Oligomerization route to Py-Im polyamide macrocycles

Article abstract of DOI:10.1021/ol901311m

Image Persented Cyclic eight-ring pyrrole-imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solu

Full text of DOI:10.1021/ol901311m

ORGANIC
LETTERS
2009
Vol. 11, No. 16
3590-3593
Oligomerization Route to Py-Im
Polyamide Macrocycles
David M. Chenoweth, Daniel A. Harki, and Peter B. Dervan*
DiVision of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125
derVan@caltech.edu
Received June 11, 2009
ABSTRACT
Cyclic eight-ring pyrrole-imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate
endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solution-phase methods. A rapid solution-
phase oligomerization approach to eight-ring symmetrical cyclic polyamides yields 12- and 16-membered macrocycles as well. A preference
for DNA binding by the 8- and 16-membered oligomers was observed over the 12-ring macrocycle, which we attributed to a conformational
constraint not present in the smaller and larger systems.
Pyrrole-imidazole polyamides are a class of cell-permeable
oligomers that target the minor groove of DNA in a
sequence-specific manner.1,2 Antiparallel arrangements of
N-methylpyrrole (Py) and N-methylimidazole (Im) carboxa-
mides (Im/Py) recognize G•C from C•G base pairs, whereas
Py/Py specifies for both T•A and A•T.3 Hairpin Py-Im
polyamides have been programmed for a broad repertoire
of DNA sequences with high affinities.4 These cell-perme-
able5 ligands can influence gene transcription by disrupting
protein-DNA interfaces2 and have been shown to control
transcription of genes important in human disease.6 Py-Im
polyamides have also been used for a variety of applications
such as fluorescence-based DNA detection,7 transcriptional
activation with artifical transcription factor mimics,5e,8 and
self-assembly of DNA nanoarchitectures.9
We recently reported solution-phase methods for the
synthesis of hairpin10a and cyclic polyamides.11a Key to the
cyclic polyamide synthesis was a macrocyclization from an
acyclic precursor that yielded cyclic polyamide 1z (Figure
1). Activation of the C-terminal Py amino acid of 1z as a
pentafluorophenyl ester allowed efficient macrocyclization
by the γ-NH2 on the turn moiety under dilute reaction
conditions. Our studies of polyamide 1 revealed it possessed
(5) (a) Belitsky, J. M.; Leslie, S. J.; Arora, P. S.; Beerman, T. A.; Dervan,
P. B. Bioorg. Med. Chem. 2002, 10, 3313–3318. (b) Crowley, K. S.; Phillion,
D. P.; Woodard, S. S.; Scheitzer, B. A.; Singh, M.; Shabany, H.; Burnette,
B.; Hippenmeyer, P.; Heitmeier, M.; Bashkin, J. K. Bioorg. Med. Chem.
Lett. 2003, 13, 1565–1570. (c) Best, T. P.; Edelson, B. S.; Nickols, N. G.;
Dervan, P. B Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12063–12068. (d)
Edelson, B. S.; Best, T. P.; Olenyuk, B.; Nickols, N. G.; Doss, R. M.; Foister,
S.; Heckel, A.; Dervan, P. B. Nucleic Acids Res. 2004, 32, 2802–2818. (e)
Xiao, X.; Yu, P.; Lim, H. S.; Sikder, D.; Kodadek, T. Angew. Chem., Int.
Ed. Engl. 2007, 46, 2865–2868. (f) Nickols, N. G.; Jacobs, C. S.; Farkas,
M. E.; Dervan, P. B. Nucleic Acids. Res. 2007, 35, 363–370. (g) Dose, C.;
Farkas, M. E.; Chenoweth, D. M.; Dervan, P. B. J. Am. Chem. Soc. 2008,
130, 6859–6866. (h) Hsu, C. F.; Dervan, P. B. Bioorg. Med. Chem. Lett.
2008, 18, 5851–5855.
(1) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215–2235
.
(2) Dervan, P. B.; Edelson, B. S. Curr. Opin. Struct. Biol. 2003, 13,
284–299
.
(3) (a) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382,
559–561. (b) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan,
P. B. Nature 1998, 391, 468–470. (c) Kielkopf, C. L.; Baird, E. E.; Dervan,
P. B.; Rees, D. C. Nat. Struct. Biol. 1998, 5, 104–109. (d) Kielkopf, C. L.;
White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B.;
Rees, D. C. Science 1998, 282, 111–115.
(4) Hsu, C. F.; Phillips, J. W.; Trauger, J. W.; Farkas, M. E.; Belitsky,
J. M.; Heckel, A.; Olenyuk, B. Z.; Puckett, J. W.; Wang, C. C.; Dervan,
P. B. Tetrahedron 2007, 63, 6146–6151.
10.1021/ol901311m CCC: $40.75
Published on Web 07/23/2009
 2009 American Chemical Society

