Novel phthalimide nucleosides for the specific recognition of a CG Watson-Crick base pair

Article abstract of DOI:10.1016/S0040-4039(00)02256-5

In an effort to extend the triple helix recognition code we have synthesized various substituted phthalimide-derived nucleosides that can recognize a CG Watson-Crick base pair. NMR experiments performed on the free nucleosides in methylene chloride at lowered temperatures indicate the strength and extent of H-bonding to the cytosine amino group of a CG base pair which strongly depend on the substituent of the phthalimide nucleobase. Consistent with the formation of an additional hydrogen bond to N7 of guanine, ureido-substituted nucleoside analogs show a higher overall affinity as compared to the 3-aminophthalimide nucleoside.

Full text of DOI:10.1016/S0040-4039(00)02256-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1479–1481
Novel phthalimide nucleosides for the specific recognition of a
CG Watson–Crick base pair
David Lengeler and Klaus Weisz*
Institut fu¨r Chemie der Freien Universita¨t Berlin, Takustraße 3, D-14195 Berlin, Germany
Received 15 November 2000; accepted 4 December 2000
Abstract—In an effort to extend the triple helix recognition code we have synthesized various substituted phthalimide-derived
nucleosides that can recognize a CG Watson–Crick base pair. NMR experiments performed on the free nucleosides in methylene
chloride at lowered temperatures indicate the strength and extent of H-bonding to the cytosine amino group of a CG base pair
which strongly depend on the substituent of the phthalimide nucleobase. Consistent with the formation of an additional hydrogen
bond to N7 of guanine, ureido-substituted nucleoside analogs show a higher overall affinity as compared to the 3-aminophthal-
imide nucleoside. © 2001 Elsevier Science Ltd. All rights reserved.
Triple helix formation through selective binding of a
single-stranded oligonucleotide to regions of double-
stranded DNA has long been recognized as a powerful
tool for various applications. Examples include the
site-specific inhibition of transcription or the develop-
ment of sequence-specific artificial nucleases. Third
strand binding is based on the formation of specific
hydrogen bonds between nucleobases of the single
strand and the purine bases of the Watson–Crick
duplex in its major groove.1 Thus, in the pyrimidine
motif formation of Hoogsteen hydrogen bonds between
third strand pyrimidine and Watson–Crick purine bases
results in isomorphous C+·GC and T·AT base triplets.
As a consequence of this binding mode, the recognition
by third strand bases without severe distortions of the
oligonucleotide backbone is mostly restricted to GC
and AT Watson–Crick base pairs in homop-
urine·homopyrimidine sequences. In contrast, CG and
TA interruptions within the duplex target are not effec-
tively recognized by natural nucleobases.
a CG Watson–Crick base pair. The 3-aminophthal-
imide riboside 1 is expected to interact with a CG base
pair by forming two hydrogen bonds with the 4-amino
group of C and the 6-carbonyl oxygen of G (Fig. 1).
Molecular modeling studies suggest that a 1·CG base
triplet should be closely isomorphous to the canonical
C+·GC and T·AT base triplets, thus minimizing any
distortions of the sugar-phosphate backbone when
incorporated in a triplex-forming oligonucleotide. In
addition, the phthalimide heterocycle is expected to be
less prone to intercalation, a problem often encountered
for oligonucleotide-directed triplex formation using
more extended aromatic ring systems as base
surrogates.3
The synthesis of 1 is outlined in Scheme 1. Due to the
poor nucleophilicity of the phthalimide nitrogen, a
Vorbrueggen-type coupling of sugar and base is not
To overcome this major limitation in the sequence-spe-
cific triplex formation, much work has been devoted in
the past to the design of non-natural nucleobases for
extending the triple helix recognition code.2 Here we
describe the synthesis of novel nucleoside analogs which
are based on the easily accessible amino-substituted
phthalimide derivative 1 for the selective recognition of
Keywords: triple helix; hydrogen bond; nucleoside; base pair.
