Conformation-specific cleavage of antisense oligonucleotide-RNA duplexes by RNase H

Article abstract of DOI:10.1039/b006476i

The North-form (3'-endo) constrained 1-(1',3'-O-anhydro-β-D-psicofuranosyl)thymine block, <*>, was systematically incorporated at various sites, one at a time, into a set of four antisense oligonucleotides (AONs). The hybrids of these AONs with a matched

Full text of DOI:10.1039/b006476i

View Article Online / Journal Homepage / Table of Contents for this issue
Conformation-specific cleavage of antisense oligonucleotide-RNA
duplexes by RNase H
Pushpangadan I. Pradeepkumar, Edouard Zamaratski, András Földesi and
Jyoti Chattopadhyaya*
2
Department of Bioorganic Chemistry, Box 581, Biomedical Center, S-75123 Uppsala,
University of Uppsala, Sweden. E-mail: jyoti@bioorgchem.uu.se; Fax: ϩ4618-554495;
Tel: ϩ4618-4714577
Received (in Cambridge, UK) 8th August 2000, Accepted 12th December 2000
First published as an Advance Article on the web 5th February 2001
The North-form (3Ј-endo) constrained 1-(1Ј,3Ј-O-anhydro-β--psicofuranosyl)thymine block, T, was systematically
incorporated at various sites, one at a time, into a set of four antisense oligonucleotides (AONs). The hybrids of these
AONs with a matched 15mer RNA target were subjected to the RNase H cleavage reaction, and compared with that
of the native counterpart, in order to probe how far the local influence of a single North-locked sugar is transmitted
in steering conformational changes in the neighbouring nucleotides. It was found that the introduction of a single
North-sugar locked T nucleotide in the AONs makes up to four of the neighbouring nucleotides at the 5Ј-end of the
modification site resistant to the RNase H cleavage reaction. This suggests that a stretch of 5-nucleotides, including
the T nucleotide, in the AON strand adopts a North-type conformation, giving a local RNA/RNA type hybrid
structure instead of a regular DNA/RNA type duplex structure. Although these 5-nucleotide regions were completely
resistant to RNase H promoted hydrolysis, they could serve as the binding site for the enzyme. Interestingly, none
of these local adaptations of the RNA/RNA type structure were observable by CD spectroscopy, showing it to be
an unsuitable means of monitoring any subtle alteration of the local structure. This work, therefore, constitutes an
example of how the engineered conformation of a substrate can be used to exploit the stereochemical sensitivity of
an enzyme to map local microscopic conformational changes. The other implication of this work is that it provides
a new tool to gather local structural information, which may help to optimize the number of constrained residues
which need to be incorporated to induce the antisense strand to adopt either A- or B-type geometry in the hybrid
duplex, with or without the loss of RNase H recognition and/or cleavage properties.
destabilizing effect was observed. It was later found that
the introduction of multiple (N)-methanocarbathymidines,
Introduction
although increasing the thermodynamic stability of the AON/
The recruitment of RNase H, an endogenous enzyme that
specifically degrades the target RNA part of DNA/RNA hybrid
duplexes, is an important pathway for antisense action, besides
translational arrest.1 To elicit the RNase H activity, the
antisense oligonucleotide (AON) part of the AON/RNA hybrid
should retain the B-type DNA conformation with a 2Ј-endo
sugar (South-type, S), while the RNA moiety should retain its
A-type helix character with a 3Ј-endo sugar (North-type, N).2
To fulfill these requirements, various modifications of the sugar
and nucleobase, as well as of the phosphate backbone, have
been investigated, and numerous reports are available about
these modified AONs and their antisense action.3 The AONs
with one or more conformationally N-4 or S-5 form locked
nucleoside residues have also been an area of considerable
interest. The N-form constrained nucleoside-containing AONs
have been, however, found to be most promising because they
exhibit high affinity to the target RNA.6 Recently, the locked
nucleic acid (LNA, in which the sugar moiety is locked in
the North conformation) has shown unprecedented affinity
towards RNA.4e,h,j LNA (and other modifications which have
the fixed North-sugar moiety) drive the AON helix to the A-
type, resulting in an RNA/RNA-type duplex, which accounts
for their higher binding affinity; this, however, leads to the loss
of RNase H activity.7 The introduction of the conformation-
ally constrained (N)-methanocarbathymidine (4Ј,4Јa-methano-
4Јa-carbathymidine) residue in the North-form4a has been
shown to increase the thermodynamic stability of the
AON/RNA duplex, whereas with (S)-methanocarbathymidine
RNA duplex, failed to recruit any RNase H activity.4c
It is now quite clear6a,7 that all modifications that lead prefer-
entially to a North-type sugar, including its constrained form,
in an RNA-type AON result in the loss of RNase H activity,
because they resemble the RNA/RNA duplex, except when they
appear at the termini or in the middle in the gapmer-AON.8
It has been so far assumed that probably three or four
North-type conformational repeats are necessary to enhance the
thermal stability of an RNA-type AON/RNA duplex.6a Clearly,
the introduction of a few North-type constrained nucleosides in
an AON is perhaps necessary to enhance the binding specificity
to the target RNA, yet their numbers have to be somehow
restricted in order to elicit an RNase H response. At this time,
however, nobody knows where the balance lies between
accomplishing tighter binding and target specificity to the
RNA and loosing the conformational tolerance of the RNase
H recognition and its substrate specificity, owing to the local
structural perturbances in an RNA-type AON/RNA hybrid.
We show here, by a combination of CD spectroscopy and
RNase H degradation studies on a set of hybrid AON/RNA
duplexes (consisting of a series of analogous single bicyclic
psiconucleoside-modified AONs (3)–(6) with fixed North-sugar
conformation and the complementary target RNA (7)), that the
local structure perturbance upon introduction of a single North-
type constrained nucleoside spreads up to 4 nucleotides toward the
5Ј-end of the modification site, producing neither any global
helical conformational change nor reducing any overall RNase
H activity compared to the native hybrid duplex: [(2) ϩ (7)].
