A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules

Arcamone F.

Belitsky J. M.

Best T. P.

Boger D. L.

Bremer R. E.

Chenoweth D. M.

Daniel A. Gubler

Dervan P. B.

Doss R. M.

Edelson B. S.

Fiedler H. P.

Jevgenij A. Raskatov

Jordan L. Meier

Katy A. Muzikar

Kopka M. L.

Marques M. A.

Matsuda H.

Mrksich M.

Muzikar K. A.

Nickols N. G.

Olenyuk B. Z.

Pelton J. G.

Peter B. Dervan

Pilch D. S.

Renneberg D.

Schneider K.

Stover J. S.

Wade W. S.

White S.

Wolter F. E.

Woods C. R.

English

PubMed

Muzikar, Katy A.

Meier, Jordan L.

Gubler, Daniel A.

Raskatov, Jevgenij A.

Dervan, Peter B.

Caltech Authors - Main

Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

Crossref

Organic Letters

10.1021/ol202285y r 2011 American Chemical Society
Published on Web 09/29/2011
ORGANIC
LETTERS
2011
Vol. 13, No. 20
5612–5615
Expanding the Repertoire of Natural
Product-Inspired Ring Pairs for Molecular
Recognition of DNA
Katy A. Muzikar, Jordan L. Meier, Daniel A. Gubler, Jevgenij A. Raskatov, and
Peter B. Dervan*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125, United States
dervan@caltech.edu
Received August 30, 2011
ABSTRACT
A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin
polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with
N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate
sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules.
Small molecules capable of perturbing gene expression
by the selective modulation of proteinDNA interfaces
may have important applications in biology and human
medicine. Hairpin pyrrole-imidazole polyamides are a
class of synthetic minor groove-binding ligands that can
be programmed to bind a broad repertoire of DNA
sequences with affinities and specificities comparable to
those of DNA-binding proteins (Figure 1A).1 Cell-perme-
able polyamides have been shown to localize to the nuclei
of living cells2,3 and regulate endogenous gene expression by
interfering with transcription factor/DNA interfaces.48
The structures of these programmable oligomers were
inspired by the crescent-shaped natural products netropsin
and distamycin (Figure 1B), which are comprised of two
and three aromatic N-methylpyrrole heterocyclic rings,
respectively.9 Distamycin is known to bind the minor
groove of A,T tracks of DNA with both 1:1 and 2:1
ligand/DNA stoichiometries.10,11 Guided by structural
and biophysical studies, synthetic analogues of distamycin
were developed which applied single-atom changes to the
N-methylpyrrole ring in order to rationally alter the A,T
(1) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215–2235.
(2) Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 12063–12068.
(3) 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.
(4) Olenyuk, B. Z.; Zhang, G. J.; Klco, J. M.; Nickols, N. G.; Kaelin,
W.G., Jr.; Dervan, P. B.Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16768–
16773.
(5) Matsuda, H.; Fukuda, N.; Ueno, T.; Tahira, Y.; Ayame, H.;
Zhang, W.; Bando, T.; Sugiyama, H.; Saito, S.; Matsumoto, K.;
Mugishima, H.; Serie, K. J. Am. Soc. Nephrol. 2006, 17, 422–432.
(6) Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 10418–10423.
(7) Muzikar, K. A.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad.
Sci. U.S.A. 2009, 106, 16598–16603.
(8) Nickols, N. G.; Jacobs, C. S.; Farkas, M. E.; Dervan, P. B. ACS
Chem. Biol. 2007, 2, 561–571.
(9) Arcamone, F.; Penco, S.; Orezzi, P.; Nicolella, V.; Pirelli, A.
Nature 1964, 203, 1064–1065.
(10) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson,
R. E. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376–1380.
(11) Pelton, J. G.;Wemmer,D. E.Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 5723–5727.
Org. Lett., Vol. 13, No. 20, 2011 5613
binding specificity of the natural product.12,13 These efforts
resulted in a set of pairing rules, in which amino acid
oligomers composed of heterocyclic analogues of N-
methylpyrrole oriented in a hairpin configuration can be
combined as unsymmetrical ring pairs in order to read the
minor groove of DNA.14 Specifically, side-by-side stacked
N-methylimidazole (Im) and N-methylpyrrole (Py) car-
boxamides (Im/Py pairs) distinguish G 3C from C 3G base
pairs, whereasN-methyl-3-hydroxypyrrole (Hp)/Py shows
specificity for T 3A over A 3T. Finally, Py/Py pairs specify
for both T 3A and A 3T (Figure 1A).
