PNA-directed triple-helix formation by N7-xanthine

Article abstract of DOI:10.1055/s-2005-868503

We report the first example of alkylation of underivatized xanthine with chloroacetic acid to yield a separable mixture of N⁷- and N ⁹-(methylenecarboxyl)xanthine and its conversion to a peptide nucleic acid monomer compatible with Fmoc-based oligomerization chemistry. Additionally, we have simultaneously prepared the N⁷- and N ⁹-PNA monomers of guanine by alkylation of 2-N-isobutyrylguanine which were subsequently separated. Molecular modeling of the nucleobase base triplets indicates that N⁷-xanthine and N⁷-guanine form isomorphous triplets with adenine and guanine, respectively. We also show that polyamides containing N⁷-xanthine are compatible with triple-helix formation. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-868503

1442
LETTER
PNA-Directed Triple-Helix Formation by N7-Xanthine
P
N
A
-DirectedTr
o
iple-Helix
F
o
e
N
-Xanthin
r
e
t H. E. Hudson,* Mykhaylo Goncharenko, Andrew P. Wallman, Filip Wojciechowski
Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada
Fax +1(519)6613022; E-mail: rhhudson@uwo.ca
Received 16 August 2004
the distance and orientation of the site of attachment of the
N7- or N1-methylenecarbonyl group is different relative to
the purine target (Figure 1).
Abstract: We report the first example of alkylation of underiva-
tized xanthine with chloroacetic acid to yield a separable mixture of
N7- and N9-(methylenecarboxyl)xanthine and its conversion to a
peptide nucleic acid monomer compatible with Fmoc-based oligo-
merization chemistry. Additionally, we have simultaneously pre-
pared the N7- and N9-PNA monomers of guanine by alkylation of 2-
N-isobutyrylguanine which were subsequently separated. Molecu-
lar modeling of the nucleobase base triplets indicates that N7-xan-
thine and N7-guanine form isomorphous triplets with adenine and
guanine, respectively. We also show that polyamides containing N7-
xanthine are compatible with triple-helix formation.
Based on examination of molecular models, we reasoned
that N7-linked xanthine would be an excellent candidate
for recognition of adenine and is isomorphous with N7-
guanine (Figure 1, c).9 In addition, the purine base may
deliver a stabilizing effect based on stacking interactions
within the Hoogsteen strand.
Due to the number of potentially nucleophilic sites of xan-
thine, direct alkylation usually shows poor regioselective-
ly. Regioselective monoalkylation has been described
when protecting groups were used,10,11 via trimethylsily-
lated intermediates,12 or by use of a (tri-n-butylphos-
phine)cobalt(III) complexes.13 As well, there are limited
examples of regioselective alkylation to produce the N9-
isomer.14 Finally, the reported reference to PNA contain-
ing N9-xanthine should rather indicate hypoxanthine
(inosine).15
Key words: xanthine, peptide nucleic acid, isomorphous, triplex
PNA is a synthetic mimic of DNA in which the sugar-
phosphate backbone of the natural nucleic acid has been
replaced with a polyamide backbone but maintains the
natural nucleobases.1 Possessing an uncharged polyamide
backbone, PNA avidly binds to complementary nucleic
acids and may form PNA:DNA:PNA triple helices depen-
dent on the sequence context.2 In order to favor triplex for-
mation for amenable sequences, bisPNAs that contain two
PNA domains covalently connected are commonly used.3
Thus, one domain is responsible for Watson–Crick
recognition4 of a polypurine sequence, while the other is
