ARYL-β-C-LOCKED NUCLEIC ACID AS UNIVERSAL HYBRIDIZATION PROBES1
B Ravindra Babu and Jesper Wengel
Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, DK-5230, Odense M, Denmark
Universal bases are able to bind isoenergically with each of the natural nucleotides. Almost all Universal
bases reported are DNA analogues, which are known to adopt S-type furanose conformation predominantly
due to the lack of an anomeric effect. A series of Aryl-2’-O,4’-C-methylene-ß-D-ribofuranosides, which are
known to preorganise in locked C3’-endo (N-type) RNA like furanose conformation, were synthesised to
study the effect of structural organisation on universal hybridization.
OH
OH
OH
Base
Base
Base
O
HO
O
O
HO
OH
OH
S-type conformation
(B-form duplexes)
Selective irradiation of the H3’ proton of compounds 13a-13e
gave enhancement of signals of the aromatic aglycon (3.0% for
13a, 2.7% for 13b, 6.2% for 13c, 7.0% for 13d and 6.8% for
13e) which confirms the cis-positioning of the H3’-proton and
the aglycon on the furanose ring and furthermore supports an Ntype furanose conformation. In addition, a single-crystal X-ray
diffraction study was performed of the phenyl analogue 14a.
O
N-type conformation
(A-form duplexes)
Molecular structure (ORTEP-plot) of the
Phenyl bicyclic C-glycoside14a.
Locked Nucleic Acid
(N-type conformation)
Thermal denaturation studies
Introduction
A Universal base analogue forms base pairs with each of the natural DNA/RNA bases with little
discrimination. Most of these analogues are non-hydrogen bonding, hydrophobic, aromatic bases which
stabilize duplex DNA by stacking interactions.2 Such bases have attracted much attention due to their
potential utility in the design of oligonucleotide primers or hybridization probes when the identity of one or
more base in the target sequence is unknown. Promising universal bases with a 2-deoxy-β-D-ribofuranosyl
moiety have been reported, e.g. 3-nitropyrrole, 5-nitroindole, isocarbostyril, 8-aza-7-deazaadenine and
pyrene derivatives.2 There has been some success in the design of universal base analogues but examples
which are able to hybridize without significant duplex destabilization are rare. Moreover, all of the universal
bases that have been described are DNA analogues and very little work has been reported on
ribonucleosides.
Stimulated by the work of Kool and collaborators on hybridization using non-polar aromatic moieties as
replacements of the natural bases3 and the desire to obtain improved binding affinity for universal
hybridization,2 we became interested in studying LNA-type derivatives of aryl C-nucleosides containing
various planar aromatic moieties as aglycons. The furanose ring of LNA (locked nucleic acid) monomeric
nucleotide is known to be locked in North (3E) RNA like furanose conformation and it is characterized by
very high binding affinity and efficient Watson-Crick discrimination when hybridized with single stranded
DNA or RNA targets.4
5’-d(GTGAXATGC)
DNA target 3’-d(CACTYTACG)
X
Ar
A
C
G
T
12
5
6
7
15
7
6
8
ON3 (17c)
15
7
6
9
ON4 (17d)
18
17
18
19
ON5 (17e)
13
6
6
7
ON6 (Abasic LNA)
<3
-
-
-
Reference (X=T)
28
11
12
ON1 (17a)
F
ON2 (17b)
19
14
• LNA-type C-glycoside monomers (17a-e, ON1-ON5) stabilize the duplex when compared to corresponding LNA-type abasic monomer (ON6) .
• Unlike Phenyl-RNA monomer10, the Phenyl-LNA monomer (17a) didn’t behave in universal way, but showed preference for Adenine.
• Similar results were obtained for the 4-fluoro-3-methylphenyl (17b), 1-naphthyl (17c) and 2,4,5-trimethylphenyl (17e) derivatives (ON2,ON3, and ON5).
• The pyrenyl LNA nucleotide 17d displays more encouraging properties in relation to universal hybridization (Tm values all being within 17-19 oC).
