Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage

Article abstract of DOI:10.1080/15257770.2011.581259

A complete series of the 2'-5' and 3'-5' regioisomeric types of r(ApA) and 2'-d(ApA) analogues with the-hydroxy-phosphonate C3'-O-P-CH(OH)-C4" internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5'-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5'-hydroxyl of the-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3'(2')-O-P-CH₂-C4" linkage lacking the 5'-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5'-carbon atom configuration. Potential usefulness of the-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.581259

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Synthesis and Properties of ApA
Analogues with Shortened Phosphonate
Internucleotide Linkage
Dr Šárka Králíková a , Dr Miloš Buděšínský a , Dr Ivan Barvík b , Dr
Milena Masojídková a , Dr Zdeněk Točík a & Ivan Rosenberg a
a Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic , Prague , Czech Republic
b Charles University, Faculty of Mathematics and Physics, Institute of
Physics , Prague , Czech Republic
Published online: 02 Sep 2011.
To cite this article: Dr Šárka Králíková , Dr Miloš Buděšínský , Dr Ivan Barvík , Dr Milena Masojídková
, Dr Zdeněk Točík & Ivan Rosenberg (2011) Synthesis and Properties of ApA Analogues with Shortened
Phosphonate Internucleotide Linkage, Nucleosides, Nucleotides and Nucleic Acids, 30:7-8, 524-543,
DOI: 10.1080/15257770.2011.581259
To link to this article: http://dx.doi.org/10.1080/15257770.2011.581259
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Nucleosides, Nucleotides and Nucleic Acids, 30:524–543, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.581259
SYNTHESIS AND PROPERTIES OF ApA ANALOGUES WITH
SHORTENED PHOSPHONATE INTERNUCLEOTIDE LINKAGE
1
1
2
1
ˇ
Sa´rka Kra´l´ıkova´, Milosˇ Budeˇsˇ´ınsky´, Ivan Barv´ık, Milena Masoj´ıdkova´,
Zdeneˇk Tocˇ´ık,1 and Ivan Rosenberg1
1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Prague, Czech Republic
2Charles University, Faculty of Mathematics and Physics, Institute of Physics, Prague,
Czech Republic
A complete series of the 2 ꢁ–5 ꢁ and 3 ꢁ–5 ꢁ regioisomeric types of r(ApA) and 2 ꢁ-d(ApA) analogues
2
with the α-hydroxy-phosphonate C3 ꢁ-O-P-CH(OH)-C4 ꢁꢁ internucleotide linkage, isopolar but non-
isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU
studied. Due to the chirality on the 5 ꢁ-carbon atom of the modified internucleotide linkage bearing
phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs.
To examine the role of the 5 ꢁ-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization,
the appropriate r(ApA) analogues with 3 ꢁ(2 ꢁ)-O-P-CH2-C4 ꢁꢁ linkage lacking the 5 ꢁ-hydroxyl were
synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the mod-
ified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable
differences in the stability of complexes with polyU, depending on the 5 ꢁ-carbon atom configura-
tion. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is
discussed.
Keywords α-Hydroxy-phosphonate linkage; ApA analogues; phosphonate internu-
cleotide bond; modified sugar-phosphate backbone; NMR conformational study; MDS
study
Received 16 March 2011; accepted 12 April 2011.
Part of the special issue in honor of Vladimir Efimov.
Support by the grant 202/09/0193 (Czech Science Foundation) and Research Centers
KAN200520801 (Acad. Sci. CR) and LC060061 (Ministry of Education, CR), under the Institute Research
Project Z40550506 is gratefully acknowledged, as well as the support by Project No. MSM 0021620835
(Ministry of Education, CR) and the META Centrum supercomputing facilities provided under the re-
search intent MSM 6383917201. The authors would also like to thank the staff of the Mass Spectrometry
Group of IOCB AS, v.v.i. (Dr. Josef Cvacˇka, Head) for HR-MS spectra.
Address correspondence to Ivan Rosenberg, Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo na´m. 2, 166 10 Prague 6, Czech Republic.
E-mail: ivan@uochb.cas.cz
524

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
INTRODUCTION
525
The synthesis of structurally diverse types of nucleoside phosphonic
acids, as well as phosphonate oligonucleotides with the P–C bridging link-
age, have been in focus of our attention for a long time.[1–10] We have
already reported on the synthesis of a novel type of isopolar non-isosteric
5ꢁ-nucleotide analogues[4,11,12] in which the 5ꢁ-phosphoryl group is replaced
with 5ꢁCH(OH)-P(O)(OH)2, a chiral α-hydroxy-phosphonate moiety.
Therefore, in order to obtain the basic data giving information about the
properties of the modified internucleotide linkage, in comparison with other
relevant types of linkages, we opted for the study of the thermal stability of
duplex- (or triplex-) like structures of the appropriately modified “dimers”
(i.e., diribonucleoside monophosphates) with polynucleotides using spec-
troscopic methods in view of the fact that a variety of modified dinucleoside
monophosphates[13] and their enantiomers,[14] neutral phosphotriester[15]
and methylphosphonate[16] dimers, and a series of the isopolar internu-
cleotide linkage-modified ApAs[17–20] have been employed as model com-
pounds to study changes in hybridization properties due to the presence of
particular modifications. These studies encouraged us to undertake a study
dealing with the following specific tasks: (i) the synthesis of a unique set
of phosphonate ApA analogues in ribo and 2ꢁ-deoxyribo series with chiral
α-hydroxy-phosphonate C3ꢁ-O-P-CH(OH)-C4ꢁꢁ internucleotide linkage and
non-chiral C3ꢁ(2ꢁ)-O-P-CH2-C4ꢁꢁ one, (ii) the hybridization of the modified
ApAs with polyU, (iii) the nuclear magnetic resonance (NMR) conforma-
tional investigation of the modified sugar-phosphate backbone, and (iv) the
structural characteristics obtainable on this topics from the evaluation by
molecular dynamics simulation (MDS).
We also expected the sum of obtained information to help decide on
the plausibility of a further extension of the project into syntheses of mod-
ified oligonucleotides that would require laborious synthesis of protected
ribonucleoside 5ꢁ-hydroxy-phosphonates, the monomeric units for the re-
verse order (5ꢁ to 3ꢁ end) oligonucleotide assembly. In addition, this work
is also experimentally related to our earlier reported study[21] dealing with
the kinetics and mechanisms of the isomerization of internucleosidic 3ꢁ-O-
P-CH2-5ꢁ and 3ꢁ-O-P-CH(OH)-5ꢁ linkages to their 2ꢁ,5ꢁ-counterparts.
RESULTS
Chemistry
The ribo-ApA phosphonates (R)-5, (S)-5, (R)-6, (S)-6 (Scheme 1) were syn-
thesized by the phospho-diester condensation of the protected phosphonic
acid 2 (R/S 19/81) with adenosine derivatives 3 and 4 in the presence N,Nꢁ-
dicyclohexylcarbodiimide (DCC) for 9 days. The high performance liquid