involved in Hoogsteen binding.5
N
H
N
H
N
N
O
N
H
N
H
O
H
O
O
N
(a)
N
N
N
N
H
N
O
H
Various modified nucleobases have been incorporated
into both DNA and PNA oligomers to investigate and op-
timize recognition of natural nucleic acids by triplex for-
mation. The most studied triplex, the pyrimidine motif,
has the requirement for protonation of cytosine residues in
the Hoogsteen strand. Consequently, such triple-stranded
complexes are most stable in an environment that is gen-
erally below physiological pH. In order to overcome the
pH dependence of triplex stability, nucleobase analogs of
protonated cytosine such as N7-guanine have been utilized
in DNA chemistry6 and more recently in PNA.7 As well,
the synthetic pyrimidine pseudoisocytosine, which is also
a protonated cytosine analog, has been successfully
utilized in PNA oligomers.8
N
H
O
N
O
O
H
H
N
H
O
N
O
N
N
N
N
H
(b)
N
O
N
O
O
N
NH
N
O
N
O
O
H
H
N
The use of the synthetically more accessible N7-guanine in
conjunction with thymine in the Hoogsteen strand is not
optimal because the N7-G*G:C and T*A:T base triplets
are not isomorphous. This will presumably cause a distor-
tion in the backbone of the third strand polymer because
O
H
N
N
(c)
N
N
N
H
O
N
O
O
Figure 1 Proposed nucleobase interactions for a (PNA)2:DNA
triple helix. (a) Base triplet with N7-guanine; (b) nonisomorphous
base triplet with N1-thymine; (c) base triplet with N7-xanthine.
SYNLETT 2005, No. 9, pp 1442–1446
Advanced online publication: 29.04.2005
DOI: 10.1055/s-2005-868503; Art ID: S07604ST
© Georg Thieme Verlag Stuttgart · New York
0
1.
0
6.
2
0
0
5

LETTER
PNA-Directed Triple-Helix Formation by N7-Xanthine
1443
O
O
For this work, the xanthine-7-acetic acid derivative 1 was
required (Scheme 1). Our attempt to adapt the procedure
of Müller et al.10 to produce N7-xanthine led exclusively to
the N3,N7-bis(carboxymethylene) product. Although
simple alkylated derivatives of xanthine are well known,
no direct monoalkylation method to form xanthine-7-ace-
tic acid has been described. In fact, this relatively simple
compound has not been previously reported.
O
O
N
N
H
N
HN
OH
1
HN
5
a
7
N
N
HN
8
+
O
N
H
N
4
O
O
N
H
O
N
H
9
3
OH
xanthine
1, 38%
2, 21%
O
HN
N
We have developed a technically simple alkylation of un-
modified xanthine by using chloroacetic acid in aqueous
base. The reaction afforded a mixture of the monoalkylat-
ed N7- and N9-regioisomers 1 and 2 where required N7-de-
rivative 1 prevailed in the ratio 38:21, respectively.
Fractional recrystallization from water was used to sepa-
rate these isomers on a 5 g scale resulting in ca. 98% pu-
rity (N7-isomer). The overall chemical yield of
approximately 60% is likely due to hydrolysis of chloro-
acetic acid, which is employed at a 1:1 stoichiometry with
xanthine. This moderate yield is acceptable in view that
xanthine may be recovered from the reaction mixture and
the other starting materials are inexpensive. Since the pro-
cedure is very simple and inexpensive, no further attempts
at optimization of the reaction yield were considered
necessary.
N
NH
d, 57%
e, 60%
O
O
O
N
Fmoc
N
OH
H
5
b, 98%
c, 60%
Cl
O
H
O
H
H
N
N
Fmoc
N
H
O
H2N
OH
4
3
Scheme 1 Reagents and conditions: (a) ClCH2CO2H, NaOH, H2O,
reflux 5 h, key HMBC correlations shown by double-headed arrows;
(b) allyl alcohol, HCl (g), reflux 2.5 h; (c) i. Fmoc-NHS, DIEA,
CH2Cl2, 0–20 °C, 4 h; ii. HCl–Et2O, 0 °C; (d) DCC, HOBt, DMSO–
DMF, 18 h; (e) i. Pd[(PPh3)]4, MeOH–THF, p-CH3C6H4SO2Na,
20 °C, 17 h; ii. HCl, H2O.
