Consequences of P-chirality in chimeric 2′-O- methyloligoribonucleotides with stereoregular methylphosphonothioate linkages

Article abstract of DOI:10.1002/ejoc.200500395

Stereoregular chimeric oligonucleotides modified with diastereomerically pure methylphosphonothioate linkages interact with complementary DNA or RNA templates in a stereo-dependent manner. There is an unprecedented large difference in the stability of duplexes of oligonucleotides modified with alternating methylphosphonothioate linkages (ΔT_m = 34°C) that is dependent only on the stereochemistry of the methylphosphonothioate phosphorus centres. The nature of these interactions was studied by circular dichroism and NMR spectroscopy, and a thermodynamic analysis was performed. The collected data indicate that the absolute configuration at the P-chiral methylphosphonothioate linkages in chimeric 2′-O-methylribonucleotides could, in some cases, determine the global conformation of single-strand oligonucleotides and constrain the conformation of hybrid duplexes formed with RNA. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500395

FULL PAPER
DOI: 10.1002/ejoc.200500395
Consequences of P-Chirality in Chimeric 2Ј-O-Methyloligoribonucleotides with
Stereoregular Methylphosphonothioate Linkages
Lucyna A. Wozniak,*[a] Magdalena Janicka,[a] and Małgorzata Bukowiecka-Matusiak[a]
Dedicated to Professor Wojciech J. Stec on the occasion of his 65th birthday
Keywords: Chimeric oligonucleotides / Chirality / Conformational analysis / Circular dichroism / Nucleic acids /
Thermodynamic stability
Stereoregular chimeric oligonucleotides modified with dia-
stereomerically pure methylphosphonothioate linkages inter-
act with complementary DNA or RNA templates in a stereo-
dependent manner. There is an unprecedented large differ-
ence in the stability of duplexes of oligonucleotides modified
NMR spectroscopy, and a thermodynamic analysis was per-
formed. The collected data indicate that the absolute config-
uration at the P-chiral methylphosphonothioate linkages in
chimeric 2Ј-O-methylribonucleotides could, in some cases,
determine the global conformation of single–strand oligonu-
cleotides and constrain the conformation of hybrid duplexes
formed with RNA.
with alternating methylphosphonothioate linkages (∆Tm
=
34 °C) that is dependent only on the stereochemistry of the
methylphosphonothioate phosphorus centres. The nature of
these interactions was studied by circular dichroism and
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
internucleotide backbones, including those with dia-
stereomerically pure P-chiral non-ionic modifications, are
of special interest.[14,15] It has previously been established
that P-chiral oligonucleotide methylphosphonates (Oligo-
MePO) (1) interact with complementary DNA targets in a
highly stereodependent manner. The profound effect of the
P-chirality of methylphosphonates incorporated into Oligo-
MePO on their physicochemical properties was first de-
scribed by Miller et al.[16] and further confirmed by Lesni-
kowski and co-workers[17–19] in their studies following the
first stereocontrolled synthesis of Oligo-MePO. Recently
chimeric methylphosphonate oligonucleotides were success-
fully used to study the structures of the active sites and the
mechanisms of the action of enzymes in detail.[20] Hecht
and co-workers not only identified key determinants of the
stability of enzyme–substrate complexes but demonstrated
that the introduction of (SP)-methylphosphonate linkages
at the cleavage site could transform topoisomerase IB into
a potent endonuclease.[21] Kurpiewski et al. used chimeric
oligonucleotides modified both with diastereomerically
pure (RP)- and (SP)-phosphorothioates and methylphos-
phonates to revise the mechanisms for the coupling between
DNA recognition specificity and the catalysis in EcoRI en-
donuclease.[22] These studies allowed the roles of individual
phosphoryl oxygen atoms in catalysis to be delineated and
led to the conclusion that a “crosstalk ring” in the complex
couples recognition to catalysis and connects the two cata-
lytic sites to each other.
Introduction
There is an increasing body of data that demonstrates
the broad applicability of synthetically modified oligonucle-
otides that have altered internucleotide linkages.[1–3] Such
oligonucleotides have proven to be suitable tools to study
the consequences of sequence-specific inhibition of gene ex-
pression,[4,5] including potential therapeutic (antisense or
antigene strategies) applications,[6] or the mechanisms of the
action of rybozymes.[7] It was recently demonstrated that
modified oligonucleotides can also deliver precise infor-
mation on protein functions and interactions with DNA
provided that detailed information on their intrinsic struc-
ture is available.[8–11] The physicochemical and biological
properties of modified oligonucleotides can be widely tuned
since numerous procedures have been elaborated that enable
various modifications to internucleotide phosphodiester
bonds, sugar or base moieties.[12]
Modification of the internucleotide phosphorus centre
allows the synthesis of oligonucleotides with enhanced af-
finity towards complementary templates, increased enzy-
matic stability, and improved membrane permeability.[13]
The chimeric constructs consisting of various mixed
[a] Centre of Molecular and Macromolecular Studies,
Department of Bioorganic Chemistry,
Polish Academy of Sciences,
Sienkiewicza 112, 90-363 Lodz, Poland
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Recently, we reported the large-scale synthesis of dinucle-
For the model structural studies we designed several
oside (3Ј,5Ј)-methylphosphonothioates and their separation homopyrimidine chimeric 2Ј-O-methyloligoribonucleotides
into diastereomerically pure isomers,[23,24] which could be (2) modified with increasing numbers of methylphos-
considered as substrates for P-chiral dimeric building phonothioate internucleotide bonds. The unusual differ-
blocks in the synthesis of chimeric oligonucleotides (Oligo- ences in the affinity of stereoregular chimeric 2Ј-O-methyl-
MePS) of predetermined P-chirality.[25] We demonstrated oligoribonucleotides towards DNA and RNA templates is
that Oligo-MePS,[26] considered as isosteric analogues of discussed in the context of the predominant conformations
Oligo-MePO,[27] exhibit stereodependent binding properties of these hybrid duplexes and possible different solvation
towards complementary DNA or RNA targets. During our patterns.[38,39]
studies on the stereocontrolled synthesis of Oligo-
MePO[28,29] we also evaluated stereospecific methods for PS
Ǟ PO oxidation reactions[30] allowing conversion of Oligo-
Synthesis
MePS to Oligo-MePO.
