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Streamlined High-Throughput Screening of GLP-1 Analogues using Automated Parallel Peptide Synthesis
Authors:
T.M. Angermeier, C.L. Simpson, S.K. Singh, J.M. Collins
Company / Academic Institution
CEM Corporation, 3100 Smith Farm Road, Matthews, NC 28104, USA
Peptides acting as GLP-1 analogues—such as liraglutide and semaglutide—have gained significant attention for their therapeutic roles in diabetes management and weight loss. As new analogues and applications emerge, the utility of this peptide class continues to expand. High-throughput screening via automated parallel peptide synthesis has become a valuable tool in analogue development, though it has traditionally been limited to shorter sequences (≤20 amino acids).1 In this study, we present an approach for synthesizing and screening a library of GLP-1 analogues using liraglutide, a 31-residue peptide, as the model peptide sequence. Our methodology utilizes 96-well plate synthesis at elevated temperatures on an automated parallel peptide synthesizer, enabling simultaneous synthesis of multiple analogues. This methodology optimizes solvent consumption, total synthesis time, and improves product purity.
Numaswitch ─ A novel biochemical platform for peptide and protein production
Authors:
Dr. Christian Schwarz, Dr. Roland Hecht, Dr. Jitin Bali
Company / Academic Institution
Numaferm GmbH
Peptides and proteins are essential biomolecules with broad applications across various industries, including pharmaceuticals, agriculture, veterinary medicine, generics, and cosmetics. However, the development of efficient production processes at an industrial scale remains challenging, as traditional methods such as chemical synthesis and recombinant expression often fail to meet the growing demand.
To address these challenges, Numaferm has introduced a novel biochemical production platform known as Numaswitch. This platform is designed to produce peptides and proteins of all lengths and functionalities with high yield and quality. The Numaswitch approach involves fusing target peptides or pepteins to Switchtag proteins, which facilitate the production of fusion proteins as inclusion bodies in Escherichia coli cells. Following extraction, Switchtags play a crucial role in promoting the correct refolding of the targets in the presence of Ca²⁺ ions, effectively overcoming the common issue of low refolding efficiencies associated with conventional IB methods. Additionally, the platform utilizes a specially engineered Numacut TEV protease, which enables precise, scarless cleavage of the Switchtag, resulting in the release of target peptides or proteins with a native N-terminus and no additional amino acids.
Numaswitch is a highly reliable and universal platform for peptide and protein production aligned with the principles of green chemistry. It significantly reduces the use of hazardous raw materials, improving the safety of both the production process and the final product. Numaswitch offers a cost-effective, efficient, and sustainable alternative to traditional methods like chemical synthesis and other recombinant expression systems.
ONE-POT SOLUTION-PHASE STRATEGY FOR THE SYNTHESIS OF BICYCLIC PEPTIDES USING THE T3P/[PD(PPH3)4] SYSTEM
Authors:
Abate Luigi, Pasquini Nicolò Maria, Della Valle Alice, Novelli Federica, De Simone Daniele, Cupioli Emilia, Orvieto Federica, Branca Danila
Company / Academic Institution
IRBM S.p.A.
In recent years, macrolactam peptides have gained considerable attention as a novel therapeutic modality, owing to their potential to target challenging biological interfaces. Technologies such as RNA display and Phage Display have played a central role in advancing this field by enabling the identification of high-affinity peptide macrocycle ligands.
Introducing conformational constraints in macrocycle peptides via cross-linking is a valuable strategy for stabilizing the active conformation. These constraints enhance biological activity, receptor affinity, and proteolytic resistance.
In this study, we describe a novel solution-phase method for the synthesis of bicyclic peptides in a one-pot fashion. The approach enables the simultaneous removal of orthogonal side-chain protecting groups and in situ formation of macrolactam cross-links. Several conditions were tested, and the combination of T3P/[Pd(PPh3)4] was identified as the most successful system for promoting deprotection and dual cyclization in a single step.