526
S. Kra´l´ıkova´ et al.
chromatography (HPLC) analyses of both reaction mixtures revealed, after
deprotection of products, the presence of all four isomers (R)-5, (S)-5, (R)-6,
(S)-6 in each reaction mixture, suggesting a rapid 2ꢁ ≈ 3ꢁ isomerization dur-
ing removal of the acid-labile protecting groups. Therefore, both mixtures
were combined and the pool of isomers (R)-5, (S)-5, (R)-6, and (S)-6 subjected
to separation by reversed phase HPLC.
SCHEME 1 The synthetic route to 2ꢁ,5ꢁꢁ and 3ꢁ,5ꢁꢁ r(ApA) containing the chiral C3ꢁ-O-P-CH(OH)-C4ꢁꢁ
linkage.
The synthesis of the 2ꢁ-deoxy-ApA phosphonates (R)-11, (S)-11 was
performed via nucleophilic addition of methyl-(H)-phosphonate 8 to
nucleoside-5ꢁ-aldehyde 9 (Scheme 2) in refluxing dichloromethane in the
presence of triethylamine. A similar type of the addition reaction has already
been published.[22] The obtained, protected dimer 10a was deprotected, and
the epimers (R)-11 and (S)-11 were resolved by reversed phase HPLC. Con-
figuration on the C5ꢁ was assigned based on the NMR spectra.
SCHEME 2 The synthetic route to 3ꢁ,5ꢁꢁ d(ApA) containing the chiral C3ꢁ-O-P-CH(OH)-C4ꢁꢁ linkage.
For the comparative study on the hybridization properties of polyU with
ApA analogs 5 and 6, the 2ꢁ–5ꢁꢁ and 3ꢁ–5ꢁꢁ ApA dimers 17 and 18 with
the 2ꢁ(3ꢁ)-O-P-CH2-5ꢁꢁ phosphonate internucleotide linkage lacking the 5ꢁꢁ-
hydroxy group (Scheme 3) were prepared. The key nucleoside phosphonate
15 was synthesized in a high yield by the nucleosidation reaction of silylated
6-N -benzoyladenine 13 with diethyl-[1-O-acetyl-2,3-di-O-benzoyl-5-deoxy-D-
5-ribofuranosyl]phosphonate[23] (12) in the presence of tin tetrachloride,
instead of trimethylsilyl triflate, as a Lewis acid catalyst. The transesterifi-
cation reaction of phosphonate 14 with bromotrimethylsilane followed by
hydrolysis of trimethylsilyl ester group under mild conditions afforded the
phoshonic acid 15, which was condensed with the 2ꢁ(3ꢁ)-O-acetyl derivatives

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
527
SCHEME 3 The synthetic route to 2ꢁ,5ꢁꢁ and 3ꢁ,5ꢁꢁ r(ApA) containing the non-chiral C3ꢁ-O-P-CH2-C4ꢁꢁ
linkage.
16 in the presence of DCC. The deprotection procedure followed by the
separation of crude dimers on Dowex 1 × 2 in acetate form afforded the
desired regioisomers with the 2ꢁ–5ꢁꢁ (17) and 3ꢁ–5ꢁꢁ (18) linkage, respectively.
Thermal Measurements
All prepared dimers were subjected to a study on their complexation
properties with polyU. The mixing curves with polyU and the respective
phosphonate analogue of ApA, measured at 3◦C and 260 nm, exhibited
their maximal hypochromicities at the 2U : 1A ratio in all cases (data not
shown). The obtained Tm values for all prepared dimers (R)-5, (S)-5, 17,
(R)-6, (S)-6, 18, (R)-11, and (S)-11 are summarized in Table 1.
DISCUSSION
NMR Conformational Study
Assignment of the configuration at C5ꢁ in the ribo compounds 5 and 6
and the deoxyribo epimers 11 by interpretation of NMR data is is closely con-
nected with the conformation around the C4ꢁ-C5ꢁ bond. The high value of
J (P,C3ꢁ) = 12.4–13.7 Hz, together with the low value of J (P,H4ꢁ) = 2.6–3.5 Hz
in one of the epimers 5, 6, and 11, indicate the trans-arrangement of P and
TABLE 1 Comparison of Tm and ꢀTm values for 2:1complexesa of polyU with ApA analogues
ribo 2ꢁ–5ꢁꢁ
ribo 3ꢁ–5ꢁꢁ
2ꢁ-deoxy 3ꢁ–5ꢁꢁ
(R)-5 (S)-5
17
r(ApA) (R)-6 (S)-6
18
r(ApA) (R)-11 (S)-11 d(ApA)
Tm (◦C)
5.2 20.3 15.6
12.6
—
15.6
10.6
8.2
15.7
—
3.2b
−3.8
7.2
+0.2
7.0
—
ꢀ Tm (◦C) −7.4 +7.7 +3.0
−0.1 −5.0 −4.4
a 0.15 mM (a total base concentration) solution in 50 mM-Tris/HCl buffer pH 7.6 with 10 mM Mg2+
b Tm value for polyU itself is about 3 ◦C.
.