15 However, while Py/
Py and Py/Im represent the most robust sets of sequence-
specific pairs, continued efforts to optimize polyamide
affinity and specificity have led to the evaluation of several
additional amino acid pairings.16,17 For example, incor-
poration of the fused heterocycles hydroxybenzimidazole
(Hz) and imidazopyridine (Ip) within the hairpin poly-
amide scaffold produces Py/Hz and Py/Ip ring pairs cap-
able of functionally replacing Hp/Py and Py/Im in T 3A
and C 3G recognition.
18 The continued exploration of new
ring pairs is essential in order to further refine the ability of
these molecules to recognize the sequence-dependent mi-
crostructures of DNA, and importantly to optimize their
pharmacokinetic properties for biological applications.
Recently Sussmuth and co-workers revealed the discov-
ery of the natural products proximicins AC, which
contain a central core of 2,4-disubstituted furan amino
acid dipeptides (Figure 1C).19 While these molecules show
structural similarity to netropsin and distamycin, 4-amino-
furan-2-carboxylates represent a new heterocyclic amino
acid which had not been previously reported in the
literature.2022 Notably, the proximicins show consid-
erably greater antitumor activity and cytotoxicity than
Figure 1. Natural and engineered small molecules for binding
the minor groove of DNA.
Figure 2. DFT-optimization (B3LYP/6-31G(d)) of the PyPyPy
and PyFnPy trimers in the conformation relevant for DNA
recogntion. The angle X-Y-Z defines the trimer curvature.
Comparison with Im and Hp is provided in the Supporitng
Information.
(12) Wade, W. S.; Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc.
1992, 114, 8783–8794.
(13) Mrksich, M.; Wade, W. S.; Dwyer, T. J.; Geierstanger, B. H.;
Wemmer, D. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
7586–7590.
(14) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan,
P. B. Nature 1998, 391, 468–471.
(15) Dervan, P. B.; Edelson, B. S. Curr. Opin. Struct. Biol. 2003, 13,
284–299.
(16) Marques, M. A.; Doss, R. M.; Urbach, A. R.; Dervan, P. B.
Helv. Chim. Acta 2002, 85, 4485–4517.
(17) Doss, R. M.; Marques, M. A.; Foister, S.; Chenoweth, D. M.;
Dervan, P. B. J. Am. Chem. Soc. 2006, 128, 9074–9079.
(18) Renneberg, D.; Dervan, P. B. J. Am. Chem. Soc. 2003, 125,
5707–5716.
(19) Fiedler, H. P.; Bruntner, C.; Riedlinger, J.; Bull, A. T.; Knutsen,
G.; Goodfellow, M.; Jones, A.; Maldonado, L.; Pathom-aree, W.; Beil,
W.; Schneider,K.;Keller, S.; Sussmuth,R.D. J.Antibiot. (Tokyo) 2008,
61, 158–163.
(20) Woods, C. R.; Faucher, N.; Eschgfaller, B.; Bair, K. W.; Boger,
D. L. Bioorg. Med. Chem. Lett. 2002, 12, 2647–2650.
(21) Boger,D.L.; Fink,B. E.;Hedrick,M.P. J.Am.Chem.Soc. 2000,
122, 6382–6394.
(22) Stover, J. S.; Shi, J.; Jin, W.; Vogt, P. K.; Boger, D. L. J. Am.
Chem. Soc. 2009, 131, 3342–3348.
5614 Org. Lett., Vol. 13, No. 20, 2011
netropsin and distamycin, and the two families of mole-
cules appear to exert their influence on different cellular
targets.23However, in contrast tonetropsin anddistamycin,
the proximicins do not stabilize the thermal melting of
DNA duplexes. The recent development of a synthetic
route to the proximicin core allowed synthesis of furan-
based netropsin and distamycinmimics (Figure 1C). These
cationic proximicin analogues show greatly reduced ther-
mal stabilization of DNA relative to netropsin and
distamycin.24 Molecular modeling indicates replacement
of the central Py of a PyPyPy trimer by furan (Fn) is
accompanied by a significant reduction in overall molecu-
lar curvature, likely due to reduced repulsion of the amide
carbonyl group by the Fn oxygen compared to the N-Me
group of Py (Figure 2). This raises the question of how the
Fn amino acidmight behave in the context of ring pairs, as
enforced in a hairpin polyamideDNA complex. Specifi-
cally, doesFn/Pymimic the sequence preferences of Py/Py,
and does Fn/Im mimic the sequence preferences Py/Im?
To this end, we synthesized Py/Im hairpin polyamide 1,
which targets the sequence 50-WGGWCW030 (where W=
A or T), as well as three analogues (24) with furan rings
substituted at varying positions within the polyamide
hairpin structure (Figure 3). This particular core sequence
allows comparisonof all threePy/Im/Fn ringpairswithin a
single series of analogues: by altering which position of the
parent Py-Im polyamide 1 is substituted with a furan we
