Synthesis of oligoribonucleotides with phosphonate-modified linkages

Article abstract of DOI:10.1039/c1ob05488k

Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2′-O-benzoyloxymethoxymethyl protected monomers is presented in both 3′→5′ and 5′→3′ directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3′- and 5′-phosphonate linkages are evaluated. The introduction of the 5′-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔT_m -1.8 °C/mod.), whereas the introduction of the 3′-phosphonate units resulted in considerable destabilisation of the duplexes (ΔT_m -5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.

Full text of DOI:10.1039/c1ob05488k

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PAPER
Synthesis of oligoribonucleotides with phosphonate-modified linkages†
Ondrˇej Pa´v,a Ivana Kosˇiova´,a Ivan Barv´ık,b Radek Pohl,a Milosˇ Budeˇsˇ´ınsky´a and Ivan Rosenberg*a
Received 29th March 2011, Accepted 14th June 2011
DOI: 10.1039/c1ob05488k
Solid phase synthesis of phosphonate-modified oligoribonucleotides using
2¢-O-benzoyloxymethoxymethyl protected monomers is presented in both 3¢→5¢ and 5¢→3¢ directions.
Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3¢-
and 5¢-phosphonate linkages are evaluated. The introduction of the 5¢-phosphonate units resulted in
moderate destabilisation of the RNA/RNA duplexes (DTm -1.8 ◦C/mod.), whereas the introduction of
the 3¢-phosphonate units resulted in considerable destabilisation of the duplexes (DTm -5.7 ◦C/mod.).
Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate
linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and
phosphodiesterase II.
The latter three groups are examples of a new trend in RNA
synthesis – the use of a base-labile 2¢-protecting group that allows
for the one-pot deprotection of the 2¢-hydroxyl and nucleobases
Introduction
The unique properties of RNA have stimulated the development
of a variety of applications in diagnostics and therapeutics and
and cleavage from the solid support.
in basic research where RNA can be used as a catalytic agent
Although there are several protecting groups available for
(ribozymes),1 an affinity ligand (aptamers)2 or an agent to induce
gene silencing (RNA interference, micro RNA).3,4 Because of
this usefulness, the demand for modified RNA has increased
dramatically in the past decade.
synthesis in 3¢→5¢ direction, there is also a demand for 5¢→3¢
synthesis tailored, for example, to the synthesis of RNA with 5¢-
modified internucleotide linkages or for convenient introduction
of ligands and labels at the 3¢-end.
In our case, the need to develop new methodology for the
The chemical synthesis of RNA is a more difficult task than
the synthesis of DNA because the 2¢-hydroxy group of the ribose
needs to be protected. The correct choice of a 2¢-O-protecting
group can considerably influence the length of the coupling time,
the coupling yield and the quality of the final product. Thus, this
protecting group must be stable throughout the cycles of solid
phase synthesis, sterically-unhindered so as to not interfere with
the chain growth and readily removable under conditions where
the final RNA is completely stable.
The tert-butyldimethylsilyl (TBDMS) group5 was the first
2¢-protecting group used in commercial RNA synthesis. Since
then, several protecting groups have been proposed in place
of TBDMS, such as triisopropylsilyloxymethyl (TOM),6 bis(2-
acetoxyethyloxy)methyl (ACE),7 tert-butyldithiomethyl (DTM),8
2-cyanoethoxymethyl (CEM),9 2-(4-toluylsulfonyl)ethoxymethyl
(TEM),10 pivaloyloxymethyl (POM),11 1,1-dioxo-1l6-thiomor-
pholine-4-carbothioate (TC),12 or acetal levulinyl ester (ALE).13
synthesis of RNA in the reverse direction has originated from our
intention to synthesise oligoribonucleotides that are modified by
various nucleoside-5¢-phosphonates. These compounds have been
of interest to us for many years.14
Until now, only the TBDMS protecting group has been reported
for RNA synthesis in the reverse direction using phosphoramidite
condensation method.15 Silyl protecting groups are removed by flu-
oride anions. However, this deprotection requires time-consuming
desalting steps before the final purification of an oligoribonu-
cleotide by HPLC. Moreover, 5¢-phosphonate monomers are
introduced into the oligonucleotide chain by the phosphotriester
condensation method using pyridine as the coupling solvent. Thus
during the phosphotriester coupling step, the use of the 2¢-O-
TBDMS group in the elongated unit might not be applicable
because of possible migration of TBDMS to free 3¢-hydroxy group
under basic conditions.
