Recognition of nine base pairs in the minor groove of DNA by a tripyrrole peptide - Hoechst conjugate

Article abstract of DOI:10.1021/ja003095d

A tripyrrole peptide - Hoechst conjugate (FPH-1) has been designed which recognizes nine dA/dT base pair A/T rich dsDNA sequences at subnanomolar concentrations and complexes its targets at near diffusion controlled rates to form a fluorescent product. Sp

Full text of DOI:10.1021/ja003095d

VOLUME 123, NUMBER 11
MARCH 21, 2001
© Copyright 2001 by the
American Chemical Society
Recognition of Nine Base Pairs in the Minor Groove of DNA by a
Tripyrrole Peptide-Hoechst Conjugate
Alexander L. Satz and Thomas C. Bruice*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California at
Santa Barbara, Santa Barbara, California 93106
ReceiVed August 21, 2000
Abstract: A tripyrrole peptide-Hoechst conjugate (FPH-1) has been designed which recognizes nine dA/dT
base pair A/T rich dsDNA sequences at subnanomolar concentrations and complexes its targets at near diffusion
controlled rates to form a fluorescent product. Spectrofluorometric titrations show the stoichiometry of the
complex to be (FPH-1)2:dsDNA. Spectrofluorometric titrations were also employed to determine the product
of the equilibrium constant for complexation (K1K2) of dsDNA by two FPH-1 molecules for 35 different
oligomeric duplexes. Single base pair mismatches in the FPH-1 binding site were found to cause significant
decreases in K1K2 of 18- to 2300-fold. Thermal denaturation experiments provided similar results. Arguments
are presented which favor the structure of the (FPH-1)2:dsDNA minor groove complex to involve the two
FPH-1 molecules in a slightly staggered, side-by-side, and antiparallel arrangement such that the bis-
benzimidazole moiety of one FPH-1 molecule lies adjacent to the tripyrrole moiety of the second FPH-1
molecule.
Introduction
the control of expression of specific genes has spurred efforts
in the development of new minor groove binding agents. It is
expected that those capable of recognizing longer DNA
sequences will exhibit the greatest specificity; however those
few agents targeted to longer DNA sequences have generally
lacked binding specificity.10-19
Organic compounds which bind the minor groove of B-DNA
with sequence selective recognition have drawn considerable
attention.1-6 Studies indicate that these agents may influence
the regulation of gene expression by inhibiting the binding of
regulatory proteins to their DNA binding sites. 1,7-9 Interest in
Minor groove binders such as Hoechst 33258 and tripyrrole
(1) Kielkopf, C. L.; Bremer, R. E.; White, S.; Szewczyk, J. W.; Turner,
J. M.; Baird, E. E.; Dervan, P. B.; Rees, D. C. J. Mol. Biol. 2000, 295,
557-567.
(8) Chiang, S. Y.; Bruice, T. C.; Azizkhan, J. C.; Gawron, L.; Beerman,
T. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2811-2816.
(9) Simon, H.; Kittler, L.; Baird, E.; Dervan, P.; Zimmer, C. FEBS Lett.
2000, 471, 173-176.
(2) Sharma, S. K.; Tandon, M.; Lown, J. W. J. Org. Chem. 2000, 65,
1102-1107.
(10) Trauger, J. W.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1998,
120, 3534-3535.
(3) Xuereb, H.; Maletic, M.; Gildersleeve, J.; Pelczer, I.; Kahne, D. J.
Am. Chem. Soc. 2000, 122, 1883-1890.
(4) Zimmer, C.; Wa¨hnert, U. Prog. Biophys. Mol. Biol. 1986, 47, 31-
112.
(5) Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. Engl. 1996, 35,
1438-1474.
(6) Bailly, C.; Chaires, J. B. Bioconjugate Chem. 1998, 9, 513-538.
(7) Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E. E.; Dervan, P.
B. Nature 1997, 387, 202-205.
(11) Kissinger, K. L.; Dabrowiak, J. C.; Lown, J. W. Chem. Res. Toxicol.
1990, 3, 162-168.
(12) Chen, Y. H.; Yang, Y. W.; Lown, J. W. J. Biomol. Struct. Dyn.
1996, 14, 341-355.
(13) Turner, J. M.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1997,
119, 7636-7644.
(14) Trauger, J. W.; Baird, E. E.; Mrksich, M.; Dervan, P. B. J. Am.
Chem. Soc. 1996, 118, 6160-6166.
10.1021/ja003095d CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/17/2001

2470 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Satz and Bruice
Figure 1. The chemical structures of (FPH-1)2:dsDNA complex, Hoechst 33258, and distamycin.
peptides bind in the minor groove of dsDNA at runs of four or
more A/T base pairs (Figure 1).4 Regions of DNA that are
unusually rich in A/T base pairs play key roles in the structure
and function of the eukaryotic genome. This is partly attributable
to the ability of AT-rich sequences to produce bent DNA or be
readily unwound and unpaired in supercoiled DNA. Importantly,
many nuclear proteins specifically recognize AT-rich DNA
sequences, for example, mammalian HMGI protein and TATA-
box binding protein TBP.20
We report here the study of a tripyrrole peptide-Hoechst
conjugate (FPH-1) designed as a minor groove binding agent
capable of recognizing nine base pair A/T rich DNA sequences
(Figure 1). FPH-1 is fluorescent when bound in the minor groove
of dsDNA. The agent was designed, with the aid of molecular
modeling, to bind DNA as a side-by-side staggered antiparallel
dimer as depicted in Figures 2 and 3.2,21,22 We have investigated
the binding of FPH-1 and Hoechst 33258 to the 18 base pair
double-stranded DNA oligomer 5′-GCGGTATAAAATTC-
GACG-3′ (1) and the 17 base pair oligomer 5′-GCGAATT-
TAATTCGACG-3′ (12). Oligomer 1 contains the TATA box,
which in eukaryotes consists of the consensus sequence 5′-
TATAAAA-3′, and is recognized by the TBP (TATA binding
protein) subunit of TFIID of the RNA polymerase II transcrip-
tion initiation complex.23 Oligomer 12 demonstrates the effect
of substituting adenine and thymine bases within the FPH-1
binding site. Interactions between FPH-1 and 18 different
oligomeric duplexes containing single base pair mismatches of
1 and 12 were investigated to determine sequence selectivity.
Also investigated are the effect of double base pair mismatches
as contained within oligomeric duplexes 11 and 22-24 (Table
1). Last, kinetic investigations show FPH-1 to complex its
dsDNA target at a near diffusion controlled rate.