(1Ј,4Јa-methano-4Јa-carbathymidine) in the South-form,5a
a
402
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b006476i

View Article Online
Table 1 The Tms and thermodynamic parameters of the duplexes formed by the RNA target (7) and the AONs (2)–(6)
AONs (2)–(6)/
RNA (7) hybrids
Ϫ∆HЊ/
ϪT∆SЊ/
Ϫ∆GЊ298
/
∆∆GЊ298
/
Tm/ЊC
∆Tm/ЊC
kJ molϪ1
Ϫ∆SЊ/e.u.
kJ molϪ1
kJ molϪ1
kJ molϪ1
Native AON (2)
AON (3)
44.5
37.7
39.5
39.7
39.3
494.3 ( 32.4)
413.1 ( 39.7)
501.6 ( 27.6)
465.2 ( 14.5)
437.9 ( 44.3)
1.44 ( 0.10)
1.21 ( 0.13)
1.48 ( 0.09)
1.37 ( 0.05)
1.28 ( 0.14)
429.1
359.9
442.1
408.2
381.4
65.2
53.2
59.5
57.0
56.5
Ϫ6.8
Ϫ5
Ϫ4.8
Ϫ5.2
12.0
5.7
8.2
8.7
AON (4)
AON (5)
AON (6)
Scheme 1 Reagents and conditions: (i) 4-toluoyl chloride, pyridine, rt, overnight; (ii) silylated base, TMSOTf, acetonitrile, 4 ЊC, 1 h, rt, 18 h;
(iii) MsCl, pyridine, 4 ЊC, overnight; (iv) 90% aq. CF3COOH, rt, 20 min.; (v) NaH, DMF, 4 ЊC, 9 h; (vi) methanolic NH3, rt, 2 d; (vii) DMTrCl,
pyridine, rt, overnight; (viii) 2-cyanoethyl-N,N-diisopropylchlorophosphonamidite, N,N-diisopropylethylamine, acetonitrile, rt, 2 h.
Fig. 1 Sequences of the AONs containing the conformationally restricted nucleoside T, their native counterpart and their RNA target.
(2)–(6) (CAUTION: opening of the oxetane ring in T takes
place upon ammonia treatment at 55 ЊC).
Results and discussion
We have synthesized a bicyclicpsiconucleoside, 1-(1Ј,3Ј-O-
anhydro-β--psicofuranosyl)thymine (compound 1 in Scheme
1), in which the sugar is fixed in the 3Ј-endo conformation
(J4Ј,5Ј = 8.4 Hz).9 This 1Ј,2Ј-oxymethylene bridged nucleoside
has been systematically incorporated at different sites (Fig. 1) in
a set of test antisense sequences [AON (3)–(6)] targeted to the
coding region [oligo-RNA (7)] of the SV-40 large T antigen.10
All oligodeoxynucleotides have been prepared by the phos-
phonamidite approach11 on an automated DNA/RNA syn-
thesizer and deprotected at room temperature to give the AONs
The thermal meltings (Tms) and the thermodyanamic prop-
erties12 of the native and modified-AON/RNA hybrid duplexes
are shown in Table 1. It can be seen that the Tms of the
modified-AON (3)–(6)/RNA (7) hybrid duplexes are 4.8–6.8 ЊC
lower than the native counterpart, (2) ϩ (7). Interestingly, the
Tm differences of the AONs (4)–(6)/RNA (7), having modifi-
cations around the middle are within 5 0.2 ЊC whereas modi-
fication at the 3Ј-terminus, in AON (3), results in a Tm reduction
of 6.8 ЊC. It is noteworthy that a slight gain in enthalpy is well
compensated by a loss of entropy owing to the restricted
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408
403

View Article Online
Fig. 3 RNase H hydrolysis of the target RNA (7) when hybridized to
Fig. 2 CD spectra of the duplexes formed by the AONs (2)–(6) and
the RNA target (7). For comparison, typical B-type and A-type spectra
are presented: DNA/DNA duplex formed by AON (2) and comple-
mentary DNA: 5Ј-d(GAAGAAAAAATGAA)-3Ј, and RNA/RNA
duplex formed by self-complementary 17mer RNA: 5Ј-r(UAACAU-
GUUUGGACUCU)-3Ј.
the AONs (2) (native) and (4): Lane 1 represents the snake venom
phosphodiesterase ladder of the target RNA; Lanes 2, 3 and 4 corre-
spond to the cleavage reaction promoted by the native AON (2), after
30, 60 and 120 min, respectively; Lanes 5, 6 and 7 correspond to the
cleavage reaction promoted by the AON (4), after 30, 60 and 120 min,
respectively.
motion of the constrained T block in the hybrid duplexes of the
AONs (3)–(6) and the target RNA, resulting in an overall loss
of free energy (7–12 kJ molϪ1) of stabilization (Table 1) relative
to the native AON (2)/RNA (7) duplex at 298 K. This may
suggest that the 1Ј,2Ј-oxymethylene bridged nucleoside moiety
(1) has introduced a local conformational heterogeneity6a in the
AON/RNA hybrid structure (vide infra) (Note: a mismatch in a
DNA/RNA duplex normally results in a 12–18 ЊC loss of Tm).4e
Fig. 2 shows superimpositions of the CD spectra of all four
single modified AONs (3)–(6) along with the native AON (2)/
RNA, typical RNA/RNA and DNA/DNA duplexes.13 All
the modified AON/RNA hybrid duplexes (3)–(6) exhibited CD
spectra intermediate between those of the native A-type RNA/
RNA and B-type DNA/DNA duplexes, mimicking those of
the natural AON (2)/RNA hybrid duplex. The spectra of the
modified AONs (3)–(6)/RNA and the native AON/RNA hybrid
duplexes had a positive band at 263–267 nm, a negative band at
240–245 nm and a crossover point at 247–250 nm. These data
suggested that the single modification, the incorporation of the
conformationally constrained T nucleotide in various sites of
the AONs (3)–(6), did not alter the global helical conformation
of the resulting hybrid compared to the native DNA/RNA
counterpart. This is also an indication of the potential ability of
the AON (3)–(6)/RNA hybrids to recruit RNase H (see below)
as does the native structure.
Fig. 4 RNase H hydrolysis of the target RNA (7) when hybridized to
the AONs (3), (5) and (6): Lane 1 represents the snake venom phos-
phodiesterase ladder of the target RNA; Lanes 2, 3 and 4 correspond to
the cleavage reaction promoted by the AON (5), after 30, 60 and 120
min, respectively; Lanes 5, 6 and 7 correspond to the cleavage reaction
promoted by the AON (6), after 30, 60 and 120 min, respectively; Lanes
8, 9 and 10 correspond to the cleavage reaction promoted by the AON
(3), after 30, 60 and 120 min, respectively.
This also seems to indicate that there are no significant con-
formational differences on average, that affect base stacking in
the AON (3)–(6)/RNA hybrids. If a single nucleotide is N-type,
then the resultant spectral change would indeed be small, but
if 5 nucleotides became N-type, one might well expect a larger
difference in the CD spectrum, towards the A-form. On the
other hand, it is not clear how much the sugar conformation
has to be altered to the N-type, or near N-type, in all four
nucleotides next to the constrained T to alter the base stacking
and helicity of the hybrid duplex. So the apparent absence of a
sugar-induced co-operative effect (although observable by the
RNase H assay) on the conformation of the stacked helices
shows the insensitive nature of CD (only reflecting base stacking
geometry). The lack of CD spectroscopic evidence presented,
therefore, either argues against base-stacking induced conform-
ational changes orchestrated by the sugar modification, or else
change of the sugar puckers simply does not induce any alter-
ation of the base stacking geometry, meaning that the sugar
pucker change is uncoupled from the base-stacking. It is also
possible that a subtle change of hydration characteristics (not
detectable by CD) in the minor groove might inhibit the Mg2ϩ
hydrate complex of RNase H acting on the neighboring nucleo-
tides of the T residue in the AON (3)–(6)/RNA hybrids.