Therefore, we report here a new RNA synthesis strategy based
on the base-labile 2¢-O-benzoyloxymethoxymethyl (BOMOM)
group. Using this protecting group, we have synthesised two
sets of oligoribonucleotides modified by regioisomeric 3¢- and 5¢-
phosphonate linkages in both the 3¢→5¢ and 5¢→3¢ directions,
respectively (Fig. 1). It is worth noting that only a few mod-
ifications of internucleotide linkages have been reported in the
ribo series so far, and among these reports, phosphorothioate and
aInstitute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo
nam. 2, 16610, Prague, Czech Republic. E-mail: ivan@uochb.cas.cz;
Fax: +420 220 183 531; Tel: +420 220 183 381
bInstitute of Physics, Faculty of Mathematics and Physics, Charles Univer-
sity, Ke Karlovu 5, 121 16, Prague 2, Czech Republic. E-mail: ibarvik@
karlov.mff.cuni.cz; Fax: +420 224 922 797; Tel: +420 221 911 450
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05488k
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phosphate groups. Moreover, the phosphonate monomers are
introduced into the oligonucleotide chain by the phosphotriester
condensation method using 4-methoxy-1-oxido-2-pyridylmethyl
group (MOP) as an intramolecular coupling catalyst.18,19 Since
both methyl and MOP groups are removed by treatment with
benzenethiol, we obtained uniform phosphate/phosphonate de-
protection protocol. Therefore, compounds 3a–d were converted
to the corresponding methyl phosphoramidites 7a–d using methyl
N,N,N¢,N¢-tetraisopropylphosphordiamidite.
In the 5¢-series, the synthetic path proceeded in a similar
manner. 5¢-O-TBDPS derivatives 2a–d were protected with the
BOMOM group, and after separation of the regioisomers on
silica gel, the desired isomers 4a–d were dimethoxytritylated with
dimethoxytritylchloride in the presence of silver triflate. Once
again, we did not observe formation of 3¢-O-BOMOM derivative
6b in the case of the guanosine derivative. Cleavage of the 5¢-O-silyl
group with TBAF (8a–d) followed by the phosphitylation reaction
afforded 5¢-phosphoramidites 9a–d in good yields.
Fig.
1 Regioisomeric 3¢-phosphonate (Bpc-B) and 5¢-phosphonate
(B-pcB) linkages.
boranophosphate oligoribonucleotides are the most widely used
as research and diagnostic tools.16
Results and discussion
The stability of the BOMOM group in compounds 4 was
examined in the presense of 3% DCA in DCM (DMTr deblocking
conditions) and pyridine (phosphonate coupling conditions) by
the NMR experiments. The BOMOM group was found completely
stable under these conditions, neither migration, cleavage nor
formation of the 2¢,3¢-O-cyclic formal was observed.
BOMOM-Cl 10 was prepared by the alkylation of sodium
benzoate with one equivalent of bis(chloromethyl)ether.
Next, we prepared the appropriate 3¢- and 5¢-phosphonate
monomers according to the method shown in Scheme 2.
In the 3¢-series, 2¢,5¢-bis-DMTr nucleosides 11a–d were phos-
phonylated with diisopropyl tosyloxymethylphosphonate in the
presenceofsodium hydride. Uponacidic deprotection, these nucle-
osides provided 3¢-phosphonates 12a–d. 5¢-Dimethoxytritylation
of these compounds followed by benzoylation of the 2¢-hydroxy
group afforded fully protected phosphonates 13a–d. Note that
in the case of 3¢-phosphonate monomers 2¢-O-Bz group was
used instead of 2¢-O-BOMOM. The reason for this change is
that all attempts to introduce BOMOM group selectively on
Synthesis of monomeric building blocks
First, we prepared appropriate 3¢- and 5¢-phosphoramidite build-
ing blocks as shown in Scheme 1.
