Nucleoside phosphitylation using ionic liquid stabilised phosphorodiamidites and mechanochemistry

Article abstract of DOI:10.1039/c2cc36367d

A range of nucleoside phosphoramidites incorporating small amino substituents have been readily synthesised using ionic liquid stabilised phosphorodiamidites coupled with mechanochemistry. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2cc36367d

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COMMUNICATION
Nucleoside phosphitylation using ionic liquid stabilised
phosphorodiamidites and mechanochemistrywz
Kerri Crossey,a Christopher Hardacre*a and Marie E. Migaud*b
Received 31st August 2012, Accepted 24th October 2012
DOI: 10.1039/c2cc36367d
A range of nucleoside phosphoramidites incorporating small
amino substituents have been readily synthesised using ionic liquid
stabilised phosphorodiamidites coupled with mechanochemistry.
which incorporate small amino functionalities have been used for
the rapid preparation of deoxynucleoside phosphoramidites, the
methodology is not straightforward.7 Firstly, the method requires
strictly anhydrous conditions with respect to both reagents and
glassware. Secondly, the nucleoside phosphoramidites are pre-
pared in situ and are not isolated. These were, however, shown
to provide an efficient route towards oligonucleotides. Thus, in
order to provide a balance between hydrolytic stability and to allow
for effective oligomerisation, bulky diisopropylamino derivatives
are the preferred reagents.8 2-Cyanoethyl-N,N0-diisopropylchloro-
phosphoramidite has been used in this regard; however, this reagent
is expensive, moisture sensitive and highly unstable at room
temperature.9 Consequently, 2-cyanoethyl-N,N,N0,N0-tetraisopro-
pylphosphorodiamidite is commonly used as an alternative reagent
in conjunction with a proton-source activator.10 Although this
provides a route to access relatively stable nucleoside phosphor-
amidites, the presence of the diisopropylamino groups means that
their chemical reactivity is reduced by virtue of steric hindrance.11
Despite extensive optimisation of the current solution-based
methodologies for the effective syntheses of deoxy- and ribo-
nucleoside phosphoramidites, the procedures still suffer from a
number of issues including low yield and low stability of the
resulting nucleoside phosphoramidites. These issues can be
associated with the poor solubility of the nucleosides in apolar
organic solvents. This makes it necessary to conduct the
phosphitylation reactions in polar solvents such as dichloromethane
(DCM), pyridine or dimethylformamide (DMF). However, the use
of polar solvents increases the probability of hydrolysis of
the reagents or products due to the increased hydrophilicity of
the solvents.
Within the field of carbohydrate and nucleotide chemistry, novel
methods are currently being widely explored for the derivatisation
of sugar moieties where traditional methods are hampered by
challenging syntheses which require multi-step processes and
selective functional group manipulations.1 In this regard, mechano-
chemistry has been used to overcome issues such as reagent
solubility and reactivity,2 while ionic liquids (ILs) have been shown
to stabilise hydrolytically sensitive reagents.3 Herein, we have
combined these approaches to enable the phosphitylation of
deoxyribo- and ribo-nucleosides using hydrolytically unstable
phosphorodiamidite reagents in high yields. Furthermore, we have
negated the need for the expensive activator 1H-tetrazole by
using a laboratory-friendly alternative in the form of pyridinium
trifluoroacetate (PyꢀTFA).
Nucleoside phosphoramidites are key building blocks for the
automated, solid supported syntheses of oligonucleotide-based
therapeutics. Many of these antisense oligonucleotide-based drugs
are being evaluated for the treatment of a variety of disease states.4
Variations at the phosphorus centre whereby small amino
substituents are utilised to access chemically adaptable nucleo-
side phosphoramidites are limited5 despite the notion that they
improve the rates of the coupling process and increase oligo-
merisation yields. For example, Chamberlain has demonstrated
that in automated RNA oligonucleotide synthesis, nucleo-
side phosphoramidites incorporating small amines were more
readily oligomerised.6 However, access to oligonucleotides via
these small amino derivatives is more challenging due to hydrolytic
instability of both the phosphitylation reagents as well as
the resulting nucleoside phosphoramidite building blocks.