528
S. Kra´l´ıkova´ et al.
FIGURE 1 The preferred C4ꢁ-C5ꢁ rotamers of epimeric 5ꢁ-hydroxy-phosphonate dimers 5, 6, and 11.
C3ꢁ which, together with a gauche relationship of H4ꢁ/H5ꢁ (as indicated by
small J (H4ꢁ,H5ꢁ) = 1.1–1.3 Hz), establishes the (S)-configuration at C5ꢁ in
these epimers (Figure 1). On the other hand, low values of J (P,C3ꢁ) = 1–2.4
Hz, J (P,H4ꢁ) = 2.1–3.2 Hz, and J (H4ꢁ,H5ꢁ) = 2.0–3.1 Hz in the second
epimer of 5, 6 and 11 show the gauche arrangement of all coupled nuclei
(P/C3ꢁ, P/H4ꢁ, and H4ꢁ/H5ꢁ) in agreement with (R)-configuration at C5ꢁ
and preferred conformation shown in Figure 1. The (S) and (R)-epimers
also differ remarkably in the geminal coupling constant of J (P,C4ꢁ) which
is very small (<1 Hz) for (S)- and large (≈10.8–12.2 Hz) for (R)epimer,
probably due to the different orientation (gauche and trans, respectively) of
the phosphorus atom versus the ring oxygen.
It is well known that in solution, the furanose ring in ribo- and deoxyri-
bonucleotides exists as an equilibrium mixture of conformers commonly
denoted as N-type (C3ꢁ-endo) and S-type (C2ꢁ-endo).[24,25]
Using a model assuming two rapidly interconverting conformers and
a generalized Karplus equation,[26] one can calculate the pseudorotation
parameters (phase angle P, puckering amplitude ꢁm, and molar fraction x)
of both conformers present[27,28] by means of the PSEUROT program.[29]
The calculated parameters for dimers 5, 6, 11, 20, and 21 are shown in Table 2
(data for the natural phosphodiester 2ꢁ–5ꢁꢁ and 3ꢁ–5ꢁꢁ ApAs are included for
comparison).
Remarkable differences in the values obtained for molar fractions of the
N-type furanose conformer in both 5ꢁ- and 2ꢁ(or 3ꢁ)-residues of dimers (xN
values ranging from 0.78 to 0.08) were checked in view of their possible
relation to the respective Tm characteristics. Interestingly, for all dimers
except for (R)-6 a good linear correlation (r2 = 0.94) was found between a
sum of molar fractions of the N-type furanose conformer of both residues,
on one hand, and the corresponding Tm values on the other, as it is shown
in Figure 2. The reason for anomalous behavior of (R)-6 is not clear. For
instance, molar fractions of sugar residues of (R)-6 can differ in the free
dimer and in a complex with polyU. Detailed analysis of conformational

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
529
FIGURE 2 The relation between a sum of N-type conformers in both residues of dimers. ApA(2ꢁ–5ꢁ),
ApA(3ꢁ–5ꢁ), 5, 6, 11, 17, 18, and corresponding Tm values.
properties of the structurally modified ApA-type dimers based on NMR data
at different temperatures is in progress. Nevertheless, the obtained data
confirmed the known fact that the higher is the content of furanose N-type
conformers, the higher are the related Tm values of complexes of ApA dimers
with polyU.
TABLE 2 Calculated pseudorotation parameters of furanose rings in dimers 5, 6, 11, 17, 18 (two-state
model; phase angle PN −36◦ to 72◦ (step 9◦) and PS 108◦ to 216◦; pucker amplitude φm(N) and φm(S)
in the range 34◦ to 42◦ (step 2◦); molar fractions xN and xS 0 to 1 (step 0.02))
3ꢁ-residue
xN PS
5ꢁ-residue
xN PS
Compound
PN
φ
m(N)
φ
m(S)
xS
PN
0
φ
m(N)
φ
m(S)
xS
ApA(2ꢁ–5ꢁ)
ApA(3ꢁ–5ꢁ)
(S)-5
(R)-5
(S)-6
9
40
36
38
34
42
38
42
42
42
38
0.42 162
0.60 180
0.78 180
0.20 162
0.14 162
0.24 153
0.10 180
0.18 180
0.46 153
0.16 153
34
36
36
38
40
38
42
42
34
40
0.58
0.40 18
0.22
36
34
36
36
40
42
42
42
40
40
0.58 162
0.58 162
0.74 162
0.10 162
0.48 171
0.08 162
0.34 180
0.18 189
0.54 153
0.34 135
36
40
40
36
40
36
34
42
40
36
0.42
0.42
0.26
0.90
0.52
0.92
0.66
0.82
0.46
0.66
−9
18
9
27
27
0
−9
−9
0
−18
0.80 −9
0.86 36
(R)-6
0.76
9
(S)-11
(R)-11
17
0.90 −9
0.88 −9
0.54 18
18
0.84
0