In the 3¢-series, 2¢-O-BOMOM (3a–d) and 3¢-O-BOMOM (5a–
d) derivatives were prepared by the reaction of 5¢-O-DMTr nucleo-
sides 1a–d and benzoyloxymethoxymethyl chloride 10 (BOMOM-
Cl)17 in the presence of dibutyltindichloride, followed by the
separation of the regioisomers on silica gel. By this method,
the desired 2¢-O-BOMOM derivatives 3a–d, eluting as an TLC
faster isomer, were obtained in 32–48% yield. In case of the
guanosine derivative, we did not observe formation of 3¢-O-
BOMOM derivative 5b.
In our preliminary experiments, we found the cyanoethyl
(CE) protection of the phosphate groups incompatible with
BOMOM group. During the basic deprotection step of a model
oligoribonucleotide, we observed simultaneous cleavage of both
groups which resulted in an undesired cleavage of internucleotide
linkages. Therefore we decided to use methyl protection of the
Scheme 1 Reagents and conditions: (i) BOMOM-Cl, Bu2SnCl2, DIPEA, DCE, 80 ◦C(ii) CH3O-P(NiPr2)2, tetrazole, ACN, r.t. (iii) TBAF, THF, r.t. (iv)
DMTr-Cl, silver triflate, py, r.t.
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Scheme 2 Reagents and Conditions: (i) TsOCH2PO(OiPr)2, NaH, DMF, r.t. (ii) 80% AcOH, r.t. (iii) DMTr-Cl, py, r.t. (iv) BzCN, Et3N, ACN, r.t. (v)
Me3SiBr, 2,6-lutidine, ACN, r.t. (vi) MOP, DCC, py, r.t. (vii) BOMOM-Cl, Bu2SnCl2, DIPEA, DCE, 80 ◦C(viii) DMTr-Cl, silver triflate, py, r.t.
2¢-hydroxyl failed and the alkylation reactions in the presence of
tin derivatives, DIPEA, NaH, DBU, Cs2CO3 and Ag2O did not
afford the desired product (data not shown). Therefore Bz group
which is in this particular case fully compatible with the solid
phase and deprotection cycle was used.
ence of DCC to afford appropriate phosphonomethyl monomers
19a–d.
Synthesis and deprotection of modified oligoribonucleotides
The deprotection of isopropyl ester groups with bro-
motrimethylsilane and the subsequent esterification of free phos-
phonic acid with 4-methoxy-1-oxido-2-pyridylmethanol (MOP-
OH) in the presence of DCC afforded the appropriate phospho-
nomethyl monomers 14a–d.
In the 5¢-series, ethoxymethylidene derivatives 15a–d were phos-
phonylated with diisopropyl tosyloxymethylphosphonate in the
presence of sodium hydride. 5¢-Phosphonates 16a–d were obtained
upon acidic deprotection of the ethoxymethylidene group. These
compounds were protected with the BOMOM group. Because
we did not observe any separation of regioisomers on silica gel,
the mixture of BOMOM phosphonates was dimethoxytritylated
with dimethoxytritylchloride in the presence of silver triflate to
afford a mixture of regioisomers, 17a–d and 18a–d, which was
then separated by preparative HPLC. The desired regioisomers
17a–d were treated with bromotrimethylsilane and subsequently
esterified with 4-methoxy-1-oxido-2-pyridylmethanol in the pres-
Once all monomeric building blocks were prepared, various
oligoribonucleotides were synthesised in both the 3¢→5¢ (20, 21,
22, 24, 26, 28, 30, 32) and the 5¢→3¢ directions (20, 21, 23, 25, 27,
29, 31, 33) (Table 1). Solid-phase synthesis was performed on a
1 mmol scale. Phosphoramidite units were incorporated using the
phosphoramidite condensation method with a 3-minute coupling
time and using 0.5 M ethylthiotetrazole (ETT) as the activator.
Phosphonate units were incorporated using the phosphotriester
condensation method with a 10-minute coupling time and using
0.3 M 2,4,6-triisopropylbenzenesulfonylchloride (TIPS) as the
activator.
