Recognition of the minor groove of DNA by hairpin polyamides containing α-substituted-β-amino acids

Article abstract of DOI:10.1021/ja000509u

Incorporation of the flexible amino acid β-alanine (β) into hairpin polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids is required for binding to DNA sequences longer than seven base pairs with high affinity and sequence selectivity. Pairing the α-substituted-β- amino acids (S)-isoserine ((S)Is), (R)-isoserine ((R)Is), β-aminoalanine (Aa), and α-fluoro-β-alanine (Fb) side-by-side with β in hairpin polyamides alters DNA binding affinity and selectivity relative to the parent polyamide containing a β/β pairing. Quantitative DNase I footprinting titration studies on a restriction fragment containing the sequences 5'- TGCNGTA-3' (N = A, T, G, and C) show that the polyamide ImPy(S)IsImPy-γ- PyPyβImPy-β-Dp (S)Is/β pairing) binds to N = T (K(a) = 4.5 x 10⁹ M^-1) in preference to N = A (K(a) = 6.2 x 10⁸ M^-1). This result stands in contrast to the essentially degenerate binding of the parent ImPyβImPy-γ- PyPyβImPy-β-Dp (β/β pairing) to N = T and N = A, and to the slight preference of ImPyβImPy-γ-PyPy(S)IsImPy-β-Dp (β/(S)Is pairing) to N = A over N = T. Additionally, this study reveals that incorporation of (R)Is, Aa, and Fb into polyamides significantly reduces binding affinity. Therefore, DNA binding in the minor groove is sensitive to the stereochemistry, steric bulk, and electronics of the substituent at the α-position of β-amino acids in hairpin polyamides containing β/β pairs.

Full text of DOI:10.1021/ja000509u

6342
J. Am. Chem. Soc. 2000, 122, 6342-6350
Articles
Recognition of the Minor Groove of DNA by Hairpin Polyamides
Containing R-Substituted-â-Amino Acids
Paul E. Floreancig, Susanne E. Swalley, John W. Trauger, and Peter B. Dervan*
Contribution from the DiVision of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, California 91125
ReceiVed February 10, 2000
Abstract: Incorporation of the flexible amino acid â-alanine (â) into hairpin polyamides composed of
N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids is required for binding to DNA sequences
longer than seven base pairs with high affinity and sequence selectivity. Pairing the R-substituted-â-amino
acids (S)-isoserine (SIs), (R)-isoserine (RIs), â-aminoalanine (Aa), and R-fluoro-â-alanine (Fb) side-by-side
with â in hairpin polyamides alters DNA binding affinity and selectivity relative to the parent polyamide
containing a â/â pairing. Quantitative DNase I footprinting titration studies on a restriction fragment containing
the sequences 5′-TGCNGTA-3′ (N ) A, T, G, and C) show that the polyamide ImPySIsImPy-γ-PyPyâImPy-
â-Dp (SIs/â pairing) binds to N ) T (Ka ) 4.5 × 109 M-1) in preference to N ) A (Ka ) 6.2 × 108 M-1).
This result stands in contrast to the essentially degenerate binding of the parent ImPyâImPy-γ-PyPyâImPy-
â-Dp (â/â pairing) to N ) T and N ) A, and to the slight preference of ImPyâImPy-γ-PyPySIsImPy-â-Dp
(â/SIs pairing) to N ) A over N ) T. Additionally, this study reveals that incorporation of RIs, Aa, and Fb into
polyamides significantly reduces binding affinity. Therefore, DNA binding in the minor groove is sensitive to
the stereochemistry, steric bulk, and electronics of the substituent at the R-position of â-amino acids in hairpin
polyamides containing â/â pairs.
Introduction
recognizes a C‚G base pair.4 A Py/Py pair binds with equal
affinity to A‚T and T‚A and in preference to G‚C and C‚G.4,5
However, the unsymmetrical Hp/Py pair distinguishes T‚A from
A‚T.3 Covalently linking the antiparallel polyamide subunits into
a hairpin structure in the N f C orientation with γ-aminobutyric
acid (γ)7 increases both binding affinity2,7 and, through the
suppression of slipped binding motifs,8 sequence selectivity.
Small molecules which permeate cells and bind to predictable
sequences of DNA can potentially control the expression of
specific genes.1 Polyamides containing N-methylpyrrole (Py),
N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp)
amino acids are synthetic ligands that have an affinity and
sequence specificity for DNA which rival naturally occurring
DNA binding proteins.2,3 DNA recognition depends on side-
by-side amino acid pairings in the minor groove.2-8 Antiparallel
pairing of imidazole opposite pyrrole (Im/Py) selectively
recognizes a G‚C base pair, while a Py/Im combination
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10.1021/ja000509u CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/23/2000

DNA Recognition by Hairpin Polyamides
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6343
An analysis of an X-ray crystal structure of a polyamide/
DNA complex4f reveals that the curvature of polyamides
comprised solely of Py, Im, and Hp rings does not perfectly
match the curvature of B-form DNA, creating an upper limit
of 4-5 contiguous aromatic rings for minor groove binding.8a
Incorporation of the flexible amino acid â-alanine (â) into
polyamides relaxes the curvature, allowing for selective recogni-
tion of significantly longer sequences of DNA.8c-f,9 The â/â
pair binds to A‚T and T‚A in preference to C‚G and G‚C8c-f
but, like the Py/Py pair does not distinguish A‚T from T‚A.
Since the â/â pair will be a pivotal component in polyamides
designed to bind DNA sequences longer than 7 base pairs the
question arises as to whether the A‚T/T‚A degeneracy of the
â/â pair can be broken.
This work addresses the feasibility of altering the molecular
recognition properties of the â/â pair through the incorporation
of functionalized â-amino acids. For reasons of chemical
stability functionalization was limited to the R-position of
â-alanine. An examination of a model of a DNA/polyamide
complex containing a â/â pair indicates that the hydrogens at
the R-carbon reside in a sterically hindered environment.
Additionally, the environment around each enantiotopic hydro-
gen at the R-position is quite different, with the pro-R hydrogen
projecting toward the wall of the minor groove and the pro-S
hydrogen projecting toward the floor of the minor groove.
Therefore binding to DNA should be affected by both the size
of the substituent and the stereochemistry of the R-carbon. To
test this, the R-substituted â-amino acids (S)-isoserine (SIs), (R)-
isoserine (RIs), (S)-R-fluoro-â-alanine (Fb), and (S)-â-aminoala-
nine (Aa) have been incorporated into polyamides and the
binding of these polyamides to DNA has been studied (Figure
1). These substituted â-amino acids differ from â not only in
size but in hydrogen bonding ability and polarity, offering the
possibility for altered molecular recognition properties.
Because hairpin polyamides keep the side-by-side pairing
units in register and disfavor slipped motifs seen in unlinked
homo- and heterodimers, the relative binding affinities of the
four unsymmetrical aliphatic pairs â/SIs, â/RIs, â/Fb, and â/Aa
were characterized within the context of a 2â2 hairpin motif.