The dinucleoside (3Ј,5Ј)-methylphosphonothioate di-
meric building blocks (4) were prepared from the corre-
sponding dinucleotides (UMePSC)2Ј-OMe (3) in good yield
Results and Discussion
(61%) using the triazolidite one-pot method described pre-
We now report the synthesis of chimeric 2Ј-O-methyloli- viously (Scheme 1).[25,40]
goribonucleotides (Oligo-MePS, 2) with diastereomerically
The assignment of the (SP) and (RP) absolute configura-
pure dinucleoside (3Ј,5Ј)-methylphosphonothioates (3) of a tions to FAST- and SLOW-eluting isomers 3, respectively,
1
predefined absolute configuration incorporated at the phos- by means of X-ray analysis[23] and H ROESY NMR spec-
phonothioate centre. and the properties of hybrid duplexes troscopy has previously been reported.[24]
formed with complementary DNA and RNA targets. It is
The diastereomerically pure isomers 3 were 3Ј-O-phos-
known that the incorporation of particular electron-with- phitylated with (CNCH2CH2O)(iPr2N)2P/1H-tetrazole in
drawing (e.g. O-alkyl, O-alkoxyalkyl) 2Ј-substituents can MeCN to give (RP)- or (SP)-5Ј-O-DMT-[2Ј-OMe-uridin-3Ј-
augment the RNA-like C3Ј-endo sugar conformation of oli- yl (3Ј,5Ј)-5Ј-O-2Ј-OMe-cytidine methylphosphonothioate]-
goribonucleotides, the modified oligomers pre-organising 3Ј-O-(β-cyanoethyl)-N,N-diisopropylphosphoramidites (4)
into the A conformation adopted by RNA duplexes.[31–34] and used as dimeric building blocks for condensation by
Such modifications generally lead to a considerable increase the phosphoramidite method on a solid support (Table 1).
in the stability of heteroduplexes with RNA, which is spe-
Modified chimeric oligonucleotides 2, the phosphodies-
cially required for efficient antisense activity (steric block- ters d(AG)5A (9) and r(AG)5A (10) were prepared as com-
ing).[35] The highly enhanced plasma stability and the in- plementary templates and the isosequential phosphodies-
creased in vitro potency of entirely 2Ј-O-methyl-modified ters d(TC)5T (11) and (2Ј-OMe)(UC)5U (12) used as refer-
siRNA[36] and their potential as valuable tools to study ence oligomers (Table 1) were prepared on a 1 µmol scale.
siRNA mechanisms has also been demonstrated.[37]
Chimeric oligonucleotides with a single dinucleoside (3Ј,5Ј)-
Scheme 1.
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P-Chirality in Methyloligoribonucleotides with Methylphosphonothioate Linkages
FULL PAPER
Table 1. MALDI-TOF MS analysis of chimeric 2Ј-O-methyloligoribonucleotides 2 and 13 and reference oligomeric phosphodiesters 9–
12.
Oligonucleotide
m/z[a]
5Ј-r(UMePSC UC UC UC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UMePSC UC UC UC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePSC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePSC UC UC U)2Ј-OMe-3Ј9
5Ј-r(UMePSC UMePSC UMePSC UMePSC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UMePSC UMePSC UMePSC UMePSC UMePSC U)2Ј-OMe-3Ј
5Ј-d(AG.AG.AG.AG.AG.A)-3Ј
(RP,RP)-2a
3479 (3478)
3481 (3478)
3467 (3463)
3466 (3463)
3521 (3523)
3523 (3523)
3459 (3460)
3616 (3616)
3305 (3306)
3450 (3453)
3452 (3447)
3452 (3447)
(SP,SP-2b
(RP)-2c
(SP)-2d
(RP,RP,RP,RP,RP)-2e
(SP,SP,SP,SP,SP)-2f
9
5Ј-r(AG.AG.AG.AG.AG.A)-3Ј
10
5Ј-d(TC TC TC TC TC T)-3Ј
5Ј-r(UC UC UC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePOC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePOC UC UC U)2Ј-OMe-3Ј
11
12
Oxo-(RP)-13a
Oxo-(SP)-13b
[a] Calculated molecular weights are given in brackets.
methylphosphonate linkage [(RP)-13a and (SP)-13b] were phonothioates (3b) has proved diagnostic for the assign-
prepared by stereospecific oxidation of the internucleotide ment of the absolute configurations and conformations of
methylphosphonothioate centres of the corresponding the corresponding dinucleotides.[24] Analogously, fully de-
methylphosphonothioate oligonucleotides (RP)-2c and (SP)- protected (SP)-OH-2Ј-OMe-uridin-3Ј-yl (3Ј,5Ј)-5Ј-O-2Ј-
2d, respectively, with Oxone®.[29]
OMe-cytidine methylphosphonothioate (3a) was analyzed
by H NMR spectroscopy in order to assign the conforma-
1
tion of both 2Ј-O-methylribose rings in water (Figure 1).
Formation of Hybrid Duplexes with Complementary DNA
and RNA Templates
Extensive studies and theoretical analyses have been fo-
cused on the factors contributing to the thermal stability
of the duplexes. Hydrogen-bonding architecture, π–π base-
stacking interactions and hydration networks are among
the major stabilizing interactions in double-helical struc-
tures. Since duplex formation is an enthaply-driven process,
it was concluded that entropy-driven hydrophobic effects
are hidden by the unfavourable entropy of bond rotations.
However, electrostatic or van der Waals interactions, which Figure 1. Representations of the S-type (DNA-like) and N-type
(RNA-like) structures of the sugar conformations.
are enthaply-driven effects, may be more important in nu-
cleic acids than solvent-induced interactions. Moreover,
The conformation of the ribose rings was studied using
there is no unified picture as to the relative importance of
a two-state model and described in terms of an equilibrium
solvophobic, electrostatic or van der Waals effects on the
stability of hybrid duplexes of modified oligonucleotides.
Modifications of the internucleotide phosphorus centre, in
particular those leading to changes in lipophilic/hydro-
between N and S conformers in solution.[41,42] The 3Ј-endo
pucker of the ribose ring is typical of A-RNA and the intro-
duction of an electronegative 2Ј-O-methyl group leads to
enhanced 3Ј-endo pucker stabilization.[34] From the 1H
phobic properties (reduction of total negative charge), will
NMR spectra (2D ROESY and NOESY, 31P NMR/1H
influence the relative energies of solvation of the bases when
NMR correlations) it became evident that unlike in crystal-
stacked or unstacked and the surface area desolvated on
line (RP)-3b,[23] both in organic solution and particularly in
stacking.[39,41]
D2O (10 m Tris-HCl, 100 m NaCl, and 10 m MgCl2
Here we analyze the factors that contribute to the sta-
pH = 7.4), a considerable fraction of the uridine moiety is
bility of hybrid duplexes formed between the chimeric oli-
present as the S conformer with the more populated 2Ј-endo
gonucleotides 2 and 13 and complementary DNA and
fraction observed for (SP)-3b (N: 45% at 25 °C in CDCl3).
