Increased RNA affinity of HNA analogues by introducing alkoxy substituents at the C-1 or C-3 position

Article abstract of DOI:10.1081/NCN-100002429

1,5-Anhydrohexitol nucleoside congeners with alkoxy substituents, were prepared, resulting in a further improvement of their RNA affinity and antisense potential.

Full text of DOI:10.1081/NCN-100002429

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INCREASED RNA AFFINITY OF HNA ANALOGUES BY
INTRODUCING ALKOXY SUBSTITUENTS AT THE C-1 OR
C-3 POSITION
A. Van Aerschot a , M. Meldgaard a , F. Volders a , G. Schepers a , J. Rozenski a & P.
Herdewijn a
a Rega Institute for Medical Research, Katholieke Universiteit Leuven ,
Minderbroedersstraat 10, Leuven, 3000, Belgium
Published online: 07 Feb 2007.
To cite this article: A. Van Aerschot , M. Meldgaard , F. Volders , G. Schepers , J. Rozenski & P. Herdewijn (2001)
INCREASED RNA AFFINITY OF HNA ANALOGUES BY INTRODUCING ALKOXY SUBSTITUENTS AT THE C-1 OR C-3 POSITION,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 781-784, DOI: 10.1081/NCN-100002429
To link to this article: http://dx.doi.org/10.1081/NCN-100002429
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 781–784 (2001)
INCREASED RNA AFFINITY OF HNA ANALOGUES
BY INTRODUCING ALKOXY SUBSTITUENTS AT
THE C-1 OR C-3 POSITION
A. Van Aerschot,∗ M. Meldgaard, F. Volders, G. Schepers,
J. Rozenski, and P. Herdewijn
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium
ABSTRACT
1,5-Anhydrohexitol nucleoside congeners with alkoxy substituents, were pre-
pared, resulting in a further improvement of their RNA affinity and antisense
potential.
The strong hybridizing potential of anhydrohexitol nucleic acids (HNA) by
virtue of its pre-organisation by now is well documented (1–3), and some interesting
biological antisense effects have been reported (inhibition of Ha-ras and ICAM-1
(4), and antimalarial activity (5)). To further augment the affinity for target RNA
structures, two different roads can be explored by looking for analogues which
either increase the conformational preorganisation of the monomeric structures,
or which alternatively augment the hydration potential. In addition, cost and ease
of synthesis need to be considered. Consequently, 3ꢀ-O-methylated altrohexitol
analogues and 1ꢀ-O-methylglycosidic analogues were envisaged and incorporated
into HNA sequences.
Synthesis of the 3ꢀ-O-methylated analogue followed the route previously de-
scribed for preparation of the altrohexitol monomers (ANA) (6). Chemoselective
methylationwithouttemporaryprotectionofthenucleobasegavethemethylatednu-
cleoside. Further modification yielded the desired phosphitylated building block 1*.
∗Corresponding author.
781
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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VAN AERSCHOT ET AL.
The 1ꢀ-O-methylglycosidic analogues were obtained starting from ubiquitous
methylglucopyranoside. Openingofthe2,3-allo-epoxide(7,8), wasfollowedbyde-
oxygenation of the 3-position. Removal of the benzylidene position is less straight-
forward because of the glycosidic linkage, but can be accomplished alternatively
via hydrogenation in almost quantitative yield. Further modification yielded the
desired phosphitylated building block 2*.
All oligos were assembled on a propanediol containing universal support,
obviating the need of modified supports (9). With a coupling time of 3 minutes,
coupling yields were consistently over 95% and higher. Electrospray ionization
mass spectrometry (ESI-MS) in negative mode indicated all monoisotopic masses
to be consistently within 0.5 Da of the calculated masses.
Scheme 1. a) 3.2 eq. uracil, 3 eq. NaH, DMF 120◦C 24h (86%); b) 5 eq. NaH, 3 eq. CH3I, THF,
7h 0◦C (38%); c) 90% TFA (74%); d) MMTrCl, pyridine (89%); e) DIEA, CH2Cl2, (iPr)2N(CN)PCl
(90%); f) 3 eq. thymine, 2.8 eq. NaH, DMF, 96 h, 120◦C (71%); g) 2 eq. CSCl2, 7 eq. DMAP, CH2Cl2
at −40◦C followed by 4 eq. 2,4-Cl2C6H3OH at RT for 1 h (85%); h) 1.5 eq. Bu3SnH, AIBN, toluene
80◦C (85%); i) 10% TFA-MeOH 3 h (55%); alternatively H2, Pd/C in MeOH-HOAc 98:2 for 18 h
(90%); j) DMTrCl, pyridine (85%); k) DIEA, CH2Cl2, (iPr)2N(CN)PCl (67%).
When hybridised with complementary HNA, the introduction of the 3ꢀ-O-
methylated uridine nucleoside analogue (1) into a HNA strand results in an in-
creased thermal stability of the duplex compared to the unmodified HNA:HNA
duplex (ꢀTm = +0.6◦C/modification) (entry A and B, Table 1). However, this
increase is less pronounced than the increase in thermal stability obtained by mod-
ifying the nucleobase with a methyl substituent in the 5-position (ꢀTm = +1.1◦C/