Five polyamides which differ at a single â-alanine position were
synthesized by solid-phase methodology (Figure 2).10 The
binding affinities of polyamides ImPyâImPy-γ-PyPyâImPyâDp
(1), ImPySIsImPy-γ-PyPyâImPyâDp (2), ImPyRIsImPy-γ-
PyPyâImPyâDp (3), ImPyâImPy-γ-PyPySIsImPyâDp (4), Im-
PyAaImPy-γ-PyPyâImPyâDp (5), and ImPyFbImPy-γ-PyPy-
âImPyâDp (6) (Dp ) dimethylaminopropylamine) to the seven
base pair sequence 5′-TGCNGTA-3′ (Figure 1), where N ) A,
T, G, or C, have been studied by quantitative DNase I
footprinting.11 The binding of polyamide 2 and its Fe(II)/EDTA
analogue 2E to their target sequences has also been studied by
MPE‚Fe(II)12 footprinting and affinity cleaving.13 MPE‚Fe(II)
footprinting affords information about the exact size and location
Figure 1. Binding model for the complexes formed between the DNA
target sequences and ImPyAbImPy-γ-PyPyâImPy-â-Dp (Ab ) R-sub-
stituted-â-amino acid). Circles with dots represent lone pairs of N3 of
purines and O2 of pyrimidines. Circles containing an H represent the
N2 hydrogen of guanine. Putative hydrogen bonds are illustrated by
dotted lines. A ball-and-stick model is also shown. Shaded and
nonshaded circles represent imidazole and pyrrole carboxamides,
respectively. Nonshaded diamonds represent â-alanine, and the shaded
diamond represents the R-substituted-â-amino acid.
of the binding sites, and affinity cleaving studies determine the
binding orientation and stoichiometry of the polyamide/DNA
complex. Quantitative DNase I footprinting titrations provide
equilibrium association constants (Ka) for the polyamides with
match and mismatch binding sites.
Results and Discussion
Monomer Synthesis. The protected isoserine monomer (S)-9
was synthesized through a variation of the route developed by
Swindell and co-workers (Scheme 1).14 (S)-glycidyl p-methoxy-
phenyl ether was prepared in enantiomerically pure form through
a hydrolytic kinetic resolution15 of the racemic epoxide 7. The
epoxide was opened with NaN3 and the resulting azido alcohol
was protected as its benzyl ether (8). The azide was reduced
with Ph3P and H2O16 and, in the same step, converted to a Boc-
carbamate. The p-methoxyphenyl ether was removed with ceric
(9) 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.
(10) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141-
6146.
(11) (a) Brenowitz, M.; Senear, D. F.; Shea, M. A.; Ackers, G. K.
Methods Enzymol. 1986, 130, 132-181. (b) Brenowitz, M.; Senear, D. F.;
Shea, M. A.; Ackers, G. K. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8462-
8466. (c) Senear, D. F.; Brenowitz, M.; Shea, M. A.; Ackers, G. K.
Biochemistry 1986, 25, 7344-7354.
(12) (a) Van Dyke, M. W.; Dervan, P. B. Biochemistry 1983, 22, 2373-
2377. (b) Van Dyke, M. W.; Dervan, P. B. Nucleic Acids Res. 1983, 11,
5555-5567.
(13) (a) Taylor, J. S.; Schultz, P. G.; Dervan, P. B. Tetrahedron 1984,
40, 457-465. (b) Dervan, P. B.; Science 1986, 232, 464-471.
(14) Swindell, C. S.; Krauss, N. E.; Horwitz, S. B.; Ringel, I. J. Med.
Chem. 1991, 34, 1176-1184.
(15) Tokunga, M.; Larrow, J. F.; Kakiuchi, F.; Jacobsen, E. N. Science
1997, 277, 936-938.
(16) Vaultier, M.; Knouzi, N.; Carrie´, R. Tetrahedron Lett. 1983, 24,
763-764.

6344 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Floreancig et al.
Figure 2. Polyamides synthesized for this study.
Scheme 1a
a
(i) (R,R)-N,N′-bis(3, 5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II), H2O, p-dioxane. (ii) NaN3, NH4Cl, 95% EtOH. (iii) NaH,
DMF, then BnBr. (iv) Ph3P, THF, then H2O, then (Boc)2O, i-Pr2NEt. (v) CAN, NaHCO3, CH3CN, H2O. (vi) PDC, DMF. (vii) BnCl, KOH, EtOH,
H2O. (viii) Me3SiCHN2, MeOH, CH2Cl2. (ix) Et2NSF3, THF. (x) NH4O2CH, 10% Pd/C, MeOH, then (Boc)2O, i-Pr2NEt, p-dioxane. (xi) KOH,
THF, H2O.
ammonium nitrate,17 and the primary alcohol was oxidized to
carboxylic acid 9 with PDC in DMF.18 The R-enantiomer of 9
(R-9) was prepared in an identical manner except that the
opposite enantiomer of the Co(II)‚salen catalyst was employed
for the hydrolytic kinetic resolution of 7. The products of this
route were shown to have enantiomeric excesses of >98% based
on an 1H NMR analysis of the R-methylbenzyl amide derivatives
of (S)-9 and (R)-9. Orthogonally protected â-aminoalanine 10
(Scheme 2) was prepared from Boc-L-serine as reported by Roy
and Imperiali.19
Boc-protected R-fluoro-â-alanine (13) was prepared through
a variation of the sequence developed by Young and co-workers
(Scheme 1).20 (R)-serine (11) was converted to its N,N-
dibenzylamine by treatment with BnCl and NaOH, and the
product of this reaction was esterified by adding TMSCHN2.
The resulting dibenzylamino alcohol was converted to fluoride
12 with migration of the amino group and inversion of
stereochemistry through the action of Et2NSF3. The benzyl
groups were removed with hydrogenolysis, and the crude amine
was directly protected as a Boc-carbamate. Carboxylic acid 13
was formed by saponification of the resulting methyl ester with
(17) Fukuyama, T.; Laird, A. A.; Hotchkiss, L. M. Tetrahedron Lett.
1985, 26, 6291-6292.
(18) Corey, E. J.; Schmidt, G. Tetrahedron Lett. 1979, 20, 399-402.
(19) Sinha Roy, R. Ph.D. Thesis, California Institute of Technology, 1996.
We thank Grant Walkup and Barbara Imperiali for helpful discussions.
(20) Gani, D.; Hitchcock, P. B.; Young, D. W. J. Chem. Soc., Perkin
Trans. 1 1985, 1363-1372.

DNA Recognition by Hairpin Polyamides
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6345
Scheme 2: Solid-Phase Synthesis of Polyamidesa
a
For details see text and Experimental Section.
aqueous NaOH. The enantiomeric purity of 13 was shown to
be >98% through an 19F NMR analysis of its R-methylbenzyl
amide.
incorporation of the Aa module into the polyamide. Alloc-
protecting group removal was achieved with Pd2dba3‚CHCl3,
Ph3P, and BuNH2‚HCO2H in THF.23 The resulting amine was
coupled to Fmoc-Py24 with HBTU and i-Pr2NEt. After depro-
tection of the Fmoc group with piperidine the resin was capped
with N-methylimidazole carboxylic acid and HBTU, the Boc
group was removed under standard conditions, and the poly-
amide was cleaved from the resin with Dp to provide 5.
Coupling of 13 with 15 was accomplished with an HBTU
coupling, and the synthesis of 6 was completed with standard
conditions.
The synthesis of 4 (not shown in Scheme 2) required the
coupling of ImPy-â-Pam-resin with 9. Standard conditions were
employed to complete the synthesis of the benzyl ether of 4,
which was converted to 4 according to the method used for the
synthesis of 2 and 3.