RNA templates by CD spectroscopy and thermodynamic
analysis and assume that additional information can be
gained from the conformational analysis of dia-
The small coupling constant (J1Ј,2Ј = 2.9 Hz) for cytidine
indicates that this sugar moiety prefers the N conformation,
whereas a value of J1Ј,2Ј = 5.85 Hz for the uridine moiety is
indicative of the greater conformational flexibility of this
stereomerically pure dinucleoside (3Ј,5Ј)-methylphos-
1
phonothioate (3a) by H NMR spectroscopy.
moiety in solution and a significant content of the 2Ј-endo
puckered conformer in dynamic equilibrium between the 2Ј-
NMR Analysis of Dinucleoside (3Ј,5Ј)-Methylphosphono-
endo and 3Ј-endo conformations.[43]
thioate (3a) in Water
The conformational analysis based on individual coup-
NMR analysis of the partially protected, dia- ling constants J1Ј2Ј, J2Ј3Ј, and J3Ј4Ј was performed using the
stereomerically pure dinucleoside (3Ј,5Ј)-methylphos- PSEUROT 6.2 program and confirmed that a relatively
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FULL PAPER
high proportion of the uridine moiety adopts the S con- gonucleotides 2a–2d and 12 are similar to the correspond-
former (62%). These results indicate that the (SP)-methyl- ing hybrid duplexes with RNA 10 in having a negative mo-
phosphonothioate modification of the internucleotide bond lar ellipticity of moderate magnitude at wavelengths above
in 2Ј-O-methylribonucleotides has significantly increased 220 nm, a crossover point between 245 and 249 nm, and a
the population of the S conformers relative to the unmodi- large positive broad band from 250 to 300 nm with a maxi-
fied internucleotide phosphodiester bonds in 2Ј-O-methylri- mum at 266 nm characteristic of the C3Ј-endo pucker. In
bonucleotides.
contrast, the spectrum of the reference oligodeoxynucleo-
tide d(TC)5T (11) exhibits a relatively low and flat band
with a maximum at 274 nm and as such is considered as a
reference for the B-like (C2Ј-endo) DNA structure.[45]
From the spectra of the duplexes (Figure 2, b) we con-
cluded that (RP) modifications [vide (RP,RP)-2a and (RP)-
2c], a single (SP) modification in (SP)-2d, or the flanking
modifications of (SP,SP)-2b do not cause significant changes
in the total conformation of the hybrid duplexes with RNA
templates. A major change is observed, however, for the hy-
brid duplex formed between (SP,SP,SP,SP,SP)-2f and an
RNA template. The positive band at 268 nm for this duplex
is less intense with the crossover point shifted to 249 nm,
implying an increasing distortion of the global conforma-
Conformation of Chimeric Oligonucleotides as Determined
by Circular Dichroism
Although quantitative structural information cannot be
gained from analysis of the CD spectra of oligonucleotides,
these spectra can provide reliable characteristics of their
global conformation in solution if compared with the CD
spectra of reference samples.[44]
To compare the properties of the chimeric oligonucleo-
tides with different modifications we recorded the CD spec-
tra of single-stranded oligonucleotides and hybrid duplexes
(Figure 2). The CD spectra of single-stranded chimeric oli-
Figure 2. Circular dirchroism (CD) spectra of a) free single strands of oligonucleotides 2: 2a (᭹), 2b (+), 2c (᭺), 2d (∆), 2e (᭡), 2f (ᮀ),
11 (×), and 12 (−); b) heteroduplexes of chimeric oligonucleotides 2 with RNA 10: 2a/RNA (᭹), 2b/RNA (+), 2c/RNA (᭺), 2d/RNA
(∆), 2e/RNA (᭡), 2f/RNA (ᮀ), 11/RNA (×), and 12/RNA (−). Reagents and conditions: 10 m Tris-HCl, 100 m NaCl, and 10 m
MgCl2 (pH = 7.4), T = 20 °C, concentration = 4 µ. See Table 1 for base sequences of the corresponding oligonucleotides.
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P-Chirality in Methyloligoribonucleotides with Methylphosphonothioate Linkages
Table 2. Melting temperatures of hybrid duplexes: chimeric oligomers 2 and 13 with complementary DNA 9 and RNA 10.
FULL PAPER
Oligonucleotide
Tm [°C] of duplexes[a]
DNA 9
RNA 10
5Ј-r(UMePSC UC UC UC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UMePSC UC UC UC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePSC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePSC UC UC U)2Ј-OMe-3Ј
5Ј-r(UMePSC UMePSC UMePSC UMePSC UMePSC U)2Ј-OMe-3Ј
5Ј-r(UMePSC UMePSC UMePSC UMePSC UMePSC U)2Ј-OMe-3Ј
5Ј-d(TC TC TC TC TC T)-3Ј
5Ј-r(UC UC UC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePOC UC UC U)2Ј-OMe-3Ј
5Ј-r(UC UC UMePOC UC UC U)2Ј-OMe-3Ј
(RP,RP)-2a
(SP,SP)-2b
(RP)-2c
40.4
28.1
41.4
29.0
41.0
n.d.
44.0
41.0
43.8
36.0
75.5
65.9
76.3
71.3
71.2
37.0
57.7
74.9
74.4
70.0
(SP)-2d
(RP,RP,RP,RP,RP)-2e
(SP,SP,SP,SP,SP)-2f
11
12
Oxo-(RP)-13a
Oxo-(SP)-13b
[a] Tm calculated from the first derivative;[46] the error in the temperature measurement did not exceed 0.1 °C.