can assess base pair specificity of both a Fn/Im (2) pair as
well as a Fn/Py (3) pair. Furthermore, by incrementally
increasing the number of furan rings from zero (1) to two
(4) we can begin to assess the effects overall Fn content will
have on the energetics of Py/Im/Fn polyamide DNA-
binding.
Polyamides 14 were synthesized by solid-phase meth-
ods using Boc-protectedmonomeric units onKaiser oxime
resin. Incorporation of Boc-protected Py and Im rings
followed established methods (Scheme S1).25 However,
while the reported route to 4-tert-butoxycarbonylamino-
furan-2-carboxylic acid (Fn monomer) proved robust,24
incorporation of the Fn monomer unit into eight-ring Py-
Im polyamides was complicated by the sensitivity of the
furan monomer to the repeated application of the harsh
acidic conditions used for Boc removal. Increasing decom-
position of the growing oligomer chain was observed
following each trifluoroacetic acid (TFA) deprotection.
Attempts to use Lewis acid deprotection strategies (boron
trifluoride diethyl etherate or trimethylsilyl chloride) were
also unsuccessful, resulting in negligible recovery of poly-
amides from the resin. Hairpins 24 represent a minimal
panel of furan-containing polyamides that require (at
most) only two cycles of exposure to TFA, and thus were
accessible by this route, although in overall yields (0.57%)
approximately 50% lower than those previously reported
for polyamide oxime synthesis. Therefore we note that
for future syntheses of hairpin polyamides incorporat-
ing Fn rings it may be prudent to utilize Fmoc-based
peptide synthesis methodologies due to the milder condi-
tions required in removal of the Fmoc protecting group.
Critically, polyamides containingFn rings, once isolated,
appear to have photochemical and thermal stabilities iden-
tical to those for standard Py-Im polyamides and remain
purebyHPLCanalysis evenafter being in solution at room
temperature for several days.
Figure 3. Chemical and ball and stick structures of polyamides
containing furan amino acids. Ball and stick symbols are defined
as follows: N-methylpyrrole is denoted by an open circle, N-
methylimidazole is denoted by a filled circle, and furan is
denoted by open circles with “F” inside.
(23) Schneider, K.; Keller, S.; Wolter, F. E.; Roglin, L.; Beil, W.;
Seitz, O.; Nicholson, G.; Bruntner, C.; Riedlinger, J.; Fiedler, H. P.;
Sussmuth, R. D. Angew. Chem., Int. Ed. 2008, 47, 3258–3261.
(24) Wolter, F. E.; Schneider, K.; Davies, B. P.; Socher, E. R.;
Nicholson, G.; Seitz, O.; Sussmuth, R. D. Org. Lett. 2009, 11, 2804–
2807.
(25) Belitsky, J. M.; Nguyen, D. H.; Wurtz, N. R.; Dervan, P. B.
Bioorg. Med. Chem. 2002, 10, 2767–2774.
(26) Pilch, D. S.; Poklar, N.; Gelfand, C. A.; Law, S. M.; Breslauer,
K. J.; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
8306–8311.
(27) Bremer, R. E.; Szewczyk, J. W.; Baird, E. E.; Dervan, P. B.
Bioorg. Med. Chem. 2000, 8, 1947–1955.
Org. Lett., Vol. 13, No. 20, 2011 5615
Following synthesis, polyamides 14 were analyzed for
DNA binding affinity and specificity by melting tempera-
ture analysis26 of 12-mer duplex DNA oligonucleotides
containing the sequence 50-TGGXCA-30 where X was
cycled through all four base pair possibilities (Table 1).
Spectroscopic analyses show that all four hairpins con-
siderably increase the thermal stability of each DNA
duplex, indicative of polyamide-DNA binding. The rela-
tive shifts inDNAmelting temperature induced by 13 for
each of the two match DNA duplexes tested are similar
(ΔTm ≈ 1518 C), indicating that the binding affinity of
hairpin polyamides containing Fn/Py or Fn/Im ring pairs
remains intact relative to the traditional Py/Im scaffold
(Table 1). This thermal stabilization is reduced when X is
mutated to aC 3GorG 3C (ΔΔTm≈ 57 C), demonstrat-
ing polyamide specificity is also unaffected by Fn substitu-
tion. Furthermore, multiple Fn pairs are tolerated when
housed in the hairpin polyamide scaffold, as indicated by
the marked stabilization of duplex DNA by polyamide 4.
Thus, despite the differences in Fn and Py curvature
predicted from modeling studies, our experimental results
indicate that 2,4-substituted furan amino acids can func-
tion as Py-mimics when paired in the hairpin polyamide
DNA complex.
A more subtle finding of the melting temperature study
