Investigation on condensing agents for phosphinate ester formation with nucleoside 5′-hydroxyl functions

Article abstract of DOI:10.1002/ejoc.200700528

Condensation of a uridine 3′-deoxy-3′-C-methylenephosphinate with thymidine and guanosine derivatives to form methylenephosphinate esters was investigated. A number of different condensing agents were compared, and these include pivaloyl chloride, triisopropylbenzenesulfonyl chloride (TPSCl), phosphonium and uronium derivatives, numerous chlorophosphates and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (OXP). The phosphonium derivatives gave slow condensations or oxidative side reactions (hydroxybenzotriazole derivatives) during preactivation of the methylenephosphinate. Pivaloyl chloride gave long coupling times, and competing 5′-O-pivaloylation was detected. TPS-Cl gave rapid condensation but also rapid oxidation of the product. Most chlorophosphates gave competing 5′-O-phosphorylation of the nucleoside component, as well as base phosphorylation. However, 2-chloro-5,5-dimethyl-2- oxo-1,3,2-dioxaphosphorinane (DMOCP) gave a rather efficient formation of dinucleoside methylenephosphinates at a decent rate. However, O ⁶-protection of guanines could become necessary with this reagent, since upon extended reaction time traces of O⁶-phosphorylation were detected even with a low concentration (60 mM) of DMOCP (2 equiv. to phosphinate). Bis(2-oxo-3-oxazolidinyl) phosphinic chloride (OXP) can, unlike DMOCP, be used in nearly equimolar amounts to phosphinate. Under such conditions OXP gives virtually quantitative condensation at a rate comparable to that of 2 equiv. of DMOCP and with no side reactions detected. We could also not detect any decomposition of OXP-preactivated phosphinate. Nucleophilic catalysts, more powerful than pyridine (N-methylimidazole, iodide and 4-methoxypyridine), accelerated the reactions with OXP, but preactivation in the absence of the 5′-OH component led to decomposition of the activated phosphinate. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700528

FULL PAPER
DOI: 10.1002/ejoc.200700528
Investigation on Condensing Agents for Phosphinate Ester Formation with
Nucleoside 5Ј-Hydroxyl Functions[‡]
Anna Winqvist[a] and Roger Strömberg*[a]
Keywords: Oligonucleotides / Nucleotides / Phosphinates / Phosphonates / Methylenephosphonates / Condensation
reactions
Condensation of a uridine 3Ј-deoxy-3Ј-C-methylenephosphin- tection of guanines could become necessary with this rea-
ate with thymidine and guanosine derivatives to form meth-
ylenephosphinate esters was investigated. A number of dif-
ferent condensing agents were compared, and these include
pivaloyl chloride, triisopropylbenzenesulfonyl chloride (TPS-
Cl), phosphonium and uronium derivatives, numerous chlo-
rophosphates and bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
ride (OXP). The phosphonium derivatives gave slow conden-
sations or oxidative side reactions (hydroxybenzotriazole de-
rivatives) during preactivation of the methylenephosphinate.
Pivaloyl chloride gave long coupling times, and competing
5Ј-O-pivaloylation was detected. TPS-Cl gave rapid conden-
sation but also rapid oxidation of the product. Most chloro-
phosphates gave competing 5Ј-O-phosphorylation of the nu-
cleoside component, as well as base phosphorylation. How-
ever, 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane
(DMOCP) gave a rather efficient formation of dinucleoside
gent, since upon extended reaction time traces of O6-phos-
phorylation were detected even with a low concentration
(60 mM) of DMOCP (2 equiv. to phosphinate). Bis(2-oxo-3-ox-
azolidinyl)phosphinic chloride (OXP) can, unlike DMOCP, be
used in nearly equimolar amounts to phosphinate. Under
such conditions OXP gives virtually quantitative condensa-
tion at a rate comparable to that of 2 equiv. of DMOCP and
with no side reactions detected. We could also not detect any
decomposition of OXP-preactivated phosphinate. Nucleo-
philic catalysts, more powerful than pyridine (N-methylimid-
azole, iodide and 4-methoxypyridine), accelerated the reac-
tions with OXP, but preactivation in the absence of the 5Ј-
OH component led to decomposition of the activated phos-
phinate.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
methylenephosphinates at a decent rate. However, O6-pro- Germany, 2008)
ported a trinucleotide containing 3Ј-deoxy-3Ј-C-methylene-
Introduction
phosphonates, and recently Collingwood et al.[9] reported
incorporation of these linkages into oligodeoxyribonucleo-
tides.
A large number of modified nucleic acid fragments have
been developed in the past. This number increased drasti-
cally due to the development of antisense therapy.[1–3] The
interest in modified RNA has increased further by the po-
tential of using siRNA for therapeutics.[4,5] Recently, modi-
fied RNA has also become interesting for the development
of the potential use of siRNA in disease treatment. In ad-
dition, modified di- and oligonucleotides have been used as
potential enzyme inhibitors and in investigations of enzy-
matic mechanisms for a number of decades, and the use
has increased with the number of analogues available.[6] An
interesting modification that was introduced in dinucleo-
tides already in 1970[7] is internucleoside 3Ј-deoxy-3Ј-C-
methylenephosphonate linkages. Since then little has been
reported on this type of modification. Mazur et al.[8] re-
The early work by Moffatt and later by Mazur occurred
before the development of the H-phosphonate ap-
proach[10–15] to oligonucleotide synthesis. After the intro-
duction of this methodology, it seemed likely that an analo-
gous approach via alkylhydrogenphosphinates should be
possible for incorporation of methylenephosphonate link-
ages. An early attempt at the basic concept demonstrated
efficient coupling of methylphosphinate with a nucleoside
to give the corresponding phosphinate ester, which was fur-
ther converted into the phosphonate and thiophosphonate
analogues.[16] More recently, thymidine 3Ј-deoxy-3Ј-C-
methylenephosphinate building blocks, together with piva-
loyl chloride as condensing agent, were used for introduc-
tion of methylenephosphonate linkages into oligodeoxyri-
bonucleotides.[7] The ISIS Pharmaceuticals group has since
also reported on the synthesis of nucleoside 3Ј-deoxy-3Ј-C-
methylenephosphinate building blocks[17] and use of four
different coupling agents[18] for internucleoside linkage for-
mation, although these coupling agents were only com-
pared with a deoxyribonucleoside 3Ј-deoxy-3Ј-C-methylene-
[‡] Nucleoside 3Ј-Deoxy-3Ј-C-Methylenephosphinates, 3. Part 1:
A. Winqvist, R. Strömberg, Eur. J. Org. Chem. 2002, 1509–
1515. Part 2: A. Winqvist, R. Strömberg, Eur. J. Org. Chem.
2002, 3140–3144.
[a] Department of Biosciences and Nutrition, Novum, Karolinska
Institutet,
17177 Stockholm, Sweden
E-mail: Roger.Stromberg@biosci.ki.se
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FULL PAPER
phosphinate which is sterically considerably less hindered casional syntheses of short oligonucleotide analogues, it
than the corresponding protected ribonucleoside 3Ј-deoxy- would be generally preferred if an oligonucleotide analogue
3Ј-C-methylenephosphinate.
of, e.g., 20–24 units could be synthesized from one day to
We have recently reported on the development of syn- another. For a high coupling yield, reagents that can com-
thetic methods for key steps towards ribonucleoside 3Ј-de- pete with the activated phosphinate for the nucleoside 5Ј-
oxy-3Ј-C-methylenephosphinates, i.e., 3Ј-carbon extension hydroxyl function should be avoided. In addition, side reac-
at the nucleoside level,[19] synthesis of methylenephos- tions at the lactam functions of the nucleobases are best
phinate building blocks[20] and oxidation of methylenephos- kept at a minimum.
phinate linkages.[21] In this paper we report on an evalu-
ation of condensing agents for the formation of the methyl- ing block 1 was condensed with two different 5Ј-hydroxyl
To investigate the above aspects, the phosphinate build-
enephosphinate internucleoside linkage.
components, thymidine derivative 2a and guanosine deriva-
tive 2b, to give the 3Ј-deoxy-3Ј-C-methylenephosphinate es-
ters 3a and 3b, respectively (Scheme 1). A number of con-
densing agents were evaluated (Figure 1) using 31P NMR
Results
In the condensation of a nucleoside 3Ј-deoxy-3Ј-C-meth- spectroscopy to monitor the course of the reactions. The
ylenephosphinate building block with the 5Ј-hydroxyl func- products 3a and 3b were isolated and characterised, and as
tion of a nucleoside (or elongating oligonucleotide) there these were isomeric mixtures they were also further con-
are a number of aspects to consider. The formation of the verted into the achiral compounds 4a and 4b, respectively,
internucleoside linkage should be sufficiently fast for practi- by oxidation with iodine in pyridine/water.[19] The first con-
cal usage. Although a relatively long condensation time can densing agent investigated was pivaloyl chloride, which is
be acceptable for the formation of dinucleotides and in oc- the most commonly used in H-phosphonate-based oligonu-
Scheme 1.