* Corresponding author. Fax: +49-30-838-55310; e-mail: weisz@
chemie.fu-berlin.de
Figure 1. Canonical C+·GC base triplet (left) and 1 binding a
CG base pair to form a 1·CG base triplet (right).
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02256-5

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D. Lengeler, K. Weisz / Tetrahedron Letters 42 (2001) 1479–1481
Scheme 1. (i) Bu3SnOMe, C2H4Cl2, 32%; (ii) PPh3, DEAD, THF, 37%; (iii) Fe3(CO)12, toluene, MeOH, 86%.
ation. Unfortunately, observation of the cytosine amino
successful. However, deacetylation of 1-O-acetyl-2,3,5-
proton NHb is severely hampered under such condi-
tions due to spectral overlap. However, employing spe-
cifically 4-15N amino labeled cytidine enables easy
detection of the amino proton even in the presence of
strong overlapping proton resonances of the excess
nucleoside analog by means of a 1H-15N filter, as imple-
mented in a one-dimensional HMQC experiment.8
tri-O-benzoyl-b-D-ribofuranose at the 1-position with
Bu3SnOMe4 and reaction with 3-nitrophthalimide
under Mitsunobu conditions leads to N-(2%,3%,5%-tri-O-
benzoyl-b-D-ribofuranosyl)-3-nitrophthalimide. Subse-
quent reduction of the nitro group to give 1 is
accomplished under mild conditions with Fe3(CO)12
and methanol.5
Due to the large association constant (Kass>104–105 M−1
in CHCl3 at 293 K) a 1:1 mixture of C and G in
CD2Cl2 fully associates to form a CG Watson–Crick
base pair that may interact with a third base by its
residual hydrogen bond donor and acceptor sites. Thus,
a downfield shift of the C Hb signal upon addition of a
tenfold excess of 1, 2, 3 or 4 to a 1:1 mixture of
4-15N-dC and dG in CD2Cl2 at 240 K indicates the
formation of ternary complexes with the cytosine
amino proton involved in a hydrogen bond with the
phthalimide derivatives (Fig. 3).9 In contrast, no signifi-
cant changes in their chemical shift were observed for
G H1 and C Ha resonances consistent with their partic-
ipation in a Watson–Crick hydrogen bond that is not
disrupted in the presence of the nucleoside analog.
For increasing the acidity of the NH group and for
extending possible hydrogen bond contacts to N7 of the
guanine base we have synthesized additional derivatives
2, 3 and 4 (Fig. 2). Reaction of 1 with benzoylchloride
in THF/pyridine gives N-benzoylated 2 (82% yield).
The urea group in compounds 3 and 4 was introduced
through the reaction of 1 with chlorosulfonylisocyanate
in CH2Cl2 and with n-butylisocyanate in THF in 83
and 64% yield, respectively.6
To study hydrogen bond interactions between our
nucleoside analogs and a CG base pair, we have per-
formed NMR measurements on a 1:1 mixture of 3%,5%-
di-O-(triisopropylsilyl) protected 2%-deoxycytidine and
2%-deoxyguanosine in the presence of the free tri-O-ben-
zoylated ribonucleosides 1, 2, 3 or 4 in CD2Cl2. Hydro-
gen bond formation is easily followed by a downfield
shift of the corresponding proton resonance involved in
hydrogen bonding. The observed chemical shift
depends on: (i) the association constant for complex
formation; (ii) on the limiting chemical shift of the
hydrogen-bonded proton in a complex which is a mea-
sure of the geometry and strength of the hydrogen
bond;7 (iii) on the limiting chemical shift of the non-
hydrogen-bonded proton in a monomer which is influ-
enced by its individual chemical environment. In order
to minimize contributions due to different proton
chemical environments, we have recorded chemical
shifts of the same non-Watson–Crick hydrogen-bonded
cytosine amino proton NHb in the presence of each of
the four synthetic nucleosides. Clearly, this proton is
always expected to be engaged in a hydrogen bond for
a ternary complex.
