, 1996
Wiederholt et al.
Scheme 1a
of a new peak (retention time ∼9 min). The bromoacetylated Hoechst
derivative (5 or 9) in DMF was added to this reaction mixture to a
concentration of 15 mM (40% DMF). The reaction mixture was shaken
at ambient temperature for 48 h (a precipitate appeared during this
period). An equal amount of formamide was added to the reaction
mixtures, and they were heated to 90 °C (to dissolve aggregates) and
loaded directly onto the gel for isolation by electrophoresis.
The conjugated product was purified on a 20% denaturing (7 M
urea) polyacrylamide gel. A fluorescent band (365 nm excitation) was
present whose position was retarded slightly with respect to the oligomer
containing the free thiol linker. This band was excised from the gel,
crushed, and soaked in 0.3 M sodium acetate, pH 6. The gel was
removed and the product desalted using a C18 Sep-pak column and a
water/methanol gradient. The conjugated oligomer was eluted with
approximately 80% methanol in water. Yields varied, typically 0.3-
0.5 A260 unit. The product exhibited a UV/vis spectrum having
characteristics of both DNA (λmax ) 260 nm) and the Hoechst
fluorophore (λmax ) 342 nm).
Nucleoside Analysis. Nucleoside composition was determined after
nuclease P1/snake venom phosphodiesterase/bacterial alkaline phos-
phatase hydrolysis: A 20 µL reaction mixture containing 0.5 A260 unit
of oligomer in 25 mM sodium acetate, pH 5.3, was incubated for 1 h
at 37 °C with nuclease P1. The reaction mixture was then rebuffered
to pH 8 with a solution of 100 mM Tris‚HCl, pH 8.0; 10 mM MgCl2,
3 units of snake venom phosphodiesterase, and 2 units of alkaline
phosphatase were added, and the mixture was incubated for an
additional 2 h at 37 °C. An aliquot containing approximately 0.2 A260
unit of material was analyzed by HPLC using a 4.6 × 250 mm column
of ODS-Hypersil in 20 mM potassium phosphate, pH 5.5, and a gradient
of 0-70% methanol (60 min) followed by isocratic elution with solvent
B. The following retention times were observed (260 nm): (dC) 6.4,
(dG) 10.4, (dT) 11.1, (dA) 14.2, (d[Tp(NHCH2CH2SH)T]) 29.8 and
30.5, (d[Tp(NHCH2CH2S-Hoechst)T]) 62 and 63.5 min (both Hoechst
conjugates exhibited similar retention times).
Tm Values. Tm values were obtained for complexes containing a
1:1 mixture of the conjugate and its complementary sequence in 21
mM HEPES, pH 7.5, containing 100 mM NaCl and 20 mM MgCl2 at
duplex concentrations in the low micromolar range. Solutions were
heated in 0.5 or 1.0 °C steps, and absorbance readings were taken after
a period of temperature stabilization. Absorbance and temperature
readings were plotted using Igor software. Tm values were determined
from first- and second-order derivatives, as well as graphically from
the absorbance vs temperature plots.
Fluorescence Measurements. Fluorescence measurements were
made in solutions typically containing ∼1 µM DNA-Hoechst conjugate
in 21 mM HEPES, pH 7.5, containing 100 mM NaCl and 20 mM
MgCl2. All emission measurements were made with the following list
of parameters: slit width Ex/Em ) 5 nm/5 nm, high sensitivity, fast
speed. Samples were introduced into a 1.25 mL cell thermally isolated
with a water jacket. Temperature was controlled with a recirculating
water bath. Fluorescence emissions were measured at 450 nm with
an excitation wavelength of 342 nm. The ∆F values were ratios
obtained at 450 nm. This emission wavelength represented the emission
maximum for the duplex conjugates and was slightly off the emission
maximum for the single-stranded conjugates.
a Conditions: (i) CH3COOH, heating. (ii) H2/Pd-C. (iii) (BrCH2CO)2/
DMAP, -78 °C.
be employed as a site for attachment of the desired linker
without interfering with the fluorescent characteristics. Attempts
to directly functionalize the phenolic hydroxyl of the native
Hoechst 33258 fluorophore were unsuccessful. For this reason
we chose to synthesize the bisbenzimidazole ring system in order
to vary the functionality of the fluorophore as needed.
