The role of nucleophilic catalysis in chemistry and stereochemistry of ribonucleoside H-phosphonate condensation

Article abstract of DOI:10.1039/b812780h

The efficiency and stereoselectivity of condensation of ribonucleoside 3′-H-phosphonates with alcohols were investigated as a function of amines used for the reaction. It was found that irrespective of the presence or absence of nucleophilic catalysts, th

Full text of DOI:10.1039/b812780h

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PAPER
www.rsc.org/njc | New Journal of Chemistry
The role of nucleophilic catalysis in chemistry and stereochemistry of
ribonucleoside H-phosphonate condensationw
Michal Sobkowski,*a Jacek Stawinskib and Adam Kraszewskia
Received (in Montpellier, France) 24th July 2008, Accepted 10th September 2008
First published as an Advance Article on the web 29th October 2008
DOI: 10.1039/b812780h
The efficiency and stereoselectivity of condensation of ribonucleoside 30-H-phosphonates with alcohols were
investigated as a function of amines used for the reaction. It was found that irrespective of the presence or absence
of nucleophilic catalysts, the Dynamic Kinetic Asymmetric Transformation (DYKAT) was the major factor
responsible for the stereoselective formation of the DP(SP) isomers of the H-phosphonate diesters, and a
mechanistic rationalization of this observation was proposed. In addition, studies on the reactions carried out in
the presence of various bases led to the conclusion that certain sterically hindered pyridines, e.g. 2,6-lutidine, may
act as nucleophilic catalysts in the condensation of ribonucleoside 30-H-phosphonates with alcohols.
Recently, we have proposed a Dynamic Kinetic Asymmetric
Introduction
Transformation (DYKAT) as a possible mechanism for the
stereoselectivity observed in these reactions.1 According to this
P-Chiral oligonucleotide analogues (e.g. phosphorothioates,2
phosphoramidates,3 methylphosphonates,4 or boranophos-
model, diastereomers of nucleoside H-phosphonic–pivalic
phates5) having defined configuration at the phosphorus atom
mixed anhydrides 2 exist in a rapid equilibrium, and one of
find diverse applications in investigations of nucleic acid inter-
them, namely the LP(SP) diastereomer, is significantly more
actions with other biologically important molecules, for exam-
reactive towards nucleosides (or alcohols) than the other one
ple proteins, RNA, and DNA.6 Such P-chiral oligonucleotides
(Fig. 1 and Chart 1). To simplify mechanistic considerations,
may also be considered as potential drugs for nucleic acid-based
in our earlier studies the role of nucleophilic and base catalysis
therapies,7 that could permit a more precise tuning of oligo-
by the amines was consciously neglected. However, since the
nucleotide interactions with the biological targets than is possible
participation of nucleophilic catalysis in condensation of
H-phosphonates is a well-established phenomenon,11–15 it
with the currently used pools of P-diastereomers. This should
also relieve problems of potential variation of therapeutic and
was important to examine and to assess its impact on the
toxic effects resulting from different ratios of P-diastereomers
asymmetric induction in the reactions investigated. In this
produced in various batches of oligonucleotide drugs.
paper we present studies on the role of nucleophilic catalysis
There are several strategies to stereocontrolled synthesis of
P-chiral oligonucleotides.8 One of them, stereoselective (or more
in the chemistry and stereochemistry of condensation of
ribonucleoside H-phosphonates with alcohols.
precisely, diastereoselective) condensation of ribonucleoside H-phos-
phonates,9 attracted our attention due to its simplicity and high
efficiency. It makes use of commercially available H-phosphonate
synthons which are condensed with nucleosides under standard
reaction conditions commonly used for the synthesis of H-phos-
phonate diesters to provide DP diastereomersz as major products.
Results and discussion
In routine condensations of nucleoside H-phosphonates
pyridine or quinoline (either neat or diluted with non-basic
solvent) is used as a basic component of the reaction mixture.
Both of these weakly basic heterocyclic amines (pKa 5.2 and
4.9, respectively) secure fast and quantitative formation
of H-phosphonate diesters due to their ability to act as
nucleophilic catalysts.11,12 In contrast to this, in the presence
of more powerful nucleophilic catalysts, e.g. NMI or DMAP,y
nucleoside H-phosphonates are prone to P-acylation that
compromises the diester formation.12 Also strongly basic
tertiary amines (e.g. TEA, pKa 11.0) are usually avoided since
these can promote undesired base-catalysed bis-acylation of
H-phosphonate monoesters,13,15 while in the presence of less
basic tertiary amines (e.g. DMA, pKa 5.1) the condensations
a Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Noskowskiego 12/14, 61-704 Poznan, Poland.
