Xylo-configured oligonucleotides (XNA, xylo nucleic acid): Synthesis of conformationally restricted derivatives and hybridization towards DNA and RNA complements

Article abstract of DOI:10.1016/S0960-894X(03)00441-4

Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 2′-deoxy-2′-fluoro-β-D-xylofuranosyl nucleotide monomer, a novel conformationally locked 2′-amino-2′-deoxy-2′-N,4′-C-methylene-β-D- xylofuranosyl nucleotide monomer, and

Full text of DOI:10.1016/S0960-894X(03)00441-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2285–2290
Xylo-Configured Oligonucleotides (XNA, Xylo Nucleic Acid):
Synthesis of Conformationally Restricted Derivatives and
Hybridization Towards DNA and RNA Complements
Nicolai E. Poopeiko,a Martin Juhl,b Birte Vester,c Mads D. Sørensenb
and Jesper Wengela,*
aNucleic Acid Center, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
cNucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
Received 6 February 2003; revised 2 April 2003; accepted 24 April 2003
Abstract—Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 20-deoxy-20-fluoro-b-d-xylofur-
anosyl nucleotide monomer, a novel conformationally locked 20-amino-20-deoxy-20-N,40-C-methylene-b-d-xylofuranosyl nucleotide
monomer, and a known 20-deoxy-b-d-xylofuranosyl nucleotide monomer (XNA monomers) have been synthesized and their
hybridization towards DNA and RNA complements studied. Thermal denaturation studies of nine-mer mixed-base sequences
composed of a mixture of XNA monomers and DNA monomers revealed preferential hybridization towards RNA complements
relative to DNA complements. For 14-mer homo-thymine XNAs containing thirteen XNA monomers, stable complexes towards
single-stranded DNA and RNA were formed at pH 7. Gel-shift experiments revealed these complexes to involve at least two XNA
strands per DNA or RNA target strand.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
monomer A, Fig. 1), Seela et al. showed that 20-deoxy-
xylonucleosides predominantly adopt an N-type
furanose conformation.16 The hybridization properties
of xylo-DNA have been intensively studied towards
DNA complements and towards xylo-DNA comple-
ments, but not towards RNA complements.12ꢀ17 Incor-
poration of a single xylo-DNA monomer into a DNA
In the design of antisense oligonucleotides several
aspects have to be considered, for example, resistance
towards degradation by nucleases and efficient hybridi-
zation towards RNA targets.1ꢀ4 In general, the most
efficient RNA binding has been obtained with antisense
oligonucleotides structurally mimicking RNA, for
example, containing furanose rings restricted or locked
in an N-type (north type, C30-endo type) con-
formation.1ꢀ4 This has convincingly been demonstrated
for bicyclic oligonucleotides, for example, LNA (locked
nucleic acid),3,5ꢀ8 and by the introduction of electro-
negative groups at the 20-position of the furanose
ring.1ꢀ4,9ꢀ11
In a comprehensive study12ꢀ16 of oligonucleotides con-
taining xylo-configured 20-deoxynucleotides (‘xylo-DNA’;
*Corresponding author. Tel.: +45-6650-2510; fax: +45-6615-8780;
e-mail: jwe@chem.sdu.dk
Figure 1. Structures of XNA monomers.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00441-4

2286
N. E. Poopeiko et al. / Bioorg. Med. Chem. Lett. 13(2003) 2285–2290
strand significantly reduced the binding affinity towards
the complementary DNA target.12ꢀ16,18 An almost fully
modified homo-thymine xylo-DNA formed a complex
with complementary DNA of comparable thermal stabi-
lity as the reference DNA:DNA duplex,12,17 and fully
modified self-complementary xylo-DNA formed a duplex
of similar stability to that formed by the corresponding
DNA.16 An almost fully modified homo-thymine xylo-
DNA was shown to form a complex with com-
plementary RNA of similar thermal stability as the
reference duplex.17 Methylene-extended xylo-DNA
(monomer B, Fig. 1) has been synthesized and was
found to induce limited, but significant, destabilization
when hybridized towards DNA complements.19 Nota-
bly, monomers A and B were both found to induce
protection against 30-exonucleolytic degradation.12,19
Whereas the introduction of a few xylo-LNA monomers
(monomer C, Fig. 1) into a homo-thymine DNA strand
had a very negative influence on the hybridization
properties, an almost fully modified homo-thymine
xylo-LNA displayed high-affinity recognition of both
complementary DNA and RNA.20-22 Due to the meth-
ylene linkage between the O20 and the C40 atoms of
xylo-LNA monomers these are effectively locked in an
N-type furanose conformation.
