POSTER BOARDS

P1: 01

Towards comprehensive tandem mass spectrometry analysis of peptidomimetics

Erckes Vanessa, Misiek Aleksandra, Streuli Alessandro, Steuer Christian

Company / Academic Institution

ETH Zurich, Institute of Pharmaceutical Sciences, Laboratory of Pharmaceutical Analytics, Zurich, Switzerland

Peptides, whether derived from natural sources or chemically modified for pharmaceutical applications, exhibit immense structural diversity. For structure elucidation and sequence confirmation, the application of tandem mass spectrometry (MS/MS) offers a powerful technique generating sequence-specific fragmentation spectra only requiring low sample amounts. Despite these advantages, the complexity and volume of MS/MS data often hinders complete interpretation while making manual evaluation time-consuming. Commercially available MS/MS data analysis softwares are usually restricted to linear natural amino acid sequences.

To address these limitations, our goal was to develop a program for MS/MS data evaluation capable of analyzing a broad range of modified peptides. As a first step, we built the open-source tool PICKAPEP, which enables the computational representation of diverse peptidomimetic structures, including custom amino acids and multiple state-of-the-art cyclization and modification strategies.1 Secondly, we created an algorithm for theoretical fragment calculation of previously described fragmentation patterns for peptidomimetic structures generated by PICKAPEP combined with automated MS/MS data processing. To validate our approach, we used experimental MS/MS data from exemplary peptidomimetics with head-to-tail or side-chain modifications, such as cyclosporin and semaglutide, respectively. Fragmentation was performed using collision-induced dissociation (CID) and electron transfer dissociation (ETD) on an ion trap mass spectrometer. Applying our workflow for automated data analysis, we successfully conducted high-throughput evaluation and confirmed literature-reported fragmentation patterns. However, we also observed that these known patterns accounted for only a small fraction of the total fragments detected in MS/MS analysis.

Our work enables the computational generation of a broad range of peptidomimetics and accelerates their MS/MS data evaluation compared to manual interpretation. Building on our tool and associated results, future refinements in structure-based fragmentation calculations will hopefully contribute to a more comprehensive MS/MS data analysis for complex peptidomimetics and pave the way for the elucidation of completely unknown peptide-based structures by MS/MS analysis.

1.              Erckes, V.;  Hilleke, M.;  Isert, C.; Steuer, C., PICKAPEP: An application for parameter calculation and visualization of cyclized and modified peptidomimetics. Journal of Peptide Science 2024, 30 (12), e3646.

P1: 02

Linear peptides as IgG-epitope mimetic for allergic immunotherapy against birch pollen

Alessandro Streuli¹, Lara Šošić², Marta Paolucci², Agathe Duda², Klaus Eyer³, Pål Johansen², Christian Steuer¹

Company / Academic Institution

1ETH Zurich, Institute of Pharmaceutical Sciences, Laboratory of Pharmaceutical Analytics, Zurich, Switzerland

2Department of Dermatology, University Hospital Zurich, Zurich, Switzerland

3Department of Biomedicine, Aarhus University, Aarhus, Denmark

Allergies affect nearly one billion people globally and pose a growing health burden. While symptoms are managed with pharmacotherapy, allergen immunotherapy (AIT) remains the only disease-modifying treatment, stimulating allergen-specific immune cells such as B-cells. Allergen-specific IgG antibodies are known to play a protective role in the disease,however, their levels do not consistently correlate with treatment success, implying that isotype, neutralization, and binding affinity may also be critical. Effective desensitization may therefore depend on the formation of allergen-neutralizing IgG antibodies. In this context, short peptides mimicking IgG-binding epitopes may bridge the gap between effective induction of protective IgG responses and improved AIT safety.

We characterized the serological and cellular antibody repertoires of AIT-treated and untreated birch pollen allergic patients and assessed neutralization capacity against Bet v 1, a 159-amino acid protein. Epitope mapping of patient sera revealed four main IgG-binding regions. Six corresponding linear peptides (25 - 34 amino acids) were synthesized viaFmoc solid-phase peptide synthesis on Rink amide resin with N-terminal acetylation. Peptides were purified (>95% purity), and TFA^- exchanged for Cl^- as counterion. Characterization was performed by LC-MSⁿ for sequence confirmation, LC-UV for purity and ¹⁹F-NMR for residual TFA content. Peptide secondary structure was analyzed by CD spectroscopy, and serum stability assessed in mouse and human serum using LC-MS. In vitro, allergenicity was assessed using Bet v 1-primed rat basophile cells and patient leukocytes, while in vivo immunogenicity was tested in female CH3 mice. 

All peptides mimicking the newly determined IgG-epitopes showed minimal to no allergenicity in degranulation assays, of which two induced Bet v 1-specific seroconversion in mice. No correlation could be established between the induced immune response and the determined secondary structure or serum stability. These results provide a first proof of concept combining epitope mapping with synthetic peptides to develop safer, novel AIT approaches.

