The mechanism of the phosphoramidite synthesis of polynucleotides

Article abstract of DOI:10.1039/b808999j

The mechanism of the coupling step in polynucleotide synthesis using 5′-4,4′-dimethoxytritylthymidine-3′-β-cyanoethyl-N, N-diisopropylphosphoramidite as the phosphitylating agent and catalysed by the salt of saccharin and N-methylimidazole in acetonitrile

Full text of DOI:10.1039/b808999j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
The mechanism of the phosphoramidite synthesis of polynucleotides†
Mark A. Russell, Andrew P. Laws, John H. Atherton and Michael I. Page*
Received 28th May 2008, Accepted 2nd July 2008
First published as an Advance Article on the web 29th July 2008
DOI: 10.1039/b808999j
The mechanism of the coupling step in polynucleotide synthesis using
5ꢀ-4,4ꢀ-dimethoxytritylthymidine-3ꢀ-b-cyanoethyl-N,N -diisopropylphosphoramidite as the
phosphitylating agent and catalysed by the salt of saccharin and N-methylimidazole in acetonitrile has
been studied by 31P NMR. Pre- and post-equilibria between the activator salt and released
diisopropylamine have been examined by 1H NMR and ITC, which show that the salt between
saccharin and diisopropylamine will be present in acetonitrile. Activation of the phosphoramidite by
the salt of saccharin and N-methylimidazole involves nucleophilic catalysis and the formation of a
reactive saccharin adduct bonded through its carbonyl oxygen to phosphorus. The rate constants for
the reaction of the 4-methoxyphenol with 5ꢀ-4,4ꢀ-dimethoxytritylthymidine-3ꢀ-b-cyanoethyl-N,N -
diisopropylphosphoramidite in the presence of saccharin–N-methylimidazole salt show a non-linear
dependence on phenol concentration, becoming independent at high phenol concentrations, compatible
with a change in rate limiting step from the alcoholysis step to the activation step.
Introduction
There is an increasing need to synthesise large quantities of high
purity oligonucleotides for use in nucleic acid research and for
the manufacture of anti-sense oligonucleotides.1–3 While there
have been numerous studies centred around finding new synthetic
routes and on improving existing methods to increase yield and
product purity, detailed kinetic and mechanistic studies on the
individual steps involved in these syntheses are largely lacking.
Arguably the most important step in oligonucleotide synthesis
is the coupling of the nucleotide units. One such method that
has shown great efficiency is the phosphoramidite route and is,
today, the most widely used. The phosphoramidite method utilises
solid phase methodologies, where the growing oligonucleotide is
covalently anchored to a solid matrix within a column and reagents
are washed down for reaction.3–8 The nucleotide to be coupled to
the growing oligomer is often presented as a phosphoramidite
containing a diisopropylamine leaving group and cyanoethyl and
4,4ꢀ-dimethoxytrityl (DMTr) as P–O and 5ꢀ hydroxy protecting
groups, respectively (Scheme 1).
The phosphoramidite coupling reaction (often incorrectly re-
ferred to as phosphorylation rather than phosphitylation) is the
nucleophilic substitution of the amine moiety of the nucleosidic
phosphoramidite by the 5ꢀ hydroxy function of the solid support
bound nucleoside. The reaction must be performed in the presence
of a suitable acid/base activator as the reactants are otherwise inert
(Scheme 1).
Scheme 1
There have been many studies involving screening of
numerous activators to measure their relative efficien-
cies, examples include: 1H-tetrazole;9,10 2,4-dinitrophenol;11 2-
bromo-4,5-dicyanoimidazole;12 various carboxylic acids;13 4,5-
dicyanoimidazole;14 5-phenyltetrazole;15 arylsulfonyl-tetrazoles;16
which are used for activation in phosphoramidite method-
ologies and benzylthiotetrazole and ethylthiotetrazole17 for H-
phosphonate routes. One of the most widely used activators is
1H-tetrazole, first used by Caruthers et al.9,10 Studies by Dahl
et al.,18 and later by Nurminen et al.,19–22 of the phosphitylation
reaction suggested that 1H-tetrazole has a dual role. Firstly,
the activator acts as an acid to protonate the nitrogen of the
amine leaving group. Secondly, it acts as a nucleophile to displace
isopropylamine from the protonated amidite to form a highly
reactive tetrazolide intermediate. The tetrazolide intermediate
then undergoes nucleophilic attack by the nucleosidic alcohol
to produce the phosphite product, one equivalent of amine and
tetrazole. The difference in pKa between the departing amine and
tetrazole means that the final acid base products react to generate
a salt (Scheme 2).