Experimental Section
Materials. Purified DNA oligomers were purchased from the
Biomolecular Resource Center, University of California at San Fran-
cisco. Hoechst 33258 and 0.05 wt % 3-(trimethylsilyl)propionic-2,2,3,3-
d4 acid, sodium salt in D2O were purchased from Aldrich and used
without further purification. Solvents, and most reagents including
triisopropylsilane (TIS), trifluoroacetic acid (TFA), dichloromethane
(DCM), dimethylformamide (DMF), dicyclohexylcarbodiimide (DCC),
diisopropylethylamine (DIPEA), acetonitrile (ACN), and hydroxyben-
zotriazole (HOBt), were purchased from Aldrich Chemical Co. Some
reagents used for solid-phase chemistry including Rink amide MBHA
resin and PyBOP24 were bought from Novabiochem. The synthesis of
34 has been previously published.25
(15) Kelly, J. J.; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 6981-6985.
(16) Singh, M. P.; Plouvier, B.; Hill, G. C.; Gueck, J.; Pon, R. T.; Lown,
J. W. J. Am. Chem. Soc. 1994, 116, 7006-7020.
(17) Beerman, T. A.; Woynarowski, J. M.; Sigmund, R. D.; Gawron, L.
S.; Rao, K. E.; Lown, J. W. Biochim. Biophys. Acta 1991, 1090, 52-60.
(18) Filipowsky, M. E.; Kopka, M. L.; Brazilzison, M.; Lown, J. W.;
Dickerson, R. E. Biochemistry 1996, 35, 15397-15410.
(19) Lown, J. W.; Krowicki, K.; Balzarini, J.; Newman, R. A.; Declercq,
E. J. Med. Chem. 1989, 32, 2368-2375.
Procedures (DNA Binding Experiments). Oligomeric duplexes
were formed by annealing complementary oligomers by heating equal
molar mixtures to 95 °C for 10 min and slowly cooling to ambient
temperature. Molar extinction coefficients for oligomeric duplexes were
(23) Latchman, D. S. Gene regulation: a eukaryotic perspectiVe, 3rd ed.;
Stanley Thornes: Cheltenham, 1998.
(20) Forde, B. G. Plant promoters and transcription factors; Nover, L.,
Ed.; Springer-Verlag: Berlin; New York, 1994; pp 87-103.
(21) Kopka, M. L.; Goodsell, D. S.; Han, G. W.; Chiu, T. K.; Lown, J.
W.; Dickerson, R. E. Structure 1997, 5, 1033-1046.
(24) Coste, J.; Lenguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205-
208.
(25) Satz, A. L.; Bruice, T. C. Bioorg. Med. Chem. 2000, 8, 1871-
1880.
(22) Wemmer, D. Nat. Struct. Biol. 1998, 5, 169-171.

Base Pair Recognition by Peptide-Hoechst Conjugate
Table 1. Product of Equilibrium Association Constants for
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2471
Complexation (K1K2 × 10-14 M-2
)
FPH-1
Ht33258
5200
A. 18 Base Pair dsDNA Oligomersa,b
5′-GCGGTATAAAATTCGACG-3′ (1)
37 000
1400
940
900
2100
16
1300
620
620
1300
2
5′-GCGGCATAAAATTCGACG-3′ (2)
5′-GCGGTGTAAAATTCGACG-3′ (3)
5′-GCGGTACAAAATTCGACG-3′ (4)
5′-GCGGTATGAAATTCGACG-3′ (5)
5′-GCGGTATAGAATTCGACG-3′ (6)
5′-GCGGTATAAGATTCGACG-3′ (7)
5′-GCGGTATAAAGTTCGACG-3′ (8)
5′-GCGGTATAAAACTCGACG-3′ (9)
5′-GCGGTATAAAATCCGACG-3′ (10)
5′-GCGGTATAGGAATTCGCG-3′ (11)
1634
3800
3100
B. 17 Base Pair dsDNA Oligomers
5′-GCGAATTTAATTCGACG-3′ (12)
5′-GCGGATTTAATTCGACG-3′ (13)
5′-GCGAGTTTAATTCGACG-3′ (14)
5′-GCGAACTTAATTCGACG-3′ (15)
5′-GCGAATCTAATTCGACG-3′ (16)
5′-GCGAATTCAATTCGACG-3′ (17)
5′-GCGAATTTGATTCGACG-3′ (18)
5′-GCGAATTTAGTTCGACG-3′ (19)
5′-GCGAATTTAACTCGACG-3′ (20)
5′-GCGAATTTAATCCGACG-3′ (21)
5′-GCGAATTCCAATTGACG-3′ (22)
5′-GCGACTTCAATTCGACG-3′ (23)
5′-GCGGATTGAATTCGACG-3′ (24)
225 000
7500
1800
1900
321
512
400
290
150
500
25
2500
9200
31
31
a Determined by nonlinear least-squares fitting of an isothermal
binding curve. Equilibrium constants are given as the product of K1K2
for 2:1 ligand:DNA stoichiometries since separation of the individual
equilibrium constants is not possible. The stated equilibrium constants
have a standard deviation of (60%. b The FPH-1 binding site is
underlined. Mismatch sites are typed in bold face.
approximated using A260 ) 16 800 M-1 (G/C base pair)-1 and A260
)
13 600 M-1 (A/T base pair)-1. Solutions of known ligand concentrations
were prepared via peak integration of the ligand’s NMR spectra where
the ligand samples used contained a known quantity of the internal
standard 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt.25 UV/
vis spectra were acquired on a Cary 100 Bio UV-vis spectrophotometer
equipped with a temperature programmable cell block. All thermal
melting experiments were carried out using 10 mM potassium
phosphate, 150 mM, pH 7.0 buffer. For each series of thermal melting
curves acquired using a particular oligomeric duplex (for example,
1 + no ligand 1 + Ht33258 1 + FPH-1), dsDNA concentrations were
kept constant and 2 equiv of ligand was added. The dsDNA concentra-
tions employed ranged from 0.25 to 0.42 µM. Data points were taken
every 1 °C with a temperature ramp of 0.5 °C min-1. Thermal melting
temperatures were calculated by first-derivative analysis. Fluorescence
spectra were obtained on a Perkin-Elmer LS50B fluorimeter equipped
with a constant temperature water bath set at 26 °C. Solutions were
excited at 345 nm. Emissions were monitored at 450 or 470 nm.
Spectrofluorometric titrations were obtained by titrating a constant
concentration of DNA, usually between 1 and 500 nM, with a relatively
concentrated solution of ligand.
Figure 2. Computer-generated model of proposed (FPH-1)2:dsDNA
side-by-side antiparallel staggered complex.