P1: 03

From HPLC to FPLC and back again- Challenges, pitfalls and opportunities in purification and purity determination of synthetic peptides

Alessandro Streuli¹, Vanessa Erckes¹, Brunello Nardone², Vincent Bedard², Francois Beland², Christian Steuer¹

Company / Academic Institution

¹ETH Zurich, Institute of Pharmaceutical Sciences, Laboratory of Pharmaceutical Analytics, Zurich, Switzerland

² SiliCycle Inc., 2500, Boulevard du parc technologique, Quebec City, Canada

Synthetic peptides are increasingly utilized in therapeutic development due to their ability to target hard-to-access protein–protein interactions. However, during their production via solid-phase peptide synthesis (SPPS), various unwanted reactions can occur. These include deamidation, isoaspartate formation, truncations, and amino acid residue misincorporation - each modification potentially arising at specific positions along the growing peptide chain. Although these modifications are often minor, they can significantly impact peptide function and are notoriously difficult to detect due to their subtle mass and physicochemical differences.

Accurate characterization and quality control of synthetic peptides therefore depend heavily on the choice of the chromatographic system. In this study, we systematically evaluated three reversed-phase (RP) columns - SiliaChrom® InnoPep, SiliaChrom® ResiPure™ Advanced C18 and Waters® SunFire C18 - as both scouting and quality control tools, along with key chromatographic parameters including column temperature, gradient steepness, and organic modifier. A library of 38 model peptides was synthesized via SPPS to simulate three critical modifications: amide-acid variants, residue misincorporation, and isoaspartate formation. First, we addressed the often-overlooked challenge of transferring HPLC-based purity methods to preparative flash chromatography (FPLC). By incorporating key parameters from the analytical system - such as column and dwell volume – we reduced prediction deviations of product elution in FPLC from 17% to under 3% enabling first-pass purifcation with >90% purity in all cases. Secondly, we assessed the separation efficacy of the RP scouting columns for detecting closely related impurities.

Our findings provide a practical framework for method transfers from HPLC to FPLC without additional experiments, allowing effective first-pass purifcation. Gradient-steepness and the choice of the modifier were identified as the most impactful factors on separation efficacy. Notably, both scouting columns with 10 µm particle size performed comparably to the established reference column with 3 µm particle size routinely used in our laboratory.

P1: 04

Next-Gen Solutions in Silica-Based Peptide Synthesis and Downstream Processing

Brunello Nardone, Mathieu Simard, Steeves Potvin, Vincent Bédard, Georges Thibaut Koumba, Denis Boudriau, François Béland

Company / Academic Institution

SiliCycle

Polymeric resins have long been the industry standard for solid-phase peptide synthesis (SPPS) due to their ability to swell in solvents, mechanical strength, acid resistance (e.g., TFA), and scalability [1,2]. However, these materials contribute significantly to environmental impact, with peptides showing an average process mass intensity (PMI) of around 13,000 kg of waste per kg of active pharmaceutical ingredient (API). For comparison, small molecule syntheses typically fall between 168 and 308 kg/kg API [3,4].

To address this challenge, we developed SiPPS, a novel silica-based resin with minimal swelling designed to reduce solvent usage and overall waste while maintaining high performance in peptide synthesis. We will share successful examples of SiPPS used in synthesizing reference peptides and introduce the latest updates in our proprietary reverse-phase media for peptide purification. Together, these technologies offer a more sustainable approach to peptide manufacturing without compromising quality or scalability.

P1: 05

Fine-tuning oncogenic signaling: Regulation of β-catenin by phosphorylation

Tobias Gökler, Anne C. Conibear

Company / Academic Institution

Technical University of Vienna

The β-catenin protein is a master regulator in the Wnt signaling pathway that drives many biological processes such as cellular proliferation and differentiation. This role requires strict control of its transcriptional potential, translocation, and subsequent degradation, which are orchestrated by post-translational modifications (PTMs). These PTMs are primarily located at the intrinsically disordered termini of β-catenin. Herein, the four consecutive phosphorylation events (S33, S37, T41, S45) at the N-terminus trigger E3 ligase recruitment, causing ubiquitination and subsequent proteasomal degradation. This process is key for maintaining cellular homeostasis and ultimately suppresses the oncogenic potential of β-catenin. The degradation mechanism on a molecular level is still highly debated, moreover, the impact of phosphorylation on the protein structure has been neglected due to the proposed disorder of the β-catenin N-terminus. The highly dynamic nature of disordered protein regions, especially if post-translationally modified, challenges structural predictions. In this study, we show the chemical synthesis of site-specifically phosphorylated variants of the β-catenin N-terminus involved in proteasomal degradation. The combined experimental spectroscopic methods (circular dichroism and NMR spectroscopy) with structure prediction tools allow us to investigate underlying degradation mechanisms based on local conformational changes and dynamic transitions induced by phosphorylation. Changes in the protein structure can reveal new binding sites for previously unknown interaction partners, or even define target proteins for novel therapeutical approaches.

P1: 06

Alchemist – Powering StreaMLine to Accelerate Peptide Drug Discovery

Martin Kræmer, Ulrike Leurs, Kristoffer Rigbolt, Louise S. Dalbøge, and Jens Christian Nielsen.