Recently, an alkylated hexylureido phthalimide was
used as a synthetic receptor for a CG base pair and
found to bind via three hydrogen bonds with high
affinity in chloroform (Kassꢀ103 M−1 at room tempera-
ture).10 Assuming only minor influences of different
residues and solvent, it is expected that under our low
temperature conditions all of the CG base pairs have
associated with the urea analogs 3 and 4 being in
excess.11 Consequently, the C Hb chemical shift should
directly reflect the strength of the hydrogen bond
between the cytosine amino and phthalimide carbonyl
Our strategy also makes use of low temperatures and
an excess of the nucleoside surrogate to promote associ-
Figure 2. Nucleoside 1 and derivatives 2, 3 and 4.

D. Lengeler, K. Weisz / Tetrahedron Letters 42 (2001) 1479–1481
1481
2. (a) Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillespie,
P.; Dervan, P. B. J. Am. Chem. Soc. 1992, 114, 7976–
7982; (b) Huang, C.-Y.; Cushman, C. D.; Miller, P. S. J.
Org. Chem. 1993, 58, 5048–5049; (c) Sasaki, S.;
Nakashima, S.; Nagatsugi, F.; Tanaka, Y.; Hisatome,
M.; Maeda, M. Tetrahedron Lett. 1995, 36, 9521–9524;
(d) Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc.
1995, 117, 10769–10770; (e) Huang, C.-Y.; Bi, G.; Miller,
P. S. Nucleic Acids Res. 1996, 24, 2606–2613; (f) Leh-
mann, T. E.; Greenberg, W. A.; Liberles, D. A.; Wada,
C. K.; Dervan, P. B. Helv. Chim. Acta 1997, 80, 2002–
2022; (g) Lecubin, F.; Benhida, R.; Fourrey, J.-L.; Sun,
J.-S. Tetrahedron Lett. 1999, 40, 8085–8088.
Figure 3. 1H chemical shift l for the cytosine amino proton
Hb in a mixture of [4-15N]-3%,5%-di-O-(triisopropylsilyl)-2%-de-
oxycytidine, 3%,5%-di-O-(triisopropylsilyl)-2%-deoxyguanosine
and 1–4 (1:1:10) in CD2Cl2 at 240 K; cCG=5.45 mM.
3. Koshlap, K. M.; Gillespie, P.; Dervan, P. B.; Feigon, J. J.
Am. Chem. Soc. 1993, 115, 7908–7909.
4. Nudelman, A.; Herzig, J.; Gottlieb, H. E.; Keinan, E.;
Sterling, J. Carbohydr. Res. 1987, 162, 145–152.
group, pointing to a slightly stronger hydrogen bond
for 3 with a non-alkylated urea substituent. Compared
to 3 and 4, Hb resonances in complexes of 1 and 2 with
only two potential hydrogen bonds are less downfield
shifted. Excluding any cooperative effects, loss of an
additional hydrogen bond contact is always expected to
improve the geometry and strength of residual contacts
unless there is a perfect steric fit. Thus, the available
chemical shift data can only be rationalized by lower
association constants with only partial CG complexa-
tion by 1 and 2 even at lower temperatures. Compared
to 2, 1 shows stronger binding towards the cytosine
amino group. This might be due to steric hindrance of
the bulky benzoyl group. In addition, a stronger hydro-
gen bond involving the more acidic amide proton of 2
might compromise the strength of the NHbꢀO hydro-
gen bond by geometric readjustments.
5. Landesberg, J. M.; Katz, L.; Olsen, C. J. Org. Chem.
1972, 37, 930–936.