Synthesis of the Hoechst Fluorophores. The terminal
phenol residue is introduced relatively late in the described
synthetic procedure.33 We simply altered the final portions of
the known synthetic pathway as needed to modify the character
of this terminal phenol. For example, after preparation of 2-(3,4-
diaminophenyl)-6-(1-methyl-4-piperazinyl)benzimidazole (1;
Scheme 1),33 we introduced a terminal anilino residue to provide
a site to tether a bromoacetamido linker (see 5; Scheme 1). The
synthesis of 5 proceeded without difficulty; we simply altered
the final cyclization step by incorporating a nitrophenyl moiety
in place of the more common phenol to generate 3 (Scheme 1).
The nitro group was subsequently reduced to the corresponding
amino group which could then be acylated with bromoacetic
anhydride to generate the Hoechst derivative 5.
The related Hoechst derivative 9 extends the bromoacetamide
linker away from the chromophore by inserting an additional
two carbons between the phenylbisbenzimidazole chromophore
and the bromoacetamido moiety. This derivative increases the
length of the linker between the fluorophore and its site of
attachment to the DNA. The two-carbon linker may also
insulate the fluorescent bisbenzimidazole from any undesirable
electronic effects resulting from the presence of the bromo-
acetamido group attached to the terminal aromatic ring of the
chromophore. Compound 9 was prepared by a procedure
similar to that described for 5, and the route is outlined in
Scheme 2. It involves the condensation of 1 with the imino
methyl ester 6. After removal of the trifluoroacetamide, the
aminoethoxy derivative 8 was bromacetylated to generate 9.
Synthesis of the DNA-Hoechst Conjugates. With both
Hoechst derivatives 5 and 9 we have employed a terminal
bromoacetamido moiety to permit covalent attachment to the
DNA sequence. The bromoacetamido group has functioned well
in previous studies as a thiol-specific alkylating agent for
postsynthetic conjugation by a variety of fluorophores, or other
agents, to thiol linkers present in DNA sequences.28
Results
We have prepared oligonucleotides tethering the fluorescent
minor-groove-binding agent Hoechst 33258. This dye exhibits
both favorable helix-stabilizing properties41 and enhanced
fluorescence characteristics12,16 upon binding to double-stranded
DNA sequences. Both Hoechst 33258 and the related Hoechst
33342 derivative (differing only in the nature of the terminal
phenol moiety) are similarly fluorescent. A variety of structur-
ally related derivatives in which the terminal phenol moiety has
been replaced with other functionality retain both the DNA-
binding characteristics and the desired fluorescence properties.20
Those observations suggested that the terminal phenol ring could
From the wide variety of techniques available to covalently
attach reporter groups to DNA sequences,22,28,29,40,42 we chose
a backbone-labeling procedure40,42 in which the reporter group
is tethered to an internucleotide phosphorus residue. The
(42) (a) Letsinger, R. L.; Schott, M. E. J. Am. Chem. Soc. 1981, 103,
7394-7398. (b) Yamana, K.; Letsinger, R. L. Nucleic Acids Symp. Ser.
1985, 16, 169-173. (c) Letsinger, R. L.; Bach, S. A.; Eadie, J. S. Nucleic
Acids Res. 1986, 14, 3487-3497. (d) Agrawal, S.; Tang, J.-Y. Tetrahedron
Lett. 1990, 31, 1543-1546.