To check its ability to elicit RNase H, the 5Ј-32P labeled
15mer RNA target (7) was hybridized with the complementary
AONs (2)–(6) and incubated with Escherichia coli RNase H1 at
21 ЊC. Aliquots were taken after 30, 60 and 120 min, analysed
by PAGE, and the extent of the hydrolysis was estimated from
the residual full length RNA left after a given incubation time.
The cleavage sites and the products were identified by com-
parison with the partial snake venom phosphodiesterase digest
ladder of the target RNA. Results are shown in Figs. 3–5: after
RNase digestion for 60 min, the PAGE showed that all the
modified AONs (3)–(6) were hydrolysed at similar rates com-
pared to the native counterpart (2). The target RNA digestion
404
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408

View Article Online
this hybrid, allowing cleavage of the RNA both at the A5 site as
well as the A10/U11 sites.
(iv) This kind of RNase H recognition of the structural per-
turbances was also found in the cleavage behaviour of the AON
(5)/RNA hybrid (modification opposite A8 site in RNA) where
further shift of the cleavage sites was observed. The cleavage
sites after 120 min were found to be at A5, A6, and A7. How-
ever after digestion for 30 min, cleavage at G12 and A13 was
revealed, showing that the region A13–G15 is accessible for
cleavage, which again proves that the A8–G12 region affected
by the modification is not accessible for cleavage, but can serve
as a binding site for the enzyme.
(v) As expected, the AON modification opposite A10 of
the complementary RNA, as in the AON (6), resulted in the
absence of any cleavage sites in the region A10–G15, while
regaining all the cleavage sites from A5–A9 present in the native
hybrid duplex. Interestingly, the AON (6) retains the major
cleavage sites of the native duplex, while the 5 basepairs from
A10 to A14 were insensitive to RNase H promoted cleavage.
That there was no detectable difference in the hydrolysis rates
of the native and modified AON/RNA duplexes shows that the
enzyme binds to the region affected by the modification almost
as well as to the native hybrid, although the former has both
globally and locally a DNA/RNA type structure and the latter
produces a local distortion giving an RNA/RNA type structure.
However, absolutely no cleavage of the RNA occurred within
the modified region (spanning a stretch of 5 basepairs)
although the enzyme can bind to it, which means that the struc-
tural requirements of enzyme binding and catalytic cleavage
are very different.
The above results suggest the following: (a) that the cleavage
activity of the enzyme was suppressed within a region, about 5
basepairs long, towards the 3Ј-end of the RNA in the AON/
RNA hybrid, starting from the base opposite the modified T
nucleotide in the AON strand. The neighboring sites beyond
this 5 basepair region were fully susceptible to hydrolysis by the
enzyme. (b) That the binding site and the cleavage site in the
substrate–enzyme complex are different14 and that the binding
site is at least five nucleotides away towards the 5Ј-end of the
RNA from the cleavage site. (c) It is also interesting to note that
the structural requirements for substrate binding and substrate
cleavage by RNase H appear to be different. RNase H could
bind to the A3–A7 region in the AON (3)/RNA duplex to
produce hydrolysis at A7, the cleavage at position A5 (which is
present in the native duplex) is absent.
One of the interesting aspects of this work is how the con-
formational transmission from the T residue to the neighbour-
ing nucleotides in the AONs controls the enzyme recognition
and function. The structural distortions caused by the con-
strained North-type sugar conformation of T in any of the
AONs (3)–(6) makes the neighbouring 4 basepairs at the 5Ј-end
of the AON strand (i.e., the 3Ј-end of the RNA strand) com-
pletely inactive to the catalytic cleavage reaction, although good
for enzyme binding, showing how a conformationally con-
strained nucleotide can enforce the neighbouring nucleotide
structure locally to adopt a specific RNA/RNA duplex type
conformation13 (compared to the normal B-DNA/A-RNA type
conformation). This work, therefore, is a direct example of how
the conformation of a substrate can be used to exploit the
stereochemical sensitivity of an enzyme to map local micro-
scopic conformational change. The second implication of this
work is that the above information may help to optimize the
number of constrained residues that need to be incorporated
to induce the antisense strand to adopt either A- or B-type
geometry in the hybrid duplex, with or without the loss of
RNase H recognition and/or cleavage properties.
Fig. 5 RNase H cleavage pattern of the hybrid duplexes formed by
the AONs (2)–(6) and the RNA target (7). Solid and dashed arrows
represent the major and the minor cleavage sites, respectively, at com-
plete degradation (2 h of incubation). Short arrows demonstrate the
sites accessible for cleavage at the initial reaction times (30 min of incu-
bation). Boxes represent the parts of the RNA sequence insensitive to
RNase H cleavage as the result of the AON modification.
was complete after 120 min for the native AON/RNA as well
as for the modified AON/RNA hybrids, despite the fact that
the AONs (3)–(6)/RNA (7) had 4.8–6.8 ЊC lower Tms than the
native counterpart (2).
Comparison (see Fig. 5) of the cleavage sites of the AON/
RNA hybrids with those of the native counterpart sheds light
on how local structural differences dictate the substrate
specificity, recognition and cleavage properties of degradation
by RNase H:
(i) In the native hybrid duplex, [(2) ϩ (7)], the major cleavage
sites (after 120 min) were found to be at the 3Ј-phosphate of A5
(i.e. A5–A6) and A8 (i.e. A8–A9) (shown by arrow in Fig. 5),
and the minor cleavage sites were at the 3Ј-phosphate of A6 and
A7. However after digestion for 30 min, longer 9–11mer RNA
species were detected as a result of cleavage at A9, A10 and
U11, which were later degraded further to shorter fragments.
This shows that the whole region from A5 to U11 in the RNA
of the native duplex was accessible for RNase H promoted
cleavage.
(ii) In the case of AON (3), the modification opposite A3 of
the complementary RNA, made the region A3–A7 inaccessible
for RNase H cleavage, which resulted in the loss of the cleavage
sites at A5 and A6. Instead major sites at A10 and U11
appeared, in addition to the preserved sites A7, A8 and A9.
This shift of the cleavage site shows that the A3–A7 region,
although inaccessible to the cleavage, can serve as a binding site
for the enzyme.
(iii) A modification opposite A6 of RNA in the AON (4)
resulted in complete loss of RNase H promoted cleavage in the
region A6–A10; the only sites accessible for cleavage were at
A5, A10 and U11, situated on the edges of the A6–A10 region.