Figure 1. Condensing agents used for the coupling of 1 and 2 (for R see text or Table 1).
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Eur. J. Org. Chem. 2008, 1705–1714

Condensing Agents for Phosphinate Ester Formation with Nucleoside 5Ј-Hydroxyl Functions
cleotide synthesis.[22,23] Coupling of 1 (30 m) with 2a
Reactions were carried out with 1, 2a and 60 m or
(28.5 m) in pyridine/acetonitrile (1:3, v/v) in the presence 120 m of chlorophosphate in pyridine/acetonitrile (1:3,
of 60 m pivaloyl chloride gave 3a. The rate of condensa- v/v). Several tested diaryl chlorophosphates (diphenyl, di-o-
tion was relatively low, especially when compared to the tolyl, or di-o-chlorophenyl chlorophosphate) proved to be
corresponding reaction with H-phosphonates. After 11 min, too reactive, and although coupling was fast, competing 5Ј-
about 50% of 3a was formed, whereas the corresponding O-phosphorylation of 2a (6–40%) occurred. In addition,
reaction for H-phosphonates is generally complete at the substantial amounts of phosphorylation at the lactam func-
time needed to record the first spectrum (less than 2– tionalities (the O4-position of uracil or thymidine) were ob-
4 min).[24] The reaction did not give more than approxi- served also with the less reactive diethyl chlorophosphate.
mately 95% product, and TLC analysis revealed that minor To verify that this kind of side products forms under these
amounts of 5Ј-O-pivaloylated 2a were formed.
specific conditions, we carried out additional experiments
The reagent 2,4,6-triisopropylbenzenesulfonyl chloride where 2Ј,3Ј,5Ј-tri-O-butyryluridine was treated with 120 m
(TPS-Cl) has also been reported to give efficient condensa- diethyl chlorophosphate in pyridine/acetonitrile (1:3, v/v).
tion with H-phosphonates, although oxidation of both H- Mass spectral analysis of this reaction mixture after 24 h
phosphonate monoesters[25] and diesters[26] occurred. Con- revealed the presence of both a phosphorylated species
densation of 1 and 2a with 60 m TPS-Cl in pyridine/aceto- (m/z = 591 [M – H+]) and the product [the 6-(N-pyridi-
nitrile (1:3, v/v) gave complete and rapid conversion into 3a nium)-2-aminopurine derivative[30]] of its subsequent reac-
(31P NMR: δ = 37.0, 37.7 ppm) in less than the time re- tion with pyridine (m/z = 516). More hindered dialkyl
quired to record the first spectrum. However, a product (31P chlorophosphates gave higher selectivity. Diisopropyl
NMR: δ = 42.3, 42.6 ppm) from a subsequent reaction was chlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphorinane
detected, amounting to 30% after about 3 h. The chemical (OCP) and 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphos-
shift alone is not definite evidence, but this together with phorinane (DMOCP) seemed promising, at least at 60 m
the rapid conversion to the phosphonate anion 4a upon ad- concentration (Table 1). At this lower concentration no
dition of water makes it a reasonable suggestion that the competing side reactions were detected with diisopropyl
compound formed is the corresponding chlorophos- chlorophosphate, OCP and DMOCP. DMOCP proved less
phonate. A similar reaction has previously been reported reactive and did not give side products, even at 120 m, in
with H-phosphonate diesters.[24]
the reaction of 1 and 2a. However, subsequent side reac-
Chlorophosphates can be efficient condensing agents and tions were observed at the O6-position of guanosine, when
were introduced early in H-phosphonate couplings[8,27] and the DMOCP coupling was carried out with 2b. The reaction
have later been used at low temperature in H-phosphonate- of 1, 2b and 120 m DMOCP gave 15% O6-phosphoryla-
based larger scale oligonucleotide synthesis in solu- tion after leaving the condensation mixture for about 3 h.
tion.[28,29] A chlorophosphate was also used in condensa- Even though the phosphodiester formed from the reagent,
tion of methylphosphinate with a nucleoside.[14] In addition, in the use of 60 m DMOCP, seemed to trap all excess chlo-
2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane rophosphate, we observed that this trapping is somewhat
(DMOCP) has been used for methylenephosphinate linkage slower than the coupling. This in turn leaves some chloro-
formation with deoxyribonucleotide 3Ј-deoxy-3Ј-C-methyl- phosphate to react with the base. In fact, trace amounts
enephosphinate building blocks.[16]
(Յ1%) of O6-phosphorylation were detected even before
Table 1. Reaction of 1 with 2a in the presence of dialkyl chlorophosphates.[a]
[a] Reaction carried out in acetonitrile/pyridine (3:1, v/v). [b] Signals of two isomeric products observed at δ = 37 and 37.7 ppm. [c] n.e.
= not estimated, since no data could be collected by NMR spectroscopy before a reaction time of 4 min. [d] The amount of competing
phosphorylation of 2a was estimated from the NMR spectra after the coupling reaction was complete. [e] n.o. = not observed.
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A. Winqvist, R. Strömberg
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completion of the condensation reaction, implying that it is Table 2. Condensations of 1 with 2a (to 3a) as aided by carbonium
or phosphonium salts.[a]
difficult to completely avoid this side reaction. The amount
of the side product increased slightly upon standing (still
within Յ1%) until all chlorophosphate had been converted
into pyrophosphate (by reaction with the formed phosphate
anion) after which no further significant increase was de-
tected upon standing overnight. As this condensing agent
otherwise seemed promising, further analysis of the product
of this side reaction was carried out. First 2Ј,3Ј,5Ј-tri-O-
butyryl-N2-phenoxyacetylguanosine was treated with
120 m DMOCP in pyridine/acetonitrile (1:3, v/v), and MS
analysis after 48 h revealed the presence of both a phos-
phorylated adduct (m/z = 776 [M – H+]) and the product
from subsequent reaction with pyridine (m/z = 689). In ad-
dition MS analysis of the reaction mixture of 1, 2b and
Entry
Coupling agent
Conc. Time for the formation of
[m] 75% product 90% product
2–1
2–2
2–3
2–4
2–5
2–6
2–7
HBTU
HATU
BOP
PyFOP
PyFOP
60
60
60
60
120
60
3.3 h
10 min
1.1 h
4.6 h
18 min
3.0 h
Ͻ4 min
Ͻ4 min
–
n.e.[c]
n.e.[c]
50% in 1.5 h
–
PyClOP
PyClOP/N-methylimidazole[b]
60
0.5 h
[a] Reaction carried out in acetonitrile/pyridine (3:1, v/v). [b] The
concentration of N-methylimidazole was 0.5 . [c] n.e. = not esti-
mated, since no data could be collected by NMR spectroscopy be-
fore a reaction time of 4 min.
DMOCP was carried out which revealed the presence of erate the reaction, catalysis by N-methylimidazole was ex-
a DMOCP adduct of the U-G dimer (m/z = 1380.5 [M – amined. Coupling of phosphinate 1 with alcohol 2a using
H+]).