(41) Wilson, W. D.; Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin,
D. Biochemistry 1993, 32, 4098-4104.

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DNA-Tethered Hoechst GrooVe-Binding Agents
J. Am. Chem. Soc., Vol. 118, No. 30, 1996 7059
Scheme 2a
Scheme 3a
a Conditions: (i) CH3COOH, heating. (ii) K2CO3/CH3OH. (iii)
(BrCH2CO)2/DMAP, -78 °C.
a Conditions: (i) DTT. (ii) 5, 48 h, ambient temperature, PAGE.
3), was followed by reduction of the unsymmetrical disulfide
with tris(2-carboxyethyl)phosphine44 or DTT to eliminate the
triphenylacetyl moiety and unmask the thiol labeling site (11).
For each diastereomer, the free thiol of 10 (or corresponding
dA sequence) was reacted with an excess of 5 or 9 in an
aqueous/DMF (60:40) solution at pH 8.0. The conjugation
reaction functioned best with concentrations of DNA and
reporter group in the low millimolar range, but the reaction was
complicated by the fact that Hoechst 33258 is known to
aggregate in aqueous solutions at concentrations near or above
30 µM.16 The use of high concentrations of N,N-dimethyl-
formamide reduced such aggregation and maximized the
solubility of the fluorophore without precipitating the thiol-
containing DNA. To compensate for aggregation problems
present during isolation of the DNA conjugate, it was necessary
to resolve the product from the heterogeneous reaction mixture
by employing a denaturing (7 M urea) polyacrylamide gel. Each
conjugate could be excised from the gel, extracted, and desalted
to generate a purified DNA-Hoechst conjugate (e.g., 12 in
Scheme 3). The purified conjugates were solubilized in aqueous
solution at concentrations of not more than ∼10 µM, and
analyzed by reversed phase HPLC. Such analyses resulted in
the elution of a single peak (see the supporting information).
The isolated materials exhibited absorption characteristics for
both the DNA (λmax ) 260 nm) and the Hoechst groove binder
(λmax ) 342 nm). Digestion of the conjugates by a mixture of
nucleases and alkaline phosphatase, followed by HPLC analysis,
resulted in a chromatogram indicating the presence of the
common nucleosides, and a strongly retained species corre-
sponding to the material obtained when one isomer of the dimer
d[Tp(NHCH2CH2SH)T] was conjugated to 5 or 9. Using this
approach, both phosphorus diastereomeric conjugates of 12
(Scheme 3) were prepared, as were both diastereomers of the
corresponding dA rich sequence d[CpGpCAp(NHCH2CH2S-
Hoechst)ApApApApApGpCpG] (13). Each of the diastereo-
meric pairs are differentiated only by their relative elution time
from the HPLC column. For example, 12a represents the early
eluting diastereomer for the conjugate prepared from 10a, while
12b represents the later eluting isomer prepared from 10b. Each
pair of diastereomeric conjugates, upon complexing to the
complementary target sequence, is provided with a 5-6 base
pair binding site composed of dA-dT base pairs. Attachment
of the groove binding agent to either the dT-containing (12a/
12b) or the dA-containing (13a/13b) sequence has the effect
of placing the covalent tether in one of the two possible
reasons for this choice were fourfold: (i) Locating the tether
on the internucleotide phosphorus places the groove-binding
agent on the outer surface of the nucleic acid duplex structure
where it is unlikely to interfere with base-base hydrogen-
bonding interactions between the probe and the target sequence.
(ii) By using the internucleotide phosphorus as the site of
attachment, the tether can be easily moved from one inter-
nucleotide linkage to another to explore variations in the
positioning of the tethered groove-binding agent within a given
sequence. (iii) Tethering the reporter group to an internucleotide
phosphorus residue places it in two diastereomeric positions
about the internucleotide phosphorus; one diastereomer may be
more effective in directing the Hoechst derivative toward the
minor-groove binding site. (iv) In principle, the groove-binding
agent could have been simply attached to one terminus of the
probe sequence, but positioning the groove-binding agent near
the end of a dA-dT rich duplex might be deleterious in that
only a portion of the groove-binding agent would be able to
effectively interact within the groove structure due to potential
end-fraying effects. More efficient groove binding might result
if such derivatives were attached to the DNA backbone near
the center of the short duplex sequence, at the point where the
duplex exists as a well-formed structure.