Company / Academic Institution

Gubra

StreaMLine is Gubra´s innovative platform for peptide drug discovery that greatly shortens the time from initial hit to clinical drug candidate. By synthesis, screening, and AI-assisted data analysis of thousands of peptides, the iterative process of peptide optimization is shortened considerably. Liquid chromatography coupled to mass spectrometry (LC-MS) is a cornerstone in this process, but the extensive hands-on time for processing and analysis of LC-MS data remains a challenge. To alleviate this and enable high-throughput profiling of peptide drug candidates by LC-MS, we have developed the Alchemist software.

Alchemist fully automates peak detection, quantitation and data processing of high-resolution LC-MS data, enabling analysis of thousands of peptide samples to reliably detect synthesis byproducts, as well as degradation products from accelerated stability experiments.

   By leveraging machine learning, including random forest models, on large sets of Alchemist-identified synthesis byproducts, actionable insights are obtained and challenging coupling reactions identified. This accelerates data validation and quality assurance, providing medicinal chemists with a comprehensive, rapid, and unbiased analysis of each synthesis.

From a typical parallel synthesis of 192 peptides, around 2.000.000 MS1 and MS2 scans are condensed to 200.000 LC-MS features. Alchemist processing further compresses this list to ~6000 quantified features and matches commonly detected synthesis byproducts (SBPs) such as missed couplings, tert-butylation, and oxidation. In addition, the data is screened for unexpected side reactions by aggregating SBPs across all peptides to a list of approximately 20 side reactions.

Critically, Alchemist integrates seamlessly with our modelling workflows, ensuring that only robust, high-quality data informs compound progression. With Alchemist at the heart of StreaMLine, we have turned the analytical bottleneck into a powerful driver of accelerated drug discovery, empowering chemists with the data they need to optimize syntheses and advance clinical candidates faster.

P1: 07

Morpholino nucleo- β-amino acids as building blocks for the preparation of functional Nucleo-peptides with protein-targeting capability

Michela Pesenti, Enrico Mario Alessandro Fassi, Kaliroi Peqini, Giulia Castiglioni, Marco Albani, Giovanni Grazioso, Sara Pellegrino.

Company / Academic Institution

Università degli Studi di Milano

The design and synthesis of nucleic acid analogues has emerged as an expanding field, with applications in chemical biology and gene therapy. In this context, nucleo-peptides represent a versatile class of analogues compared to traditional oligonucleotides, peptide nucleic acids (PNAs), or phosphorodiamidate morpholino oligomers (PMOs). They offer several advantages, e.g. greater synthetic flexibility and stability, and the capacity to merge nucleic acid recognition and peptide-like functionality1. Recently, we developed morpholino nucleo- β-amino acids as innovative building blocks for nucleo-peptides2. Their distinctive structure, i.e. a morpholine ring fused to a β-amino acid backbone, confers remarkable chemical and conformational stability. Moreover, these systems allow for tunable architectures and side-chain modifications, thereby unveiling new possibilities for targeted molecular interactions and functional versatility. 

In this work, we have optimised the synthesis of morpholino β-amino acids with the aim of improving yields and addressing the challenge of poor solubility that is associated with these structures. This has been achieved by employing fully protected nucleosides, specifically adenosine, cytidine, guanosine and thymidine, as starting materials. The morpholine ring was formed through the Summerton “one-pot” oxidative ring-opening and reductive amination, starting from nucleosides in which the secondary hydroxyl group of the sugar moiety was protected as a silyl ether. In the case of adenine, guanine, and cytidine, the exocyclic amine was also protected. This strategy enabled efficient and selective ring closure under mild conditions, facilitating the preparation of high-purity monomers. The resulting Fmoc-derivatives morpholino β-amino acids were used in a tailored solid-phase peptide synthesis protocol for the preparation of novel nucleo-peptides designed to interact selectively with HMGB1, a nuclear protein involved in chromatin organization and inflammatory processes3.

1. Roviello et al. (2010), Amino Acids, 39(1), 45–57. 

2. Bucci et al. (2020), Scientific Reports, 10(1), 19331.

3. Ugrinova et al. (2017), Advances in Protein Chemistry and Structural Biology 107:37-76

P1: 08

Streamlined High-Throughput Screening of GLP-1 Analogues using Automated Parallel Peptide Synthesis

 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.

P1: 09

No-Wash Peptide Synthesis (UE-SPPS) with TBEC as a Replacement for DIC

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

P1: 10

High-Throughput Peptide Library: Rapid, Purification-Free Synthesis of Linear and Cyclic Peptides

Yuyan Chen, Fengping Xiao

Company / Academic Institution

BioDuro

Peptide therapeutics are a rapidly expanding drug class, yet traditional library synthesis methods are slow, costly, and hampered by purification bottlenecks. This significantly hinders early drug discovery. Therefore, innovative and efficient methodologies are critically needed to rapidly and cost-effectively generate and screen diverse peptide libraries, accelerating the identification of promising therapeutic candidates.

This High-Throughput Peptide Library Platform enables up to 45-fold increase in speed compared to traditional peptide synthesis methods. Furthermore, the platform reduces the cost per peptide by over 70%.

The platform exhibits high success rates, achieving ~95% success for linear peptide synthesis and ~80% for cyclic peptide synthesis at a 384-peptide scale.