very high DNA binding affinities and could modulate gene
expression in cell culture from which we infer eight-ring
cycles are cell permeable.11a
An orthogonal polymerization/oligomerization strategy for
the synthesis of 1 and related polyamides is reported here.
This method affords symmetrical Py-Im polyamide mac-
rocycles from simple Py-Im building blocks in a convergent
(6) (a) Olenyuk, B. Z.; Zhang, G. J.; Klco, J. M.; Nickols, N. G.; Kaelin,
W. G.; Dervan, P. B Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16768–16773.
(b) Kageyama, Y.; Sugiyama, H.; Ayame, H.; Iwai, A.; Fujii, Y.; Huang,
L. E.; Kizaka-Kondoh, S.; Hiraoka, M.; Kihara, K. Acta Oncol. 2006, 45,
317–324. (c) Nickols, N. G.; Jacobs, C. S.; Farkas, M. E.; Dervan, P. B.
ACS Chem. Biol. 2007, 2, 561–571. (d) Nickols, N. G.; Dervan, P. B. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 10418–10423. (e) Matsuda, H.; Fukuda,
N.; Ueno, T.; Tahira, Y.; Ayame, H.; Zhang, W.; Bando, T.; Sugiyama,
H.; Saito, S.; Matsumoto, K.; others, O. J. Am. Soc. Neph. 2006, 17, 422–
432. (f) Yao, E. H.; Fukuda, N.; Ueno, T.; Matsuda, H.; Matsumoto, K.;
Nagase, H.; Matsumoto, Y.; Takasaka, A.; Serie, K.; Sugiyama, H.;
Sawamura, T. Hypertension 2008, 52, 86–92. (g) Takahashi, T.; Asami,
Y.; Kitamura, E.; Suzuki, T.; Wang, X.; Igarashi, J.; Morohashi, A.;
Shinojima, Y.; Kanou, H.; Saito, K.; Takasu, T.; Nagase, H.; Harada, Y.;
Kuroda, K.; Watanabe, T.; Kumamoto, S.; Aoyama, T.; Matsumoto, Y.;
Bando, T.; Sugiyama, H.; Yoshida-Noro, C.; Fukuda, N.; Hayashi, N. Chem.
Biol. 2008, 15, 829–841. (h) Hochhauser, D.; Kotecha, M.; O’hare, C.;
Morris, P. J.; Hartley, J. M.; Taherbhai, Z.; Harris, D.; Forni, C.; Mantovani,
R.; Lee, M.; Hartley, J. A. Mol. Cancer Ther. 2007, 6, 346–354.
(7) (a) Rucker, V. C.; Foister, S.; Melander, C.; Dervan, P. B. J. Am.
Chem. Soc. 2003, 125, 1195–1202. (b) Chenoweth, D. M.; Viger, A.;
Dervan, P. B. J. Am. Chem. Soc. 2007, 129, 2216–2217. (c) Fujimoto, J.;
Bando, T.; Minoshima, M.; Kashiwazaki, G.; Nishijima, S.; Shinohara, K.;
Sugiyama, H. Bioorg. Med. Chem. 2008, 16, 9741–9744.
Figure 1. Structures of macrocyclic polyamides 1z-3z and 1-3
and their ball-and-stick models. Polyamide shorthand code: closed
circles, Im monomer; open circles, Py monomer.
(8) (a) Arndt, H. D.; Hauschild, K. E.; Sullivan, D. P.; Lake, K.; Dervan,
P. B.; Ansari, A. Z. J. Am. Chem. Soc. 2003, 125, 13322–13323. (b) Kwonj,
Y.; Arndt, H. D.; Qian, M.; Choi, Y.; Kawazoe, Y.; Dervan, P. B.; Uesugi,
M. J. Am. Chem. Soc. 2004, 126, 15940–15941. (c) Hauschild, K. E.;
Metzler, R. E.; Arndt, H. D.; Moretti, R.; Raffaelle, M.; Dervan, P. B.;
Ansari, A. Z. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5008–5013. (d)
Stafford, R. L.; Arndt, H. D.; Brezinski, M. L.; Ansari, A. Z.; Dervan, P. B.
J. Am. Chem. Soc. 2007, 129, 2660–2668. (e) Stafford, R. L.; Dervan, P. B.
J. Am. Chem. Soc. 2007, 129, 14026–14033.
manner (Scheme 1). As serendipitous minor products, higher-
order oligomers such as the 12-membered (2) and 16-
membered (3) cyclic polyamides are also produced by this
method. In addition to describing the synthetic chemistry to
prepare 1-3, we examined the DNA binding properties of
such expanded polyamide macrocycles.
(9) (a) Cohen, J. D.; Sadowski, J. P.; Dervan, P. B. Angew. Chem., Int.
Ed. 2007, 46, 7956–7959. (b) Schmidt, T. L.; Nandi, C. K.; Rasched, G.;
Parui, P. P.; Brutschy, B.; Famulok, M.; Heckel, A. Angew. Chem., Int.
Ed. 2007, 46, 4382–4384. (c) Cohen, J. D.; Sadowski, J. P.; Dervan, P. B.
J. Am. Chem. Soc. 2008, 130, 402–403.
Our strategy for this oligomerization route relied on the
palindromic nature of polyamide 1. Disconnection of 1 at