The DNA containing the thiol tether was synthesized using
standard phosphoramidite-based procedures until the site for
attachment of the tether was reached. At this point, a nucleoside
residue containing an H-phosphonate internucleotide linkage43
was incorporated into the growing oligonucleotide chain and
immediately oxidized in the presence of carbon tetrachloride/
pyridine and N-(triphenylacetyl)cystamine.40 This procedure
results in the incorporation of a phosphoramidate internucleotide
linkage at the site of choice, tethering a masked thiol. Owing
to the presence of two prochiral nonbridging oxygens, the
phosphoramidate is formed as a diastereomeric pair. The bulky
triphenylacetyl residue attached to the tether (see 10; Scheme
3) results in differential chromatographic mobilities sufficient
to permit effective resolution of the two isomeric phosphor-
amidates. To date we have not been able to unambiguously
assign the absolute configurations of the two phosphoramidate
stereocenters, and have simply labeled them according to their
relative elution order from a reversed phase column using HPLC
analyses. For example, 10a elutes from the HPLC column
“earlier” than 10b.
Synthesis and isolation of a single isomer of a dodecamer
containing a masked thiol tether, for example (see 10; Scheme
(44) Burns, J. A.; Butler, J. C.; Moran, J.; Whitesides, G. M. J. Org.
Chem. 1991, 56, 2648-2650.
(43) Froehler, B. C. Tetrahedron Lett. 1986, 27, 5575-5578.

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7060 J. Am. Chem. Soc., Vol. 118, No. 30, 1996
Wiederholt et al.
Table 1. Helix Stabilization and Fluorescence Characteristics of
DNA-Hoechst Conjugates
13a and 13b result in the Hoechst derivative being tethered at
the opposite end of the minor-groove-binding site. This
variation in location should have the effect of reversing the
orientation of the groove-binding fluorophore within the minor
groove. The Tm values measured for complexes containing 13a
and 13b were 74 and 63 °C, respectively, similar in both cases
to the values observed for the corresponding diastereomers of
12 (Table 1). With both conjugates 12 and 13, one diastereomer
(isomer a) resulted in a higher Tm value for the duplex.
Hoechst
site of
derivative conjugation isomera Tmb (°C) ∆Fc
Conjugates 14 and 15 were prepared from 9, which contains
a linker lengthened by two additional carbon atoms. This
increase in length should provide more flexibility for the Hoechst
fluorophore. As noted for the conjugates 12 and 13, conjugates
14 and 15 are designed as positional isomers, with the Hoechst
derivative being tethered at either end of a stretch of dA-dT
base pairs. Similar to the results noted for 12 and 13, both
DNA duplexes containing diastereomers 14a and 15a exhibited
Tm values increased by 18-19 °C relative to that of the native
duplex. By comparison, 14b and 15b exhibited more moderate
increases of 6 °C. A diastereomeric mixture of 14a/14b (40:
60) resulted in a Tm increase of intermediate value (∼13 °C).
conjugate
native duplex
55 ( 1
51
51
74
59
74
63
73
61
nfd
nf
nf
6
6
7
10
10
12
12
13
13
14
14
15
15
1
1
1
1
2
2
1
1
2
2
a
b
a
b
a
b
a
b
a
b
5
5
5
5
9
9
9
9
4
23
12
12
11
74
61
a Isomer designations “a” and “b” were assigned according to the
relative elution order of the phosphorus diastereomers of 10 (or the
corresponding dA-containing sequence) during HPLC isolation proce-
dures. In each case, the corresponding Hoechst conjugation isomer
results from the reaction of the same isomer of 10 (or the corresponding
dA-containing sequence). For example, 12a was generated from 10a,
while 12b resulted from 10b). b Values are reported in °C for the
conjugate shown complexed to its native complementary sequence, 5′-
d(CpGpCpApApApApApApGpCpG) or 5′-d(CpGpCpTpTpTpTpTp-
TpGpCpG), and were obtained in 21 mM HEPES, pH 7.0, containing
100 mM NaCl and 20 mM MgCl2. Each value is the average of two
or more determinations, and the precision is estimated to be (1 °C.
c Values represent the fluorescence enhancement ratio observed at 450
nm upon adding 1 equiv of the complementary native sequence. d nf
) no fluorescence.
