Synthetic receptors for CG base pairs.

Article abstract of DOI:10.1021/ol006157d

Hydrogen-bond-mediated complexation of a CG base pair by a hexylureido phthalimide and a hexylureido isoindolin-1-one was studied by (1)H NMR spectroscopy in an organic solvent. Chemical shift data indicate that both receptors effectively bind the CG base pair from the major groove side.

Full text of DOI:10.1021/ol006157d

ORGANIC
LETTERS
2000
Vol. 2, No. 19
2931-2934
Synthetic Receptors for CG Base Pairs
Eric Mertz, Sebastiano Mattei, and Steven C. Zimmerman*
Department of Chemistry, 600 South Mathews AVenue, UniVersity of Illinois,
Urbana, Illinois 61801
sczimmer@uiuc.edu
Received June 4, 2000
ABSTRACT
Hydrogen-bond-mediated complexation of a CG base pair by a hexylureido phthalimide and a hexylureido isoindolin-1-one was studied by 1H
NMR spectroscopy in an organic solvent. Chemical shift data indicate that both receptors effectively bind the CG base pair from the major
groove side.
The numerous examples of biomolecular recognition medi-
ated through hydrogen bonds have inspired the design of
nonnatural hydrogen-bonding systems capable of functioning
as receptors for molecules and assemblies of biological
interest. Because DNA and RNA contain multiple hydrogen-
bonding sites on the edges of the nucleic acid bases,
individual bases as well as oligonucleotides are ideal targets
for recognition through hydrogen bonding.1,2 The major
groove side of a cytosine-guanine (CG) base pair presents
a noncontiguous donor-acceptor-acceptor (DAA) hydrogen-
bonding array (Figure 1). This immediately suggests that a
receptor capable of binding the base pair could be designed
from a complementary hydrogen-bonding module. In addi-
tion to strong binding, the three hydrogen bonds formed
between such a receptor and the CG base pair should provide
selective binding because the thymine-adenine, guanine-
(1) Previous examples of host-guest chemistry involving recognition
of nucleic acid bases include the following: (a) Feibush, B.; Saha, M.;
Onan, K.; Karger, B.; Giese, R. J. Am. Chem. Soc. 1987, 109, 7531-7533.
(b) Adrian, J. C., Jr.; Wilcox, C. S. J. Am. Chem. Soc. 1989, 111, 8055-
8057. (c) Zimmerman, S. C.; Wu, W. J. Am. Chem. Soc. 1989, 111, 8054-
8055. (d) Rebek, J., Jr. Acc. Chem. Res. 1990, 23, 399-404. (e) Hamiliton,
A. D. J. Chem. Educ. 1990, 67, 821-828. (f) Hisatome, M.; Maruyama,
N.; Furutera, T.; Ishikawa, T.; Yamakawa, K. Chem. Lett. 1990, 2251-
2254. (g) Seel, C.; Vo¨gtle, F. Angew. Chem., Int. Ed. Engl. 1991, 30, 442-
444. (h) Furuta, H.; Magda, D.; Sessler, J. L. J. Am. Chem. Soc. 1991, 113,
978-985. (i) Ogoshi, H.; Hatakeyama, H.; Kotani, J.; Kawashima, A.;
Kuroda, Y. J. Am. Chem. Soc. 1991, 113, 8181-8183. (j) Inouye, M.; Kim,
K.; Kitao, T. J. Am. Chem. Soc. 1992, 114, 778-780. (k) Schwartz, E. B.;
Knobler, C. B.; Cram, D. J. J. Am. Chem. Soc. 1992, 114, 10775-10784.
(l) Nowick, J. S.; Chen, J. S.; Noronha, G. J. Am. Chem. Soc. 1993, 115,
7636-7644. (m) Eliseev, A. V.; Schneider, H.-J. J. Am. Chem. Soc. 1994,
116, 6081-6088. (n) Cˇ udic´, P.; Zˇinic´, M.; Tomisˇic´, V.; Simeon, V.;
Vigneron, J.-P.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1995, 1073-
1075. (o) Bell, T. W.; Hou, Z.; Zimmerman, S. C.; Thiessen, P. A. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2163-2165. (p) Mathew, J.; Buchardt, O.
Bioconjugate Chem. 1995, 6, 524-528. (q) Kickham, J. E.; Loeb, S. J.;
Murphy, S. L. Chem. Eur. J. 1997, 3, 1203-1213. (r) Spivak, D.; Gilmore,
M. A.; Shea, K. J. J. Am. Chem. Soc. 1997, 119, 4388-4393. (s) Asanuma,
H.; Ban, T.; Gotoh, S.; Hishiya, T.; Komiyama, M. Macromolecules 1998,
31, 371-377. (t) Piantanida, I.; Tomisˇic´, V.; Zˇinic´, M. J. Chem. Soc., Perkin
Trans. 2 2000, 375-383.
Chim. Acta 1997, 80, 2002-2022. (c) Kool, E. T. Chem. ReV. 1997, 97,
1473-1487. (d) Doronina, S. O.; Behr, J.-P. Chem. Soc. ReV. 1997, 63-
71. (e) Kool, E. T. New J. Chem. 1997, 21, 33-45. (f) Nielsen, P. E. Chem.
Eur. J. 1997, 3, 505-507. (g) Dervan, P. B.; Bu¨rli, R. W. Curr. Opin.
Chem. Biol. 1999, 3, 688-693. (h) Nielsen, P. E. Acc. Chem. Res.1999,
32, 624-630.
(3) Zimmerman, S. C. Schmitt, P. J. Am. Chem. Soc. 1995, 117, 10769-
10770.
(4) (a) Sasaki, S.; Nakashima, S.; Nagatsugi, F.; Tanaka, Y.; Hisatome,
M.; Maeda, M. Tetrahedron Lett. 1995, 36, 9521-9524. (b) Cho, Y. L.;
Jeong, K.-S. Tetrahedron Lett. 1997, 38, 8337-8340. (c) Lecubin, F.;
Benhida, R.; Fourrey, J.-L.; Sun, J.-S. Tetrahedron Lett. 1999, 40, 8085-
8088. (d) Saito, A.; Kuroda, R. Tetrahedron Lett. 1999, 40, 4837-4840.
(e) Lo´pez de la Paz, M.; Gonza´lez, C.; Vicent, C. Chem Commun. 2000,
411-412.
(2) Selected reviews and reports on artificial ligands for sequence specific
recognition of nucleic acids include the following: (a) Thuong, N. T.;
He´le`ne, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 666-690. (b) Lehmann,
T. E.; Greenberg, W. A.; Liberles, D. A.; Wada, C. K.; Dervan, P. B. HelV.
10.1021/ol006157d CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/19/2000

