(596e) Iterative Synthesis of Heteropolymers Using Organic Solvent Nanofiltration

Authors: 
Liu, R., Imperial College London
Gaffney, P., Imperial College London
Marchetti, P., Imperial College London
Livingston, A. G., Imperial College London
Schaepertoens, M., Imperial College London
 

Iterative Synthesis of Heteropolymers Using Organic Solvent Nanofiltration

 

Ruiyi Liu, Piers Gaffney, Patrizia Marchetti, Marc Schaepertoens, Ruijiao Dong, Andrew Livingston

 

Barrer Centre, Department of Chemical Engineering, Imperial College London


Sequence-defined heteropolymers underpin much of biology in the forms of proteins/ peptides and oligonucleotides (DNA and RNA). The pharmaceutical industry is investing heavily in the potential of these species to push through the bottleneck in the drug discovery pipeline. Such polymers feature a repeating backbone motif, but each monomer possesses a variable side-chain. It is the order and chemical nature of these side-chain residues that confers the function of the final heteropolymer. Beyond such familiar biomolecules, heteropolymers present opportunities in chemical bar-coding and security, and even for long-term information storage. Looking still further ahead, and taking a cue from Nature, engineered heteropolymers of non-biological monomers might provide uniquely tailored and functional engineering materials, if only they could be flexibly prepared at scale.

Oligonucleotides (oligos) and peptides are the dual test-beds upon which heteropolymer synthesis is founded. They are prepared by iterative synthesis, a process in which one monomer at a time is coupled to the terminus of a growing chain - like differently colored beads threaded onto a string. To ensure error-free sequence synthesis, other reactive groups are masked by protecting groups; permanent protecting groups block the side-chains throughout the synthesis, and a temporary protecting group is removed in the final stage of each chain extension cycle to expose a new reactive chain terminus. To simplify purification after chain extension, and to make the process easily generalizable, both oligos and peptides are widely synthesized on solid phase supports, where excess reagents are simply flushed from a packed bed or column of support beads.

Solid phase synthesis protocols are intrinsically difficult to up-scale. Furthermore, for the successful iterative synthesis of long sequences to succeed, the coupling efficiency must be as near to quantitative as possible, but mass transfer from the bulk liquid to the solid support is the rate-determining step for all but the very slowest coupling reactions. We overcome both these roadblocks to scalable iterative synthesis by performing coupling reactions in the bulk liquid phase and separating reaction debris by organic solvent nanofiltration (OSN).

OSN membranes separate solutes principally on the basis of size selectivity. During liquid phase iterative synthesis the growing heteropolymer is the largest solute present, whilst the monomer debris is the next largest. Therefore, the ability of the membrane to retain the growing heteropolymer, but to permeate the monomer debris, is the critical separation criterion. The molecular weight of the monomer debris from oligo or peptide synthesis falls in the range 400-700 Da. Indeed, it is hard to conceive of functional heteropolymers, either natural or man-made, with smaller monomer debris. Thus the development of OSN membranes with permeation pathways open enough to filter such large species, and yet maintain their physical and chemical integrity during repeated synthesis cycles, was an essential enabling technology for liquid phase heteropolymer synthesis.

We have previously shown that short peptides and oligos could be prepared by means of liquid phase synthesis and purification by single-stage OSN. We have now achieved pharmaceutical length oligo synthesis (20-mer) using a two-stage membrane purification apparatus that takes the cycle yield to near-quantitative recovery of the growing polymer. Despite harsh solvents (DMF and acetonitrile), and both acidic and basic treatments, the OSN membranes maintained constant performance over multiple cycles (> 19).

The same principles have been applied to the synthesis of poly-ethers. Uniform (or monodisperse) poly-ethylene glycols up to 60-mer were constructed by the stepwise concatenation of short units, purifying the growing chain by OSN after each cycle. We are now extending these principles to the synthesis of engineered heteropolymers with a chemically robust poly-ether backbone and functionalised chiral side-chains.