0.5Kl[L]f + K1K2[L]f2
F ) ΣΦf
(1)
(2)
2
(
)
1 + K1[L]f + K1K2[L]f
[L]Bound
f n[DNA]T
F ) ΣΦ
In eq 1 ΣΦf is the total fluorescence intensity upon saturation of dsDNA
binding sites with ligand, K1 and K2 are the equilibrium association
constants for the first and second binding events, and [L]f is the
concentration of ligand free in solution. In eq 2 [L]Bound is the
concentration of ligand bound to dsDNA, n is the stoichiometry of
binding, and [DNA]T is the total concentration of duplex DNA in the
sample.
For generation of meaningful isothermal binding curves, the general
rule is that [DNA]T ) 1/K1, where K1 is the equilibrium constant for
complexation of dsDNA in cases where the binding stoichiometry is
1:1. With 2:1 ligand:dsDNA stoichiometries, the appropriate concentra-
tion of dsDNA can be roughly approximated by taking the square root
of the product of the equilibrium association constants (K1K2), such
that [DNA] ) 1/(K1K2)1/2. That the concentration of dsDNA employed
is appropriate must be verified visually by plotting the fluorescence
signal vs [ligand]t, where [ligand]t is the total concentration of ligand
added to the dsDNA solution. The plot must show a significant
curvature to be a usable isothermal binding curve. At higher concentra-
tions of dsDNA, these plots effectively yield two straight lines which
are useful in determining ligand:dsDNA stoichiometries but not
equilibrium constants. Isothermal binding curves for dsDNA oligomers
such as 1 and 12, which contain the preferred binding site for FPH-1,
Following determination of ligand:dsDNA complex stoichiometries,
equilibrium constants for dsDNA complexation were investigated by
generating isothermal binding curves via spectrofluorometric titrations
(titration of a dilute dsDNA solution with ligand). Equilibrium constants
for 2:1 ligand:dsDNA stoichiometries were calculated by fitting
isothermal binding curves using eqs 1 and 2 (Figure 5).25 Equation 1
was employed to fit plots of fluorescence vs concentration of unbound
ligand ([L]f), where [L]f is calculated by eq 2. The derivation and use
of eqs 1 and 2 have been discussed by our laboratory.25,26 Table 1 lists
the calculated equilibrium constants for complexation.
(26) Browne, K. A.; He, G. X.; Bruice, T. C. J. Am. Chem. Soc. 1993,
115, 7072-7079.

2472 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Satz and Bruice
product was then further purified by flash chromatography (silica, 1:1
DCM:hexanes) to provide 26 with only minor impurities as judged by
NMR (267 mg, 20% yield): TLC (1:1 DCM/hexanes) Rf 0.2; 1H NMR
(CDCl3) δ 1.55 (s, 9H, (CH3)3C), 3.9 (s, 3H, CH3-NPy), 7.33 (bd, 1H,
Ar H), 7.55 (m, 1H, Ar H); LREI m/e 226 (M•+ 226.23), 170, 153,
140.
tert-Butyl 4-Amino-1-methylpyrrole-2-carboxylate. Without fur-
ther purification 26 (230 mg, 1 mmol) was dissolved in 30 mL of
methanol to which 60 mg of 10% palladium on charcoal catalyst was
added. The solution was stirred underneath an atmosphere of hydrogen
gas (∼1 atm) for 3 days by which time TLC (silica, DCM) showed no
remaining starting material. The solution was filtered through Celite
and the filtrate evaporated to give crude amine with only minor
impurities and no left over starting material as judged by 1H NMR. 1H
NMR (CDCl3) δ 1.52 (s, 9H, (CH3)3C), 3.78 (s, 3H, CH3-NPy), 6.30
(d, J ) 2.2 Hz, 1H, Ar H), 6.40 (d, J ) 2.2 Hz, 1H, Ar H) ppm.
tert-Butyl 4-[(9-Fluorenylmethoxycarbonyl)amino]-1-methylpyr-
role-2-carboxylate (29). Without further purification tert-butyl 4-amino-
1-methylpyrrole-2-carboxylate was then dissolved in 40 mL of dioxane,
20 mL of saturated NaHCO3, and 5 mL of methanol to which was
added FMOC-Cl (259 mg, 1 mmol). The colloidal solution was stirred
at room temperature for 24 h. The product was extracted into diethyl
ether and washed several times with water. The crude product was
purified by flash chromatography (silica, 2.5:97.5 ethyl acetate:DCM)
to provide 29 as a shiny tan/white solid (130 mg, 55% yield): TLC
(2.5:97.5 ethyl acetate/DCM) Rf 0.4; 1H NMR (CDCl3) δ 1.55 (s, 9H,
(CH3)3C), 3.85 (s, 3H, CH3-NPy), 4.26 (t, J ) 6.59 Hz, 1H, Ph2-CHR),
4.48 (d, J ) 6.78 Hz, 2H, -CH2OC(dO)N-), 6.51 (bs obscured by
CHCl3 absorption, -(OdC)NH-), 6.94 (s, 1H, pyrrole Ar H), 7.00
(s, 1H, pyrrole Ar H), 7.312 (t, J ) 6.78 Hz, 2H, FMOC Ar H), 7.41
(m, 2H, FMOC Ar H), 7.61 (bd, J ) 7.33 Hz, 2H, FMOC Ar H), 7.77
(d, J ) 7.69 Hz, 2H, FMOC Ar H) ppm; IR (type 61 3M IR card)
2957, 2911, 1697, 1588, 1440, 1244, 1153 cm-1; HRMS (FAB)
418.1886 (418.1892 calcd for C25H26N2O4).
Phenyl 4-Nitro-1-methylpyrrole-2-carboxylate (27). Compound 25
(2 g, 11.8 mmol), phenol (1.1 g, 11.8 mmol), and DCC (2.6 g, 12.6
mmol) were dissolved in 20 mL of DMF and stirred at room
temperature for 24 h. Precipitated dicyclohexylurea was removed by
filtration and solvent was then removed by evaporation under vacuum.
Product was then dissolved in DCM and washed several times with
saturated NaHCO3. The organic layer was then dried with Na2SO4
before evaporation of solvent to give crude product mixture. Product
was purified by flash chromatography (silica, 90:10 DCM/ethyl acetate)
to provide 27 as tan powder (664 mg, 23% yield): TLC (90:10 DCM/
ethyl acetate) Rf 0.30; 1H NMR (CDCl3) δ 4.04 (s, 3H, CH3-NPy), 7.17-
7.2 (m, 2H, Ar H), 7.28-7.32 (m, 1H, Ar H), 7.42-7.47 (m, 2H, Ar
H), 7.68-7.70 (m, 2H, Ar H); LREI m/e 246 (M•+), 153, 137, 107,
79.