catalyst afforded the protected anomeric nucleosides
which were separated by column chromatography to
give the a-anomer 4a and the b-anomer 4b in yields of
24 and 49% respectively. Nucleosides 4a and 4b were
deprotected using saturated methanolic ammonia to
afford the a-anomer 5a and the b-anomer 5b26 in yields
of 75% and 83%, respectively. The site of attachment of
fluorine at C2(0) was confirmed by the large geminal
2
0
coupling constants JH2( ),F of ca. 48-51 Hz for com-
pounds 2–5 and the large geminal coupling constants
JC2( ),F of ca. 184–191 Hz (13C NMR).27,28 The
1
0
anomeric configurations of 5a and 5b were assigned by
1H NMR. Thus, firm evidence for the b-configuration
of 5b was obtained from the appearance of the signal of
H10 (d 6.02) as a doublet with a large coupling constant
(3JH1 ,F=21.4 Hz). In contrast, the signal for H10 in the
0
spectrum of 5a appeared as doublet of doublets with a
larger coupling constant between the H10 and H20 pro-
tons (3JH1 ,H2 =3.3 Hz). The lack of a significant cou-
pling between protons H10 and H20 in the case of 5b is
characteristic of a trans-10,20-configuration in furanose
derivatives,29 and hence of b-configuration of com-
pound 5b. It should be noted that in the spectrum of
5a, the signal for H40 is shifted ꢁ0.4 ppm down field
in comparison with the corresponding signal in the
spectrum of 5b which further supports the anomeric
assignments.30
0
0
In order to further study and understand the influence of
preorganization of xylo-configured monomers (XNA-
monomers23) on the hybridization towards RNA and
DNA complements, we herein describe synthesis, binding
properties and binding modes of various XNAs,23 i.e.,
xylo-DNA12ꢀ17 (monomer A, Fig. 1), the novel con-
formationally restricted 20-fluoro-xylo-DNA (monomer
D, Fig. 1), and the novel conformationally locked
20-amino-xylo-LNA (monomer E, Fig. 1). We speculated
that the introduction of an electronegative 20-fluoro sub-
stituent at the 20-position of xylo-DNA monomers could
increase the population of the N-type furanose con-
formation, and that the presence of the secondary amine
functionality in 20-amino-xylo-LNA would be of potential
interest as a conjugation site and as a protonation site.
Using standard transformations in satisfactory yields,
nucleoside 5b was, via the DMT (4,40-dimethoxytrityl)
protected nucleoside 6, converted into the desired phos-
phoramidite derivative 731 (Scheme 1) which was used
for incorporation of monomer FxT (Fig. 1, monomer D,
Base=thymin-1-yl) into XNAs (Table 1).
Synthesis of 1-(2-amino-2-deoxy-2-N,4-C-methylene-2-
N-trifluoroacetyl-b-d-xylofuranosyl)thymine (19) and
the corresponding 20-amino-xylo-LNA phosphoramidite
building block 21 starting from nucleoside 8 was carried
out in thirteen steps in an overall yield of 5.2% (Scheme 2).
The novel nucleoside 8 was prepared from 3-O-benzyl-
1,2-di-O-acetyl-4-C-mesyloxymethyl-5-O-mesyl-l-threo-
Results and Discussion
Synthesis of XNA monomers
Phosphoramidite monomers were used on an auto-
mated DNA synthesizer for the synthesis of all XNAs
studied (ON2–ON7 and ON9–ON15; Table 1). The
known xylo-DNA phosphoramidite building block 1
(Scheme 1) was prepared from thymidine essentially as
described12,24 and used for incorporation of monomer
dxT (Fig. 1, monomer A, Base=thymin-1-yl).