Although bis(dialkylamino)methoxyphosphines, P(OMe)(NR2)2,
The use of phosphorodiamidite reagents require the use of a
weakly acidic in situ activator to allow for activation of the
P–N bond. 1H-Tetrazole is the most widely used activator for
this transformation; yet it possesses a number of practical
issues. Firstly, 1H-tetrazole is a classified explosive and highly
toxic.12 Secondly, 1H-tetrazole is expensive and, therefore, the
overall phosphitylation process is not cost effective.13
a QUILL/School of Chemistry and Chemical Engineering, Queen’s
University, Belfast, UK. E-mail: c.hardacre@qub.ac.uk;
Fax: +44 (0)2890974687
ILs have been shown to provide a unique medium for the
synthesis, stabilisation and reactivity of a range of hydrolytically
unstable phosphorus reagents, including chlorophosphoramidites,14
while mechanochemistry is being used increasingly for a wide array
of organic transformations.15 This solvent free technique has been
applied to enable nucleoside silylation in one pot using solvent-free
ball milling conditions.16 More recently, it was demonstrated
b School of Pharmacy, Queen’s University, Belfast, UK.
E-mail: m.migaud@qub.ac.uk; Fax: +44 (0)28 90247794
w This article is part of the ChemComm ‘Mechanochemistry’ web
themed issue.
z Electronic supplementary information (ESI) available: Experimental
details and NMR spectra of stabilised phosphorous reagents 1–4 and
nucleoside phosphoramidites. See DOI: 10.1039/c2cc36367d
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11969–11971 11969

reactions. Even though the use of NH4Cl failed to yield 1a, it is
important to note that even after 30 min ball milling, only a small
amount of degradation of 1 was observed by 31P NMR, thus
highlighting the stabilising nature of the IL. However, due to the
high hygroscopic nature of the other activators investigated, an
optimal 2 : 1 mole ratio of phosphorodiamidite : nucleoside was
implemented. This was to ensure that reaction completion was not
compromised by degradation of the starting phosphorodiamidite
due to the activator-promoted water content of the reaction
mixture over time. Even after drying the activator under
high vacuum, it was difficult to remove all traces of water as
evidenced by the presence of a small amount of decomposed
phosphorodiamidite in the crude reaction mixture. This observation
was complemented by Karl-Fischer data obtained before and after
drying of the activator. For example, prior to drying, the water
content of PyꢀTFA was 10 wt%. After drying under vacuum this
decreased to 1 wt%. In comparison, the water content of NH4Cl
was 0.3 wt% even prior to drying. Despite the high water contents,
the reactions were high yielding and clean with only the product
and easily separable hydrolysed phosphorodiamidite present as
shown by the NMR data of the crude mixtures (see ESIz).
Fig. 1 Schematic representation of the reaction of phosphorodiamidites
to form nucleoside phosphoramidites using mechanochemistry and a
stabilising IL.
that IL stabilised chlorophosphoramidites in conjunction with
ball milling, mediated the successful phosphitylation of nucleosides
and 2-deoxynucleosides even when the chlorophosphoramidite
incorporated small amino functionalities.14c Herein, we show that
phosphorodiamidites in the presence of a stabilising IL environment
and a weakly acidic activator could be ball-milled with both
deoxyribo- and ribo-nucleosides leading to the production of
phosphitylated nucleosides in high yields.
The phosphorodiamidites used, herein, (1–3, Fig. 1) were
prepared according to the IL procedure previously reported.17 In
this instance, they were extracted into a small amount of 1-hexyl,
3-methyl imidazolium tris(pentafluoroethyl)trifluorophosphate
([C6mim][FAP]) which acted as a stabiliser prior to concentration.