Extensively studied Universal base
O
HO
N
N
O
NO2
NO2
NH
N
HO
O
OH
N
HO
N
O
Hypoxanthine deoxyribofuranoside
(naturally occuring DNA)
6
N
N
O
O
OH
5-Nitroindole deoxyribofuranoside
(destabilizing, Tm~2-5 oC)
3-Nitropyrrole deoxyribofuranoside
(destabilizing, Tm~11-14 oC)
• not indiscriminate in all sequence context
• non.analysable data in primers containing multiple substitution
HO
OH
OH
5
N
7
Isocarbostyril deoxyribofuranoside
(destabilizing, Tm~2 oC)
8
In order to study the effect of the LNA-type β-C-aryl nucleotide monomers in A-type duplexes we
synthesized ON7-ON9. The reference strand which, being composed entirely of 2’-OMe-RNA monomers,
is known to structurally mimic an RNA strand.15 As mentioned above, increased binding affinity of
universal hybridization probes is considered important and we therefore constructed ON7-ON9 as a
mixture of six 2’-OMe-RNA monomers, one central LNA-type β-C-aryl glycoside monomer (17b, 17c or
17d), and two affinity-enhancing LNA thymine monomers TL.
H
N
H
N
HO
O
HO
N
HO
O
OH
OH
8-Aza-7-deazaadenine deoxyribofuranoside9
(Hydrogen bonding base analogue)
OH
The pyrenyl LNA nucleotide [∆Tm ~ -10 oC for 17d (ON5 relative to ON1)] is more destabilizing when
compared to a pyrenyl DNA monomer11 [∆Tm ~ -5 oC]. It therefore appears that stacking (or intercalation)
by the pyrene moiety is disfavoured by conformationally locking the furanose ring into an N-type (3E)
conformation. However, comparison of the thermal stabilities of ON1, ON2, ON3 and ON5 with ON4
strongly indicates some productive interaction of the pyrenyl moiety with the helix, e.g. intercalation.
O
OH
Pyrenyl ß-C-deoxyribofuranoside11
(destabilizing, Tm~5 oC)
Phenyl-ß-C-ribofuranoside10
(destabilizing, Tm~16 oC)
5’-d(GOMeTLGOMeAOMeXAOMeTLGOMeCOMe)
DNA target 3’-d(CACTYTACG)
X
Ar
A
C
G
T
ON7 (17b)
31
25
26
27
ON8 (17c)
34
27
27
32
39
38
37
40
35
14
19
21
Synthesis of Aryl-2’-O,4’-C-methylene-ß-D-ribofuranosides
F
The key intermediate, aldehyde 11, was synthesized from the known furanoside 112 following two different
routes. Coupling of the aldehyde 11 with different aryl Grignard reagents yielded stereoselectively one
epimer of each of the compounds 12a-12e in good yields13. Subsequently, cyclization, debenzylation,
dimethoxytritylation followed by phosphitylation afforded the phosphoramidite building blocks 16a-16e in
satisfactory yields.
O
O
i, ii
O
HO
HO
HO
iii
O
O
HO
O
O
MPMO
MsO
iv
O
HO
MPMO
O
1
O
MsO
MPMO
O
O
MsO
v
O
5’-d(G
O
MsO
MPMO
O
2
ON9 (17d)
OMe OMe
T
OMe
G
OMe OMe
A
T
OMe OMe
A
T
G
OMe OMe
C
)
OCH3
OH
o
Melting temperatures (Tm values/ C) measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt buffer (10 mM
sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5 µM concentrations of the two strands; A = adenin-9-yl monomer, C = cytosin-1-yl
monomer, G = guanin-9-yl monomer, T = thymin-1-yl; AOMe, COMe, GOMe, TOMe are corresponding 2’-OMe-RNA monomers and TL = LNA thymine monomers.