Department of Chemical and Biological Sciences, University of Huddersfield,
Queensgate, Huddersfield, UK HD1 3DH
† Electronic supplementary information (ESI) available: Example of the
31P NMR spectra for rate determination of thymidine phosphoramidite
with 4-methoxyphenol catalysed by one molar equivalent of saccharin–N-
methylimidazole. See DOI: 10.1039/b808999j
Although it has been suggested that protonation of the phos-
phoramidite occurs on phosphorus,18,21,23,24 it seems likely that for a
reaction to occur, nitrogen protonation is required.21,25,26 Evidence
supporting this has been reported by Korkin and Tvetkov27,28 who
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Results and discussion
Pre- and post-equilibria
Initially it was important to establish details of the equilibria
between saccharin and N-methylimidazole, and also that with the
amine generated during phosphitylation–diisopropylamine. The
phosphoramidites used in these studies all contained diisopropy-
lamine due to its preferred use in oligonucleotide manufacture.
1H NMR studies performed in deuterated acetonitrile of
mixtures of saccharin (SH) and N-methylimidazole (NM) indicate
full proton transfer from saccharin to N-methylimidazole. In
the presence of diisopropylamine (DIA), full proton transfer
from both saccharin and the protonated N-methylimidazle to
diisopropylamine occurs (Table 1). Thus, similar to the order
in water, the increase in basicity in acetonitrile is in the or-
der of: saccharin anion < N-methylimidazole < diisopropy-
lamine. Isothermal titration calorimetery was used to calculate
protonation equilibrium constants between saccharin and N-
methylimidazole, and between saccharin and diisopropylamine.
These also indicate the same order of basicity as determined by 1H
NMR with equilibrium constants of 1.27 × 104 (SH + NM) and
5.59 × 105 (SH + DIA) respectively. In terms of salt formation
between all three components, that between saccharin anion and
diisopropylaminium will be predominant in acetonitrile by a factor
of 44 (Scheme 3).
Scheme 2
showed, by molecular modelling of H2P-NH2, that protonation
on nitrogen lengthens and weakens the phosphorus nitrogen
bond, whereas phosphorus protonation shortens and strengthens
the phosphorus nitrogen bond. Nurminen21,22 has claimed that
phosphorus protonation could be achieved with strong acids,
although subsequent nucleophilic substitution of the amine was
much slower than with the corresponding nitrogen protonated
species.
Efficient activation requires an acid to protonate the phospho-
ramidite and a base to act as both a good nucleophile, to facilitate
rapid conversion to the activated intermediate, and as a good leav-
ing group to enable formation of the phosphite triester. HX type
activators, such as 1H-tetrazole, do not meet these requirements
of strong acid and good nucleophile; as a strong acid is likely
to generate a weakly nucleophilic conjugate base whereas a strong
nucleophile is likely to be derived from a weak acid.5,29,30 There have
been a number of studies undertaken to find alternative activators
to these HX types with salts of strong acids and nucleophilic bases
showing great potential as effective promoters of phosphitylation.
Examples of these activators include: salts of benzimidazole with
trifluoroacetic acid, tetrafluoroboric acid, hexafluorophosphoric
acid and trifluoromethansulfonic acid;31,32 imidazolium triflate;33
N-methylimidazolium triflate;34 salts of 4-dimethylaminopyridine
with 5-(o-nitrophenyl)tetrazole and 5-(p-nitrophenyl) tetrazole;35
pyridinium trifluoroacetate;36 N-methylimidazolium triflate and
trifluoroacetate; N-methylbenzoimidazolium triflate; and N-
phenylimidazolium triflate.29
Scheme 3
Herein we report mechanistic studies on the phosphitylation
of nucleosidic species in acetonitrile using saccharin and N-
methylimidazole as an activator, which is currently used as part of
the manufacture of oligonucleotides on an industrial scale.
