Stereochemistry of internucleotide bond formation by the H-phosphonate method. 1. Synthesis and 31P NMR analysis of 16 diribonulceoside (3′-5′)-H-phosphonates and the corresponding phosphorothioates

Article abstract of DOI:10.1080/15257770500265729

□ Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ³¹

Full text of DOI:10.1080/15257770500265729

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Stereochemistry of Internucleotide Bond Formation by
the H-Phosphonate Method. 1. Synthesis and 31P Nmr
Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates
and the Corresponding Phosphorothioates
Michal Sobkowski a , Jadwiga Jankowska a , Adam Kraszewski a & Jacek Stawinski b c
a Institute of Bioorganic Chemistry, Polish Academy of Sciences , Poznan, Poland
b Institute of Bioorganic Chemistry, Polish Academy of Sciences , Poznan, Poland
c Department of Organic Chemistry , Arrhenius Laboratory, Stockholm University ,
Stockholm, Sweden
Published online: 16 Aug 2006.
To cite this article: Michal Sobkowski , Jadwiga Jankowska , Adam Kraszewski & Jacek Stawinski (2005) Stereochemistry
of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside
(3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12,
1469-1484
To link to this article: http://dx.doi.org/10.1080/15257770500265729
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1469–1484, 2005
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500265729
STEREOCHEMISTRY OF INTERNUCLEOTIDE BOND FORMATION BY
THE H-PHOSPHONATE METHOD. 1. Synthesis and 31P NMR
Analysis of 16 Diribonulceoside (3 -5 )-H-Phosphonates and the
Corresponding Phosphorothioates
ᮀ
MichalSobkowski, Jadwiga Jankowska, and Adam Kraszewski
Institute
of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
ᮀ
Jacek Stawinski
Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Poznan, Poland, and Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, Stockholm, Sweden
Sixteen diribonucleoside (3ꢀ-5ꢀ)-H-phosphonates were synthesized via condensation of the
ᮀ
protected ribonucleoside 3ꢀ-H-phosphonates with nucleosides, and the influence of a nucleoside
sequence on the observed stereoselectivity was analyzed. 31P NMR spectroscopy was used to evaluate
a relationship between chemical shift and absolute configuration at the phosphorous center of the
H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the
most cases such correlation was found, there was however several exceptions to the rule where the
relative positions of resonances arising from RP and SP diastereomers were reversed.
Keywords H-Phosphonates; Phosphorothioates; Nucleotides; Stereoselective coupling;
31P NMR spectroscopy
INTRODUCTION
One of the fundamental questions of nucleoside and nucleotide chem-
istry is to find out how a nucleobase may influence a chemical or stereo-
chemical outcome of the reactions. In this context, understanding factors
governing stereoselectivity during condensations of suitably protected ribo-
In honor and celebration of the life and career of John A. Montgomery.
Received 1 February 2005; accepted 23 March 2005.
The financial support from the State Committee for Scientific Research, Republic of Poland
(Grant No. 4 T09A 100 23) and the Swedish Research Council, is gratefully acknowledged.
Some parts of this publication were presented at the XVInI ternational Roundtable on Nucleo-
sides, Nucleotides, and Nucleic Acids, Minneapolis, Minnesota, Sept. 12–16, 2004. See Ref. [1].
Address correspondence to Michal Sobkowski, Institute of Bioorganic Chemistry, Polish Academy
of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland. E-mail: msob@ibch.poznan.pl
1469