tion from C3Ј-endo which can only be caused by multiple with Tm = 71.2 °C and the complex formed between phos-
alternating (SP)-methylphosphonothioate modifications. phodiester 2Ј-O-methyloligonucleotide 12 and RNA 10 pro-
The smaller amplitude of the 2f/RNA CD spectrum is vides a complex of similar thermal stability. Analogous be-
probably due to a similar conformation deviation to that haviour was observed for the hybrid duplexes (RP,RP)-2a/10
seen for single-strand 2f with alternating (SP) modifications, and (RP)-2c/10. In this context, a destabilizing effect of the
as presented in Figure 2 (a). Such an effect is not observed (SP)-methylphosphonothioate function should be pointed
for the alternating (RP) modifications of 2e (Figure 2, a). out. The modifications located at the flanking positions of
This can be rationalized in terms of a different pre-organi- Oligo-MePS (2a and 2b) also contribute to the stability of
zation caused by steric effects and/or the solvation of single- the hybrid duplexes in a stereodependent manner. The sta-
strand oligonucleotides with alternating (RP) and (SP) bility of duplexes formed by (RP,RP)-2a and (SP,SP)-2b is
modifications.[31,33] The process of duplex formation is ac- similar to that formed by (RP)-2c and (SP)-2d and is in
companied by changes in the solvation pattern, which are agreement with the results of studies of chimeric oligonucle-
manifested in a more intense CD spectrum for 2c/RNA otides modified with incorporated methylphosphonates re-
than for 2d/RNA and a less intense spectrum for duplex ported by Reynolds et al.[14]
13a/RNA than for 13b/RNA. This correlates with a signifi-
From the data collected in Table 2 and Table 3 it is evi-
cant decrease in the amplitude at 274 nm for (RP)-13a com- dent that several combinations of ∆H° and ∆S° elicit the
pared with (SP)-13b and has significant thermodynamic same binding affinity, indicating that different factors gov-
consequences (vide infra).
ern a particular duplex formation process. Regardless of the
position of the methylphosphonothioate modification in
chimeric oligonucleotides 2, the hybrid duplexes consisting
of oligonucleotides with incorporated (RP)-MePS [(RP,RP)-
2a, (RP)-2c, and (RP,RP,RP,RP,RP)-2e] and a DNA/RNA
template are thermally more stable than their (SP)-Oligo-
MePS–DNA/RNA counterparts, that is, they have higher
melting temperatures and lower ∆G37° values. The thermo-
dynamic properties of the duplexes formed by stereoregular
chimeric oligonucleotides 2 strongly depend on the nature
of the template. 2Ј-O-Methyloligoribonucleotides are con-
sidered as structural analogues of RNA with a C3Ј-endo
pucker[32,38] and therefore possess an increased affinity
towards RNA templates.[34,36] Note that in the case of a
single P-chiral modification incorporated into the middle of
Oligo-MePS [(RP)-2c and (SP)-2d], the difference in stability
caused by the opposite absolute configuration [(RP)-2c/
DNA vs. (SP)-2d/DNA] is ∆Tm = 12.4 °C for DNA tem-
plate 9, and ∆Tm = 5 °C for duplexes (RP)-2c/RNA and
(SP)-2d/RNA. These differences in duplex stabilities are not
reflected in the CD spectra of single-strand 2c and 2d,
either in the shape or intensity of the spectra of the duplexes
formed by both oligonucleotides. Oligonucleotide (RP)-13a,
with a single methylphosphonate modification, forms a hy-
brid duplex with a stability comparable to that determined
for the corresponding methylphosphonothioate analogue,
Thermodynamic Studies
The data collected in Table 2 reveal a strong relationship
between the absolute configuration at the phosphorus atom
of the internucleotide methylphosphonothioate linkage and
the stability of the hybrid duplexes with complementary
DNA and RNA templates.
In all cases, the chimeric oligonucleotides with (RP)-
methylphosphonothioates (2a, 2c, and 2e) elicit stronger af-
finity towards complementary DNA and RNA than those
with the (SP) modifications. Oligomers oxo-13a and oxo-
13b, modified with a single methylphosphonate linkage, ap-
pear to form hybrid duplexes with a stability close to those
determined for methylphosphonothioates. The differences
in the binding stabilities caused by incorporation of a single
modification into chimeric oxo-(RP)-13a or oxo-(SP)-13b
(∆Tm = 4.4 °C for RNA and ∆Tm = 7.8 °C for DNA) is in
this case smaller than the corresponding difference between
2c and 2d. The Tm values of the duplexes formed with the
target RNA 10 and the (RP)-modified chimeric oligomers
2a and 2c are about 18 °C higher than those formed with
RNA 10 and the reference phosphodiester 11. Interestingly,
the chimeric 2e with alternating (RP)-methylphospho-
nothioate modifications forms a complex with RNA 10
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Table 3. Standard thermodynamic parameters[46] for hybrid duplexes of chimeric oligonucleotides and DNA/RNA templates.
∆H° [kcal/mol] ∆S° [cal/molK] ∆G37° [kcal/mol]
DNA 9
RNA 10
DNA 9 RNA 10
DNA 9
RNA 10
(RP,RP)-2a
–46.0 0.2
–30.6 0.4
–45.9 0.2
–38.5 0.3
–44.5 0.6
n.c.
–43.4 0.4
–62.2 0.6
–54.2 0.3
–46.8 0.3
–74.2 1.4
–66.6 0.9
–84.6 1.7
–78.2 1.1
–63.5 0.8
–42.9 1.1
–75.6 0.9
–64.0 1.0
–89.2 0.8
–70.9 0.3
–121
–77
–120
–102
–116
1
1
1
1
2
–188
–170
–218
–202
–158
–112
–192
–168
–231
–179
4
3
5
3
2
4
3
3
2
1
–8.5 0.3
–6.7 0.5
–8.6 0.3
–7.0 0.5
–8.7 0.9
n.c.
–8.4 0.6
–8.4 0.4
–9.0 0.3
–7.6 0.3
–16.01 1.8
–13. 9 1.2
–17.1 2.3
–15.5 1.4
–14.6 1.0
–8.2 1.6
–16.2 1.3
–11.9. 1.4
–17.5 1.1
–15.4 0.4
(SP,SP)-2b
(RP)-2c
(SP)-2d
(RP,RP,RP,RP,RP)-2e
(SP,SP,SP,SP,SP)-2f
Ref. 2Ј-OMe 12
Ref. deoxy 11
Oxo-(RP)-13a
Oxo-(SP)-13b
n.c.