60 m PyClOP and 0.5  N-methylimidazole in pyridine/
Sekine et al.[31] introduced a number of uronium and acetonitrile (1:3, v/v) gave ca 90% conversion into products
phosphonium reagents as condensing agents for H-phos- in about 30 min. Thus, the reaction rate was significantly
phonates. A drawback in the use of some of these reagents increased, but still considerably lower than with the least
was that the preactivation of H-phosphonates, in the ab- reactive chlorophosphates.
sence of alcohol, resulted in oxidative decomposition. For
The reagent bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
the present study a few reagents, i.e., HBTU, HATU, BOP, ride (OXP) has been used as a condensing reagent in forma-
PyFOP, PyAOP and PyClOP were selected (Figure 1). The tion of carboxylic acid derivatives[32,33] and has also been
condensations of 1 with 2a were carried out by the use of reported for H-phosphonate couplings[25] as well as for the
60 m of the various uronium and phosphonium reagents condensation of methylphosphinate[14] and thymidine 3Ј-de-
in pyridine/acetonitrile (1:3, v/v). The two uronium rea- oxy-3Ј-C-methylenephosphinate,[16] although in the last
gents, HBTU and HATU, gave relatively slow reactions. case the efficiency reported was discouraging. Coupling of
With HATU a higher rate was obtained than with HBTU, phosphinate 1 with 2a was carried out at various concentra-
giving 90% conversion into the product in about 18 min. tions of OXP (36–120 m) in neat pyridine or pyridine/ace-
Major differences in reactivity were also observed among tonitrile (1:3, v/v), as shown in Table 3. The rate was some-
the phosphonium reagents (Table 2). The use of PyFOP or what affected by the solvent composition, being slightly
PyAOP as condensing agents gave fast coupling reactions. faster in a pyridine/acetonitrile mixture (1:3, v/v) than in
However, PyAOP gave detectable amounts of oxidation neat pyridine. No side reactions were observed in the coup-
products during the condensation time. PyFOP gave fast ling to 2a. However, when phosphinate 1 was condensed
couplings (both at concentrations of 60 m or 120 m of with 2b by the use of 60 m OXP in pyridine or a mixture
the condensing reagent) in which reactions were nearly of pyridine/acetonitrile, traces of a side product were ob-
complete upon recording of the first spectrum (Ն90% con- served after 1 h (presumably from reaction with the lactam
version after about 4 min). No side reactions were observed function of the guanine base). MS analysis after prolonged
during these condensations, i.e., with alcohol present from treatment revealed the presence of the pyridinium adduct
the beginning. However, when phosphinate 1 was treated (m/z = 1293.5). A positive finding was then that the reaction
with 60 m PyFOP in pyridine/acetonitrile (1:3, v/v), in the rates were independent of the concentration of OXP, unlike
absence of an alcohol component, the activated phosphi- the reaction with DMOCP, which suggests a fast activation
nate underwent further conversions relatively fast (within step that is followed by a rate-limiting substitution step.
5 min) into an intermediate that decomposed to give the When the coupling between 1 and 2a was carried out by
corresponding C-phosphonate. Addition of an alcohol to the use of 36 m (1.2 equiv.) OXP in pyridine/acetonitrile
this intermediate, before it decomposed, did not result in (1:3, v/v), conversion into the product occurred at a rate
the formation of a phosphinate ester. Hence, the oxidative similar to that of the reaction with 60 m OXP (about 90%
side reaction can be a drawback in the use of condensing in 11 min). In the coupling of 1 to the guanosine derivative
reagents such as PyFOP or PyAOP when these reagents are 2b, we also did not observe any side reactions when the
used in a synthesis that involves premixing of the condens- reaction was carried out with 36 m OXP (1.2 equiv. to 1).
ing agent with the phosphinate before addition of the The reaction went to completion, and no O6-phosphoryla-
alcohol.
tion was detected even after leaving the mixture overnight.
To avoid the use of hydroxybenzotriazole derivatives, the Interestingly, the internucleoside linkage formation oc-
coupling of phosphinate 1 with alcohol 2a using 60 m Py- curred at a higher rate with 2b than with 2a; at a concentra-
ClOP in pyridine/acetonitrile (1:3, v/v) was performed. This tion of 36 m OXP the coupling was more than 90% com-
gave about 50% conversion into products in 1.5 h. To accel- plete when the first spectrum was recorded (4 min).
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Condensing Agents for Phosphinate Ester Formation with Nucleoside 5Ј-Hydroxyl Functions
Table 3. Formation of 3a in the condensations of 1 with 2a, aided by OXP.
Entry
Coupling agent (solvent system)
Conc.
[m]
Time for the formation of
75% product
90% product
3–1
3–2
3–3
3–4
3–5
3–6
3–7
3–8
OXP (pyridine)
OXP (pyridine)
OXP (pyridine/CH3CN)
60
120
60
36
36
36
36
36
11 min
11 min
6 min
6 min
n.e.[a]
6 min
4 min
n.e.[a]
17 min
17 min
11 min
11 min
Ͻ4 min
12 min
7 min
OXP (pyridine/CH3CN)
OXP/N-methylimidazole[b] (pyridine/CH3CN)
OXP/2,6-lutidine[b] (pyridine/CH3CN)
OXP/4-methoxypyridine[b] (pyridine/CH3CN)
OXP/sodium iodide[b] (pyridine/CH3CN)
Ͻ4 min
[a] n.e. = not estimated since no data could be collected by NMR before 4 min. reaction time. [b] The concentrations of N-methylimidazole,
2,6-lutidine, 4-methoxypyridine and sodium iodide were all 0.5 .
Treatment of phosphinate 1 with OXP in the absence of of the initial intermediate was rapid and essentially over
the 5Ј-hydroxyl component, using similar conditions, gave within 10 min, but with 4-methoxypyridine the first active
an activated 3Ј-deoxy-3Ј-C-methylenephosphinate deriva- intermediate had a longer life time (ca 50% remaining after
tive that was stable (several signals at δ = 33–34 ppm). Sub- about 20 min).
sequent addition of the 5Ј-hydroxyl component 2a to the
preactivated phosphinate building block gave 3a, at a rate
comparable to that obtained when 2a was present before
the addition of OXP. Attempts to further increase the reac-
Discussion
tion rate in the condensation between 1 and 2a were carried
Condensation of methylenephosphinate 1 with nucleo-
out using nucleophilic assistance and/or base catalysis. In side 2a by the aid of the investigated phosphonium deriva-
the first instance N-methylimidazole was utilized for this tives was either slow or (with hydroxybenzotriazole deriva-
purpose. Indeed, reaction of 1 with 2a using 36 m OXP tives) gave oxidative side reactions during condensation or
and 0.5  N-methylimidazole in pyridine/acetonitrile (1:3, preactivation of 1. Some of these reagents, in particular Py-
v/v) gave a significant increase in the reaction rate. Com- FOP, could be useful in solution synthesis but seem less
plete conversion into the product occurred within the time suitable for situations where preactivation of phosphinate
required to record the first spectrum. However, when the with the condensing reagent typically is preferred, e.g. in
treatment of 1 with OXP in the presence of 0.5  N-methyl- solid-phase oligo(nucleoside methylenephosphonate) syn-
imidazole was performed in the absence of the 5Ј-hydroxyl thesis via phosphinate intermediates. Acyl chlorides as con-
component, the activated phosphinate building block was densing reagents give side reactions at the lactam function
not stable. Side reactions took place forming unidentified of guanosine; however, the resulting side product is re-
products that failed to give 3a upon addition of 2a. Instead, ported to be cleaved by ammonolysis (as commonly used in
we considered the use of a less basic nucleophile (4-meth- the final deprotection of oligonucleotides) to regenerate the
oxypyridine), a poorly nucleophilic base (2,6-lutidine) and original structure of the nucleobase.[24] Nevertheless, piva-
a nonbasic nucleophile (iodide) (Table 3). Treatment of 1 loyl chloride is less than ideal since competing 5Ј-O-acyl-
and 2a with 36 m OXP and 2,6-lutidine (0.5 ) in pyr- ation leads to non-quantitative coupling. TPS-Cl gives fast
idine/acetonitrile (1:3, v/v) gave a rate similar to that of the condensation but causes relatively fast oxidation of the
reaction with only pyridine present, suggesting no strong product. TPS-Cl could in principle still be used as a coup-
base catalysis. On the other hand, similar reactions with ling agent, but adds little advantage compared to several
added 4-methoxypyridine or iodide were faster. The reac- other investigated alternatives. For oligonucleotide synthe-
tion in the presence of 4-methoxypyridine gave 90% prod- sis, further studies would be also needed to see whether the
uct in 7 min, and the reaction with added sodium iodide oxidized intermediates would cause further problems that
was complete when recording the first spectrum (less than could not be readily circumvented.