Assignment of N7- and N9-isomers 1 and 2 was made by
use of 2D NMR (gHMBC) spectroscopy. This experiment
clearly showed a difference between the N7- and N9-iso-
mers with respect to the coupling of methylene protons of
the -CH2-COOH fragment with C4 and C5 positions.
Methylene protons of N7-derivative 1 (singlet at d = 4.99
ppm) are coupled to both C5 and C8 carbons and not to
C4. The analogous methylene protons of N9-isomer 2 (sin-
glet at d = 4.44 ppm) showed coupling to C8 and C4 but
not C5. Although xanthine may be alkylated at the N3 po-
sition, this possibility was discounted by comparison to
the available data for N3-xanthine acetic acid.16
The monomer synthesis was next advanced by DCC/
HOBt-mediated condensation between acid 1 and back-
bone submonomer 4. However, due to poor solubility of
xanthine-7-acetic acid a 1:1 DMSO–DMF solvent system
was employed. Once the nucleobase derivative was con-
densed with the backbone submonomer, the solubility im-
proved substantially. The Fmoc-protected monomer allyl
ester was transformed into target acid 5 by cleavage of
allyl ester with palladium triphenylphosphine complex in
the presence of sodium p-toluenesulfinate, as previously
reported.19 The sodium salt of 5 was surprisingly well
soluble in water.20
Initially, we chose to condense compound 1 with methyl
N-(2-Fmoc-aminoethyl)glycinate (10, Scheme 2) based
upon ongoing work in our laboratory. However, the poor
solubility of xanthine derivatives led to poor selectivity in
the saponification of the methyl ester versus elimination
of the Fmoc-protecting group. Thus, we changed to the al-
lyl ester (4, Scheme 1), which affords orthogonal removal
condition to Fmoc-deprotection and also increases the sol-
ubility of the backbone submonomer in organic solvents.
While this compound has been previously synthesized by
transesterification from tert-butyl ester,17 we have em-
ployed a convenient 2-step method starting from 2-N-
aminoethylglycine18 (3) followed by carbamylation with
Fmoc-N-hydroxysuccimide (Fmoc-NHS). We have found
compound 3 to be a very useful precursor that may be pro-
duced and esterified easily on large scale (ca. 20 g) for
synthesis of Boc-, Mmt- and Fmoc- backbone submono-
mers. When possible, we prefer to use the methyl ester, as
it is less susceptible than the allyl ester to 2-oxopiperazine
formation during the carbamylation reaction as observed
by Seitz and coworkers.17
Next we turned our attention to the preparation of the gua-
nine PNA monomers. The alkylation of guanine is known
to give a mixture of N9/N7-regioisomers where N9-isomer
is the main product. With respect to formation of N-glyco-
sides, the N9-isomer may be favored by use of a bulky O6-
protecting group such as the diphenylcarbamoyl deriva-
tive,21 but this effect can be less reliable for alkylation re-
actions.22 Alternatively, 6-chloro-2-aminopurine is often
used to access the N9-guanine derivatives selectively.23
Since we would find both N7- and N9-carboxymethylgua-
nine useful, we pursued the synthesis starting with un-
modified guanine. An isobutyryl group was installed on
the 2-amino position in 90% yield by reaction with isobu-
tyryl anhydride in DMF at 150 °C (Scheme 2).22 Reaction
of 2-N-isobutyrylguanine with tert-butyl bromoacetate at
ambient temperature in the presence of sodium hydride af-
forded a mixture of N9/N7-isomers 6 and 7 with overall
94% yield and 62:32 ratio, respectively, whereas reaction
at elevated temperatures favored the formation of the N9-
regioisomer. Compounds 6 and 7 were separated by ex-
Synlett 2005, No. 9, 1442–1446 © Thieme Stuttgart · New York