Although these equilibria are expected on the basis of the pKa’s
in water, this order of basicity is contrary to that indicated by
pKa values in acetonitrile reported in the literature, which puts the
Table 1 1H chemical shifts of N-methylimidazole (NMI), saccharin and diisopropylamine (DIA) in deuterated acetonitrile
d NMI H2
(ppm)
d NMI H4
(ppm)
d NMI H5
(ppm)
d NMI CH3
(ppm)
d DIA CH
(ppm)
d DIA CH3
(ppm)
Sample composition
NMI
NMI + saccharin
DIA
DIA + saccharin
NMI + saccharin + DIA
7.39
8.43
—
—
7.50
6.95
7.39
—
—
6.98
6.90
7.27
—
—
6.93
3.63
3.81
—
—
3.64
—
—
2.84
3.45
3.48
—
—
0.95
1.30
1.31
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Table 2 pKa values of aliphatic ammonium ions in water and
To distinguish between these possibilities, studies were
conducted of the reaction of the phosphoramidite, saccha-
rin and N-methylimidazole in the absence of an alcohol.
Addition of increasing molar equivalents of saccharin–N-
methylimidazole salt to 5ꢀ-4,4ꢀ-dimethoxytritylthymidine-3ꢀ-b-
cyanoethyl-N,N -diisopropylphosphoramidite in acetonitrile gave
31P NMR spectra that showed a decrease in the signal due to the
starting amidite, and an increasing proportion of a relatively broad
signal centred at d 134 ppm, which is assigned to the activated
species. There is also the presence of two small additional signals,
of equal peak area, at d 7 ppm due to the formation of diastereoiso-
mers of the H-phosphonate species produced by reaction of traces
of water in the system with the activated species. There is also
a set of three small signals at approximately d 125 ppm, which
are due to the formation of a P–O–P anhydride type species
arising from reaction of the H-phosphonate/phosphite and the
activated species.22 Addition of excess N-methylimidazole and/or
diisopropylamine prior to or after initial activation showed an
increase in the concentration of reactants, demonstrating a true
equilibrium between the species.
Saccharin is a strong enough acid to cause detritylation of the 5ꢀ
protecting group of the phosphoramidite and the presence of N-
methylimidazole as a base also helps to reduce the acidity of the
medium to prevent unwanted detritylation. To study activation
with saccharin in the absence of N-methylimidazole, a sim-
ple phosphoramidite (dimethyl N,N -diisopropylphosphoramidite)
was used. Addition of increasing molar equivalents of saccharin to
the phosphoramidite in both acetonitrile and chloroform showed,
from 31P NMR spectra, an increase in the activated species at the
expense of starting amidite.
The same signal for the activated species was observed upon
the addition of N-methylimidazole–saccharin to the amidite
in chloroform, indicating that the activated species must be a
saccharin adduct. In acetonitrile, there was a small shift in the
position of the signal for the activated species, which is probably
due to a medium effect rather than to the formation of a different
adduct.