Polyamide Synthesis. Polyamides 1-6 were prepared using
standard solid-phase synthesis starting with Boc-â-alanine Pam-
resin using the monomers shown in Scheme 2. Resins 14 and
15 were prepared according to established protocols.10 For
polyamide 1 coupling proceeded smoothly from 15 using Boc-
â-alanine and HBTU.10,21 The synthesis of 1 was completed
according to standard protocols.10 Coupling of (S)-9 and (R)-9
to 15 was also accomplished with HBTU. The synthesis of the
resins bearing the benzyl ethers of the polyamides was
completed according to established procedures. The resins were
cleaved with Dp and the benzyl ethers were removed with
TMSBr and PhSMe in anhydrous TFA22 to provide 2 and 3.
The resin containing 9 was also cleaved with 3,3′-diamino-N-
methyldipropylamine. After removal of the benzyl ether the
polyamide was coupled with EDTA dianhydride to provide 2E.
Monomer 10 did not couple well to the imidazole amine of
15. Therefore, dimer 21 was synthesized in solution from 10.10
HBTU-mediated coupling of 21 to resin 14 resulted in efficient
Quantitative DNase I Footprinting. Equilibrium association
constants (Ka) between polyamides 1-6 and the 3′-32P-end-
labeled 294 bp restriction fragment from the cloned plasmid
pSES 19-1 were determined with quantitative DNaseI foot-
(23) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem.
Soc. 1990, 112, 1691-1696.
(24) (a) Wurtz, N. R.; Dervan, P. B. Unpublished results. (b) Vasquez,
E.; Caamano, A. M.; Castedo, L.; Mascaren˜as, J. L. Tetrahedron Lett. 1999,
40, 3621-3624.
(21) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gilleson, D. Tetrahedron
Lett. 1989, 30, 1927-1930.
(22) Fujii, N.; Otaka, A.; Sugiyama, N.; Hatano, M.; Yajima, H. Chem.
Pharm. Bull. 1987, 35, 3880-3883.

6346 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Floreancig et al.
Table 1. Effects of Stereochemistry on Equilibrium Association
a,b
Constants (M-1
)
polyamide pairing
Ka(T)c
Ka(A)d
specificitye
1
2
3
4
â/â
1.6 × 1010 (0.5) 1.4 × 1010 (0.3)
1.1
7.2
-
SIs/â
4.5 × 109 (1.3) 6.2 × 108 (2.3)
RIs/â <3 × 108
<1 × 108
â/SIs
1.6 × 109 (0.3) 3.2 × 109 (0.3)
0.5
a Values reported for polyamides 1, 2, and 4 are the mean values
from at least three DNase I footprinting titration experiments. Due to
non-specific ligand binding exact values for Ka could not be determined
for polyamide 3. Numbers in parentheses are the standard deviations
for each value. b The assays were performed at 22 °C at pH 7.0 in the
presence of 10 mM Tris‚HCl, 10 mM KCl, 10 mM MgCl2, and 5 mM
CaCl2. c Ka(T) is the equilibrium association constant for the sequence
5′-TGCTGTA-3′. d Ka(A) is the equilibrium association constant for
the sequence 5′-TGCAGTA-3′. e Specificity is defined as Ka(T)/Ka(A).
Figure 4. Quantitative DNase
I
footprinting titration with
ImPyRIsImPy-γ-PyPyâImPy-â-Dp (3, left) and ImPySIsImPy-γ-
PyPyâImPy-â-Dp (2, right) on the 292-bp restriction fragment from
the plasmid pSES 19-1: lane 1, A reaction; lane 2, DNase I control;
lanes 3-9, 100 pM, 200 pM, 500 pM, 1 nM, 2 nM, 5 nM, and 10 nM
3; lane 10, A reaction; lane 11, DNase I control; lanes 12-21, 10 pM,
20 pM, 50 pM, 100 pM, 200 pM, 500 pM, 1 nM, 2 nM, 5 nM, and 10
nM 2; lane 22, intact DNA. The four putative binding sites are shown
on the right of the autoradiograms. All reactions contain a 15 kcpm
restriction fragment, 10 mM Tris‚HCl (pH 7.0), 10 mM KCl, 10 mM
MgCl2, and 5 mM CaCl2.
Figure 3. Binding isotherms for the DNase I footprinting titrations of
polyamides 1-4 at the 5′-TGCTGTA-3′ site of the 292 base pair 3′-
32P-labeled restriction fragment from the plasmid pSES 19-1. Open
squares represent polyamide 1, filled diamonds represent polyamide
2, filled circles represent polyamide 3, and open circles represent
polyamide 4.
printing titrations (10 mM Tris‚HCl, 10 mM KCl, 10 mM
MgCl2, 5 mM CaCl2, pH 7.0, 22 °C). This restriction fragment
contains the four possible 5′-TGCNGTA-3′ binding sites with
identical intervening sequences, providing binding affinity
information for the functionalized â-amino acid/â couples
against all four base pairs in a single experiment.
Effects of Stereochemistry on Binding. The binding affini-
ties of polyamides 1-4 were studied in order to determine the
effects of introducing a hydroxyl group and altering the
stereochemistry at the R-position of the â-amino acid (Table 1
and Figure 3). As expected polyamide 1, containing a â/â
pairing, bound with essentially equal affinity to the sequences
5′-TGCTGTA-3′ and 5′-TGCAGTA-3′.25 Virtually no binding
of this polyamide (or of 2-6) was observed at the sequences
5′-TGCCGTA-3′ and 5′-TGCGGTA-3′. Incorporation of the
SIs/â pair (polyamide 2), however, demonstrated a preference
for binding to 5′-TGCTGTA-3′ over 5′-TGCAGTA-3′. Inversion
of the stereochemistry of the R-carbon in the isoserine residue
(polyamide 3, RIs/â pairing) resulted in a substantial loss of
binding affinity at all sites (Figure 4). Precise determinations
of equilibrium association constants for 3 were impossible due
to significant nonspecific ligand binding at concentrations
required to observe a footprint. Noteworthy in these experiments
was that polyamide 4, which contains a â/SIs pairing, binds to
the sequence 5′-TGCAGTA-3′ preferentially with respect to 5′-
TGCTGTA-3′.25 Although the preference is modest, this result
demonstrates that the T‚A/A‚T binding degeneracy normally
observed with the â/â pairing can be altered in a predictable
S
manner through the incorporation of either the Is/â or â/SIs
pairings. The association constants of 1, 2, and 4 reveal that,
although the introduction of a hydroxyl group destabilizes
binding to both the T and A sites, the destabilization is greater
at the A site than at the T site.
Effects of Substituent on Binding. The binding affinities
of polyamides 2, 5, and 6, were compared to evaluate the effects
of changing the substituent of the R-substituted-â-amino acid
(Table 2). Polyamide 5, containing the Aa/â pairing and
polyamide 6, containing the Fb/â pairing, showed remarkably
weak binding to the A‚T and T‚A sequences.
(25) DNase I footprinting gels of polyamides 1, 4, 5, and 6 with the
3′-32P-labeled restriction fragment from pSES 19-1, the MPE footprinting
gel of 2 with the 5′-32P-labeled restriction fragment, and the affinity cleaving
gel of 2E with the 5′-32P-labeled restriction fragment are included in the
Supporting Information.