Upon completion of the synthesis, oligoribonucleotides an-
chored to the LCAA-CPG were first treated with a mixture of
benzenethiol, TEA and DMF to remove the methyl and MOP
protecting groups from the phosphate and phosphonate groups,
respectively. Next, the oligoribonucleotides were treated with
gaseous ammonia to remove the BOMOM and acyl protecting
Table 1 Hybridisation properties of phosphonate-modified oligoribonucleotides
3¢-Phosphonate
5¢-Phosphonate
Tm (◦C) (DTm/mod.)
Strands No.
Strands No.
Tm (◦C) (DTm/mod.)
37.1
20/21
22/21
24/21
20/26
20/28
30/21
20/32
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢ r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
5¢-r(GCA UAU CAC)-3¢/5¢-r(GUG AUA UGC)-3¢
20/21
23/21
25/21
20/27
20/29
31/21
20/33
37.1
18.1 (-6.3)
26.2 (-5.5)
24.2 (-6.5)
23.3 (-4.6)
no Tm
31.3 (-1.9)
34.2 (-1.5)
34.4 (-1.4)
32.3 (-1.6)
19.3 (-2.2)
19.9 (-2.2)
no Tm
Modified units are underlined. RNA/RNA duplexes (50 mM TRIS-HCl pH 7.2, 1 mM EDTA, 100 mM Na+). Total strand concentration 4 mM. Tm
0.5 ◦C.
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groups and to release the final product from the solid support.
In the end, the RNA was washed from the column by TEAA
buffer and purified using a Poly-Pak purification protocol (Glen-
Research, DMTr ON mode). Using gaseous ammonia (0.7 MPa),
we observed neither chain cleavage nor formation of guanosine
formaldehyde adducts reported by Lavergne et al.11
The mechanism of BOMOM deprotection is very likely similar
to that of POM,11 consisting of the cleavage of the ester group,
followed by the formation of 2¢-O-formaldehydehemiacetal, which
is completely stable and temporarily protects 2¢-hydroxy group
under basic conditions. This hemiacetal is hydrolysed under the
conditions of the aqueous workup.
Concerning the coupling time and the stepwise yield, we did
not observe any significant difference between the synthesis in the
3¢→5¢ or 5¢→3¢ direction. Nonamers 20 and 21 were isolated in
75–80% yield. The phosphonate-modified nonamers 22–33 were
isolated in 55–71% yield. The phosphoramidite stepwise yield was
96–99%. The phosphonate stepwise yield was 93–96%.
Hybridisation properties of modified oligoribonucleotides
The use of modified oligonucleotides as therapeutic and diagnostic
tools is based on the hybridisation of the oligonucleotide with a
target sequence. Thus, determination of hybridisation properties
is one of the basic experiments used to evaluate the examined
modification.
The thermal stabilities of duplexes formed by phosphonate-
modified oligoribonucleotide and complementary RNA strands
have been evaluated by UV melting experiments. The obtained Tm
values are summarised in Table 1.
Fig. 2 Conformation of 3¢-phosphonate (top) and 5¢-phosphonate (bot-
The methylene group insertion into the internucleotide link-
age caused in both cases a decrease in the melting temper-
ature of the phosphonate-modified duplexes when compared
to the unmodified duplex. The introduction of two or three
5¢-phosphonate units in an alternate mode (23, 25, 27, 29)
resulted in moderate destabilisation of the RNA/RNA duplexes
(approximately -1.6 ◦C/mod.), whereas the introduction of 3¢-
phosphonate units (22, 24, 26, 28) resulted in considerable
destabilisation of the duplexes (approximately -5.7 ◦C/mod.).
Fully 5¢-phosphonate-modified nonamers 31 and 33 still retained
the hybridisation ability (DTm -2.2 ◦C/mod.), whereas nonamers
that were fully modified by 3¢-phosphonate units 30 and 32
showed no hybridisation. Thus, it seems that the lengthening
of the internucleotide linkage brought the additional degree of
freedom into the conformation of the sugar-phosphate backbone,
resulting in a decrease in the hybridisation ability. Surprisingly,
there has been a remarkable difference between the hybridisation
ability of oligoribonucleotides modified by regioisomeric 3¢- and
5¢-phosphonate linkages, which can be most clearly seen in case of
fully modified nonamers 30, 32 and 31, 33.