both γ-amino turns affords two identical halves of the
molecule. Bimolecular coupling between two molecules,
followed by intramolecular ring closure, delivers cyclic
Py-Im polyamides. Bifunctional oligomer 4 contains every
atom needed to construct cyclic polyamides 1-3 by this
process (Scheme 1).
(10) (a) Chenoweth, D. M.; Harki, D. A.; Dervan, P. B. J. Am. Chem.
Soc. 2009, 131, 7175–7181. For additional solution-phase polyamide
synthesis studies see: (b) Mrksich, M.; Parks, M. E.; Dervan, P. B. J. Am.
Chem. Soc. 1994, 116, 7983–7988. (c) Xiao, J.; Yuan, G.; Huang, W.; Chan,
A. S.; Lee, K. L. J. Org. Chem. 2000, 65, 5506–5513. (d) Harris, D.; Stewart,
M.; Sielaff, A.; Mulder, K.; Brown, T.; Mackay, H.; Lee, M. Heterocycl.
Commun. 2007, 13, 17–23. (e) Xiao, J.-H.; Huang, W.-Q.; Tang, F.-L.;
Yuan, G.; Chan, A. S. C.; Lee, K-L. D Chin. J. Chem. 2000, 18, 603–607.
(f) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122,
6382–6394. (g) Mamidyala, S. K.; Firestine, S. M. Tetrahedron Lett. 2006,
47, 7431–7434. (h) Sinyakov, A. N.; Feshchenko, M. V.; Ryabinin, V. A.
Russ. J. Bioorg. Chem. 2004, 30, 98–99. (i) Yuan, G.; Tang, F. ARKIVOC
2003, ii, 32–37. (j) Xiao, J. H.; Yuan, G.; Huang, W. Q.; Tang, F. L.; Chan,
A. S. C.; Lee, K. L. D. Chin. Chem. Lett. 2000, 11, 207–208. (k) For
additional solid-phase hairpin polyamide synthesis studies see: Baird, E. E.;
Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141–6146. (l) Belitsky, J. M.;
Nguyen, D. H.; Wurtz, N. R.; Dervan, P. B. Bioorg. Med. Chem. 2002, 10,
2767–2774. (m) Wurtz, N. R.; Turner, J. M.; Baird, E. E.; Dervan, P. B.
Org. Lett. 2001, 3, 1201–1203. (n) Krutzik, P. O.; Chamberlin, A. R. Bioorg.
Med. Chem. Lett. 2002, 12, 2129–2132. (o) Krutzik, P. O.; Chamberlin,
A. R. Methods Mol. Biol. 2002, 201, 77–92. (p) Ayame, H.; Saito, T.; Bando,
T.; Fukuda, N.; Sugiyama, H. Nucleic Acids. Res. Suppl. 2003, 67–68. (q)
Moore, M. J. B.; Cuenca, F.; Searcey, M.; Neidle, S. Org. Biomol. Chem.
2006, 4, 3479–3488.
The pentafluorophenyl ester 4 was prepared in one step
from the previously reported carboxylic acid of 4.11a Acidic
deprotection of the γ-amino functionality of 4 followed by
drying in vacuo yields intermediate 5 which is the substrate
for the homodimerization/oligomerization reaction. To initiate
this sequence, the protected trifluoroacetate salt 5 was diluted
with DMSO, then treated with an organic base (DIEA) to
unmask the nucleophilic primary γ-amine. The ensuing
oligomerization/macrocyclization process provides benzyl-
carbamate protected cyclic polyamides 1z, 2z, and 3z and
trace amounts of unisolated higher-order oligomers. A
distribution of uncyclized intermediates corresponding to the
dimer (8-ring cycle, 1z), trimer (12-ring cycle, 2z), tetramer
(16-ring cycle, 3z), and higher-order adducts can be observed
at early time points, as evidenced by HPLC analysis at 2 h
(Figure S1, Supporting Information). Extended reaction times
(20 h) reveals cyclized polyamides 1z, 2z, and 3z in a ratio
of 6.6:2.6:1 almost exclusively (Figure 2). Isolation of 1z
(11) (a) Chenoweth, D. M.; Harki, D. A.; Phillips, J. W.; Dose, C.;
Dervan, P. B. J. Am. Chem. Soc. 2009, 131, 7182–7188. For additional
solid-phase cyclic polyamide synthesis studies, see: (b) Cho, J.; Parks, M. E.;
Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10389–10392. (c)
Zhang, Q.; Dwyer, T. J.; Tsui, V.; Case, D. A.; Cho, J.; Dervan, P. B.;
Wemmer, D. E. J. Am. Chem. Soc. 2004, 126, 7958–7966. (d) Herman,
D. M.; Turner, J. M.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1999,
121, 1121–1129. (e) Melander, C.; Herman, D. M.; Dervan, P. B.
Chem.sEur. J. 2000, 6, 4487–4497.
Org. Lett., Vol. 11, No. 16, 2009
3591