is that polyamides incorporating the Fn/Im pair (2,4)
exhibit slightly decreased thermal stabilization of DNA
duplexeswhen compared topolyamides containing aPy/Im
pair at this position (1, 3). While the magnitude of this
decrease is close to the limit of error of the melting
temperature assay, a consistent trend was observed for
all four duplexes (Table 1). It was unclear whether this
reduction in DNA-binding affinity is an inherent property
of the Fn/Im pair, a positional effect of Fn-incorporation
adjacent to the turn residue, or an indication of repulsive
effects between the Fn oxygen, which projects outward
from the minor groove, and the negatively charged DNA
backbone.24 We explored this by synthesizing polyamides
inwhich theFn rings of 24were replacedwith desmethyl-
pyrrole (Ds) aminoacids (FigureS1, 57), whichprovide a
more polar, less bulky alternative to Py but offer a hydro-
gen instead of a lone pair for interaction with the DNA
backbone.27 Thermal denaturation analysis indicates Ds-
containing polyamides stabilize duplex DNA with similar
magnitudes and specificity as Py/Im and Py/Im/Fn-poly-
amides 14 (Table S1). As with the Fn series, Ds/Im
containing polyamide 5 exhibits slightly decreased thermal
stabilization relative to Py/Im polyamide 1, despite its
drastically different electronic and hydrogen bonding
properties. Therefore, the small loss in affinity for Fn/Im
at this position is likely a consequence of loss of the
hydrophobic N-methyl of Py and not any repulsive inter-
actions of the Fn monomer.
Recent in vitro ADMET analysis of a hairpin Py/Im
polyamide revealed high levels of plasma protein binding,
an oftentimes negative pharamacokinetic determinant
characteristic of a highly lipophilic compound.28 This led
us to briefly explore the ability of alternative ring pairs to
modulate polyamide lipophilicity. Our anticipation was
that incorporation of themore polar Fn ring (and removal
of the corresponding N-methyl group) would decrease
lipophilicity. However, substitution of Py by one or two
Fn rings (compounds 3, 4) does not significantly alter the
lipophilicity of parent polyamide 1 as measured by its
LogD in a miniaturized shake-flask octanol/water parti-
tion assay (Table S2). In contrast, compounds 57demon-
strated a considerable decrease inLogDvalue compared to
parent compound 1, representative of increased water
solubility. While the greater polarity of the Ds-containing
polyamides is consistent with previous observations,27 the
lack of effect of Fn incorporation on LogD is surprising.
The difficulty in predicting such properties a priori is an
indication that DNA-binding polyamides may benefit
from a systematic effort to define their pharmacokinetic
properties, as has been done for cellular uptake, and is thus
an area of active research.
Acknowledgment. We are grateful to the National In-
stitutes ofHealth (GM27681) for research support. J.L.M.
(PF-10-015-01-CDD) and D.A.G. (PF-09-262-01-CDD)
were supported by postdoctoral fellowships from the
American Cancer Society. J.A.R. was supported by an
Alexander von Humboldt postdoctoral fellowship.
Supporting Information Available. Supplementary data
associated with this article, including synthetic proce-
dures and characterization data, thermal DNA stabiliza-
tion data, computational analyses, and polyamide LogD
values can be found in the Supporting Information. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Table 1.Melting Temperatures of DNA/Polyamide Complexes
for Cognate and Mismatch Duplexesa
match DNA mismatch DNA
X-Y A-T T-A G-C C-G
DNA 58.0 ( 0.2 58.2 ( 0.5 59.6 ( 0.1 59.8 ( 0.3
1 76.2 ( 1.9
[18.2]
76.2 ( 2.3
[17.9]
71.2 ( 0.2
[11.7]
72.3 ( 0.3
[12.4]
2 73.8 ( 0.3
[15.8]
75.2 ( 1.5
[17.0]
69.9 ( 0.1
[10.3]
70.0 ( 0.4
[10.1]
3 76.6 ( 1.1
[18.6]
77.1 ( 0.8
[18.9]
70.8 ( 0.6
[11.9]
72.9 ( 0.3
[13.1]
4 73.2 ( 1.3
[15.2]
74.6 ( 1.0
[16.3]
68.5 ( 0.5
[8.6]
69.1 ( 0.3
[9.3]
aAll values are derived from at least three independent experiments.
Shift in melting temperature temperature (ΔTm) is reported in brackets
below the absolute value.
(28) Chenoweth, D. M.; Harki, D. A.; Phillips, J. W.; Dose, C.;
Dervan, P. B. J. Am. Chem. Soc. 2009, 131, 7182–7188.


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Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

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