4 min). This clearly suggests that nucleophilic catalysis is
Most chlorophosphates gave competing 5Ј-O-phosphor-
operating. Although this can occur by displacing the chlo- ylation of the nucleoside component 2a, in addition to the
ride on OXP, as reported for several chlorophosphates,[34] known problem of O4- and O6-phosphorylation of the ura-
this would not affect the rate since the reaction is indepen- cil/thymine and guanine bases.[35–38] However, a couple of
dent on the OXP concentration. Presumably then, nucleo- the least reactive chlorophosphates (diisopropyl chloro-
philic catalysis takes place on the intermediate(s) formed phosphate, OCP and DMOCP) can be of interest for the
from the reaction of OXP with 1. When 1 was activated formation of internucleoside methylenephosphinate link-
with OXP in the presence of both iodide and 4-methoxypyr- ages. There are some limitations, though. Competing 5Ј-O-
idine, but in the absence of alcohol, the same problem as phosphorylation of 2a or 2b could be avoided, but O6-phos-
with N-methylimidazole appeared, i.e., consumption of the phorylation of the guanine base was still observed in all
initially formed intermediate. Again, this led to incomplete cases even at 60 m concentrations. However, with 60 m
coupling upon addition of 2a. With iodide the consumption DMOCP this side reaction is relatively minor which sug-
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A. Winqvist, R. Strömberg
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gests that this reagent could be suitable in the synthesis caution is needed though, since the lactam functionalities of
where low levels of O6-phosphorylation are acceptable. guanosine and uridine/thymidine (in the case of diisopropyl
In the oligo(nucleoside methylenephosphinate) synthesis, chlorophosphate and OCP) do not stay completely intact,
where repeated coupling takes place, it should be used with and further O4 (of U or T) and O6 (of G) protection may
caution since concomitant O6-phosphorylation may be- be necessary if one wishes to avoid this completely.
come too prominent if longer sequences are to be made. It
The use of OXP for the formation of internucleoside
has been reported that in the presence of various nucleo- methylenephosphinate linkages seems to be the most gene-
philes (pyridine or 1,2,4-triazole derivatives) further substi- ral solution. Although several of the other investigated con-
tution of the O6-phosphorylated species occur.[35,36] Studies densing agents can be considered, OXP in nearly equimolar
on the cleavage of these groups under ammonolytic condi- amounts to the phosphinate building block seems to be the
tions, which are commonly used for the deprotection of oli- safest choice with respect to minimisation of side reactions
gonucleotides, showed that both the regenerated original while still retaining a reasonable condensation time. With
structure of the nucleobase and the corresponding amino OXP as coupling reagent no side reactions could be de-
derivative could be formed. The latter reaction which leads tected at near equimolar amounts of the reagents. The
to a modification of the base is most pronounced with gua- phosphinate 1 was also stable after activation by OXP prior
nosine (converted into 2,6-diaminopurine), and the dif- to coupling which could make the reagent useful not only
ferent tested deblocking conditions gave substantial for solution synthesis where the alcohol component is pres-
amounts of base modification.[36] To avoid this problem, ent from the start, but also when phosphinate is preacti-
O6-protection of guanosine[39,40] would be recommended. If vated in the absence of alcohol. It certainly seems worth to
O6-protection is employed together with O4-protection of try to optimise coupling time and other conditions with
uridines (or thymidines), diisopropyl chlorophosphate or OXP in the synthesis of oligonucleoside (methylenephosphi-
OCP could perhaps be advantageous, since condensations nate)s.
are more rapid with these reagents than with DMOCP.
The use of OXP for the formation of oligo(nucleoside
methylenephosphinate)s seems to be the most general solu-
tion, at least if complete avoidance of side reactions is de-
sired. Due to that the rate is independent on the concentra-
Experimental Section
tion of OXP, this reagent can be used at near equimolar
amounts to the phosphinate building block and still give a
decent coupling time. Under such conditions we could not
observe any side reactions, neither during condensation nor
in the reaction mixture afterwards. The OXP-promoted
condensation can be catalysed by nucleophiles such as N-
methylimidazole, iodide and 4-methoxypyridine. However,
the use of these nucleophilic catalysts is less suitable if pre-
activation of the phosphinate is done, since the initially
formed active intermediate decomposes relatively fast,
which leads to incomplete condensation. On the other
hand, in the use of 36 m OXP in pyridine/acetonitrile (1:3,
v/v) without any additional nucleophilic catalyst, gave no
detectable side reactions and a reasonable time of conden-
sation also upon preactivation of the phosphinate.
General Remarks and Methods: NMR spectra were recorded with
a
Bruker Avance DRX-400 instrument (400.13 MHz in 1H,
162.00 MHz in 31P, and 100.62 MHz in 13C). 13C and H chemical
shifts are given in ppm, downfield from external TMS. 31P chemical
shifts are given downfield from external H3PO4 (2%, v/v) in D2O.
Most assignments of 1H and 13C NMR signals were made by stan-
1
dard 1H-1H COSY and H-13C HMQC experiments. Silica gel col-
1
umn chromatography was generally carried out using Matrex silica,
60 Å (35–70 µm, Amicron). TLC analysis was carried out on pre-
coated plates Silica Gel 60 F254 (Merck), with detection by UV light
and/or by charring with 8% sulfuric acid in methanol. Solutions
were concentrated under reduced pressure at temperatures not ex-
ceeding 40 °C. Pivaloyl chloride was purchased from Fluka and
distilled before use. PyAOP and PyFOP[41] as well as OCP and
DMOCP[42] were synthesized essentially according to the published
methods, the two latter ones were additionally recrystallised from
dichloromethane/toluene or dichloromethane/hexane. OXP was
purchased from Aldrich. 2-N-Phenoxyacetylguanosine was pre-
pared essentially according to a published method[43] using the acid
anhydride for acylation, and the crude product was further purified
by crystallization from methanol. Other reagents and solvents were
of ordinary commercial grade unless otherwise stated. Diisopropyl
chlorophosphate was purchased from Toronto Research Chemicals
Inc. Diethyl chlorophosphate was purchased from Aldrich. BOP
and HBTU were purchased from Nova Biochem, and HATU was
purchased from Perseptive Biosystems. Acetonitrile (p.a.) was dried
with molecular sieves (3 Å). Pyridine (p.a.) was dried with molecu-
lar sieves (4 Å). The phosphinate building block 1 was prepared as
described previously.[18]
Conclusions
In the present study a large number of condensing rea-
gents were evaluated for coupling of the phosphinate build-
ing block 1 to 5Ј-hydroxyl components 2a or 2b, to achieve
the 3Ј-deoxy-3Ј-C-methylenephosphinate esters 3a or 3b.
Several of the condensing reagents that were examined were
most effective in the synthesis of internucleoside methylene-
phosphinate linkages. Condensations using PyFOP were
fast and efficient, but only if premixing of coupling agent
and phosphinate in the absence of hydroxyl component can
be avoided. Couplings using diisopropyl chlorophosphate,
OCP and especially DMOCP proceeded at acceptable reac-
3Ј-O-(4-Methoxytrityl)thymidine
(2a):[44]
Thymidine
(5 g,
20.6 mmol) was dried by evaporation of added pyridine (25 mL)
and was dissolved in dry pyridine (50 mL). The solution was cooled
tion times and could find all use in solution syntheses. Some to 0 °C (ice bath), and pivaloyl chloride (2.8 mL, 22.7 mmol,
1710
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1705–1714

Condensing Agents for Phosphinate Ester Formation with Nucleoside 5Ј-Hydroxyl Functions
1.1 equiv.) was added. The reaction mixture was left in a slowly
melting ice bath for 1 h and then stirred at room temperature for
an additional 3 h. 4-Methoxytrityl chloride (12.7 g, 41.3 mmol,
2 equiv.) was added, and the reaction mixture was stirred at 20 °C
for 85 h. The reaction mixture was diluted with toluene (200 mL)
and washed with saturated aqueous NaHCO3 (2ϫ 200 mL). The
combined water layers were washed with CH2Cl2 (100 mL). The
combined organic layers were dried (Na2SO4) and concentrated.