To identify the position of the 31P NMR chemical shift of
P–O–C bonds, acetic acid was used instead of saccharin–N-
methylimidazole to activate the thymidine phosphoramidite in
acetonitrile. Addition of increasing molar equivalents of acetic
acid showed an increase in the proportion of a pair of signals at d
131.7 and 131.1 ppm due to the two diastereoisomers formed upon
substitution of diisopropylamine with acetic acid. The chemical
acetonitrile.38
Acid
pKa in water
pKa in acetonitrile
Diisopropylammonium
Di-n-butylammonium
Diethylammonium
Tri-n-butylammonium
Triethylammonium
Tri-n-propylammonium
Diisobutylammonium
11.0537
11.25
10.98
10.89
10.65
10.65
10.50
16.522
18.31
18.75
18.09
18.46
18.1
17.88
acidity of the conjugate acid of N-methylimidazole (pKa 17.1)22
above that of diisopropylamine (pKa 16.5).22 An inspection of the
pKa values (Table 2) for a range of aliphatic ammonium ions show
a good correlation between those in water and in acetonitrile. The
reported pKa of diisopropylammonium ion in acetonitrile of 16.5
is almost two units lower than the average for ammonium ions
and lies well outside the pKa range. It therefore seems likely that
the reported pKa value of 16.5 is incorrect and its pKa should
be approximately 18.5. This would place the relative basicity of
saccharin, N-methylimidazole and diisopropylamine as similar to
that determined by our experiments.
As the released diisopropylamine is the most basic component
of the phosphitylation reaction mixture, a salt will be formed
between saccharin and the displaced diisopropylamine, which will
contribute to the driving force for the reaction. As well as salt
formation, there will be a large thermodynamic driving force due
to the positive enthalpy change accompanying the formation of a
strong phosphorus to oxygen bond in the phosphite triester prod-
uct versus the phosphorus nitrogen bond in the phosphoramidite
reactants. This is illustrated by P–O and P–N bond dissociation
energies of 360 and 230 kJ mol−1, respectively.39,40
Phosphoramidite activation
If the phosphitylation of alcohols occurs by activation of
the phosphoramidite with saccharin–N-methylimidazole salt via
nucleophilic catalysis and a two step mechanism (Scheme 2), then
there are three possibilities for the activating nucleophile. After
initial protonation of the diisopropylamine group, substitution of
the protonated amine could occur by either N-methylimidazole to
form a charged intermediate or by the saccharin anion, through
either its carbonyl oxygen or nitrogen atom, to give two possible
neutral intermediates (Scheme 4).
Scheme 4
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Table 3 Summary of the 31P NMR chemical shifts of phosphorus III species in deuterated acetonitrile
d 149 ppm
d 149 ppm
d 134 ppm
d 150-162 ppma
d 142 ppm
d 139 ppm
d 132 ppm
d 138 ppm
a Varies with NMI concentration.
shift of 132 ppm can be compared with the d 134/139 ppm
observed with saccharin and saccharin–N-methylimidazole, com-
patible with P–O bond formation. To confirm these obser-
vations and to determine the 31P NMR chemical shift of
P–N–C bonds, the reaction between diethylchlorophosphite and
N-methylimidazole was studied. Addition of N-methylimidazole
to the diethylchlorophosphite in acetonitrile showed formation
of a broad 31P NMR signal, which showed a large up-field
movement in chemical shift on increasing the concentration of
N-methylimidazole from d 162 ppm with one molar equivalent to
d 151 ppm with five molar equivalents. The shape of the signal
became broader and less symmetrical on addition of excess N-
methylimidazole. This is thought to be due to association of
N-methylimidazole molecules to the charged activated species
reducing the amount of positive charge on the phosphorus
compatible with the observed change in chemical shift. Reducing
the temperature of the five molar equivalent sample showed a
further up-field shift in the signal and a sharpening in its shape.
Addition of dimethylaminopyridine to diethylchlorophosphite in
acetonitrile gave an activated species with a 31P NMR signal at d
142 ppm, corresponding to an adduct with a more delocalised
charge. Table 3 shows a summary of the 31P NMR chemical
shifts in acetonitrile of the relevant adducts with the chemical
shifts of P–N species being between d 142–162 ppm whereas the
P–O phosphite ester species resonate further up-field at d 132–
139 ppm. The chemical shifts of the P–saccharin adduct resonate
at approximately d 135 ppm, compatible with bonding through
the saccharin’s carbonyl oxygen atom.
This is compatible with studies performed by Sekine et al., who,
in a not too dissimilar reaction, demonstrated the reaction of
activated phosphonates with an oxygen nucleophile in the presence
of nitrogen nucleophiles.17
A function of N-methylimidazole in the reaction is to reduce
the acidity of the reaction mixture so as to prevent unwanted
side reactions, such as de-purination and premature detritylation.