In contrast to the variation in Tm values observed for
diastereomeric pairs of conjugates, the position of the conjugated
dye, with respect to its orientation in the minor groove, had
little impact upon the Tm values for the conjugated duplexes.
The two conjugate pairs 12a and 13a and then 14a and 15a
position the Hoechst derivatives in opposite relative orientations
within the DNA minor groove [the same relationship is present
for the pair 12b and 13b (or 14b and 15b)]. However,
regardless of such orientation effects, the Tm values remained
largely unchanged for various positional isomers. Both 12a and
13a exhibited the same 19 °C increase in Tm values, and
corresponding similarities were observed for 14a and 15a. In
a similar manner, 12b and 13b (as well and 14b and 15b) all
exhibited Tm values largely independent of the position of the
Hoechst derivative.
diastereomeric positions, as well as at two different positions
on the DNA (at opposite ends of the minor-groove-binding site).
In similar fashion, we prepared the diastereomeric conjugates
14 and 15, again beginning with 10 (or the dA-containing
sequence) but using the Hoechst derivative 9 containing the two-
carbon spacer separating the terminal phenyl ring from the
reactive bromoacetamido linker. The isolation, analysis, and
identification of the diastereomeric pairs 14a/14b and 15a/15b
proceeded in a manner strictly analogous to that described above
for 12 and 13.
Helix-Stabilizing Properties. Absorbance vs temperature
plots for the native dodecameric duplex (see Table 1) (∼1 µM)
in the presence of free Hoechst 33258, or either of the analogues
5 and 9, at a concentration of 1.2, 5, or 10 µM, all resulted in
very similar Tm values varying from 62 to 65 °C. These Tm
values, measured in the presence of the various free fluoro-
phores, were 7-10 °C higher than the Tm value observed for
the native duplex (Tm ) 55 °C).
The helix-stabilizing properties of the conjugated sequences
were evaluated by adding between 1.0 and 1.2 equiv of the
native complementary sequence and then analyzing the variation
in Tm values under a common set of buffer and salt conditions.
Conjugating a large bulky group to a DNA duplex, in the
absence of any specific binding effects, can result in helix
destabilization as noted for 10a/10b (Table 1). Complexes
containing one of the conjugated dodecameric sequences all
exhibited increased Tm values (Table 1). The DNA duplex
formed from 12a and its complement exhibited a 19 °C increase
in Tm relative to that of the unmodified duplex. By comparison,
the duplex containing the corresponding diastereomer 12b
resulted in a more moderate increase of 4 °C. Relative to the
diastereomeric complexes of 12, the DNA duplexes formed from
Fluorescence Characteristics. We initiated our fluorescence
studies by examining the characteristics of a 1 µM solution of
Hoechst 33258 titrated with the native dodecameric duplex 5′-
d(CpGpCApApApApApApGpCpG)•d(CpGpCpTpTpTpTp-
TpTpGpCpG). The 1:1 complex of DNA and dye exhibited
(λex ) 342 nm, λem ) 450 nm) an enhancement in emission
maximum (∆F) of 26, a value that is consistent with previous
studies16 employing the dodecamer [d(CpGpCpGpApApTp-
TpCpGpCpG)]2. In this related work a value for ∆F of
approximately 30 was reported. In similar experiments we
observed ∆F values of 11 and 19, for 1:1 complexes of the
native duplex dodecamer and the Hoechst derivative 5 or 9,
respectively. We recognized that at concentrations in the low
micromolar range the possibility existed for multiple modes of
binding by these bisbenzimidazoles,16 not all of which would
necessarily result in significant enhancement of fluorescence
properties, but as an important control experiment, we needed
to quantify the fluorescence characteristics for the untethered
fluorophores bound to the target duplex under conditions where
Tm values and fluorescence properties of the conjugated
complexes would be studied.