MPE‚Fe(II) Footprinting and Affinity Cleavage. MPE
footprinting studies were performed on polyamide 2 and affinity

DNA Recognition by Hairpin Polyamides
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6347
Figure 5. (a) MPE‚Fe(II) footprinting titration of 2 and affinity cleaving study of 2E on the 292-bp 3′-32P-labeled restriction fragment from the
plasmid pSES 19-1: lane 1, intact DNA; lane 2, A reaction; lane 3, MPE control; lanes 4-6, 100 pM, 1 nM, and 10 nM 2; lanes 7-9, 100 pM,
1 nM, and 10 nM 2E. The sequences 5′-TGCTGTA-3′ and 5′-TGCAGTA-3′ are shown at the right of the autoradiogram. All reactions contain a
15 kcpm restriction fragment, 20 mM HEPES buffer (pH 7.0), and 10 mM NaCl. (b) Results from MPE‚Fe(II) footprinting titration of 2. Bold
sequences represent binding sites determined by the published model. Bar heights are proportional to the relative protection from cleavage at each
band. (c) Results from the affinity cleaving study of 2E. Bold sequences represent binding sites determined by the published model. Line heights
are proportional to the relative cleavage at each band.
Table 2. Effects of Substituent on Equilibrium Association
of this study. Introducing a substituent to the pro-R R-position
a,b
Constants (M-1
)
of â causes a steric clash with the minor groove and, thereby,
diminishes binding affinity. When introduced to the pro-S
R-position of â a small substituent, such as a hydroxyl group,
can fit into the floor of the minor groove without a significant
energetic cost. The cleft of the T‚A base pair accommodates
polyamide pairing
Ka(T)c
Ka(A)d
specificitye
1
2
5
6
â/â
1.6 × 1010 (0.5) 1.4 × 1010 (0.3)
1.1
7.2
1.6
1.4
SIs/â
4.5 × 109 (1.3) 6.2 × 108 (2.3)
Aa/â 1.8 × 108 (0.1) 1.1 × 108 (0.2)
4.9 × 108 (1.0) 3.4 × 108 (0.2)
S
Fb/â
the hydroxyl group of Is, whereas H2 of adenine in the A‚T
a Values are the mean values from at least three DNase I footprinting
titration experiments. Numbers in parentheses are the standard devia-
tions for each value. b The assays were performed at 22 °C at pH 7.0
in the presence of 10 mM Tris‚HCl, 10 mM KCl, 10 mM MgCl2, and
5 mM CaCl2. c Ka(T) is the equilibrium association constant for the
sequence 5′-TGCTGTA-3′. d Ka(A) is the equilibrium association
constant for the sequence 5′-TGCAGTA-3′. e Specificity is defined as
Ka(T)/Ka(A).
base pair introduces an unfavorable interaction with the hydroxyl
group. Additionally, the possibility of a second hydrogen bond
to O2 of T exists when SIs is placed opposite T. This explanation
for the binding selectivity of the SIs/â pairing is consistent with
the conclusions drawn from an X-ray crystal structure the
complex of a polyamide containing an Hp/Py pairing in the
minor groove.3b The amino group of Aa, which is expected to
be protonated under the conditions of the assay, interacts
unfavorably with the 3′-base. The poor binding of polyamide 6
is somewhat curious given the small size of fluorine and
indicates that the electron density of fluorine could possibly
introduce an electrostatic repulsion with the electron-rich minor
groove.
Implications for the Design of Minor Groove Binding
Molecules. These results demonstrate that the incorporation of
R-substituted-â-amino acids into hairpin polyamides can sig-
nificantly alter the DNA binding properties of these molecules
relative to polyamides containing â-alanine. Binding is sensitive
to the stereochemistry, steric bulk, and electronic properties of
cleaving studies were performed on polyamide 2E with both
the 3′- and the 5′-32P-end-labeled restriction fragment 294 bp
restriction fragment from pSES 19-1 in order to confirm the
exact location of the binding site and the binding orientation
(Figure 5).25 These experiments demonstrated that the incor-
S
poration of an Is residue into a hairpin polyamide alters only
binding affinity and selectivity and not the expected binding
site or orientation.
Basis for the Substituent Effects. A recent NMR study of
the complex of a polyamide containing a â/â pairing in the
minor groove of DNA9 offers insight into the origin of the results

6348 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Floreancig et al.
the R-substituent. Polyamides with the SIs/â pairing show
moderate selectivity for binding to T‚A over A‚T and suffer a
modest loss in binding affinity compared to the parent polyamide
containing a â/â pairing. This demonstrates that binding
selectivity can be modulated through manipulation of the flexible
linker unit. The effects of incorporating the SIs/â pair on cellular
uptake and transcription inhibition remain to be studied and will
be reported in due course.
was stirred at 0 °C for 45 min. To the resulting alkoxide was added
BnBr (2.0 g, 12 mmol). The mixture was stirred for 3 h while slowly
warming to room temperature. The reaction was quenched by adding
H2O, and the organic material was extracted into EtOAc. The EtOAc
layer was washed with brine (4 × 50 mL) and was then dried (MgSO4),
filtered, and concentrated. The residue was purified by flash chroma-
tography (16% EtOAc in hexanes) to provide the benzyl ether (2.2 g,
79%). 1H NMR (CDCl3, 300 MHz) δ 3.52 (t, 2H, J ) 4.6 Hz), 3.79 (s,
3H), 3.98 (m, 3H), 4.75 (s, 2H), 6.85 (s, 4H), 7.39 (m, 5H) ppm; 13C
NMR (CDCl3, 75 MHz) δ 51.7, 55.5, 67.7, 72.3, 76.2, 114.2, 115.2,
127.7, 128.3, 137.5, 152.3, 153.9 ppm; IR (neat) νmax 3022, 2933, 2872,
2835, 2102, 1509, 1232, 1041, 825 cm-1; Exact mass calcd for
C17H19N3O3: 313.1426. Found: 313.1435 (EI).
N-[(2S)-3-(4-Methoxyphenoxy)-2-(phenylmethoxy)propyl](tert-
butoxy)carboxamide. To azide 8 (2.1 g, 6.7 mmol) in THF (50 mL)
was added Ph3P (2.1 g, 8.1 mmol). The mixture was stirred at room
temperature for 1 h. To the mixture was added H2O (2 mL). The mixture
was stirred at room temperature for 2 h. To this solution were added
Et3N (2.0 mL, 1.4 g, 14 mmol) and (Boc)2O (1.8 g, 8.1 mmol). The
mixture was stirred at room temperature for 1 h, then was partitioned
between EtOAc and H2O. The organic layer was dried (Na2SO4),
filtered, and concentrated. The residue was purified by flash chroma-
tography (15% EtOAc in hexanes) to provide the carbamate (1.2 g,
46%). 1H NMR (CDCl3, 300 MHz) δ 1.42 (s, 9H), 3.33 (m, 1H), 3.48
(m, 1H), 3.75 (s, 3H), 3.85 (m, 1H), 3.98 (d, 2H, J ) 5.1 Hz), 4.64 (d,
1H, J ) 11.7 Hz) 4.73 (d, 1H, J ) 11.7 Hz), 4.83 (br s, 1H), 6.82 (s,
4H), 7.30 (m, 5H) ppm; 13C NMR (CDCl3, 75 MHz) δ 28.9, 42.2,
56.3, 69.7, 72.9, 76.8, 115.2, 116.1, 128.4, 128.5, 129.0, 138.7, 153.3,
156.6 ppm; IR (neat) νmax 3366, 2976, 2932, 1712, 1509, 1232, 1170,
1040 cm-1; Exact mass calcd for C22H29NO5: 387.2046. Found:
387.2042 (EI).