The saccharin–N-methylimidazole salt is an effective activator of
the phosphoramidites and activation occurs rapidly and stoichio-
metrically. The saccharin anion is more nucleophilic towards the
phosphoramidite than the N-methylimidazole.
Alcoholysis
The alcoholysis of thymidine phosphoramidite in acetonitrile re-
quires only one molar equivalent of saccharin–N-methylimidazole
salt and one molar equivalent of alcohol to give quantitative
formation of the phosphite triester product. The alcoholysis
reaction could proceed by either direct displacement of the
protonated amine or by initial nucleophilic attack of the activator
and subsequent displacement of saccharin by the incoming alcohol
(Scheme 5).
Further evidence for the active intermediate being the P–O
saccharin adduct was obtained from FT-IR. The spectrum of
saccharin in acetonitrile shows the presence of a signal at 1744 cm−1
assigned to the stretching frequency of the carbonyl group, which
is lost on addition of one molar equivalent of N-methylimidazole,
indicative of proton transfer and enolate anion formation. Addi-
tion of either one molar equivalent of dimethylphosphoramidite
or one molar equivalent of both dimethylphosphoramidite and
N-methylimidazole to saccharin in acetonitrile also results in the
loss of the carbonyl signal, compatible with formation of a P–O
rather than a P–N adduct, which would retain a carbonyl stretch
(Scheme 4).
Scheme 5
The rate of the alcoholysis reaction of the thymidine phospho-
ramidite with the 5ꢀ hydroxyl group of a nucleoside is very rapid but
can be reduced by using weakly nucleophilic alcohol or phenol so
that kinetics can be monitored by rapid 31P NMR techniques (see
ESI†). The change in concentration with time for the formation of
phosphite triester from the reaction of thymidine phosphoramidite
with one molar equivalent of saccharin–N-methylimidazole shows
second order kinetics for a range of substituted phenols and alco-
hols at varying initial concentrations. However, the calculated rate
constants show a non-linear dependence upon the concentration
In summary, phosphoramidite activation with saccharin–N-
methylimidazole salt results in the formation of an activated
intermediate that is an adduct of saccharin with bonding to
phosphorus being through the carbonyl oxygen atom of saccharin.
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Table 4 Observed second order rate constants for reaction of thymidine
phosphoramidite with 4-methoxyphenol catalysed by one molar equiva-
lent of saccharin–N-methylimidazole salt in acetonitrile
Table 5 Observed second order rate constants for reaction of thymidine
phosphoramidite with various phenols and alcohols catalysed by one
molar equivalent of saccharin–N-methylimidazole salt
4-Methoxyphenol concentration (M)
k
obs/M−1 s−1
Alcohol
k
obs/M−1 s−1
0.05
0.10
0.15
0.20
0.30
0.40
0.50
0.75
0.230
0.285
0.307
0.330
0.403
0.417
0.400
0.410
4-Cyanophenol
4-Chlorophenol
Phenol
4-Methylphenol
4-Methoxyphenol
Propan-2-ol
0.237
0.227
0.227
0.218
0.228
0.238
0.185
t-Butanol
in a fast step to form the product, so that formation of the
intermediate is rate-limiting.
of 4-methoxyphenol (Table 4). At low phenol concentrations,
the rate of alcoholysis is dependent on the 4-methoxyphenol
concentration, but above 0.3 M 4-methoxyphenol, the rate of
phosphite triester formation becomes independent of the phenol
concentration. This change in dependence on concentration is
indicative of a change in rate limiting step and is compatible with
the presence of an intermediate on the reaction pathway.
If phosphoramidite (PA) activation involves nucleophilic catal-
ysis by saccharin to form a P–saccharin intermediate by displace-
ment of the diisopropylamine (DIA) so that alcoholysis proceeds
by a two step mechanism, there are two elementary reactions
involved in formation of the phosphite trieseter (POR) product
(Scheme 6). The rate of phosphite triester formation is given by eqn
(1), but at low concentration of alcohol, where k−1[NMI][DIA] >
k2[ROH], this simplifies to eqn (2) and the rate is dependent on
alcohol concentration and the rate limiting step is k2, the rate of
breakdown of the intermediate.
