Overcoming hydrolytic sensitivity and low solubility of phosphitylation reagents by combining ionic liquids with mechanochemistry

Article abstract of DOI:10.1039/c1cc11025j

Ionic liquids have been used in combination with ball milling on a range of chlorophosphoramidite reagents to phosphitylate nucleosides and 2-deoxynucleosides. The enhanced stability offered by the ionic liquid mediated processes combined with efficient mass transfer induced by ball milling has enabled excellent yields to be obtained even when using small dialkyl amino groups as well as the more commonly used diisopropylamino protection.

Full text of DOI:10.1039/c1cc11025j

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COMMUNICATION
Overcoming hydrolytic sensitivity and low solubility of phosphitylation
reagents by combining ionic liquids with mechanochemistryw
Christopher Hardacre,*a Haifeng Huang,a Stuart L. James,a Marie E. Migaud,*b
Sarah E. Normana and William R. Pitnerc
Received 22nd February 2011, Accepted 4th April 2011
DOI: 10.1039/c1cc11025j
Ionic liquids have been used in combination with ball milling on a
range of chlorophosphoramidite reagents to phosphitylate
nucleosides and 2-deoxynucleosides. The enhanced stability offered
by the ionic liquid mediated processes combined with efficient mass
transfer induced by ball milling has enabled excellent yields to be
obtained even when using small dialkyl amino groups as well as the
more commonly used diisopropylamino protection.
this limitation and provide means to access oligonucleotides
which are sufficiently reactive and yet hydrolytically stable to
allow for effective oligomerisation, bulky diisopropylamino
derivatives are utilised in phosphoramidite nucleoside chemistry.
These stable and commonly used diisopropylamino chloro-
phosphoramidite reagents have been optimised for use in
DNA synthesis. In order to achieve oligomerisation yields
comparable with that of DNA synthesis, RNA-synthetic
chemists have manipulated the nature of the protecting groups
at the C20 position of the nucleoside phosphoramidites in order
to increase reactivity and overcome steric hindrance which limits
the phosphitylation process. However, an alternative, and
potentially more versatile methodology, has been proposed
and demonstrated by Chamberlin and co-workers.5b This
method uses nucleoside phosphoramidites incorporating less
hindered amino groups. Unfortunately, it is limited by the poor
accessibility to its phosphitylating reagents. Although the
presence of a less hindered amine at the phosphorus centre has
been shown to enhance coupling rates, the reagents with smaller
amines are more susceptible to decomposition. Considering the
numerous emerging technologies and medicines relying on
effective oligonucleic acid chemistry, development of more
versatile reagents than those commonly in use would be timely.
Current methods of synthesising ribonucleoside phosphor-
amidites have a number of issues including low yield, in terms
of utilisation of the nucleoside via the phosphodiamidites, and
low stability of the ribonucleoside phosphoramidites.
Two common obstacles to performing efficient chemical transfor-
mations are insolubility of reagents and decomposition of
sensitive reagents/products. Ionic liquids (ILs), even when used
as an additive rather than a solvent, can provide a protective
medium in which hydrolytically sensitive species can be con-
veniently handled,1 whilst mechanochemistry provides a way
to perform reactions on poorly soluble species.2 Herein, we
demonstrate, for the first time, that the advantages of these
two approaches may be combined to enable new chemistry.
Specifically, we have combined these approaches to achieve
enhanced reactivity, improved reaction profiles and increased
yields in the phosphitylation of hindered secondary alcohols with
a range of hydrolytically labile chlorophosphoramidites.
Oligonucleotides address therapeutic needs for a variety
of disease states.3 However, methods for phosphitylation of
nucleosides (synthetic precursors to oligonucleotides) have not
changed significantly since the 1980s.4 This has limited the
availability of reactive and functionally versatile nucleoside
phosphoramidites, in particular for the preparation of the
more structurally congested ribonucleic acid species. Although
a range of alkoxy substituents have been examined, access to
chemically adaptable nucleoside phosphoramidites using
structurally diverse amino functionality has been restricted.5
This lack of scope in nucleoside phosphoramidite building
block availability is mainly due to the low stability of the
phosphitylation reagents used for their preparation. To mitigate
ILs have recently shown some success in mediating the
phosphitylation of nucleosides using the NiPr2-stabilised reagent.6
However, we show here that, in the presence of a stabilising IL
environment, mechanochemical reaction conditions (ball milling)
can also lead to high yields of dialkylamino phosphitylated
derivatives of deoxyribo- and ribo-nucleosides (Fig. 1). In these
reactions, the ionic liquid was present as 1.5 mol equivalent to the
nucleoside and chlorophosphoramidite, i.e. the IL was an additive
rather than a bulk solvent.
a School of Chemistry and Chemical Engineering, Queen’s University,
Belfast, UK. E-mail: c.hardacre@qub.ac.uk;
The chlorophosphoramidites (1–3, Fig. 1) used in this work
were prepared in high yield using the IL process previously
reported.6 In each case, these chlorophosphoramidites could be
stored in an IL in air for months without detectable degradation.