The residue was further dried by evaporation of added toluene
(100 mL) and was then dissolved in THF/ethanol (1:1, v/v;
150 mL). 2  aqueous sodium hydroxide (75 mL, 150 mmol,
7.3 equiv.) was added, and the reaction mixture was stirred at 60–
70 °C for 3 h. The mixture was cooled to 20 °C, and ammonium
chloride (8.5 g, 159 mmol, 7.7 equiv.) was added. The mixture was
concentrated to remove the organic solvents. The residual aqueous
mixture was washed with CH2Cl2 (2ϫ 150 mL). The combined or-
ganic layers were dried (Na2SO4) and concentrated. The residue
was purified by silica gel column chromatography (30–80% ethyl
acetate in toluene) to give 2a (9.2 g, 87%). 1H NMR (CDCl3,
20 °C): δ = 1.61–1.66 (m, 1 H, 2Јa-CH), 1.78 (s, 3 H, CH3), 1.82–
1.86 (m, 1 H, 2Јb-CH), 2.39 (dd, J = 4.0, 6.5 Hz, 1 H, 5Ј-OH),
3Ј-O-(4-Methoxytrityl)thymidine [2Ј-O-(tert-Butyldimethylsilyl)-3Ј-
deoxy-5Ј-O-(4-methoxytrityl)uridine 3Ј-C-methylenephosphinate]
(3a): To a solution of 2a (35.2 mg, 0.068 mmol) and phosphinate 1
(57 mg, 0.072 mmol, 1.05 equiv.) in dry pyridine/acetonitrile (6:10,
v/v; 1.6 mL) was added a solution of PyFOP (84.7 mg, 0.144 mmol,
2.10 equiv.) in dry acetonitrile (0.8 mL). The reaction mixture was
stirred at 20 °C for 30 min. The mixture was diluted with CH2Cl2
(10 mL) and washed with saturated aqueous NaHCO3 (10 mL).
The water layer was washed with CH2Cl2 (5 mL). The combined
organic layers were dried (Na2SO4), toluene (5 mL) was added, and
the solution was concentrated. The residue was further dried by
evaporation of added toluene (5 mL) and purified by silica gel col-
umn chromatography (30–100% ethyl acetate in CH2Cl2) to give
3a as an isomeric mixture (in an isomeric of ratio of 53:47 as deter-
mined by relative integrals of 1H NMR signals, 80.7 mg, 99%). The
isomeric mixture was used for the next step. 1H NMR (CD3CN,
20 °C): δ = 0.09 (s, 3 H, SiCH3), 0.20 (s, 3 H, SiCH3), 0.85 [s, 9 H,
SiC(CH3)3], 1.38–1.54 (2ϫ m, 1 H, CH2P), 1.64–1.89 (2ϫ m, 2 H,
2Ј-H(T)), 1.96–2.03 (2ϫ m, 1 H, CH2P), 2.47–2.67 (2ϫ m, 1 H, 3Ј-
2
3
H
(U)), 3.30 and 3.33 (2ϫ dd, J = 11.7, J = 2.9, 3.2 Hz, 1 H, 5Ј-
Hb(U)), 3.49 and 3.54 (2ϫ dd, 2J = 10.6, 12.9, 3J = 2.5 Hz, 1 H,
3,19–3.25 (m, 1 H, 5Ј-Ha) 3.53–3.59 (m, 1 H, 5Ј-Hb), 3.72 (s, 3 H, 5Ј-Ha(U)), 3.61–3.72 (2ϫ m, 2 H, 5Ј-Ha(T) and 5Ј-Hb(T)), 3.73 and
OCH3) 3.87–3.91 (m, 1 H, 4Ј-H), 4.29 (d, J = 6.3 Hz, 1 H, 3Ј-H), 3.75 (2ϫ s, 6 H, 2 ϫ OCH3), 3.84–3.88 and 3.92–3.95 (2ϫ m, 1 H,
6.06 (dd, J1Ј,2Ј = 5.8, 8.8 Hz, 1 H, 1Ј-H), 6.77 (d, J = 8.9 Hz, 2 H,
MMT) 7.07–7.28 (m, 9 H, 6-H, MMT), 7.38 (d, J = 7.5 Hz, 4 H,
MMT) 8.53 (s, 1 H, NH) ppm.
4Ј-H(T)), 3.97–4.07 (2ϫ m, 1 H, 4Ј-H(U)), 4.16–4.20 and 4.25–4.28
(2ϫ m, 1 H, 3Ј-H(T)), 4.43 and 4.46 (2ϫ d, J = 4.2, 4.4 Hz, 1 H,
2Ј-H(U)), 5.17 and 5.18 (2ϫ d, J = 8.1 Hz, 1 H, 5-H(U)), 5.61 and
5.62 (2ϫ s, 1 H, 1Ј-H(U)), 6.09–6.17 (2ϫ m, 1 H, 1Ј-H(T)), 6.83–
6.90, 7.12–7.36 and 7.40–7.50 (3ϫ m 29 H, 6-H(T), 2ϫ MMT),
6.94 and 7.02 (2ϫ dd, 1JP,H = 546, 548 Hz, 3JP,H = 2 Hz, 1 H, PH),
7.90 and 7.93 (2ϫ d, J = 8.4 Hz, 1 H, 6-H(U)), 8.97 (br. s, 1 H,
NH), 9.15 and 9.22 (2ϫ br. s, 1 H, NH) ppm. 31P NMR (CD3CN,
20 °C): δ = 37.8, 37.9 ppm. C66H73N4O13PSi (1189.38): calcd. C
66.65, H 6.19, N 4.71; found C 66.47, H 6.24, N 4.76.
2Ј,3Ј-Di-O-butyryl-2-N-(phenoxyacetyl)guanosine (2b): 5Ј-O-(4-
Methoxytrityl)-2-N-phenoxyacetylguanosine[45] (1 g, 1.45 mmol)
was dried by evaporation of added pyridine (2ϫ 5 mL) and dis-
solved in dry pyridine (2 mL). Butyric anhydride (0.52 mL,
3.2 mmol, 2.2 equiv.) was added, and the reaction mixture was
stirred at 20 °C for 20 h. The mixture was concentrated and the
residue dissolved in CH2Cl2 (60 mL). The solution was washed
with saturated aqueous NaHCO3 (30 mL). The water layer was
washed with CH2Cl2 (20 mL). The combined organic layers were
dried (Na2SO4), diluted with toluene (20 mL) and concentrated.
The residue was further dried by evaporation of added toluene
(10 mL) and dissolved in CH2Cl2 (5 mL). 1-Butanol (2.7 mL,
29.0 mmol, 20 equiv.) was added followed by p-toluenesulfonic acid
monohydrate (0.83 g, 4.35 mmol, 3 equiv.), and the reaction mix-
ture was stirred at 20 °C for 30 min. The mixture was diluted with
CH2Cl2 (50 mL) and washed with saturated aqueous NaHCO3
(50 mL). The water layer was washed with CH2Cl2 (20 mL). The
combined organic layers were dried (Na2SO4), diluted with toluene
(20 mL) and concentrated. The residue was purified by recrystalli-
sation (CH2Cl2/diethyl ether) to give 2b (0.65 g, 80%). 1H NMR
2Ј,3Ј-Di-O-butyryl-2-N-(phenoxyacetyl)guanosine [2Ј-O-(tert-Butyl-
dimethylsilyl)-3Ј-deoxy-5Ј-O-(4-methoxytrityl)uridine 3Ј-C-methyl-
enephosphinate] (3b): To a solution of 2b (20 mg, 0.036 mmol) and
phosphinate 1 (30 mg, 0.038 mmol, 1.05 equiv.) in dry pyridine/ace-
tonitrile (6:10, v/v; 0.8 mL) was added a solution of PyFOP (44 mg,
0.075 mmol, 2.1 equiv.) in dry acetonitrile (0.4 mL). The reaction
mixture was stirred at 20 °C for 30 min. The mixture was diluted
with CH2Cl2 (10 mL) and washed with saturated aqueous NaHCO3
(10 mL). The water layer was washed with CH2Cl2 (5 mL). The
combined organic layers were dried (Na2SO4), toluene (5 mL) was
added, and the solution was concentrated. The residue was further
dried by evaporation of added toluene (5 mL) and purified by silica
gel column chromatography (2–5% methanol in CH2Cl2) to give
(CDCl3, 20 °C): δ = 0.89 [t, J = 7.4 Hz, 3 H, C(O)CH2CH2CH3], 3b as an isomeric mixture (in an isomeric ratio of 52:48 as deter-
1
1.00 [t, J = 7.4 Hz, 3 H, C(O)CH2CH2CH3], 1.58 [sext, J = 7.4 Hz, mined by relative integrals of H NMR signals, 41 mg, 93%). The
2 H, C(O)CH2CH2CH3], 1.70 [sext, J = 7.4 Hz, 2 H, C(O)CH2- isomeric mixture was used for the next step. 1H NMR (CD3CN,
CH2CH3], 2.26 [t, J = 7.4 Hz, 2 H, C(O)CH2CH2CH3], 2.39 [t, J
20 °C): δ = 0.07 and 0.13 (2ϫ s, 3 H, SiCH3), 0.20 and 0.22 (2ϫ
= 7.4 Hz, 2 H, C(O)CH2CH2CH3], 3.86–4.02 (m, 2 H, 5Ј-Ha and s, 3 H, SiCH3), 0.86–1.01 [15 H, 2ϫ C(O)CH2CH2CH3 and
5Ј-Hb), 4.33 (s, 1 H, 4Ј-H), 4.71 (s, 2 H, CH2OPh), 4.95 (d, J =
8.7 Hz, 1 H, OH), 5.71 (d, J = 5.3 Hz, 1 H, 3Ј-H), 5.85–5.89 (m, 1
H, 2Ј-H), 5.95 (d, J1Ј,2Ј = 7.3 Hz, 1 H, 1Ј-H), 7.05 (d, J = 8.3 Hz,
2 H, Ph), 7.10 (t, J = 7.4 Hz, 1 H, Ph), 7.37 (t, J = 7.9 Hz, 2 H,
Ph), 7.77 (s, 1 H, 8-H), 9.30 (s, 1 H, NH), 11.8 (s, 1 H, NH) ppm.