(13.9%), 2z (5.5%), and 3z (2.1%) by preparative HPLC,
followed by Cbz-deprotection under acidic conditions (solu-
tion of CF3CO2H and CF3SO3H), provides polyamide mac-
rocycles 1-3.
geometry, with two adjacent, antiparallel ImPyPyPy strands
followed by an identical repeat of this motif linked through
two intervening turn units (Figure S2, Supporting Informa-
tion).
Scheme 1. Synthesis of Macrocyclic Polyamides 1-3 and
Higher-Order Cycles by Oligomerization of Bifunctional
Intermediate 5
Figure 2. Reverse-phase HPLC analysis (20 h) of the oligomer-
ization reaction revealing products 1z, 2z, and 3z. Peaks were
identified by high-resolution mass spectrometry following isolation
and Cbz-deprotection.
Table 1. Tm Values for Cycles 1-3 in the Presence of DNAa
Quantitative DNase I footprint titrations have historically
been utilized to measure polyamide-DNA binding affinities
and specificities.12 However, this method is limited to
measuring Ka values e2 × 1010 M-1, which invalidates this
technique for quantifying the exceptionally high DNA-
binding affinities of cycles 1 and 3. The magnitude of DNA
thermal stabilization (∆Tm) of DNA-polyamide complexes
has been utilized to rank order polyamides with high DNA
binding affinities,5g,11a and we have employed melting
temperature analysis (∆Tm) for evaluating DNA-binding
ability in this study. Polyamide 1, which targets the DNA
sequence 5′-WGWWCW-3′ (W ) A or T) through pairing
of two antiparallel ImPyPyPy strands, increases the melting
temperature of 14-mer dsDNA containing one binding site
by 23.6 °C.11a With polyamide macrocycles 2 and 3 in hand,
we evaluated their ability to bind duplex DNA relative to
cycle 1. Trimeric macrocycle 2 failed to bind its target
double-stranded DNA sequence as evidenced by the complete
lack of ligand-promoted thermal stabilization of duplex DNA
melting (Table 1). This result is presumably due to inherent
geometrical constraints of 2, preventing the side-by-side
antiparallel alignment of the ImPyPyPy strands, a motif well
accommodated by the DNA minor groove. Remarkably, in
the case of tetrameric macrocycle 3, dsDNA binding and
thermal stabilization was restored to a comparable value to
dimer 1. We hypothesize that an even number of ImPyPyPy
strands allows 3 to possess a collapsed or folded tetramer
a All values reported are derived from at least three melting temperature
experiments with standard deviations indicated in parentheses. ∆Tm values
are given as Tm(DNA/polyamide) - Tm(DNA). The propagated error in
∆Tm measurements is the square root of the sum of the square of the standard
deviations for the Tm values.
In summary, we have demonstrated that macrocyclic
polyamides can by synthesized by oligomerization of a
bifunctional polyamide to yield a distribution of cyclic
polyamide oligomers. Additionally, we show that certain
large cyclic polyamide geometries are able to bind dsDNA,
a result which could be utilized in the design of highly
specific molecules for targeting larger binding site sizes. Due
to the size of the 16-ring cycle, the larger series are likely
(12) Trauger, J. W.; Dervan, P. B. Methods Enzymol. 2001, 340, 450–
466.
3592
Org. Lett., Vol. 11, No. 16, 2009

not cell permeable and therefore have limited utility in gene
regulation studies. Rather, 16-ring cycles may add to the
repetoire of DNA binding motifs for use in DNA nanotech-
nology.9
the California Tobacco-Related Disease Research Program
(16FT-0055) for a postdoctoral fellowship.
Supporting Information Available: Compound charac-
terization, Figure S1, Figure S2, and experimental proce-
dures. This material is available free of charge via the Internet
at http://pubs.acs.org.
Acknowledgment. This work was supported by the
National Institutes of Health (GM27681). D.M.C. is grateful
for a Caltech Kanel predoctoral fellowship. D.A.H. thanks
OL901311M
Org. Lett., Vol. 11, No. 16, 2009
3593