Using the [C6mim][FAP]-stabilised chlorophosphoramidites,
the phosphitylations of partially protected nucleosides were
attempted by adding dichloromethane (DCM) to solubilise the
Fax: +44 (0)28 90974687
b School of Pharmacy, Queen’s University, Belfast, UK.
E-mail: m.migaud@qub.ac.uk; Fax: +44 (0)28 90247794
c Merck KGaA, PC R & D Ionic Liquids, Frankfurter Str. 250, 64271,
Darmstadt, Germany
w Electronic supplementary information (ESI) available: Experimental
details and NMR spectra of stabilised chlorophosphoramidites 1–3
and nucleoside phosphoramidites 1a–3c. See DOI: 10.1039/c1cc11025j
c
5846 Chem. Commun., 2011, 47, 5846–5848
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Table 1 Comparision of the yields from the reaction of diethylamino
chlorophosphoramidite (2) with 2-deoxy adenosine (a) under various
reaction conditions
DCM
(24 h)
Sonication
(30 min)
Ball milling
(30 min)
Ionic liquid
[C2mim][FAP] 65%
[C4mim][FAP] 40%
[C6mim][FAP] 15%
Decomposition of 2 Decomposition of 2
40%
48%
65%
82%
Fig. 1 Schematic showing the reaction of chlorophosphoramidites to
form phosphitylated nucleosides using IL media. For synthetic details
see ESI.w
In order to improve the dissolution of the nucleosides in the
IL, sonication and ball milling were investigated in the absence of
DCM using the IL stabilised chlorophosphoramidite (Table 1).
The ball milled reactions were performed using the procedure
outlined recently for mechanochemical nucleoside silylation by
Giri et al.9 Remarkably, using ball milling, in the presence of only
very small amounts of [C4mim][FAP] and [C6mim][FAP] as
stabilisers (1.5 : 1 mole ratio IL : chlorophosphoramidite), much
higher reaction rates and product yields resulted than under
stirring or sonication. After 30 min, isolated yields of 40 to 82%
were obtained with little decomposition (compared with o40%
isolated yield for standard stirring after 24 h). For all reactions,
the order of reagent addition was critical, with the best yields
obtained when the chlorophosphoramidite was added to the
partially protected nucleoside and Hunig’s base with stirring. It
¨
should be noted that, without DCM, there was less than 5%
conversion after 24 h. The diisopropyl amino derivatives showed
excellent conversion with isolated yields of 84–92% for the
2-deoxy-ribose based nucleosides but only 46% conversion for
the ribose parent, even when catalysed by the addition of
4-dimethylaminopyridine (DMAP). More surprisingly, only low
yields were obtained using diethylamino and methylethyl amino
chlorophosphoramidites. 2a was only obtained in 15% yield after
several days and still lower yields (o1%) were found for the
methylethyl amino derivative (3a). This is unexpected because
reactivity of the chlorophosphoramidite commonly increases on
decreasing the size of the amine substituent.5a Furthermore,
neither the addition of DMAP nor higher temperatures signifi-
cantly improved the yield. In all cases, the main species found in
the reaction mixture was the unreacted phosphitylation reagent,
with o10% decomposition. Given the generally high reactivity of
chlorophosphoramidites, the kinetics are thought to be mass
transfer limited. This is supported by the fact that low concentra-
tions of o50 mM for the protected nucleosides have been
reported in [NTf2]ꢀ based ILs and o10 mM in [FAP]ꢀ based
ILs.7 Clearly, good contact between the chlorophosphoramidite
and the nucleoside must occur for high yields to be obtained
and this is difficult to achieve due to the low solubility of the
nucleoside in the IL phase.
Hunig’s base and nucleoside mixture. If the base was not present
¨
before chlorophosphoramidite addition, cleavage of the DMT
protecting group was observed. The most promising results
occurred using the [C6mim][FAP] stabilised materials under
ball milling conditions. The overall yields were high and the
reactions were clean with only the product and excess chloro-
phosphoramidite being detected in the crude mixture. Further-
more, during the reaction of 2a and 2b, no unreacted nucleosides
1
were found by H NMR even in the crude product.
The ball milled reactions were pastes, i.e. viscous liquid phases
(primarily the IL, chlorophosphoramidite and Hunig’s base)
¨
containing solid particles (primarily the nucleoside reagent and/
or product). These reactions can be thought of as unusual liquid-
assisted grinding (LAG) reactions2d,10 in which the liquid may
accelerate the reaction by aiding transport as well as provide a
protective medium for sensitive species. This approach, as well as
LAG with ILs, appears to be novel in mechanochemistry.