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M. Sobkowski et al.
nucleoside 3ꢁ-H-phosphonates with various hydroxylic components,[2–8] may
have a pivotal role in controlling a ratio of the diastereomers of the H-
phosphonate diesters formed.
In reactions of ribonucleoside H-phosphonates with nucleosides, only
the structure of a nucleotidic component (an H-phosphonate monoester)
was considered to be responsible for the observed stereoselectivity.[5,7,8]
Although in the deoxy series the stereoselectivity is usually rather low (if
any),[3,6] some reports indicated that in certain instances it was a nucleosidic
component that directed the stereochemical course of the reaction.[3] All
these data were, however, fragmentary and incomplete, making it difficult
to draw more general conclusions concerning sources of stereoselectivity in
H-phosphonate diester synthesis.
To fill up this gap, we carried out syntheses of 16 possible diribonucleo-
side (3ꢁ-5ꢁ)-H-phosphonates to assess the influence of both the nucleotidic
and nucleosidic component on the stereoselectivity of the internucleoside
H-phosphonate bond formation.
Having at hand a set of diastereomeric diribonucleoside (3ꢁ-5ꢁ)-H-phos-
phonates and a possibility to convert them stereospecifically into the cor-
responding P-chiral dinucleoside phosphorothioates,[5,9,10] another objec-
tive of this work was to evaluate a correlation between configuration at the
phosphorous center and the 31P NMR chemical shifts of the investigated
compounds.
Absolute configuration of P-chiral dinucleoside phosphorothioates can
be unambiguously determined by enzymatic methods, by taking advantage
of the fact that snake venom phosphodiesterase (SVPD) digests preferen-
tially the RP diastereomers,[11,12] while nuclease P1, the SP isomers.[13] This
enzymatic method is reliable but rather time consuming. To facilitate the
configurational assignment, Eckstein postulated that absolute configuration
at the phosphorous center of di(deoxyribonucleoside) phosphorothioates
could be correlated with 31P NMR chemical shifts of these compounds.[14]
According to this correlation rule (referred further to as the Eckstein’s rule),
the RP diastereomers of deoxy-[14] and ribonucleoside[5] phosphorothioates
resonate at lower field in the 31P NMR spectroscopy, relatively to the corre-
sponding SP diastereomers. For other classes of nucleotide analogues, e.g.,
phosphoranilidate monoesters,[15] aryl phosphoranilidate diesters,[15] aryl
phosphorothioate triesters,[15] tervalent phosphoramidites,[16] and oxathia-
phospholanes,[17] an analogous correlation between configuration at the
phosphorous center and the 31P NMR chemical shifts, was observed.
There are, however, notable exceptions from the Eckstein’s rule, for
which the positions of 31P NMR resonances of SP and RP diastereomers
are reversed. For example, SP diastereomers of protected ribo- and deoxyri-
bonucleoside H-phosphonate diesters,[5,18] H-phosphonothioates,[18,19] and
dinucleoside phosphorothioates[5] do not resonate upfield but downfield,

Stereochemistry of Internucleotide Bond Formation
1471
relatively to the corresponding RP diastereomers. Nevertheless, in all in-
stances, within a given class of nucleotide analogues, no variations in the
order of chemical shifts between RP and SP diastereomers were observed.
Thus, until recently, for closely related compounds, a correlation between
31P NMR shifts and the sense of chirality seemed to be a reliable means for
the assignment of absolute configuration at the phosphorous center for P-
chiral nucleotide analogues. The present studies, however, have shown that
one should exercise some caution, since even for structurally closely related
compounds, this correlation rule may not hold.
RESULTS AND DISCUSSION
This paper addresses two important issues of nucleotide chemistry: (a) an
influence of nucleotidic and nucleosidic components on stereoselectivity
during formation of P-chiral diribonucleoside H-phosphonates and (b) a
generality of a relationship between 31P NMR chemical shifts and absolute
configurations of the phosphorous center of ribonucleoside H-phosphonate
and phosphorothioate diesters.
To get some insight into these problems, sixteen diribonucleoside (3ꢁ-
5ꢁ)-H-phosphonates of type 4 were synthesized via condensation of 5ꢁ-
O-dimethoxytrityl-2ꢁ-O-t-butyldimethylsilyl ribonucleoside H-phosphonates
(1) with four 2ꢁ,3ꢁ-O-dibenzoyl ribonucleosides (2) in the presence of
pivaloyl chloride as a condensing agent (Figure 1). For comparison, also
ethanol was used as a hydroxylic component. The reactions were carried out
either in neat pyridine or in methylene chloride (DCM) containing limited
amount of pyridine (3 equiv., relatively to H-phosphonate) and progress of
the condensations was monitored by 31P NMR spectroscopy.
Degree of stereoselectivity in the investigated reactions was assessed
by integration of the resonance signals of the corresponding RP and SP
diastereomers formed. The obtained H-phosphonate diesters 4aa-de[20]
were then stereospecifically sulfurized with elemental sulfur[5,9,10] to the
corresponding phosphorothioates 5aa-de and the reaction mixtures were
again analyzed by 31P NMR. The stereochemical results of these experiments
are presented in Table 1.
As it is apparent from the above data, stereoselectivity in the reactions
investigated was controlled primarily by a nucleotidic component 1 as
the stereochemical outcomes of the condensations with nucleosides and
ethanol (or other aliphatic alcohols, e.g., isopropanol, t-butanol; results not
shown), in most cases were similar. Some fluctuations in stereoselectivity
related to the nature of a hydroxylic component occurred, however, these
did not exceed ca. 10% (in absolute values). Usually, in couplings with
pyrimidine nucleosides (e.g., 2b, 2d), the stereoselectivity was higher than
for purine ones (e.g., 2a, 2c). The highest stereoselectivity was observed for