Experimental Section
General. Dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole
(HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HBTU), Boc-â-alanine, Boc-γ-aminobutyric acid, and
0.2 mmol/g Boc-â-alanine-(4-carboxamidomethyl)-benzyl-ester-copoly-
(styrene-divinylbenzene) resin (Boc-â-Pam-resin) were purchased from
Peptides International. N,N-diisopropylethylamine, N,N-dimethylform-
amide (DMF), N-methylpyrrolidone (NMP), and acetic anhydride were
purchased from Applied Biosystems. Reagent grade dichloromethane
and triethylamine were purchased from EM. Biograde trifluoroacetic
acid (TFA) was purchased from Halocarbon. All other chemicals were
purchased from Aldrich. All reagents were used without further
purification. A shaker for manual solid-phase synthesis was obtained
from Thermolyne. Screw-cap glass peptide synthesis reaction vessels
(5 mL and 20 mL) with a no. 2 sintered glass frit were made as
described by Kent.26 1H NMR spectra were recorded on a General
Electric-QE NMR 300 MHz spectrometer. Infrared spectra were
recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer. UV
spectra were measured in water on a Hewlett-Packard model 8452A
diode array spectrophotometer. Matrix-assisted, laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF) was per-
formed at the Protein and Peptide Microanalytical Facility at the
California Institute of Technology. HPLC analysis was performed on
either an HP 1090M analytical HPLC or a Beckman Gold system using
a RAINEN C18, Microsorb MV, 5 µm, 300 × 4.6 mm reversed phase
column in 0.1% (wt/v) TFA with acetonitrile as eluent and a flow rate
of 1.0 mL/min. Preparatory reverse phase HPLC was performed on a
Beckman HPLC with a Waters DeltaPak 25 × 100 mm, 100 µm C18
column equipped with a guard, 0.1% (wt/v) TFA, 0.25% acetonitrile/
min. 18Ω water was obtained from a Millipore MilliQ water purification
system, and all buffers were filtered through a 0.2 µm membrane.
Polyamide 1 and resins 14 and 15 were prepared as previously
described.10
N-[(2S)-3-Hydroxy-2-(phenylmethoxy)propyl](tert-butoxy)car-
boxamide. To the p-methoxyphenyl ether (1.2 g, 3.1 mmol) in CH3-
CN (40 mL) and H2O (10 mL) were added NaHCO3 (2.1 g, 25 mmol)
and ceric ammonium nitrate (4.5 g, 8.2 mmol). The mixture was stirred
at room temperature for 15 min and then was diluted with EtOAc and
washed with brine. The organic layer was dried (Na2SO4), filtered, and
concentrated. The residue was purified by flash chromatography (30%
EtOAc in hexanes to 50% EtOAc/50% hexanes) to provide the alcohol
(630 mg, 72%). 1H NMR (CDCl3, 300 MHz) δ 1.44 (s, 9H), 3.25 (m,
3H), 3.40 (dd, 1H, J ) 3.3, 7.0 Hz), 3.57 (m, 2H), 4.58 (s, 2H), 4.95
(m, 1H), 7.36 (m, 5H) ppm; 13C NMR (CDCl3, 75 MHz) δ 28.1, 39.9,
60.8, 71.5, 76.4, 79.6, 127.5, 127.7, 128.3, 137.8, 156.8 ppm; IR (neat)
ν
max 3359, 2976, 2932, 1691, 1513, 1366, 1252, 1170 cm-1; Exact mass
(S)-Glycidyl p-Methoxyphenyl Ether ((S)-7). To (R, R)-N,N′-bis-
(3, 5-di-tert-butylsalicylidene)-1, 2-cyclohexanediaminocobalt(II) (120
mg, 0.2 mmol) in toluene (2 mL) was added glacial HOAc (24 mg,
0.4 mmol). The mixture was stirred at room temperature for 1 h. The
toluene was removed under reduced pressure. To the brown residue
was added (()-glycidyl p-methoxyphenyl ether (3.6 g, 20 mmol) and
H2O (210 mg, 12 mmol) in p-dioxane (2 mL). The reaction was stirred
at room temperature for 18 h. The mixture was purified by flash
chromatography (30% EtOAc in hexanes) to provide the enantiomeri-
cally pure epoxide (1.2 g, 33% recovery, 83% of the theoretical yield).
calcd for C15H23NO4: 282.1627. Found: 282.1696 (EI).
(2S)-3-[(tert-Butoxy)carbonylamino]-2-(phenylmethoxy)propano-
ic Acid (9). To the alcohol (580 mg, 2.1 mmol) in DMF (15 mL) was
added PDC (3.0 g, 8.0 mmol). The mixture was stirred at room
temperature for 16 h and was then diluted with EtOAc and washed
several times with brine. The organic layer was dried (Na2SO4), filtered,
and concentrated. The residue was purified by flash chromatography
(100% EtOAc to 10% MeOH in EtOAc) to provide the carboxylic acid
1
(230 mg, 38%). H NMR (CDCl3, 300 MHz) δ 1.39 (s, 9H), 3.44 (br
m, 1H), 3.56 (br m, 1H), 4.03 (br s, 1H), 4.47 (d, 1H, J ) 11.5 Hz),
4.76 (d, 1H, J ) 11.5 Hz), 5.03 (br s, 1H), 7.30 (m, 5H) ppm; 13C
NMR (CDCl3, 75 MHz) δ 28.9, 42.9, 73.0, 80.3, 128.6, 128.7, 129.0,
130.6, 137.7, 156.5, 174.2 ppm; IR (neat) νmax 3352, 2978, 1715, 1515,
1367, 1252, 1168, 1122 cm-1; [R]22D ) -47.1° (c 0.31, acetone); Exact
mass [M + H] calcd for C16H21NO5: 296.1498. Found: 296.1499
(DCI).
Determination of the enantiomeric purity of (S)-9 and (R)-9. To
(S)-9 or (R)-9 (50 mg, 0.17 mmol) in DMF (2.5 mL) were added HBTU
(250 mg, 0.7 mmol) and i-Pr2NEt (0.5 mL). The mixture was stirred at
room temperature for 15 min. To the mixture was added (S)-R-
methylbenzylamine (125 mg, 1.0 mmol). The mixture was stirred at
room temperature for 5 h, then was diluted with EtOAc. The organic
layer was washed successively with 10% aqueous citric acid and brine.
The organic material was dried (Na2SO4), filtered, and concentrated.
The residue was filtered through a short plug of silica gel (40% EtOAc
in hexanes) to provide the amide (64 mg, 88%). The final products
were analyzed by 1H NMR (300 MHz, CDCl3). The amide from (S)-9
[R]22 ) +9.4° (c 0.16, acetone).
D
(2S)-1-Azido-3-(4-methoxyphenoxy)propan-2-ol.14 To the glycidyl
ether (4.5 g, 25 mmol) in 95% EtOH (250 mL) were added NaN3 (8.2
g, 125 mmol) and NH4Cl (6.7 g, 125 mmol). The mixture was stirred
at room temperature for 14 h and was then filtered and concentrated.