pyrimidine-purine-pyrimidine triplex motif.2b,6 To find base
pair receptors capable of stronger binding to CG, the 4-(N-
hexylureido)-phthalimide 2 and the 4-(N-hexylureido)-isoin-
dolin-1-one 3 were designed to form three hydrogen bonds
with the CG base pair (Figure 1). Molecular modeling
suggested a better fit for 2‚CG and 3‚CG in comparison to
1‚CG. The synthesis of receptors 2 and 3 and recognition
studies with CG in chloroform solution are detailed herein.
Phthalimide receptor 2 was synthesized through N-
alkylation of 4-nitrophthalimide7 with the dendritic bromide
4 (Scheme 1). This Fre´chet-type dendron8 provides solubility
Scheme 1
in organic solvents and a simple 1H NMR spectrum, making
it ideal for NMR binding analyses. Subsequent Raney nickel
mediated hydrogenation of the nitro adduct 5 followed by
condensation with triphosgene and hexylamine produced the
desired urea 2. The synthesis of 3 proceeded through a key
cyclization of nitrobenzoate 8 with dendritic amine 9
(Scheme 2).9 Base precursor 8 was synthesized by conversion
of methyl 6-methyl-3-nitrobenzoate (6)10 to the corresponding
4-nitrophenyl ester 7. Free radical bromination of 7 afforded
8 in moderate yield. Treatment of 8 with 9 and base afforded
10, which was readily converted into the desired urea 3 using
the methods described for the synthesis of 2.
Figure 1. The major groove side of the CG base pair as a donor-
acceptor-acceptor (DAA) hydrogen-bonding array and the comple-
mentary phthalimide (2) and isoindolin-1-one (3) receptors com-
plexed to CG by the formation of three hydrogen bonds.
cytosine, and adenine-thymine base pairs present different
arrays of hydrogen-bond donor and acceptor sites. We have
1
previously used H NMR complexation studies as a conve-
nient method for quantifying binding between the ureido
naphthimidazole receptor 1 and a CG base pair.3 Other
Scheme 2
groups have subsequently used this approach to examine the
binding of other artificial and natural base pair binders.4
Receptor 1 was found to complex CG with an association
constant (Kassoc) of 160 M-1 through formation of three
hydrogen bonds; however, this Kassoc value is relatively low
considering the strength of some triply hydrogen bonded
complexes having a DAA-ADD motif.5
Furthermore, we viewed the studies in organic solvents
as a logical first step toward developing nucleotide bases
capable of recognizing CG base pairs within triplex DNA.
The nucleotide analogue of 1 was incorporated into oligo-
nucleotides, but it did not effectively bind CG in the
(5) (a) Kelly, T. R.; Zhao, C.; Bridger, G. J. J. Am. Chem. Soc. 1989,
111, 3744-3745. (b) Murray, T. J.; Zimmerman, S. C. J. Am. Chem. Soc.
1992, 114, 4010-4011. (c) Zimmerman, S. C.; Corbin, P. S. Struct. Bonding
(Berlin) 2000, 96, 64-94.
(6) Zimmerman, S. C.; Schmitt, P., unpublished results
2932
Org. Lett., Vol. 2, No. 19, 2000