This shows that there are at least two binding sites available in
Synthesis of 1-(1Ј,3Ј-O-anhydro-ꢀ-D-psicofuranosyl)thymine (1)
The title compound (1) was prepared from 1,2:3,4-di-O-
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408
405

View Article Online
isopropylidene-β--psicofuranose (8) (Scheme 1), which was
synthesized from -fructose.15 Protection of 8 with a 4-toluoyl
group gave 9, which was coupled with O,O-bis(trimethyl-
silyl)thymine, in the presence of TMSOTf as Lewis acid and
acetonitrile as solvent to furnish an (1:1) anomeric mixture of
the protected psiconucleosides 10a and 10b in 67% yield.16
These were separated by careful column chromatography and
the stereochemistry of C2Ј in 10 was confirmed by means of
NOE measurements. Methanesulfonylation of the β-anomer
10b afforded the 1Ј-mesylate 11 (98%). Deprotection of the
isopropylidine group using 90% aqueous CF3COOH yielded 12
(92%). The oxetane ring formation was achieved by treatment
of 12 with NaH in DMF at 0 ЊC for 9 h to give 13 (60%).
Removal of the 4-toluoyl group from 13 furnished the desired
1-(1Ј,3Ј-O-anhydro-β--psicofuranosyl)thymine (1),17 which
was converted into the phosphonamidite building block 15
(90%) via the 6Ј-O-(4,4Ј-dimethoxytrityl) derivative 14. The
phosphonamidite 15 was then used for incorporation of the T
residue into AONs (3)–(6).
1-[3Ј,4Ј-O-Isopropylidene-6Ј-O-(4-toluoyl)-ꢁ-D-psicofuran-
osyl]thymine (10a) and 1-[3Ј,4Ј-O-isopropylidene-6Ј-O-(4-
toluoyl)-ꢀ-D-psicofuranosyl]thymine (10b). Thymine (3.7 g, 29.6
mmol) was suspended in hexamethyldisilazane (35 ml) and
trimethylchlorosilane (5.6 ml) was added. The reaction mixture
was stirred at 120 ЊC under a nitrogen atmosphere for 16 h. The
volatile material was evaporated and the residue was kept on
an oil pump for 20 min. The sugar 9 (7.0 g, 18.5 mmol) was
dissolved in dry acetonitrile and added to the persilylated
nucleobase. The mixture was cooled to 4 ЊC and trimethylsilyl
trifuromethanesulfonate (4.3 ml, 24 mmol) was added dropwise
under a nitrogen atmosphere. After being stirred at 4 ЊC for 1 h,
the mixture was stirred at room temperature for 18 h. Saturated
NH4Cl was added to the reaction mixture and with stirring for
30 min. The organic layer was decanted and the aqueous layer
was extracted 3 times with diethyl ether. The combined organic
phase was washed first with saturated sodium bicarbonate
solution and then with brine, then dried over MgSO4, filtered
and evaporated. The resultant oil was carefully chromato-
graphed using 0–3% MeOH–CH2Cl2 yielding 10a and 10b
(5.5 g, 12.3 mmol, 67%).
Experimental
(10a): RF: 0.5 (System B). 1H-NMR (CDCl3): 8.8 (s, 1H,
NH), 7.95 (d, J = 8.2 Hz, 2H, 4-toluoyl), 7.5 (s, 1H, H-6), 7.28
(d, J = 8.4 Hz, 2H, 4-toluoyl), 5.22 (d, J3Ј,4Ј = 5.9 Hz, 1H, H-3Ј),
4.83 (t, J4Ј,5Ј = 4.7 Hz, 1H, H-4Ј), 4.71 (dd, Jgem = 13.1 Hz,
J5Ј,6Ј = 7 Hz, 1H, H-6Ј), 4.55–4.38 (m, 2H, H-5Ј, H-6Љ), 4.29
(dd, Jgem = 11.8 Hz, J1Ј,1ЈOH = 7.9 Hz, 1H, H-1Ј), 3.79 (dd,
J1Љ,1ЈOH = 6.7 Hz, 1H, H-1Љ), 3.34 (t, 1H, 1Ј-OH), 2.43 (s, 3H,
4-toluoyl), 1.92 (s, 3H, CH3), 1.39, 1.34 (s, 2 × 3H, CH3);
13C-NMR (CDCl ): 166.6 (C᎐O, 4-toluoyl); 164.1 (C-4); 150
All solvents were dried and distilled according to the reported
procedures.18 Chromatographic separations were performed on
Merck G60 silica gel. Thin layer chromatography (TLC) was
performed on Merck pre-coated silica gel 60 F254 glass-backed
plates developed in the following dichloromethane–methanol
mixtures: (A) 98:2 (v/v), (B) 95:5 (v/v), (C) 93:7 (v/v) and (D)
90:10 (v/v). 1H-NMR spectra were recorded on JEOL GX 270
(if nothing else is indicated) and Bruker DRX 600 spectro-
meters at 270.1 MHz and 600 MHz, respectively, using TMS
(0.0 ppm) or methanol (3.4 ppm) as internal standards.
13C-NMR spectra were recorded on a JEOL GX 270 spectro-
meter at 67.9 MHz using the central peak of CDCl3 (76.9 ppm)
as an internal standard. 31P-NMR spectra were recorded on a
JEOL GX 270 spectrometer at 109.4 MHz using 85% phos-
phoric acid as an external standard. Chemical shifts (δ scale)
are reported in ppm. Thermal denaturation experiments were
performed on a PC computer interfaced with a Perkin-Elmer
UV/VIS spectrophotometer Lamda 40 with PTP-6 peltier tem-
perature contoller. The CD spectra were recorded using a
JASCO J41-A spectropolarimeter. Fast deprotecting phos-
phonamidites were purchased from Amersham Pharmacia
Biotech (Sweden). T4 polynucleotide kinase, E. coli RNase H
(5units/5 µL) and [γ-32P] ATP were purchased from Amersham
᎐
3
(C-2); 144.3 (4-toluoyl); 135.1 (C-6); 129.6, 129.2, 126.2
(4-toluoyl); 113.8 (C-5); 108.9 (C-Me2); 99.7 (C-2Ј); 83.7 (C-5Ј);
82.5 (C-3Ј); 80.7 (C-4Ј); 65.1 (C-1Ј); 63.7 (C-6Ј); 27, 25.3 (CH3,
isopropyl); 21.5 (OCH3); 12.5 (CH3, C-5 CH3). 1D Diff. NOE
shows 1.6% NOE enhancement for H6–H5Ј and no other
NOEs between the other endocyclic-sugar protons and H6,
which are present in the β-anomer (see below).