E-mail: msob@ibch.poznan.pl; Fax: +48 61 8520 532;
Tel: +48 61 852 8503
b Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, S-106 91 Stockholm, Sweden
w Stereochemistry of internucleotide bond formation by the H-phos-
phonate method. Part 4.1
z For the compounds presented in this paper the DP descriptor refers
to a structure in which the P–H bond is directed to the right in the
Fischer projection, and in the LP one, to the left. The full DP/LP
notation is described in ref. 10.
y Abbreviations: DABCO, 1,4-diazabicyclo[2.2.2]octane; DIPEA, di-
isopropylethylamine; DMAP, 4-(N,N-dimethylamino)pyridine; DMA,
N,N-dimethylaniline; DTBP, 2,6-di-tert-butylpyridine; EDIPP,
4-ethyl-2,6-diisopropyl-3,5-dimethylpyridine; HMTA, hexamethylene-
tetramine; Lut, 2,6-lutidine; MPO, 4-methoxypyridine N-oxide; NMI,
N-methylimidazole; PvCl, pivaloyl chloride; Py, pyridine; TEA,
triethylamine.
ꢀc
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Fig. 1 Putative routes of the reaction during stereoselective condensation of ribonucleoside H-phosphonate monoester 1 with alcohols and
nucleosides according to the DYKAT mechanism in the absence (curved arrows) and in the presence (central pathways) of a nucleophilic catalyst.
solely as base catalysts or as base and nucleophilic catalysts
during H-phosphonate condensations. To this end, the reac-
tions of H-phosphonate 1 with ethanol were carried out in the
presence of selected tertiary amines, various pyridine deriva-
tives, and strong nucleophilic catalysts. The obtained data
(Table 1) showed that irrespective of significant differences in
the yields and stereoselectivity observed for different amines,
the same DP(SP) diastereomer of diester 4 was always formed
as the main product. In the light of our earlier studies,1,18 these
results might suggest that in the absence of nucleophilic
catalysts, the previously described DYKAT mechanism oper-
ated at the level of the mixed anhydride 2 (Fig. 2, Path B),
while in the nucleophile-catalyzed reactions, an analogous
DYKAT took place at the level of adducts of type 3 (Fig. 2,
Path A2).z
Additionally, these experiments confirmed the earlier find-
ings11–15 that neither powerful nucleophilic catalysts nor
strongly basic tertiary amines could promote quantitative
condensations of H-phosphonates with alcohols. However,
in contrast to the literature data,11–15 there was no (or very
little) side product formation, and the 31P NMR spectra of the
reaction mixtures revealed only presence of the expected
diester 4 and unreacted monoester 1 (Fig. 3). This lack of
by-product formation was tentatively attributed to low
Chart 1
are effective but sluggish (at least 10 times slower than those
for pyridine), presumably due to lack of nucleophilic cata-
lysis.12 Thus, it was somewhat surprising that 2,6-lutidine
(pKa 6.7), which is usually considered as poorly nucleophilic
base,16,17 promoted condensations of ribonucleoside H-phos-
phonates with similar efficiency as pyridine or quinoline.1
Moreover, the stereochemistry of the reactions performed in
the presence of 2,6-lutidine was the same as that with pyridine.
The above called into question the commonly accepted
non-nucleophilic character of 2,6-lutidine and prompted us
to consider the involvement of nucleophilic catalysis as an
additional process in the DYKAT mechanism (Fig. 1).
Since the involvement of P–N+ adducts of type 3 (Fig. 1) in
ribonucleoside H-phosphonate diester formation has to be
crucial for the rate of condensation as well as for stereo-
chemical outcome of the reaction (Fig. 2), we undertook
investigations to pinpoint the cases in which amines acted
z Involving a rapid 3-DP " 3-LP equilibrium in which the more
reactive diastereomer 3-DP was esterified preferentially.
ꢀc
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Fig. 2 Possible stereochemistry of esterification of the more reactive LP(SP) diastereomer of ribonucleoside H-phosphonic—pivalic mixed
anhydride 2 in the presence (Path A) and absence (Path B) of nucleophilic catalysts.