Each conjugate 12-15 was titrated with the corresponding
complementary sequence (for example, see Figure 1), and the
emission enhancement characteristics (∆F) of the conjugated
dyes were examined (Table 1). Both 12a and 12b exhibited
∆F values that were virtually identical, but only 60% of the
value obtained for untethered 5 in the presence of the duplex
dodecamer. The regioisomeric complexes 13a and 13b were
similarly fluorescent, with 13a exhibiting the more pronounced

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DNA-Tethered Hoechst GrooVe-Binding Agents
J. Am. Chem. Soc., Vol. 118, No. 30, 1996 7061
Figure 2. Fluorescence vs temperature plot for conjugate 14b
complexed to the complementary sequence 5′-d(CpGpCpTpTpTpTpTp-
TpGpCpG). Inset: absorbance vs temperature plot for the same
complex.
Figure 1. Fluorescence emission spectrum for a ∼1 µM solution of
14a titrated with aliquots of the complementary sequence 5′-d(CpGp-
CpApApApApApApGpCpG).
Hoechst dyes upon binding to double-stranded DNA sequences.
The combination of a hybridization-triggered binding event, with
the generation of a strong fluorescent signal, should provide
the basis for the development of a DNA-based diagnostic, or
in situ hybridization probe.
effects, but the 7-fold enhancement must still be considered only
a moderate effect. By comparison, 14a with a nine-carbon
linker resulted in a 23-fold enhancement in quantum yield
(Figure 1, Table 1), slightly greater than the effect observed
for untethered 9 in the presence of the dodecamer, and similar
to the value observed for the untethered Hoechst 33258 (∆F )
26). Emission characteristics for the complementary diastereo-
mer 14b were not as dramatic as those observed for 14a, but
this conjugate still exhibited a ∆F of 12, 65% of the value
observed for untethered 9. The regioisomeric conjugates 15a
and 15b both exhibited significant ∆F values (12 and 11), but
neither exhibited the dramatic enhancement observed for 14a.
A mixture of 14a/14b (40:60) resulted in an intermediate value
(∆F ≈ 15).
In the present work we have employed the internucleotide
phosphate as the site for attachment of the groove-binding
fluorophore in order to reduce any interference by the tethered
fluorophore in the hybridization event. We note that conjugates
such as 10, carrying a large bulky substituent that is incapable
of binding to the DNA sequence, can result in moderate
reductions in the stability of the hybridization complex.
However, conjugates 12-15 all resulted in increases (4-19 °C)
in the Tm values for the hybridization complexes. A potential
disadvantage in using the internucleotide phosphate as the tether
site is that incorporation of the tether generates two diastereo-
mers (Rp and Sp) about phosphorus. We had anticipated that
this potential difficulty might be exploited in an advantageous
manner if one of the two diastereomeric conjugates resulted in
more effective helix stabilization or fluorescence characteristics.
The four “a” diastereomers (Table 1) all result in nearly a 20
°C increase in their Tm values relative to that of the simple native
duplex. The “b” diastereomers are also capable of enhancing
the Tm values of the dodecamer duplexes, but only at more
moderate levels (4-8 °C). This consistent increase in Tm values
for the a diastereomers suggest that the positioning in the a
diastereomers results in the fluorophore being more effectively
oriented toward the minor groove (Figure 3) and, therefore,
tentatively being assigned as the Rp diastereomers.45 The b
stereocenters are then the complementary Sp diastereomers. We
note that the pro-S oxygens of the phosphodiester linkages (in
both strands) are oriented toward the minor groove side of the
phosphoribose backbone, while the pro-R oxygens are oriented
toward the major groove side of the phosphoribose backbone.