The residue was partitioned between EtOAc and H2O. The organics
were dried (Na2SO4), filtered, and concentrated. The residue was
purified by flash chromatography (30% EtOAc in hexanes) to provide
the azido alcohol (5.5 g, 98%). 1H NMR (CDCl3, 300 MHz) δ 2.50 (d,
1H, J ) 5.0 Hz), 3.50 (m, 2H), 3.76 (s, 3H), 3.95 (m, 2H), 4.13 (m,
1H), 6.83 (s, 4H) ppm; 13C NMR (CDCl3, 75 MHz) δ 54.0, 56.3, 70.0,
70.4, 115.3, 115.5, 152.9, 154.9 ppm; IR (neat) νmax 3432, 2934, 2102,
1508, 1230, 1043, 825 cm-1
.
(2S)-3-(4-Methoxyphenoxy)-2-(phenylmethoxy)propylazide (8).
To NaH (60%, 1.0 g, 25 mmol) in DMF (30 mL) at 0 °C was added
the azido alcohol (2.0 g, 8.3 mmol) in DMF (20 mL). The mixture
(26) Kent, S. B. H. Annu. ReV. Biochem. 1988, 57, 957-989.

DNA Recognition by Hairpin Polyamides
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6349
showed a triplet (J ) 6.0 Hz) at δ 3.89 ppm and the amide from (R)-9
showed a triplet (J ) 6.0 Hz) at δ 3.93 ppm. In both spectra no evidence
of diastereomeric contamination (<3%) was observed.
propylamino)-N-methylamine (1 mL). The mixture was shaken at 37
°C for 10 h and then was filtered. The residue was purified by reverse
phase HPLC and was lyophilized. To the product of the cleavage in
TFA (0.5 mL) at 0 °C were added thioanisole (100 mg, 0.8 mmol) and
TMSBr (100 mg, 0.7 mmol). The mixture was stirred at 0 °C for 2 h
and then was purified by reverse phase HPLC. The product was
lyophilized and then was dissolved in DMSO (1 mL). To this solution
was added EDTA dianhydride (50 mg, 0.2 mmol) in DMSO (0.5 mL)
and NMP (0.5 mL). The mixture was shaken at 37 °C for 3 h. To the
mixture was added aqueous NaOH (1 M, 1 mL). The mixture was
shaken an additional 30 min at 37 °C. The mixture was purified by
reverse phase HPLC to provide 2E (0.2 mg, 1% recovery from resin).
Methyl (2S)-3-[(tert-Butoxy)carbonylamino]-2-fluoropropanoate.
To 1220 (500 mg, 1.7 mmol) in MeOH (15 mL) were added 10% Pd/C
(75 mg) and ammonium formate (1.5 g, 24 mmol). The mixture was
immersed in a preheated oil bath (80 °C). The mixture was stirred at
reflux for 1.75 h. The mixture was filtered over Celite and the filtrate
was concentrated. The residue was swirled in EtOAc, and the solution
was decanted away from the solids. The solution was concentrated to
provide the amine. To the crude amine in p-dioxane (12 mL) were
added Boc2O (750 mg, 2.9 mmol) and i-Pr2NEt (1 mL, 740 mg, 5.7
mmol). The mixture was stirred at room temperature for 2 h, then was
concentrated. The residue was purified by flash chromatography (33%
MALDI-TOF-MS (monoisotopic) 1698.8 (1698.8, calcd for C75H100
N27O20).
-
1
ImPyRIsImPy-γ-PyPyâImPy-â-Dp (3). The preparation of 3 from
15 was conducted in a manner identical to that of the synthesis of 2.
MALDI-TOF-MS (monoisotopic) 1381.6 (1381.7, calcd for C63H81-
EtOAc in hexanes) to provide the protected amine (90 mg, 24%). H
NMR (CDCl3, 300 MHz) δ 1.41 (s, 9H), 3.65 (m, 2H), 3.78 (s, 3H),
4.88 (m, 1.5H), 5.04 (m, 0.5H) ppm; 13C NMR (CDCl3, 75 MHz) δ
28.8, 42.7, 43.0, 53.1, 87.0, 89.4, 156.1, 168.7 ppm; IR (neat) νmax
N24O13); [R]22 ) +2.7° (c 0.04, H2O).
D
3376, 2979, 1766, 1715, 1520, 1368, 1251, 1170, 1109 cm-1
.
ImPyâImPy-γ-PyPySIsImPy-â-Dp (4). To (S)-9 (400 mg, 1.4
mmol) in DMF (2 mL) were added HBTU (500 mg, 1.3 mmol) and
i-Pr2NEt (1 mL). The mixture was agitated for 30 s and then was
allowed to stand for 5 min. The mixture was added to ImPy-â-Pam
resin (700 mg, 0.14 mmol, prepared according to established protocols10)
and the reaction was shaken at 37 °C for 10 h. The completion of the
synthesis of benzylated 4 proceeded according to established protocols.
To benzylated 4 (50 mg, 0.03 mmol) in TFA (1 mL) at 0 °C were
added thioanisole (300 mg, 2.4 mmol) and TMSBr (300 mg, 2.0 mmol).
The mixture was stirred at 0 °C for 3 h. The residue was purified by
reverse phase HPLC and was lyophilized to provide 4 (15 mg, 9%
recovery from resin). MALDI-TOF-MS (monoisotopic) 1381.9 (1381.7,
(2S)-3-[(tert-Butoxy)carbonylamino]-2-fluoropropanoic Acid (13).
To Boc-R-fluoro-â-alanine methyl ester (90 mg, 0.4 mmol) in MeOH
(5 mL) was added aqueous NaOH (1.0 M, 2 mL, 2 mmol). The mixture
was stirred at room temperature for 1.5 h. The MeOH was removed
under reduced pressure, and the aqueous solution was acidified to pH
1 with aqueous HCl (1 M). The mixture was extracted with EtOAc to
1
provide the carboxylic acid. H NMR (MeOH-d4, 300 MHz) δ 1.47
(s, 9H), 3.55 (m, 2H), 4.96 (ddd, 1H, J ) 49.0, 6.6, 2.6 Hz) ppm; 13
C
NMR (MeOH-d4, 75 MHz) δ 38.4, 52.7, 90.2, 97.5, 99.9, 167.9, 181.1,
181.4 ppm; IR (neat) νmax 3369, 2990, 1740, 1698, 1525, 1254, 1160,
1104 cm-1; [R]22 ) -6.0° (c 0.23, acetone); Exact mass [M + H]
D
1
calcd for C8H15FNO4: 208.0985. Found: 208.0988 (DCI).
calcd for C63H81N24O13); H NMR (DMSO-d6) δ 1.71 (m, 4H), 2.35
Determination of the Enantiomeric Purity of 13. To (S)-13 or
(R)-13 (9 mg, 0.044 mmol) in DMF (1.0 mL) were added HBTU (17
mg, 0.14 mmol) and i-Pr2NEt (10 µL). The mixture was stirred at room
temperature for 15 min. To the mixture was added (S)-R-methylben-
zylamine (12 mg, 0.1 mmol). The mixture was stirred at room
temperature for 5 h and then was diluted with EtOAc. The organic
layer was washed successively with 10% aqueous citric acid and brine.
The organic material was dried (Na2SO4), filtered, and concentrated.