In this case, the [C6mim][FAP] and phosphorodiamidite
were present as a 1 : 1 mole ratio. Although ILs with smaller
imidazolium alkyl chain lengths (ethyl or butyl) allow for successful
phosphitylation, the reduced interaction of the phosphorus
reagent with the smaller alkyl chain means hydrolytic stability is
compromised.14c Hence, in this study [C6mim][FAP] was used for
all the reactions undertaken.
This mechanochemical approach was readily extended to include
the phosphitylation of 1 with 2-deoxycytidine, b, and 2-deoxy-
adenosine, c, in addition to the IL-stabilised ethyl methyl phos-
phorodiamidite, 2, with nucleosides a–c (Table 2). For all reactions
performed, PyꢀTFA was the activator of choice. The isolated yields
given in Table 2 are comparable to those previously reported under
similar reaction conditions,14c but with greatly reduced reaction
times and purification protocols compared with the solvent-based
procedures of the diisopropylamino substrates. For all systems in
Table 2, again a simple workup via filtration through silica could be
applied. The simplicity of the overall procedure allows for the
inclusion of small amino functionalities as well as eliminating
the need for the traditionally implemented aqueous work-up.
The co-existence of the H-phosphonate with the nucleoside phos-
phoramidites does not affect the purification of the product since it
is inert and non-reactive. Furthermore removing the need for an
aqueous work-up means a significant portion of the phosphitylated
nucleoside is not lost via hydrolysis. This is particularly important
as the hydrolytic instability of nucleoside phosphoramidites are well
documented, particularly with respect to the smaller amino
substituents.18a Importantly, in this case, the nucleoside phosphor-
amidites are isolated in a pure form which is a significant advantage
over solvent based protocols where the nucleoside phosphorami-
dites must be generated in situ.7 In addition, unlike for the methoxy
groups used in previously reported protocols for small amino-
substituents,7 the cyanoethyl group can be readily removed via
b-elimination using a mild base. Due to the high hydrolytic
instability of these materials, a stabilising amount of IL could be
Using the [C6mim][FAP]-stabilised phosphorodiamidites, the
ball milled reactions were performed using the mechanochemical
procedure outlined by Norman et al.14c However, in this instance
an in situ activator was used instead of the Hunig’s base. The yields
¨
of 1a from the IL-stabilised bis-morpholino phosphorodiamidite, 1,
with a range of activators are summarised in Table 1. Although 1a
was obtained in high yield (80%) using 1H-tetrazole, a replacement
for this hazardous substance was explored as the explosive nature
of this material means it would not be suitable for ball milling at
larger scales. Both N-methylimidazolium triflate (NMIꢀOTf)
and PyꢀTFA are readily prepared from the corresponding
acid and have been shown to be effective, cheap alternatives to
1H-tetrazole.18 Using PyꢀTFA, 1a could be isolated in a yield of
85% after purification. The purification was performed simply via
filtration through silica thus negating the need for extensive and
time consuming workup procedures. The same work-up procedure
was used when NMIꢀOTf was used as the activator, but as PyꢀTFA
resulted in slightly enhanced yields it was used in all subsequent
Table 1 Comparison of the yields from the reaction of bis-morpholino
phosphorodiamidite (1) with partially protected guanosine (a) using
various activators
Table 2 Yields for the nucleoside phosphitylation ball milling reactions
using PyꢀTFA
Protected nucleoside
Amine
Yield (%)
Entry
Activator
Yield (%)
Guanosine (a)
–N(Morpholino)2 (1)
–NEtMe (2)
–N(Morpholino)2 (1)
–NEtMe (2)
–N(Morph olino)2 (1)
–NEtMe (2)
85
80
83
74
86
75
1
2
3
4
NH4Cl
0
80
83
85
1H-Tetrazole
NMIꢀOTfa
PyꢀTFA
2-Deoxy cytidine (b)
2-Deoxy adenosine (c)
a
N-Methylimidazolium triflate.
c
This journal is The Royal Society of Chemistry 2012
11970 Chem. Commun., 2012, 48, 11969–11971

protocol to effect nucleoside phosphitylation with enhanced yields
and reactivities. Overall, the method provides significant potential
to extend the use for phosphitylation chemistry by increasing the
ease of reaction and workup procedures.