530
S. Kra´l´ıkova´ et al.
Thermal Stability
All the prepared dimers (R)-5, (S)-5, 17, (R)-6, (S)-6, 18, (R)-11, and (S)-11
exhibited, in complex with polyU, a maximum hyperchromicity at the 2U:1A
stoichiometry, suggesting the presence of triplex-like complexes. Similar
results, in fact, were also obtained with both natural ApAs and also with a
series of another, earlier studied phosphonate analogues of ApA.[18–21] In
all cases, the thermal characteristics showed single transition profiles only
(data not shown), with considerably different Tm values (Table 1).
The measurements of thermal stability of the 2U:1A complexes revealed
that in the 2ꢁ–5ꢁꢁ series, the complex stability expressed in Tm value strongly
depended on the configuration of the 5ꢁꢁ carbon atom bearing the hydroxy
group and the phosphorus moiety. Whereas the (S)-5 epimer formed a much
more stable complex with polyU than the natural r(ApA), indeed the most
stable one of all the dimers studied, the (R)-5 epimer, formed a very weak
complex at the limit of detectability.
Surprisingly, in the 3ꢁ–5ꢁꢁ series, the complex of the (R)-6 epimer with
polyU was found as stable as that of natural r(ApA) itself, whereas the (S)-6
epimer formed a much weaker complex with polyU.
Concerning these latter findings, it is worth mentioning that with the
2ꢁ-deoxyoligonucleotides (15 mers) incorporating the same internucleotide
linkage modifications in the chain, the presence of (S)-configured 3ꢁ–5ꢁꢁ
linkage had a very strong stabilizing effect upon the complex stability whereas
the presence of (R)-configured one destabilized the complex totally.[10]
The complicated relationships in the ribo series might be explained,
just intuitively, by a steric conflict between the 5ꢁꢁ hydroxy group of the
hydroxyphosphonate linkage and the 2ꢁ (or 3ꢁ) hydroxyls. A more detailed
study will be attempted in this matter.
Concerning the complexation properties of the ribo ApA phosphonate
analogues 17 (2ꢁ–5ꢁꢁ) and 18 (3ꢁ–5ꢁꢁ) containing the non-chiral 3ꢁ(2ꢁ)-O-P-
CH2-5ꢁꢁ internucleotide linkage (i.e., lacking the 5ꢁꢁ hydroxyl), the dimer
17 (2ꢁ–5ꢁꢁ) exhibited, in contrast to 18 (3ꢁ–5ꢁꢁ), the Tm value identical with
that of the natural ApA (3ꢁ–5ꢁꢁ). Lower Tm values obtained for the reference
dimers 17 and 18, in comparison with their “hydroxy” congeners (S)-5 and
(R)-6, respectively, demonstrated clearly the importance of the 5ꢁꢁ hydroxy
group of the hydroxyphosphonate linkage, as was anticipated.
With the deoxyribo counterparts, both 3ꢁ–5ꢁꢁd(ApA) analogues, that
is, the epimers (R)-11 and (S)-11, exhibited a very low thermal stability in
comparison with that of the r(ApA) analogues 6 but still comparable with
that of the natural d(ApA). On the other hand, the dimer (R)-11 with a Tm
value of 3.2◦C did not form any detectable complex with polyU, as the Tm
value of polyU itself is just about 3◦C.

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
531
Last, the role of counterions in measuring the thermal characteristics
must be mentioned. Obviously, the formation of triplex-like complexes be-
tween all the respective dimers and polyU strongly depends on the Mg2+
ions concentration. We found that below a 10 mM concentration (at total
base concentration of 0.15 mM), no sharp transition profiles could be ob-
tained. The replacement of Mg2+ ions for a concentration 10 times higher in
monovalent sodium ions, completely disabled the formation of complexes.
The same effect was observed when Mg2+ and Na+ ions were used jointly
at the concentrations of 10 and 100 mM, respectively; sodium ions strongly
compete with Mg2+ ones.
Molecular Dynamics Simulations
Molecular dynamics (MD) simulations were run for two model systems
to shed some light on the experimental results. We focused on comparison
of the 2ꢁ–5ꢁ (S)-5 and (R)-5 ApA dimers, where the largest differences in
melting temperatures were observed. Each model system consisted of two
anti-parallel uridine dodecamers and a pseudostrand created by six ApA
analogues (either (R)-5 or (S)-5), which were bound by Watson-Crick and
Hoogsteen hydrogen bonds to the first and second rU12 strands, respectively
(see Figure 3).
Three MD simulations at different temperatures of 300 K, 350 K, 400 K
were run for each of the two model systems. MD simulations reached a length
of 100 ns through the use of computing power of NVIDIA graphics process-
ing units (GPUs) in connection with the ACEMD software package.[30,31]
The results of MD simulations have proved to be in excellent agreement
with experimental results (NMR, melting temperatures). All MD simula-
tions were launched from a conformer with the trans arrangement of P and
C3ꢁ atoms (see Figures 1 and 4), which predominantly persisted in the case of
(S)-5 modified internucleotide linkages (partly due to stabilization through
the intramolecular hydrogen bond between the O3ꢁ atom and 5ꢁꢁ hydroxyl
group). Nevertheless, in the case of (R)-5 modified internucleotide linkages
transitions to the gauche arrangement of P and C3ꢁ atoms usually occurred
(see Figures 1 and 4). Transitions of modified internucleotide linkages from
trans to gauche were accompanied by interconversions of riboses from the
north (C3ꢁ-endo) to south (C2ꢁ-endo) conformations.
Both simulated systems behaved qualitatively similarly, considering their
in silico thermal stability, but the trans arrangement of modified internu-
cleotide linkages was undoubtedly a little bit more compatible with the
triple helical structure. All MD simulations were carried out above the ex-
perimental melting point. Nevertheless, simulated systems remained more
or less stable at 300 K on the 100 ns time scale. In contrast, both triple helical
complexes were almost completely disintegrated at 400 K. This happened
faster for the (R)-5 than for (S)-5 simulated system.

532
S. Kra´l´ıkova´ et al.
FIGURE 3 Model system consisting of two anti-parallel uridine dodecamers and a pseudostrand created
by six (S)-5 ApA analogues (alternatively depicted in MAGENTA and YELLOW for clarity), which are
bound by Watson-Crick and Hoogsteen hydrogen bonds to the first (CYAN) and second (GREEN) rU12
strands.
FIGURE 4 The preferred conformers of (S)-5 (left) and (R)-5 (right) ApA analogues. Note that the
conformation of C4ꢁ-C5ꢁ rotamers are in agreement with those determined by NMR (see Figure 2).