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INCREASED RNA AFFINITY OF HNA ANALOGUES
783
Table 1. Hybridisation Data for Hexameric Hexitol Sequences (6ꢀ→4ꢀ) with Incorporation of
3 Methylated Building Blocks 1 or 2
HNA
ANA
RNA
DNA
Sequence
Complement
Complement
Complement
Complement
A
B
C
D
E
1 C 1 CC 1 (HNA)
UCU CCU (HNA)
UCU CCU (ANA)
TCT CCT (HNA)
2 C 2 CC 2 (HNA)
52.4 (64)
50.7 (61.2)
54
54
56.7
58.8 (71)
55#
61.8 (71.2)
60.6
31 (42)
no Tm
no Tm
no Tm
no Tm
no Tm
30.5 (40)
38.4 (47.6)
39 (48)
62.7
39.9 (49.5)
Tm’s in a buffer of 0.1 M NaCl and 20 mM phosphate, pH 7.4 with a duplex concentration of 4 µM.
Numbers in brackets are Tm’s in a high salt buffer (1.0 M NaCl). 1 denotes a 3ꢀ-O-methylated ANA
monomer, 2 denotes a 1ꢀ-O-methylated HNA monomer.
# lit data incorrect (in ref. 12, the duplex was HNA-HNA, instead of HNA-ANA.
modification) (compare entry A and D). Hybridising the modified ONs with com-
plementary RNA corroborates this pattern of thermal stabilisation of the duplexes.
As expected, none of the hexitol based ONs hybridised with complementary DNA.
Clearly, for this series the results indicate that a 5-methyl is more important than a
3ꢀ-O-methyl and that a methylation of the 3ꢀ- hydroxyl group in ANA is destabilising
when pairing to an RNA sequence is envisaged.
For the hexitol analogue 2 comparison is more straightforward, and the ther-
mal stabilisation is of the same order as for 1 when compared to HNA, as well in
its pairing with hexitol oligonucleotides as with RNA sequences. Therefore, intro-
duction of 2 seems to be slightly more favorable over addition of a HNA monomer.
Incorporation of respectively 1 or 2 into octameric (GCGUAGCG) HNA se-
quences and hybridisation with RNA, yielded an analogous picture with a small
increase for introduction of 1 and a solid increase of 1.6◦C in Tm for introduction of
2. However, thermal unwinding of a self-complementary HNA duplex gave another
pattern, where incorporation of 1 resulted in considerable stabilisation of the duplex
(ꢀTm = +3◦C/modification) exceeding the stabilisation obtained for substitution
of uracil for thymine (ꢀTm = +2.4 ◦C/modification) (Table 2).
Table 2. Thermal Stability of Self Complementary HNA Sequences
Containing 3ꢀ-O-methyl or 1ꢀ-O-methyl Modifications (1 or 2)
Sequence
Tm/◦C
ꢀTm/◦Ca
GUGU ACAC
G 1 G 1 ACAC
GTGT ACAC
G 2 G 2 ACAC
65.0
76.7
74.5
76.9
ref.
+3
+2.4/ref.
+0.6
Tm’s obtained in 0.1 M NaCl, 20 mM phosphate, pH 7.4 with an oligo
concentration of 8 µM (4 µM of duplex).
aꢀTm/modification.

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VAN AERSCHOT ET AL.
Generally, the ONs containing the 3ꢀ-O-methyl derivative (1) showed a small
increase in thermal stability towards complementary sequences as compared to
HNA, except in the case of a self-complementary sequence for which an increase
in thermal stability of 3◦C per modification was observed. Compared to ANA, how-
ever, the 3ꢀ-O-methylation caused a small decrease in thermal stability of duplexes
between a modified ON and a complementary target, especially when targeting
RNA. The methyl glycosidic analogues 2, however, seem to be endowed with
higher affinity for RNA in comparison with well-known HNA, while at the same
time having economically more favorable monomers. These compounds therefore
could have strong potential for antisense purposes and need to be evaluated further.
In addition, it proved possible to incorporate the new modified monomers into
RNA without compromising their affinity for their respective RNA target.
ACKNOWLEDGMENT
This work was supported by a grant from the K.U. Leuven (GOA 97/11).
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