Cyanuryl-PNA monomer: Synthesis and crystal structure

Article abstract of DOI:10.1021/ol006257j

The chemical synthesis and crystal structure of the peptide nucleic acid (PNA) monomer 11 having cyanuric acid as the nucleobase is reported. The crystal structure of 11 shows molecular tapes arising from continuous intermolecular dimeric hydrogen bonding

Full text of DOI:10.1021/ol006257j

ORGANIC
LETTERS
2000
Vol. 2, No. 18
2825-2828
Cyanuryl-PNA Monomer: Synthesis and
Crystal Structure
,†
Gangadhar J. Sanjayan, V. R. Pedireddi,* and Krishna N. Ganesh*
DiVision of Organic chemistry (Synthesis), National Chemical Laboratory,
Pune 411008, India
kng@ems.ncl.res.in
Received June 26, 2000
ABSTRACT
The chemical synthesis and crystal structure of the peptide nucleic acid (PNA) monomer 11 having cyanuric acid as the nucleobase is
reported. The crystal structure of 11 shows molecular tapes arising from continuous intermolecular dimeric hydrogen bonding, with successive
tapes held by single hydrogen bonds in the backbone.
Study of nucleic acid mimics through the design and
synthesis of DNA analogues has assumed interest not only
from a structural point but also from their applications as
new medicinal agents.1 Among a wide variety of structurally
modified nucleic acids synthesized over the past decade,
peptide nucleic acids (1, PNA) have come to the fore because
they recognize the complementary nucleic acids through
Watson-Crick base pairing and exhibit strand invasion.2
Employing nonnatural nucleobase ligands in place of natural
nucleobases would help in understanding the recognition
process in terms of various factors contributing to the
complementation events such as hydrogen bonding and
internucleobase stacking.3 There is increasing interest in
modulating and expanding the recognition motifs of standard
base pairs as this may have potential applications in
diagnostics and nanomaterial chemistry.2,4 The nonstandard
nucleobases employed so far with PNA include 2-amino-
purine5 2, 2,6-diaminopurine6 3, æ-isocytosine7 4, E-base8
5, hypoxanthine9 6, 2-thiouracil10 7, and 6-thioguanine11 8,
and each offered specific effects on the stability of the
derived PNA:DNA hybrids.
A basic requirement for triplex formation is that the central
base of the triad must be able to form hydrogen bonds from
both sides, and purine bases are ideal for this purpose.12
Cyanuric acid, 9, a six-membered cyclic imide with alternate
arrangement of hydrogen bond donors and acceptors is
potentially well suited for such a purpose. It forms a network
(4) (a) Luyten, I.; Herdewijin, P. Eur. J. Med. Chem. 1998, 33, 515-
576. (b) Rana, V. S.; Ganesh, K. N. Nucleic Acids Res. 2000, 28, 1162-
1169.
(5) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Chem. Commun.
1997, 1913-1914.
(6) Haaima, G.; Hansen, H. F.; Christensen, L.; Dahl, O.; Nielsen, P. E.
Nucleic Acids Res. 1997, 25, 4639-4643.
(7) Egholm, M.; Christensen, L.; Dueholm, K.; Buchardt, O.; Coull, J.;
Nielsen, P. E. Nucleic Acids Res. 1995, 23, 217-222.
(8) Eldrup, A. B.; Dahl, O.; Nielsen, P. E. J. Am. Chem. Soc. 1997, 119,
11116-11117.
† E-mail: pediredi@ems.ncl.res.in.
(1) Bennett, C. F. In Applied Antisense Oligonucleotide Technology;
Stein, C. A., Craig, A. M., Eds.; Wiley-Liss Inc.: New York, 1998.
(2) (a) Nielsen, P. E. Curr. Opin. Biotechnol. 1999, 10, 71-75. (b)
Nielsen, P. E. Curr. Opin. Struct. Biol. 1999, 9, 353-357.
(3) (a) Jeffrey, G. A.; Saenger, W. In Hydrogen Bonding in Biological
Structures; Springer-Verlag: Berlin, 1991. (b) Guckian, K. M.; Schweitzer,
B. A.; Rex X.-F.; Sheils, C. J.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem.
Soc. 2000, 122, 2213-2222.
(9) Timar, Z.; Bottka, S.; Kovacs, L.; Penke, B. Nucleosides Nucleotides
1999, 18, 1131-1133.
(10) Lohse, J.; Dahl, O.; Nielsen, P. E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 11804-11808.
(11) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem.,
Int. Ed. 1998, 37, 2796-2823.
(12) Soyfer, V. N.; Potaman, V. N. Triple helical Nucleic Acids;
Springer: New York, 1996.
10.1021/ol006257j CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/12/2000

hexagonal network13d (Scheme 1b) instead of the anticipated
pattern shown in Scheme 1c. It appears that leaving a
potential hydrogen-bonding moiety such as keto oxygen
unsatisfied in the crystal structure owing to steric factors
leads to an improper packing arrangement, which precluded
the formation of continuous hydrogen-bonded tapes. Cya-
nuric acid nucleosides have been shown to originate in
radiation-exposed deoxyguanosine samples,14 and DNA
oligomers containing this base have been synthesized.15
The backbone moiety of nucleic acids, in the form of
sugar-phosphate linkage in DNA/RNA or peptide in PNA,
plays a crucial/indirect role in the formation of dimeric
hydrogen bonds.16 It would therefore be interesting to study
the effect of replacing the N-CH3 group with the putative
PNA backbone on its hydrogen-bonding propensity. We
report here the chemical synthesis of PNA monomer ethyl
N-(2-Boc-aminoethyl)-N-(cyanuric-1-ylacetyl)glycinate 11
having cyanuric acid as the base and which can be directly
used for the solid phase synthesis of PNA oligomers. Its
crystal structure interestingly shows a distinct difference from
10 in the pattern of intermolecular hydrogen bonding and
crystal packing, with the involvement of the backbone as
well.
of well-defined robust hydrogen-bonded systems arranged
on a molecular tape,13 with tapes held together by single
hydrogen bonds as shown in Scheme 1a. Monosubstitution
of cyanuric acid perturbs this hydrogen-bonding network,
as shown for N-methylcyanuric acid 10 which forms a
Selective mono N-alkylation of 9 is not an efficient
process, unlike that of other nucleobases. Though N-
carboxymethylcyanuric acid 15 is known to be one of the
products in the hydrolysis of N-methylenecarboxy melamine,17
the reported failure to isolate the pure product prompted us
to explore an alternative method of its synthesis as the
benzylester 14 (Scheme 2). The reaction of nitrobiuret 12
Scheme 1. Hydrogen Bonding in (a) Cyanuric Acid 9, (b)
N-Methylcyanuric Acid 10 (Found), (c) 10 (Expected)
Scheme 2. Synthesis of Cyanuric Acid PNA Monomer 11a
a Reagents: (i) glycine benzyl ester toluene-4-sulfonate, Et3N,
DMF, 80 °C, 6 h; (ii) CDI, pyridine, reflux, 30 min; (iii) KOH,
aqueous MeOH, reflux, 45 min; (iv) ethyl N-(2-Boc-aminoethyl)-
glycinate, HOBT, 0 °C, DCC, DMF.
with the benzyl ester of glycine gave the N-benzyloxycar-
bonylmethyl biuret 13. The next step involved construction
(13) (a) Coppens, P.; Vos, A. Acta Crystallogr. 1971, B27, 146-158.
(b) MacDonald, J. C.; Whitesides, G. M. Chem. ReV. 1994, 94, 2383-
2420. (c) Ranganathan, A.; Pedireddi, V. R.; Rao, C. N. R. J. Am. Chem.
Soc. 1999, 120, 1752-1753. (d) Ranganathan, A.; Pedireddi, V. R.;
Sanjayan, G.; Ganesh, K. N.; Rao, C. N. R. J. Mol. Struct. 2000, 522, 87-
94.
(14) Raoul, S.; Cadet, J. J. Am. Chem. Soc. 1996, 118, 1892-1898.
(15) Gasparutto, D.; Da Cruz, S.; Bourdat, A.-G.; Jaquinod, M.; Cadet,
J. Chem. Res. Toxicol. 1999, 12, 630-638.
2826
Org. Lett., Vol. 2, No. 18, 2000

Compound 11 crystallizes20 in an orthorhombic space
group, Pbca, with one molecule in the asymmetric unit as
shown in Figure 1. A packing analysis, shown in Figure 2,
reveals that 11 has the anticipated centrosymmetric hydrogen-
bonded dimeric structure with an H‚‚‚O distance of 2.07 Å
(Figure 2a). In comparison with the corresponding hydrogen
bond distance in the crystal structure of 9 (H‚‚‚O, 1.78 Å),
the strength of the hydrogen bond in 11 is certainly weaker.
However, this is not surprising since the presence of the
bulky substituent at one of the heterocyclic nitrogens affects
the acidity of the remaining protons. It is worth mentioning
that the hydrogen bond distance noted in compound 11 is
comparable with such distances found in the various mo-
lecular complexes of 9.13c,d Furthermore, the adjacent mol-
ecules are held together by N-H‚‚‚O hydrogen bonds formed
between the keto oxygen of imide and the -NH of the
backbone, with an H‚‚‚O distance of 2.14 Å (Figure 2b).
Such an arrangement ultimately leads to the formation of
chains of molecules arranged in a helical mode. In the
3-dimensional lattice, the arrangement of the helices viewed
down the [010] axis is shown in Figure 2c. Since such an
interaction was not feasible in the crystal structure of 10, it
could not form the required dimeric hydrogen bond. It would
be interesting to compare the structural features of cyanuryl
PNA monomer 11 in solution (1H NMR) and crystals. Earlier
structural analyses of PNA monomers and dimers21 have
indicated high barriers of rotation (10-25 kcal M-1) and a
low rate of exchange (0.5-2 s-1, 37 °C) around the tertiary
amide bond (ø1), resulting in a cis:trans ratio of 30:70. In
the major trans form, the methylene protons next to the
nucleobase are toward the 2-aminoethyl unit, while in the
minor cis isomer, they are on the side of the glycyl unit.
The DQF-COSY assignment and observance of diagnostic
cross-peak between R-CH2 of 2-aminoethyl and CH2 adjacent
Figure 1. ORTEP drawing of an asymmetric unit in the crystal
structure of 11.
of the heterocyclic ring by carbonyl insertion-cyclization
of 13. It is known that refluxing an equimolar mixture of
N-monosubstituted biuret and diethyl carbonate with sodium
ethoxide leads to N-monosubstituted cyanurates.18 However,
this reaction with 13 led to an intractable mixture of products,
perhaps due to the presence of the base-sensitive N-
methylenecarboxy substituent. The procedure was hence
modified by use of the more reactive carbonyl diimidazole
in the presence of pyridine as base to obtain 14 in good
yields. This was saponified to the free acid 15 followed by
DCC-mediated coupling with ethyl N-(2-Boc-aminoethyl)-
glycinate to give the desired product 11. All products gave
satisfactory analytical and spectroscopic data for unambigu-
ous characterization.19
Figure 2. (a) Dimeric hydrogen bond pattern between the cyanuric acid moieties in the crystal structure of 11. (b) Helical arrangement of
adjacent molecules along [100] connected together by a N-H‚‚‚O hydrogen bond formed between keto oxygen of cyanuric acid moiety and
-NH of the backbone. (c) Three-dimensional arrangement of helices viewed down [010] in the crystal structure of 11.
Org. Lett., Vol. 2, No. 18, 2000
2827

to cyanuryl residue in the NOESY 1H NMR of 11 indicated
the major rotamer to be the trans isomer,21 identical to that
of the crystal structure.
angle γ on the aminoethylene side suggests opposite
conformations. The bulky tert-Boc group perhaps forces
different values in the torsion angle R for 11 as compared
to the oligomers.
In conclusion, we have presented a method for synthesis
of cyanuryl-PNA monomer 11 that is useful in the prepara-
tion of new PNA analogues. The structural features found
in the crystal data of cyanuryl monomer 11 shows confor-
mational similarity to PNA oligomeric structures, around
bonds encompassing the tertiary amide group. The results
indicate a preferred trans orientation of the side chain
carrying base in the monomer, identical to that found in the
oligomeric PNA complexes. Future efforts are directed
toward the synthesis of cyanuric PNA oligomers for studying
the consequences on DNA recognition.
To our knowledge, the structure of 11 reported here is
the first crystal structure of any PNA monomer, with the
other crystal structures known being those of a T-T
photodimer,22 a PNA2:DNA triplex,23 and a PNA:PNA
duplex.24 Table 1 shows a comparison of some of the torsion
Acknowledgment. We thank Professor C. N. R. Rao,
FRS, JNCASR, Bangalore, for helpful suggestions. K.N.G.
is a Senior Honorary Faculty of JNCASR. G.J.S. thanks
CSIR, New Delhi, for a Research Associateship.
Table 1. Torsion Anglesa in Known PNA Crystal Structures
OL006257J
compd
R
â
γ
δ
ø1
ø2
(19) 11 and 14 are present in solution as mixtures of rotamers in ratios
of 7:3 and some NMR signals are in multiples on this account. ma, major
isomer; mi, minor isomer. N-Benzyloxycarbonylmethyl cyanuric acid
14: mp 242-244 °C; 1H NMR (DMSO-d6, 200 MHz) δ 11.75 (s, 1H),
9.70 (mi), 8.85 (mi), 8.15 (ma), 7.70 (ma), 6.75 (ma) (s, 1 H), 7.35 (s, 5H),
5.15 (m, 2H), 4.45-4.56 (m, 2H); 13C NMR (DMSO-d6, 75.5 MHz) δ 41.62,
41.87, 42.45, 66.13, 66.25, 66.80, 118.78, 122. 07, 128.05, 128.18, 128.73,
135.78, 136.11, 148.62, 149.75, 155.00, 155.64, 158.45, 159.06, 167.97,
168.83, 170.35. Anal. Calcd for C12H11N3O5 (277.23): C, 51.98; H, 3.99;
N, 15.16. Found: C, 51.63; H, 4.32; N, 15.41. Ethyl N-(2-Boc-aminoethyl)-
11
-77
-88
-103
-60
54
73
-86
-111
70
118
-108
93
1
142
166
-175
PNA dimer22
PNA2:DNA23
PNA-PNA24
strand 1
-171
1
-112
-118
56
69
73
65
114
106
5
6
-176
-179
strand 2
§ Defined as in ref 24.
1
N-(cyanuric-1-ylacetyl)glycinate 11: mp 178-80 °C; H NMR (DMSO-
d6/CDCl3, 500 MHz) δ 11.48 (ma) and 11.13 (mi) (s, 1H), 6.56 (ma) and
6.37 (mi) (br, 1 H), 4.57 (ma) and 4.43 (mi) (s, 2 H), 4.14-4.17 (mi) and
4.05 (ma) (m, 4 H), 3.40 (ma) and 3.33 (mi) (m, 2 H), 3.15 (ma), 3.03 (mi)
(m, 2 H), 1.35 (s, 9 H), 1.22 (mi) and 1.17 (ma) (t, 3 H); 13C NMR (DMSO-
d6/CDCl3, 125.75 MHz) δ 167.35, 165.26, 165.17, 154.34, 147.99, 147.00,
59.74. Anal. Calcd for C16H25N5O8 (415.40): C, 46.26; H, 6.06; N, 16.86.
Found: C, 46.49; H, 5.74; N, 16.56.
(20) Crystal data for 11: (C16H25N5O8), orthorhombic, space group, Pbca,
a ) 18.779(1), b ) 10.669(1), and c ) 20.715(1) Å, V ) 4150.3(1) Å3, Z
) 8, Dc ) 1.330 Mg m-3, λ(Mo KR) ) 0.107 mm-1, F(000) ) 1760, λ )
0.71073 Å, 1 < θ < 24° (-17 e h e 20, -11 e k e 11, -23 e l e 22),
15009 total reflections, 2985 independent reflections which were used in
the refinement. The structure was solved and refined (Sheldrick, G. M.) to
R1 ) 0.046 and wR2 ) 0.133. Hydrogen atoms were obtained from
difference Fourier maps. Structure factors available on request from the
author.
angles found in these structures. It is shown that ø1 in 11 is
similar to that in the PNA duplex and triplex and corresponds
to the trans form, while in the photodimer it is locked in the
cis form. While the torsion angle ø2 in the PNA oligomers
is close to zero (planar), considerable departure occurred in
11, perhaps to relieve the mutual repulsion of the amide
carbonyl and cyanuryl ring carbonyls. While the torsion angle
δ on the glycyl side was similar in both the monomer 11
and the PNA oligomers, the relative sign and magnitude of
(21) Torres, R. A.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
649-653.
(16) (a) Saenger, W. Principles of Nucleic acid Structure; Springer-
Verlag: New York, 1984. (b) Nielsen, P. E.; Egholm, M. Peptide Nucleic
Acid (PNA). Protocols and Applications; Horizon Scientific Press: Norfolk,
1999.
(17) Kruger, R. J. Prakt. Chem. 1890, 42, 473-477.
(18) Dunnigan, D. A.; Close, W. J. J. Am. Chem. Soc. 1953, 75, 3615-
3616.
(22) Clivio, P.; Guillaume, D.; Adeline, M.-T.; Hamon, J.; Riche, C.;
Fourrey, J.-L. J. Am. Chem. Soc. 1998, 120, 1157-1166.
(23) Betts, L.; Josey, J. A.; Veal, J. M.; Jordan, S. R. Science 1995,
270, 1838-1841.
(24) Ramussen, H.; Kastrup, J. S.; Nielsen, J. N.; Nielsen, J. M.; Nielsen,
P. E. Nature Struct. Biol. 1997, 4, 98-101.
2828
Org. Lett., Vol. 2, No. 18, 2000