Synthesis of 1-(2-deoxy-2-fluoro-b-d-xylofuranosyl)-
thymine (5b) and the corresponding phosphoramidite
building block 7 was performed as depicted in Scheme 1.
Methyl 2-deoxy-3,5-di-O-benzoyl-2-fluoro-b-d-xylofur-
anoside (2) was obtained from d-xylose as described25
and was then by acetolysis converted into 1-O-acetyl
furanose 3 in excellent yield. Condensation of com-
pound 3 with silylated thymine using TMS-triflate as
Scheme 1. Reagents and conditions: (i) AcOH/Ac2O (3.8/1, v/v),
concd H2SO4, 75 min, 97%; (ii) persilylated thymine, TMS–triflate,
1,2-dichloroethane, reflux, 3 h; (iii) satd. NH3 in MeOH, 20 h; (iv)
DMTCl, pyridine, 20 h, 90%; (v) NCCH2CH2OP(Cl)N(i-Pr)2, (i-
Pr)2NEt, CH2Cl2, 30 min, 81%.

N. E. Poopeiko et al. / Bioorg. Med. Chem. Lett. 13(2003) 2285–2290
2287
Table 1. XNAs synthesized and hybridization dataa
give nucleoside 10 which by treatment with DBU effi-
ciently was converted into the 2,20-anhydro nucleoside
11 in 89% yield. Opening of intermediate 11 with
retention of configuration at C20 by refluxing in a mix-
ture of aqueous sulfuric acid (0.4 M) and acetone (1/1,
v/v) resulted in near quantitative formation of the ery-
thro-configurated nucleoside 12. Triflylation of 12
proved troublesome and the optimal result was achieved
with 1.1 equiv of Tf2O, 10 equiv of pyridine and 4 equiv
of DMAP in dichloromethane. However, triflate 13
could not be separated from an unidentified byproduct,
and this mixture was reacted with sodium azide in DMF
for 18 h furnishing C20-inverted 20-azido-20-deoxy-
nucleoside 14 in 65% yield (from 12). The reduction of
the azido group was carried out using a modified Stau-
dinger reaction with trimethylphosphine in a mixture of
THF and aqueous NaOH affording the bicyclic nucleo-
side 15 in 81% yield. Demesylation of 15 was carried
out by a substitution reaction using sodium benzoate
and 15-crown-5 in DMF at 120 ꢂC under high dilution
conditions. Benzoate 16 could not be separated from 15-
crown-5 and therefore the mixture was subjected to
debenzoylation affording nucleoside 17 in 48% yield
(two steps). Protection of the secondary amino group
went smoothly using ethyl trifluoroacetate and DMAP,
ON1: 50-d(GTGATATGC)
ON2: 50-d(GTGAdxTATGC)
ON3: 50-d(GTGAFxTATGC)
ON4: 50-d(GTGANHxTLATGC)
ON5: 50-d(GdxTGAdxTAdxTGC)
ON6: 50-d(GNHxTLGANHxTLANHxTLGC)
ON7: 50-d(GꢀLTLGANHxTLAꢀLTLGC)
ON8: 50-T14
ON9: 50-T7dxTT6
ON10: 50-T7FxTT6
ON11: 50-T7NHxTLT6
ON12: 50-(dxT)13
ON13: 50-(FxT)13
T
T
ON14: 50-(dxT)2(FxT)(dxT)3(FxT)(dxT)3(FxT)(dxT)2T
ON15: 50-(dxT)3[(dxT)(NHxTL)]4(dxT)2T
Hybridization data- ÁTm values relative to reference ON1/ON8
Complementary DNA
Complementary RNA
ON1b
XNA
28 ꢂC/30 ꢂC/31 ꢂCb
26 ꢂC/27 ꢂC/29 ꢂCb
DTm valuesb
DTm valuesb
ON2
ON3
ON4
ON5
ON6
ON7
ꢀ6 ꢂC
ꢀ5 ꢂC
ꢀ3 ꢂC
No Tm
No Tm
ꢀ3 ꢂC
ꢀ1 ꢂC
+1 ꢂC
+1 ꢂC
No Tm
ꢀ10 ꢂC
+8 ꢂC
ON8b
XNA
30 ꢂC/33 ꢂC b
DTm valuesb
29 ꢂC/30 ꢂCb
DTm valuesb
ON9
ꢀ10 ꢂC
ꢀ10 ꢂC
ꢀ7 ꢂC
ꢀ4 ꢂC
ꢀ4 ꢂC
ꢀ3 ꢂC
+9 ꢂC
+7 ꢂC
+9 ꢂC
+16 ꢂC
ON10
ON11
ON12
ON13
ON14
ON15
+/ꢀ0 ꢂC
+3 ꢂC
+1 ꢂC
+9 ꢂC