Table 1 Diastereomeric excess (de) of the DP(SP) diastereomer of the H-phosphonate diester 4b (Fig. 1, B = Ura) formed in the presence of
various amines
pKa (H2O)a
pKa (DMSO)
pKa (ACN)
pKHB
dec,d (DP)
Yield of diester (%)d
b
Entry
Amine
Strong nucleophilic catalysts
MPO
1
2
3
4
5
2.119
5.2
7.0
8.7
3.520
12.421
62%
57%
60%
53%
53%
27
66
84
42
85
HMTAe
NMI
1.922
2.724
2.622
2.829
14.323
18.326
17.728
DABCOe
DMAP
8.925
7.927
9.7
Heteroaromatic amines
Pyrazine
Pyrimidine
6
7
8
9
0.7
1.2
3.6
4.9
4.9
5.030
5.2
5.9
6.0
6.2
6.4
6.5
6.7
6.7
7.5
(7.6)f
8.0
8.0
1.229
1.429
39%
39%
59%
63%
66%
47%
62%
69%
68%
64%
68%
63%
70%
70%
68%
52%
65%
64%
55
70
100
100
100
70
100
100
100
100
100
100
100
100
100
92
Tetramethylpyrazine
Quinoline
12.028
1.929
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1,10-Phenanthrolinee
2,6-Di-tert-butyl-pyridine
Pyridine
1.031
3.227
4.027
3.827
12.528
13.932
14.532
1.929
2.029
2.129
2-Picoline
4-Picoline
Neocuproinee
2,5-Lutidine
3,4-Lutidine
2,6-Lutidine
2,4-Lutidine
2,4,6-Collidine
EDIPP
4.327
4.427
4.527
14.732
14.432
15.032
15.028
2.229
2.129
2.329
(ꢁ)-Nicotine
(ꢂ)-Nicotine
Tertiary amines
DMA
N-Methylmorpholine
TEA
DIPEA
100
100
24
25
26
27
5.1
7.4
11.0
11.4
2.533
9.036
11.428
15.635
18.828
0.534
1.722
2.022
1.122
56%
70%
75%
71%
100
91
74
89
a
b
Aqueous pKa data, unless otherwise indicated, are taken from ref. 37. Hydrogen bonding basicity. pKHB = logK(formation of HB complex); larger
values correspond to greater basicity.38 c One should note that the difference between de values, for instance de 52% and de 75%, corresponds to
d
over two-fold increase of the stereoselectivity measured as a ratio of diastereomers (i.e. B3 : 1 vs. B7 : 1, respectively). Determined via
integration of the corresponding 31P NMR signals. For structure, see Chart 1. Estimated, assuming an additive and similar methyl and ethyl
e
f
39
40
groups effect on the pKa and a linear correlation between a,a0-steric hindrance and pKa of substituted pyridines.
concentration of the amines in the reaction mixtures (0.3 M or
ca. 2.5%).
sumed in the acylation of 50-OH or N3-H functions of
uridine.41 These side reactions could compete with the forma-
tion of the mixed anhydride 2 and, at least partly, could
be responsible for incomplete condensations. However,
H-phosphonate condensations were also not quantitative in
the presence of tertiary aliphatic amines alone (Table 1, entries
25–27), i.e. under the conditions in which the acylation of
nucleoside components was negligible.41 This issue was
addressed in additional experiments, which indicated that
the mixed anhydride 2 might undergo deacylation by pivalic
In order to find sources for the incomplete condensations
that have been carried out in the presence of the amines
examined herein, the reactivity of pivaloyl chloride towards
nucleosides was investigated in separate experiments. It was
found that TEA and pyridine derivatives when used alone did
not promote significant acylation of nucleosides, however, in
the presence of strong nucleophilic amines (e.g. DMAP) or
TEA–pyridine mixtures, pivaloyl chloride was rapidly con-
ꢀc
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Fig. 4 A possible participation of general acid catalysis in deacyla-
tion of the mixed anhydride 2 by pivalic acid.
unhindered endocyclic nitrogen atoms (i.e. having at least one
a position unsubstituted), the question might arise, whether
this could hold also for a,a0-dimethylpyridines?
Fig. 3 31P NMR spectra of the reaction of H-phosphonate 1 with
ethanol (3 equiv.) promoted by PvCl (1.5 equiv.) in DCM containing
3 equiv. of 2,6-lutidine or TEA. The minor signal (ca. 1.5%) at ꢁ3.2 ppm
in the upper spectrum is in the region of P-acylated compounds.
Although 2,6-lutidine and its derivatives are usually con-
sidered as poor nucleophiles,16,17 they can act as nucleophiles
under mild conditions undergoing, for instance, N-alkylation
with alkyl halides,44 alkyl iodonium triflate45 or radical
cations,46 or N-sulfonation with triflic anhydride.47 Notably,
in phosphorus chemistry the lack of nucleophilic properties of
2,6-lutidine was observed for P(V) compounds, e.g. for phos-
phoroiodidates,16 while whether nucleophilic catalysis by this
base may operate for H-phosphonates, remains to be deter-
mined. Since P(V) and P(III) compounds differ significantly in
electrophilicity,48 and the steric hindrance around the phos-
phorus atom in H-phosphonates is clearly lower than that
in P(V) compounds, significant differences in their reactivity
towards hindered pyridine derivatives cannot be excluded.