Greening Peptide Purification Through Replacement of Acetonitrile with Ethanol
Authors:
C.L. Simpson, S.T. Payne, D.J. Cesta, S.K. Singh, J.M. Collins
Company / Academic Institution
CEM Corporation, 3100 Smith Farm Road, Matthews, NC 28104, USA
Increased global demand for peptide therapeutics has put a renewed emphasis on improving the efficiency and sustainability of peptide production and have included efforts to identify greener alternatives to acetonitrile for HPLC purification. Ethanol has been identified as an ideal alternative, based on both a low UV-cutoff and similar separation characteristics, but its higher viscosity has hindered routine use due to higher system backpressures.1 This work describes the use of an optimized approach (using a novel integrated heating system) for elevated temperature HPLC purification that enables routine access to ethanol even with 5 µm particle size columns. Additionally, an assessment of the potential for esterification side-reactions known to occur with methanol was undertaken with ethanol to establish the robustness of the approach.2
Ultra-Efficient Solid-Phase Peptide Synthesis of Pharmaceutical Peptides
Authors:
M.D. Kessler, D.J. Cesta, C.L. Simpson, S.K. Singh, J.M. Collins
Company / Academic Institution
CEM Corporation, 3100 Smith Farm Road, Matthews, NC 28104, USA
The surge in use of GLP-1 agonists and other therapeutic peptides has increased the demand for solid-phase peptide synthesis (SPPS). At the same time, sustainability concerns around the waste and environmental impact of the process have grown. Recently we reported an ultra-efficient methodology for automated SPPS that eliminates up to 95% of the solvent waste from the process by eliminating resin washing steps after each coupling and deprotection.1 One key advantage of this wash-free method, is that it does not require the use of specialty activators or amino acids, and instead focuses on minimizing the usage of reagents that are abundantly available and widely used. This feature makes the methodology easy to implement into existing peptide research and production programs.
No-Wash Peptide Synthesis (UE-SPPS) with TBEC as a Replacement for DIC
Authors:
T.M. Angermeier, C.L. Simpson, S.K. Singh, J.M. Collin
Company / Academic Institution
CEM Corporation, 3100 Smith Farm Road, Matthews, NC 28104, USA
Diisopropylcarbodiimide (DIC) and ethyl (hydroxyimino) cyanoacetate (Oxyma) are widely used for amino acid activation in peptide synthesis. It was recently reported that a reaction between DIC and Oxyma generates hydrogen cyanide (HCN).1,2 1-tert- Butyl-3-ethylcarbodiimide (TBEC) has been reported as a viable substitute for DIC that eliminates the formation of HCN in presence of Oxyma.3,4 In-situ carbodiimide-based coupling has shown minimal epimerization and side product formation at elevated temperature.5 It was of interest to conduct a comparison study on the use of DIC and TBEC under elevated temperature coupling using microwave irradiation with recently described ultra-efficient “no-wash” (UE-SPPS) conditions between all reactions.6
Towards the total synthesis of the bacteriocin Nisin A on solid support
Authors:
Ilia Perov1, Bethan Donnelly1,, Tyler Mallett1, John Vederas1
Company / Academic Institution
1 University of Alberta, Edmonton, Canada
Nisin A is a natural antimicrobial peptide produced by the lactic acid bacterium Lactococcus lactis. Nisin is mainly used as a food preservative, although it has underexplored potential biomedical applications as an alternative to conventional antibiotics and cancer therapeutics.1
Nisin’s low stability under physiological conditions hampers it from becoming a drug – it quickly decomposes in the basic media and has multiple protease cleavage sites.2
Modifications in the nisin structure can address these challenges and potentially enhance its activity against a broader range of bacteria. The chemical synthesis of nisin might be advantageous to recombinant expression as it allows to make various nisin analogs bearing modifications in their backbone and side chains that are not accessible otherwise. After the report by the Shiba group on the total synthesis of nisin in solution3, we are developing a procedure relying on solid-phase peptide synthesis. We investigated strategies to synthesize dehydrated residues and lanthionine rings chemically, the central motifs responsible for nisin biological activity. So far, we have completed the solid phase synthesis of disconnected segments of nisin, which we want to connect using native chemical ligation with non-canonical thiolated amino acids. The chemical synthesis of nisin may serve as a basis for developing and studying new nisin analogues. It may ultimately lead to the discovery of potent and stable nisin variants for use as antibiotics.
1. Field et al. EMS Microbiology Reviews, 2023, 47(3), fuad023.
2. Musiejuk et al. Pharmaceuticals, 2023, 16(8), 1058.
3. Fukase et al. Tetrahedron Letters, 1988, 29 (7), 795–798.