We thank QUILL and DEL for funding (KC) and Merck
KGaA for the donation of ILs.
Fig. 2 Synthesis of 4a from partially protected guanosine and
phosphoramidite 4 using ball milling conditions.
Notes and references
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added to allow for storage and further chemical manipulation. For
example, in the presence of the IL, the ethyl methyl derivatives can
be stored at low temperatures (ꢁ20 1C) for several months. In the
absence of the IL, rapid degradation of the product occurs within a
few hours.
The nucleoside phosphoramidites prepared, herein, are a
mixture of diastereoisomers at the phosphorus centre, which is
consistent for all commercially available nucleoside phosphor-
amidites. The isolated yields and diastereomeric ratios
reported for 1a–2c using our newly developed ball milling
procedure are in good agreement with the solvent based
protocols for phosphitylated nucleosides via the favoured
diisopropylamino derivatives, where the yields range from
60–90% depending on the protected nucleoside used.18a,19
The ball milling procedures documented, herein, have significant
advantages over solvent based protocols including increased
reaction rate taking minutes vs. hours for complete conversion,
as well as improved reaction profiles.
3 E. J. Amigues, C. Hardacre, G. Keane and M. E. Migaud, Green
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In contrast to the dimorpholino and ethylmethyl derivatives,
the dipyrrolidino derivative 3 was found to be highly unstable
under the ball milling conditions used for the preparation of
1a–c and 2a–c. The phosphorodiamidite 3 rearranged rapidly to
the corresponding alkylphosphonic diamide as shown by the
peak at 26 ppm in the 31P NMR spectra of the reaction’s crude.17
Interestingly, when the [C6mim][FAP] : phosphorodiamidite
mole ratio was increased to 2 : 1, successful phosphitylation
by 3 was observed. In contrast to 1a–c and 2a–c, some
decomposition of 3a was evident by the 31P NMR of the crude
mixture. However, 3a could still be isolated after purification in
the presence of [C6mim][FAP], albeit in much lower yields (15%)
than for the other phosphorodiamidites studied.
13 The bulk price of 1H-tetrazole is $700/kg from ChemImpex
International, Chicago, IL, USA.
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The study has also been extended to examine the phosphityla-
tion of partially protected guanosine, a, with bis-(2-cyanoethyl)-
N,N-diisopropylphosphoramidite 4 in order to demonstrate the
applicability of this newly developed ball-milling protocol
to include phosphoramidites (Fig. 2). Phosphoramidites are
used as phosphitylation reagents for a wide range of chemical
transformations such as the derivatisation of alcohols20 and
peptides.21 Using the reaction conditions developed, herein,
the [C6mim][FAP]-stabilised phosphoramidite 4 was used to phos-
phitylate partially protected guanosine a in high yields. Again, the
reaction was clean and purification was performed via filtration
through silica which allowed the isolation of 4a in a yield of 87%.
The continued success of clinical programs incorporating
oligonucleotide based therapeutics means that an environmentally
benign, clean and cost efficient synthesis of these molecules is
highly desirable. By combining ball milling with IL stabilisation
of reactive phosphorus species, we have developed an efficient
20 P. B. Brady, E. M. Morris, O. S. Fenton and B. R Sculimbrene,
Tetrahedron Lett., 2009, 50, 975–978.
21 S.-B. Chen, Y.-M. Li, S.-Z. Luo, G. Zhao, B. Tan and Y.-F. Zhao,
Phosphorus, Sulfur and Silicon Relat. Elem., 2000, 164, 277–291.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11969–11971 11971