1444
R. H. E. Hudson et al.
LETTER
ploiting their differential solubility and column chroma- protons of N9-derivative 6 with singlet at d = 4.86 ppm
tography and were subsequently identified by 2D-NMR have mutual coupling signals with carbonyl of COOt-Bu
experiments.24
fragment, C4 and C8 carbons. However, while analogous
methylene protons of N7-isomer 7 (singlet at d = 5.06
ppm) showed similar coupling with COOt-Bu carbonyl
and C8 carbon, they did not show interaction with C4 car-
bon but instead showed coupling to C5. In summary, these
compounds were found to be consistent with those recent-
ly reported.22
Regiochemical assignment of 13C signals in substituted
purines is known from INEPT experiments.25 Thus, the
signal for C5 was identified as the lone upfield peak at
d = 120.3 ppm (N9-isomer 6) and d = 112.4 ppm (for N7-
isomer 7). Signals at d = 141.0 ppm (6) and d = 145.5 ppm
(7) in carbon spectra were assigned to C8 on the basis of
the residual signal from attached protons. The same effect Once purified, N9- and N7-isomers 6 and 7 were used sep-
was used for assignment of signals in carbon spectra for arately in further transformations. Cleavage of tert-butoxy
methylene, tert-butoxy and isobutyryl groups in the high group was made by the treatment in TFA–CH2Cl2 fol-
field area of spectra. Interaction of attached protons of iso- lowed by DCC/HOBt-mediated coupling of carboxy-
butyryl and tert-butoxy groups gave the ground for as- methyl guanines 8 and 9 with methyl N-(2-Fmoc-
signment of signals at d = 180.9 ppm (6) and d = 180.7 aminoethyl)glycinate (10). This reaction afforded esters
ppm (7) as the carbonyl carbon of i-PrCONH-fragment 11 and 12, which were purified chromatographically. Sa-
and d = 167.4 ppm (6) and d = 167.5 (7) as for carbonyl ponificaton of Fmoc-protected monomer methyl esters re-
carbon of COOt-Bu groups. Given that signals of C2 and vealed the monomer acids 13 and 14.26 The hydrolysis
C6 in the purine ring8 are expected in the range of d = was completed by using eight-fold excess of NaOH in
150–160 ppm, we assigned to these atoms the peaks at H2O–THF medium at 0 °C for five minutes. Prolonged
d = 149.6 ppm and d = 155.5 ppm (6) and the peaks at d = reaction time or poorly soluble monomer esters led to an
153.3 ppm and d = 157.5 ppm (7) accordingly. Thus, the unacceptable degree of Fmoc removal, in which case the
signals at d = 148.8 ppm (6) and d = 147.8 ppm were as- allyl ester was employed.
signed for C4 atoms in accordance with literature data for
Since the use of N7-guanine derivatives in triplex forma-
purines.23 As done for the substituted xanthines, gHMBC
tion is well established, we have first tested the ability of
NMR spectroscopy was used to assign the regioisomers
N7-xanthine to form triple helices. We synthesized Ac-T6-
based on coupling of the CH2COOt-Bu fragment methyl-
lys-NH2 (15) and Ac-X6-lys-NH2 (16) by standard
ene protons with the C4 and C5 atoms. Thus, methylene
methods27 and investigated their hybridization with
c
O
N
OH
O
O
N
Ot-Bu
a, 90%
b, 96%
O
8, 84%
9, 71%
N
N
O
1
HN
HN
N
N
5
7
O
O
N
O
N
HN
O
8
+
+
N
N
N
HN
O
N
H
O
HN
N
N
9
H
H2N
4
3
O
H
N
N
N
N
N
O
H
H
t-BuO
guanine
OH
6 (64%)
8
7 (32%)
9
H
N
H
O
O
N
d
O
N
N
11, 52%
12, 38%
O
H
N
N
N
HN
O
N
O
+
O
Fmoc
N
N
N
H
O
H
N
O
OH
Fmoc
N
O
e
H
8, 9
10
11, 12
13, 64%
14, 56%
O
H
N
N
O
N
N
HN
N
HN
O
N
+
N
H
N
O
O
O
O
O
N
N
Fmoc
N
H
OH
Fmoc
N
H
OH
14
13
Scheme 2 N9/N7-Guanine Fmoc/acyl monomers. Reagents and conditions: (a) (i-PrCO)2O, DMF, 150 °C, 7 h, 90%; (b) NaH, DMF,
BrCH2COOt-Bu, 0–20 °C, 17 h; (c) CF3COOH, CH2Cl2, (C2H5)3SiH, 18 h; (d) DCC, HOBt, DMF, 18 h; (e) THF, H2O, NaOH, 0 °C, 5 min.