To get a better insight into this problem, condensations of
H-phosphonate 1 were performed in the presence of EDIPP
(a peralkylated 2,6-diisopropylpyridine derivative, pKa
B7.6)49 and DTBP (2,6-di-tert-butylpyridine, pKa 5.0) for
which the nucleophilicity might be safely excluded on steric
grounds. The yields of H-phosphonate diester 4 obtained in
these reactions (92 and 70%) were similar to those found for
trialkyl amines, while low stereoselectivity (de ca. 50%) was
similar to that observed for tertiary aniline derivatives
(e.g. DMA, de 56%). In contrast, all the other pyridine
derivatives, including a,a0-dimethylpyridines, differed only
slightly in stereoselectivity and invariably gave quantitative
condensations of H-phosphonate 1. Thus, it seems reasonable
to assume that the main route for H-phosphonate diester
formation in the presence of 2,6-lutidine derivatives could still
involve the nucleophilic catalysis (preventing in this way
deacylation of the mixed anhydride 2, and in consequence,
the yield deterioration), and that only bulky alkyl substituents
in a,a0 positions were able to suppress the nucleophilic proper-
ties of pyridine.
acid with regeneration of the starting H-phosphonate mono-
ester 1 and formation of pivalic anhydride (a poor activator of
H-phosphonates42). The rate of deacylation was found to
correlate well with the ability of an amine conjugated acid to
form hydrogen bonds (quantified as a pKHB value8) rather
than with the amine basicity expressed by pKa. A plausible
rationale, which could account for the obtained results in-
volved an increased contribution of general acid catalysis
during decomposition of the mixed anhydride 2 by amines
having high pKHB (Fig. 4).43
Thus, it can be tentatively concluded that pivaloyl chloride
promoted coupling of H-phosphonates with alcohols in the
presence of strongly nucleophilic amines, and/or those of high
H-bonding basicity, did not go to completion due to con-
sumption of the condensing agent (PvCl) in the acylation
of nucleosides or due to formation of unreactive pivalic
anhydride via a partial deacylation of the mixed anhydride 2.
In contrast, pyridine and most of its derivatives investigated
secured quantitative condensations (with an exception of
pyridine derivatives bearing branched substituents in both
a positions)** despite significant differences in their pKa
(3.6–8.0) and considerably high pKHB values (1.9–2.3).
Although it might be argued that the high pKHB of pyridines
should be associated with high catalytic activity of their
conjugate acids which should lead to deacylation of the mixed
anhydride 2 (Fig. 4), apparently it was not the case in the
reactions discussed. A plausible explanation of the excellent
yields obtained for the most of the pyridines examined could
be the participation of nucleophilic catalysis, i.e. the involve-
ment of intermediate phosphonopyridinium adducts of type 3
(Fig. 2) which, as monofunctional entities, should undergo a
nucleophilic attack at the phosphorus centre only. While this is
readily understandable in the case of pyridine derivatives with
In additional experiments the condensations of uridine
H-phosphonate 1 with ethanol performed in the presence of
mixtures of 2,6-lutidine with more nucleophilic amines
(pyridine, NMI, DMAP, MPO) were investigated (Fig. 5). In
neither case were any specific effects due to the nucleophilic
amine noted, and the yields and stereoselectivity of the con-
densations were proportional to the weighted average of the
values obtained for each amine used separately. This lent
support to the aforementioned assumption that the same
mechanism (i.e. nucleophilic catalysis) was operating for 2,6-
lutidine and for other amines of known nucleophilic character.
8 The pKHB measures the relative strength of the acceptor in hydro-
gen-bonded complex formation with a reference acid (H-bonding
basicity). pKa and pKHB may be unrelated.38
** Two heteroaromatic amines, pyrazine and pyrimidine, were appar-
ently too weakly basic (pKa 0.7 and 1.2, respectively) to be efficient
promoters of the condensations investigated since a significant detri-
tylation was observed during the course of reactions, even in the
presence of 6 equiv. of an amine (c E 5%). These amines were thus
excluded from further investigations.