The mechanochemically promoted phosphitylation in
[C6mim][FAP] was readily extended to the other IL-stabilised
diethyl and methylethyl chlorophoramidites as well as 2-deoxy-
cytidine and guanosine nucleosides (b and c) (Table 2). All
reactions under mechanochemical conditions gave high isolated
yields (73–91%) compared with stirring in DCM where
The unusual reactivity trend (1) c (2) > (3) observed under
these DCM-based conditions is consistent with the qualitative
visual observation that (1) has a significantly lower solubility
in the ILs than either (2) or (3). Liquid mixtures often have
complex heterogeneous structures on the molecular scale8 and,
therefore, it is possible that the IL–chlorophosphoramidite
interaction is maintained even in DCM, thus decreasing the
concentration of free, reactive phosphitylating reagents. In this
case, as the solubility of the nucleoside in the IL is very low, its
interaction with 2 or 3 will be significantly reduced compared
with 1, thereby decreasing the reaction rate. In order to vary
the strength of IL interaction with 2, a range of [FAP]ꢀ based
ILs with differing imidazolium alkyl chain lengths (ethyl, butyl
and hexyl) was investigated. Yields of 2a from the IL stabilised
diethylamino chlorophosphoramidite (2) under the standard
Table 2 Yields for the nucleoside phosphitylation reactions for the
DCM and ball milled reactions
Protected nucleoside
Amine
DCM (%)
Ball mill (%)
2-Deoxy adenosine (a)
–NiPr2 (1)
–NEt2 (2)
–NEtMe (3)
–NiPr2 (1)
–NEt2 (2)
–NEtMe (3)
–NiPr2 (1)
–NEt2 (2)
–NEtMe (3)
92
15
0
84
9
80
82
79
80
91
74
89
82
73
reaction conditions (DCM, Hunig’s base, stirring) are given in
¨
Table 1. As expected, and following from the observation that
the solubility of 2 in the IL decreases as the IL alkyl chain
length is shortened, the reaction yield was correspondingly
greater with shorter IL alkyl chains (ethyl > butyl > hexyl).
However, as expected, the reduced interaction of the phosphorus
reagent with the shorter alkyl chain length based ILs also
increased its hydrolytic sensitivity as shown by the presence of
decomposition products in 31P NMR spectra.
2-Deoxy cytidine (b)
Guanosine (c)
5
46a
8
5
a
Addition of DMAP to the reaction mixture.
c
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Fig. 2 1H NMR spectrum of compound 3c after purification. The corresponding 31P NMR spectrum is shown as the inset.
isolated yields ranged between 0 and 15% for the IL-stabilised
diethyl and methylethyl chlorophoramidites. Remarkably,
31P NMR revealed only the product and unreacted chloro-
phosphoramidite in all cases. In addition, simple workup
by filtration through silica could also be applied, thereby,
avoiding the need for aqueous workup and extensive purifi-
cation methods (Fig. 2). For all systems, the nucleoside
phosphoramidite was isolated in pure form and, for the more
hydrolytically unstable derivatives, a stabilising amount of IL
was then added to allow for storage and further chemical
manipulations. As found for all commercially available nucleo-
side phosphoramidites, those prepared, herein, were a mixture
of diastereoisomers at the phosphorus centre.
chemically labile, irrespective of the amine functionality. The
method, therefore, will enable more efficacious RNA and DNA
oligomerisation protocols to be developed.
We acknowledge funding from Merck and EPSRC.
Notes and references
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As expected from the increased stability of hydrolytically
sensitive phosphorus reagents using ILs, following isolation,
the phosphitylated nucleosides could be stored at room tempera-
ture, without the need for an inert gas atmosphere and showing
no degradation after 5 days. This demonstrates the utilisation of
ILs as stabilising/storage media for phosphitylated nucleoside
derivatives and other reactive analogues currently under-utilised
in oligonucleotide syntheses. Therefore, this approach not only
allows facile access to diverse reactive building blocks, it also
provides means to store them effectively.
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Importantly, although ball milling has been used to enable
solventless persilylation of nucleosides,9,11 these reactions are
much less air and moisture sensitive than those reported in this
communication. Here, we describe the first combination of IL
stabilisation with mechanochemical reaction conditions to
simultaneously overcome problems of hydrolytic sensitivity
and low reactant solubility. Specifically, we have combined
these techniques to achieve enhanced reactivity, improved
reaction profiles and increased yields in the phosphitylation
of partially protected nucleosides with a range of hydrolytically
labile chlorophosphoramidites. This represents a simple method
to access, isolate and store reliably reagents known to be
8 (a) I. Bako, T. Megyes, T. Grosz, G. Palinkas and J. Dore, J. Mol. Liq.,
´ ´ ´ ´
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c
5848 Chem. Commun., 2011, 47, 5846–5848
This journal is The Royal Society of Chemistry 2011