Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis

Article abstract of DOI:10.1081/NCN-200060150

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep

Full text of DOI:10.1081/NCN-200060150

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LARGE-SCALE SYNTHESIS OF “Cpep” RNA MONOMERS
AND THEIR APPLICATION IN AUTOMATED RNA SYNTHESIS
R. T. Pon a , S. Yu a , T. Prabhavalkar b , T. Mishra b , B. Kulkarni b & Y. S. Sanghvi c
a Department of Biochemical and Molecular Biology , University of Calgary , Calgary, Canada
b Innovassynth Technologies (India) Ltd. , Khopoli, India
c Rasayan Inc. , Encinitas, California, USA
Published online: 15 Nov 2011.
To cite this article: R. T. Pon , S. Yu , T. Prabhavalkar , T. Mishra , B. Kulkarni & Y. S. Sanghvi (2005) LARGE-SCALE SYNTHESIS
OF “Cpep” RNA MONOMERS AND THEIR APPLICATION IN AUTOMATED RNA SYNTHESIS, Nucleosides, Nucleotides and Nucleic
Acids, 24:5-7, 777-781, DOI: 10.1081/NCN-200060150
To link to this article: http://dx.doi.org/10.1081/NCN-200060150
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):777–781, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060150
LARGE-SCALE SYNTHESIS OF ‘‘Cpep’’ RNA MONOMERS AND THEIR
APPLICATION IN AUTOMATED RNA SYNTHESIS
5
R. T. Pon and S. Yu
Department of Biochemical and Molecular Biology, University
of Calgary, Calgary, Alberta, Canada
5
T. Prabhavalkar, T. Mishra, and B. Kulkarni
Innovassynth Technologies (India)
Ltd., Khopoli, India
5
Y. S. Sanghvi
Rasayan Inc., Encinitas, California, USA
5
Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics.
Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We
believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2’-protecting group for large-
scale syntheses. Unlike 2’-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2’-
deprotection. The syntheses of 2’-Cpep protected nucleosides (A, C, G, and U) has been accomplished on
a 0.5 Kg scale. The 2’-Cpep monomers were transformed into 3’-O-phosphoramidites for conventional
automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and
initial automated solid-phase synthesis are demonstrated.
Keywords 2’-Protecting Groups, Phosphoramidites, Oligoribonucleotides
INTRODUCTION
Oligonucleotides that modulate gene expression are recognized as powerful
research tools and potential therapeutic agents. The antisense oligonucleotides are
the most advanced, with one product on the market, but other oligonucleotides
technologies, such as ribozymes, immunostimulatory CpG sequences, aptamers,
miRNA and RNAi are emerging rapidly. With the advent of these products in the
clinic and market, there will be a huge manufacturing demand. While the synthesis
of DNA has become routine and manufacturing costs have dropped significantly,
the synthesis of RNA remains a challenge. Herein, we present our efforts toward
large-scale synthesis of 2’-Cpep RNA monomers as the first step in our program to
reduce large-scale RNA production costs.
Address correspondence to R. T. Pon, Department of Biochemical and Molecular Biology, University of
Calgary, Calgary, Alberta, Canada T2N 4N1.
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R. T. Pon et al.
The synthesis of RNA using solid-phase methods on small-scales (ꢀ1 mmol) has
been routine for a number of years. Three 2’-protecting groups: t-butyldimethyl-
silyl[1] (TBDMS); [(triisopropylsilyl)oxy]methyl[2] (TOM); and bis(2-acetoxyethoxy)-
methyl[3] (ACE) have been successfully commercialized for the synthesis of RNA,
both in large number and large scale. Among these groups, the 2’-TBDMS[1]
chemistry is the oldest and most commonly used, but it has not provided the quality
and yield of oligonucleotide possible in DNA synthesis. As a result, in 1998, 2’-
TOM[2] and 2’-ACE[3] groups were introduced as more satisfactory alternatives.
Although oligoribonucleotide synthesis with either 2’-TOM and 2’-ACE chemistry is
commercially available, the monomeric reagents are not accessible at reasonable
cost, especially in large quantities.
We believe that low-cost RNA monomers will be crucial to the success of
RNA based therapeutics. In this regard, we believe that the 2’-Cpep RNA monomers
reported by Reese[4] may ultimately provide cost and chemistry advantages cur-
rently unattainable with 2’-TOM or 2’-ACE groups. The Cpep reagent is crystalline,
easy to prepare in large scales, and it has unique hydrolysis properties, which avoid
problems with intemucleotide linkages migration during acidic 2’-deprotection.
RESULTS AND DISCUSSION
Synthesis of Cpep Reagent
The Friedel-Crafts acylation of ethylene with 3-chloropropionyl chloride 1 in
DCM furnished excellent yield of 1,5-dichloropentan-3-one[5] 2 (Figure 1). Next,
FIGURE 1 Cpep synthesis on Kg scale.