Experimental
1H, 13C, 31P and 15N NMR spectra were recorded on a Bruker
400 MHz or Bruker 500 MHz spectrometer. Coupling constants
J are quoted in Hz.
Isothermal titration calorimetry was performed on a MicroCal
VP-ITC microcalorimeter. For determination of the saccharin–
diisopropylamine equilibrium constants, a solution of saccharin
(0.25 mM) in acetonitrile was placed in the cell block and the
titrating syringe was loaded with diisopropylamine (7.3 mM) in
acetonitrile. Titre volumes of 4 ll were used in this experiment with
a 150 second delay between additions. For the determination of the
saccharin–N-methylimidazole equilibrium constants, saccharin
(1 mM) in acetonitrile was placed in the cell block and N-
methylimidazole (14.6 mM) in acetonitrile was loaded into the
titration syringe. In this case, titre volumes of 2 ll were titrated
with a 150 second delay between injections.
(1)
(2)
Infra-red experiments were performed on a Thermo Nicolet 380
FT-IR. All reagents were pre-prepared in deuterated acetonitrile
˚
and stored over 4 A molecular sieves under dry argon for at
least 24 hours before use. In a typical experiment, saccharin and
dimethyl N,N -diisopropylphosphoramidite were mixed together
in acetonitrile under a dry argon atmosphere and the resulting
mixture injected into a 1 mm quartz cell, which had been flushed
with dry acetonitrile and sealed with rubber septa; the spectrum
was then recorded.
The kinetic measurements were carried out using rapid 31P
NMR techniques on a Bruker 400 spectrophotometer. All reagents
were pre-prepared in deuterated acetonitrile and stored over
Scheme 6
Conversely, at high concentration of alcohol, where k2[ROH] >
k-1[NMI][DIA], the rate of formation of the intermediate, k1 is rate
limiting and the reaction is independent of alcohol concentration,
eqn (3).
˚
4 A molecular sieves under dry argon for at least 24 hours
prior to use. In a typical kinetic run, two identical samples
of saccharin–N-methylimidazole salt and 4-methoxyphenol in
deuterated acetonitrile were prepared in sealed/argon flushed
NMR tubes. To one of these samples, the NMR shimming
and deuterium locking protocols were performed after addition
of thymidine phosphoramidite. Thymidine phosphoramidite was
then added to the other sample, which was placed in the NMR
magnet as quickly as possible and the experiment was started.
31P NMR spectra were recorded successively at intervals of 23
seconds. This time base was chosen to gain the maximum number
of data points over the kinetic run, but to also give adequate signal
resolution. Rate constants were obtained by fitting concentration
versus time data to either first or second order exponential decays
(3)
There is little dependence on the nature of the phenol or alcohol
on the rate of phosphite triester formation (Table 5) as expected if
the rate limiting step is formation of the intermediate.
In conclusion, phosphoramidite activation with saccharin–N-
methylimidazole salt results in the formation of an activated
phosphite ester intermediate that is an adduct of saccharin with
bonding to phosphorus being through the carbonyl oxygen atom
of saccharin. In the presence of alcohols, this intermediate reacts
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using Scientist software. Errors in rate constants are typically
below 5%, data with errors above this were discarded.
20 E. J. Nurminen, J. K. Mattinen and H. Lonnberg, J. Chem. Soc., Perkin
Trans. 2, 1999, 2551.
21 E. J. Nurminen, J. K. Mattinen and H. Lonnberg, J. Chem. Soc., Perkin
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22 E. J. Nurminen, J. K. Mattinen and H. Lonnberg, J. Chem. Soc., Perkin
Trans. 2, 2001, 2159.
23 T. A. Van Der Knaap and F. Bickelhaupt, Phosphorus and Sulfur, 1984,
21, 227.
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