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
533
Detailed analysis of MD simulations including ion stabilization of triple
helical structures will be published elsewhere.
CONCLUSION
Studies on thermal stabilities of complexes of polyU with a series of
structurally similar phosphonate analogues of ApA dimer containing chiral
3ꢁ(2ꢁ)-O-P-(HO)CH-5ꢁꢁ linkage showed remarkable differences in Tm values.
They correlate to a very high extent with the sum of molar fractions of the
N-type furanose conformer of both residues: the higher Tm value, the higher
sum of the N-type conformers. These findings were consistent with those ob-
tained from the molecular dynamic simulations. Comparing hybridization
abilities of the 2ꢁ,5ꢁ and 3ꢁ,5ꢁ regioisomeric dimers, quite opposite differences
in affinity towards polyU were found in the dependence on chirality on the 5ꢁ
carbon atom. Similarly, in the 2ꢁ-deoxy series the C5ꢁ chirality also played cru-
cial role in the affinity d(ApA) toward polyU. We may conclude, therefore,
that it is worth devoting an effort to the synthesis of protected nucleoside
5ꢁ-hydroxy-phosphonates and, thus, opening the way for the preparation of
chimeric oligonucleotides in the ribo and deoxyribo series bearing both
phosphodiester and the 3ꢁ(2ꢁ)-O-P-(HO)CH-5ꢁꢁ internucleotide linkages. We
believe that superior resistance of the linkage against nucleases, together
with its potential to increase hybridization, could bring a new quality into
the area of oligonucleotides usable in antisense technologies. The synthe-
sis of suitably protected reversed monomers in deoxyribo and ribo series,
compatible with the solid-phase oligonucleotide synthesis, is underway.
MATERIALS AND METHODS
Chemistry
The solvents were evaporated at 40◦C and 2 kPa and the products were
dried over phosphorus pentoxide at 50–70◦C and 13 Pa. The course of the
reactions was checked by thin layer chromatography (TLC) cards (Fluka,
Switzerland) and the products were detected by UV monitoring. For flash col-
umn chromatography, silica gel 40–60 µm (Fluka) was used. TLC and silica
gel chromatography were carried out in the following solvent systems (v/v):
chloroform–ethanol 9:1 (C1); ethyl acetate-acetone-ethanol-water 4:1:1:1
(H1); 2-propanol—concentrated aqueous ammonia—water 7:1:2 (I), for
charged compounds; EtOAc-toluene 1:1 (T1); EtOAc-toluene 1:4 (T2). An-
alytical HPLC was performed on Nucleosil 100-5 C18 (4.6 × 150 mm col-
umn; Macharey-Nagel) using a linear gradient of methanol in 0.1M TEAA.
Preparative reversed phase chromatography was carried out on octadecyl sil-
ica column (25 × 250 mm, 20 µm, IOCB Prague); compounds were eluted
with a linear gradient of methanol in 0.1M-TEAA at 10 ml/min. UV spectra

534
S. Kra´l´ıkova´ et al.
and thermal characteristics were taken on Cary Bio 100 (Varian, Australia)
spectrophotometer. High-resolution FAB mass spectra were recorded on the
ZAB-EQ (VG Analytical, UK) instrument with glycerol and thioglycerol as
matrices. NMR spectra were measured on a Varian UNITY-500 spectrometer
(1H at 500 MHz; 13C at 125.7 MHz frequency) in d6-DMSO and/or D2O at
20◦C. The signals were referenced either to solvent signal (converted to δ
scale using relations δH(DMSO) = 2.50 and δC(DMSO) = 39.7 ppm) or to
DSS (in D2O). Proton 2D-COSY spectra were used for the structural assign-
ment of coupled protons and 2D-ROESY spectra for detection of the NOE
contacts. Carbon-13 chemical shifts and coupling constants J (C,P) were ob-
tained from broadband proton-decoupled spectra using standard and APT
pulse sequences.
The structure of all compounds was confirmed by NMR spectra. 1H NMR
data of monomers 2, 12, 14, 17, and 18a are presented in the Experimental
1
section, together with the syntheses of these compounds while the H and
13C NMR parameters of dimers 5, 6, 11, 17 and 18 are summarized in Tables 3
and 4. The 2ꢁ–5ꢁꢁ and 3ꢁ–5ꢁꢁ regioisomers could be distinguished easily either
in 1H NMR spectra on the basis of vicinal coupling of phosphorus to H2ꢁ(in
5) and/or H3ꢁ (in 6, 11) (see Table 3) or in 13C NMR spectra from vicinal
coupling of phosphorus to carbon atoms C1ꢁ (in 5) and/or C4ꢁ (in 6, 11)
(see Table 4).
5ꢀ-O-Acetyl-6-N-benzoyl-2ꢀ,3ꢀ-O-isopropylideneadenosin-5ꢀ-
C-ylphosphonic acid (2)
Dimethylphosphonate 1a (4) (R/S 17/83) (1.268 g, 2.44 mmol) was
treated with acetic anhydride (1.15 ml, 12.20 mmol) and DMAP (50 mg,
0.41 mmol) in pyridine (20 ml) at room temperature under exclusion of
moisture until the starting compound disappeared (15 hours; TLC in C1
and H1). The reaction mixture was diluted with 1M-triethylammonium hy-
drogen carbonate (TEAB, 20 ml), extracted with chloroform (100 ml), and
the organic layer was washed with 1M TEAB (3 × 50 ml) and dried over an-
hydrous Na2SO4. The solvent was evaporated in vacuo and the residue was
co-distilled with toluene (2 × 20 ml) and treated with bromotrimethylsilane
(1.61 ml, 12.20 mmol) and 2,6-lutidine (1.42 ml, 12.20 mmol) in acetoni-
trile (25 ml) for 20 hours (TLC in C1 and H1). Triethylamine (0.68 ml,
4.88 mmol) was added, the mixture was evaporated to dryness, and the
product was purified by reversed phase chromatography (elution with linear
gradient of methanol in water). Yield, 934 mg (60%) of triethylammonium
salt of 2 (R:S = 19:81). HR FAB calcd for C22H24N5O9PNa (M + Na)+
556.12095, found 556.12161.
(R)-2: 1H NMR (DMSO, 500 MHz) δ 11.00 (1H, bs, NH), 8.53 and 8.14
(2 × 1H, 2 x s, H-2 and H-8), 7.97 (2H, m, Ar-H), 7.61 (2H, m, Ar-H), 7.51
(1H, m, Ar-H), 6.09 (1H, d, J(1ꢁ,2ꢁ) = 2.0 Hz, H-1ꢁ), 5.32 (1H, dd, J(2ꢁ,1ꢁ) =