Synlett 2005, No. 9, 1442–1446 © Thieme Stuttgart · New York

LETTER
PNA-Directed Triple-Helix Formation by N7-Xanthine
1445
poly(rA).28 Thermal melting analysis (Tm = 62.0 °C) and
determination of the stoichiometry of binding confirmed
the expected triplex formation for (15) with poly(rA).
Xanthine oligomer 16 also showed a single cooperative
transition (Tm = 59.5 °C, pH 7.0) that was slightly stabi-
lized at acidic pH (Tm = 66.0 °C, pH 5.5). A Job plot re-
vealed that oligomer 16 binds in a 2:1 fashion with
poly(rA), consistent with the supposition that N7-xanthine
is a suitable mimic of thymine (Figure 2).
empirical methods (PM3), as previously described: Fenyö,
R.; Tímár, Z.; Pálinkó, I.; Penke, B. J. Mol. Struc.–
Theochem. 2000, 496, 101.
(10) Müller, C. E.; Deters, D.; Dominik, A.; Pawlowski, M.
Synthesis 1998, 1428.
(11) Bridson, P. K.; Richmond, G.; Yeh, F. Synth. Commun.
1990, 20, 2459.
(12) Müller, C. E.; Shi, D.; Manning, M. Jr.; Daly, J. W. J. Med.
Chem. 1993, 36, 3341.
(13) Marzilli, L. G.; Epps, L. A.; Sorrell, T.; Kistenmacher, T. J.
J. Am. Chem. Soc. 1975, 97, 3351.
(14) For example: Hoffmann, M. F. H.; Brückner, A. M.; Hupp,
T.; Engels, B.; Diederichsen, U. Helv. Chim. Acta 2000, 83,
2580.
(15) (a) Sanjayan, G. J.; Pedireddi, V. R.; Ganesh, K. N. Org.
Lett. 2000, 2, 2825. (b) Timár, Z.; Bottka, S.; Kovács, L.;
Penke, B. Nucleosides Nucleotides 1999, 18, 1131.
(16) N7- and N9-xanthine acetic acids were separated by
differential solubility in water, the N7-derivative being less
soluble. N7-isomer: white solid, mp 320 °C (dec). 1H NMR
(400 MHz, DMSO): d = 13.30 (br s, 1 H), 11.61 (s, 1 H),
10.91 (s, 1 H), 7.91 (s, 1 H), 4.99 (s, 2 H). 13C NMR (100
MHz, DMSO): d = 169.8, 156.3, 151.9, 149.6, 143.9, 107.3,
47.7. HRMS (EI): m/z calcd for C7H6N4O4: 210.0389; found:
210.0397. N9-isomer: white solid, mp >290 °C (dec). 1H
NMR (400 MHz, DMSO): d = 10.82 (s, 1 H), 7.60 (s, 1 H),
4.84 (s, 2 H). 13C NMR (100 MHz, DMSO): d = 170.1,
158.9, 152.2, 142.8, 138.5, 115.7, 48.4. HRMS (EI): m/z
calcd for C7H6N4O4: 210.0389; found: 210.0451. N3-isomer:
see ref.11
(17) Seitz, O.; Köhler, O. Chem.–Eur. J. 2001, 7, 3914.