The generated crude peptide libraries are validated for extensive direct compatibility with a range of bioassays.

Our High-Throughput Peptide Library Platform leverages an advanced methodology centered on optimized Solid-Phase Peptide Synthesis (SPPS). This includes rapid synthesis protocols performed in pre-activated resin plates, significantly reducing synthesis time.

The platform employs a dual-use synthetic strategy, enabling the generation of both linear peptides via standard amide coupling and cyclic peptides through specialized cyclization techniques (thioether, disulfide, lactam).

The platform provides LCMS and reliable peptide concentration data. Conduct qualitative and rough quantitative analyses.

The platform can achieve crude peptide purity of 70~80% UV purity at 214nm checking peptide content by BCA or Bradford method, enabling their direct use in biological assays without additional purification。

This direct-to-biology approach supports diverse binding assays (SPR, radioisotope binding) and functional assays (fluorescence, absorbance, luminescence, FP, HTRF, FLIPR, etc.)

This novel High-Throughput Peptide Library Platform represents a significant advancement, effectively addressing critical bottlenecks in peptide drug discovery.

The rapid, cost-effective screening approach, which requires no further purifications, streamlines workflows and accelerates the identification of bioactive peptides. Critically, this platform bridges the gap between combinatorial synthesis and functional validation, reducing discovery timelines from months to weeks.

P1: 11

Comparative Analysis of Cyclolinopeptide Content in Various Flaxseed Cultivars

Oyunchimeg Sharav, and Martin JT Reaney

Department of Food and Bioproduct Sciences, University of Saskatchewan

Company / Academic Institution

University of Saskatchewan, SK, Canada

Flaxseed oil, a rich source of α-linolenic acid (up to 55%), reached a global production volume of 3.4 million tons in 2024, with Canada contributing approximately 40%. This study investigates the distribution of linusorbs (LOs)—cyclic peptides derived from flaxseed oil—across oil, foot, and meal fractions from 13 Canadian flax cultivars, using environmentally friendly solvents. Based on LO profiles, cultivars were classified into five distinct groups. Groups I and II exhibited similar ratios of tryptophan-containing LOs but differed in total LO content, while Groups III to V displayed unique compositional patterns.

LO concentrations varied significantly among fractions: the foot contained 1–4 times more LOs than the oil, while the meal consistently showed the lowest levels. Correlation analysis indicated a weak to moderate positive relationship between α-linolenic acid content and non-tryptophan LOs. These findings highlight the potential for targeted breeding and processing strategies to enhance the functional properties of flaxseed-derived products.

P1: 12

Benchtop 19F NMR for Detection and Analysis of Disease Biomarkers

Adam Schofield, Matthew Reed, Dave Ellis, Chris R. Coxon

Company / Academic Institution

The University of Edinburgh

Biological samples such as blood, urine, and cerebrospinal fluid contain numerous 'biomarkers’, which may indicate health and disease. Detecting multiple biomarkers in the same test using established methods can be challenging. We propose utilising fluorine nuclear magnetic resonance (19F NMR) spectroscopy as a simple, rapid, non-invasive and cost-effective simultaneous test for multiple disease biomarkers. A key advantage of 19F NMR is that bodily fluids do not contain organic fluorine atoms, resulting in no background signal. Thus, only artificially introduced, biomarker-specific fluorine-containing probes are detectable, providing their own unique signals that allow the simultaneous detection and quantification of multiple biomarkers.

Sepsis is a critical health emergency that results in 11 million deaths globally each year. Early and accurate diagnosis can prevent unnecessary death; however, it is often difficult to diagnose due to the similarity of symptoms with other diseases. Here, we discuss the application of our technology by showcasing the development of a fluorine-containing probe designed to detect dipeptidyl peptidase III (DPP3), a zinc-based metallopeptidase associated with septic shock. High blood DPP3 levels indicate a poor prognosis.

Using the same technology, we aim to target multiple diseases, such as non-small cell lung cancer (NSCLC), which shows that high levels of aminopeptidase N in NSCLC is linked to poor prognosis.

P1: 13

Hot Spot-Enriched Library: A Targeted Approach for Inhibiting Protein-Protein Interactions

Shirley Meyer, Alexandre Murza, Michel Grandbois, Pierre-Luc Boudreault

Company / Academic Institution

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada

Protein-protein interactions (PPIs) play a critical role in many biological processes, including cell signaling and immune responses, making them promising therapeutic targets for applications such as cancer and neurodegenerative diseases. With approximately 650,000 identified PPIs compared to only 20,000 coding genes, modulating these interactions offers significant therapeutic potential. However, PPI interfaces, often flat and lacking cavities, pose challenges for the development of effective modulators. The identification of "hot spots"—residues that significantly contribute to binding energy—opens new avenues for developing PPI modulators.

This project aims to develop a "hot spot" enriched library to maximize the chances of discovering modulators capable of inhibiting PPIs compared to commercial library. Our approach consists of three main steps: (1) the design and synthesis of small molecules, (2) the design and synthesis of peptide macrocycles, and (3) the biological evaluation of these compounds. Small molecules are synthesized using combinatorial chemistry, while peptide macrocycles are synthesized on solid phase. Finally, compound activity will be evaluated using a bioluminescence-based technology that enables real-time monitoring of protein interactions in living cells.