Figure 3. View down the long axis of the (FPH-1)2:dsDNA complex
shown in Figure 2. The dsDNA atoms are not shown in order to
illustrate the curvature of the two side-by-side binding FPH-1 molecules.
were generated at dsDNA concentrations as low as 1 nM. Equilibrium
constants are given as the product of K1K2 since separation of the
individual equilibrium constants is not possible (due to their similarity
in magnitude). Multiple assays gave a standard deviation of roughly
(60% for those values stated in Table 1. Titrations of the 18-mer (Table
1a) and 17-mer (Table 1b) oligomeric duplexes were accomplished in
150 and 10 mM NaCl buffers, respectively.
Stop-flow fluorimetry was accomplished using an Applied Photo-
physics stop-flow spectrophotometer. Samples were excited at 260
nm. Fluorescence emission was monitored by employing a 320 nm
interference filter. Measurements were taken in 10 mM potassium
phosphate, 10 mM NaCl, pH 7.2 buffer. Each data point shown in
Figure 7 is the average of at least three trial runs. Second-order rate
constants were determined as the slope through plots of kobs vs [ligand]
(see Figure 8a). Error values were determined by first calculating the
second-order rate constants for each individual data point and then
finding the standard deviation of those values from the slope of the
plots of kobs vs [L]. First-order off rates were determined from the
y-intercept at [L] ) 0 (Figure 8a).
Phenyl 4-[(9-Fluorenylmethoxycarbonyl)amino]-1-methylpyrrole-
2-carboxylate (30). Compound 27 (134 mg, 0.54 mmol) was dissolved
in 20 mL of ethyl acetate to which 30 mg of 10% palladium on carbon
catalyst was added. The solution was stirred under an atmosphere of
hydrogen gas (∼1 atm) for 48 h at which time, as estimated by TLC
(silica, DCM/methanol), the reaction was mostly complete. The solution
was filtered though Celite and evaporated to give crude product. The
1H NMR of the crude product indicated minor impurities with very
little left over starting material. TLC (100% DCM) Rf ∼ 0.00; LREI
m/e 216 (M•+ 216.23, calcd for C12H12N2O2), 123, 95. Without further
purification the crude product was dissolved in 2 mL of saturated
NaHCO3 and 4 mL of dioxane to which was added FMOC-Cl (112
mg, 0.43 mmol). The colloidal reaction mixture was stirred for 24 h.
Product formation was monitored by TLC (silica, 6:0.5:3.5 DCM/ethyl
acetate/hexanes). The reaction mixture was diluted with diethyl ether
and washed several times with water. The organic layer was then dried
using Na2SO4 and evaporated. The crude product was then purified by
flash chromatography (silica, 6:0.5:3.5 DCM/ethyl acetate/hexanes) to
give 30 (124 mg, 52% yield): TLC (silica, 6:0.5:3.5 DCM/ethyl acetate/
Procedures (Organic Synthesis). General. 1H and 13C NMR spectra
were obtained on a Varian Unity Inova 400 or 500 spectrometer at
400 MHz and 100 or 150 MHz, respectively. TLC was carried out on
silica gel (KIESELGER 60 F254) glass backed commercial plates and
visualized by UV light. Fast atom bombardment mass spectra, HRMS
and LRMS, were obtained on a VG analytical, VG-70E double focusing
mass spectrometer, with an Ion Tech Xenon Gun FAB source, and an
OPUS/SIOS data interface and acquisition system. High-pressure liquid
chromatography was accomplished using a Hewlett-Packard Series 1050
HPLC equipped with a diode array detector. For preparative separations
an Alltech Macrosphere 300A, C8, silica, 7 µm, 250 mm × 10 mm
reverse phase column was used. For analytical separations an Alltech
Macrosphere 300A, C18, silica, 7 µm, 250 mm × 4.6 mm reverse phase
column was used.
tert-Butyl 4-Nitro-1-methylpyrrole-2-carboxylate (26). Compound
25 (1 g, 5.9 mmol) was added to 40 mL of diethyl ether and 2 mL of
concentrated sulfuric acid in a round-bottom flask. The colloidal solution
was cooled to -40 °C, and a steady stream of isobutylene was bubbled
through the solution for several minutes. The solution was capped tightly
with a rubber septa and copper wire, allowed to warm to room
temperature, and stirred for 24 h. The crude reaction mixture was
washed repeatedly with saturated NaHCO3 and 1 M Na2CO3. Crude
1
hexanes) Rf 0.55; H NMR (CDCl3) δ 3.90 (s, 3H, CH3-NPy), 4.29 (t,
J ) 6.41 Hz, 1H, Ph2-CHR), 4.54 (d, J ) 6.59 Hz, 2H, -CH2OC-
(dO)N-), 6.74 (s, 1H, pyrrole Ar H), 6.91 (s, 1H, pyrrole Ar H), 7.17-
7.19 (m Ar H), 7.23-7.28 (m obscured by CHCl3 absorption, Ar H),

Base Pair Recognition by Peptide-Hoechst Conjugate
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2473
7.32-7.37 (m, Ar H), 7.39-7.47 (m, Ar H), 7.64 (m, 2H, FMOC Ar
H) ppm, 7.80 (d, J ) 7.50 Hz, 2H, FMOC Ar H) ppm; LRMS (CI/
CH4) m/e 438 (M•+).
318 nm, λ(min) ) 284 nm (FPH-1 was observed to obey the Beer-
Lambert law at µM concentrations in contrast to Hoechst 33258 which
does not27).
Pentafluorophenyl 4-Nitro-1-methylpyrrole-2-carboxylate (28).
Compound 25 (1 g, 5.9 mmol), pentafluorophenol (1.08 g, 5.9 mmol),
and DCC (1.4 g, 5.9 mmol) were dissolved in 20 mL of DMF and
stirred at room temperature for 4 h. The disappearance of compound
25 was monitored by TLC (100% DCM). The solvent was removed
by evaporation under vacuum and the crude product mixture recon-
stituted in diethyl ether. Precipitate, including dicyclohexylurea, was
removed by filtration, and the solvent was evaporated to give a sticky
brown mixture. Purification was achieved by flash chromatography
(silica, 1:1 DCM/hexanes) to provide 28 as a fluffy white powder (1.4
g, 71% yield): TLC (silica, 1:1 DCM/hexanes) Rf 0.4; 1H NMR (CDCl3)
δ 4.05 (s, 3H, CH3-NPy), 7.75 (bd, 1H, pyrrole Ar H), 7.77 (m, 1H,
pyrrole Ar H); 13C NMR (CDCl3) δ 38.297 (C-NPy), 3 signals were
detected in the aromatic region 115.793 + 119.799 + 129.579, 155.950
(Py-C(dO)-) ppm; IR (type 61 3M IR card) 3140, 2918, 2847, 1758,
1519, 1322, 1042 cm-1; LRMS (FAB) 336 (M + H)+.