aMelting temperatures (Tm values) were obtained from the maxima of
the first derivatives of the melting curves (A260 versus temperature)
recorded in medium salt buffer (10 mM sodium phosphate, 100 mM
sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5 mM concentra-
tions of the two complementary strands (assuming identical extinction
coefficients for all modified and unmodified oligonucleotides). A, C, G
and T are DNA monomers, dxT=thymin-1-yl xylo-DNA monomer,
FxT=thymin-1-yl 20-fluoro-xylo-DNA monomer, NHxTL=thymin-1-
yl 20-amino-xylo-LNA monomer, aLTL=thymin-1-yl a-L-LNA
monomer; ‘no Tm’ indicates the absence of a cooperative transition
above 5 ꢂC.
bShown are changes in Tm values (ÁTm values) compared with the
reference Tm values obtained for the reference oligonucleotides ON1
and ON8 (reference Tm values from different experimental series are
given). All transitions were significant and monophasic, and no tran-
sitions were detected in experiments conducted without com-
plementary strands.
pentofuranose32 by condensation with silylated thymine
using TMS–triflate as catalyst (90% yield). Deacetyla-
tion of 8 using saturated ammonia in MeOH produced
nucleoside 9 in an excellent yield of 93%. In order to
obtain the (20R)-20-azido configured nucleoside 14 a
double inversion strategy was applied. The first inver-
sion was carried out by mesylation of 9 in 86% yield to
Scheme 2. Reagents and conditions: (i) satd. NH3 in MeOH, rt (93%);
(ii) MsCl, pyridine, rt (86%); (iii) DBU, CH3CN (89%); (iv) 0.4 M aq
H2SO4/acetone (1/1, v/v), reflux (98%); (v) Tf2O, pyridine, DMAP,
CH2Cl2, 0 ꢂC; (vi) NaN3, DMF, rt (65%, 2 steps); (vii) P(CH3)3, 2 M
aq NaOH, THF, rt (81%); (viii) BzONa, 15-crown-5, DMF, 120 ꢂC;
(ix) satd. NH3 in MeOH, rt (48%, 2 steps); (x) CF3CO2Et, DMAP, rt
(93%); (xi) 10% Pd/C, H2, rt (96%); (xii) DMTCl, pyridine, rt (94%);
(xiii) NC(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2NEt, CH2Cl2, rt (35%).

2288
N. E. Poopeiko et al. / Bioorg. Med. Chem. Lett. 13(2003) 2285–2290
producing 18 in 93% yield, which was followed by a
high-yielding debenzylation reaction affording diol
19.33 The results of an NOE experiment of diol 19
support the configuration assigned to the prepared
nucleosides (mutual NOE effects between H10 and the
H500 protons (1%/1%), between H30 and the H500 pro-
tons (2%/1%) and between the H6 proton of the thy-
mine moiety and H50 (3%/<1%) were observed).
Standard 50-O-DMT protection (94% yield of 20) fol-
lowed by 30-O-phosphitylation furnished the desired
phosphoramidite building block 2134 (35% yield) sui-
table for incorporation of 20-amino-xylo-LNA mono-
mer NHxTL (Fig. 1, monomer E, Base=thymin-1-yl)
into XNAs (Table 1).
Hybridization properties of XNA towards DNA comple-
ments
The strongly destabilizing effect of incorporating a few
isolated xylo-DNA monomers into a DNA strand with
regard to hybridization towards DNA complements is
confirmed in the present study [monomer dxT;12ꢀ16 ON2
and ON5 (relative to ON1) and ON9 (relative to ON8)].