6. Analytical and spectral data for the nucleoside analogs.
Compound 1: 1H NMR (250 MHz, CD2Cl2): l (ppm)
4.54–4.75 (m, 3H; H4%, H5%, H5¦), 6.06 (d, 1H; H1%), 6.19
(app. t, 1H; H3%), 6.33 (dd, 1H; H2%), 7.36–8.09 (m, 18H;
ArH). Anal. calcd for C34H26N2O9: C, 67.32; H, 4.32; N,
1
4.62; found: C, 66.57; H, 4.21; N, 4.38. Compound 2: H
NMR (250 MHz, CDCl3): l (ppm) 4.56–4.81 (m, 3H;
H4%, H5%, H5¦), 6.09 (d, 1H; H1%), 6.17 (app. t, 1H; H3%),
6.28 (dd, 1H; H2%), 7.29–8.25 (m, 23H; ArH), 8.62 (s, 1H;
NH). Anal. calcd for C41H30N2O10: C, 69.29; H, 4.25; N,
1
3.94; found: C, 68.35; H, 4.43; N, 3.72. Compound 3: H
NMR (250 MHz, CD2Cl2): l (ppm) 4.56–4.82 (m, 3H;
H4%, H5%, H5¦), 5.48 (s, br, 2H; NH2), 6.12 (d, 1H; H1%),
6.19 (app. t, 1H; H3%), 6.29 (dd, 1H; H2%), 7.27–8.03 (m,
18H; ArH), 8.21 (s, 1H; NH). Anal. calcd for
C35H27N3O10: C, 64.71; H, 4.19; N, 6.47; found: C, 63.97;
H, 4.32; N, 6.36. Compound 4: 1H NMR (250 MHz,
CDCl3): l (ppm) 0.87 (t, 3H; CH3), 1.22–1.50 (m, 4H;
CH2CH2), 3.22 (t, 2H; N-CH2), 4.54–4.79 (m, 3H; H4%,
H5%, H5¦), 6.08 (d, 1H; H1%), 6.19 (app. t, 1H; H3%), 6.29
(dd, 1H; H2%), 7.22–8.09 (m, 18H; ArH). Anal. calcd for
C39H35N3O10: C, 66.38; H, 5.00; N, 5.95; found: C, 66.31;
H, 5.03; N, 5.56.
In summary, we have synthesized four differently sub-
stituted phthalimide-derived nucleoside analogs that
can bind a CG base pair via specific hydrogen bonds.
By employing selectively 15N amino labeled cytosine as
a local probe, NMR experiments may not only provide
rapid information on their affinity towards a CG base
pair but also on the strength of individual hydrogen
bonds, thus complementing future studies on the ther-
modynamics of binding. It is expected that such system-
atic investigations will yield more insight into the
binding of a CG base pair with novel nucleobases and
constitute a first step in understanding the complex
interplay of the different interactions effective in a triple
helical oligonucleotide.
7. (a) Golubev, N. S.; Shenderovich, I. G.; Smirnov, S. N.;
Denisov, G. S.; Limbach, H.-H. Chem. Eur. J. 1999, 5,
492–497; (b) Dingley, A. J.; Masse, J. E.; Peterson, R.
D.; Barfield, M.; Feigon, J.; Grzesiek, S. J. Am. Chem.
Soc. 1999, 121, 6019–6027; (c) Dunger, A.; Limbach,
H.-H.; Weisz, K. J. Am. Chem. Soc. 2000, 122, 10109–
10114.
8. For the preparation of 4-15N labeled 2%-deoxycytidine,
see: Kupferschmitt, G.; Schmidt, J.; Schmidt, T.; Fera,
B.; Buck, F.; Ru¨terjans, H. Nucleic Acids Res. 1987, 15,
8225–6241.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft for
financial support.
9. Note that care was taken to ensure that nucleoside con-
centration and molar ratios were equal for all samples.
10. Mertz, E.; Mattei, S.; Zimmerman, S. C. Org. Lett. 2000,
2, 2931–2934.
11. Self-association of the phthalimide nucleoside 4 in meth-
ylene chloride was found to be much smaller than the
expected binding to a CG base pair (Kass,self=13 M−1 at
room temperature).
References
1. Thuong, N. T.; He´le`ne, C. Angew. Chem., Int. Ed. Engl.
1993, 32, 666–690.
.