13C NMR (CDCl3, 20 °C): δ = 13.7 [C(O)CH2CH2CH3], 13.8
SiC(CH3)3], 1.49–1.71 [5 H, CH2P and 2ϫ C(O)CH2CH2CH3],
2.14 (m, 1 H, CH2P), 2.24–2.43 [4 H, 2ϫ C(O)CH2CH2CH3], 2.63–
2.75 (2ϫ m, 1 H, 3Ј-H(U)), 3.30–3.40 (m, 1 H, 5Ј-Hb(U)), 3.49–3.58
(m, 1 H, 5Ј-Ha(U)), 3.74 and 3.77 (2ϫ s, 3 H, CH3O), 4.01–4.07
(2ϫ m, 1 H, 4Ј-H(U)), 4.35–4.56 (4 H, 2Ј-H(U), 5Ј-Ha(G), 5Ј-Hb(G)
and 4Ј-H(G)), 4.69–4.78 (2 H, CH2OPh), 5.16 and 5.18 (2ϫ d, J =
[C(O)CH2CH2CH3], 18.3 [C(O)CH2CH2CH3], 18.5 [C(O)- 6.6 Hz, 1 H, 5-H(U)), 5.62–5.84 (3 H, 2Ј-H(G), 3Ј-H(G) and
CH2CH2CH3], 35.6 [C(O)CH2CH2CH3], 36.0 [C(O)CH2CH2CH3], 1Ј-H(U)), 5.93 and 5.97 (2ϫ d, J = 5.4 and 6.6 Hz) (1 H, 1Ј-H(G)),
1
62.6 (C-5Ј), 66.6 (CH2OPh), 71.9 (C-3Ј), 72.7 (C-2Ј), 85.8 (C-4Ј), 7.12 and 7.14 (2ϫ d, JP,H = 554 and 556 Hz for the respective
88.1 (C-1Ј), 115.0, 123.4, 125.2, 130.1, 139.1 (C-8), 146.7, 146.8, isomer, 1 H, PH), 6.84–7.46 (19 H, MMT and Ph), 7.76 and 7.81
155.0, 156.4, 169.7, 171.9, 172.6 ppm. C26H31N5O9 (557.56): calcd.
C 56.01, H 5.60, N 12.56; found C 55.94, H 5.63, N 12.60.
(2ϫ s, 1 H, 8-H), 7.94 and 7.97 (2ϫ d, J = 6.6 Hz, 1 H, 6-H(U)),
9.25 (br. s, 1 H, NH), 10.7–11.2 (2ϫ br. s, 1 H, NH), 11.8 (br. s, 1
Eur. J. Org. Chem. 2008, 1705–1714
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1711

A. Winqvist, R. Strömberg
FULL PAPER
H, NH) ppm. 31P NMR (CD3CN, 20 °C): δ = 39.7 and 40.3 ppm.
C(O)CH2CH2CH3], 2.47–2.56 (m, 1 H, 3Ј-H(U)), 3.02 [q, J =
C62H74N7O16PSi (1232.36): calcd. C 60.43, H 6.05, N 7.96; found 7.4 Hz, 1 H, 2ϫ N(CH2CH3)3], 3.31 (br. d, J = 10 Hz, 1 H, 5Ј-
2
C 60.25, H 6.03, N 8.01.
Ha(U)), 3.44 (dd, J = 11, J4Ј,5Ј = 3.7 Hz, 1 H, 5Ј-Hb(U)), 3.69 (s, 3
H, OCH3), 3.82–3.88 (m, 1 H, 5Ј-Ha(G)), 4.12–4.27 (3ϫ m, 1 H,
4Ј-H(U), 1 H, 5Ј-Hb(G), 1 H, 4Ј-H(G)), 4.53 (dd, J2Ј,3Ј = 5.0 Hz, 1 H,
2Ј-H(U)), 4.78 (d, 2J = 16.2 Hz, 1 H, CH2OPh), 4.89 (d, 2J =
16.2 Hz, 1 H, CH2OPh), 5.10 (d, J = 8.9 Hz, 1 H, 5-H(U)), 5.59
Triethylammonium 3Ј-O-(4-Methoxytrityl)thymidine [2Ј-O-(tert-Bu-
tyldimethylsilyl)-3Ј-deoxy-5Ј-O-(4-methoxytrityl)uridine 3Ј-C-meth-
ylenephosphonate] (4a): Compound 3a (50 mg, 0.042 mmol) was
dissolved in pyridine (1.25 mL), and a solution of iodine (50 mg,
197 mmol, 4.7 equiv.) in pyridine/water (96:4, v/v; 1.25 mL) was
added. The reaction mixture was stirred at 20 °C for 24 h. The mix-
ture was diluted with CH2Cl2 (20 mL) and washed with a combined
solution of aqueous sodium thiosulfate (10%, w/w; 10 mL) and 2 
aqueous TEAB (10 mL). The water layer was washed with CH2Cl2
(10 mL). The combined organic layers were washed with 2  aque-
ous TEAB (10 mL). The resulting aqueous layer was washed with
CH2Cl2 (2ϫ 5 mL). The combined organic layers were dried
(Na2SO4) and concentrated. The residue was further dried by evap-
oration of added toluene (5 mL) and purified by silica gel column
chromatography (5–20% methanol in CH2Cl2 containing 0.1% tri-
ethylamine) to give 4a (48 mg, 95%). 1H NMR (CD3OD, 20 °C): δ
= 0.19 (s, 3 H, SiCH3), 0.21 (s, 3 H, SiCH3), 0.92 [s, 9 H, SiC-
(CH3)3], 1.25–1.42 [10 H, CH2P and N(CH2CH3)3], 1.74–1.82 (5
H, 2Ј-Ha(T), 2Ј-Hb(T) and CH3(T)), 1.87–2.00 (m, 1 H, CH2P), 2.57–
2.66 (m, 1 H, 3Ј-H(U)), 3.14 [q, J = 7.3 Hz, 6 H, N(CH2CH3)3],
3.40–3.53 (3 H, 5Ј-Hb(T), 5Ј-Ha(U) and 5Ј-Hb(U)), 3.71 (s, 6 H, 2ϫ
CH3O), 3.79 (2 H, 4Ј-H(T) and 5Ј-Ha(T)), 4.11–4.17 (m, 1 H, 4Ј-
H(U)), 4.27–4.32 (m, 1 H, 3Ј-H(T)), 4.61–4.65 (m, 1 H, 2Ј-H(U)), 5.13
(d, J = 8.0 Hz, 1 H, 5-H(U)), 5.76 (d, J1Ј,2Ј = 2.2 Hz, 1 H,
1Ј-H(U)), 6.26 (dd, J1Ј,2Јa = 6.1, J1Ј,2Јb = 8.1 Hz, 1 H, 1Ј-H(T)), 6.82–
7.45 (28 H, 2ϫ MMT), 7.59 (s, 1 H, 6-H(T)), 8.05 (d, J = 8.0 Hz,
3
(dd, J = 4.2 and 2.3 Hz, 1 H, 3Ј-H(G)), 5.68 (d, J1Ј,2Ј = 2.8 Hz, 1
H, 1Ј-H(U)), 6.01–6.05 (2 H, 1Ј-H(G) and 2Ј-H(G)), 6.82–7.38 (19 H,
2
MMT and Ph), 7.73 (d, J = 8.1 Hz, 1 H, 6-H(U)), 8.12 (s, 1 H, 8-
H(G)) ppm. 13C NMR ([D6]DMSO/D2O, 4:1, v/v; 60 °C): δ =
–5.18 (SiCH3), –5.08 (SiCH3), 8.60 [N(CH2CH3)3], 13.0 [C(O)-
CH2CH2CH3], 13.3 [C(O)CH2CH2CH3], 17.56 and 17.58
[SiC(CH3)3 and C(O)CH2CH2CH3], 17.8 [C(O)CH2CH2CH3], 22.4
1
(d, JC,P
= 135 Hz, CH2P), 25.5 [SiC(CH3)3], 34.9 [C(O)-
CH2CH2CH3], 35.2 [C(O)CH2CH2CH3], 38.4 (C-3Ј(U)), 46.2
[N(CH2CH3)3], 55.0 (OCH3), 62.5 (C-5Ј(U)), 63.7 (C-5Ј(G)), 66.1
3
(CH2OPh), 71.3 (C-3Ј(G)), 71.8 (C-2Ј(G)), 76.9 (d, JC,P = 6.8 Hz,
3
3
C-2Ј(U)), 82.6 (d, JC,P = 7.3 Hz, C-4Ј(G)), 83.3 (d, JC,P = 11.3 Hz,
C-4Ј(U)), 86.5 and 86.8 [C(Ar)3 and C-1Ј(G)], 89.6 (C-1Ј(U)), 100.9
(C-5(U)), 113.2, 114.5, 121.2, 121.3, 126.9, 127.00, 127.8, 127.9,
128.1, 129.5, 130.1, 134.7, 140.0 (C-6(U) and C-8(G)), 143, 8, 143.9,
147.2, 148.5, 150.3, 157.6, 158.3, 163.2, 171.6, 171.7, 172.1 ppm.