–113
–173
–146
–126
1
1
1
1
albeit the value of Tm for 2c/RNA (76.3 °C) is 2.8 °C higher This difference is even more profound for the (SP)-modified
than that for the corresponding (RP)-13a/RNA (74.4 °C). A hybrid duplexes with values of –T∆S = –202 cal/molK for
comparison of the CD spectra of oligonucleotides with a (RP)-2d, and –T∆S = –112 cal/molK for (SP,SP,SP,SP,SP)-2f,
single modification, (RP)-13a and (SP)-13b [∆H° = –89 kcal/ respectively. Generally, in all cases but 2f, the increase in
mol for (RP)-13a vs. ∆H° = –71 kcal/mol for (SP)-13b], with entropy can be interpreted to result from a change in the
the corresponding (RP)-2c and (SP)-2d oligonucleotides solvation pattern of the hydrophobic groups.[47]
[∆H° = –85 kcal/mol for (RP)-2c and ∆H° = –78 kcal/mol
Since the CD spectrum of the single-stranded oligonucle-
for (SP)-2d] (methylphosphonate vs. methylphosphonothio- otide (SP,SP,SP,SP,SP)-2f is significantly distinct from those
ate) reveals a stronger influence of the (RP) configuration of the other chimeric oligonucleotides 2, it can be con-
in 13a on the shape of the CD spectrum, manifested as a cluded that this modification causes a major conforma-
significant decrease in amplitude at 266 nm in comparison tional change. Hybrid duplex (SP,SP,SP,SP,SP)-2f/RNA is
to that for 2d. It is possible that the difference in the hybrid- the least stable (since hybrid duplex 2f/DNA was not de-
ization stability of the (RP) and (SP) chimeric oligonucleo- tected under the experimental conditions), with a relatively
tides 2 is a result of unfavourable steric interactions between low negative entropy (∆S° = –112 cal/molK) and enthalpy
the methyl group of the (SP) isomer and the flanking resi- (∆H° = –43 kcal/mol). These data together with NMR
dues in the major groove (Figure 1, b), as was postulated studies of the (SP) dinucleotide 3a imply that the unique
for methylphosphonate analogues.[31] For the (RP) dia- behaviour of the oligonucleotide 2f with alternating (SP)
stereomer the methyl groups are directed away from the modifications is a result of a major difference in the confor-
neighbouring residues in the major groove (Figure 1, a). mation of this oligonucleotide, which is shifted toward a B-
Hydration of the axial projecting methyl group and less ef- like structure with a main C2Ј-endo pucker. According to
fective exposure to the solvent of the equatorial methyl the thermodynamic criterion, the hybrid duplex 2f/RNA is
group in the (SP) diastereomer add to the relative destabili- very similar to the one formed between reference r(UC)5-
zation of the hybrid duplexes formed with the target DNA/ U2Ј-OMe (11) and DNA template 9, that is, a duplex of
RNA strand. Previously, the favourable contribution of sol- DNA/RNA type (Figure 3).
vent interactions to the stability of the (RP) isomers over
Regardless of the number of (RP) or (SP) modifications
the (SP) diastereomers in Oligo-MePO was supported by ab in both types of hybrid duplexes (with RNA and DNA tem-
initio and free-energy perturbation calculations for oligonu- plates), the enthalpy factor is higher for the (SP)-modified
cleoside (3Ј,5Ј)-methylphosphonates.[31]
oligonucleotides than for the corresponding (RP) diastereo-
For oligonucleotides 2a, 2c, and 2e containing (RP)- isomers and this can be rationalized in terms of the domi-
MePS moieties, more stable duplexes are formed with nant enthalpy of solvation responsible for the global con-
smaller changes in both enthalpy and entropy than is the formation of the duplexes.[47] On the other hand, the lower
case for the hybrid duplexes formed between unmodified stability of hybrid duplexes with (SP) modifications is to a
oligonucleotides 12 and RNA or DNA templates. For (SP)- greater extent controlled by an entropy factor. The presence
MePS modified oligonucleotides (SP,SP)-2b, (SP)-2d, and of non-ionic hydrophobic internucleotide methylphospho-
(SP,SP,SP,SP,SP)-2f, smaller changes in the thermal stability nothioate centres with sulfur fixed between neighbouring
per modification and the formation of less stable duplexes nucleosides makes the solvation entropy larger and positive.
are observed.
In contrast, the entropy of the (RP)-modified oligonucleo-
An increase in the number of modifications results in a tides (2a and 2c) is lower than the entropy of the (SP) coun-
decrease in the enthalpy of the duplexes with (RP) [from terparts (2b and 2d). Note that the number of (RP) modifi-
∆H° = –84.6 kcal/mol for (RP)-2c to ∆H° = –63.5 kcal/mol cations is not a crucial factor determining the stability of
for 2e] and (SP) modifications (∆H° = –78.2 kcal/mol for 2d duplexes with DNA and RNA, but an increasing number
and ∆H° = –42.9 kcal/mol for 2f). The same effect is ob- of (SP) modifications rapidly reduces the stability of du-
served for the entropy contribution [–T∆S = –218 cal/molK plexes with both DNA and RNA templates. Interestingly,
for (RP)-2c, increasing to –T∆S = –158 cal/molK for 2e]. modifications located at the flanking positions of Oligo-
5194
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5189–5197

P-Chirality in Methyloligoribonucleotides with Methylphosphonothioate Linkages
FULL PAPER
tions[39] responsible for the decrease in the stability of (SP)-
modified oligonucleotides should clarify to what extent
conformational effects induced by the (SP)-methylphospho-
nothioate linkages are significant factors affecting the sta-
bility of hybrid duplexes.
The reported differences in the stability of the hybrid du-
plexes described herein illustrate the potential for possible
tuning of the properties of chimeric oligonucleotides modi-
fied with diastereomerically pure dimeric methylphospho-
nothioate building blocks. These results may provide inter-
esting data in respect of the potential application of this
type of oligonucleotide as therapeutic agents,[25] for study-
ing and controlling oligonucleotide expression (antisense
approach, RNase H independent or by steric blocking of
translation machinery, siRNA), and as precise stereochemi-
cal tools to study the interactions of oligonucleotides with
other biomolecules.
Figure 3. Energy-minimized structures of the hybrid duplexes a)
(RP,RP,RP,RP,RP)-2e/RNA and b) (SP,SP,SP,SP,SP)-2f/RNA. The du-
plex structures were generated with the HyperChem. 7.5 molecular
modelling program[49] and optimized using the AMBER96 force
field method with default parameters.[50]
Experimental Section
Reactions were carried out under dry argon. Solvents and reagents
were purified according to standard laboratory techniques and dis-
tilled directly into reaction vessels. Column chromatography and
TLC analyses were performed on silica gel (Kieselgel 60, 240–
400 mesh, E.Merck Inc.) and silica gel HP TLC precoated F254
plates (purchased from E. Merck Inc.), respectively. NMR spectra
were recorded with a Bruker Advance DRX 500 spectrometer op-
erating at 500.13 MHz (1H) and 202.46 MHz (31P). Chemical shifts
MePS [2a and (SP,SP)-2b] also contribute to the stability of
the hybrid duplexes in a stereodependent manner. The ef-
fect of stabilization of duplexes formed by chimeric oligo-
nucleotides 2a and 2b modified at the flanking positions
(two modifications) is similar to that of oligonucleotides 2c
and 2d with a single modification in the middle of the oligo-
nucleotides. Moreover, Oligo-MePS 2e with alternate (RP) (δ) are reported relative to TMS (1H) and 80% H3PO4 (31P) as
external standards. Mass spectra were recorded with Finnigan Mat
95 (NBA, Cs+ gun operating at 13 keV) and Voyager Elite
(MALDI-TOF) spectrometers.
modifications (five methylphosphonothiates) forms with the
RNA template 10 a relatively stable duplex with Tm
=
71.2 °C, which is of comparable stability to the duplexes
formed between the target RNA 10 and (RP)-modified
chimeric oligomers 2a and 2c, which are about 18 °C
(∆∆G37° = 7.6 kcal/mol) more stable than those formed be-
tween RNA 10 and the reference deoxyribonucleoside
phosphodiester 11.