Likewise, incorporation of one or three 20-fluoro-xylo-
DNA monomers (FxT) or 20-amino-xylo-LNA mono-
mers (NHxTL) leads to decreased affinity towards DNA
with a tendency towards less detrimental effect for the
conformationally locked 20-amino-xylo-LNA monomer.
The (almost) fully modified homo-thymine XNAs
ON12–ON15 display Tm values in the same range as
that of the reference ON8 with a moderate affinity-
enhancing effect of the 20-amino-xylo-LNA monomer
NHxTL. Notably, it is possible to combine different
XNA monomers and obtain satisfactory results as
shown for the chimeric XNAs ON14 and ON15.
Conformational restriction of 20-deoxy-20-fluoro mono-
mer xT relative to 20-deoxy monomer xT was verifie0d
F
d
1
by comparing H NMRdata for the corresponding 5 -
O-DMT protected nucleosides. Thus, whereas the signal
of the H10 proton for 1-(2-deoxy-5-O-(4,40-dimethoxy-
trityl)-b-d-xylofuranosyl)thymine appears as a double
doublet (3JH1 ,H2 =2.7 and 6.2 Hz), the signal of the
12
0
0
Hybridization properties of XNA towards RNA comple-
ments
H10 proton of 1-(2-deoxy-2-fluoro-5-O-(4,40-dimethoxy-
trityl)-b-d-xylofuranosyl)thymine (6) appears as
a
doublet (3JH1 ,F=21.9 Hz) indicating strong N-type
conformational restriction35,36 of the furanose ring of 6,
Direct comparison between the Tm values obtained
towards the DNA and RNA complements clearly
demonstrates the RNA selective hybridization of XNA.
Remarkably, taking the different configurations of the
DNA and XNA monomers into consideration, the ste-
reoirregular XNAs ON2–ON4 display unchanged
RNA-binding relative to reference ON1.
0
and accordingly also xT. For the 20-amino-xylo-LNA
F
nucleosides (including monomer NHxTL) having the
bicyclo[2.2.1]heptane constitution, a locked N-type fur-
anose conformation was validated by the appearance of
the signal for H10 in the H NMRspectrum of nucleo-
1
side 1933 as a singlet as has been reported also for other
LNA derivatives.5ꢀ7
Incorporation of three XNA monomers (ON5 and
ON6) abolishes, or significantly reduces, the affinity
towards RNA, but by combining a-l-LNA, DNA and
20-amino-xylo-LNA monomers (ON7), high-affinity
recognition of RNA is achieved. Notably, by combining
these three differently configured monomers a positive
effect only on RNA binding affinity is induced (compare
ON4 and ON7). The preferential RNA hybridization of
XNA is substantiated by the significantly increased
thermal stabilities obtained with the (almost) fully
modified homo-thymine XNAs ON12–ON15 with the
highest Tm value achieved for ON15 containing 20-
amino-xylo-LNA monomers. The ÁTm value obtained
for ON12 (+9 ꢂC) is higher than the ÁTm value repor-
ted for an analogous homo-thymine XNA [(dxT)12T]
(+3 ꢂC).17 The use of slightly different hybridization
conditions is a likely explanation for this difference.