Preliminary results from the small molecule library reveal a high degree of structural diversity and molecular descriptors that align with those of reported PPI inhibitors. In particular, TPSA (≈85), cLogP (≈5), % of aromatic atoms (≈25%), and number of rings (≈4) are almost identical. The Glo (0.24) falls somewhere between the globularity of FDA-approved drugs (0.14) and known PPI modulators (0.34). This highlights the library's potential to generate effective inhibitors in high-throughput screenings.

By incorporating critical residues from "hot spots," this library design offers a promising alternative to traditional libraries optimized for conventional targets. Moreover, we anticipate these libraries to give superior hit rates compared to standard high-throughput screening libraries on PPI targets (usually below 0.01%) based on the above design elements and a unique 3D conformational approach.

P1: 14

Molecular basis and regulation of Ankyrin-mediated ligand-recruitment to neuronal axonal versus synaptic sites

Omkar Rajendra Valanju, Kendall Dean, Vinzenz Gerstmeir, Kristin Johnson, Lars Schönemann, Sonja Kalcher, Paul Jenkins, Hans Michael Maric

Company / Academic Institution

Rudolf Virchow Center - Center for Integrative and Translational Bioimaging, University of Würzburg, Germany

Neuronal excitability and the initiation of action potential depends on specialized neuronal structures the axon initial segment (AIS) and dendritic synapses. Ankyrin G and B are critical determinants for the formation and organisation of these structures by promoting transport and stabilisation. Alteration of their function has been linked to numerous neurodevelopmental disorders including autism spectrum disorder. Numerous cell adhesion molecules and ion channels have been shown to engage via short linear motifs into direct interactions with two distinct binding sites at the N-terminal domain of Ankyrin B and G. However, the molecular details in terms of Post Translational Modifications remains to be resolved. 

Our previous studies highlight the power of peptide microarrays studying neuronal scaffold interactions and the use of the gained insights for the development of probes and inhibitor leads with unique specificity profiles.[1][2] Using same method we systematically screened putative Ankyrin G and B interaction partners. This was followed deep-mutational scans probing all possible point mutants revealing ligand-wide binding requirements. The data redefines the core binding motives of previously reported binders, rationalizes the mode of action of disease-causing mutations and identify sequences expected to exert a dominant negative effect on specific recruitment sites. Follow-up analysis including post-translational modifications pinpointed dominant effects of conserved post translational modifications underlying the scaffolding. Analysis of the corresponding mutations in neurons highlights the fundamental importance of the identified regulation sites and provide into the pathology underlying previously reported and related disease mutations.[3]



[1] Maric et al. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.

[2] Khayenko et al. A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity 

[3] Valanju at al. Molecular basis and regulation of Ankyrin-mediated ligand-recruitment to neuronal axonal versus synaptic sites. In preparation

P1: 15

Tag-Assisted Peptide Synthesis (TAPS) – Development of a continuous liquid-phase process

Dr. Stefan Lukas Peters, Dr. Tim Kramer, Dr. Mareike Helmdach, Dr. Fabian Hogenkamp, Dr. Andreas Hans Heindl

Company / Academic Institution

CordenPharma International

Solid Phase Peptide Synthesis (SPPS), invented by Merrifield in 1963, is the current state of the art for manufacturing of peptide therapeutics. The peptide chain is built up by iterative coupling and deprotection steps using the Fmoc-protected amino acids on solid-support, enabling the removal of reagents and byproducts by washing the resin with excess solvent. However, in the context of green chemistry the enormous amount of waste and the high consumption of hazardous solvents, such as DMF, NMP and DCM, leads to a low PMI of SPPS and demands a more sustainable alternative. In contrast, Tag-Assisted Peptide Synthesis (TAPS) represents an approach which is compatible with these requirements, since the reactions are carried out in solution using soluble tags as anchor molecules, which are attached to the growing peptide chain. TAPS can be carried out using less solvent and excess of reagents, since the reactions are similar to classic solution-phase chemistry. Previous examples of this technology mainly used precipitation or nanofiltration to purify the tagged peptide after each coupling/deprotection cycle, leading to a discontinuous labour-intensive process. Attempts to design a more continuous process, using liquid-liquid extraction for intermediate purification, however used halogenated solvents[6] or DMF mixtures as process solvents.

In our continuous TAPS process the TAG peptide is maintained in the organic phase (green solvent mixture) for coupling/deprotection cycles, while excess reagents and byproducts are removed by simple aqueous wash steps. The amount of organic solvent used in this process could be reduced by 90% compared to SPPS, while the excess of amino acids and coupling reagents could also be decreased by 50%, leading to a comparable quality of the target molecule. Therefore, TAPS can be considered as a highly efficient and sustainable alternative to classic SPPS.