In order to explain this unexpected behaviour, we performed
molecular dynamics (MD) simulations at the atomic level with
the explicit inclusion of water molecules into the simulated
system. MD simulations uncovered substantial differences in the
conformational preferences of modified internucleotide linkages.
In the duplex, 5¢-phosphonate linkages adopted a conformation
that was strongly stabilised by a 2¢OH-5¢¢O hydrogen bond
contact. In contrast, 3¢-phosphonate linkages adopted various
conformations without any specific stabilisation (Fig. 2).
tom) linkages in the duplex.
Thus in the duplex, the strand modified by the 5¢-phosphonate
linkages was able to adopt a more stable and favourable con-
formation and thus exhibited significantly better hybridisation
properties than the strand modified by 3¢-phosphonate linkages.
The NMR studies performed on 3¢-phosphonate (Apc-A, 34) and
5¢-phosphonate (A-pcA, 35) dimers (Fig. 1, B = A) showed agree-
ment in the internucleotide linkage conformation of comparable
torsion angles (z¢, b, g) of the Apc-A dimer 34 with those obtained
by MD simulations. There were, however, significant differences in
comparable torsion angles (e, a¢, g) of A-pcA dimer 35. Therefore,
it seems that the 5¢-phosphonate internucleotide linkage adopts a
conformation involving the 2¢OH-5¢¢O hydrogen bond under the
influence of the interaction between two strands in the RNA/RNA
duplex.
The NMR studies on furanose ring conformation also revealed
that both adenosine units in A-pcA dimer exhibited equal
population of N and S conformers whereas 3¢-end adenosine unit
in Apc-A dimer exhibited population shifted to the S conformer
(70% S). This result could also partially explain the lower binding
affinity of the 3¢-phosphonate-modified oligonucleotides to the
RNA strand.
For more details about the MD and NMR studies, see the ESI.†
Nuclease stability of modified oligoribonucleotides
Synthesis of modified oligonucleotides is also closely connected to
the search for modifications with enhanced nuclease stability. A
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rapid degradation of phosphodiester oligonucleotides by nucleases
in body fluids is one of the most important limitations to their
in vivo use. The increase of the half-life of these compounds could
thus bring remarkable benefits, including prolonged antiviral or
diagnostic effects.
Experimental
Synthesis of monomers - general methods
Method A - Preparation of nucleosides 3, 4, 5 and 6. Dibutyltin
dichloride (1.1 mmol) was added to a solution of 5¢-protected
derivative 1 or 2 (1.0 mmol) and DIPEA (2.2 mmol) in DCE
(10 mL). The mixture was stirred 1 h at r. t. After this, the reaction
mixture was warmed to 85 ◦C, and benzoyloxymethoxymethyl
chloride 10 (1.1 mmol) was added. The reaction mixture was
heated 3 h at 85 ◦C to afford a mixture of 2¢-O-BOMOM and 3¢-O-
BOMOM derivatives. The regioisomers were separated by silica
gel chromatography (elution with gradient of 0–50% acetone in
toluene, guanosine derivatives used 0–100% acetone in toluene).
The faster TLC eluting isomer was the 2¢-O-BOMOM derivative
(3, 4) and the slower TLC eluting isomer was the 3¢-O-BOMOM
derivative (5, 6).
In connection with this, the nuclease stability of phosphonate
dimers 34 and 35 and nonamers 26–29 has been examined in the
presence of ribonuclease A (EC 3.1.27.5), ribonuclease T2 (EC
3.1.27.1), phosphodiesterase I (EC 3.1.4.1) and phosphodiesterase
II (EC 3.1.16.1). Ribonuclease A is an endoribonuclease that pref-
erentialy cleaves the 3¢-end of uridine and cytidine residues, leaving
a 3¢-phosphorylated product, via a 2¢,3¢-cyclic monophosphate.
Ribonuclease T2 is also an endoribonuclease which preferentialy
cleaves the 3¢-end of adenosine residues by the same mechanism.
Phosphodiesterase I and II are exonucleases. Phosphodiesterase I
cleaves from the 3¢-end, releasing 5¢-nucleotide units. Phosphodi-
esterase II cleaves from the 5¢-end, releasing 3¢-nucleotide units.
