Liquid-Phase Synthesis of 2′-Methyl-RNA on a Homostar Support through Organic-Solvent Nanofiltration

Significance Statement

In the highlighted paper we describe for the first time a new approach to the iterative synthesis of oligonucleotides (oligos): Liquid Phase Oligo Synthesis with Organic Solvent Nanofiltration, LPOS-OSN. In this pilot project we prepared a 2’-methyl RNA phosphorothioate nonamer (9-mer), but we expect the method to be applicable to almost all medicinal oligos.

The pipeline of classical small molecule drugs is drying up. Therefore Big Pharma is turning to primary metabolites and their analogues as potential classes of new drugs. However, peptides, oligos and oligosaccharides are poorly suited to classical batch manufacturing techniques. Instead they may be synthesised in high yield by solid phase synthesis.

We focus on oligos which have re-emerged as a promising avenue of pharmaceutical research. This was stimulated by the discovery of RNA interference (Fire and Mello, Nobel prize 2006), which underlies several mechanisms of targeted protein suppression by templated mRNA cleavage. From this it was quickly realised that modified RNA oligomers (18- to 25-mers) might provide exquisitely selective medicinal agents, if only they could be delivered to the intra-cellular environs. It has recently become clear that this is indeed possible, with many companies having oligo formulations in phase III trials.

Solid phase synthesis involves attaching the growing polymer chains to solid support beads that may be separated simply from excess monomer and other reactants by filtration and washing of the beads. Repeated cycles of first chain extension, then unblocking of a temporary protecting group that prevents multiple couplings from occurring, are used to grow biopolymers of any given sequence, one monomer at a time (see Figure, top left). This iterative method is attractive because it avoids the need to isolate and purify the large number of chain growth intermediates. However, solid phase synthesis is fundamentally un-scalable due to the challenge of completely reacting large volumes of support beads with limited amounts of expensive monomers. Currently, the largest batch of oligo prepared by solid phase oligo synthesis (SPOS) is ca. 2Kg. This scale is large enough to support clinical trials, but totally impractical for preparing the 100s of kilograms, or even tons, of oligo that will be required annually for a widely used drug.

In our paper we identify the emerging field of organic solvent nanofiltration (OSN) as a key separation technology that may unblock this bottleneck. Unlike SPOS, the combination of liquid phase oligo synthesis (LPOS) with organic solvent nanofiltration is compatible with standard industrial plant, except that the solution of growing oligo will need to be purified by filtration through a spiral-wound module of organic solvent nanofiltration membrane. Because of their high reactivity and firmly established chemistry, we have chosen to use commercially available nucleoside phosphoramidite monomers, as developed for SPOS, in an LPOS setting. We have also used the same activators, sulphur-transfer, and 5’-unblocking reagents as in SPOS.

Another key aspect of our LPOS-OSN approach is the adoption of a mono-disperse, three-armed synthetic support – a homostar. This much increases the size of the supported oligo which, along with the branched structure, maximises the rejection by OSN membranes (and therefore the isolated yield) of even the shortest dinucleotidyl homostar (see Figure, central molecular structure). Furthermore, it also assists in HPLC and mass spectral analyses, increasing their sensitivity to incomplete reactions and side reactions.

We envisioned that the same basic steps as are used in the SPOS cycle could be translated to LPOS-OSN (see Figure, top right), in which the separation of large monomer debris (MW ~ 600 g.mol-1) from the growing oligo was seen as the critical process. Although we have not quite achieved the idealised process, with both reactions being conducted outside the organic solvent nanofiltration rig, and residual Dmtr debris (which had quite high membrane rejection) having to be removed by precipitation, we successfully prepared a 9-mer sequence.

Despite some limitations, this pilot project has demonstrated the potential for LPOS-OSN to provide scalable industrial oligo production, validated the basic separation principle, and identified a route to debug this strategy. In the wider context, the success of this project strengthens our assertion that OSN will in future facilitate the scalable liquid phase production of other primary metabolites (for peptides see S. So, L. G. Peeva, E. W. Tate, R. J. Leatherbarrow, A. G. Livingston, Chem. Commun. 2010, 46, 2808-2810), as well as other high value polymers (for mono-disperse PEGs see G. Szekely, M. Schaepertoens, P. R. J. Gaffney, A. G. Livingston, Polymer Chem. 2014, 5, 694-697). We hope ultimately that organic solvent nanofiltration, along with crystallization, distillation, chromatography etc., will become a go-to separation technique for organic synthesis. 

Journal Reference

Chemistry. 2015;21(26):9535-43

Gaffney PR1, Kim JF1, Valtcheva IB1, Williams GD2, Anson MS2, Buswell AM2, Livingston AG3.

[expand title=”Show Affiliations”]
  1. Department of Chemical Engineering, Imperial College, South Kensington Campus, London, SW7 2AZ (UK).
  2. GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY (UK).
  3. Department of Chemical Engineering, Imperial College, South Kensington Campus, London, SW7 2AZ (UK). [email protected].
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Abstract

Due to the discovery of RNAi, oligonucleotides (oligos) have re-emerged as a major pharmaceutical target that may soon be required in ton quantities. However, it is questionable whether solid-phase oligo synthesis (SPOS) methods can provide a scalable synthesis. Liquid-phase oligo synthesis (LPOS) is intrinsically scalable and amenable to standard industrial batch synthesis techniques. However, most reported LPOS strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris. Precipitation can be difficult to develop and control on an industrial scale and, because many precipitations would be required to prepare a therapeutic oligonucleotide, we contend that this approach is not viable for large-scale industrial preparation. We are developing an LPOS synthetic strategy for 2′-methyl RNA phosphorothioate that is more amenable to standard batch production techniques, using organic solvent nanofiltration (OSN) as the critical scalable separation technology. We report the first LPOS-OSN preparation of a 2′-Me RNA phosphorothioate 9-mer, using commercial phosphoramidite monomers, and monitoring all reactions by HPLC, (31)P NMR spectroscopy and MS.

© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Liquid-Phase Synthesis 2'-Methyl-RNA Homostar Support through Organic-Solvent Nanofiltration. Advances In Engineering

 

 

 

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