3
ix
vi
MPMO
MPMO
v
O
MsO
MPMO
MsO
MPMO
10
MPMO
iv
O
OCH3
OH
O
9
vi
MsO
O
HO
MPMO
O
O
OCH3
O
O
8
4
The Aryl-C-bicyclic nucleosides 14a-e were evaluated for antiviral activity16 against HIV-1 in MT-4 cells
and were found to be inactive against HIV-1 at 300µM.
vii
MPMO
Ar
OH
OH
xi
O
MPMO
MPMO
x
OH
HO
OCH3
O
MPMO
O
OCH3
viii
AcO
O
6
OCH3
O
MPMO
O
MPMO
7
11
12
ix
O
O
O
MPMO
MPMO
5
xii
MPMO
Ar
O
O
MPMO
13
xiii
HO
Ar
O
xiv, xv
DMTO
HO
14
Ar
O
O
N
O
P
xvi
O
16
O
O
Ar
O
O
O P O
12-17
a
b
c
d
e
O
17
Ar
phenyl
4-fluoro-3-methylphenyl
1-naphthyl
1-pyrenyl
2,4,5-trimethylphenyl
CN
Reagents and conditions (and yields):
i. p-methoxybenzyl chloride, NaH, DMF (90%),
ii. 80% aq. acetic acid (72%),
iii a) sodium periodate, THF, H2O; b) formaldehyde, aq NaOH (1M), THF (88%),
iv MsCl, pyridine (2: 93%; 9: 89%)
v. H2O, HCl, CH3OH (1:1.5:8.5) (3: 86%; 10: 74%),
vi. NaH, DMF [4: major isomer (74%) + minor isomer (13%); synthesis of 7 from 10: 85%],
vii. KOAc, dioxane, 18-crown-6 (91%; 85%),
viii. saturated methanolic ammonia (88%; 92%),
• Preferential hybridization towards the target DNA containing the central adenine monomer for the 4-fluoro-3-methylphenyl (17b) and 1-naphthyl (17c)
monomers, and universal hybridization for the pyrenyl monomer (17d) (Tm values for ON9: 37-40 oC towards the four targets).
• The affinity-enhancing effect of the LNA thymine monomer TL is reflected in the satisfactory thermal stabilites obtained for ON7-ON9.
O
MPMO
ix. p-methoxybenzyl chloride, NaH, THF [(synthesis of 7 from 6: 80%; 69%); 8:52%]
x. 50% aq acetic acid (82%),
xi. ArMgBr, THF (12a: 88%; 12b: 85%, 12c: 95%); 12d: 89%; 12e: 88%),
xii. TMAD, Bu3P, C6H6 (13a: 77%; 13b: 84%; 13c: 78%; 13d: 79%; 13e: 80%),
xiii. DDQ, CH2Cl2, H2O (14a: 66%; 14b: 67%; 14c: 67%; 14d: 75%; 14e: 65%),
xiv. DMTCl, pyridine (15a: 71%; 15b: 61%; 15c: 60%; 15d: 61%; 15e: 78%),
xv. NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2 (16a: 66%; 16b: 66%; 16c: 60%; 16d: 68%; 16e: 63%),
xvi. DNA synthesizer
Conclusion
• Synthesis of a series of LNA-type β-configured C-aryl nucleosides has been accomplished as has their
efficient incorporation into short DNA and 2’-OMe-RNA/LNA strands using the phosphoramidite
approach.
• It has been shown that universal hybridization is achievable with a conformationally restricted monomer
as demonstrated for the pyrene LNA monomer 17d, both in a DNA context (ON4) and in an RNA-like
context (ON9).
• Importantly, the problem of decreased affinity of the known universal hybridization probes can be solved
by the incorporation of high-affinity monomers, e.g. 2’-OMe-RNA and/or LNA monomers
Acknowledgements
We thank the Danish Research Agency, the Danish National Research Foundation and Exiqon A/S for
financial support. Ms Britta M. Dahl is thanked for oligonucleotide synthesis, and Dr Carl E. Olsen for
MALDI-MS analyses.
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