ꢀc
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highly basic amines (e.g. TEA, pKa 11.0; entries 7 & 8), the rate
of epimerization was always significantly higher than that of
esterification, even in the presence of large excess of an
alcohol. Interestingly, it seems that the behaviour of a given
amine in a kinetic quenching experiment might be exploited as
a marker of its nucleophilic properties towards H-phospho-
nates, according to the following rule of thumb: the higher the
stereoselectivity of ribonucleoside H-phosphonate conden-
sation in neat methanol, the lower the nucleophilicity of the
amine used for the reaction.
Conclusions
Fig. 5 Diastereomeric excess (solid bars) of the DP(SP) diastereomer
of H-phosphonate diester 4b formed in the presence of mixtures of
amines, and the total yield of diester 4b (a sum of diastereomers, open
bars). Reaction conditions: 0.05 mmol of 1 (B = Ura) + EtOH
(3 equiv.) + amines (the number of molar equivalents specified on the
x axis) + PvCl (1.5 equiv.) in DCM (0.5 mL).
In the previous paper in this series we reported that stereo-
selectivity in condensations of ribonucleoside H-phosphonates
1 with alcohols originated from the Dynamic Kinetic Asym-
metric Transformation (DYKAT).1 The data presented in this
paper confirmed this conclusion and suggested that the equili-
brium between the diastereomers of nucleoside H-phosphonic—
pivalic mixed anhydride (2-DP " 2-LP) was significant
for the stereochemical outcome of the reaction only in the
absence of nucleophilic catalysis. In the presence of nucleo-
philic amines, however, the DYKAT mechanism was
governed most likely by the 3-DP " 3-LP equilibrium between
the putative P–N+ intermediates. In most instances this path
was also essential for quantitative yield of the condensation.
Pyridine derivatives (excluding those with a large steric
hindrance around the nitrogen atom) secured practically
quantitative yields of the condensations along with reasonable
high stereoselectivity (de 60–70%). Noteworthy, pyridine
derivatives with methyl groups in the a positions (e.g. 2,6-
lutidine) also provided fast, clean and highly stereoselective
condensations, and thus indicated that the esterification of
H-phosphonate monoesters in the presence of these bases
might proceed with the intermediacy of the P–N+ adducts
of type 3 (i.e. involving nucleophilic catalysis; Fig. 1 and 2). To
the best of our knowledge this would be the first documented
example of manifestation of nucleophilic properties of
2,6-dimethylpyridines in SN2(P) reactions.
Kinetic quenching experiments
To probe the involvement of nucleophilic catalysis in the
DYKAT mechanism, kinetic quenching experiments for
various amines were carried out using large excess of methanol.
Under such reaction conditions we expected to observe
significant changes in the ratio of diastereomers (with a
possible reversal of stereoselectivity1) of the produced
H-phosphonate diester 4a as a result of substantial increase
in the rate of esterification of the reactive intermediates (mixed
anhydride 2 and amine adduct 3). Indeed, a remarkable
decrease in stereoselectivity was observed for the reactions
involving pyridine or 2,6-lutidine as bases (Table 2).
Such results can be interpreted as a partial change of the
DYKAT into the Dynamic Thermodynamic Resolution
(DYTR) mechanism of the asymmetric induction due to
acceleration of the esterification at high concentration of
MeOH.1 In contrast, in the presence of the poorly nucleophilic
amines, the stereoselectivity under the kinetic quenching
conditions decreased only slightly.
Thus, it seems that the nucleophilic catalysis (Table 2,
entries 1 & 2) speeded up the esterification of intermediates 3
more efficiently than their epimerization, while for the base
catalysed reactions (entries 3–6 & 9) or in the presence of
For practical purposes, among investigated bases, 2,6-luti-
dine was found to be the amine of choice (quantitative yield of
condensations, high stereoselectivity, and easy availability)
Table 2 Comparison of the yield and the ratio of diastereomers of the methyl uridine H-phosphonate diester 4a (Fig. 1) formed under standard
and kinetic quenching conditions
‘‘Standard’’ 3 equiv. of MeOH 3 equiv.
of amine [1] = 100 mM
‘‘Kinetic quenching’’ 2500 equiv. of MeOH,
30 equiv. of amine [1] = 10 mM
de (DP)a
Yield of diester (%)a
de (DP)a
Yield of diester (%)a
Entry
Amine
1
2
3
4
5
6
7
8
9
Pyridine
63
68
47
56
69
49
62
68
60
100
100
100
96
73
95
89
80
84
ꢁ10b
100
100
100
100
93
95
88
91
94
2,6-Lutidine
EDIPP
DMA
12
66
42
51
45
60
61
58
TEA
Proton sponge
Pyridine + TEA 1:1c
2,6-Lutidine + TEA 1:1c
DMA + TEA 1:1c
a
b
c
Determined via integration of the corresponding 31P NMR signals. Advantage of the LP diastereomer. 3 + 3 equiv. or 15 + 15 equiv.
