Xylo-Configured Oligonucleotides (XNAs, Xylo Nucleic Acids): Synthesis and Properties
Nicolai E. Poopeikoa, Jesper Wengela, Britta M. Dahlb
aNucleic
Acid Centre, Department of Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Chemistry, University of Copenhagen,
Universitetparken 5, DK-2100, Denmark
Introduction
Synthesis of XNAs and Hybridization Studies
Among modified oligonucleotides the ones with altered 3’,4’-(threo)-configuration of phosphodiester backbone
have attracted only limited attention.1-4 In general, the monomers of the structure A – D (Figure 1) were included
into oligonucleotides. The ones containing xylonucleosides of the structure A and D have shown interesting
properties.1,4 Ribozymes containing of monomer of the structure C have been prepared, but no hybridization data
for xylo-RNA has so far reported.3 Methylene-extended xylo-DNA (monomer B) has been synthesised and found
to induce limited, but significant, destabilization when hybridized towards DNA complements.2 Here we report
data on the synthesis and binding properties of various XNAs in which monomers of the structure A, C and E are
incorporated.
The reference DNA oligos ON1 and ON7 and XNAs ON2-ON6 and ON8-ON13 (Table 2A) were synthesised
using standard phophoramidate approach.11 The coupling yields for DNA phosphoramidate were > 99%, for
xylo-configured amidates 5, 12 and 21 - > 90 %. Deprotection, purification and isolation of XNAs followed
standard procedures. Hybridization data are shown in Table 2B.
O
O
Base O
O
Base O
O
O
O P O
-
-
-
O P O
OH
Base O
O
O
O P O
B
A
Base O
O
O
O
O
-
O P O
-
O P O
F
Figure 1
Figure 1. Structures of different monomeric constituents of XNAs (xylo nucleic acids)
Synthesis
Scheme 1
HO
Thy
O
RO
Thy
O
i
DMTrO
Thy
O
iii
DMTrO
1
ii
O
P
N
4
2 R=DMTr, R 1=H
3 R=DMTr, R 1=M s
Thy
O
iv
OH
R 1O
HO
CN
O
5
Reagents and conditions: i) DMTrCl, Py, RT, 20h; ii) MsCl, Py, RT, 20h, Σ 87.8%; iii) 1M
NaOH, EtOH, 60oC, 20h, 88.9%; iv) chloro(β-cyanoethoxy)(N,N-diisopropylamino)phosphine, (i-Pr)2NEt, CH2Cl2, RT, 30 min, 93%.
Scheme 2
B zO
R e f. 6
D -xylose
OMe
O
B zO
i
O Bz
O
OAc
OBz
RO
Thy
O
OR
D M T rO
F
D M T rO
Thy
O
vi
11
9a R = B z, α−a n om er (2 3.5 % )
9b R = B z, β-a no m e r (49 .3 % )
1 0a R = H , α−a no m e r (7 4.8 % )
1 0b R = H , β-an o m e r (83 .3 % )
iii
Thy
O
OH
O
F
P
N
F
O
12
No report exists on the hybridization of XNAs towards RNA complements. From the data shown in Table 2B is
appear that XNAs is a class of molecules able to efficiently bind to complementary RNA, in some cases with
increased Tm values to reference duplexes. Especially XNAs with large portion of XNA monomers (ON10,
ON11 and ON12) display high-affinity recognition of RNA, but also a few XNA modification in an oligo
otherwise containing, e.g., DNA monomers (A), allow satisfactory binding properties towards RNA (see ON2
and ON3). These results suggest XNA to be interesting for applications within, e.g., molecular diagnostics and
antisense therapy.
CN
iii
Reagents and conditions: i) AcOH-Ac2O (3.8:1 v/v), conc. H2SO4, 75 min, 97%; ii)
(TMSO)2Thy, TMSTf, 1,2-DCE, reflux, 3h; iii) NH3, MeOH, 20h; v) DMTrCl, Py, RT,
20h, 90%; vi) chloro(β-cyanoethoxy)(N,N-diisopropylamino)phosphine, (i-Pr)2NEt,
CH2Cl2, RT, 30 min, 81%.
Conclusions
A viable synthetic routes for the XNA monomers containing at 2’-position proton, F-atom or acethoxy-group
have been developed. It was shown that XNAs have high affinity to RNA complements than to DNA ones.