The course of the cleavage reaction has been monitored by
LC-MS.
Phosphodiester-linked ApA dimer was rapidly degraded by
ribonuclease T2, phosphodiesterase I and II under experimental
conditions (with a half-life less than one minute). In contrast,
both types of phosphonate-modified dimers, 34 and 35, exhibited
exceptional nuclease resistance and no cleavage of internucleotide
linkages was observed within two hours of incubation with these
nucleases. Similar results were observed during the cleavage of
modified nonamers 26–29 by ribonuclease A and phosphodi-
esterase I and II. Neither 3¢-phosphonate (28) nor 5¢-phosphonate
(27) linkages were cleaved by ribonuclease A. We observed only
complete cleavage of phosphodiester linkages. In the case of
phosphodiesterase I and II, we observed an interesting cleavage
pattern. The one-by-one cleavage of units occurred until the
modified linkage was met. After that, the modified unit was
omitted and the phosphonate dimer was cleaved, but neither
the 3¢-phosphonate nor 5¢-phosphonate linkages were cleaved.
Moreover, after this unexpected cleavage step, further cleavage
did not continue in most cases (26–29). The cleavage profiles are
enclosed in the ESI.†
Method B – Preparation of nucleoside 8. BOMOM derivatives
4 or 6 (1.0 mmol) in pyridine (10 mL) was added to a 30 min pre-
stirred mixture of silver triflate (2.0 mmol) and dimethoxytrityl
chloride (2.0 mmol) in pyridine (10 mL). The reaction mixture
was stirred 16 h at r.t. The reaction was quenched by the addition
of methanol (5 mL), the silver chloride was filtered off, and the
mixture was concentrated under reduced pressure. The product
was purified by chromatography on silica gel (elution with a
gradient of 0–50% ethyl acetate in toluene).
To cleave the 5¢-O-TBDPS group, fully protected nucleoside
(1.0 mmol) was dissolved in 0.5 M TBAF in THF (10 mL), and
the reaction mixture was stirred 2 h at r.t. After that, the mixture
was diluted with toluene and concentrated under reduced pressure.
The product was purified by chromatography on silica gel (elution
with a gradient of 0–50% acetone in toluene, guanosine derivatives
used 0–100% acetone in toluene).
Method C – Preparation of phosphoramidites 7 and 9. Tetrazole
(1.1 mmol) was added to a solution of derivative 3, 8 (1.0 mmol)
and
methyl
N,N,N¢,N¢-tetraisopropylphosphordiamidite
(2.0 mmol) in DCM (10 mL). The reaction mixture was
stirred 2 h at r.t. After that, TEA (1 mmol) was added and the
reaction mixture was evaporated. The product was purified by
chromatography on silica gel (elution with a gradient of 0–50%
ethyl acetate in toluene, guanosine derivatives used 0–50% acetone
in toluene). In addition, the guanosine derivative were dissolved
in DCM and precipitated from hexane. Lastly, phosphoramidites
were freeze-dried from benzene.
Conclusions
In conclusion, we present here a new base-labile 2¢-protecting
group usable for the solid phase synthesis of oligoribonucleotides
in both the 3¢→5¢ and 5¢→3¢ directions. To the best of our knowl-
edge, benzoyloxymethoxymethyl is the first non-silyl protecting
group used in the solid phase synthesis of oligoribonucleotides
in the reverse direction. Moreover, post-synthetic deprotection of
oligoribonucleotides using gaseous ammonia allows for a simple
on-column deprotection protocol.
Utilising this novel methology, we prepared oligoribonu-
cleotides modified by regioisomeric 3¢- and 5¢-phosphonate
linkages in good yields and purity. Both linkages exhibited
extraordinary stability against cleavage by nucleases. The 5¢-
phosphonate linkage proved to have sufficient hybridisation
potential to be used in biochemical applications, such as siRNA
or miRNA. Another application of phosphonate linkages could
occur at specific sites of modified oligonucleotides, which require
conformational flexibility such as loops or hairpins, or nuclease
stability.