The domination of the enthalpy factor is directly ob-
served in the CD spectra of the single-stranded oligoncleot-
ides or duplexes, even in the significant difference in ampli-
tude of the positive band at 268 nm for single-strand
(RP,RP,RP,RP,RP)-2e compared to its hybrid duplex with
RNA. The incorporation of the alternating (SP)-methyl-
phosphonothioate modifications into (SP,SP,SP,SP)-2f re-
sults in a chimeric oligonucleotide structure that forms a 2f/
RNA hybrid duplex with the lowest entropy ∆S° = –112
cal/molK. Moreover, analogous to the Oligo-MePS/DNA
duplexes, the unmodified DNA/RNA duplexes are less
stable than the corresponding Oligo-MePS/RNA heterodu-
plexes and their association results in a greater change in
both enthalpy and entropy.
(RP)- and (SP)-5Ј-O-DMT-2Ј-OMe-uridin-3Ј-yl (3Ј,5Ј)-3Ј-O-(N4-
Isobutyryl 2Ј-OMe-cytidine) 3Ј-Methylphosphonothioates (3): Dinu-
cleotides 3 were obtained according to previously described pro-
cedures.[23]
FAST (SP)-3: Rf = 0.5, CHCl3/EtOH, 9:1; 31P NMR (202.46 MHz,
1
CDCl3): δ = 100.1 ppm. H NMR (500 MHz, CDCl3): δ = 1.59 [t,
2
3JHH = 7.59 Hz, 6 H, CH(CH3)2], 1.94 (d, JPH = 15.61 Hz, 3 H,
P-CH3), 2.1 [dq, 1 H, CH(CH3)2], 2.56 (m, 1 H), 3.44 (dd, 1 H),
3.6 [s, 3 H, (C)CH3O-C2Ј], 3.74 [s, 3 H, (Ura)CH3O-C2Ј], 3.8 [s, 6
H, (Ura)CH3O-DMT], 3.92 (m, 1 H), 4.18 (dd, 1 H,), 4.26 [d, 3JHH
= 6.56 Hz, 1 H, (Ura)H2Ј], 5.36 [m, 1 H, (C)H2Ј], 5.87 [s, 1 H, (C)
3
H1Ј], 6.05 [d, JHH = 3.41 Hz, 1 H, (Ura)H1Ј] ppm. FAB MS: m/z
= 962.6 [M – H]+ (C46H55N5O14PS: calcd. 963.99).
SLOW (Rp)-3: Rf = 0.45, CHCl3/EtOH, 9:1; 31P NMR (CDCl3): δ
= 98.26 ppm. 1H NMR (CDCl3): δ = 1.59 [t, 3JHH = 7.59 Hz, 6 H,
2
CH(CH3)2], 1.76 (d, JPH = 15.32 Hz, 3 H, P-CH3), 2.1 [dq, 1 H,
CH(CH3)2], 2.56 (m, 1 H), 3.44 (dd, 1 H), 3.55 [s, 3 H, (C)CH3O-
C2Ј], 3.74 [s, 3 H, (Ura)CH3O-C2Ј], 3.81 [s, 6 H, (Ura)CH3O-
3
DMT], 3.92 (m, 1 H), 4.18 (dd, 1 H,), 4.26 [d, JHH = 6.56 Hz, 1
H, (Ura)H2Ј], 5.36 [m, 1 H, (C)H2Ј], 5.95 [s, 1 H, (C)H1Ј], 6.02 [d,
3
3JHH = 3.43 Hz, 1 H, (Ura)H1Ј], 7.42 [d, JHH = 7.4 Hz, 1 H, (C)
Direct comparison of the thermodynamic parameters
(Table 3) additionally suggests duplexes (SP,SP,SP,SP,SP)-2f/
RNA 10 and the reference 2Ј-O-methyloribonucleotide
phosphodiester 12/DNA 9 have similar structures.
Further studies, including NMR experiments and mo-
lecular dynamics simulations, which are in progress, on spe-
H5], 7.92 [d, 3JHH = 8.2 Hz, 1 H, (Ura)H6], 8.16 [d, 3JHH = 7.5 Hz,
1 H, (C)H6] ppm. FAB–MS: m/z = 962.4 [M – H]+ (calcd. 963.99).
5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-methyluridyl (3Ј,5Ј)-3Ј-O-[β-Cyano-
ethyl-N,N-diisopropylaminophosphoramidityl]-2Ј-(N4-isobutyryl-2Ј-
O-methylcytidine) Methylphosphonothioate
(4): The dia-
cific solute–solvent hydrogen-bond acceptor–donor interac- stereomerically pure dinucleosides 3 (600 mg) were coevaporated
Eur. J. Org. Chem. 2005, 5189–5197
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5195

L. A. Wozniak, M. Janicka, M. Bukowiecka-Matusiak
FULL PAPER
twice with dry benzene (5 mL) and CH2Cl2 (5 mL) and kept over-
night in a desiccator under high vacuum (r.t., 0.01 Torr), dissolved
in CH2Cl2 (dried with CaH2, and stored under argon with 4 Å
molecular sieves) (3 mL), and treated with diisopropylethylamine
(4.4 equiv.) and β-cyanoethyl N,N-diisopropylchlorophosphoram-
idite (2.2 equiv.) with vigorous stirring. The reaction progress was
monitored by TLC (HP TLC plate analysis; chloroform/MeOH,
9:1 v/v). When the reaction was complete, the reaction mixture was
loaded directly into a silica gel column equilibrated with an ethyl
acetate/CH2Cl2/MeCN (3:1:1 v/v) mixture containing 1% of Et3N.