Results
The synthesis of FPH-1 was accomplished in a stepwise
manner from MBHA rink amide resin by employing FMOC
chemistry and standard manual solid-phase synthetic techniques
(Scheme 1).28 It should be noted that the synthesis of 32 and
its use in the solid-phase synthesis of a polyamide has been
previously communicated.29 However, we were unable to
synthesize the FMOC-protected monomer 32 via the reported
catalytic hydrogenation of 25. Esterified products 26 and 27,
unlike 25, were found to reduce cleanly by catalytic hydrogena-
tion. Amino pyrroles are well known for their instability, making
it difficult to purify them.30 The unpurified amines of 26 and
27 were protected with FMOC-Cl, giving 29 and 30. Unfortu-
nately, it was not possible to hydrolyze either molecule. The
FMOC protecting group was not stable to the harsh conditions
needed to hydrolyze 30. Deprotection of 30 with hydrogen
peroxide was also unsuccessful.31 The tert-butyl esters of pyrrole
carboxylates such as 26 and 29 are sometimes easily removed
by trifluoroacetic acid.30,32 However, deprotection of 29 occurred
only in concentrated sulfuric acid and in extremely low yields
due to decomposition of starting material or possibly decar-
boxylation of the product. Pyrrole carboxylic acids are known
to readily decarboxylate when heated.30,33 Pentafluorphenyl
esters are widely used in the solid-phase synthesis of peptides;
still the pentafluorphenyl pyrrole carboxylate 28 was found to
be fairly stable, reducing cleanly without any traces of hydrolysis
or polymerization.34 Coupling of 31 to the primary amine of
the MBHA rink amide resin proceeded very slowly, giving onlya
10% yield after 24 h. Yields were increased greatly by the
addition of HOBt. Coupling reactions were monitored by the
UV absorption of the deprotected FMOC. The traditional Kaiser
test is not compatible with the aromatic amine of the pyrrole
ring.35 Coupling yields between pyrrole units were observed to
be between 80 and 100%.
Pentafluorophenyl 4-[(9-Fluorenylmethoxycarbonyl)amino]-1-
methylpyrrole-2-carboxylate (31). Compound 28 (700 mg, 2.1 mmol)
was dissolved in 100 mL of 1:1 ethyl acetate/ethanol. To this solution
was added 2 mL of saturated NaHCO3 and 80 mg of 10% palladium
on carbon catalyst. The solution was stirred under an atmosphere of
hydrogen gas (∼1 atm) for 48 h. Reaction progress was monitored by
TLC. The solution was filtered through Celite and the filtrate reduced
to a volume of ∼10 to 20 mL. The reduced filtrate was combined with
25 mL of dioxane, an additional 5 mL of saturated NaHCO3, and
FMOC-Cl (596 mg, 2.2 mmol). The solution was stirred for 16 h at
room temperature. Product was extracted into diethyl ether and the
organic layer washed several times with water. Product was purified
by flash chromatography (silica, 4:6:0.5 DCM/hexanes/ethyl acetate)
to give 31 as a shiny white powder (837 mg, 75% yield). TLC (silica,
1
4:6:0.5 DCM/hexanes/ethyl acetate) Rf 0.2; H NMR (CDCl3) δ 3.90
(s, 3H, CH3-NPy), 4.27 (t, J ) 6.41 Hz, 1H, Ph2-CHR), 4.53 (d, J )
6.59 Hz, 2H, -CH2OC(dO)N-), 6.56 (s, 1H, pyrrole Ar H), 6.96 (s,
1H, pyrrole Ar H), 7.27 (bs obscured by CHCl3 absorption, -(OdC)-
NH-), 7.33 (m, 2H, FMOC Ar H), 7.42 (t, J ) 7.51 Hz, 2H, FMOC
Ar H), 7.61 (bd, J ) 7.50 Hz, 2H, FMOC Ar H), 7.78 (d, J ) 7.51 Hz,
2H, FMOC Ar H) ppm; 13C NMR (CDCl3) δ 37.06 (C-NPy), 47.33
(-COC(dO)N-), 67.23 (-CCOC(dO)N, the following 10 signals
were detected in the aromatic region 110.775 + 116.784, 120.266 +
122.739 + 123.088 + 125.106 + 127.337 + 128.027 + 141.562 +
143.868, 153.898 (-C(dO)N-), 156.348 (Py-C(dO)-) ppm; IR (type
Ligand:dsDNA complex stoichiometries were determined by
spectrofluorometric titration.25 Similar to Hoechst 33258, the
fluorescence emission of FPH-1 also increases greatly upon
complexation of dsDNA. Upon excitation at 345 nm, FPH-1:
dsDNA complexes emit a broad fluorescence signal centered
at 470 nm, red shifted ∼20 nm with respect to Hoechst 33258.
Generally Hoechst 33258:dsDNA complexes are excited near
350 nm and emit a broad fluorescence signal centered at 450
nm.26,27,37-40 All of the oligomeric duplexes investigated (Table
61 3M IR card) 2920, 1743, 1589, 1520, 1448, 1221, 1032, 742 cm-1
HRMS (FAB) 528.1105 (528.1108 calcd for C27H17N2O4F5 ).