Synthesis of XNA oligomers
The reference DNA strands ON1 and ON8 and the
XNAs ON2–ON7 and ON9–ON15 (Table 1) were pre-
pared on an automated DNA synthesizer using the
phosphoramidite approach (0.2 mmol scale, CPG solid
supports).37 The xylo-configured phosphoramidites 1, 7
and 21, the known a-L-LNA thymine amidite21,38 (used
for incorporation of monomer aLTL into ON7) and
commercially available DNA phosphoramidites were
applied. The coupling yields were >99% for DNA
phosphoramidites (using 2 min coupling time and 1H-
tetrazole as catalyst), ꢁ99% for phosphoramidite 1,
89–96% for phosphoramidite 7, and 99% for phos-
phoramidite 21. Extended coupling time (10–30 min)
and a modified procedure with pyridine hydrochloride
as catalyst21 were used for the three xylo-configured
phosphoramidites 1, 7 and 21. The satisfactory coupling
yields obtained for these xylo-configured phosphor-
amidites are noteworthy as low coupling yields for xylo-
configured phosphoramidites led Seela et al. to use the
alternative H-phosphonate method for automated
synthesis of xylo-DNA.12ꢀ14,16 After standard depro-
tection and cleavage from the solid support using 32%
aqueous ammonia (16 h, 55 ꢂC), the oligomers were
prepared for use by desalting. The purity of all oligo-
mers (>80%) was verified by capillary gel electrophor-
esis and the composition of representative oligomers by
MALDI-MS analysis.39
Hybridization characteristics of XNA
The thermal denaturation studies show that conforma-
tional restriction of XNA monomers leads to improved
binding affinity as most clearly observed for XNAs
ON4, ON11 and ON15 containing the locked 20-amino-
xylo-LNA type monomer NHxTL (compare with XNAs
ON2, ON9 and ON12, respectively, containing xylo-
d
DNA monomer xT). Importantly, different monomeric
nucleotides, including different XNA type monomers, can
be combined allowing fine-tuning of the hybridization
properties of XNA (see ON7, ON14 and ON15). In melt-
ing experiments with one mis-match centrally positioned

N. E. Poopeiko et al. / Bioorg. Med. Chem. Lett. 13(2003) 2285–2290
2289
in the complementary DNA/RNA, XNA-mediated
hybridization (ON2, ON3, ON4 and ON7) was found to
follow the Watson–Crick base pairing rules (data not
shown).
xylo-LNA and a known xylo-DNA monomer have been
synthesized. Thermal denaturation studies revealed pre-
ferential hybridization towards RNA complements for
9-mer mixed-base XNAs and 14-mer homo-pyrimidine
XNAs, composed of a mixture of XNA and DNA
monomers (and a-l-LNA monomers for ON7). Stable
complexes towards single-stranded DNA and RNA
targets were formed at pH 7 for 14-mer homo-thymine
XNAs containing 13 XNA monomers. Gel-shift experi-
ments revealed these complexes to involve at least two
XNA strands per target strand. The binding affinity of
XNA can be tuned by combining different types of
nucleotide monomers and conformational restriction of
XNA monomers clearly leads to improved binding. We
are currently exploring the binding mode of homo-pyr-
imidine XNA and optimizing the hybridization proper-
ties of XNA.
For the mixed-sequence 9-mer XNAs containing a mix-
ture of XNA and DNA monomers, hybridization is
assumed to take place via duplex formation. However,
as triplex formation between homo-pyrimidine XNA
(xylo-DNA; mixed T/C sequences) and single-stranded
DNA complements has earlier been demonstrated,40 we
decided to investigate the binding mode of XNAs
ON12, ON13 and ON15 towards single-stranded DNA
(dA14) and RNA (rA14) complements (Fig. 2).41
As expected, the complex formed between ON8 and
dA14 is a duplex (Fig. 2, upper right panel; ON8 and
rA14 are not shown because of co-migration of single
strands and complex). In accordance with earlier stud-
ies,40 the gel mobilities shown in Figure 2 reveal that
two XNA strands per dA14 target strand are involved in
complex formation both for ON12 and ON15 (and also
for ON13, not shown). It should be noted that analo-
gous hybridization behavior has been demonstrated for
other nucleic acid analogues, for example, peptide
nucleic acid.42
Acknowledgements
The Danish National Research Foundation and the
Danish Research Agency are thanked for financial sup-
port. Ms. Britta M. Dahl, University of Copenhagen, is
thanked for oligonucleotide synthesis and Dr. Michael
Meldgaard, Exiqon A/S, for MALDI-MS analyses.
With the RNA target rA14 the results indicate the for-
mation of a mixture of complexes, including one or
several with XNA:rA14 ratio(s) ꢃ2. Further preliminary
experiments43 indeed indicate changes in the distribu-
tion among complexes during incubation for several h.
Thus, whereas complexes of higher-order structure
apparently are involved, we anticipate that hybridiza-
tion between ON12, ON13 and ON15 and rA14, as with
dA14, initially involves triplex formation.
References and Notes
1. Herdewijn, P. Liebigs Ann. 1996, 1337.
2. Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,
4429.