P1: 16

Development of new chiral contrast agents by solid-phase synthesis

Rosaria Schettini1*, Matteo Magliaro1, Irene Izzo1, Roberta Napolitano2, Francesco De Riccardis1

1 - (University of Salerno, Department of Chemistry and Biology “A. Zambelli”, Via Giovanni Paolo II, 132, 84084, Fisciano (SA) Italy)

2 – (Bracco Research Center, Bracco Imaging S.p.A, 10010 – Colleretto Giacosa (TO) Italy)

Company / Academic Institution

UNIVERSITY OF SALERNO/DEPARTMENT OF CHEMISTRY AND BIOLOGY "A. ZAMBELLI"

Nowaday, magnetic resonance imaging (MRI) is a crucial diagnostic technique in clinical radiology and has become a highly significant tool for biological research. [1]

Standard solution phase chemistry is the most often used technique for MRI contrast agent production. However, the need for newly designed CAs that may be used to investigate certain biological processes is growing as the science of molecular imaging continues to advance. [2]

Therefore, considering the ‘gold standard’ Gd3+-based contrast agents, new chelators able to improve kinetic inertness, high relaxivity values and thermodynamic stability have been flourished over the last years. [3]

The long-term safety profile of GBCAs has drawn more attention recently, especially in relation to gadolinium-induced nephrogenic systemic fibrosis (NSF). [4]

Starting from 2019, using a modular solid-phase synthetic approach it has been possible to tune the design of various azamacrocycles bearing different side chains, cavity sizes and chiral pendant arms, exploiting the peptoid chemistry. [5]

Additionally, it has been shown by researchers that chiral DOTA chelators could provide a useful platform for the creation of new contrast agents with high stability and relaxivity. [6]

Considering also the concrete possibility to introduce various stereocenters on the cyclic peptoid backbone, in this communication, the design, the synthesis and the complexation properties of new potential magnetic resonance imaging probes will be elucidated.

[1] J-H. Tang et al. Dalton Trans., 2025, 54, 6741.

[2] J. Kaur et al. ChemMedChem 2024, 19, e202300521.

[3] P. Caravan et al. Chem. Rev. 2019, 119, 957.

[4] J. Endrikat et al. Investig. Radiol. 2018, 53, 541.

[5] F. De Riccardis et al. Org. Lett. 2019, 21, 7365; (b) F. De Riccardis et al. Org. Biomol. Chem. 2021, 19, 7420.

[6] P. Caravan, G.-L. Law et al. Nat. Commun 2018, 9, 857.

P1: 19

Naturally inspired peptaibols as potential 5-lipoxygenase inhibitors

Renato B. Pereira, Letícia Pinto, Marie Bisback, Maria João Araújo, Paula Gomes

Company / Academic Institution

LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, P-4169-007 Porto, Portugal

Inflammation is a protective response to infection or injury, but if left untreated, it can lead to chronic tissue damage. Non‑steroidal anti‑inflammatory drugs (NSAIDs) and selective cyclooxygenase‑2 inhibitors (COXIBs) are commonly used treatments; however, their long-term use is associated with gastrointestinal and cardiovascular side effects, respectively. Moreover, complete inhibition of the COX1/COX2 pathways can redirect arachidonic acid toward the 5-lipoxygenase (5-LOX) pathway, potentially increasing leukotriene production, which are pro-inflammatory mediators involved in the pathogenesis of various chronic conditions. Therefore, directly targeting 5-LOX offers a promising alternative strategy.

Peptaibols, a distinctive family of protease‑resistant peptides rich in hydrophobic residues, have been relatively poorly explored for anti‑inflammatory activity. In this study, a library of over 30 natural and naturally inspired peptaibols was screened at 100 µM for their ability to inhibit LOX in a cell-free assay. In general, natural peptaibols exhibited weak LOX inhibitory activity, reducing enzyme activity to ca. 70%. Among the semi-synthetic derivatives, those incorporating a Lys-Lys-Lys-βAla motif at the N-terminus showed enhanced LOX inhibition. These modifications significantly improved not only the inhibitory activity but also the water solubility of the peptides. Remarkably, the addition of caprylic acid to the modified N-terminal motif led to a further increase in LOX inhibition, with most derivatives achieving near-complete enzyme inhibition. For the most promising naturally inspired peptaibols, dose–response experiments were conducted to determine their IC₅₀ values, using zileuton, the only FDA-approved 5-LOX inhibitor, as a benchmark. In the model system employed, the optimized peptaibols displayed IC₅₀ values that were ca. 5-fold lower than that of zileuton, suggesting that these molecules hold strong potential as leads for the development of novel anti-inflammatory agents.

This research was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES), through project KIWIBOL (PTDC/ASP-PLA/2440/2021), and received support from project UID/50006 -LAQV/REQUIMTE.

P1: 20

Molecular design and synthesis of self-assembling peptide nanostructures responsive to prostate-specific antigen (PSA) for triggered drug delivery in prostate cancer

Bárbara Matos, Helena S. Azevedo

Company / Academic Institution

INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen, 208, Porto, 4200-135, Portugal

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, Porto, 4200-135, Portugal

Despite the approval of multiple new drugs in recent years, prostate cancer (PCa) remains a global health concern, accentuating the need for innovative therapeutic approaches. Peptides have emerged as promising therapeutics for cancer treatment, with more than 15 peptides available in the market and many more currently in preclinical and clinical evaluation. Peptides have been recognized as multifunctional therapeutics in cancer treatment, serving as cytotoxic agents or targeted drug delivery vehicles. In the context of drug delivery, harnessing the activity of overexpressed enzymes within the tumor microenvironment presents a promising strategy for enabling the precise delivery of anticancer agents. In PCa, several enzymes have been explored for designing enzyme-responsive peptide-drug conjugates. Notably, prostate-specific antigen (PSA) has garnered significant attention due to its exclusive activity within the PCa microenvironment.