1472
M. Sobkowski et al.
FIGURE 1 Substrates, intermediates, and products during synthesis of H-phosphonate diesters and
the corresponding phosphorothioates.
the formation of GPHC diester (4cb), while the lowest one for dinucleoside
H-phosphonate CPHG (4bc). The reason for these remains obscure and is
a subject of further studies in our laboratories.
As to the role of the solvent composition, amounts of pyridine had
rather moderate effect on the stereoselectivity of the condensations of
nucleoside H-phosphonates 1a, 1c, and 1d, and only for the reactions of
cytidine H-phosphonate (CPH, 1b), a distinct increase in stereoselectivity
with decreasing concentration of pyridine was observed.[8] In contrast to the
previous report,[8] we did not observe an exceptionally high stereoselectivity
for condensations involving guanosine H-phosphonate (GPH, 1c).

1473

1474
M. Sobkowski et al.
For most of the reactions investigated, the main diastereomer of H-
phosphonate diesters 4 resonated at lower field, while that of the corre-
sponding phosphorothioates at higher field. This was in agreement with
the previously reported data.[5,8,18,19] However, in several cases (the values
underlined in Table 1) the intensity of the low field signals of H-phospho-
nate diesters 4 were significantly smaller than those at high field, which
might have indicated that the diastereomers with opposite configuration
were formed as major products.[21] This phenomenon was observed in con-
densations involving both nucleosides and ethanol, and was also reflected
in some reaction mixtures after sulfurization.
A change in stereoselectivity in these particular instances would be an in-
teresting exception to the commonly observed preference for the formation
SP diribonucleoside (3ꢁ-5ꢁ)-H-phosphonate diesters in the condensation re-
actions. However, since the anomalies observed for H-phosphonate diesters
4 did not coincide with those for the corresponding phosphorothioates 5,
we assumed that the phenomenon was probably due to changes in rela-
tive positions of the 31P NMR signals of the phosphorous diastereomers of
compounds 4 and 5. This seemed plausible, although it clashed with the
aforementioned Eckstein’s rule.
To substantiate this assumption, a more detailed 31P NMR analysis was
carried out for the four representative cases, for which anomalous patterns
of 31P NMR signals were observed, namely the condensations UPH
U,
CPH G, GPH U, and GPH EtOH. These reactions were carried out in
acetonitrile (ACN) containing 3 equiv. of pyridine and after completion,
ACN was evaporated and replaced, consecutively, with DCM, pyridine,
and toluene. For the reaction mixtures in various solvents, the 31P NMR
spectra were recorded. The same protocol was used after sulfurization of
the corresponding H-phosphonate diesters 4 (Table 2).
For some of the investigated diastereomers of 4 and 5, the order of the
31P NMR resonances was independent of the solvent used, showing either
typical (UPHU, UPSU, CPHG) or an anomalous (GPHU, GPHEt) pattern
of signals. Thus, on this basis it was not possible to conclude what was
responsible for the observed pattern of signals in the 31P NMR spectra:
a change in stereoselectivity or a change in chemical shifts. Fortunately,
for three phosphorothioates investigated, namely CPSG, GPSU, GPSEt, the
order of the 31P NMR signals varied with solvents and indicated that a
relative position of the resonances of diastereomeric compounds in 31P
NMR spectra depended not only on the stereochemistry at the phosphorous
center but also on the solvent used. Further studies showed that even for
deprotected dinucleoside phosphorothioates the order of the 31P signals of
the corresponding diastereomers followed the Eckstein’s rule only in water,
while in ACN, the positions of the signals were reversed (Figure 2).