1
(10b): RF: 0.45 (System B). H-NMR (CDCl3): 9.2 (s, 1H,
NH), 7.71 (d, J = 8.2 Hz, 2H, 4-toluoyl), 7.5 (s, 1H, H-6), 7.18
(d, J = 7.9 Hz, 2H, 4-toluoyl), 5.44 (d, J3Ј,4Ј = 6.2 Hz, 1H, H-3Ј),
4.87 (d, 1H, H-4Ј), 4.85–4.82 (m, 1H, H-5Ј), 4.65 (dd,
Jgem = 12.6 Hz, J5Ј,6Ј = 2.4 Hz, 1H, H-6Ј), 4.3–4.2 (m, J5Ј,6ЈЈ = 3.7
Hz, 2H, H-6Љ and H-1Ј), 3.8 (dd, J1Љ,1ЈOH = 6.4 Hz, Jgem = 12.4
Hz, 1H, H-1Љ), 3.27 (t, 1H, 1Ј-OH), 2.4 (s, 3H, CH3, 4-toluoyl),
1.6 (s, 1H, CH3, thymine), 1.56, 1.4 (s, 2 × 3H, CH3, isopropyl);
13C-NMR (CDCl ): 165.6 (C᎐O, 4-toluoyl); 164.3 (C-4);
Pharmacia Biotech (Sweden), phosphodiesterase
I from
᎐
3
Crotalus adamanteus venom was purchased from SIGMA.
150.1 (C-2); 144.6 (4-toluoyl); 137.3 (C-6); 129.2, 128.9, 125.9
(4-toluoyl); 113.4 (C-5); 108.6 (C-Me2), 101.2 (C-2Ј); 86.1
(C-3Ј); 83.4 (C-5Ј); 81.7 (C-4Ј); 64.2 (C-6Ј); 63.7 (C-1Ј); 25.6,
24.1 (CH3, isopropyl); 21.4 (CH3, 4-toluoyl); 11.9 (CH3,
thymine). 1D Diff. NOE shows 0.21% NOE enhancement for
H6–H6Ј, 0.08% NOE for H6–H3Ј and 0.4% NOE for H6–H4Ј.
These are consistent with the β-anomer.
Preparation of the phosphonamidite (15)
6-O-(4-Toluoyl)-1,2:3,4-di-O-isopropylidene-D-psicofuranose
(9). The psicofuranose (8) (5.9 g, 22.5 mmol) was coevaporated
with pyridine 3 times and dissolved in 100 ml of the same
solvent. The solution was cooled in an ice bath and 4-toluoyl
chloride (3.3 ml, 24.8 mmol) was added dropwise under a nitro-
gen atmosphere, with continued stirring at the same temper-
ature for 2 h. Saturated sodium bicarbonate solution was
added and stirring was continued for a further 2 h; the mixture
was then extracted with CH2Cl2. The organic phase was
washed with brine and dried over MgSO4 and evaporated, then
coevaporated with toluene. Recrystallisation from methanol
furnished 9 (7.7 g, 20.2 mmol, 90%). RF: 0.75 (System A).
1H-NMR (CDCl3): 7.9 (d, J = 8 Hz, 2H, 4-toluoyl), 7.3 (d,
J = 7.9 Hz, 2H, 4-toluoyl), 4.8 (d, J3,4 = 5.7 Hz, 1H, H-4), 4.7 (d,
1H, H-3), 4.48–4.35 (m, 3H, H-5, H-6, H-6Ј), 4.33 (d, J1,1Ј = 9.7
Hz, 1H, H-1), 4.1 (d, 1H, H-1Ј), 2.41 (s, 3H, CH3, 4-toluoyl),
1.46 (s, 3H), 1.44 (s, 3H), 1.35, 1.33 (s, 2 × 3H, CH3, isopropyl);
13C-NMR (CDCl ): 166.3 (C᎐O, 4-toluoyl); 143.7, 129.8, 128.9,
1-[1Ј-O-Methylsulfonyl-3Ј,4Ј-O-isopropylidene-6Ј-O-(4-tolu-
oyl)-ꢀ-D-psicofuranosyl]thymine (11). Compound 10b (1.6 g, 3.5
mmol) was coevaporated with pyridine three times and dis-
solved in 25 ml of the same solvent. The mixture was cooled in
an ice bath and methanesulfonyl chloride (0.75 ml, 9.7 mmol)
was added dropwise to the mixture, with continued stirring for
15 min at the same temperature. The reaction was kept in at
4 ЊC for 12 h, then poured into cold saturated sodium
bicarbonate solution and extracted with CH2Cl2. The organic
phase was washed with brine, dried over MgSO4, filtered and
evaporated, then coevaporated with toluene giving compound
11 (1.89 g, 3.5 mmol, 98%). RF: 0.7 (System B). 1H-NMR
(CDCl3): 7.75 (d, J = 8.3 Hz, 1H, 4-toluoyl), 7.38 (d, J = 1.3 Hz,
1H, H-6), 7.22 (d, J = 8.4 Hz, 1H, 4-toluoyl), 5.39 (d, J3Ј,4Ј = 6
Hz, 1H, H-3Ј), 4.96 (d, Jgem = 11.4 Hz, 1H, H-1Јa), 4.94–4.88
(m, 2H, H-4Ј and H-5Ј), 4.7 (dd, Jgem = 12.6 Hz, J5Ј,6Ј = 2.5 Hz,
᎐
3
126.8 (4-toluoyl); 133.6, 112.7, 111.6; 85.2 (C-3); 82.9 (C-5);
82.3 (C-4); 69.7 (C-1); 64.5 (C-6); 26.4, 26.2, 24.8 (CH3,
isopropyl); 21.2 (CH3, 4-toluoyl).
406
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408

View Article Online
1H, H-6Ј), 4.39 (d, 1H, H-1Љ), 4.3 (dd, J5Ј,6Љ = 3.4 Hz, 1H, H-6Љ),
2.98 (s, 3H, CH3, OMs), 2.4 (s, 3H, CH3, 4-toluoyl), 1.7, 1.66 (s,
2 × 3H, CH3, isopropyl); 13C-NMR (CDCl ): 165.7 (C᎐O, 4-
toluoyl); 162.9 (C-4); 150.2 (C-2); 145.1 (4-toluoyl); 135.5 (C-6);
129.1, 128.7, 125.6, (4-toluoyl); 114.2 (C-5); 110.1 (C-Me2); 98.3
(C-2Ј); 87.1 (C-3Ј); 84.2 (C-5Ј); 81.7 (C-4Ј); 69.9 (C-1Ј); 64.1 (C-
6Ј); 37.4 (CH3, 4-toluoyl); 25.8, 24.3 (CH3, isopropyl); 21.3
(CH3, mesyl); 12.3 (CH3, thymine).