R. T. Pon et al.
779
crude 2 was refluxed with 4-chloroaniline in presence of PTSA followed by
addition of triethyl orthoformate. Upon completion of the reaction, 4,4-
diethoxypiperdine analog 3 was isolated as a crystalline product in >90% yield.
Reaction of Hu¨nigs base and BF3 Á Et2O in DCM at 0°C furnished the desired
Cpep reagent 4 in >90% yield as a crystalline product.
Synthesis of Cpep-Protected Nucleoside 9
Appropriately base protected ribonucleosides were treated with 1.2
equivalents of triisopropylsilyl chloride (TIPSCI) to furnish 6 in >80% yield.
The isolation of 6 was significantly simplified by using a mixture of ACN/pyridine
during 3’- to 5’-protectiOn. The reaction of Cpep reagent 4 with 6 in the presence
of DCA provided 7 in >70% yield. Hygroscopic PTSA was replaced with DCA as
a more easily manageable acid during scale-up studies. Deprotection of TIPS
from 7 was carried out with TEAF in THF to furnish 8 in >75% yield. TBAF,
traditionally used, was replaced with TEAF as a cheaper and easily separable
deblocking reagent in the scale-up process. Lastly, treatment of 8 with 1.1 eq.
of DMT-C1 in pyridine provided 9 as an amorphous powder after column
chromatography. This series of reactions were carried out on >200 g scale to
establish the scale-up route (Figure 2).
Phosphitylation Reaction of 9
Phosphitylation of 9 with 2-cyanoethyl diisopropyl-chlorophosphoramidite
resulted in faster reaction, however with formation of byproducts. Use of 2-
cyanoethyl-N,N,N’,N’-tetraisopropylphosphorodiamidite (Phos) instead furnished a
FIGURE 2 Synthesis of 2’-O-Cpep phosphoramidites.

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R. T. Pon et al.
much cleaner reaction with no byproducts formed. Due to its inherent safety, the
Phos reagent seems to be the right choice for phosphitylation on large scales.
The purity of phosphoramidites 10 prepared from Pharma grade Phos reagent
(Digital Specialty Chemicals, Canada) was >98% (31P NMR).
Oligonucleotide Synthesis
A test sequence rU20 was synthesized on CPG solid-support (1 mmol) using an
ABI 394 DNA synthesizer. A 0.1 M solution of rU phosphoramidite 10 was
employed with 0.25 M ethylthiotetrazole (ETT) solution as activator and a 10 min
coupling time. Standard RNA synthesis cycles were utilized for deprotection (3%
DCA in DCM), capping (Ac2O/2,6-lutidine/THF) and oxidation (I2/H2O/pyridine/
THF). The oligonucleotide was cleaved from the solid-support and base
deprotection was done by treatment with 3:1 NH4OH/EtOH (RT, 48 h). The
deprotection of the 2’-Cpep groups was accomplished in 4:6 formate buffer (0.5 M,
pH 2.5)/dimethylacetamide (40°C, 5 h). CGE analysis showed >55% product
(Figure 3). This was slightly less than using 2’-TBDMS but protocol optimization has
not been completed.
The synthesis and scale-up of the four Cpep monomers 9 (B = U, CBz, Apiv and
G
pac) has been accomplished on a large scale. During scale-up, several process
improvements have been made to yield a safe, environmentally clean, robust and
economical synthesis of these new RNA monomers. Presently, the cost and
accessibility of 2’-ACE and 2’-TOM RNA monomers are limitations to large-scale
drug production. We have shown that 2’-Cpep monomers can be prepared in large
scale at low cost. The synthesis of mixed RNA sequences on a large scale is
currently under investigation and the results will be published in due course.
FIGURE 3 Analysis of 2’-Cpep and TBDMS rU 20-mers.

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