Stereochemistry of Internucleotide Bond Formation
1475
TABLE 2 Positions and Values of 31P NMR Resonances of Protected H-Phosphonate Diesters of
Type 4 and the Corresponding Phosphorothioates of Type 5 in Various Solvents. The Anomalies
Are Marked with Grey Background
aThe inserts are symbolic representations of the signals pattern, not the actual spectra.
bDue to poor solubility of the diester in neat toluene, a mixture containing 20% of DCM and 80%
(v/v) of toluene was used.
FIGURE 2 The 31P NMR spectra of unprotected diester GPS U in (a) H20 and (b) ACN.

1476
M. Sobkowski et al.
Finally, for one of the investigated dinucleoside phosphorothioate that
showed a solvent-dependent relative position of the 31P NMR signals (GPSU,
5cd), the absolute configurations of the separate diastereomers were deter-
mined by enzymatic methods (see the Experimental). As expected, only the
major diastereomer of 5cd was the substrate for SVPD (Figure 3, chromato-
grams c and d). This confirmed its RP configuration and excluded the pos-
sibility of a change in stereochemical preference during condensation in
this, and most likely also in other analogous instances.
A solvent-dependence of 31P NMR chemical shifts was further investi-
gated by recording the spectra of the fully protected phosphorothioate di-
ester GPSU (5cd) in ACN and in DCM containing various amounts of pyri-
dine (0, 25, 50, 75, and 100%; Figure 4). Since for both ACN and DCM,
the presence of more than 50% pyridine caused change in the order of the
31P NMR signals of the diastereomers, one can suppose that a relative po-
sition of the signals is mainly determined by the major component of the
solvent system. For the DCM-Py mixtures the diagram consists of roughly
two intersecting lines, while for the ACN-Py mixtures, two concave curves
with minima at different pyridine concentrations indicate probably more
complex interactions with the solutes in this case.
Also, variable-temperature 31P NMR spectra of the selected derivatives
were recorded to find out the influence of temperature on a relative position
of the resonances of P-diastereomers of the investigated compounds. To
this end the samples in toluene were warmed up between 20 and 70◦C,
and 31P NMR spectra were recorded every 10◦C. For the H-phosphonate
diester UPHEt (4de) no significant changes could be detected, neither
in the signals positions nor in the ratio of the diastereomers. For the
phosphorothioate diester UPSEt (5de), a moderate downfield shift (0.35
ppm) for both diastereomers in the investigated range of temperatures
was observed. In contrast to this, the chemical shifts of the diastereomers
of the mixed anhydride UPHPv (3d) were much more sensitive to the
temperature (Figure 5). Although signals from both diastereomers showed
upfield shifts with temperature, at t > 50◦C the order of the resonances
changed, indicating higher sensitivity to temperature of the diastereomer
3d resonating at lower field.
EXPERIMENTAL
31P NMR spectra were recorded at 121 MHz on a Varian Unity BB VT
spectrometer. 31P NMR experiments were carried out in 5 mm tubes using
0.1 M concentrations of phosphorous-containing compounds in appropri-
ate solvents (0.5 mL) and the spectra were referenced to 2% H3PO4 in
D2O (external standard). Pyridine, dichloromethane (POCh, Poland), and