;
FPH-1. The solid-phase synthesis of FPH-1 was accomplished using
MBHA rink amide resin (53 mg, 0.0265 mmol loading sites) and
standard manual solid-phase FMOC techniques. Coupling reactions
were accomplished using 2.5 equiv of 31, 2 equiv of HOBt, and 4
equiv of DIPEA in anhydrous DMF and were run for 24 h. High
coupling yields between 70 and 100% were measured by absorption at
290 nm of deprotected FMOC after resin was treated with a 20%
piperidine/DMF solution. Coupling of 34 to the growing polyamide
chain was accomplished using 2.5 equiv of 34, 2.5 equiv of PyBOP,
and 8 equiv of DIPEA for 24 h. Resin cleavage was achieved in 4 h
using a 95% TFA, 2.5% water, and 2.5% TIS solution. Product was
purified by HPLC with an increasing gradient of acetonitrile in 0.1%
aqueous TFA solution. After purification the product was precipitated
out of a methanol/ether solution with anhydrous HCl gas. Product purity
was shown by analytical HPLC. Yield was determined by NMR
spectroscopy (as mentioned above) to be 3.0 × 10-6 mol (11% yield):
1H NMR (CDCl3) δ 2.12 (m, 2H, -CH2COPh), 2.5 (overlapped by
DMSO solvent peak, -CH2OPh), 2.90 (s, 3H, CH3-NR2), 3.82 (s,
overlaps HOD absorption, CH3-NPy), 3.85 (s, overlaps HOD absorption,
CH3-NPy), piperazine ring CH2 signals are hidden by HOD absorption,
4.18 (t, overlaps HOD absorption, J ) 5.74 Hz, NC(dO)CH2-), signals
detected between 7 and 9 ppm are due to Ar H and carbamate protons,
7.15 (m) + 7.23 (m) + 7.53 (bt) + 7.66 (m) + 7.85 (s) + 8.11 (m) +
8.52 (s) ppm; LRMS (FAB) 878 (M + H)+1. Fluorescence emission
spectrum: broad peak centered at 475 nm. UV spectrum: λ(max) )
(27) Loontiens, F. G.; Regenfuss, P.; Zechel, A.; Dumortier, L.; Clegg,
R. M. Biochemistry 1990, 29, 9029-9039.
(28) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-
214.
(29) Vazquez, E.; Caamano, A. M.; Castedo, L.; Mascarenas, J. L.
Tetrahedron Lett. 1999, 40, 3621-3624.
(30) Jones, R. A. Pyrroles; John Wiley and Sons, Inc.: New York, 1992;
Vol. 48, pp 315-316.
(31) Kenner, G. W.; Seely, J. H. J. Am. Chem. Soc. 1971, 94, 3259-
3260.
(32) Crook, P. J.; Jackson, A. H.; Kenner, G. W. J. Chem. Soc. C 1971,
474-487.
(33) Joule, J. A.; Mills, K.; Smith, G. F. Heterocyclic Chemistry, 3rd
ed.; Chapman and Hall: London, 1995; pp 244-245.
(34) Kisfaludy, L.; Schon, I. Synthesis 1983, 325-327.
(35) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal.
Biochem. 1981, 117, 147-157.
(36) Chadwick, D. J.; Chambers, J.; Meakins, D. G.; Snowden, R. L. J.
Chem. Soc., Perkin Trans. I 1973, 1766-1773.
(37) He, G. X.; Browne, K. A.; Blasko, A.; Bruice, T. C. J. Am. Chem.
Soc. 1994, 116, 3716-3725.

2474 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Satz and Bruice
Scheme 1
Figure 5. Isothermal binding curve generated by spectrofluorometric
titration of 3 nM 12 with FPH-1. Plots show relative fluorescence
intensity in arbitrary units vs concentration unbound FPH-1 as calculated
by eq 2. Data points were fit using eq 1.
Figure 4. Titration of 33 µM 12 with FPH-1. Plots shows relative
fluorescence intensity at 475 nm in arbitrary units vs ligand:DNA ratio.
The two straight lines were generated by linear least squares fitting of
data points early and late in the titration. The point of intersection of
the two lines over the x-axis provides the stoichiometry of the ligand:
DNA complex.
Equilibrium association constants for complexation of dsDNA
were determined via spectrofluorometric titrations (see Table 1
and Figure 5). FPH-1 is highly selective for its nine base pair
target site, whereas Hoechst 33258 shows no such selectivity.
For example, whereas equilibrium association constants (K1K2)
for formation of Hoechst 33258 complexes with 1 and 11 are
nearly equal (oligomers 1 and 11 both contain two separate and
nonoverlapping binding sites for Hoechst 33258), the equilib-
rium constants for formation of FPH-1 complexes with 1 and
11 differ by greater than 4 orders of magnitude. The same is
true for oligomers 12 and 22 whose equilibrium constants for
complexation by Hoechst 33258 are again nearly equal while
those for complexation of 12 and 22 by FPH-1 differ by 9000-
fold (oligomers 11 and 22 both contain double base pair
mismatches). Similar comparison of equilibrium constants for
single base pair mismatch oligomers 6 and 17 also demonstrate
the selectivity of FPH-1. All single base pair mismatches of
oligomers 1 and 12 cause significant decreases in K1K2 ranging
from 18- to 2300-fold.
1) were determined to form (FPH-1)2:dsDNA complexes.
Additionally, all oligomeric duplexes whose equilibrium as-
sociation constants for complexation by Hoechst 33258 are listed
in Table 1 were also found to form (Ht33258)2:dsDNA
complexes. In contrast, the oligomeric duplex 23 was determined
to form a Ht33258:23 complex with a 1:1 stoichiometry (no
equilibrium association constant is given in Table 1 for the
Ht33258:23 complex). Figure 4 shows a representative example
of a plot used to determine FPH-1:dsDNA stoichiometry.
(38) Bostock-Smith, C. E.; Searle, M. S. Nucl. Acids Res. 1999, 27,
1619-1624.
(39) Haq, I.; Ladbury, J. E.; Chowdhry, B. Z.; Jenkins, T. C.; Chaires,
J. B. J. Mol. Biol. 1997, 271, 244-257.
(40) Loontiens, F. G.; McLaughlin, L. W.; Diekmann, S.; Clegg, R. M.
Biochemistry 1991, 30, 182-189.

Base Pair Recognition by Peptide-Hoechst Conjugate
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2475
Figure 6. Ionic strength dependence of the equilibrium constants for
association of FPH-1 (4) and Hoechst 33258 (O) with oligomeric
duplex 1.
Table 2. Melting Temperatures for Ligand:DNA Complexes (°C)a
∆tm
∆tm
DNA oligomers
t0
m
FPH-1 Ht33258
5′-GCGGTATAAAATTCGACG-3′ (1)
5′-GCGGCATAAAATTCGACG-3′ (2)
5′-GCGGTGTAAAATTCGACG-3′ (3)
5′-GCGGTACAAAATTCGACG-3′ (4)
5′-GCGGTATGAAATTCGACG-3′ (5)
5′-GCGGTATAGAATTCGACG-3′ (6)
5′-GCGGTATAAGATTCGACG-3′ (7)
5′-GCGGTATAAAGTTCGACG-3′ (8)
5′-GCGGTATAAAACTCGACG-3′ (9)
5′-GCGGTATAAAATCCGACG-3′ (10) 61
5′-GCGGTATAGGAATTCGCG-3′ (11) 60
5′-GCGAATTTAATTCGACG-3′ (12)
5′-GCGAATTCCAATTGACG-3′ (22)
60
63
62
61
62
61
60
61
62
10
6
4
4
2
1
3
2
2
6
0
13
1
5
3
3
3
3
2
2
2
2
2
3
6
3
Figure 7. Thermal melting curves for oligomers 1 (0.28 µM, panel
A) and 11 (0.37 µM, panel B) and their ligand (2 equiv) complexes:
DNA + FPH-1 ()), DNA + Hoechst 33258 (×), and DNA with no
ligand (O).