535

536

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
537
2.0 Hz, J(2ꢁ,3ꢁ) = 6.3 Hz, H-2ꢁ), 5.21 (1H, dd, J(3ꢁ,2ꢁ) = 6.3 Hz, J(3ꢁ,4ꢁ) = 2.7
Hz, H-3ꢁ), 5.16 (1H, dd, J(5ꢁ,4ꢁ) = 7.6 Hz, J(5ꢁ,P) = 11.2 Hz, H-5ꢁ), 4.42 (1H,
ddd, J(4ꢁ,3ꢁ) = 2.7 Hz, J(4ꢁ,5ꢁ) = 7.6 Hz, J(4ꢁ, P) = 4.5 Hz, H-4ꢁ), 1.81 (3H, s,
OAc), 1.51 and 1.30 (2 × 3H, 2 x s, 2 x CH3).
(S)-2: 1H NMR (DMSO, 500 MHz) δ 11.00 (1H, bs, NH), 8.51 and 8.05
(2 × 1H, 2 x s, H-2 and H-8), 7.97 (2H, m, Ar-H), 7.52 (1H, m, Ar-H), 7.43
(2H, m, Ar-H), 5.91 (1H, d, J(1ꢁ,2ꢁ) = 3.9 Hz, H-1ꢁ), 5.37 (1H, dd, J(2ꢁ,1ꢁ) =
3.9 Hz, J(2ꢁ,3ꢁ) = 6.3 Hz, H-2ꢁ), 5.16 (1H, dd, J(3ꢁ,2ꢁ) = 6.3 Hz, J(3ꢁ,4ꢁ) = 2.7
Hz, H-3ꢁ), 5.12 (1H, dd, J(5ꢁ,4ꢁ) = 3.2 Hz, J(5ꢁ,P) = 12.2 Hz, H-5ꢁ), 4.50 (1H,
td, J(4ꢁ,3ꢁ) = 2.7 Hz, J(4ꢁ,5ꢁ) = J(4ꢁ, P) = 3.2 Hz, H-4ꢁ), 1.93 (3H, s, OAc),
1.52 and 1.30 (2 × 3H, 2 x s, 2 x CH3).
2ꢀ-Deoxyadenosin-2ꢀ-yl adenosin-5ꢀꢀ-C-ylphosphonate (5)
and 3ꢀ-deoxyadenosin-3ꢀ-yl adenosin-5ꢀꢀ-C-ylphosphonate (6)
Method A
A mixture of triethylammonium salt of phosphonate 2 (R:S = 19:81)
(450 mg, 0.71 mmol) and 6-N -benzoyl-3ꢁ,5ꢁ-O-(tetraisopropyldisiloxan-1,3-
diyl)adenosine (3) (523 mg, 0.85 mmol) was treated with DCC (0.732 g,
3.55 mmol) in pyridine (4 ml) at room temperature overnight, and then
concentrated to a thick oil which was set aside at room temperature for
6 days (disappearance of the starting phosphonic acid 2; TLC in H1). Re-
action mixture was diluted with 60% aqueous pyridine (10 ml) and, after
2 hours of standing, concentrated in vacuo. Purification by silica gel chro-
matography (elution with a linear gradient of H3 in ethyl acetate, followed
by H1 in H3) afforded a protected dimer that was treated with ethanol-conc.
aqueous ammonia mixture (100 ml, 50/50) for 48 hours to remove acetyl
and benzoyl protecting groups. After evaporation of the solvent, the residue
was co-distilled with ethanol (3 × 25 ml) and dry toluene (2 × 15 ml) and
finally treated with 0.5M TBAF in tetrahydrofurane (8.5 ml, 4.25 mmol).
The solution was kept at room temperature under exclusion of moisture
(TLC in systems H1 and I) for 24 hours, then diluted with an equal volume
of water, treated with Dowex 5 (Et3N+ form) in 50% aqueous ethanol to
remove tetra-n-butylammonium cations, and evaporated. The solid product
was set aside for 18 hours with 0.025M sulfuric acid (100 ml) to remove the
isopropylidene protecting group, and sulfuric acid was removed on Dowex
50 × 2 column (H+ form) by elution with water. The ion exchanger was then
eluted with 2.5% aqueous ammonia to release the mixture of compounds 5
and 6.
Method B
Triethylammonium salt of 2 (R:S = 19:81) (465 mg, 0.73 mmol) and
6-N -benzoyl-2ꢁ-O-(1-(2-chloroethoxy)ethyl)-5ꢁ-O-dimethoxytrityladenosine

