Date: 21 July 2025 | Category: News
Straightforward, scalable, solution-phase synthesis of peptide bonds in flow.
Authors: Zoe E. Wilson, Enol Lopez, Nils J. Flodén, Charis Watkins, Giulia Bianchini, Steven V. Ley
Dr Zoe Wilson (University of Auckland) and Ley group (University of Cambridge) [1] have used simple solution-phase flow conditions for scalable synthesis of peptides. This was achieved using in-situ activation of the C-terminus as a mixed anhydride, Figure 1. Both the Vapourtec R-Series and E-Series were used effectively, resulting in gram-scale synthesis of the hexapeptide linear precursor of bioactive cyclic peptide Segetalin A.
Figure 1: General scheme showing peptide-coupling. Use of in-line IR monitoring allowed efficient collection of the entire plug and facilitated rapid screening of reaction conditions. NMM is N-methylmorpholine.
Rise of the peptides
Peptides are garnering increasing attention due to their wide-capabilities and rising therapeutic use [2]. In fact, recent analysis shows that amide bond formation is the mostly commonly used reaction in medicinal chemistry programmes [3]. Flow chemistry lends itself well to peptide synthesis through the advent of solid phase peptide synthesis (SPPS) and innovative instrumentation, for example, the Vapourtec Peptide-Builder, the Peptide-Explorer and the Peptide-Scaleup. The Ley group are leaders in this field, through development of multi-step processes using solid-supported reagents and scavengers [4], automated solution phase flow synthesis of secondary amides [5], and using Ghosez’s halogenation reagent to effect coupling of sterically hindered N-methyl amino acids through acid chloride intermediates [6].
This recently published work [1] focuses on the synthesis of secondary amides, which represent the majority of naturally derived amides, directly from amino acids without the requirement for solid supported reagents.
Mixed anhydride-mediated peptide coupling
Usually, formation of mixed anhydrides for peptide coupling is achieved at initial low temperatures, followed by warming of the reaction to ambient temperature, allowing coupling to occur. In this case, implementation of isobutyl chloroformate (IBCF) activated the carboxylic acid and formed the mixed anhydride rapidly at room temperature, then a stream of amine was introduced to affect the requisite bond formation. If protecting groups on the peptide were stable to acidic conditions, in-line purification was used: immobilised sulfonic acid removed any residual base, and immobilised dimethylamine removed any residual IBCF.
This approach was successful in both plug flow and continuous flow modes (< 10 mmol scale), with activation times under 4 minutes and coupling times of 30 – 40 minutes, giving 84 – 98% yields when primary amine-containing amino acids were used. This contrasts with previous couplings in batch mode using IBCF, where considerably lower yields were observed by the group.
The utility of the mixed anhydride approach culminated in the preparation of hexapeptide 1, the linear precursor to the natural product Segetalin A, on 1.2 g scale, Figure 2.
Figure 2: Hexapeptide Trp-Ala-Gly-Val-Pro-Val (1) was prepared in 52% overall yield (based on lowest yielding parallel steps), with no evidence of racemisation. Yields include relevant deprotection steps where required.
Dr Zoe Wilson, lead author, says, “The Vapourtec systems proved to be invaluable in this work. The consistent and accurate flow rates delivered by the R-Series system allowed us to use plug flow to carry out the optimisation of reaction conditions and determination of scope, readily enabling the union of multiple plugs of reagents with minimal waste. The three chemical resistant channels of the E-Series system then allowed us to reliably carry out the developed coupling continuously in a small footprint on scales of up to 10 mmol.”
Summary
The Vapourtec flow systems were effectively utilised to develop and optimise a solution-phase amide coupling that uses a mixed anhydride. A range of amino acids with varied protecting groups were incorporated, and in some cases, products were amenable to in-line purification through scavenging. A Boc/benzyl ester protecting group strategy then allowed preparation of 1.2 g of hexapeptide 1, a precursor to the bioactive natural product segetalin A.
References:
[1] Straightforward, scalable, solution‑phase synthesis of peptide bonds in flow. (Z. E. Wilson, E. Lopez, N. J. Flodén, C. Watkins, G. Bianchini, S. V. Ley, J. Flow Chem., 2025, 15, 67 – 77). https://doi.org/10.1007/s41981-025-00347-2
[2] Therapeutic peptides: current applications and future directions. (L. Wang, N. Wang, W. Zhang et al., Signal Transduct. and Target Ther., 2022, 7, 48). https://doi.org/10.1038/s41392-022-00904-4
[3] Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone? (D. G. Brown, J. Boström, J. Med. Chem., 2016, 59, 10, 4443 – 4458). https://doi.org/10.1021/acs.jmedchem.5b01409
[4] A flow reactor process for the synthesis of peptides utilizing immobilized reagents scavengers and catch and release protocols. (I. R. Baxendale, S. V. Ley, C. D. Smith, G. K. Tranmer, Chem. Commun., 2006, 46, 4835 – 4837). https://doi.org/10.1039/B612197G
[5] Fully Automated Sequence-Specific Synthesis of α-Peptides Using Flow Chemistry. (K. R. Knudsen, M. Ladlow, Z. Bandpey, S. V. Ley, J. Flow Chem., 2014, 4, 1, 18 – 21). https://doi.org/10.1556/JFC-D-13-00033
[6] Synthesis of Natural and Unnatural Cyclooligomeric Depsipeptides Enabled by Flow Chemistry. (D. Lücke, T. Dalton, S. V. Ley, Z. E. Wilson, Chem. Eur. J., 2016, 22, 4206 – 4217). https://doi.org/10.1002/chem.201504457
