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M. D. Sùrensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1853±1856
other nucleotides. A separate end-capping cycle (con-
sisting of detritylation and acetylation) was used to
block further elongation at the 50-end, thus preparing
for branching. The support was subsequently treated
with a 0.5 M solution of hydrazine hydrate in a pyr-
idine:acetic acid:water buer (4:3:0.35, v:v:v) for 5 min
to remove the levulinoyl protecting group. The ®rst
nucleotide in the branch was subsequently attached at
the 300-hydroxy group (in the case of branching nucleotide
X) or at the 200-hydroxy group (in the case of branching
nucletide Y) by 5 min couplings at standard amidite
concentration whereupon the remaining part of the
branch was synthesized using standard conditions. The
50-O-DMT group of the latest incorporated nucleotide
was removed as the last step on the synthesizer for ONs
A±F whereas the 50-O-DMT group was kept on for ONs
G and H. The coupling yields determined spectro-
photometrically at 498 nm (quanti®cation of the amount
of DMT cation released during each detritylation step)
were >99% for commercial amidites, ꢁ95±98% for the
modi®ed amidite 5, and ꢁ76% for amidite 6. The ONs
were cleaved from the solid support and deprotected by
treatment with 32% aqueous ammonia for 16 h at room
temperature. The crude ONs were precipitated from
ethanol to give pure ONs A±F (Table 1)11 or reversed
phase puri®ed (COP columns, Cruachem; procedure
includes detritylation) to give pure ONs G and H.11 The
composition of the ONs was con®rmed by matrix-assisted
laser desorption mass spectrometry.12
e
Figure 1. Structure of branching nucleotides V,1c X and Y, and
structure of an LNA thymine monomer (TL). U=uracil-1-yl. T=thy-
min-1-yl. R=H or CH3.
in the presence of N,N-diisopropylethylamine in anhy-
drous dichloromethane followed by column chromato-
graphy.7 Using similar strategies for levulinoylation of
the 200-hydroxy group and phosphitylation of the 30-
hydroxy group, the 30-C-levulinoyloxyethyl branched
LNA phosphoramidite derivative 6 was synthesized.7,8
The hybridization properties of ONs A±H towards single
stranded DNA (A12) were evaluated (Table 1). The
melting temperatures (Tm values) of the non-branched
duplexes A:A12 (31.5 ꢀC) and G:A12 (31.0 ꢀC) were used
as references. The branched ONs B, C, E, F and H were
designed to bind complementary ONs through the for-
mation of bimolecular complexes as shown in Figure 2.
In all cases, only one melting transition (hyperchromic
shift) was detected and the formation of a structure
involving a triple helical segment was con®rmed if a
melting transition was detected not only at 260 nm but
also at 284 nm.13
The ONs B, C and H have the potential of forming
eight Hoogsteen basepairs in addition to the Watson±
Crick duplex. In analogy with our earlier results, and the
results of Kool et al.14 on circular DNA, it was expected
that only one melting transition, i.e., cooperativity
between the two binding modes, would be observed
leading to stabilization. Compared to the reference
duplexes A:A12 and G:A12, the Y-shaped ONs B, C and
H showed slightly improved binding anities towards
A12 as indicated by the Tm values increased by +1,
+1.5 and +3.0 ꢀC, respectively. The involvement of a
triple helix in the complexes formed with ONs B, C and
H, but not with the references A and G, was con®rmed by
transitions observed at 284 nm.13 It appears from the Tm
results that the presence of a C2-linker has no signi®cant
in¯uence on the thermal stability (B:A12 versus C:A12).
The geometry for formation of the bimolecular complexes
is apparently slightly more favourable with the branching
point Y than with X, but for synthetic reasons the latter
was evaluated further in this preliminary study.
Scheme 1. (i) Levulinic anhydride, DMAP, pyridine; (ii) H2, Pd(OH)2/
C, EtOH; (iii) DMTCl, pyridine; (iv) 2-cyanoethyl N,N-diisopropyl-
phosphoramidochloridite, N,N-diisopropylethylethylamine, CH2Cl2.
DMT=4,40-dimethoxytrityl. Lev=levulinoyl. T=thymin-1-yl.
The ONs A±H (Table 1) were synthesized using standard
phosphoramidite chemistry9 with cycles including detri-
tylation, coupling, capping and oxidation. The syntheses
were initiated by constructing the linear strand using
standard amidite concentration (0.033 M in anhydrous
acetonitrile) and coupling time (1 min). However, when
the amidite 5 was incorporated, a 0.042 M solution and
a coupling time of 30 min were applied. For incorpora-
tion of amidite 6, a 0.050 M solution and a coupling
time of 2Â60 min were applied (coupling, washing,
coupling with a fresh solution of amidite, capping, oxi-
dation and detritylation). The oxidation was performed
with tert-butyl hydroperoxide for the amidites 5 and 6
to prevent strand cleavage,10 and with iodine for the