538
S. Kra´l´ıkova´ et al.
(4) (684 mg, 0.88 mmol) were treated and worked up according to Method
A excluding TBAF treatment.
Separation
The epimeric mixtures of compounds 5 and 6 obtained in both Methods
A and B were combined and separated on C-18 HPLC column by a gradient
elution of 0–50% methanol in 0.1M-TEAA). All epimers, (R)-5, (S)-5 and
(R)-6, (S)-6 were obtained in a HPLC pure form. Each single epimer was first
desalted on C-18 column (elution with water) and then converted into the
sodium salt on Dowex 50×2 (Na+ form). Freeze-drying from water afforded
(R)-5 (36 mg, 4%), (S)-5 (35 mg, 4%), (R)-6 (101 mg, 12%) and (S)-6 (62 mg,
7%). HR FAB calcd for C20H26N10O10P (M + H)+ 597.15713, found for (R)-5
597.15656, for (S)-5 597.15666, for (R)-6 597.15697, and for (S)-6 597.15618.
2ꢀ,3ꢀ-Dideoxyadenosin-3ꢀ-yl deoxyadenosine-5ꢀꢀ-C-ylphosphonate
((R)-11) and ((S)-11)
6-N -Benzoyl-5ꢁ-O-tert-butyldiphenylsilyl-2ꢁ-deoxyadenosine (28) (594 mg,
1 mmol) and diisopropylethylamine (0.684 ml, 4 mmol) in tetrahydrofurane
(5 ml) were treated with chloro-N ,N -diisopropylamino methoxyphosphine
(29) (0.384 ml, 2 mmol) under argon (TLC in a system of 10% triethylamine
in T1, or in C1). After 30 minutes, the reaction mixture was diluted with
ethyl acetate (30 ml), and the organic layer was washed with a cold saturated
solution of sodium hydrogen carbonate (4 × 10 ml) and dried over Na2SO4.
The solvent was evaporated, the residue was co-distilled with toluene (3 ×
20 ml) to remove diisopropylethylamine, and then treated with tetrazole
(containg 10% of water) (0.77 g, 10 mmol) in acetonitrile (20 ml). After
20 minutes, the reaction mixture was diluted with ethyl acetate (100 ml),
the organic layer was washed with water (3 × 100 ml) and then dried over
Na2SO4. The solvent was evaporated and methyl H-phosphonate 8 was dried
by co-distillation with toluene (2 × 10 ml). This product was used for further
reaction without characterization.
Reaction of Nucleoside 5ꢀ-aldehyde with H-phosphonate 8
6-N -Benzoyl-3ꢁ-O-tert-butyldiphenylsilyladenosine-5ꢁ-aldehyde (9), pre-
pared from 6-N -benzoyl-3ꢁ-O-tert-butyldiphenylsilyladenosine[4] (594 mg,
1 mmol), was dried by co-distillation with toluene (2 × 10 ml) and dissolved
in dichloromethane (5 ml) under exclusion of moisture. This solution was
added via septum under argon to methyl H-phosphonate 8 followed by tri-
ethylamine (0.139 ml, 1 mmol). The reaction mixture was heated at 50◦C
for 3 days (TLC in C1 and H1). Then it was concentrated and the residue
was treated with 60% aqueous pyridine (20 ml) at 50◦C for 2 days (TLC in
H1). The solution was then concentrated and treated with Dowex 50 × 2
(pyridinium form) in 60% aqueous pyridine to remove N -methylpyridinium

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
539
cations. The suspension was filtered, the filtrate was evaporated, and the
residue was co-distilled with ethanol (3 × 20 ml). The product was purified
on silica gel (elution with a linear gradient of H3 in ethyl acetate followed
by H1 in H3) to afford 1.04 g (83%) of a TLC-pure, protected dimer 10b
(R/S 1/6). The removal of N -benzoyl and O-silyl protecting groups was per-
formed as described for compounds 5 and 6. The deprotected crude product
11 was purified on DEAE–Sephadex (HCO3− form) by elution with a linear
gradient of 0–0.2 M TEAB in water, followed by preparative reversed phase
HPLC (elution with linear gradient of 0–50% methanol in 0.1 M TEAA)
to afford both epimers (R)-11 and (S)-11. Each epimer was desalted on a
reversed phase column (elution with water) and converted into the sodium
salt on Dowex 50 × 2 (Na+ form). Freeze drying from water afforded 245 mg
(43%) of (S)-11 and 43 mg (8%) of (R)-11 (yields are related to the starting
nucleoside). HR FAB calcd for C20H26N10O8P (M + H)+ 565.16730, found
565.16801.
2ꢀ-Deoxyadenosin-2ꢀ-yl deoxyadenosin-5ꢀꢀ-C-ylphosphonate (17)
and 3ꢀ-deoxyadenosin-3ꢀ-yl adenosin-5ꢀꢀ-C-ylphosphonate (18)
Reaction of 12 with Silylated 6-N-Benzoyladenine (13)
6-N -Benzoyladenine (239 mg, 1 mmol) in hexamethyldisilazane (10 ml,
47 mmol) was refluxed in the presence of trimethylsilyl chloride (1 ml,
8 mmol) at 150◦C under stirring and exclusion of moisture for 10 hours.
Volatiles were removed under reduced pressure and the resulting oil was
co-distilled with xylene (2 × 10 ml) and acetonitrile (2 × 10 ml) to remove
traces of HMDS. A solution of diethyl-1-O-acetyl-2,3-di-O-benzoyl-5-deoxy-
D-ribofuranos-5-C-yl-phosphonate (12)[32](521 mg, 1 mmol) was added via
septum under argon to the silylated base followed by the addition of tin
tetrachloride (0.24 ml, 2 mmol). The mixture was kept at room tempera-
ture for 24 hours (TLC in C1, H1). The reaction mixture was then diluted
with pyridine (1 ml) and toluene (10 ml) and stirred overnight. The thick
suspension (a precipitated complex of tin tetrachloride with pyridine) was
filtered through Celite, the precipitate was washed with chloroform, and the
solvents were removed under diminished pressure. The product was puri-
fied by reversed phase chromatography (elution with a linear gradient of
methanol in water). Yield, 625 mg (89%) of diethyl 2ꢁ,3ꢁ,6-tri-N,O-benzoyl-5ꢁ-
deoxyadenosin-5ꢁ-C-ylphosphonate (14). HR FAB calcd for C35H34N5O9PNa
(M + Na)+ 722.19920, found 722.19849. 1H NMR (DMSO, 500 MHz) δ 11.30
(1H, bs, NH), 8.82 (2H, s, H-2 and H-8), 8.05 (2H, d, Ar-H), 8.01 (2H, d,
Ar-H), 7.78 (2H, d, Ar-H), 7.70 (1H, t, Ar-H), 7.65 (1H, t, Ar-H), 7.61 (1H,
t, Ar-H), 7.56 (2H, t, Ar-H), 7.53 (2H, t, Ar-H), 7.39 (2H, t, Ar-H), 6.64
(1H, d, J(1ꢁ,2ꢁ) = 5.6 Hz, H-1ꢁ), 6.56 (1H, bt, J(2ꢁ,1ꢁ) = J(2ꢁ,3ꢁ) = 5.7 Hz,
H-2ꢁ), 6.03 (1H, dd, J(3ꢁ,2ꢁ) = 5.7, J(3ꢁ,4ꢁ) = 4.3 Hz, H-3ꢁ), 4.73 (1H, dtd,
J(4ꢁ,3ꢁ) = 4.3, J(4ꢁ,5ꢁ) = J(4ꢁ,5ꢁꢁ) = 6.9, J(4ꢁ,P) = 9.4 Hz, H-4ꢁ), 3.95 (4H,