The binding studies were performed in CDCl3 solutions
by titrating receptors 2 or 3 with a 1:1 mixture of tri-O-
acetylcytidine and tri-O-acetylguanosine and observing the
Such minimal self-association precludes dimer formation at
1.9 mM, the fixed concentration of 2 used in the complex-
ation analysis. Interestingly, 3 was found to self-associate
much more strongly than 2, possibly due to the increased
hydrogen-bond-acceptor potential of the lactam carbonyl
group in 3 compared to the imide carbonyl group in 2.
Accordingly, binding studies of 3‚CG were conducted with
a sufficiently low fixed concentration of 3 (0.130 mM) so
that the 3‚3 dimer could be neglected.
The downfield shifts in both urea protons of the two
receptors indicate the formation of the two hydrogen bonds
between the urea group and the base pair. The weaker
complexation observed for the amide analogue 11‚CG
complex (Kassoc ) 70 M-1) confirms the importance of both
urea group hydrogen bonds (Table 1). The downfield shift
1
changes in H NMR chemical shifts of H-1, H-2, and H-5
(Figures 2 and 3). Strong binding between C and G (Kassoc
∼ 105 M-1)11 allowed a 1:1 mixture of the two nucleosides
to be treated as a single component.3 In a representative study
of the 2‚CG complex, increasing [CG] from 0.54 to 6.7 mM
resulted in significant downfield shifts in the 1H NMR signals
for H-1 (∆δ ) 2.1 ppm), H-2 (∆δ ) 1.5 ppm), and H-5
(∆δ ) 0.37 ppm) of receptor 2. Titration of 3 with CG
(0.26-6.5 mM) resulted in similar downfield shifts in signals
for H-1 (∆δ ) 2.4 ppm), H-2 (∆δ ) 1.5 ppm), and H-5
(∆δ ) 0.40 ppm) of receptor 3. For both receptors, plots of
∆δ vs [CG] for the aforementioned protons were fit to a
nonlinear binding equation appropriate for a 1:1 binding
model.12,13 Thus, association constants (Kassoc) of 1016 ((57)
M-1 and 736 ((155) M-1 were determined for 2‚CG and
3‚CG, respectively (Figure 2).
Table 1. Summary of Association Constants for Complexation
of Synthetic Receptors with CG
Figure 2. Representative plot of ∆δ vs [CG] for titration of a
synthetic receptor with CG. Shown is titration to form the complex
3‚CG. ∆δ for only H-1, H-2, and H-5 are shown; data for these
three protons were fit to a nonlinear binding equation to calculate
in H-5 observed for both 2 and 3 is also consistent with
complexation occurring as shown in Figure 1. Uncomplexed
2 and 3 both have free rotation about the C-N bond linking
the aromatic ring and the urea group. Such free rotation
allows conformations 3a and 3b to be populated in the case
of uncomplexed 3 (Figure 3); similar conformations can exist
for uncomplexed 2. Formation of the three hydrogen bonds
Kassoc
.
In the case of phthalimide 2, self-association studies
showed the receptor to weakly dimerize (Kdimer ) 18 M-1).
(7) Huntress, E. H.; Shriner, R. L. In Organic Syntheses; Blatt, A. H.,
Ed.; Wiley and Sons: New York, 1943; Collect. Vol. 2, p 459.
(8) (a) Hawker, C. J.; Fre´chet, J. M. J. J. Am. Chem. Soc. 1990, 112,
7638-7647. (b) Zeng, F.; Reichert, D. E. C.; Kolotuchin, S. V.; Zimmerman,
S. C. Science 1996, 271, 1095-1098.
(9) This cyclization approach is analogous to those used in the follow-
ing: (a) Almansa, C.; Gomez, L. A.; Cavalcanti, F. L.; Rodriguez, R.;
Carceller, E.; Bartroli, J.; Garcia-Rafanell, J.; Forn, J. J. Med. Chem. 1993,
36, 2121-2133. (b) Egbertson, M. S.; Naylor, A. M.; Hartman, G. D.; Cook,
J. J.; Gould, R. J.; Holahan, M. A.; Lynch, J. J., Jr.; Lynch, R. J.; Stranieri,
M. T.; Vassallo, L. M. Bioorg. Med. Chem. Lett. 1994, 4, 1835-1840.
(10) Phadnis, A. P.; Nanda, B.; Patwardhan, S. A.; Gupta, A. S. Indian
J. Chem.; Sect. B 1984, 23, 1098-1102.
(11) Kyogoku, Y.; Lord, R. C.; Rich, A. Biochim. Biophys. Acta 1969,
179, 10-17.
(12) Wilcox, C. S. In Frontiers in Supramolecular Organic Chemistry
and Photochemistry; Schneider, H. J., Durr, H., Eds.; VCH: New York,
1991; pp 123-143.
(13) Job’s plot analyses for the 2‚CG and 3‚CG complexes were
consistent with 1:1 stoichiometry. Job, P. Ann. Chim. Ser. 10 1928, 9, 113-
134.
Figure 3. Formation of the 3‚CG complex by freezing of the anti
conformation.
Org. Lett., Vol. 2, No. 19, 2000
2933