With respect to the position of the nitrogen in the corresponding
phosphoramidate, these relative assignments are reversed45
owing to atom priority in the stereocenter assignments.
In contrast to the differences in Tm values observed with the
two diastereomeric conjugates, the positional differences had
little effect upon the stability of the hybridization products.
To confirm that the observed fluorescence properties were
the result of binding to the duplex DNA, we examined the
temperature effects of the emission spectra. In all cases the
fluorescence signal of the duplex conjugate was initially
observed to decrease only marginally with temperature. But
this initial effect was then followed by a more dramatic nonlinear
decrease in quantum yield until a value was reached that was
similar to that measured for the single-stranded conjugate. The
transitions obtained for the various conjugated duplexes mirrored
the transitions observed for a thermally induced helix-to-coil
transition (Figure 2, and inset).
Discussion
The use of a site-specific thiol tether introduced to an
internucleotide phosphate residue permits the postsynthetic
conjugation of DNA sequences by a variety of agents. In the
present work we have used fluorophores, analogues of the
Hoechst minor-groove-binding bisbenzimidazoles, to prepare
covalent DNA-Hoechst conjugates. These conjugates have
been designed to permit normal hybridization between the
conjugate DNA sequence and the target sequence. Hybridiza-
tion by the conjugate to a target sequence converts a single-
stranded species to a double-stranded complex and generates
the B-form secondary structure with both the major and the
minor grooves. In this respect, the hybridization event triggers
a binding event by the tethered Hoechst fluorophore within the
generated minor groove structure. The Hoechst fluorophores
were chosen as a class of reagents for two reasons, the first
being their affinity for the minor groove of duplex DNA
containing dA-dT base pairs, and the second being the dramatic
enhancement in emission maxima (>20-fold) observed with the
(45) Owing to the vagaries of the Cahn-Ingold-Prelog system in
assigning stereocenters, the Sp-phosphoramidate places the nitrogen of the
P-N bond in the position occupied by the pro-R-oxygen of the inter-
nucleotide phosphodiester. Similarly, the absolute configurations of the Sp-
phoshphoramidate and the Rp-phosphorothioates have the nitrogen and sulfur
atoms in the same abolute positions. Correspondingly, the Rp-phosphor-
amidate then places the nitrogen into the position of the pro-S-oxygen of
the internucleotide phosphate, and corresponds in configuration to the Sp-
phosphorothioate.

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7062 J. Am. Chem. Soc., Vol. 118, No. 30, 1996
Wiederholt et al.
structure. Consistent with the first explanation is the observation
that 1 equiv of the Hoechst derivative 5 in the presence of duplex
DNA resulted in only a moderate 9-fold enhancement in
fluorescence. However, by adding to derivative 5 a short two-
carbon linker (generating 9), at least nominally to insulate the
bromoacetamido linker from the bisbenzimidazole ring system,
more dramatic fluorescence effects were observed. One equiva-
lent of derivative 9 in the presence of duplex DNA now results
in a 19-fold enhancement in fluorescence.
Lengthening the linker of the Hoechst derivative results
overall in a longer tether between the dye and the DNA
sequence. The addition of these two carbons increases the tether
length to nine atoms and appears to permit optimal binding by
the tethered fluorophore; both diastereomers of 14 and 15
(prepared from 9) now exhibit at least an 11-fold increase in
fluorescence when bound to their hybridization targets. The
observation that the double-stranded complex formed from 14a
now exhibits the most dramatic effects, with a 23-fold enhance-
ment in fluorescence, is also likely the result of an increased
linker length. This difference in fluorescence effects for 14a
and 15a suggests that one orientation of the bisbenzimidazole
within the minor groove (that present in 14a) is preferred with
regard to attendant fluorescence effects. Orientation effects are
difficult to correlate with the reported crystal structures4 since
self-complementary sequences are typically employed.
