Angewandte
Chemie
(Asp-amAla), tagged with 6-cPOO and 6-cPNN as recognition
elements (Figure 1b). On the basis of qualitative conforma-
tional analysis,[7] we anticipated these oligomers would form
base pairs since their overall backbone/base-pairing axes are
similar to that of Asp-Glu oligo-dipeptide tagged with 2,4-
dioxo-5-aminopyrimidine (5-aPOO; Figure 1c), which does
exhibit strong base pairing.[2] The effect on base pairing
when orotic acid is incorporated through its N1-position at
selective locations within a PNA2:NA triplex, has been
studied.[8]
Since the goal of the study was to assess the base-pairing
properties of the oligomers, we prepared the required Fmoc-
protected (Fmoc = 9-fluorenylmethoxycarbonyl) monomers
for the machine assisted synthesis of 6-cPOO-tagged Asp-
amAla oligo-dipeptides.[9] The ion-exchange HPLC purifica-
tion was challenging. In the case of the hexamer we obtained
homogeneous material, whereas in the case of the dodecamer
we could only isolate mixtures containing the 11- and 12-mer;
for the hexadecamer we isolated a mixture of 14-, 15-, and 16-
mer (see Table S1 in the Supporting Information),[9] and used
them in this study. The cross-pairing of Asp-amAla(6-cPOO)
oligo-dipeptides with adenine containing DNA and RNA
sequences were investigated using temperature dependant
UV and CD spectroscopy; the results are summarized in
Figure 2 (and Table S2, entries 1–16 in the Supporting Infor-
mation). Strikingly, all of the data consistently point to no (or
very weak) base-pairing interactions (Figure 2a,b), which is
supported by the negligible effects that the variations in
concentration of oligomers, salt, and length of the pairing
strands have on the base-pairing behavior. That there is
interaction was shown by a Job plot (Figure 2c) which, not
surprisingly, shows a triplex with a 2:1 ratio of 6ÀcPOO-tagged
dodecapeptide with poly-r(A).
Figure 2. Weak base pair interactions of Asp-amAla(6-cPOO) oligomers.
UV spectroscopic Tm curves of Asp-amAla(6-cPOO) oligomers docu-
menting a) no pairing with DNA oligomers and b) very weak pairing
with RNA oligomers. c) Job plot showing the 2:1 ratio of the pairing
partners in the homo-duplex formed at 08C. Measurements were
made with total concentration of approximately 10 mm (1:1) in 1m
NaCl, 10 mm aq. NaH2PO4, 0.1 mm Na2EDTA, pH 7.0. No self pairing
was observed for individual partner strands.
We checked if the weak pairing behavior of the orotamide
unit was consistent with a correlation between the DpKa and
the base-pairing strength.[2] The pKa value of the orotamide
unit in dipeptide S21[9] was determined to be 6.6,[9] lower than
that of either orotic acid (pKa2 = 9.45)[10a] or of N(1)-methyl
orotamide (pKa = 9.1)[5a], but in agreement with the pKa of
methyl orotate (7.93)[5a]
. The difference in pKa values
between deoxyadenosine (3.8)[10a] and an orotamide deriva-
tive (6.6) is less (DpKa ca. 3) when compared with the
This interpretation finds support in observations that the
5-aPOO-tagged (pKa = 8.9) Asp-Glu oligo-dipeptide, which
exhibits base pairing, has the same overall backbone/base-
pairing axes (Figure 2c).[2b]
difference (DpKa ca. 5) between deoxyadenosine and deox-
[11]
ythymidine (pKa ca. 9.5)[5a]
.
These results agree with the
correlation, ꢀthe smaller the DpKa of base-pairing partners,
the weaker the base-pairing strength’.[2b] That this pKa value
of 6.6 of the orotamide unit in S21 correlates with deproto-
We turned our attention to the 6-cPNN-tagged oligo-
dipeptides. Being aware that the pKa value of the heterocycle
has an effect on its base-pairing disposition, we measured the
pKa of 2,4-diaminopyrimdine-6-carboxamide derivative S22[9]
and found it to be 4.7,[9] consistent with that known for
orotamide.[5c] Therefore, an oligomer tagged with 6-cPNN
should form base pairs when partnered with thymine or uracil
(pKa ca. 9.5–9.8)[10a], which is in agreement with the correla-
tion of the DpKa and the base-pairing strength.[2b] The
required oligo-dipeptides Asp-amAla(6-cPNN)6,12&16 were pre-
pared from Fmoc-protected monomers and purified without
any difficulty by ion-exchange HPLC methods (see Table S1
in the Supporting Information).[9] Their base-pairing behavior
was investigated with thymine- (DNA) and uracil-containing
À
À
nation of its N1 H proton (and not N3 H proton) was
established by the similarity of the change in its UV spectrum
(lmax shifts from 280 nm to 300 nm by deprotonation)[9] with
that of the known[5b] N(3)-methyl orotic acid (a behavior not
shown by N(1)-methyl derivative). Similar bathochromic
shifts were also observed for the temperature dependent
UV spectrum of Asp-amAla(6c-POO)12 either alone, or in the
presence of its pairing partner, indicating a temperature
facilitated deprotonation of the orotamide unit within the
oligomer.[9] All these point to the orotamide unit existing in its
anionic form at N1, which—and not the change in the
backbone—seems to be contributing to its weak base pairing.
Angew. Chem. Int. Ed. 2009, 48, 8124 –8128
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8125