of the residue afforded 14 (647 mg, 1.13 mmol, 87%). RF: 0.5
(System B). 1H-NMR (CDCl3): 7.4–7.1 (m, 12H, aromatic
DMTr and H-6), 6.85–6.82 (m, 4H, aromatic DMTr), 5.4 (d,
J3Ј,4Ј = 4.1 Hz, 1H, H-3Ј), 5.13 (d, Jgem = 7.9 Hz, 1H, H-1Ј), 4.76
(d, 1H, H-1Љ), 4.35 (dd, J4Ј,5Ј = 8.3 Hz, 1H, H-4Ј), 4.28–4.21 (m,
J5Ј,6Ј = 2.5 Hz, J5Ј,6Љ = 4.7 Hz, 1H, H-5Ј), 3.98 (dd, Jgem = 12.4 Hz,
1H, H-6Ј), 3.81 (dd, 1H, H-6Љ), 3.8 (s, 6H, OCH3, DMTr), 1.92
(s, 3H, CH3, thymine); 13C-NMR (CDCl3): 164.23, 158.1 (C-4);
149.5, 144.5 (C-2); 135.9, 135.3, 129.8, 128.9, 127.9, 127.5,
126.4, 112.8 (DMTr); 111.6 (C-5); 90.9 (C-2Ј); 87.6 (C-3Ј); 83.6
(C-5Ј); 78.2 (C-1Ј); 69.7 (C-4Ј); 60.8 (C-6Ј); 54.9 (DMTr); 11.9
(CH3, thymine).
᎐
3
1-[1Ј-O-Methylsulfonyl-6Ј-O-(4-toluoyl)-ꢀ-D-psicofuranosyl]-
thymine (12). Compound 11 (1.9 g, 3.5 mmol) was stirred with
10.5 ml of 90% CF3COOH in water for 20 min at room
temperature. The reaction mixture was evaporated and
coevaporated with pyridine. Chromatography of the residue
furnished 12 (1.58 g, 3.3 mmol, 92.5%). RF: 0.3 (System B).
1H-NMR (CDCl3 ϩ CD3OD): 7.75 (d, J = 8.3 Hz, 1H,
4-toluoyl), 7.52 (d, J = 1.24 Hz, 1H, H-6), 7.2 (d, J = 8.4 Hz,
1H, 4-toluoyl), 4.81 (d, Jgem = 11.6 Hz, 1H, H-1Ј), 4.76 (d,
J3Ј,4Ј = 5.3 Hz, 1H, H-3Ј), 4.75 (dd, Jgem = 12.6 Hz, J5Ј,6Ј = 3.5 Hz,
1H, H-6Ј), 4.62 (dt, J5Ј,6Љ = 2.5 Hz, 1H, H-5Ј), 4.58 (d, 1H,
H-1Ј), 4.41 (dd, J4Ј,5Ј = 3 Hz, 1H, H-4Ј), 4.33 (dd, 1H, H-6Љ),
2.98 (s, 3H, CH3, OMs), 2.4 (s, 3H, CH3, 4-toluoyl), 1.73 (s, 3H,
1-{1Ј,3Ј-Anhydro-4Ј-O-[2-cyanoethoxy(diisopropylamino)-
phosphino]-6Ј-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-psicofuranosyl}thy-
mine (15). To a stirred solution of 14 (529 mg, 0.9 mmol) in
THF (5 ml), 0.8 ml of N,N-diisopropylethylamine was added
under a nitrogen atmosphere with stirring at room temperature
for 10 min. To this solution 2-cyanoethyl-N,N-diisopropyl-
chlorophosphonamidite (0.4 ml, 1.8 mmol) was added with
continued stirring for 2 h. The reaction was quenched with
methanol (3 ml) and the mixture was dissolved in CH2Cl2,
washed with saturated NaHCO3 solution and brine. The
organic layer was dried over MgSO4, filtered and evaporated.
Chromatography of the residue (30–40% EtOAc, cyclo-
hexane ϩ 2% Et3N) furnished 15 (632 mg, 0.81 mmol, 90%). RF:
0.5 (System B). The compound was dissolved in CH2Cl2 (3ml)
and precipitated from hexane at Ϫ40 ЊC. 31P-NMR (CDCl3):
150.55; 150.46.
CH3, thymine); 13C-NMR (CDCl ϩ CD OD): 165.9 (C᎐O,
᎐
3
3
4-toluoyl); 163.8 (C-4); 151.7 (C-2); 144.9 (4-toluoyl); 136.3
(C-6); 129.2, 129, 126.1 (4-toluoyl); 110.4 (C-5); 97 (C-2Ј); 83.9
(C-5Ј); 79.8 (C-3Ј); 72.2 (C-4Ј); 69.3 (C-1Ј); 63 (C-6Ј); 37.5 (CH3,
4-toluoyl); 21.3 (CH3, mesyl); 11.9 (CH3, thymine).
1-[1Ј,3Ј-O-Anhydro-6Ј-O-(4-toluoyl)-ꢀ-D-psicofuranosyl]-
thymine (13). To a stirred solution of 80% NaH (171 mg, 5.7
mmol) in 15 ml of DMF in an ice bath, a solution of compound
12 (1.3 g, 2.6 mmol) in 15 ml of DMF was added dropwise. The
reaction mixture was stirred at the same temperature for 9 h,
quenched with 10% acetic acid solution in water and evap-
orated. The residue was coevaporated with xylene and on
chromatography yielded 13 (602 mg, 1.55 mmol, 60%). RF: 0.5
(System C). 1H-NMR (CDCl3): 7.93 (d, J = 8.1 Hz, 2H,
4-toluoyl), 7.25 (d, J = 7.9 Hz, 2H, 4-toluoyl), 6.81 (s, 1H, H-6),
5.47 (d, J3Ј,4Ј = 3.9 Hz, 1H, H-3Ј), 5.15 (d, Jgem = 7.9 Hz, 1H,
H-1Ј), 4.79–4.72 (m, Jgem = 12.3 Hz, J6Ј,5Ј = 2.55 Hz, 2H, H-1Ј
and H-6Ј), 4.55–4.42 (m, J6Љ,5Ј = 2.9 Hz, J4Ј,5Ј = 8 Hz, 3H, H-4Ј,
H-5Ј, H-6Љ), 2.4 (s, 3H, CH3, 4-toluoyl), 1.8 (s, 3H, CH3,
thymine); 13C-NMR (CDCl ): 166.6 (C᎐O, 4-toluoyl); 164.3
Synthesis, deprotection and purification of oligonucleotides
All oligonucleotides were synthesizesd on 1 µmol scale with
an 8-channel Applied Biosystems 392 DNA/RNA synthesizer.
Synthesis and deprotection of the AONs, as well as the RNA
target, were performed as previously described.19 For modified
AONs fast depropecting amidites were used and they were
deprotected by room temperature treatment with NH4OH for
16 h. All AONs were purified by reversed-phase HPLC eluting
with the following systems: A (0.1 M triethylammonium
acetate, 5% MeCN, pH 7) and B (0.1 M triethylammonium
acetate, 50% MeCN, pH 7). The RNA target was purified by
20% 7 M urea polyacrylamide gel electrophoresis and its purity,
and that of all the AONs (greater than 95%) was confirmed by
PAGE. Representive data from MALDI-MS analysis: AON (4)
[M Ϫ H]Ϫ 4478.7; calcd 4478; RNA target (7) [M Ϫ H]Ϫ 4918.1;
calcd 4917.1.