Figure 3. Duplex DNA containing the hexameric (dA-dT)6 sequence
with the relative sites that would be occupied by the nitrogens of a
single phosphoramidate linkage marked as the Rp and Sp diastereomers.
This graphic has been tilted so that the view is along the minor groove.
Regardless of whether the Hoechst derivative was oriented to
follow the dT5 residues (5′ f 3′) or was reversed, to follow the
dA5 residues (5′ f 3′), the resulting Tm values were virtually
identical [compare 12a (12b) with 13a (13b), or 14a (14b) with
15a (15b)] (Table 1). These results suggest that the helix-
stabilizing effects of the Hoechst derivatives are largely
independent of the orientation of the fluorophore.
Conclusions
We have prepared two Hoechst 33258 analogues that permit
their covalent attachment to DNA sequences. The hybridization
of the DNA-Hoechst conjugates to target DNA generates the
double-stranded hybridization product and thereby triggers the
desired binding event in the minor groove by the tethered
fluorophore. This binding event both enhances the stability of
the hybridization complex and results in a fluorescent signal
that reports on the success of the hybridization event. All of
the tentatively assigned Rp diastereomeric conjugates result in
DNA duplexes of similar stability, and in all cases they are more
stable than the diastereomers tentatively assigned as the Sp
stereocenters. The fluorescence properties of the complexes are
more varied; one of the complexes exhibits a quantum yield
enhancement that is very similar to that observed for the free
untethered fluorophore. This observation suggests that the tether
permits efficient binding by the fluorophore within the minor
groove. The development of materials that are triggered by the
hybridization event, to stabilize the hybridization product, and
to signal the event with a dramatic increase in fluorescence,
should provide the foundation for the development of a new
class of DNA diagnostics or in situ hybridization probes.
By comparison, the fluorescence emission spectra vary
significantly among the various conjugated complexes. That
the fluorescence effects result from binding to the duplex DNA
structure can be inferred from the temperature studies performed.
In all cases the fluorescence of the duplex conjugate decreased
with increasing temperature, but these effects were most
dramatic in the temperature range of the helix-to-coil transition
(see Figure 2). This observation is consistent with the fluoro-
phore being tightly bound within the minor groove where the
excited state is largely protected from collisional decay processes
involving bulk solvent, and in the bound state relaxes primarily
through radiative decay.15 As the complex undergoes a
cooperative helix-to-coil transition, the excited state becomes
more accessible to collisional processes, less relaxation by
radiative decay occurs, and a more dramatic loss in quantum
yield is observed. Owing to the cooperative nature of the helix-
to-coil transition, a cooperative fluorescence vs temperature plot
is obtained (Figure 2).
The relative enhancements in fluorescence emission spectra
for the conjugates 12-15 vary depending upon the Hoechst
derivative employed, and its orientation. For example, both
diastereomers of conjugate 12, as well as both diastereomers
of 13, exhibit significant (4-fold to 7-fold) enhancements in
emission spectra, but these values are not nearly as dramatic as
the roughly 26-fold enhancement observed for the parent,
untethered Hoechst 33258, bound to the duplex dodecamer. Two
explanations might account for the moderate fluorescence effects
observed with these four diastereomeric conjugates. (i) The
presence of the bromoacetamido linker alters the fluorescence
characteristics of the dye/DNA complex. (ii) The length of the
linker between the DNA backbone and the bisbenzimidazole
will affect the positioning of the fluorophore within the groove
Acknowledgment. This work was supported by a grant from
the NIH (GM 37065). We thank Dr. Robert Kuimelis for critical
reading of the paper.
Supporting Information Available: Figures showing the
structure and UV/vis spectrum of 13, HPLC analyses of the
conjugate 12a and dodecameric sequences, and the absorbance
vs temperature plot for the duplex formed from conjugate 13a
and the complementary strand (5 pages). See any current
masthead page for ordering and Internet access instructions.
JA960948M