ꢀc
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and is advised to be used in stereoselective ribonucleoside
30-H-phosphonate diesters formation.
30 s toluene (15 mL) was added and the mixture was evapo-
rated almost to dryness under vacuum at temperature o40 1C
(such procedure was obligatory for strongly basic tertiary
amines in order to avoid transesterification52 of the product).
The oily residue was dissolved in DCM (0.5 mL) and analyzed
by 31P NMR spectroscopy.
Experimental section
Methods and materials
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 0.5 mL of the reaction
mixture and the spectra were referenced to 2% H3PO4 in
D2O (external standard). The quantities of phosphorus-
containing compounds were determined via integration of
the corresponding 31P NMR signals. Diastereomeric excess
was calculated with accuracy of ꢂ1.5 percentage points
(an average of 3 measurements).
Acknowledgements
The financial support from the Polish Ministry of Science and
Higher Education is gratefully acknowledged.
References
1 M. Sobkowski, A. Kraszewski and J. Stawinski, Tetrahedron:
Asymmetry, 2007, 18, 2336–2348.
Commercial (Sigma-Aldrich, Alfa Aesar, Merck, POCh-
Poland) reagents and were used as purchased unless otherwise
noted. Ethanol was distilled over magnesium. Dichloro-
methane and pyridine were refluxed over P2O5, distilled, and
stored over 4 A molecular sieves until they contained below
20 ppm of water (Karl Fischer coulometric titration, Metrohm
684 KF coulometer). Anhydrous triethylamine (TEA) was
distilled and kept over CaH2. Commercial p-methoxypyridine
N-oxide (MPO) hydrate was rendered anhydrous by co-
evaporation with dry acetonitrile (1ꢃ) and dry toluene (2ꢃ).
Other liquid amines were refluxed for 1 hour with 2,4,6-
triisopropylbenzenesulfonyl chloride (TPS-Cl) and distilled
under reduced pressure. 4-Ethyl-2,6-diisopropyl-3,5-dimethyl-
pyridine (EDIPP)49 was a gift from Prof. A. T. Balaban,
Texas A&M University. Uridine H-phosphonate 150 was
obtained according to the published method. Racemization
of S-(ꢁ)-nicotine was done according to ref. 51. Immediately
prior to all reactions, H-phosphonate 1 was rendered anhy-
drous by dissolving in toluene (3 mL/0.05 mmol) and evapora-
tion of this solvent under reduced pressure. After drying under
vacuum (15 min, 0.5 Torr), the flask was filled with air, dried
up by passing through Sicapent (Merck).
2 W. J. Stec and G. Zon, Tetrahedron Lett., 1984, 25, 5275–5278;
W. B. Cruse, S. A. Salisbury, T. Brown, R. Cosstick, F. Eckstein
and O. Kennard, J. Mol. Biol., 1986, 192, 891–905.
3 J. Nilsson and J. Stawinski, Chem. Commun., 2004, 2566–2567.
4 L. A. Wozniak, M. Janicka and M. Bukowiecka-Matusiak, Eur. J.
Org. Chem., 2005, 5189–5197.
5 J. Tomasz, B. R. Shaw, K. Porter, B. F. Spielvogel and A. Sood,
Angew. Chem., Int. Ed., 1992, 31, 1373–1375; Y. Jin and G. Just,
Tetrahedron Lett., 1998, 39, 6433–6436.
6 P. Guga, Curr. Top. Med. Chem., 2007, 7, 695–713.
7 J. B. Opalinska and A. M. Gewirtz, Nat. Rev. Drug Discovery,
2002, 1, 503–514.
8 Y. X. Lu, Mini-Rev. Med. Chem., 2006, 6, 319–330.
9 H. Almer, J. Stawinski and R. Stromberg, Nucleic Acids Res., 1996,
24, 3811–3820.
10 M. Sobkowski, J. Stawinski and A. Kraszewski, Nucleosides,
Nucleotides Nucleic Acids, 2005, 24, 1301–1307; M. Sobkowski,
J. Stawinski and A. Kraszewski, Nucleosides, Nucleotides Nucleic
Acids, 2006, 25, 1363–1375; M. Sobkowski, J. Stawinski and
A. Kraszewski, Nucleosides, Nucleotides Nucleic Acids, 2006, 25,
1377–1389; M. Sobkowski, J. Stawinski and A. Kraszewski,
‘A Convenient Stereochemical Notation for P-Chiral Nucleotide
Analogs’, in Current Protocols in Nucleic Acid Chemistry, ed.