᎐
3
(C-4); 149.2 (C-2); 143.8 (4-toluoyl); 135.1 (C-6); 129.5, 128.8,
126.5 (4-toluoyl); 111.6 (C-5); 90.9 (C-2Ј); 87.3 (C-3Ј); 80.9
(C-5Ј); 78.1 (C-1Ј); 70.3 (C-4Ј); 63 (C-6Ј); 21.2 (CH3, 4-toluoyl);
11.8 (CH3, thymine).
UV Melting experiments
1-(1Ј,3Ј-O-Anhydro-ꢀ-D-psicofuranosyl)thymine (1). Com-
pound 13 (570 mg, 1.5 mmol) was dissolved in methanolic
ammonia (50 ml) and stirred at room temperature for 2 days.
The solvent was evaporated and the residue, on chrom-
atography, afforded 1 (378 mg, 1.4 mmol, 96%) RF: 0.3 (System
Determination of Tms of the AON/RNA hybrids were carried
out in the same buffer as for RNase H degradation: 57 mM
Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl2 and 2 mM DTT.
Absorbance was monitored at 260 nm in the temperature range
20 to 60 ЊC with a heating rate of 1 ЊC per minute. Prior to
the measurement the samples (1:1 mixture of AON and RNA)
were preannealed by heating to 80 ЊC for 4 min followed by slow
cooling to 20 ЊC and 30 min equilibration at this temperature.
1
D) H-NMR (CD3OD, 600 MHz): 7.38 (d, J = 1.25 Hz, 1H,
H-6), 5.58 (d, J3Ј,4Ј = 3.8 Hz, 1H, H-3Ј), 5.33 (d, Jgem = 8.1 Hz,
1H, H-1Ј), 4.9 (d, 1H, H-1Љ), 4.46–4.41 (m, J4Ј,5Ј = 8.4 Hz,
J5Ј,6Ј = 2.2 Hz, J5Ј,6Љ = 5.24 Hz, 2H, H-4Ј and H-5Ј), 4.11 (dd,
Jgem = 12.4 Hz, 1H, H-6Ј), 3.9 (dd, 1H, H-6Љ), 2.1 (s, 1H, CH3,
thymine); 13C-NMR (CD3OD): 166.8 (C-4); 151.7 (C-2); 138.4
(C-6); 112.7 (C-5); 93.2 (C-2Ј); 89.3 (C-3Ј); 85.3 (C-5Ј); 79.9
(C-1Ј); 71.9 (C-4Ј); 62.7 (C-6Ј); 12.1 (CH3, thymine).
Thermodynamic calculations from UV experiments
The thermodynamic parameters characterizing helix-to-coil
transition for the DNA/RNA hybrids were obtained from Tm
measurements over the concentration range from 2 to 10 µM
(total strand concentration, CT). Values of 1/Tm were plotted
versus (CT/4) and ∆HЊ and ∆SЊ parameters were calculated
from slope and intercept of fitted line: 1/Tm = (R/∆HЊ)ln
(CT/s) ϩ ∆SЊ/∆HЊ, where s reflects the sequence symmetry of
the self (s = 1) or nonself-complementary strands (s = 4).
1-[1Ј,3Ј-Anhydro-6Ј-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-psicofur-
anosyl]thymine (14). To a solution of 1 (353 mg, 1.3 mmol) in
anhydrous pyridine (6 ml) was added 4,4Ј-dimethoxytrityl
chloride (DMTrCl; 510 mg, 1.5 mmol), and the mixture was
stirred at room temperature overnight. Saturated NaHCO3
solution was added and the mixture extracted with dichloro-
methane. The organic phase was washed with brine, dried over
MgSO4, filtered and evaporated. Column chromatography
CD Experiments
CD spectra were measured from 300 to 220 nm in a 0.2 cm path
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408
407

View Article Online
2 O. Y. Fedroff, M. Salazar and B. R. Reid, J. Mol. Biol., 1993, 233,
length cuvette. Buffer conditions were same as for the UV and
RNase H experiments. The total strand concentration used was
2.5 µM and spectra were recorded at 21 ЊC. Each spectrum was
an average of two scans with the buffer blank subtracted,
which was recorded at the same scan speed.
509.
3 (a) S. T. Crooke, Methods Enzymol., 2000, 313, 3; (b) E. Zamaratski,
D. Ossipov, P. I. Pradeepkumar, N. Amirkhanov and J.
Chattopadhyaya, Tetrahedron, 2001, 57, 593.
4 (a) K.-H. Altman, R. Kesselring, E. Francotte and G. Rihs,
Tetrahedron Lett., 1994, 35, 2331; (b) M. A. Siddiqui, H. Ford,
C. George and V. E. Marquez, Nucleosides Nucleotides, 1996, 15,
235; (c) V. E. Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ,
J. Wang, W. R. Wanger and D. M. Matteucci, J. Med. Chem., 1996,
39, 3739; (d) S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida
and T. Imanshi, Tetrahedon Lett., 1997, 38, 8735; (e) A. A. Koshkin,
S. K. Singh, P. Nielson, V. K. Rajwanshi, R. Kumar, M. Meldgaard
and J. Wengel, Tetrahedron, 1998, 54, 3607; ( f ) D. G. Schultz
and S. M. Grynazov, Nucleic Acid Res., 1996, 24, 2966; (g) G. Wang,
J.-L. Giradet and E. Gunic, Tetrahedron, 1999, 55, 7707; (h) J.
Wengel, Acc. Chem. Res., 1999, 32, 301; (i) G. Wang, E. Gunic and
J.-L. Giradet, Bioorg. Med. Chem. Lett., 1999, 9, 1147; (j) S. Obika,
D. Nanbu, Y. Hari, K. Morio, J. Andoh, K. Morio, T. Doi and
T. Imanishi, Tetrahedron Lett., 1998, 39, 5401; (k) M. Sekine,
O. Kurasawa, K. Shohda, K. Seio and T. Wada, J. Org. Chem., 2000,
65, 3571.
5 (a) K.-H. Altman, R. Imwinkelried, R. Kesselring and G. Rihs,
Tetrahedron Lett., 1994, 35, 7625; (b) S. Obika, K. Morio, Y. Hari
and T. Imanishi, Chem. Commun., 1999, 2423; (c) S. Obika,
K. Morio, Y. Hari and T. Imanishi, Bioorg. Med. Chem. Lett., 1999,
9, 515; (d) R. Steffens and C. Leumann, Helv. Chim. Acta, 1997,
80, 2426; (e) R. Buff and J. Hunziker, Bioorg. Med. Chem., 1998,
521.