31P NMR ([D6]DMSO/D2O, 4:1, v/v; 60 °C): δ = 20.51 ppm. MS
(ES+/TOF): calcd. for [C68H90N8O17PSi]+ 1349.5931; found
1349.5934.
2Ј,3Ј,4Ј-Tri-O-butyryluridine (5): Uridine (100 mg, 0.41 mmol) was
dried by evaporation of added pyridine (2ϫ 2 mL) and dissolved
in dry pyridine (4 mL). Butyric anhydride (0.23 mL, 1.43 mmol,
1 H, 6-H(U)) ppm. 13C NMR (CD3OD, 20 °C): δ = –4.4 (SiCH3), 3.5 equiv.) was added, and the reaction mixture was stirred at 20 °C
–4.3 (SiCH3), 9.2 [N(CH2CH3)3], 12.7 (CH3(T)), 19.0 [SiC(CH3)3], for 25 h. The reaction mixture was concentrated. The residue was
1
23.6 (d, JC,P = 138 Hz, CH2P), 26.5 [SiC(CH3)3], 39.6 (C-3Ј(U)), diluted with CH2Cl2 (20 mL) and washed with saturated aqueous
39.9 (C-2Ј(T)), 47.7 [N(CH2CH3)3], 55.8 (2ϫ OCH3), 64.5 NaHCO3 (10 mL). The water layer was washed with CH2Cl2 (2ϫ
3
(C-5Ј(U)), 65.1 (C-5Ј(T)), 76.6 (C-3Ј(T)), 78.5 (C-2Ј(U)), 85.1 (d, J4Ј,P 10 mL). The combined organic layers were dried (Na2SO4), diluted
= 13.3 Hz, C-4Ј(U)), 86.4 and 86.5 (C-4Ј(T) and C-1Ј(T)), 88.5, 88.8,
91.5 (C-1Ј(U)), 101.8 (C-5(U)), 111.9, 114.3, 114.4, 128.2, 128.3,
129.1, 129.7, 129.8, 131.8 131.9, 135.9, 136.9 (C-6(T)), 138.1, 142.4
(C-6(U)), 145.5, 146.1, 146.2, 152.0, 152.3, 160.3, 160.4, 166.2,
166.3 ppm. 31P NMR (CD3OD, 20 °C): δ = 22.7 ppm. MS (ES+/
TOF): calcd. for [C72H89N5O14PSi]+ 1306.5913; found 1306.5912.
with toluene (5 mL) and concentrated. The residue was purified
by silica gel column chromatography (stepwise gradient of 1–3%
methanol in CH2Cl2). The product was obtained as an oil and ly-
1
ophilised from 1,4-dioxane to give 5 as an oil (184 mg, 99%). H
NMR ([D6]DMSO, 20 °C): δ = 0.82–0.92 [9 H, C(O)CH2CH2CH3],
1.50–1.56 [6 H, C(O)CH2CH2CH3], 2.28–2.35 [6 H, C(O)-
CH2CCH3], 4.21–4.34 (3 H, 4Ј-H, 5Ј-Ha and 5Ј-Hb), 5.35 (t, J =
5.2 Hz, 1 H, 2Ј-H), 5.46 (t, J = 5.5 Hz, 1 H, 3Ј-H), 5.72 (d, J =
8.0 Hz, 1 H, 5-H), 5.88 (dd, J = 5.2 and 1.9 Hz, 1 H, 1Ј-H), 7.71
(d, J = 8.0 Hz, 1 H, 6-H), 11.5 (s, 1 H, NH) ppm. 13C NMR
Triethylammonium
2Ј,3Ј-Di-O-butyryl-2-N-(phenoxyacetyl)gua-
nosine [2Ј-O-(tert-Butyldimethylsilyl)-3Ј-deoxy-5Ј-O-(4-methoxytri-
tyl)uridine 3Ј-C-methylenephosphonate] (4b): Compound 3b (39 mg,
0.032 mmol) was dissolved in pyridine (1 mL), and a solution of
iodine (38 mg, 150 mmol, 4.7 equiv.) in pyridine/water (96:4, v/v;
1 mL) was added. The reaction mixture was stirred at 20 °C for
24 h. The mixture was diluted with CH2Cl2 (20 mL) and washed
with a combined solution of aqueous sodium thiosulfate (10%,
w/w; 10 mL) and 2  aqueous TEAB (10 mL). The water layer was
washed with CH2Cl2 (10 mL). The combined organic layers were
washed with 2  aqueous TEAB (10 mL). The resulting aqueous
layer was washed with CH2Cl2 (2ϫ 5 mL). The combined organic
layers were dried (Na2SO4) and concentrated. The residue was fur-
ther dried by evaporation of added toluene (5 mL) and purified
by silica gel column chromatography (5–20% methanol in CH2Cl2
(CDCl3, 20 °C):
δ = 13.9 [C(O)CH2CH2CH3], 14.0 [C(O)-
CH2CH2CH3], 14.1 [C(O)CH2CH2CH3], 18.6 [C(O)CH2CH2CH3],
18.68 [C(O)CH2CH2CH3], 18.71 [C(O)CH2CH2CH3], 36.0 [C(O)-
CH2CH2CH3], 36.1 [C(O)CH2CH2CH3], 36.4 [C(O)CH2CH2CH3],
63.4 (C-5Ј), 70.5 and 73.0 (C-2Ј and C-3Ј), 80.6 (C-4Ј), 87.6 (C-1Ј),
103.5 (C-5), 139.3 (C-6), 150.2, 162.6, 172.4, 172.9 ppm.
C21H30N2O9 (454.48): calcd. C 55.50, H 6.65, N 6.16; found C
55.30, H 6.70, N 6.20.
2Ј,3Ј,4Ј-Tri-O-butyryl-2-N-(phenoxyacetyl)guanosin (6): 2-N-(Phen-
oxyacetyl)guanosine (100 mg, 0.24 mmol) was dried by evaporation
of added pyridine (2ϫ 2 mL) and dissolved in dry pyridine (4 mL).
Butyric anhydride (0.16 mL, 0.96 mmol, 3.5 equiv.) was added, and
the reaction mixture was stirred at 20 °C for 25 h. The reaction
1
containing 0.1% triethylamine) to give 4b (38 mg, 89%). H NMR
([D6]DMSO/D2O, 4:1, v/v; 60 °C): δ = 0.09 (s, 6 H, 2ϫ SiCH3),
0.78 [t, J = 7.3 Hz, 3 H, C(O)CH2CH2CH3], 0.82 [s, 9 H, SiC- mixture was concentrated. The residue was diluted with CH2Cl2
(CH3)3], 0.93 [t, J = 7.4 Hz, 3 H, C(O)CH2CH2CH3], 1.18 [t, J = (20 mL) and washed with saturated aqueous NaHCO3 (10 mL).