S. L. Beaucage, D. E. Bergstrom, G. D. Glick and R. A. Jones,
John Wiley & Sons, New York, 2007, A.1E.1–A.1E.16.
11 V. A. Efimov and I. Y. Dubey, Bioorg. Khim., 1990, 16, 211–218;
P. J. Garegg, J. Stawinski and R. Stromberg, J. Chem. Soc., Perkin
Trans. 2, 1987, 1209–1214.
12 I. Y. Dubey, V. A. Efimov, T. V. Lyapina and D. M. Fedoryak,
Bioorg. Khim., 1992, 18, 911–919; V. A. Efimov, I. Y. Dubey and
O. G. Chakhmakhcheva, Nucleosides Nucleotides, 1990, 9,
473–477.
General procedure for condensation of H-phosphonates of type 1
with alcohols
13 S. Sigurdsson and R. Stromberg, J. Chem. Soc., Perkin Trans. 2,
2002, 1682–1688; S. Sigurdsson and R. Stromberg, Nucleosides,
Nucleotides Nucleic Acids, 2003, 22, 1–12.
14 A. Kraszewski and J. Stawinski, ‘Aryl Nucleoside H-Phospho-
nates. Versatile Intermediates in the Synthesis of Nucleotides and
Their Analogues’, in Trends in Organic Chemistry, Research
Trends, Trivandrum India, 2003, vol. 10, pp. 1–19.
15 P. J. Garegg, T. Regberg, J. Stawinski and R. Stromberg, Nucleo-
sides Nucleotides, 1987, 6, 655–662.
Nucleoside H-phosphonate 1 (0.05 mmol) was dissolved in
0.5 mL of DCM and amine (3 equiv.) and EtOH or MeOH
(3 equiv.) were added, followed by PvCl (1.5 equiv.). The
reaction mixture was transferred to an NMR tube and the 31
P
NMR spectra were recorded within 1 hour.
Kinetic quenching experiments
16 J. Stawinski, R. Stromberg and R. Zain, Tetrahedron Lett., 1992,
33, 3185–3188.
17 C. W. Downey and M. W. Johnson, Tetrahedron Lett., 2007, 48,
3559–3562.
Nucleoside H-phosphonate 1 (0.05 mmol) was dissolved in
DCM (0.3–0.5 mL), and TEA (0.2 equiv.) and PvCl
(1.2 equiv.) were added successively. The formation of the
mixed anhydride 2 was confirmed by 31P NMR spectroscopy
(dP 1.47 & 1.56) and the reaction mixture was utilized within
one hour (no degradation products were found within that
period).
18 M. Sobkowski, J. Jankowska, J. Stawinski and A. Kraszewski, Nucleo-
sides, Nucleotides Nucleic Acids, 2005, 24, 1469–1484; M. Sobkowski,
J. Jankowska, J. Stawinski and A. Kraszewski, in Collection Sympo-
siumSeries, ed. M. Hocek, Institute of Organic Chemistry and Bio-
chemistry, Academy of Sciences of the Czech Republic, Prague, 2005,
vol. 7, pp. 183–187.
19 V. I. Rybachenko, K. Yu Chotii, V. V. Kovalenko and G. Shreder,
Russ. J. Phys. Chem. (Engl. Transl.), 2003, 77, 1695–1698.
20 L. Chmurzynski and Z. Warnke, Aust. J. Chem., 1993, 46,
185–194.
The solution (0.3 mL) of the intermediate 2 (0.5 mmol;
generated as described above) was added dropwise by a
syringe to a septum-sealed flask containing vigorously stirred
methanol (5 mL) and an amine (2.4% v/v or w/v). After ca.
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View Article Online
21 L. Chmurzynski, G. Wawrzyniak, Z. Warnke and Z. Pawlak,
J. Heterocycl. Chem., 1997, 34, 215–220.
38 R. W. Taft, D. Gurka, L. Joris, P. von Schleyer and J. W. Rakshys,
J. Am. Chem. Soc., 1969, 91, 4801–4808.
22 J. Graton, F. Besseau, M. Berthelot, E. Raczynska and
C. Laurence, Can. J. Chem., 2002, 80, 1375–1385.
23 Z. Pawlak, J. Mol. Struct., 1986, 143, 369–374.
24 L. Cecchi, F. De Sarlo and F. Machetti, Eur. J. Org. Chem., 2006,
4852–4860.