32P Labeling of RNA
The target RNA was 5Ј-end labeled with 32P using T4 poly-
nucleotide kinase, [γ-32P]ATP using a standard procedure.20
Labeled RNA was purified by 20% denaturing PAGE and
specific activitiy was measured using a Beckman LS3801
counter.
RNase H digestion assays
DNA/RNA hybrids (0.8 µM) consisting of a 1:1 mixture of
AON and the target RNA (specific activity 50 000 cpm) were
digested with 0.6 U of RNase H in 57 mM Tris-HCl (pH 7.5),
57 mM KCl, 1 mM MgCl2 and 2 mM DTT at 21 ЊC. Total
reaction volume was 26 µL. Aliquots (7 µL) were taken at 30, 60
and 120 min and the reaction was stopped by addition of
an equal volume of 20 mM EDTA in 95% formamide. RNA
cleavage products were resolved by 20% polyacrylamide
denaturing gel electrophoresis and visualized by autoradio-
graphy. Quantitation of cleavage products was performed using
a Molecular Dynamics phosphorImager.
6 (a) P. Herdewijn, Biochim. Biophys. Acta, 1999, 1489, 167; (b)
P. Herdewijn, Liebigs Ann. Chem., 1996, 1337.
7 M. Manoharan, Biochim. Biophys. Acta, 1999, 1489, 117.
8 C. Wahlestedt, L. Salmi, J. K. Good, T. Johnsen, T. Hokfelt,
C. Broberger, F. Porreca, A. Koshkin, M. H. Jacobson and
J. Wengel, Proc. Natl. Acad. Sci. USA, 2000, 97, 5633.
9 C. Thibaudeau and J. Chattopadhyaya, Stereoelectronic Effects in
Nucleosides and Nucleotides and their Structural Implications, 1999,
Dept of Bioorganic Chemistry, ISBN 91-506–1351-0, Uppsala
University Press, Uppsala, Sweden. Fax: 004618554495.
10 R. W. Wanger, M. D. Matteucci, J. G. Lewis, A. J. Gutierrez,
C. Moulds and B. C. Froehler, Science, 1993, 260, 1510.
11 M. H. Caruthers, Acc. Chem. Res., 1991, 24, 278.
12 L. A. Marky and K. J. Breslauer, Biopolymers, 1987, 26, 1601.
13 After the completion of this work (Tetrahedron Lett., 2000, 41,
8601), we have come across the work of Bondensgaard et al. (Aug 4th
issue of Chem. Eur. J., 2000, 6, No. 15, 2687) on the structural
studies of LNA/RNA duplexes in which the 3Ј-nucleotide flanking
the modification site adopts a clear N-type conformation (NMR);
these workers however conclude that overall A-type conformation
exists in the AON strand of the duplex stretching only one
nucleotide both at the 3Ј- and 5Ј-ends of the LNA nucleotide
modified site, which means that this part of the duplex takes up a
more A-RNA/A-RNA type helix. This contradicts our present
observations, in which we see a local structural change spanning
only at the 5Ј-end of the modified nucleotide of the AON strand and
the 3Ј-end of the complementary RNA strand. Our present work
allows us to predict that one would perhaps observe a similar
absence of RNase H cleavage at those comparable sites of the
LNA/RNA duplexes owing to the double stranded RNA/RNA
conformation.
Conclusions
(1) Incorporation of the bicyclic psiconucleoside 1, in which
the sugar moiety is fixed in the North-form, into AONs (3)–(6)
resulted in decreased thermodynamic stability of the corre-
sponding duplexes with target RNA (7) compared to the native
counterpart.
(2) CD Experiments failed to detect any local structure alter-
ations in the modified AON/RNA hybrid duplexes, and showed
high similarity between their CD spectra and that of the native
counterpart.
(3) All AON/RNA hybrids formed by the modified AONs
(3)–(6) were found to be substrates for RNase H and showed
hydrolysis to a comparable extent (after 120 min of incubation)
with that of the native counterpart, in spite of their lower
thermodynamic stability.
(4) The introduction of the single conformationally con-
strained nucleotide T caused local conformational perturb-
ances in the AON/RNA hybrids spanning 4 nucleotides
towards the 5Ј-end of the AON, which was clearly reflected in
the RNase H hydrolysis pattern of all the duplexes studied.
(5) Although not susceptible to cleavage, the structurally dis-
torted 5 basepair region of the AON/RNA hybrids can serve
as a binding site for the enzyme. This means that structural
requirements for substrate binding and substrate cleavage are
different.
14 (a) E. Kanaya and S. Kanaya, Eur. J. Biochem., 1995, 231,
557; (b) W. F. Lima and S. T. Crooke, Biochemistry, 1997, 36,
390.
15 E. J. Prinsbe, J. Smejkal, J. P. H. Verheyden and J. G. Moffatt,
J. Org. Chem., 1976, 41, 1836.
16 M. S. P. Sarma, S. Megati, R. S. Klein and B. A. Otter, Nucleosides
Nucleotides, 1995, 14, 393.
(6) The binding site and the cleavage site in the substrate–
enzyme complex are different and the binding site distant by at
least five nucleotides toward the 5Ј-direction of the RNA from
the cleavage site.
17 The uracil-1-yl and adenin-9-yl derivatives of the corresponding
bicyclic psiconucleoside were reportedly formed as an unexpected
product (in 88 and 15% yield) during the alkaline methanolysis
of 1Ј-bromo-[3Ј,4Ј,6Ј-tri-O-(4-toluyl)-β--psicofuranosyl] uracil/
adenine. (a) H. Hrebabecky and J. Farkas, Collect. Czech. Chem.
Commun., 1974, 39, 1098; (b) R. D. Elliott, S. Niwas and J. M.
Riordan, Nucleosides Nucleotides, 1992, 11, 97.
Acknowledgements
Thanks are due to the Swedish Board for Technical Develop-
ment (NUTEK) to J. C., the Swedish Natural Science Research
Council (NFR contract # K-KU 12067–300 to A. F. and
K-AA/Ku04626–321 to J. C.) and the Swedish Research
Council for Engineering Sciences (TFR) to J. C. for generous
financial support.
18 D. D. Perrin, W. L. F. Armarego and D. R. Perrin, Purification of
Laboratory Chemicals, 1985, Pergamon Press, Oxford.
19 E. Zamaratski and J. Chattopadhyaya, Tetrahedron, 1998, 54,
8183.
20 F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.
Seidman, J. A. Smith and K. Struhl, Current Protocols in Molecular
Biology, 1995, 3.10.3, John Wiley & Sons, New York.
References
1 S. T. Crooke, Biochim. Biophys. Acta, 1999, 1489, 31.
408
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408