540
S. Kra´l´ıkova´ et al.
m, P(OCH2CH3)2), 2.66 (2H, dd, J(5ꢁ,4ꢁ) = 6.9, J(5ꢁ,P) = 18.4 Hz, H-5ꢁ and
H-5ꢁꢁ), 1.17 and 1.09 (2 × 3H, 2 x t, J(CH3,CH2) = 7.1 Hz, P(OCH2CH3)2).
Partial Deprotection of 14
The carefully dried phosphonate 14 was treated with bromotrimethyl-
silane (0.70 ml, 5.34 mmol) and 2,6-lutidine (0.62 ml, 5.34 mmol) in ace-
tonitrile (10 ml) at room temperature for 24 hours (TLC in C1, H1). The
reaction mixture was concentrated in vacuo, 2M-TEAB (5 ml) was added, and
the resulting solution was evaporated. The product was purified by reversed
phase chromatography (elution with linear gradient of methanol in water).
Yield, 500 mg (75%) of triethylammonium salt of 6,2ꢁ,3ꢁ-tri-N ,O-benzoyl-5ꢁ-
deoxyadenosin-5ꢁ-C-ylphosphonate (15). HR FAB calcd for C31H27N5O9P
(M + H)+ 644.15466, found 644.15553.
Condensation of 15 with 16
A mixture of triethylammonium salt of the phosphonic acid
15 (500 mg, 0.67 mmol) and 2ꢁ,(3ꢁ)-O-acetyl-6-N -benzoyl-5ꢁ-O-tert-
butyldiphenylsilyladenosine (16) (556 mg, 0.87 mmol) was treated with DCC
(10 equiv) for 9 days under conditions given for the synthesis of compounds
5 and 6. The reaction mixture was then diluted with 60% aqueous pyri-
dine (10 ml) and, after 1 hours of standing, evaporated. The residue was
co-distilled with ethanol (2 × 20 ml) and toluene (2 × 10 ml), and finally
treated with 0.5M TBAF in tetrahydrofurane (8.7 ml, 4.35 mmol) at room
temperature under stirring and exclusion of moisture for 42 hours (TLC
in H1, I). After evaporation of the solvent, the residue was treated with
Dowex 50 × 2 (Et3N+ form) in 50% aqueous ethanol to remove tetra-n-
butylammonium cations, and then with a mixture of ethanol (50 ml) and
concentrated aqueous ammonia (50 ml). The suspension was stirred for 3
days, evaporated to dryness, and the residue suspended in 3% aqueous am-
monia (50 ml). The formed dicyclohexylurea was filtered off and the filtrate
was evaporated. The crude product was purified on DEAE-Sephadex A25
(elution with a linear gradient of 0–0.2 M TEAB) and then on Dowex 1 × 2
(CH3COO− form) column (elution with a linear gradient of 0–0.5M acetic
acid in water) to resolve regioisomers. Conversion into the sodium salt on
Dowex 50 × 2 (Na+ form) followed by freeze drying from water afforded
2ꢁ–5ꢁꢁ-isomer 17 (118 mg, 30%) and 3ꢁ–5ꢁꢁ-isomer 18 (27 mg, 7%). HR FAB
calcd for C20H26N10O9P (M + H)+ 581.16222, found 581.16311.
Molecular Dynamics Simulations—Methodology
MD simulations are a well-established tool for the investigation of struc-
tural features of biomolecules at the atomic detail level. GPUs are now able to
push MD trajectories on time scales, which until recently were only available
in supercomputing facilities.[31,32]

ApA Analogues with Shortened Phosphonate Internucleotide Linkage
541
There are two model triplex structures, consisting of dodecamer nucleic
acids chains, to be studied using fully solvated MD. Each model system con-
sists of two mutually antiparallel uridine dodecamers and a pseudostrand
created by six ApA-dimers ((R)-5, (S)-5) bound by Watson-Crick and Hoog-
steen hydrogen bridges to the first and second rU12 strands, respectively.
Explicit Na+ counterions were placed at the phosphates/phosphonates of
the nucleotides by the TLEAP module of AMBER 10.0.[33,34] Nucleic acids
with Na+ counterions were surrounded by ∼4.000 TIP3P water molecules,
˚
which extended over a distance of approximately 10 A (in each direction)
from the nucleic acid atoms. This leads to a periodic box size of ∼70 ×
3
˚
∼60 × ∼55 A .
The AMBER force field does not contain the force constants needed to
describe the modified parts of the internucleotide linkages. The force field
parameters were therefore completed with those obtained from the ab initio
calculations consistently with the original approach.[35]
Simulated systems were initially equilibrated by preliminary NPT MD
runs (20 ps) computed using the NAMD2 software package and stan-
dard CPUs.[36,37] Production NVT MD runs (100 ns) were performed with
ACEMD[31,32] on NVIDIA graphics processors. The usual computational
procedures were used. The particle Mesh Ewald summation method was
employed for electrostatic interactions.[38] The hydrogen mass was reparti-
tioned to achieve a 4 fs time step without affecting the equilibrium distribu-
tion. Data were recorded every 40 ps.
MD trajectories were visualized in the VMD software package[39,40] and
quantitatively examined (interatomic distances, torsion angles, sugar puck-
ering, etc.) by using the AMBER module PTRAJ.[34,35] Figures were produced
using CHIMERA.[41,42]
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