Speciality Chemicals Magazine JAN / FEB 2022 - Page 44

Dr Michael Pennington of AmbioPharm and Jonathan Collins of CEM share a case study of microwave-assisted peptide production *

Microwave-assisted SPPS :

From discovery to commercial production

Dr Michael Pennington of AmbioPharm and Jonathan Collins of CEM share a case study of microwave-assisted peptide production *

Peptide science began with Fischer ' s discoveries in 1901 and was modernised with the seminal work of Du Vigneaud chemically synthesising oxytocin in 1953 by liquid phase peptide synthesis . 1 , 2 Perhaps the next greatest innovation was the invention of the solid-phase peptide synthesis ( SPPS ) by Merrifield in 1963 , for the first time making it possible to produce peptides in a simple , quick , and scalable manner . 3 Over the subsequent decades , improvements in protecting groups , acylation rates , side reaction suppression , automation , cleavage , purification and isolation advanced the technology into the 21st century . As of 2021 , there are now more than 75 approved peptide therapeutics . 4 , 5 The vast majority of these are produced using SPPS . Some of the advantages that SPPS capitalises on are speed and scale-up potential , which greatly accelerated the development of many of these peptides . Numerous commercial peptides are produced using optimised Fmoc-tBu methods and a few by Boc-Bzl strategies . However , the efficiency of most of these processes is environmentally unsustainable in terms of hazardous waste generation , atom efficiency and green solvents . 6 In SPPS , all reactions need to proceed cleanly and efficiently . In general , peptide sequences are synthesised smoothly , but some sequences proceed with greater difficulty . In these cases , decreases in reaction rates and the corresponding incomplete couplings have been observed . 7

Often , repeated or prolonged reaction times show little improvement in chain assembly . Optimal deprotections and couplings require the fully solvated peptide – polymer matrix that allows for efficient reagent access . 8 The difficult region for many peptides occurs eight to 20 residues into chain assembly . Mutter et al . proposed that formation of secondary structure and in particular B-sheets that result in poor solvation of the peptide – polymer matrix as a rationale for these problems . 10 As the peptide is assembled step-wise on a solid support , it can aggregate with itself or adjacent chains through backbone hydrogen bonds . Microwave energy affects a fast and efficient way to facilitate both the deprotection and coupling reactions hindered by aggregation . For peptides , the N-terminal amino group and peptide backbone are polar , causing them constantly to attempt to align with the alternating electric field of the microwave . During peptide synthesis , microwaves can disrupt the chain aggregation caused by intra- and interchain association and facilitate access to the solid phase reaction matrix . 11 Microwave methods have been used in organic synthesis to increase reaction rates up to 1,000 times ambient temperature reactions . Yu et al . first reported on an investigation of microwave-assisted synthesis for peptides using a conventional domestic microwave oven in 1992 . 12 Coupling efficiencies were improved two- to fourfold , especially for sterically hindered amino acids , as well as the speed of the reaction ( complete in four minutes ). Lack of temperature control using a domestic microwave oven limited the utility of this type of equipment . The use of a microwave organic synthesiser was reported for making small-hindered peptides in 2002 . 13 Serendipitously , in the early 2000s , CEM , a manufacturer of microwave chemical instrumentation , decided to embark on a novel adaptation of their technology applied to microwave-assisted peptide synthesis . The original Odyssey microwave synthesiser , developed at CEM , established a fully automated microwave system for small-scale synthesis of peptides . Initially , cycles were approximately ten minutes per amino acid , a nearly fivefold increase in synthesis speed of other automated Fmoc-tBu synthesisers . 14 For lead development and standard research peptides , microwave synthesis could be viewed as a major accelerator since a 30-residue peptide could be produced easily in an overnight run . At the inception of this technology , numerous technical problems had to be solved in order for it to be become useful to the