(18) Heimer, E. P.; Gallo-Torres, H. E.; Felix, A. M.; Ahmad, M.;
Lambros, T. J.; Scheidl, F.; Meienhofer, J. Int. J. Pept. Res.
1984, 23, 203.
Figure 2 Job plot for Ac-X6-lys-NH2 indicating (PNA)2:DNA triple
helix formation.
(19) Honda, M.; Morita, H.; Nagakura, I. J. Org. Chem. 1997, 62,
8932.
(20) Selected data.
Currently, we are investigating the use of monomers 14
and 5 in the Hoogsteen strand of clamp-PNAs for the
recognition of mixed pyrimidine sequence DNA.
Allyl ester precursor to 5: white solid, mp 168–170 °C (dec).
1H NMR (400 MHz, DMSO): d = 11.53 (br s, 1 H), 10.87 (br
s, 1 H), 7.87–7.20 (m, 10 H), 5.90 (m, 1 H), 5.38–5.09 (m, 4
H), 4.66–4.19 (m) and 4.09 major (s, 7 H), 3.46 major (m,
minor rotamer overlapping with H2O), 3.09 minor (m, major
rotamer overlapping with H2O). HRMS (ESI-TOF): m/z
calcd for sodium adduct C29H28N6O7Na: 595.1917; found:
595.1912.
Acknowledgment
We thank the Natural Sciences and Engineering Research Council
(of Canada) for funding this work. APW is the recipient of an
NSERC Undergraduate Summer Research Award and FW is parti-
ally supported by the Faculty of Graduate Studies, University of
Western Ontario.
Compound 5: off-white solid, mp 162–164 °C (change in
appearance), 205–208 °C (dec). 1H NMR (400 MHz,
DMSO): d = 12.81 (br s, 1 H), 11.58 major and 11.56 minor
(1 H), 10.86 major and 10.84 minor (1 H), 7.88–7.28 (m, 10
H), 5.27 major and 5.07 minor (s, 2 H), 4.34–4.20 (m)and
3.98 major (s, 5 H), 3.43 major (m, minor rotamer
overlapping with H2O), 3.10 minor (m, major rotamer
overlapping with H2O). HRMS (ESI-TOF): m/z calcd for
sodium adduct C26H24N6O7Na: 555.1604; found: 555.1602.
(21) Robins, M. J.; Zou, R.; Guo, Z.; Wnuk, S. J. Org. Chem.
1996, 61, 9207.
(22) Timár, Z.; Kovács, L.; Kovács, G.; Schmél, Z. J. Chem. Soc.,
Perkin Trans. 1 2000, 19.
(23) For PNA: 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.
References
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497.
(2) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624.
(3) Griffith, M. C.; Risen, L. M.; Greig, M. J.; Lesnik, E. A.;
Sprankle, K. G.; Griffey, R. H.; Kiely, J. S.; Freier, S. M. J.
Am. Chem. Soc. 1995, 117, 831.
(4) Watson, J. D.; Crick, F. H. Nature 1953, 171, 737.
(5) Hoogsteen, K. Acta Crystallogr. 1963, 16, 907.
(6) Hunziker, J.; Priestly, E. S.; Brunar, H.; Dervan, P. B. J. Am.
Chem. Soc. 1995, 117, 2661.
(7) D’Costa, M.; Kumar, V. A.; Ganesh, K. N. J. Org. Chem.
2003, 68, 4439.
(8) Egholm, M.; Christensen, L.; Duholm, K. L.; Buchardt, O.;
Coull, J.; Nielsen, P. E. Nucleic Acids Res. 1995, 23, 217.
(9) Molecular models were constructed using HyperChem 5.1
from existing crystallographic data. The xanthine triplet was
initially geometry optimized using molecular mechanics
(Amber force field) and subsequently refined by use semi-
(24) Data for 6 and 7.