By designing a peptide amphiphile (PA) that includes a PSA-cleavable sequence, we combine the enzyme-cleavability of the peptides with their self-assembling properties. This self-assembling peptide nanobiomaterial represents an innovative platform to improve the therapeutic efficacy and reduce the systemic toxicity of different anticancer agents by exploring the prostate tumor-specific activity of PSA. The designed PA consists of a hydrophobic alkyl tail, a short β-sheet-forming sequence (AAAA), and a PSA-cleavable peptide (RSSYRSL), previously described in the literature. The synthesis of the proposed PA, and PA variations with tweaked molecular structure (e.g. PAs with a scrambled proteolytic substrate for PSA) were synthesized using microwave-assisted solid-phase peptide synthesis. Some PAs have shown poor water solubility, hampering their purification by conventional HPLC methods and requiring molecular redesign. The self-assembly ability of synthesized PAs, their cleavability by PSA and selective delivery of anticancer agents in PCa cells are under evaluation. The findings presented in this communication underscore the potential of leveraging PSA activity and peptide self-assembly to enhance the targeted delivery of anticancer agents into prostate tumors.

P1: 21

Synthesis of Tritium and Carbon-14 Labelled Peptides for Metabolism and Distribution Studies

Thomas Gregson

Company / Academic Institution

Pharmaron

Current strategies for the Tritium and Carbon-14 labelling of peptides are described with some examples of compounds synthesized by Pharmaron. Such isotopologues prove useful as tools in the exploration of in-vivo metabolism and distribution.

P1: 22

Candida bromodomains recognise histone tail peptides bearing diverse acylation marks.

Yordan HAYAT, Zeynep KANLIDERE, Carlo PETOSA

Company / Academic Institution

Institut Biologie Structurale

Fungal diseases affect over a billion people and kill more than a million every year. The limited repertoire of antifungal drugs and alarming rise of drug-resistant strains pose an urgent need to find new therapeutic strategies. Bromodomains play a key role in gene transcription as epigenetic readers of histones bearing post translational modifications (PTMs). Bromodomains have a hydrophobic pocket that recognises acetylated lysines on histone tails. Our work focuses on finding an inhibitor molecule against bromodomains of the fungal protein Bdf1 which is essential for fungal growth. Recent findings on histone tails revealed PTMs comprising novel lysine acylation (e.g., butyryl, crotonyl) marks of varying size. Data from our lab has revealed structural differences between fungal bromodomains and their mammalian homologues. These findings made us ask whether fungal bromodomains could interact with bulkier PTMs. Our binding data have so far confirmed this hypothesis. Future work aimed at understanding the physiological relevance of this ligand selectivity will help validate bromodomain inhibition as a potential antifungal therapeutic strategy.

P1: 23

TFA-free cleavage/deprotection in Fmoc solid-phase peptide synthesis.

Re-investigation of formic acid.

Sergey Burov; Co-authors: Maria Leko, Polina Filippova, Andrey Murko, Sergey Anisimov

Company / Academic Institution

1 Cytomed JSC, Orlovo-Denisovsky pr. 14A, 197375, St-Petersburg, Russia

2 Luxembourg Bio Technologies Ltd., Nes Ziona 7403631, Israel

Solid-phase peptide synthesis is one of the main techniques for the production of active ingredients applied in the pharmaceutical and cosmetic industry. However, the synthetic process is associated with the application of trifluoroacetic acid (TFA) in the course of peptide deprotection and cleavage from the polymeric support. It creates a potential danger for the personnel and environment and additional costs due to the necessary safety measures, utilization of waste and conversion of peptide trifluoroacetate into acetate or hydrochloride.

Herein, we describe TFA-free cleavage/deprotection with formic acid in combination with protic or Lewis acid* providing the final product in pharmaceutically acceptable salt form. The reaction conditions were optimized ensuring the removal of BOC; tBu; OtBu; Trt and Pbf protecting groups from amino acid derivatives and model peptides. The investigation of peptide release from Rink Amide AM and Wang resin revealed that the purity and the yield of the crude product can be at least comparable with that of the standard TFA procedure. While the deprotection process is accompanied with partial Ser, Thr, Tyr and Trp formylation, the desired product can be easily recovered using the treatment with nucleophilic agents. The deprotection protocol implies the application of inexpensive reagents and permits to solve the problem of toxic waste utilization. The practical utility of the suggested approach is exemplified by the synthesis of a number of peptides both for pharmaceutical and cosmetic applications.

* Proprietary technology

P1: 24

Title to follow...