Stereochemistry of Internucleotide Bond Formation
1477
FIGURE 3 RP-HPLC profiles of digestion of diastereomers of the GPS U diester with SVPD (incubation
time 48 h). Minor diastereomer: (a) before and (b) after digestion; major diastereomer: (c) before
and (d) after digestion.

1478
M. Sobkowski et al.
FIGURE 4 Positions of 31P NMR signals of diastereomers of fully protected phosphorothioate diester
GPS U (5cd) in ACN or DCM mixed with various amounts of pyridine.
FIGURE 5 Positions of 31P NMR signals of the in situ produced mixed anhydride UPH Pv (3d)
diastereomers in toluene as a function of temperature.

Stereochemistry of Internucleotide Bond Formation
1479
acetonitrile (Merck) were refluxed with P2O5, distilled, and stored over
molecular sieves 4 Å until the amount of water was below 20 ppm (Karl
Fischer coulometric titration, Metrohm 684 KF coulometer.). Pivaloyl chlo-
ride (Fluka) was distilled and used within one month. Nucleoside H-phos-
phonates of type 1 were obtained according to the published method.[22]
Snake venom phosphodiesterase (SVPD, from Crotalus adamanteus) was pur-
chased from Sigma. All HPLC analyses and purifications were performed on
ODS-Hypersil column (30 cm × 4.6 mm) detecting the eluate at 256 nm.
31P NMR data of all intermediates and products are given in Table 3.
GeneralProcedure for Condensations of H-Phosphonates of
Type 1 with Nucleosides of Type 2
Nucleoside H-phosphonate 1 (0.05 mmol) and nucleoside 2 (0.06
mmol) were mixed together and rendered anhydrous by evaporation of
added pyridine (3 mL) under reduced pressure. After a period of drying
under vacuum (15 min, 0.5 Torr), the flask was filled with air dried by
passing through Sicapent (Merck). The residue was dissolved in 0.5 mL of
appropriate solvent (DCM or ACN containing 3 equiv. of pyridine, or neat
pyridine) and pivaloyl chloride (1.2 equiv) was added. After ca. 10 min a 31
P
NMR spectrum was recorded. The same mixture was sulfurized subsequently
by adding elemental sulfur (3 equiv.) and Et3N (50 µL) and after ca. 10 min
a consecutive 31P NMR spectrum was recorded.
GeneralProcedure for Condensations of H-Phosphonates of
Type 1 with Aliphatic Alcohols
The same procedure as above was used, with the exception that H-phos-
phonate was dried separately and dry alcohol was added to its solution (prior
to pivaloyl chloride).
GeneralProcedure for in situ Preparation of the Mixed
Anhydrides of Type 3
Nucleoside H-phosphonate 1 (0.05 mmol) was rendered anhydrous by
evaporation of added pyridine (3 mL) under reduced pressure. After drying
under vacuum (15 min, 0.5 Torr), the flask was filled with air, dried by
passing it through Sicapent (Merck). The residue was dissolved in 0.5 mL
of the appropriate solvent (DCM or ACN containing 3 equiv. of pyridine),
and pivaloyl chloride (1.2 equiv) was added. The 31P NMR spectra showed
that in such mixtures the mixed anhydrides of type 3 were stable for at least
a few hours.