Compound 6 (N9-isomer): white solid, mp 310–331 °C. 1H
NMR (400 MHz, DMSO): d = 12.10 (s, 1 H), 11.66 (s, 1 H),
7.94 (s, 1 H), 4.87 (s, 2 H), 2.76 (sept, 3J = 6.8 Hz, 1 H) 1.40
(s, 9 H), 1.09 (d, 3J = 6.9 Hz, 6 H). 13C NMR (100 MHz,
Synlett 2005, No. 9, 1442–1446 © Thieme Stuttgart · New York

1446
R. H. E. Hudson et al.
LETTER
DMSO): d = 180.9, 167.4, 155.5, 149.6, 148.8, 141.0, 120.3,
83.0, 45.5, 35.3, 28.4, 19.6. HRMS (EI): m/z calcd for
C15H21N5O4: 335.15937; found: 335.15937.
minor (s, 2 H), 3.46–3.12 (m, 4 H), 2.71 (m, 1 H), 1.10 major
and 1.05 minor (d, 3J = 6.8 Hz, 6 H). HRMS (ESI-TOF):
m/z calcd for sodium adduct C30H31N7O7Na: 624.2183;
found: 624.2165.
Compound 7 (N7-isomer): white solid, mp 202–204 °C. 1H
NMR (400 MHz, DMSO): d = 12.13 (s, 1 H), 11.56 (s, 1 H),
8.10 (s, 1 H), 5.06 (s, 2 H), 2.72 (sept, 3J = 6.8 Hz, 1 H), 1.40
(s, 9 H), 1.10 (d, 3J = 6.8 Hz, 6 H). 13C NMR (100 MHz,
DMSO): d = 180.7, 167.6, 157.5, 153.3, 147, 9, 145.5, 112.4,
82.7, 48.6, 35.4, 28.3, 19.6. HRMS (EI): m/z calcd for
C15H21N5O4: 335.15937; found: 335.15937.
(27) Oligomers were synthesized on Rink amide resin by an ABI
433a peptide synthesizer at the 5 mmol scale according to the
manufacturer-supplied cycles and purified by RP-HPLC.
Data for -T6-lys-NH2 (15) HRMS (MALDI-TOF): m/z calcd
for C74H101N27O26: 1783.7411; found: 1784.6012.
Data for Ac-X6-lys-NH2 (16) HRMS (ESI-TOF): m/z calcd
for C74H89N39O26: 1940.76; found: 1940.55 [MH+]
(28) Thermal denaturation was measured at strand concentration
of 1.3 mM in base pairs with 150 mM NaCl, 10 mM
Na2HPO4, 1 mM ETDA, pH 7.0 at a 2:1 PNA:RNA ratio. All
transitions were well-behaved and monophasic. The first
derivative method was used to estimate the Tm. The
stoichiometry of binding was determined by the method of
continuous variations: Job, P. Ann. Chim. (Paris) 1928, 9,
113.
(25) Osterman, R. M.; McKittrick, B. A.; Chan, T. M.
Tetrahedron Lett. 1992, 33, 4867.
(26) Data for 13 and 14.
Compound 13 (N9-isomer): white solid, mp 234–235 °C. 1H
NMR as reported in ref.22; HRMS (ESI-TOF): m/z calcd for
sodium adduct C30H31N7O7Na: 624.2183; found: 624.2213.
Compound 14 (N7-isomer): white solid, mp 188–190 °C
(dec). 1H NMR (400 MHz, DMSO): d = 12.13 (br s, 1 H),
11.59 major and 11.56 minor (s, 1 H), 8.18 minor and 8.15
major (s, 1 H), 7.87–717 (m, 10 H), 5.38 major and 5.20
Synlett 2005, No. 9, 1442–1446 © Thieme Stuttgart · New York