Table 1. Selected 1H- and 13C-NMR data for nucleosides 10a and 10b.a
Compound
H-1’, ppm
J1’,2’, Hz
J1’,F, Hz
H-4’, ppm
10a
6.24dd
3.3
21.43
4.67-4.64 m
(+ H-3’)
10b
6.02d
<1.0
a) The spectra are recorded in CD3OD.
21.43
4.23m
C-6
138.33 d
(JC-6,F=2.86)
Acnowledgements
138.52 s
The Danish Natural Science Research Council, The Danish Technical Research Council are acknowleged for
financial support. Michael Meldgaard (Exiqon A/S) is thanked for MALDI-MS analysis.
Scheme 3
HO
BzO
O
i, ii
OH
13
R2O
O
O
iii
OAc
OBz
O
14 OAc
O
Thy
viii
O
vii
17 R1=H
18 R1=Ac
HO
OR1
O
Thy
OH
19
OAc
Thy
O
vi
OR2
OR1
v
O
Table 2B – Hybridization studies
_________________________________________________________
Complementary DNA
Complementary RNA
[5’-d(CACTATACG)]
[5’-r(CACUAUACG)]
_________________________________________________________
XNA/DNA
Tm (∆Tm) / oC)
Tm (∆Tm) / oC
ON1
30
26
ON2
24 (-6)
25 (-1)
ON3
25 (-5)
27 (+1)
ON4
b)
c)
_________________________________________________________
Complementary DNA
Complementary RNA
[r(A14)]
[d(A14)]
Tm (∆Tm) / oC)
Tm (∆Tm) / oC)
_________________________________________________________
ON7
33
29
ON8
23 (-10)
25 (-4)
ON9
23.0 (-10)
25 (-4)
ON10
34 (+0.1)
38 (+0.7)
ON11
33 (0)
38 (+0.7)
ON12
36 (+0.2)
36 (+0.1)
ON13
21 (-12)
24 (-5)
_________________________________________________________
dxT = thymin-1-yl deoxyxylo-DNA monomer A, FxT = thymin-1-yl
2’-fluoro-xylo-DNA monomer E, xT = thymin-1-yl xylo-RNA monomer C;
The reported strongly destabilizing effect of incorporation of a few isolated xylo-DNA monomers A with the
regard to hybridization towards DNA complements is confirmed by present study for ON2 and ON4 (relative to
ON1) and ON8 (relative to ON7) (Table “B). Likewise, incorporation of one or three 2’fluoro-xylo-DNA- (E) or
xylo-RNA (C) monomers into 9-mer sequence or into 14-mer sequence led to decreased affinity towards DNA
(ON3, and ON9, ON13, respectively). However, a tendency towards reducing the degree in Tm value per
modification (∆ Tm) is seen and stressed by comparable Tm values of almost fully modified XNAs (ON10, ON11
and ON12) and the reference ON7.
F
8
v
ON7: T14
ON8: 5’-T7dxTT6
ON9: 5’-T7FxTT6
ON10: 5’-(dxT)2FxT(dxT)3FxT(dxT)3FxT(dxT)2T
ON 11: 5’-(dxT)13T
ON 12: 5’-(FxT)13T
ON13: 5’-T7xTT6
ii
6
7 F
ON1: 5’-d(GTGATATGC)
ON2: 5’-d(GTGAdxTATGC)
ON3: 5’-d(GTGAFxTATGC)
ON4: 5’-d(GdxTGAdxTAdxTGC)
E
D
C
Base
O
Table 2A - List of studied XNAs and reference DNAs
x
DMTrO
15 R1=Ac, R2=Bz
16 R1=R2=H
O
Thy
xi
OH
OAc
20
References
DMTrO
O
Thy
O
N P
O
OAc
CN
21
Reagents and conditions: i) BzCl, Py, RT, 20h; ii) AcOH-Ac2O (3.8:1 v/v), conc. H2SO4,
RT, 5h, Σ 95%; iii) ) (TMSO)2Thy, TMSTf, 1,2-DCE, reflux, 3h, 67.5%; v) NH3, MeOH,
20h, 66%; vi) (CH3)2CO, 2-methoxypropene, p-TsOH, RT, 3h, 99%; vii) Ac2O, Py, RT,
20h, 96.6%; viii) 80% AcOH, 65oC, 2h, then 20h at RT, 83.5%; x) DMTrCl, Py, RT, 20h,
90%; xi) chloro(β-cyanoethoxy)(N,N-diisopropylamino)phosphine, (i-Pr)2NEt, CH2Cl2,
RT, 30 min, 87%.
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