1480

1481

1482
M. Sobkowski et al.
Preparative Synthesis and Enzymatic Digestion of
Guanosin-3 -ylUridin-5 -ylPhosphorothioate (Unprotected 5cd)
(a) Synthesis. The fully protected phosphorothioate diester 4cd was
prepared according to the above procedure at the 0.25 mmol scale. The
diastereomers of the product were isolated on silica-gel column using 0–4%
gradient of MeOH in toluene containing 1% of Et3N.
The faster eluting diastereomer: 51 mg (15% yield); δP 57.98 ppm (ACN);
Rf 0.54 (toluene-ACN-Et3N 45:45:10).
The slower eluting diastereomer: 235 mg (67% yield); δP 58.67 ppm, Rf 0.41
(toluene-ACN-Et3N 45:45:10).
Each diastereomer was subjected separately to the deprotection with
conc. ammonia (48h, rt) and the tritylated product was purified on a silica-
gel column using 0–2% gradient of 25% NH3 aq. in iPrOH containing
2% of water. The dimethoxytrityl group was removed with 80% aq. AcOH.
Triethylammonium hydrofluoride (TEA·3HF)[23] was used to cleave 2ꢁ-O-
silyl ether. Purification according to the published method,[5] yielded the
corresponding unprotected dinucleoside phosphorothioate diesters: 18 mg
(68%) of the minor isomer, and 87 mg (74%) of the major isomer. The 1H
and 31P NMR spectra of both diastereomers were in agreement with the
literature data.[5] For enzymatic experiments, the samples were purified
additionally on RP HPLC yielding ca. 100 OD of each isomer (combined
fractions from three runs).
(b) Nuclease Digestion. The nuclease digestion was performed accord-
ing to Cummins et al.[24] Separate diastereomers of phosphorothioate 5cd
(0.25 OD) and SVPD (2 mg) were incubated in 50 µL of the buffer solution
(pH 8.5)[24] for 48 h at room temperature, and then analyzed directly by
RP HPLC (see Figure 2).
CONCLUSIONS
In conclusion, our data lend support to the earlier findings by Almer
et al. that the structure of an H-phosphonate monoester was crucial for
the degree of stereoselectivity observed in the condensation reactions.[8]
Although hydroxylic component had rather minor influence on the stere-
ochemistry of the condensations, purine nucleosides usually gave slightly
lower stereoselectivity than pyrimidine ones and simple alcohols. Among
the compounds investigated, H-phosphonate CPH (1b) consistently gave the
poorest results, and for the reaction in neat pyridine hardly any stereoselec-
tivity could be observed for this derivative. In all instances, decreasing of
the pyridine content in the reaction mixture had beneficial effect on the

Stereochemistry of Internucleotide Bond Formation
1483
FIGURE 6 Stereoselectivity (% of SP diastereomer of diesters 4aa-de) during condensation of ribo-
nucleoside H-phosphonates 1a-d with nucleosides 2a-d and ethanol.
stereoselectivity. For GPH (1c), although high stereoselectivity was observed
in most cases, we could not attain the value of >99% reported by Almer
et al. In all instances, the major product of the condensations had RP con-
figuration (Figure 6).
Concerning a correlation between 31P NMR chemical shifts and a sense
of chirality at the phosphorous center, it was found that the mutual posi-
tions of the resonances of P-diastereomers were governed not only by the
absolute configuration at the phosphorous center, but also by a nucleotide
sequence, solvent composition, and temperature. Anomalous positions of
the 31P NMR signals occurred mainly for nucleotide analogues bearing pro-
tecting groups, although even for unprotected phosphorothioates variation
in the order of P-diastereomers resonances was observed. Thus, one should
exercise considerable caution while using these correlation rules for assign-
ing absolute configurations at the phosphorous center.
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of the synthesized dinucleoside H-phosphonates 4 and dinucleoside phosphorothioates 5. For
example, “4bc” means that the compound was obtained from H-phosphonate 1b and nucleoside
2c, while the compound 5ad, from 1a and 2d, with intermediacy of 4ad.
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(obtained by pre-activation of H-phosphonate monoesters). In DCM the signal of the main
diastereomer of this intermediate was located at higher field, while in toluene it was at a lower
field.
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