The column was run under a positive pressure of argon using the
same eluent. Fractions containing product 4 were concentrated to
afford pure products 4 as colorless foams. These were dissolved in
dichloromethane and precipitated in cold hexane. White powders
were isolated after centrifugation, dried under vacuum, and stored
at –20 °C under argon. Yields: 80–82%. 31P NMR: (RP)-4 (CDCl3):
δ = 99.3, 99.1 [(RP)-methylphosphonothioate signals] and 149.5,
148.8 ppm (phosphoramidite signals). FAB MS: m/z = 1162.6 [M –
H]–, 1164.7 [M + H]+ (C55H71N7O15PS: calcd. 1163.42). 31P NMR:
(SP)-4 (CDCl3): δ = 97.4, 97.5 ppm (SP)-methylphosphonothioate
signals) and 151.67, 151.34 ppm (phosphoramidite signals). FAB
MS: m/z = 1164.7 [M – H]+ (C55H71N7O15PS: calcd. 1163.42).
band width, and 1–2 s integration time. Each spectrum was
smoothed with a 9- or 15-point algorithm (included in the manu-
facturer’s software, version 2.2.1) after averaging of at least three
scans.
Supporting Information: Synthesis, NMR and MS (MALDI-TOF)
data are available along with colour figures illustrating molecular
modelling results for hybrid duplexes (2f/RNA).
Acknowledgments
This work was financially assisted by the State Committee for Sci-
entific Research (KBN-Grant 4 TO9A 073 25 awarded to L. A.
W.). The authors are indebted to Prof. W. J. Stec for his interest in
this work and helpful discussions, Dr. S. Kazmierski for NMR
analysis, and T. Ke˛błowska for skillful technical assistance.
[1] C. A. Stein, A. M. Krieg (Eds.), Applied Antisense Oligonucleo-
tide Technology, Wiley-Liss, New York, 1998.
[2] M. Phillips (Ed.), “Antisense Technology” in Methods in Enzy-
mology, Academic Press, 1999, vol. 314, part B.
[3] Protocols in Nucleic Acids Chemistry (Eds.: S. L. Beaucage,
D. E. Bergstrom, G. D. Glick, R. A. Jones), John Wiley & Sons,
Inc., Hoboken, NJ, 2003, 4.17.1–4.17.25.
[4] B. H. Loyd, R. V. Giles, D. G. Spiller, J. Grzybowski, D. M.
Tidd, Nucleic Acids Res. 2001, 29, 3664–3673.
[5] S. T. Crooke, Annu. Rev. Med. 2004, 55, 61–95.
[6] E. Wickstrom (Ed.), Clinical Trials of Genetic Therapy with
Antisense DNA and DNA Vectors, Marcel Dekker, Inc., New
York, 1998.
[7] M. Warashina, B. Nawrot, S. Obika, L. A. Wozniak, T. Kuwab-
ara, T. Imanishi, W. J. Stec, K. Taira in Synthetic Nucleic Acids
as Inhibitors of Gene Expression: Mechanisms, Applications and
Therapeutic Implications (Ed.: L. M. Khachigian), CRC Press,
Boca Raton, FL, 2005, pp. 95–113.
[8] M. R. Kurpiewski, M. Koziołkiewicz, A. Wilk, W. J. Stec, L.
Jen-Jacobson, Biochemistry 1996, 35, 8846–8854.
[9] S. A. Smith, L. W. McLaughlin, Biochemistry 1997, 36, 6046–
6058.
[10] T. M. Okonogi, S. C. Alley, E. A. Harwood, P. B. Hopkins,
B. H. Robinson, Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4156–
4160.
[11] P. R. Hardwidge, J. M. Zimmerman, L. J. Maher, Nucleic Acids
Res. 2002, 30, 1879–1885.
[12] K. F. Pirollo, A. Rait, L. S. Sleer, E. H. Chang, Pharmacol.
Ther. 2003, 99, 55–77.
[13] Y. S. Cho-Chung, C. A. Stein, A. M. Gewirtz (Eds.), “Thera-
peutic Oligonucleotides: Antisense, Rnai, Triple-Helix, Gene Re-
pair, Enhancer Decoys, Cpg, and DNA Chips” in Annals of the
New York Academy of Sciences, New York Academy of Sci-
ences, New York, 2004.
[14] M. A. Reynolds, R. I. Hogrefe, J. A. Jaeger, D. A. Schwartz,
T. A. Riley, W. B. Marvin, W. J. Daily, M. M. Vaghefi, T. A.
Beck, S. K. Knowles, R. E. Klem, L. J. Arnold Jr., Nucleic Ac-
ids Res. 1996, 24, 4584–4591.
[15] P. S. Miller, N. D. Annan, S. M. McParland, K. B. Pulford,
Biochemistry 1982, 21, 2507–2512.
[16] P. S. Miller, N. S. Dreon, M. Pulford, K. B. McParland, J. Biol.
Chem. 1980, 255, 9659–9665.
[17] M. Jaworska-Maslanka, W. Kacperczyk, D. Korczynski, Z. J.
Lesnikowski, Antisense Nucleic Acid Drug Dev. 1997, 7, 23–30.
[18] Z. J. Lesnikowski, M. Jaworska, W. J. Stec, Nucleic Acids Res.
1990, 18, 2109–2115.
[19] W. J. Stec, Z. Les´nikowski in Methods in Molecular Biology,
Protocols for Oligonucleotides and Analogs (Ed.: S. Agrawal),
Humana Press, Inc., Totowa, NJ, 1993.
[20] L. Tian, Ch. D. Claeboe, S. M. Hecht, S. Shuman, Structure
2004, 12, 31–40.
Solid-phase Synthesis of Chimeric Oligonucleotides 2 Using Dimeric
Building Block 4: Oligomers 2 and 8–13 were prepared on a 1 µmol
scale using an ABI 392 Synthesizer (Applied Biosystems Inc., Fos-
ter City, USA). Incorporation of the dimeric building blocks 4 re-
quired an increase in the coupling time to 180 s without any further
modifications of the protocol. Removal of the base-protecting
groups and cleavage from the solid support was achieved by treat-
ment of the CPG-bound oligomers with NH4OH/MeOH solution
(2:1 v/v) at room temperature for 8 h followed by HPLC purifica-
tion of oligomers (DMT-ON; ODS Hypersil column, Shandon,
UK, 210×4.6 mm, gradient 2–40% MeCN, 0.1  TEAB buffer,
pH 7.5). Standard removal of the 5Ј-DMT group was followed by
RP HPLC purification (ODS Hypersil C18, gradient 2–20%
MeCN, 0.1  TEAB buffer, pH 7.5). The purity of all oligomers
was confirmed by gel electrophoresis. Their structures were con-
firmed by MALDI-TOF spectrometry (see Table 1).