The residue was filtered through a short plug of silica gel (100% EtOAc)
to provide the amide (10 mg, 73%). The final products were analyzed
by 19F NMR (470 MHz, CDCl3). The amide from (S)-13 showed a
doublet of triplets (J ) 47, 23 Hz) at δ -24.2 ppm and the amide
from (R)-13 showed a doublet of triplets (J ) 50, 23 Hz) at δ -24.4
ppm. In both spectra no evidence of diastereomeric contamination
(<3%) was observed.
(m, 4H), 2.53 (m 2H), 2.68 (s, 6H), 2.8-3.5 (m, 8H), 3.70 (s, 3H),
3.74 (s, 6H), 3.75 (s, 9H), 3.89 (s, 3H), 3.93 (s, 3H), 4.23 (m, 1H),
6.92 (s, 1H), 7.03 (s, 1H), 7.16 (s, 1H), 7.19 (s, 1H), 7.37 (s, 1H), 7.41
(s, 1H), 7.45 (s, 1H), 8.03 (m, 2H), 9.43 (s, 1H), 9.81 (s, 1H), 9.89 (s,
1H), 10.19 (s, 1H), 10.27 (s, 1H), 10.43 (s, 1H) ppm; [R]22 ) +6.8°
D
(c 0.07, H2O).
ImPyAaImPy-γ-PyPyâImPy-â-Dp (5). To 1019 (250 mg, 0.87
mmol) in DMF (3 mL) at room temperature were added HOBt (140
mg, 1 mmol) and DCC (1.0 mL, 1 M in NMP, 1 mmol). The reaction
was stirred at room temperature for 6 h. The mixture was filtered into
a solution of ethyl 4-amino-1-methylimidazole-2-carboxylate10 (320 mg,
1 mmol). To this solution was added i-Pr2NEt (1 mL). The reaction
was stirred at room temperature for 12 h and then was diluted with
EtOAc and washed (4×) with brine. The organic material was dried
(MgSO4), filtered, and concentrated. The residue was dissolved in
MeOH (5 mL). To this mixture was added aqueous NaOH (5 mL, 5
M, 5 mmol). The reaction was stirred at room temperature for 1 h and
then was acidified to pH 1 with 10% aqueous HCl. The product was
extracted into EtOAc and then was dried (Na2SO4), filtered, and
concentrated to provide crude 21 (110 mg). To 21 (110 mg, 0.3 mmol)
in DMF (2 mL) were added HBTU (200 mg, 0.6 mmol) and i-Pr2NEt
(0.8 mL). The mixture was agitated for 30 s and then was allowed to
stand for 5 min. The mixture was added to resin 14 (200 mg, 0.04
mmol) and was shaken at 37 °C for 3 h. The solvent was drained from
the vessel, and the resin was washed with THF. To the resin were added
Pd2(dba)3 (120 mg), Ph3P (500 mg), and BuNH2‚HCO2H in THF (10
mL). The mixture was shaken at room temperature for 2 h. The liquids
were drained, and the resin was washed several times with a solution
prepared from Na2S2C(NEt2) (1.0 g) and Et3N (1.0 mL) in DMF (100
mL). To the resin were added Fmoc-pyrrole (250 mg), HBTU (320
mg), and i-Pr2NEt (1 mL) in DMF (2 mL). The mixture was shaken at
37 °C for 1 h. The liquids were drained, and the Fmoc was removed
by piperidine (20% in DMF, 30 min, room temperature). The synthesis
was completed according to established protocols, except that the resin
was washed with TFA before cleavage with Dp. To the resin was added
dimethylaminopropylamine (2 mL). The mixture was shaken at 37 °C
for 10 h and then was filtered. The residue was purified by reverse
phase HPLC and was lyophilized to provide 5 (4 mg, 8% recovery).
MALDI-TOF-MS (monoisotopic) 1380.7 (1380.7, calcd for C63H82-
ImPySIsImPy-γ-PyPyâImPy-â-Dp (2). To (S)-9 (400 mg, 1.4
mmol) in DMF (2 mL) were added HBTU (500 mg, 1.3 mmol) and
i-Pr2NEt (1 mL). The mixture was agitated for 30 s and was then
allowed to stand for 5 min. The mixture was added to resin 15 (600
mg, 0.12 mmol) and then was agitated at 37 °C for 12 h. Completion
of the synthesis of resin-bound benzylated 2 proceeded according to
established protocols.10 To the resin (500 mg, 0.1 mmol) was added
3-dimthylaminopropylamine (2 mL). The mixture was shaken at 37
°C for 10 h then was filtered. The residue was purified by reverse phase
HPLC and was lyophilized to provide benzylated 2 (30 mg, 20%
recovery). To benzylated 2 (28 mg, 0.02 mmol) in TFA (1 mL) at 0
°C were added thioanisole (300 mg, 2.4 mmol) and TMSBr (300 mg,
2.0 mmol). The mixture was stirred at 0 °C for 3 h. The residue was
purified by reverse phase HPLC and was lyophilized to provide 2 (8
mg, 29% recovery). MALDI-TOF-MS (monoisotopic) 1381.7 (1381.7,
1
calcd for C63H81N24O13); H NMR (DMSO-d6, 300 MHz) δ 1.72 (m,
4H), 2.23 (m, 2H), 2.31 (m, 2H), 2.55 (m, 2H), 2.70 (s, 3H), 2.72 (s,
3H), 2.9-3.6 (m, 8H), 3.70 (s, 9H), 3.92 (s, 3H), 3.94 (s, 6H), 4.27
(m, 1H), 6.93 (s, 1H), 6.98 (s, 1H), 7.05 (s, 1H), 7.20 (s, 1H), 7.21 (s,
1H), 7.22 (s, 1H), 7.28 (s, 1H), 7.40 (s, 1H), 7.44 (s, 1H), 7.49 (s, 1H),
8.05 (m, 2H), 9.51 (s, 1H), 9.82 (s, 1H), 9.85 (s, 1H), 9.94 (s, 1H),
9.94 (s, 1H), 9.94 (s, 1H), 10.36 (s, 1H), 10.48 (s, 1H) ppm; [R]22
-2.4° (c 0.02, H2O).
)
D
ImPySIsImPy-γ-PyPyâImPy-â-Dp-EDTA (2E). To the resin con-
taining benzylated 2 (50 mg, 0.01 mmol) was added N,N,-bis(3-
1
N25O12); H NMR (DMSO-d6, 300 MHz) δ 1.72 (m, 4H), 2.31 (m,

6350 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Floreancig et al.
2H), 2.45 (m, 2H), 2.53 (m, 2H), 2.69 (s, 6H), 2.95 (m, 2H), 3.06 (m,
2H), 3.15 (m, 2H), 3.36 (m, 3H), 3.80 (s, 3H), 3.82 (s, 3H), 3.85 (s,
3H), 3.89 (s, 6H), 3.91 (s, 9H), 6.80 (s, 1H), 6.89 (s, 1H), 6.92 (s, 1H),
6.99 (s, 1H), 7.03 (s, 1H), 7.11 (s, 1H), 7.12 (s, 1H), 7.13 (s, 1H), 7.16
(s, 1H), 7.19 (s, 1H), 7.34 (s, 1H), 7.41 (s, 1H), 7.47 (s, 1H), 8.02 (m,
2H), 9.80 (s, 1H), 9.82 (s, 1H), 9.90 (s, 1H), 9.94 (s, 1H), 10.24 (s,
1H), 10.47 (s, 1H), 10.92 (s, 1H) ppm; [R]22D ) -3.1° (c 0.06, H2O).