7.3 Hz, 6 H, 2ϫ N(CH2CH3)3], 1.30–1.37 (m, 1 H, CH2P), 1.45 [h,
J = 7.2 Hz, 2 H, C(O)CH2CH2CH3], 1.59 [h, J = 7.3 Hz, 2 H, C(O)-
CH2CH2CH3], 1.81–1.91 (m, 1 H, CH2P), 2.21 [dt, J = 7.2 and
The water layer was washed with CH2Cl2 (2ϫ 10 mL). The com-
bined organic layers were dried (Na2SO4). Toluene (5 mL) was
added, and the organic layer was concentrated. The residue was
3.5 Hz 2 H, C(O)CH2CH2CH3], 2.36 [dt, J = 7.2 and 1.6 Hz, 2 H, purified by silica gel column chromatography (stepwise gradient of
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Eur. J. Org. Chem. 2008, 1705–1714

Condensing Agents for Phosphinate Ester Formation with Nucleoside 5Ј-Hydroxyl Functions
1–3% methanol in CH2Cl2). The product was obtained as an oil
and lyophilised from 1,4-dioxane to give crystalline 6 (122 mg,
81%). 1H NMR ([D6]DMSO, 20 °C): δ = 0.79–0.94 [9 H, C(O)-
CH2CH2CH3], 1.47–1.59 [6 H, C(O)CH2CH2CH3], 2.22–2.44 [6 H,
C(O)CH2CH2CH3], 4.29–4.43 (3 H, 4Ј-H, 5Ј-Ha and 5Ј-Hb), 4.88
(s, 2 H, CH2OPh), 5.55 (dd, J = 5.9 and 3.8 Hz, 1 H, 3Ј-H), 5.87
(t, J = 6.0 Hz, 1 H, 2Ј-H), 6.09 (d, J = 6.1 Hz, 1 H, 1Ј-H) 6.97–
7.01 and 7.28–7.56 (5 H, Ph), 8.27 (s, 1 H, 8-H), 11.69 (br. s, 1 H,
NH), 11.85 (br. s, 1 H, NH) ppm. 13C NMR (CDCl3, 20 °C): δ =
13.57 [2ϫ C(O)CH2CH2CH3] 13.64 [C(O)CH2CH2CH3], 18.2
[2ϫ C(O)CH2CH2CH3], 18.3 [C(O)CH2CH2CH3], 35.6 [C(O)-
CH2CH2CH3], 35.7 [C(O)CH2CH2CH3], 35.8 [C(O)CH2CH2CH3],
62.6 (C-5Ј), 66.8 (CH2OPh), 70.1 (C-3Ј), 72.9 (C-2Ј), 79.8 (C-4Ј),
86.8 (C-1Ј), 114.9, 122.7, 122.9, 129.9, 138.1 (C-8), 146.4, 147.3,
155.1, 156.4, 169.7, 172.0, 172.3, 173.1 ppm. C30H37N5O10
(627.65): calcd. C 57.41, H 5.94, N 11.16; found C 57.24, H 6.01,
N 11.14.
the NMR tube with compound 1 (8.6 mg, 10.8 µmol) and then a
solution of 120 or 240 m coupling reagent in dry acetonitrile
(180 µL, 21.6 or 43.2 mmol, 2 or 4 equiv.).
In case of using a chlorophosphate as coupling agent, the 5Ј-O-
phosphorylated side products were identified by addition of an in-
ternal standard (ca. 0.5 equiv.). The internal standard was prepared
in advance prior to use by mixing 120 m chlorophosphate in dry
acetonitrile (45 µL, 5.4 mmol) and 53 m 2a in dry acetonitrile/
pyridine (1:1, v/v; 90 µL, 4.8 mmol). Side products resulting from
O4-phosphorylation were detected by 31P NMR spectroscopy and
in several cases also by mass pectrometry (see above). Signals that
corresponded to compounds to hydrolysis, such as remaining chlo-
rophosphate, were also identified by their absence in the spectra
taken after the addition of water (10–20 µL) to the reaction mix-
ture.
Procedure for the Coupling of Phosphinate 1 to 2b and Analysis of
the Formation of Side Products by Subsequent Reaction of the Gua-
nosine Moiety by the Coupling Reagents DMOCP and OXP: The
coupling of compound 1 to 2b using 120 (or 60) m DMOCP or
120 (or 60) m OXP were carried out according to Methods B
and C, respectively. After complete coupling, the formation of side
products was monitored by 31P NMR spectroscopy. The nature of
side products resulting from O6-phosphorylation or reaction of
OXP at the O6-position of guanosine was, apart from being de-
tected by 31P NMR spectroscopy, confirmed by mass spectrometry
(ESI-TOF) after a reaction time of 6 h, which gave the correct mass
for the corresponding phosphorylated 3b in the reaction mixture
containing DMOCP (ES+/TOF: m/z = 1380.5 [C67H84N7O19-
P2Si]+) and the pyridinium adduct (which is formed in a subsequent
step after the reaction at the O6-position) in the reaction mixture
containing OXP (ES+/TOF: m/z = 1293.5 [C67H78N8O15PSi]+).
General Procedure for Analysis of the Coupling of Phosphinate 1 to
2a
Method A (for Acyl Chlorides as Coupling Reagents): A solution
of phosphinate 1 (8.6 mg, 10.8 µmol) in dry pyridine (50 µL) was
transferred to an NMR tube. The solution was concentrated and
the residue was dried at 50 mbar overnight. The NMR tube with
compound 1 was equipped with a co-axial inner tube containing
H3PO4 (2%, v/v) in D2O, and a solution of 57 m 2a in dry aceto-
nitrile/pyridine (3:1, v/v; 180 µL, 10.3 µmol, 0.95 equiv.) was added.
A 120 or 240 m solution of the coupling agent in dry acetonitrile/
pyridine (3:1, v/v; 180 µL, 21.6 or 43.2 mmol, 2 or 4 equiv.) was
added, and the reaction was monitored by 31P NMR spectroscopy.
The signals of the two isomeric products were observed at δ = 37.7
and 37.0 ppm for all reactions carried out in acetonitrile/pyridine
(3:1, v/v) with a variation of 0.1–0.2 ppm. The formation of prod-
uct was measured using the integral from the H3PO4 signal as refer-
ence. The integral of the product was plotted against time, and this
was used to obtain times for 75 and 90% completion of reactions.
When the reaction was complete an additional 1H-coupled spec-
trum was recorded.
Acknowledgments
We thank the Swedish Research Council and the Swedish Founda-
tion for Strategic Research for financial support.
Method B (for OXP as Coupling Reagent): The reaction and analy-
sis of the coupling using 36 m OXP in actonitrile/pyridine (3:1,
v/v) was carried out essentially according to Method A, except that
a reagent solution of 72 m OXP (13.0 mmol, 1.2 equiv.) in 180 µL
of dry acetonitrile/pyridine (3:1, v/v) or pyridine was used. The
reaction and analysis of the coupling using 120 or 60 m OXP
in neat pyridine or acetonitrile/pyridine (3:1, v/v) was carried out
essentially according to Method A, except that the solvent pro-
portions of the stock solutions were different. Either neat pyridine
was used as solvent or in the case where the reactions were carried
out in a mixture of acetonitrile/pyridine (3:1, v/v), the reaction mix-
ture was prepared by adding first a solution of 57 m 2a in dry
acetonitrile (180 µL, 10.3 µmol, 0.95 equiv.) to the NMR tube with
compound 1 (8.6 mg, 10.8 µmol) and then a solution of 240 or
120 m OXP in dry acetonitrile/pyridine (1:1, v/v; 180 µL, 21.6 or
43.2 mmol, 2 or 4 equiv.). The signals of the two isomeric products
were observed at δ = 37.7 and 37.0 ppm for all reactions carried
out in acetonitrile/pyridine (3:1, v/v) and at δ = 37.0 and 36.1 ppm
in pyridine, with a variation of 0.1–0.2 ppm.
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Received: June 8, 2007
Published Online: February 11, 2008
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