Two carbocyclic locked nucleic acid analogues give structural information about the role of hydration in A-type duplexes

Article abstract of DOI:10.1080/15257770701544377

Two locked nucleic acid (LNA) analogues with three-carbon 2′-4′ linkages, saturated or unsaturated, are synthesized using a ring-closing metathesis based strategy. Strongly stabilized duplexes with complementary RNA and slightly destabilized duplexes with

Full text of DOI:10.1080/15257770701544377

Nucleosides, Nucleotides, and Nucleic Acids, 26:1529–1532, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701544377
TWO CARBOCYCLIC LOCKED NUCLEIC ACID ANALOGUES GIVE
STRUCTURAL INFORMATION ABOUT THE ROLE OF HYDRATION IN
A-TYPE DUPLEXES
Nanna Albæk, Michael Petersen, and Poul Nielsen
Nucleic Acid Center,
2
Department of Chemistry and Physics, University of Southern Denmark, Odense M, Denmark
Two locked nucleic acid (LNA) analogues with three-carbon 2ꢁ-4ꢁ linkages, saturated or unsat-
2
urated, are synthesized using a ring-closing metathesis based strategy. Strongly stabilized duplexes
with complementary RNA and slightly destabilized duplexes with complementary DNA are observed.
CD-spectroscopy indicates a less pronounced shift toward A-type duplexes compared to LNA. These re-
sults combining a strong N-type conformation with the absence of a 2ꢁ-oxygen demonstrate a stronger
importance of minor groove hydration in an intermediate duplex type than in an A-type duplex.
Keywords Locked nucleic acid; RNA recognition; duplex hydration
Locked nucleic acid (LNA)[1−3] and its analogue ENA[4] are among the
most intriguing nucleic acid analogues due to their unprecedented affin-
ity for complementary RNA and DNA sequences. The LNA and ENA
monomers (Figure 1) are both locked in the N-type conformation. A sin-
gle incorporation of an LNA monomer in an oligodeoxynucleotide (ODN)
leads to an increase in the thermal stability of 3–8◦C of a DNA:RNA du-
plex and 3–5◦C of a DNA:DNA duplex.[2] This behavior is related to the
ability of LNA-monomers to conformationally steer their neighboring 2ꢁ-
deoxynucleotides into an N-type conformation and hereby to induce A-type
or A-type like duplex conformation.[3]
ENA demonstrates almost the same stabilisation of duplexes as LNA,[4]
whereas other analogues in which the 4ꢁ-2ꢁ linkage has another constitution
(i.e., a “COC” or an even longer “COCO” or “CCCO”) demonstrates some-
what less pronounced stabilization of a DNA:RNA duplex and a neutral or
even destabilizing effect on DNA:DNA duplexes.[4,5] The 4ꢁ-2ꢁ-linkage with
three carbon atoms, however, has been only briefly mentioned in a review[6]
The Danish National Research Foundation, the Danish Natural Research Council, and the Danish
Centre for Scientific Computing are thanked for financial support.
Address correspondence to Poul Nielsen, Nucleic Acid Center, Dept of Chemistry, University of
Southern Denmark, DK-5230 Odense, Denmark. E-mail: pon@ifk.sdu.dk
1529

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N. Albæk et al.
FIGURE 1 LNA and ENA and two new carbocyclic analogues.
and reported to give a destabilized DNA:RNA duplex which is a surpris-
ing result compared to the results of the other analogues. We, therefore,
decided to synthesise this analogue, X, by a ring-closing metathesis based
strategy giving access also to the unsaturated analogue Y (Figure 1).
The synthesis started from uridine following a linear approach
(Scheme 1) and took advantage of the reported selective 5ꢁ,3ꢁ-silylation[7]
SCHEME 1 a) TBSCl, DABCO, AgNO3, THF, 90%, ref. 7; b) PhOCSCl, DMAP, CH3CN, 79%; c)
AllylSnBn3, AIBN, toluene, 65%, ref. 8; d) aq. AcOH, 75%; e) i) Dess-Martin periodinane, CH2Cl2;
ii) H2CO, MeOH, aq. NaOH, dioxane then NaBH4, 52%; f) i) BzCl, pyridine, CH3CN, 65%; ii) TBSCl,
Im., DMF; iii) NaOMe, MeOH, 71%; g) Dess-Martin periodinane, CH2Cl2, 93%; h) Ph3PCH3Br, BuLi,
THF, 95%; i) A, CH2Cl2, 96%; j) TBAF, THF, 98%; k) H2, PtO2, MeOH, 100%; l) DMTCl, pyridine,
CH3CN, 78%; m) NC(CH2)2OPClN(iPr)2, DIPEA, CH2Cl2 63%; n) DMTCl, Et3N, CH2Cl2, 78%; o)
NC(CH2)2OPClN(iPr)2, DIPEA, CH2Cl2 57%.

Two Carbocyclic LNAs
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TABLE 1 Hybridization data.
T
m(ꢀTmper modification )/ ◦C
ODN sequence
Complementary DNA
Complementary RNA
5ꢁ-dGTGATATGC-3ꢁ
5ꢁ-dGTGAXATGC-3ꢁ
5ꢁ-dGXGAXAXGC-3ꢁ
5ꢁ-dGTGAYATGC-3ꢁ
5ꢁ-dGYGAYAYGC-3ꢁ
30.0
27.5
29.0 (−1.0)
27.0 (−1.0)
29.0 (−1.0)
23.0 (−2.3)
32.0 (+4.5)
38.0 (+3.5)
31.5 (+4.0)
34.5 (+2.3)
Buffer: 5 mM Na2HPO4, 10 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0; 1.5 µM
of each strand.
and 2ꢁ-allylation,[8] protecting group manipulations, a 4ꢁ-vinylation and a
very efficient ring-closing metathesis using Grubbs second generation cata-
lyst A to give the analogue Y. Hydrogenation gave X and both analogues
were converted into DMT-protected phosphoramidites and incorporated
into ODNꢁs (Table 1)
The ODNs were mixed with complementary DNA and RNA sequences
and the stability of the duplexes were determined. Significant increases in
stability of the DNA:RNA duplexes were observed. These are only slightly
smaller than the increases found for LNA and ENA. On the other hand,
slight decreases in stability of the DNA:DNA duplexes were observed. This
is different to the data obtained with LNA and ENA for which increased
stability was found also for DNA:DNA sequences. CD-spectra (not shown)
demonstrate that the shift toward A-type (for DNA:RNA) or A-type like du-
plexes (for DNA:DNA) is smaller for the duplexes containing X or Y than
for comparable LNA sequences. In other words, the conformational steer-
ing on neighbouring 2ꢁ-deoxynucleotides is smaller for X and Y than it is
for LNA (and probably also ENA) nucleotides. This might be due to the
lack of a 2ꢁ-oxygen atom in the former hampering the hydration pattern
in the minor groove of the duplexes. Apparently, this hydration pattern is
more important in intermediate type DNA:DNA duplexes with a narrow mi-
nor groove than in the compact A-type DNA:RNA duplexes with a broader
minor groove.
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