ImPyFbImPy-γ-PyPyâImPy-â-Dp (6). To 13 (75 mg, 0.4 mmol)
in DMF (1.5 mL) were added HBTU (180 mg, 0.5 mmol) and i-Pr2-
NEt (0.8 mL). The mixture was agitated for 30 s and was allowed to
stand for 5 min. The mixture was added to resin 15 (100 mg, 0.02
mmol) and was shaken at 37 °C for 12 h. The completion of the
synthesis proceeded according to standard protocols10 to provide 6 (8
mg, 30% recovery). MALDI-TOF-MS (monoisotopic) 1383.6 (1838.7,
calcd for C63H80FN24O12); 1H NMR (DMSO-d6) δ 1.72 (m, 4H), 2.5-
3.3 (m, 12H), 2.75 (s, 3H), 2.78 (s, 3H), 3.75 (s, 9H), 3.88 (s, 6H),
3.92 (s, 6H), 3.93 (s, 3H), 6.80 (s, 1H), 6.89 (s, 1H), 6.93 (s, 1H), 6.99
(s, 1H), 7.11 (s, 1H), 7.13 (s, 1H), 7.16 (s, 1H), 7.22 (s, 1H), 7.34 (s,
1H), 7.41 (s, 1H), 7.48 (s, 1H), 8.03 (m, 2H), 9.80 (s, 1H), 9.82 (s,
1H), 9.91 (s, 1H), 10.02 (s, 1H), 10.24 (s, 1H), 10.40 (s, 1H), 10.50 (s,
precipitated. The cleavage products were resuspended in 100 mM Tris-
borate-EDTA/80% formamide loading buffer, denatured at 85 °C for
10 min, and immediately loaded onto an 8% denaturing polyacrylamide
gel (5% cross-link, 7 M urea) at 2000 V for 1.5 h. The gels were dried
under vacuum at 80 °C, then quantified using storage phosphor
technology. Equilibrium association constants were determined as
previously described.8
MPE‚Fe(II) Footprinting.12 All reactions were carried out in a
volume of 400 µL. A polyamide stock solution or water (for reference
lanes) was added to an assay buffer where the final concentrations
were: 20 mM HEPES buffer (pH 7.0), 10 mM NaCl, and 15 kcpm 3′-
or 5′-radiolabeled DNA. The solutions were allowed to equilibrate for
3 h. A fresh 50 µM MPE‚Fe(II) solution was prepared from 100 µL of
a 100 µM MPE solution and 100 µL of a 100 µM ferrous ammonium
sulfate (Fe(NH4)2(SO4)2‚6H2O) solution. MPE‚Fe(II) solution (5 µM)
was added to the equilibrated DNA, and the reactions were allowed to
equilibrate for 10 min. Cleavage was initiated by the addition of
dithiothreitol (5 mM) and allowed to proceed for 14 min. Reactions
were stopped by ethanol precipitation, resuspended in 100 mM Tris-
borate-EDTA/80% formamide loading buffer, denatured at 85 °C for
10 min, and a 7 µL sample was immediately loaded onto an 8%
denaturing polyacrylamide gel at 2000 V. The gels were dried under
vacuum at 80 °C, then quantified using storage phosphor technology.
Affinity Cleaving.13 All reactions were carried out in a volume of
400 µL. A polyamide stock solution or water (for reference lanes) was
added to an assay buffer where the final concentrations were: 20 mM
HEPES buffer (pH 7.0), 20 mM NaCl, and 15 kcpm 3′- or 5′-
radiolabeled DNA. The solutions were allowed to equilibrate for 3 h.
A fresh solution of ferrous ammonium sulfate (Fe(NH4)2(SO4)2‚6H2O)
(10 µM) was added to the equilibrated DNA, and the reactions were
allowed to equilibrate for 15 min. Cleavage was initiated by the addition
of dithiothreitol (10 mM) and allowed to proceed for 30 min. Reactions
were stopped by ethanol precipitation, resuspended in 100 mM Tris-
borate-EDTA/80% formamide loading buffer, denatured at 85 °C for
6 min, and the entire sample was immediately loaded onto an 8%
denaturing polyacrylamide at 2000 V. The gels were dried under
vacuum at 80 °C, then quantified using storage phosphor technology.
1H) ppm; [R]22 ) +1.4° (c 0.08, H2O).
D
DNA Reagents and Materials. Enzymes were purchased from
Boehringer-Mannheim and used with their supplied buffers. Deoxy-
adenosine and thymidine 5′-[R-32P]triphosphates, and deoxyadenosine
5′-[γ-32P]triphosphate were purchased from New England Nuclear.
Sonicated, deproteinized calf thymus DNA was acquired from Phar-
macia. RNase free water was obtained from USB and used for all
footprinting reactions. All other reagents and materials were used as
received. All DNA manipulations were performed according to
established protocols.27
Preparation of 3′- and 5′-End-Labeled Restriction Fragments.
The plasmid pSES 19-1 was constructed according to standard
protocols. pSES 19-1 was linearized with EcoRI and PVuII restriction
enzymes, then treated with the Klenow fragment, deoxyadenosine 5′-
[R-32P]triphosphate and thymidine 5′-[R-32P]triphosphate for 3′ labeling.
Alternatively, these plasmids were linearized with EcoRI, treated with
calf alkaline phosphatase, and then 5′-labeled with T4 polynucleotide
kinase and deoxyadenosine 5′-[γ-32P]triphosphate. The 5′-labeled
fragment was then digested with PVuII. The labeled fragment (3′ or
5′) was loaded onto a 7% non-denaturing polyacrylamide gel, and the
desired 292 base pair band was visualized by autoradiography and
isolated.
DNase I Footprinting.11 All reactions were carried out in a volume
of 400 µL. A polyamide stock solution or water (for reference lanes)
was added to an assay buffer where the final concentrations were: 10
mM Tris‚HCl buffer (pH 7.0), 10 mM KCl, 10 mM MgCl2, 5 mM
CaCl2, and 15 kcpm 3′-radiolabeled DNA. The solutions were allowed
to equilibrate for 2-12 h at 22 °C. Cleavage was initiated by the
addition of 10 µL of a DNase I stock solution (diluted with 1 mM
DTT to give a stock concentration of 1.875 u/mL) and was allowed to
proceed for 7 min at 22 °C. The reactions were stopped by adding 50
µL of a solution containing 2.25 M NaCl, 150 mM EDTA, 0.6 mg/mL
glycogen, and 30 µM base-pair calf thymus DNA, and then ethanol
Acknowledgment. We are grateful to the National Institutes
of Health for research support, the National Institutes of Health
for a postdoctoral fellowship (GM18311-02) to P.E.F., the
National Institutes of Health for a research traineeship award
(GM-08501) to S.E.S., and the National Science Foundation
for a predoctoral fellowship to J.W.T. We thank Dr. Gary
Hathaway for MALDI-TOF mass spectroscopy and Ms. Meredith
Howard for assistance in the preparation of compound 13.
Supporting Information Available: Quantitative DNase I
footprinting gels of polyamides 1, 4, 5, and 6 with the 3′-32P-
labeled restriction fragment from pSES 19-1, the MPE foot-
printing gel of 2 with the 5′-32P-labeled restriction fragment,
and the affinity cleaving gel of 2E with the 5′-32P-labeled
restriction fragment (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
(27) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
JA000509U