58
59
a tm values for oligomer complexes were determined by first
derivative analysis. t0m are melting temperatures of oligomeric duplexes
in pH 7 buffer containing 150 mM NaCl in the absence of ligand. ∆tm
are differences in melting temperatures for oligomeric duplexes in the
absence and presence of ligand. Standard deviation for ∆tm values are
(1 °C.
also have ∆tm values of only 0 and 1 °C, respectively. Hoechst
33258 raises the tm for all the oligomers investigated by 2-6
°C.
Kinetics for the formation of FPH-1 and Hoechst 33258:12
complexes were investigated by stop-flow fluorimetry.41 The
reactions were determined to be first order with respect to [L]
and [12]. Reaction order was determined by plotting pseudo-
first-order rate constants (excess ligand) vs [L] or [DNA] as
shown in Figure 8. The second-order rate constants were
determined to be 6 ( 2 × 107 and 5 ( 2 × 108 s-1 M-1 for
FPH-1 and Hoechst 33258, respectively, indicating complexation
of 12 at near diffusion controlled and diffusion controlled rates,
respectively. The first-order off rates were determined to be <1
and 20 s-1 for FPH-1 and Hoechst 33258, respectively.
The ionic strength dependence of the equilibrium constant
for association of FPH-1 with the oligomeric duplex 1 was
investigated. Figure 6 shows plots of log ( K1K2)1/2 vs log [NaCl]
for both FPH-1 and Hoechst 33258. The affinity of both ligands
for 1 decreases with ionic strength: ∂ log ( K1K2)1/2/ ∂ log [NaCl]
are -0.2 and -0.3 for FPH-1 and Hoecsht 33258, respectively.
The log of the square root of K1K2 is plotted vs log [NaCl]
because the resulting slope is unitless. In constrast, a plot of
log K1K2 vs log [NaCl] results in a slope with units M-1. A
unitless slope is needed to be able to compare our values with
those previously reported for Hoechst 33258 from plots of ∂
log K/∂ log [NaCl].27
Discussion
We sought to design a minor groove binding agent which
would recognize an extended sequence of base pairs and have
ready entrance to cells and the nucleus. The design of FPH-1
allows for easy observation of invivo and invitro reactions with
dsDNA by fluorescence spectroscopy. The biological activity
of FPH-1 is currently under investigation including the use of
fluorescence microscopy to investigate cellular localization and
uptake (fluorescence microscopy has been previously employed
to show the selective localization in nuclear DNA of certain
bis-bezimidazole type compounds42). Spectrofluorometric titra-
tions were employed to investigate stoichiometries and equi-
librium constants for formation of FPH-1:dsDNA complexes.
The technical benefit gained by employing a molecule which
The thermal stability of (FPH-1)2:dsDNA complexes were
investigated by thermal denaturation experiments. As shown in
Table 2, FPH-1 forms significantly more stable complexes with
oligomers 1 and 12 than does Hoechst 33258 (Figure 7). The
effect of FPH-1 on ∆tm (the difference between tm values for
oligomeric duplexes in the absence and presence of ligand)
decreases greatly for oligomers 2-10 which all contain single
base pair mismatches. ∆tm values tend to be more stronglyaf-
fected when the base pair mismatch does not reside at either
end of the FPH-1 binding site. FPH-1 had almost no effect on
t
m for many of the oligomers listed in Table 2 which indicates
that it binds oligomeric duplexes which contain base pair
mismatches weakly. For instance, oligomer 6 which contains a
single base pair mismatch has a ∆tm value of only 1 °C.
Oligomers 11 and 22 contain double base pair mismatches and
(41) Baliga, R.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
7814-7818.
(42) Harapanhalli, R. S.; Howell, R. W.; Rao, D. V. Nucl. Med. Biol.
1994, 21, 641-647.

2476 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Satz and Bruice
oligomers 1 and 12. Generally, the negative effect of single base
pair mismatches on K1K2 is greater when the position of the
mismatched base pair resides in the center of the FPH-1 binding
site. Comparison of equilibrium constants, K1K2, listed in Table
1 shows that the overall effect of a base pair mismatch is
equivalent for oligomers 1 and 12. As shown in Table 1 FPH-1
shows a very slight preference (∼5-fold) for oligomer 12 over
1 but overall shows no general ability to differentiate between
two different A/T rich binding sites. The few K1K2 values
determined for Hoechst 33258 are similar in magnitude (within
a factor of 10). Hoechst 33258 shows no selectivity for longer
DNA binding sites. Additionally, K1K2 values for FPH-1
complexes with 1 and 12 are significantly greater than for their
respective Hoechst 33258 complexes (by factors of 10- and 70-
fold, respectively).
The ionic strength dependence of the equilibrium constants
for association of FPH-1 and Hoechst 33258 with 1 are roughly
equivalent with slopes of -0.3 and -0.2, respectively (see
Figure 6). The effect of increasing NaCl concentrations by a
factor of 10 is calculated to decrease K1K2 values by a factor
of 2 to 3 for either ligand. The ionic strength dependence of
the equilibrium constant for association of Hoecsht 33258 with
poly[d(A-T)] has been previously reported to be ∂ log K/∂ log
[NaCl] ) -0.76.27 This indicates that the ionic strength
dependence of the equilibrium constant for assocation of
Hoecsht 33258 with DNA appears to vary significantly depend-
ing upon the type of DNA being investigated.
Thermal denaturation experiments were employed as an
alternative method for investigation of FPH-1 sequence selectiv-
ity. As indicated in Table 2, FPH-1 selectively stabilizes
oligomeric duplexes which contain a nine base pair A/T rich
binding site. Parallel to decreases in K1K2 values, single base
pair mismatches within the FPH-1 binding site cause large
decreases in ∆tm values. For instance, oligomer 6 contains a
single base pair mismatch and has a ∆tm value of only 1 °C.
Additionally, oligomers 11 and 22 contain double base pair
mismatches and have ∆tm values of only 0 and 1 °C,
respectively. In contrast, Hoechst 33258 ∆tm values are fairly
similar for all the oligomers investigated. Thus, as measured
by differences in K1K2 and in ∆tm values, FPH-1 is able to
distinguish between oligomeric duplexes indistinguishable by
Hoechst 33258.