Method D – Preparation of phosphonates 12 and 16. Sodium
hydride (3.0 mmol) was added at 0 ◦C to a stirring solution
of protected nucleoside 11 or 15 (1.0 mmol) and diisopropyl
tosyloxymethylphosphonate (2.0 mmol) in DMF (10 mL). The
reaction mixture was left to warm up gradually to r.t. and then
stirred 16 h at r.t. The reaction was quenched by the addition of
glacial acetic acid (1 mL) at 0 ◦C, and the mixture was concentrated
under reduced pressure. After that, the nucleoside-phosphonate
was treated with 80% acetic acid (10 mL) for 16 h at r.t. and then
concentrated under reduced presure. The product was purified by
chromatography on silica gel (elution with a gradient of 0–10%
ethanol in chloroform).
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Table 2 Solid phase synthesis protocol
Method E – Preparation of phosphonate 13. Dimethoxytrityl
chloride (1.1 mmol) was added to a stirring solution of phos-
phonate 12 (1.0 mmol) in pyridine (10 mL), and the mixture was
stirred 16 h at r.t. The reaction was quenched by the addition of
triethylamine (1 mL) and methanol (5 mL), and the mixture was
concentrated under reduced pressure. The residue was dissolved
in chloroform (50 mL) and extracted with a saturated solution
of sodium hydrogencarbonate (3 ¥ 20 mL). The organic layer
was dried over anhydrous sodium sulfate and evaporated. After
that, benzoyl cyanide (1.5 mmol) was added to a stirring solution
of 5¢-the dimethoxytrityl phosphonate derivative (1.0 mmol) and
triethylamine (0.2 mmol) in acetonitrile (10 mL), and the mixture
was stirred 16 h at r.t. The reaction was quenched by the addition
of methanol (5 mL), and the mixture was concentrated under
reduced pressure. The product was purified by chromatography
on silica gel (elution with a gradient of 0–50% ethyl acetate in
toluene).
Phosphoramidite condensation method
1. Detritylation
2. Condensation
3% DCA in DCM
0.1 M phosphoramidite in ACN
0.5 M ETT in ACN
120 s
180 s
3. Capping
Ac2O/2,6-lutidine/THF 1 : 1 : 8
6.5% DMAP in THF
60 s
4. Oxidation
10% tBuOOH in DCM
90 s
Phosphotriester condensation method
1. Detritylation
2. Condensation
3% DCA in DCM
0.1 M phosphonate in pyridine
0.3 M TIPS-Cl in ACN
120 s
600 s
3. Capping
Ac2O/2,6-lutidine/THF 1 : 1 : 8
6.5% DMAP in THF
60 s
of 0–50% acetone in toluene, guanosine derivatives used 0–100%
acetone in toluene) as an unseparable mixture of regioisomers.
Next, the mixture was separated by preparative HPLC (elution
with a gradient of 50–100% methanol in water). The faster reverse
phase eluting isomer was 3¢-O-BOMOM derivative (18) and the
slower reverse phase eluting isomer was 2¢-O-BOMOM derivative
(17).
Method F – Preparation of MOP phosphonates 14 and 19.
Bromotrimethylsilane (4.0 mmol) was added to a solution of
diisopropyl phosphonate 13 or 17 (1.0 mmol) and 2,6-lutidine
(8.0 mmol) in acetonitrile (10 mL). The reaction mixture was
stirred 16 h at r.t. and then concentrated under reduced presure.
The residue was treated with 2 M TEAB (5 mL) and methanol
(5 mL). The solution was evaporated, and the residue was dissolved
in chloroform (50 mL) and extracted with 0.2 M TEAB (3 ¥
20 mL). The organic layer was dried over anhydrous sodium sulfate
and evaporated. The crude nucleoside phosphonic acid was used
for the further steps without purification. DCC (5.0 mmol) was
added to a solution of nucleoside phosphonic acid (1.0 mmol)
and 4-methoxy-1-oxido-2-pyridylmethanol (3.0 mmol) in pyridine
(6 mL), and the reaction mixture was stirred for 3 days at r.t.