between the receptors and the major groove side of CG
requires the conformation 3b wherein H-5 becomes fixed
within the deshielding region of the urea carbonyl group.
Consistent with this model, complexation studies also
showed upfield shifts in the signals for H-3 in both 2 and 3.
titration of 2 with CG. Data for the titration of CG with 3
did not fit a 1:1 binding model. This result may be attributed
to dissociation of the CG base pair upon addition of an excess
of 3.14 For titration of CG with both 2 and 3, the observed
downfield shifts in H-4 of C are consistent with the formation
of a hydrogen bond between the cytidine amino group and
the imide or lactam carbonyl group of the receptors.
Downfield shifts in H-5 of C also support the formation of
such a hydrogen bond as H-5 moves into the deshielding
region of the imide or lactam carbonyl group.
In summary, 1H NMR binding studies indicate that
synthetic receptors 2 and 3 can bind CG strongly through
formation of three hydrogen bonds on the major groove side
of the base pair. The stability of these base triplets is about
10-fold higher (in terms of Kassoc) than that for the CG binder
1. Complexation in organic solvents does not necessarily
guarantee that a receptor will recognize a base pair within a
target DNA double helix.2b,6 Nonetheless, the strong com-
plexation of the CG base pair by receptors 2 and 3 bodes
well for the development of nonnatural nucleosides capable
of recognizing CG base pairs in the pyrimidine-purine-
pyrimidine DNA triple helix motif.2a-e A nucleoside analogue
of receptor 3 has been successfully incorportated into
oligonucleotides to test its ability to recognize CG base pairs
within double helical DNA. The results of these studies will
be reported separately.
1
Titration of 3 with CG also caused the H NMR singlet for
the methylene group protons (H-10) to become an AB
quartet. These protons become diastereotopic upon binding
to chiral CG. As the concentration of 3 was increased from
0.27 to 6.5 mM, ∆ν for the AB quartet increased from 6.9
to 35.4 Hz. A plot of ∆ν vs [CG] was also fit to a nonlinear
binding equation. The association constant (Kassoc ) 709 M-1)
thus determined was comparable to that found through
chemical shift analysis of protons H-1, H-2, and H-5.
Further evidence of the 2‚CG and 3‚CG complexes was
obtained in reverse titrations. Thus, titration of 1.88 mM
solutions of CG with 2 (0.57-5.1 mM) resulted in a large
1
downfield shift in the H NMR signal for H-4 of C (∆δ )
0.836 ppm, see Figure 3) and a smaller downfield shift in
the signal for H-5 of C (∆δ ) 0.07 ppm). Increasing [3]
from 0.70 to 7.0 mM in solutions with [CG] fixed at 2.0
mM resulted in similar downfield shifts in H-4 of C (∆δ )
0.826 ppm) and H-5 of C (∆δ ) 0.16 ppm). A plot of ∆δ
vs [2] fit a nonlinear equation describing a 1:1 binding model
giving Kassoc ) 800 M-1, a value similar to that found for
(14) To determine the extent to which 3 can dissociate a CG base pair,
complexes of 3 and the individual nucleosides (3‚C and 3‚G) were studied
by 1H NMR. Complexation was observed in the case of 3‚C (Kassoc ) 319
M-1), but no complexation was observed for 3‚G. We have not attempted
to fit these data to a multiequilibrium model, which in any event would be
complicated by the absence of a precise dissociation constant for the CG
base pair.
Acknowledgment. We thank the National Institutes of
Health (GM38010) for funding of this work and the
University of Illinois for fellowship assistance.
OL006157D
2934
Org. Lett., Vol. 2, No. 19, 2000