3. Wengel, J. Acc. Chem. Res. 1999, 32, 301.
4. Kværnø, L.; Wengel, J. Chem. Commun. 2001, 1419.
5. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455.
6. Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetra-
hedron 1998, 54, 3607.
Conclusion
7. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi,
T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
8. Braasch, D. A.; Corey, D. R. Chem. Biol. 2000, 55, 1.
9. Guschlbauer, W.; Jankowski, K. Nucleic Acid Res. 1980, 8,
1421.
Xylo-Configured oligonucleotides (XNA) containing a
novel conformationally restricted 20-fluoro-xylo-DNA
monomer, a novel conformationally locked 20-amino-
10. Olson, W. K. J. Am. Chem. Soc. 1982, 104, 278.
11. Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik,
E. A.; Zounes, M. C.; Cummins, L. L.; Gonzales, C.; Cook,
P. D. J. Med. Chem. 1993, 36, 831.
12. Rosemeyer, H.; Seela, F. Helv. Chim. Acta 1991, 74, 748.
13. Rosemeyer, H.; Krecmerova, M.; Seela, F. Helv. Chim.
Acta 1991, 74, 2054.
14. Seela, F.; Worner, K.; Rosemeyer, H. Helv. Chim. Acta
1994, 77, 883.
¨
15. Schoppe, A.; Hinz, H.-J.; Rosemeyer, H.; Seela, F. Eur. J.
¨
Biochem. 1996, 239, 33.
16. Seela, F.; Heckel, M.; Rosemeyer, H. Helv. Chim. Acta
1996, 79, 1451.
17. Alekseev, Y. I.; Gottikh, M. B.; Romanova, E. A.; Sha-
barova, Z. A. Mol. Biol. 1996, 30, 206.
18. Changes in duplex stabilities (ÁTm values) are discussed
relative to the corresponding reference duplex formed by
hybridization between two unmodified (DNA or RNA) com-
plementary strands.
19. Svendsen, M. L.; Wengel, J.; Dahl, O.; Kirpekar, F.;
Roepstorff, P. Tetrahedron 1993, 49, 11341.
Figure 2. Non denaturing gel electrophoresis of hybridization com-
plexes. ‘*’ Indicates the 50-32P labelled oligo visible on the gel auto-
radiogram. The ratios on the top of the individual lanes are the ratios
of the two strands (indicated at the bottom of each gel).41

2290
N. E. Poopeiko et al. / Bioorg. Med. Chem. Lett. 13(2003) 2285–2290
20. Rajwanshi, V. K.; Kumar, R.; Kofod-Hansen, M.; Wen-
gel, J. J. Chem. Soc., Perkin Trans. 1 1999, 1407.
33. 1-(2-Amino-2-deoxy-2-N,4-C-methylene-2-N-trifluoroacetyl-
b-d-xylofuranosyl)thymine (19) [major rotamer (rotamer ratio
1
21. Rajwanshi, V. K.; Ha
gel, J. Chem. Commun. 1999, 1395.
22. Rajwanshi, V. K.; Hakansson, A. E.; Kumar, R.; Wengel,
˚
kansson, A. E.; Dahl, B. M.; Wen-
2:3)]: H NMR(400 MHz, ((CD ) SO) d 11.25 (bs, 1H, NH),
3 2
7.53 (d, J=1.3 Hz, 1H, H-6), 5.98 (bs, 1H, 30-OH), 5.59 (s,
1H, H-10), 5.14 (t, J=5.7 Hz, 1H, 50-OH), 4.65 (br s, 1H,
H-20), 4.18 (t, J=2.4 Hz, 1H, H-30), 3.92–3.84 (m, 2H, H-50),
3.68 (d, J=11.9 Hz, 1H, H-500), 3.42 (d, J=11.9, 1H, H-500),
1.76 (s, 3H, CH3); 13C NMR(100.6 MHz, ((CD 3)2SO)) d
164.10, 154.18 (q, J=37.4 Hz), 150.02, 136.78, 115.75 (q,
J=287.3 Hz), 106.13, 89.22, 87.88, 72.30, 60.82, 57.12, 53.19,
12.50.
˚
J. Chem. Commun. 1999, 2073.
23. The term ‘XNA’ is defined herein as an oligonucleotide
containing one or more xylo-configured nucleotide monomers
(‘XNA-monomer(s)’).