Melting Profiles and Thermodynamic Calculations: All absorption
measurements were carried out in a cell of 1 cm pathlength with a
UV/Vis 916 spectrophotometer equipped with a Peltier Thermocell
(GBC UV/Vis 916, GBC, Dandenong, Australia). The concentra-
tion of oligomers was determined spectrophotometrically by UV
absorbance at λmax in water using the extinction coefficients calcu-
lated by the published method.[48] The unmodified phosphodiester
oligonucleotides d(TC)5T (11) and r(UC)5U2Ј–OMe (12) as well as
chimeric oligonucleotides 2 were lyophilized and redissolved in
10 m Tris-HCl, 100 m NaCl, 10 m MgCl2 buffer (pH = 7.4)
and complementary templates were dissolved in the same buffer at
a total concentration of duplexes of 4 µ. Oligo-MePS 2, and the
appropriate templates were mixed together, heated at 90 °C for
5 min, and kept overnight at 4 °C. Melting profiles were recorded
between 3 and 95 °C in both directions with a temperature gradient
of 0.3 °C/min. The melting temperatures were calculated using the
first-order derivative method. Thermodynamic parameters were
obtained numerically by fitting an analytical curve resulting from
a two-state model to experimental melting profiles. The procedure
applied for fitting was a nonlinear least-squares method included
in the Sigma Plot software (version 5.01).
CD Spectra: CD spectra were recorded in the same buffer at a
concentration of 4 µ on
a CD6 dichrograph (Jobin–Yvon,
Longjumeau, France) using cells with a 5 mm pathlength, 2 nm
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P-Chirality in Methyloligoribonucleotides with Methylphosphonothioate Linkages
FULL PAPER
[21] L. Tian, Ch. D. Claeboe, S. M. Hecht, S. Shuman, Mol. Cell
2003, 12, 199–208.
[37] A. Grunweller, E. Wyszko, B. Bieber, R. Jahnel, V. A. Erd-
mann, J. Kurreck, Nucleic Acids Res. 2003, 31, 3185–3193.
[22] M. R. Kurpiewski, L. Engler, L. A. Wozniak, A. Kobylanska, [38] J. M. Kean, C. D. Cushman, H. M. Kang, T. E. Leonard, P. S.
M. Koziolkiewicz, W. J. Stec, L. Jen-Jacobson, Structure 2004,
Miller, Nucleic Acids Res. 1994, 22, 4497–4503.
[39] P. Auffinger, E. Westhoff, Angew. Chem. Int. Ed. 2001, 40,
4648–4650.
[40] L. A. Wozniak, W. J. Stec in Frontiers in Nucleosides and Nu-
cleic Acids (Ed.: R. F. Schinazi, D. C. Liotta), Informed Hori-
zons, LLC, Tucker, USA.
[41] W. Saenger, Principles of Nucleic Acid Structure, Springer-Ver-
lag, New York, 1984.
[42] S. Neidle (Ed.), Oxford Handbook of Nucleic Acid Structure,
Oxford University Press, Oxford, 1999.
[43] T. Popenda, E. Biala, J. Milecki, R. W. Adamiak, Nucleic Acids
Res. 1997, 25, 4589–4598.
[44] B. Norden, T. Kurucsev, J. Mol. Recognit. 1994, 7, 141–155.
[45] M. F. Summers, R. A. Byrd, K. A. Gallo, C. J. Samsom, G.
Zon, W. Egan, Nucleic Acids Res. 1985, 13, 6375–6386.
12, 1775–1788.
[23] L. A. Wozniak, W. Majzner, W. J. Stec, Arkivoc 2004, 101–110.
[24] S. Olejniczak, M. J. Potrzebowski, L. A. Wozniak, Eur. J. Org.
Chem. 2004, 1958–1966.
[25] L. A. Wozniak, M. Janicka, M. Bukowiecka-Matusiak, J. Or-
ganomet. Chem. 2005, 690, 2658–2663.
[26] D. Venkaterwarlu, K. E. Lind, V. Mohan, M. Manoharan,
D. M. Ferguson, Nucleic Acids Res. 1999, 27, 2189–2195.
[27] L. A. Wozniak Reviews on Heteroatom Chemistry, vol. 19 (Ed.:
Shigeru Oae), 1999, pp. 173–202.
[28] L. A. Wozniak, J. Pyzowski, M. Wieczorek, W. J. Stec, J. Org.
Chem. 1994, 59, 5843–5846.
[29] W. J. Stec, L. A. Wozniak, J. Pyzowski, W. Niewiarowski, Anti-
sense Res. Develop. 1997, 7, 381–395.
[30] L. A. Wozniak, M. Koziolkiewicz, A. Kobylanska, W. J. Stec, [46] E. M. Evertsz, R. Karsten, T. M. Jovin, Nucleic Acids Res.
Bioorg. Med. Chem. Lett. 1998, 8, 2641–2646.
[31] F. H. Hausheer, B. G. Rao, J. D. Saxe, U. C. Singh, J. Am.
Chem. Soc. 1992, 114, 3201–3206;
[32] H. C. P. F. Roelen, E. de Vroom, A. G. van der Marel, J. H.
van Boom, Nucleic Acids Res. 1988, 16, 7633–7645.
[33] D. Hamelberg, L. D. Williams, W. D. Wilson, Nucleic Acids
Res. 2002, 30, 3615–3623.
[34] M. Egli, Angew. Chem. Int. Ed. Engl. 1996, 35, 1894–1909.
[35] T. Hamma, A. Saleh, I. Huq, T. M. Rana, P. S. Miller, Bioorg.
Med. Chem. Lett. 2003, 13, 1845–1848.
1994, 22, 3293–3303.
[47] A. Velazquez-Campoy, I. Luque, E. Freire, Thermochim. Acta
2001, 380, 217–227.
[48] P. Lubini, W. Zurcher, M. Egli, Chem. Biol. 1994, 1, 39–45.
[49] Hypercube, Inc.; www.hyper.com.
[50] W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M.
Merz Jr., D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. W. Cald-
well, P. A. Kollman, J. Am. Chem. Soc. 1995, 117, 5179–5197.
Received: June 2, 2005
Published Online: October 12, 2005
[36] C. R. Allerson, N. Sioufi, R. Jarres, T. P. Prakhash, N. Naik,
A. Berdeja, L. Wanders, R. H. Gaffney, E. E. Swayze, B. Bhat,
J. Med. Chem. 2005, 48, 901–904.
Eur. J. Org. Chem. 2005, 5189–5197
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