25 R. L. Benoit, D. Lefebvre and M. Frechette, Can. J. Chem., 1987,
65, 996–1001.
26 J. F. Coetzee and G. R. Padmanabhan, J. Am. Chem. Soc., 1965,
87, 5005–5010.
39 A. S. Chia and R. F. Trimble, J. Phys. Chem., 1961, 65, 863–866;
J. Epsztajn and R. M. K. Marcinkowski, Polish J. Chem., 1979, 53,
601–609; A. Gero and J. J. Markham, J. Org. Chem., 1951, 16,
1835–1838.
40 H. C. Brown and B. Kanner, J. Am. Chem. Soc., 1966, 88, 986–992;
B. Kanner, Heterocycles, 1982, 18, 411–419.
41 M. Sobkowski, in Collection Symposium Series, ed. M. Hocek,
Institute of Organic Chemistry and Biochemistry Academy of
Sciences of the Czech Republic, Prague, 2008, vol. 10, pp. 277–281.
42 R. Stromberg and J. Stawinski, Nucleic Acids Res. Symp. Ser.,
1987, 18, 185–188.
27 D. Augustin-Nowacka, M. Makowski and L. Chmurzynski,
J. Chem. Thermodyn., 2002, 34, 391–400.
28 I. Kaljurand, A. Kutt, L. Soovali, T. Rodima, V. Maemets, I. Leito
and I. A. Koppel, J. Org. Chem., 2005, 70, 1019–1028.
29 M. Berthelot, C. Laurence, M. Safar and F. Besseau, J. Chem.
Soc., Perkin Trans. 2, 1998, 283–290.
30 H. P. Hopkins, D. V. Jahagirdar, P. S. Moulik, D. H. Aue,
H. M. Webb, W. R. Davidson and M. D. Pedley, J. Am. Chem.
Soc., 1984, 106, 4341–4348.
31 R. L. Benoit, M. Frechette and D. Lefebvre, Can. J. Chem., 1988,
66, 1159–1162.
32 D. Augustin-Nowacka and L. Chmurzynski, Anal. Chim. Acta,
1999, 381, 215–220.
33 R. L. Benoit, M. J. MacKinnon and L. Bergeron, Can. J. Chem.,
1981, 59, 1501–1504.
34 E. Marquis, J. Graton, M. Berthelot, A. Planchat and C. Laurence,
Can. J. Chem., 2004, 82, 1413–1422.
35 P. Beltrame, G. Gelli and A. Loi, Gazz. Chim. Ital., 1980, 110,
491–494.
36 I. M. Kolthoff, M. K. Chantoon and S. Bhowmik, J. Am. Chem.
Soc., 1968, 90, 23–28.
43 M. Sobkowski, J. Stawinski and A. Kraszewski, Part 5 of this
series, New J. Chem., submitted.
44 F. Yang, X. L. Xu, Y. H. Gong, W. W. Qiu, Z. R. Sun, J. W. Zhou,
P. Audebert and J. Tang, Tetrahedron, 2007, 63, 9188–9194;
M. Szafran, Z. Dega-Szafran, B. Nowak-Wydra and
M. Pietrzak, J. Mol. Struct., 2001, 563–564, 555–564.
45 T. Umemoto and Y. Gotoh, Bull. Chem. Soc. Jpn., 1991, 64,
2008–2010.
46 V. D. Parker, E. T. Chao and G. Zheng, J. Am. Chem. Soc., 1997,
119, 11390–11394.
47 R. W. Binkley and M. G. Ambrose, J. Org. Chem., 1983, 48,
1776–1777.
48 J. Stawinski and A. Kraszewski, Acc. Chem. Res., 2002, 35,
952–960.
49 A. T. Balaban, I. Ghiviriga, E. W. Czerwinski, P. De and R. Faust,
J. Org. Chem., 2004, 69, 536–542.
50 J. Jankowska, M. Sobkowski, J. Stawinski and A. Kraszewski,
Tetrahedron Lett., 1994, 35, 3355–3358.
51 Y. Tsujino, S. Shibata, A. Katsuyama, T. Kisaki and H. Kaneko,
Heterocycles, 1982, 19, 2151–2154.
37 D. D. Perrin, Dissociation Constants of Organic Bases in Aqueous
Solution, Butterworth, London, 1965; CRC Handbook of Chemis-
try and Physics, CRC Press LLC, Boca Raton, 87th edn, 2007.
52 M. Sobkowski, J. Stawinski, A. Sobkowska and A. Kraszewski,
J. Chem. Soc., Perkin Trans. 2, 1994, 1803–1808.
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