Seokin Kim

Company / Academic Institution

BeadTech. Inc. ANSAN, SOUTH KOREA

P1: 25

Chemical synthesis and evaluation of insulins derived from a venom insulin of the cone snail Conus geographus 

Nicholas Schumann, Alan Blakely, Amber Vogel, Reza Moosavi, Zildj Acyatan and Helena Safavi-Hemami 

Company / Academic Institution

The University of Utah, Department of Biochemistry 

Cone snail venoms represent a valuable source of bioactive compounds for the development of therapeutics. The venom of Conus geographus, a fish-hunting cone snail, was recently discovered to contain insulins (Con-Ins). These insulins are hypothesized to induce hypoglycemic shock in their prey, thereby facilitating prey capture. Unlike human insulin, Con-Ins don’t dimerize and are therefore very fast acting when injected in an in vivo model. Furthermore, it is hypothesized that these insulins would have very fast clearance from the body, making them a valuable platform for the development of rapid onset/offset insulins. Unfortunately, the exploration of such analogues by SAR studies has been hampered and underdeveloped due to the difficulty in synthesizing insulins. 

Herein we describe an optimized synthetic protocol for the synthesis of Con-Ins that allows for scale-up synthesis. From this, a cryo-EM structure of one of the insulins synthesized, Con-Ins G1, a native insulin from the venom of Conus geographus, was obtained. Using the cryo-EM structure in conjunction with molecular docking simulations allowed for the design of a number of analogues of Con-Ins G1. These were synthesized and tested in a human insulin receptor activation assay and a number showed enhanced activation of the receptor relative to Con-Ins G1, with potencies approaching that of human insulin. 

P1: 26

Side-Chain Stapling of Alanine-Rich Helical Peptides for Application in Chiral Induced Spin Selectivity

Aleksandr Kazimir, Tom Seeger, Lokesh Rasabathina, Georgeta Salvan, Olav Hellwig, Christina Lamers

Company / Academic Institution

Leipzig University/ Institute for Drug Discovery

Chiral-induced spin selectivity (CISS) describes electron spin polarization during transfer through chiral systems, a phenomenon particularly notable in alanine-rich α-helical peptides such as C[AAAAK]x (x = 3 or 7).1,2,3 In this work, we investigate how synthetic modifications—specifically side-chain stapling—impact the structural and electronic properties of these peptides, both central to the CISS effect.

We first optimized the synthesis of these hydrophobic α-helical alanine-rich peptides. By shifting from conventional single-residue coupling at room temperature (yield: 10%) to a blockwise assembly of [AAAAK] units on Wang resin with microwave-assisted DIC/Oxyma coupling and 4-methylpiperidine deprotection, followed by coupling of a single cysteine in the final step, we achieved a substantial increase in yield to 80% and enhanced product purity.

To further modulate the peptides' properties, we introduced a 2,2’-bipyridine (bpy) staple between i and i+7 positions by coupling 4,4’-dicarboxy-2,2’-bipyridine to diaminopimelic acid (Dap) residues during solid-phase synthesis. UV-vis spectroscopy of the stapled analogue revealed an additional absorption peak, indicating increased charge transfer attributed to the bpy linker compared to the native peptide. Circular dichroism (CD) spectra demonstrated that the stapled peptide retains the characteristic α-helicity of the parent sequence, with the additional feature potentially reflecting increased structural robustness.

Overall, our results show that side-chain stapling is an effective strategy for fine-tuning both structural and electronic properties of alanine-rich peptides relevant to CISS. These insights lay the groundwork for the design of robust chiral peptide systems for spin-selective applications and inform future directions in spin electronics based on helical peptides.

1. Moharana et al. 2025

2. Ha Nguyen et al. 2024

3. Ha Nguyen et al. 2023

P2: 01

Exploring modifications to islet amyloid polypeptide to improve its antibacterial properties.

Pei-Ya Shan; Yun-Ju Lai, and Ling-Hsien Tu

Company / Academic Institution

National Taiwan Normal University

Human islet amyloid polypeptide (IAPP), a 37-residue peptide co-secreted with insulin, forms fibrillar deposits that contribute to pancreatic β-cell death. Previous studies demonstrated IAPP's antimicrobial activity against Staphylococcus aureus. Given the similarity between the membrane-disrupting mechanism of antimicrobial peptides (AMPs) and the cytotoxic mechanism of amyloidogenic proteins, this study investigated IAPP modifications to enhance its antibacterial properties. Using FoldAmyloid, we computationally predicted the aggregation propensity of IAPP following substitution of specific residues with positively charged lysine (Lys, K). Substitutions at Glycine (Gly, G) positions 24 and 33 (yielding G33K-IAPP and G24K-IAPP) were predicted to maintain aggregation tendency. We synthesized and characterized these modified IAPP peptides using thioflavin-T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscopy. Antibacterial assays confirmed that both IAPP variants effectively inhibited Staphylococcus aureus growth, surpassing the antibacterial activity of native IAPP.

P2: 02

Screening diverse disulfide-cyclised peptide libraries by yeast display

Matilde Salvadoretti, Sara Linciano, Ylenia Mazzocato, Zhanna Romanyuk, Alessandro Scarso and Alessandro Angelini

Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Via Torino 155, 30172

Mestre, Italy

Arzanya S.r.l., Via Rezzonico 6, 35131 Padua, