Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

Article abstract of DOI:10.1016/j.tetlet.2007.12.095

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.

Full text of DOI:10.1016/j.tetlet.2007.12.095

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 1314–1318
Cyanuryl peptide nucleic acid: synthesis and DNA
complexation properties
Raman Vysabhattar a, K. N. Ganesh a,b,
*
a Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411 008, India
b Indian Institute of Science Education and Research, 900, NCL Innovation Park, Dr. Homi Bhabha Road, Pune 411 008, India
Received 14 October 2007; revised 9 December 2007; accepted 19 December 2007
Abstract
The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-
aminoethyl)-N0-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical
studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids
when the cyanuryl unit was located in the middle of the PNA oligomer.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: PNA; Cyanuryl PNA
Peptide nucleic acids1 (Fig. 1A) are a new class of DNA
(Fig. 1B) mimics that have emerged as potential antigene/
antisense agents in the field of nucleic acid-based therapeu-
tics. The chiral and negatively charged sugar-phosphate
backbone of DNA is replaced in PNA by an achiral, neu-
tral backbone composed of N-(2-aminoethyl)glycyl (aeg)
units.1 The nucleobases are attached to the backbone
through tertiary acetamide linkers, and PNA binding to
the target DNA/RNA sequences occurs with high sequence
specificity and affinity.2 PNA binds to complementary
DNA and RNA to form duplexes via Watson–Crick
(WC) base pairs and triplexes through combined WC and
Hoogsteen (HG) hydrogen bonding. PNA–DNA/RNA
hybrids exhibit greater thermal stability than analogous
DNA:DNA and DNA:RNA complexes.3 Because of this
attractive feature and stability to proteases and nucleases,
PNAs are of great interest in medicinal chemistry, with
potential for development as gene-targeted drugs and as
reagents in molecular biology and diagnostics.4
Nucleobases are attached to the PNA backbone via ter-
tiary amide bonds leading to syn and anti conformers
(Fig. 2) which are rotameric isomers as a result of the high
energy barrier for their interconversion. These two non-
interconvertible rotamers may have consequences on the
NH
O
B
O
B
NH
N
N
O
H
O
O P O
B
O
O
N
N
A
B
O
B
Fig. 1. Structures of (A) PNA and (B) DNA.
O
syn
anti
O
A
B
*
Corresponding author. Tel.: +91 20 25898021; fax: +91 20 2589 8022.
Fig. 2. The rotameric structures of aeg PNA (A) syn and (B) anti forms.
E-mail address: kn.ganesh@iiserpune.ac.in (K. N. Ganesh).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.12.095

R. Vysabhattar, K. N. Ganesh / Tetrahedron Letters 49 (2008) 1314–1318
1315
orientation of attached nucleobases, thereby affecting the
strengths of their hydrogen bonding in complementation.
The X-ray crystal structure of PNA showed a predomi-
nance of the syn conformation in the solid state,5 whereas
it exhibited both syn and anti conformers in solution.6 In
principle, it is possible to have different rotamers for each
of the bases within the same PNA strand and the ultimate
specificity and binding strength of a PNA oligomer is deter-
mined by the statistical predominance of identical, favor-
able rotamers leading to productive base pairing.
We hypothesized that a modified nucleobase with equal
propensity for hydrogen bonding from each face would
statistically double the probability of efficient hydrogen
bonding, thereby negating the effects of non-productive
rotamers. Several unnatural bases forming stable DNA tri-
plexes have been reported,7a,b and recently, base modified
PNAs have been described as well.7c Cyanuric acid (Cya)
(Fig. 3A) is a symmetric six-membered cyclic imide with
alternate arrangement of hydrogen bond donors and
acceptors with the ability to form hydrogen bonds from
both sides of the heterocyclic ring.8 The base pairing poten-
tial of cyanuric acid and its N-alkyl analog with hydrogen
bonding complements such as melamine9 (Fig. 3C) and N-
ethyladenine10 (Fig. 3D) in monomeric form in the solid
state have been well studied.
size CyaPNA oligomers containing cyanuryl units in place
of thymine at specific sites and examine their DNA com-
plexation properties. The work presented here describes
the synthesis and characterization of sequence specific
cyanuryl PNA oligomers and a study of their hybridization
properties with complementary DNA oligomers.
Synthesis of CyaPNA monomer: The synthesis of
CyaPNA monomer 6 through cyanuric acid ring construc-
tion had been reported earlier by us (Scheme 1).13 How-
ever, poor yields prompted us to search for alternative
methods to prepare 6. Instead of the ring closure approach,
we attempted N-alkylation of the commercially available
cyanuric acid to obtain directly the target CyaPNA mono-
mer 6. N-Monoalkylation of cyanuric acid with ethyl
N-(2-Boc-aminoethyl)-N-(1-bromoacetyl) glycinate 4 was
attempted in DMSO and K2CO3 (Scheme 1). Compound
4 was prepared in three steps from ethylenediamine accord-
ing to known procedures.14,15 The precursor N-Boc-amino-
ethylglycine 3 was treated with bromoacetyl chloride in
DCM in the presence of triethylamine to yield glycinate 4.
N-Alkylation of cyanuric acid with the bromoacetyl
compound 4 was carried out in DMSO at >70 °C where
cyanuric acid is more soluble in DMSO. The reaction went
to completion in 5 h to give the PNA ester 5 (Scheme 1).
Hydrolysis of ester 5 was accomplished with LiOH,
followed by neutralization with acid to yield the desired
cyanuryl PNA acid monomer 6. The structural integrity
of monomer 6 and all other intermediate compounds was
established by 1H and 13C NMR spectroscopic analysis
and confirmed by mass spectrometry. Compound 6 was
used for coupling with a solid support to synthesize the
CyaPNA oligomers.
N-Methylcyanuric acid (MCA) (Fig. 3B) with close
structural similarity to uracil forms hydrogen bonded com-
plementary pairs with N9-ethyladenine (EA),10,11 as
observed by X-ray crystallography. The interesting base
pairing potential of cyanuric acid along with the biological
properties of cyanuryl nucleotides12 inspired us to synthe-
PNA oligomers 8–13 containing CyaPNA units at spe-
cific sites in place of thymine were assembled by solid-phase
peptide synthesis on MBHA resin functionalized with
Nc-(benzyloxycarbonyl)-Na-Boc-lysine. The unmodified
PNA-T oligomer 7 and mixed base oligomer PNA 13 were
synthesized for control studies (Table 1). The chimeric
PNA oligomers containing CyaPNA units in different posi-
tions were designed to examine the effect of distance
between the two sites of modifications on the stability
of the derived CyaPNA:DNA complexes. The PNA
O
O
N
Me
B
NH2
NH2
N
H
O
H
O
H
O
H
O
N
N
N
N
N
N
N
N
H
A
N
N
H2N
N
NH2
Me
C
D
Fig. 3. Structures of (A) cyanuric acid, (B) methyl cyanuric acid, (C)
melamine, and (D) N9-ethyladenine.
H
N
O
H
H
NH2
ii
i
NH2
Br
N
OEt
N
H2N
60%
3
70%
Boc
Boc
2
1
O
O
N
HN
NH
HN
NH
v
O
O
N
O
O
iv
55%
HN
Boc
O
O
iii
O
H
N
O
O
70%
N
O
OEt
N
4
H
N
OEt
Boc
OH
N
6
5
Boc
Scheme 1. Reagents and conditions: (i) (Boc)2O, THF; (ii) Ethyl bromoacetate, Et3N, acetonitrile; (iii) Bromoacetyl chloride, Et3N, DCM; (iv) Cyanuric
acid, K2CO3, DMSO; (v) 2 N LiOH, methanol.

1316
R. Vysabhattar, K. N. Ganesh / Tetrahedron Letters 49 (2008) 1314–1318
Table 1
0.16
MALDI-TOF spectral analysis of PNA oligomers
Entry
Sequence
Mass
Massa
0.14
0.12
0.10
0.08
0.06
(calcd)
observed
PNA9:DNA14
PNA 7
PNA 8
PNA 9
PNA 10
PNA 11
PNA 12
PNA 13
H-TTTTTTTT-Lys-NH2
2273.93
2298.91b
2276.91
2276.91
2201.88b
2855.24
2864.32
2275.24
2300.64b
2276.62
2279.77
2301.56b
2858.46
2868.17
H-CyaTTTTTTT-Lys-NH2
H-TTTTTTTCya-Lys-NH2
H-TTTTCyaTTT-Lys-NH2
H-CyaTTTCyaTTT-Lys-NH2
H-GTAGATCACT-Lys-NH2
H-GCyaAGACyaCACCya-Lys-NH2
T = thyminyl PNA; Cya = cyanuryl PNA.
a
=HRMS.
=(M++Na)+.
b
0
20
40
60
80
100
oligomers were cleaved from the solid support using tri-
fluoromethanesulfonic acid (TFMSA) in the presence of
trifluoroacetic acid (TFA)16 to yield PNAs carrying lysine
at the C-terminus. All PNAs 7–13 were purified by HPLC
on an RPC-18 column. The purity of the oligomers was
confirmed by HPLC using a RPC-18 column and they were
characterized by MALDI-TOF mass spectrometry (Table
1). The mixed sequences containing both purines and
pyrimidines PNA 12 and PNA 13 were synthesized to
check the duplex stability and discrimination between
parallel and antiparallel DNA. The results of cyanuryl
PNA 13 were compared with the results of control PNA 12.
The complementary DNA oligomers for triplex studies
(DNA 14, GCAAAAAAAACG and DNA 15, GCAAAA-
CAAACG) having a CG/GC lock at the ends to prevent
slippage and DNA oligomers for the duplex studies
(DNA 16, AGTGATCTAC and DNA 17, CAT-
CTAGTGA) were synthesized on an automated DNA syn-
thesizer using standard phosphoramidite chemistry,17 and
purified by C18 RP HPLC.
UV–Tm studies on CyaPNA2:DNA triplexes: The PNA
homopyrimidine sequences with complementary homopu-
rine DNA sequences are well known to form PNA2:DNA
triplexes.18 The stoichiometry of the CyaPNA:DNA com-
plexes was found to be 2:1 by UV-mixing curves (recorded
at 260 nm) (Fig. 4).18 Keeping the total concentration con-
stant, a series of mixtures of PNA and DNA of different
relative molar equivalents of PNA and DNA were pre-
pared and the UV absorbance of each mixture was
recorded at 260 nm. The net UV absorbance steadily
decreased due to hypochromic effects upon complexation
as the PNA concentration increased, until all the strands
present were fully involved in complex formation. The
absorbance then increased upon the addition of excess
PNA. The stoichiometry of CyaPNA:DNA complexation
was derived from the minimum in the plot of absorbance
with respect to mole fraction (Fig. 4) and found to be 2:1.
The annealed PNA2:DNA triplexes were subjected to
temperature-dependent UV absorbance measurements.
The normalized absorbance versus temperature plots
derived from these experiments exhibited a single sigmoidal
transition, and the maxima in first derivative plots (Fig. 5)
indicate the melting temperature Tm (Table 2).18 The con-
Mole fraction (PNA)
Fig. 4. UV-Job plot corresponding to the PNA 9:DNA 14 mixtures in the
molar ratios of 100:0, 80:20, 60:40, 50:50, 40:60, 20:80, 0:100 (buffer,
10 mM sodium phosphate pH 7.2, 100 mM NaCl), UV absorbance at
260 nm.
0.04
0.03
0.02
0.01
0.00
9:12
10:12
8:12
7:13
7:12
11:12
9:13
10
20
30
40
50
60
70
80
Temperature (oC)
Fig. 5. UV–Tm first derivative curves of PNA2:DNA triplexes.
Table 2
UV-melting temperatures of CyaPNA:DNA 14 triplexes
Entry
Sequence
Tm (°C)
DTm (°C)
1
2
3
4
5
PNA 7, H-TTTTTTTT-Lys-NH2
PNA 8, H-CyaTTTTTTT-Lys-NH2
PNA 9, H-TTTTTTTCya-Lys-NH2
PNA 10, H-TTTTCyaTTT-Lys-NH2
PNA 11, H-CyaTTTCyaTTT-Lys-NH2
44.6
34.2
67.9
56.7
44.2
—
ꢀ10.4
+23.3
+12.1
ꢀ0.4
T = Thyminyl PNA, Cya = cyanuryl PNA, Tm = melting temperature
(measured in the buffer 10 mM sodium phosphate, 10 mM NaCl, pH 7.0).
trol PNA2:DNA triplex derived from unmodified aeg
PNA-T8 (Table 2, entry 1) had a Tm of 44.6 °C, which
was increased by 23.3 °C when a CyaPNA unit was incor-
porated at the C-terminus (Table 2, entry 3). However, the
CyaPNA substitution at the N-terminus destabilized
the PNA2:DNA triplex by 10.4 °C (Table 2, entry 2).

R. Vysabhattar, K. N. Ganesh / Tetrahedron Letters 49 (2008) 1314–1318
1317
Table 4
Interestingly, substitution of a CyaPNA unit in the middle
UV-melting temperatures of CyaPNA:DNA duplexes
of the sequence stabilized the triplex by 12.1 °C (Table 2,
entry 4). PNA 11 (Table 2, entry 5) with two simultaneous
modifications at the N-terminus and middle of the
sequence formed a triplex with stability as good as that
from unmodified PNA 7, without much further stabili-
zation. The stabilization by the center modification seems
to negate the destabilization effects of N-terminus
modification.
Entry Sequence
DNA Tm
DTm
(ap-p)
1
2
16
17
51.2
48.6
PNA 12, H-GTAGATCACT-Lys-NH2
2.6
3
4
16
17
48.7
45.8
PNA 13, H-GCyaAGACyaCACCya-
2.9
Lys-NH2
T = Thyminyl PNA, Cya = cyanuryl PNA, Tm = melting temperature
(measured in the buffer 10 mM sodium phosphate, 10 mM NaCl, pH 7.0).
The sequence specificity of CyaPNA hybridization was
examined through Tm measurements with single mis-
match-containing DNA oligomer (DNA 15) (Table 3,
entries 2 and 4). The control sequence PNA 7 formed a tri-
plex with single mismatch sequence (DNA 13) with a Tm
lower by 9.6 °C (Table 3, entry 2), while cyanuryl PNA 9
exhibited destabilization by 13.2 °C. Thus CyaPNAs show
better sequence discrimination (Table 3, entry 4).
The effect of incorporation of a CyaPNA unit in place of
a thyminyl PNA unit in mixed purine-pyrimidine sequence
PNA 12 was studied to examine the consequence of substi-
tution on duplex stability. In PNA 13, all three thyminyl
PNA units in PNA 12 were replaced with a CyaPNA
monomer and annealed with corresponding DNA 16 and
DNA 17 to generate antiparallel and parallel duplexes,
respectively. The establishment of stable duplexes was indi-
cated by sigmoidal transitions in the UV–Tm curves and
confirmed by peaks in the first derivative plots (Fig. 6).
PNA 12 showed a Tm of 51.2 °C (Table 4, entry 1) for
the antiparallel duplex with DNA 16, whereas the Tm of
the corresponding parallel duplex with DNA 17 was
48.6 °C (Table 4, entry 2) which is lower by 2.6 °C. In com-
parison, the antiparallel duplex of CyaPNA 13 with DNA
16 exhibited a Tm of 48.7 °C (Table 4, entry 3) and the par-
allel duplex with DNA 17 showed a Tm of 45.8 °C (Table 4,
entry 4). The CyaPNA:DNA duplexes thus showed desta-
bilization by 2.5 °C compared to control PNA 12, but with
almost the same degree of discrimination (2.9 °C) among
parallel and antiparallel duplexes as the control PNA 12.
Thus CyaPNA modification is well tolerated within the
mixed PNA sequences for duplex formation. The insertion
of CyaPNA units within the PNA oligomer does not alter
the base stacking pattern as the CD profile of the derived
triplexes was unaffected.
Table 3
In conclusion, this work demonstrates that a cyanuryl
PNA unit with the capacity to form hydrogen bonds from
either side of the heterocyclic ring can act as a pyrimidine
mimic and stabilizes remarkably the corresponding
PNA2:DNA triplexes. Normally, base modifications incor-
porated in the middle of sequences show destabilization of
the derived triplexes and in this context, CyaPNA is inter-
esting in showing the opposite behavior. This is perhaps
due to the propensity of the cyanuryl unit to form H-bonds
from both sides, irrespective of the rotameric form. The
cyanuryl PNA also exhibits a better sequence discrimina-
tion of the derived triplexes than the unmodified PNA.
The destabilization seen with CyaPNA at the N-terminus
in triplexes arises perhaps from unfavorable base stacking
at this end, in constrast to the C-terminus or in the middle.
In the case of duplexes, even though CyaPNA 13 destabi-
lized the antiparallel duplex with complementary DNA
16, it is as good as the control PNA 12 in discriminating
the parallel and antiparallel duplexes. A study of the
hybridization properties of cyanuryl PNA with comple-
mentary RNA is in progress.
Mismatch studies of CyaPNA:DNA triplexes
Entry PNA sequence
1
DNA
Tm (°C) DTm
DNA 14 44.6
DNA 15 35.0
—
ꢀ9.6
PNA 7, H-TTTTTTTT-Lys-NH2
2
3
4
DNA 14 67.9
DNA 15 54.7
—
ꢀ13.2
PNA 9, H-TTTTTTTCya-Lys-NH2
T = Thyminyl PNA, Cya = cyanuryl PNA, Tm = melting temperature
(measured in the buffer 10 mM sodium phosphate, 10 mM NaCl, pH 7.0).
0.04
12:17
12:16
0.03
0.02
0.01
0.00
13:16
13:17
Acknowledgments
R.V. thanks CSIR, New Delhi for a research fellowship.
K.N.G. acknowledges the Department of Science and
Technology, New Delhi for the award of a J. C. Bose
Fellowship.
10
20
30
40
50
60
70
80
Temperature (oC)
Fig. 6. UV–Tm first derivative curves of PNA:DNA duplexes.

1318
R. Vysabhattar, K. N. Ganesh / Tetrahedron Letters 49 (2008) 1314–1318
9. Damodaran, K.; Sanjayan, G. J.; Rajamohanan, P. R.; Ganapathy,
References and notes
S.; Ganesh, K. N. Org. Lett. 2001, 3, 1921–1924.
10. Pedireddi, V. R.; Ranganathan, A.; Ganesh, K. N. Org. Lett. 2001, 3,
99–102.
11. (a) Ranganathan, A.; Pedireddi, V. R.; Sanjayan, G.; Ganesh, K. N.;
Rao, C. N. R. J. Mol. Struct. 2000, 522, 87–94; (b) Coppens, P.; Vos,
A. Acta Crystallogr. B 1971, 27, 146–158.
12. Gasparutto, D.; Da Cruz, S.; Bourdat, A.-G.; Jaquinod, M.; Cadet, J.
Chem. Res. Toxicol. 1999, 12, 630–638.
13. Sanjayan, G. J.; Pedireddi, V. R.; Ganesh, K. N. Org. Lett. 2000, 2,
2825–2828.
14. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen,
H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.;
Buchardt, O. J. Org. Chem. 1994, 59, 5767–5773.
15. Meltzer, P. C.; Liang, A. Y.; Matsudaira, P. J. Org. Chem. 1995, 60,
4305–4308.
16. Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202–
4207.
17. Gait, M. J. Oligonucleotide Synthesis, A Practical Approach; IRL
Limited: Oxford, 1984.
1. (a) Nielsen, P. E.; Egholm, M.; Buchardt, O. Science 1991, 254, 1497–
1500; (b) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4,
916–928.
2. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S.
M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566–568.
3. Jensen, K. K.; Qrum, H.; Nielsen, P. E.; Norden, B. Biochemistry
1997, 37, 5072–5077.
4. (a) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew.
Chem., Int. Ed. 1998, 37, 2796–2823; (b) Nielsen, P. E. Curr. Opin.
Biotech. 2001, 12, 16–20.
5. Betts, L.; Josey, J. A.; Veal, J. M.; Jordan, S. R. Science 1995, 270,
1838–1842.
6. Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G. Science 1994,
265, 777–780.
7. (a) Soyfer, V. N.; Potaman, V. N. Triple Helical Nucleic Acids;
Springer: New York, 1996; (b) Ganesh, K. N.; Kumar, V. A.;
Barawkar, K. A. In Supramolecular Control of Structure and Reactiv-
ity, Hamilton, A. D., Ed.; John Wiley & Sons: New York, 1996;
Chapter 6, pp 263–327; (c) Curr. Top. Med. Chem. 2007, 7, 667–679.
8. (a) MacDonald, J. C.; Whitesides, G. M. Chem. Rev. 1994, 94, 2383–
2420; (b) Ranganathan, A.; Pedireddi, V. R.; Rao, C. N. R. J. Am.
Chem. Soc. 1999, 120, 1752–1753.
18. Kim, S. H.; Nielsen, P. E.; Egholm, M.; Buchardt, O. J. Am. Chem.
Soc. 1993, 115, 6477–6489.