Figure 8. Plots of pseudo-first-order rate constants for complexation
of DNA by excess ligand determined via stopped-flow fluorimetry.
Panel A: addition of excess Hoechst 33258 (b) or FPH-1 ()) to 100 or
500 nm 9, respectively. Panel B: addition of 1000 nM Hoechst 33258
(b) or 3000 nM FPH-1 ()) to varying concentrations of 9.
fluoresces upon binding dsDNA is shown by the long list of
oligomeric duplexes we have been able to investigate (Table
1). In contrast, literature reports of nonfluorescent minor groove
binders are often limited to investigation of one or two dsDNA
binding sites which make it difficult to asses details of sequence
specificity.10-16,18,19
All of the oligomeric duplexes investigated (Table 1) were
determined to form (FPH-1)2:dsDNA complexes. Additionally,
all oligomeric duplexes whose equilibrium association constants
for complexation by Hoechst 33258 are listed in Table 1 were
also found to form (Ht33258)2:dsDNA complexes. The struc-
tures of the (FPH-1)2:dsDNA and (Ht33258)2:dsDNA complexes
are quite different. The observed 2:1 FPH-1:dsDNA complex
stoichiometries suggest the formation of side-by-side trimeric
complexes as depicted in Figure 2. This will be discussed later.
Consistent with known characteristics, each Hoechst 33258
molecule in a (Ht33258)2:dsDNA complex resides in a four dA/
dT base pair region such that the two Hoechst molecules are
arranged end-to-end and do not overlap each other.43,44 The
stoichiometry of Ht33258:dsDNA complexes depends on the
number of Hoechst 33258 binding sites contained within the
dsDNA oligomer. For example, the oligomeric duplex 17 which
contains two Hoechst 33258 binding sites separated by a G/C
base pair d(-AATTCAATT-) forms a (Ht33258)2:17 complex.
The oligomeric duplex 23 contains a single Hoechst 33258
binding site of d(-AATT-) and forms only the 1:1 Ht33258:23
complex.
Hoechst 33258 is not long enough to recognize sequences of
more than four DNA base pairs. Attempts to target longer
sequences have been made by synthesizing longer molecules
with a larger number of benzimidazole or pyrrole units.15,45
However, the curvature of these extended molecules fails to
match the natural curvature of the minor groove of DNA. This
problem was overcome by employing polyamide-type molecules
connected with linkers.11 These types of molecules are capable
of forming highly stable DNA complexes. Interestingly, many
of these long polyamides were never investigated for their
sequence selectivity beyond qualitative footprinting experiments
from which the authors simply concluded that these long
molecules bound both short and long A/T rich tracts. In other
cases, the molecules formed strong DNA complexes but showed
a lack of sequence selectivity.10,14
Experimental results rule out the possibility of hairpin46
formation. A FPH-1 hairpin would be expected to have a binding
site of four to five base pairs, similar to Hoechst 33258 or a
Equilibrium association constants were determined for com-
plexation of 35 different oligomeric duplexes by FPH-1 (Table
1). As shown in Table 1, FPH-1 is clearly selective for its
preferred nine dA/dT base pair binding site as contained within
(45) Pilch, D. S.; Xu, Z. T.; Sun, Q.; LaVoie, E. J.; Liu, L. F.; Breslauer,
K. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13565-13570.
(46) de Clairac, R. P. L.; Seel, C. J.; Geierstanger, B. H.; Mrksich, M.;
Baird, E. E.; Dervan, P. B.; Wemmer, D. E. J. Am. Chem. Soc. 1999, 121,
2956-2964.
(43) Neidle, S. Biopolymers 1997, 44, 105-121.
(44) Geierstanger, B. H.; Wemmer, D. E. Annu. ReV. Biophys. Biomol.
Struct. 1995, 24, 463-493.

Base Pair Recognition by Peptide-Hoechst Conjugate
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2477
tripyrrole polyamide. For example, an oligomeric duplex such
as 5 (which contains the binding site 5′-AAATT-3′) would be
expected to form a highly stable FPH-1:5 hairpin-type complex.
Investigations instead show formation of an relatively weak
(FPH-1)2:5 complex (∆tm ) 2 °C, K1K2 ) 2.1 × 1017). Basic
model building suggests that a FPH-1 hairpin (where the
tripyrrole moiety folds back over the bis-benzimidazole rings)
does not form due to steric clashes between the terminal pyrrole
unit and the bulky piperizine ring (this same steric clash is
avoided in the side-by-side antiparallel (FPH-1)2:dsDNA com-
plex shown in Figure 2 because the two FPH-1 molecules are
staggered).
even though Hoechst 33258 does not. Placing the bis-benzim-
idazole moiety of one FPH-1 molecule adjacent to the tripyrrole
moiety of the second FPH-1 molecule in an antiparallel
arrangement avoids placement of the bulky and positively
charged piperizine rings against one another. Also, slightly
staggering the two FPH-1 molecules avoids placing the terminal
primary amide of one FPH-1 molecule adjacent to the bulky
piperazine ring of the other. Staggering the molecules is
especially reasonable since structural investigations of side-by-
side complexes of polyamides with dsDNA consistently showthe
two polyamide molecules of the trimeric complex to be both
antiparallel and staggered.2,21,22,47
The ability of FPH-1 to conform to the curvature of the minor
groove of DNA is shown in the side-by-side antiparallel (FPH-
1)2:dsDNA complex of Figures 2 and 3. The structure in Figures
2 and 3 were generated via a molecular modeling program and
illustrate the curvature of the two FPH-1 molecules when placed
in a side-by-side manner within the minor groove. The anti-
parallel side-by-side complex of FPH-1 with dsDNA has
precedence since monocationic minor groove binders have a
propensity to complex dsDNA in this manner.2,21,22,47 Previous
investigations show that bis-benzimidazole compounds related
to 34 (Scheme 1) form side-by-side complexes with dsDNA25
Rate constants were determined for complexation of 12 by
FPH-1 and Hoechst 33258. The rate constant for formation of
the Hoechst 33258:12 complex is diffusion-controlled. This
result is consistent with literature reports that distamycin also
complexes DNA at diffusion-controlled rates.41 The rate constant
for complexation of 12 by FPH-1 is somewhat slower (6 × 107
M-1 s-1) although still near diffusion controlled.
Acknowledgment. This work was supported by a grant from
the National Institute of Health (5R37DK09171-36).
(47) Kielkopf, C. L.; Baird, E. E.; Dervan, P. B.; Rees, D. C. Nat. Struct.
Biol. 1998, 5, 104-9.
JA003095D