24. Kong, J. P.; Kim, S. K.; Moon, B. J.; Kim, S. J.; Kim,
H. B. Nucleosides Nucleotides 2001, 20, 1751. We used sodium
hydroxide instead of lithium hydroxide during reaction on the
30-O-mesylated intermediate.
34. 1-(2-Amino-3-O-((2-cyanoethoxy)(N,N-diisopropylamino)-
phosphino)-2-deoxy-5-O-(4,40-dimethoxytrityl)-2-N,4-C-meth-
ylene-2-N-trifluoroacetyl-b-d-xylofuranosyl)thymine (21): 31P
25. Wright, J. A.; Wilson, D. P.; Fox, J. J. J. Med. Chem.
1970, 13, 269.
NMR(400 MHz, CH CN) d 154.9, 154.2, 152.5, 152.3.
3
26. 1-(2-Deoxy-2-fluoro-b-d-xylofuranosyl)thymine (5b): 1H
NMR(300 MHz, CD 3OD) d 7.71 (s, 1H, H-6), 6.02 (d, 1H,
J=21.4 Hz, H-10), 4.95 (d, 1H, J=49.4 Hz, H-20), 4.35 (dd,
1H, J=3.3 and 10.4 Hz, H-30), 4.26–4.21 (m, 1H, H-40), 4.00–
3.86 (m, 2H, H-50), 1.86 (br s, 3H, CH3); 13C NMR
(75.5 MHz, CD3OD; selected signals) d 138.5, 111.0, 100.7
(J=184.9), 90.4 (J=38.4), 85.0, 73.8 (J=25.8), 60.7, 12.6.
27. Michalik, M.; Hein, M.; Michael, F. Carbohydr. Res.
2000, 327, 185.
28. Kowollik, G.; Langen, P. Z. Chem. 1975, 15, 147.
29. Lemieux, R. U.; Lineback, D. R. Ann. Rev. Biochem.
1963, 32, 155.
30. Mikhailopulo, I. A.; Poopeiko, N. E.; Pricota, T. I.;
Sivets, G. G.; Kvasyuk, E. I.; Balzarini, J.; De Clercq, E. J.
Med. Chem. 1991, 34, 2195 and references cited therein.
31. 1-(3-O-((2-Cyanoethoxy)(N,N-diisopropylamino)phosphino)
-2-deoxy-5-O-(4,40 -dimethoxytrityl)-2-fluoro-b-d -xylofur-
anosyl)thymine (7): 31P NMR(300MHz, CH 3CN) d 153.8 and
151.2.
35. Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972,
94, 8205.
36. Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973,
95, 2333.
37. Caruthers, M. H. Acc. Chem. Res. 1991, 51, 2155.
˚
38. Hakansson, A. E.; Koshkin, A. A.; Sørensen, M. D.;
Wengel, J. J. Org. Chem. 2000, 65, 5161.
39. MALDI-MS m/z ([M-H]ꢀ; found/calcd) ON4, 2776/2778;
ON6, 2833/2833; ON7, 2834/2834; ON9, 4191/4197; ON10,
4210/4215; ON11, 4220/4221; ON12, 4191/4197; ON14, 4247/
4251; ON15, 4300/4302.
40. Ivanov, S. A.; Alekseev, Y. I.; Gottikh, M. B. Mol. Biol.
2002, 36, 131.
41. Hybridization was performed in 100 mM NaCl, 10 mM
KCl, 20 mM Tris–HCl (pH 7.5) by heating to 65 ꢂC, slowly
cooling after 2 min to 37 ꢂC followed by incubation at 5 ꢂC for
1–2 h. The resulting complexes were then analyzed on 20%
acrylamide gels run at approximately 14 ꢂC.
42. Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H.
J. Am. Chem. Soc. 1992, 114, 1895.
43. Poopeiko, N. E.; Juhl, M.; Vester, B.; Wengel, J.
Manuscript in preparation.
32. Sørensen, M. D.; Kværnø, L.; Bryld, T.; Hakansson, A. E.;
˚
Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am.
Chem. Soc. 2002, 124, 2164.