After that, the reaction mixture was diluted with water (4 mL)
to achieve a 60% solution of pyridine in water. The solution was
then heated 16 h at 50 ◦C to cleave off one ester group before being
concentrated under reduced pressure. The product was purified by
chromatography on silica gel (elution with a gradient of 0–100% of
a mixture ethyl acetate/ethanol/acetone/water 4 : 1 : 1 : 1 in ethyl
acetate) and lyophilised from dioxane.
Synthesis and purification of oligoribonucleotides
Oligonucleotides 20–33 were synthesised using the protocol
shown in Table 2. Synthesis was performed on a 1 mmol
scale in the 3¢→5¢ direction on [2(3)-O-benzoyl-5-O-
dimethoxytrityl-1-(4-N-benzoylcytosine-1-yl)-b-D-ribofuranos-
3(2)-O-succinoyl]LCAA-CPG 36 and in the 5¢→3¢ directions
on [2-O-benzoyloxymethoxymethyl-3-O-dimethoxytrityl-1-(2-N-
isobutyrylguanin-9-yl)-b-D-ribofuranos-5-O-succinoyl]LCAA-
CPG 37 using GenSyn V02 DNA/RNA synthesiser.
Deprotection of nonamers was achieved in two steps. First, the
column with oligoribonucleotide anchored to the LCAA-CPG
was treated with a benzenethiol/TEA/DMF (1 : 1.4 : 2 v : v : v)
mixture for 6 h to remove the methyl and MOP protecting groups
from the phosphate and phosphonate groups, respectively. Then,
the column was washed with DMF, ACN and dried. Second, the
column was inserted into the pressure vessel and treated with
gaseous ammonia (0.7 MPa) for 8 h to the remove BOMOM and
acyl protecting groups,and to release the final product from the
solid support. Finally, the deprotected RNA was washed off of the
column by a 0.1 M TEAA buffer and purified using a Poly-Pak
purification protocol (GlenResearch, DMTr ON mode).
Method
G – Preparation of phosphonates 17 and 18.
Dibutyltin dichloride (1.1 mmol) was added to a solution of 5¢-
phophonomethyl derivative 16 (1.0 mmol) and DIPEA (2.2 mmol)
in DCE (10 mL). The mixture was stirred 1 h at r. t. After
that, the reaction mixture was warmed up to 85 ◦C, and
benzoyloxymethoxymethyl chloride 10 (◦1.1 mmol) was added. The
reaction mixture was heated 3 h at 85 C to afford a mixture of
2¢-O-BOMOM and 3¢-O-BOMOM derivatives. The regioisomers
were purified by chromatography on silica gel (elution with
a gradient of 0–50% acetone in toluene, guanosine derivatives
used 0–100% acetone in toluene) as an unseparable mixture
of regioisomers and used for further steps without separation.
The mixture of BOMOM derivatives (1.0 mmol) in pyridine
(10 mL) was added to a 30 min pre-stirred mixture of silver
triflate (2.0 mmol) and dimethoxytrityl chloride (2.0 mmol) in
pyridine (10 mL). The reaction mixture was stirred 16 h at
r.t. The reaction was quenched by the addition of methanol
(5 mL), the silver chloride was filtered off and the mixture
was concentrated under reduced pressure. The regioisomers were
purified by chromatography on silica gel (elution with a gradient
Tm experiments
Tm experiments were performed on a CARY 100 Bio UV Spec-
trophotometer (Varian Inc.) equipped with a Peltier temperature
controller and thermal analysis software. The samples were
prepared by mixing complementary strands of RNA together to
give a 4 mM final concentration in 50 mM TRIS/HCl pH 7.2,
1 mM EDTA and 100 mM Na+. A heating-cooling cycle in the
◦
range 5–60 ◦C with a gradient of 0.2 C min-1 was applied. Tm
values were determined from the maxima of the first derivative
plots of absorbance versus temperature (Tm 0.5 ◦C).
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Nuclease stability experiments
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Acknowledgements
The support by the grant 202/09/0193 (Czech Science Foun-
dation) and Research centers KAN200520801 (Acad. Sci. CR)
and LC060061 (Ministry of Education, CR) under the Institute
research project Z40550506 is gratefully acknowledged, as is the
support by project No. MSM 0021620835 (Ministry of Education,
CR).
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6126 | Org. Biomol. Chem., 2011, 9, 6120–6126
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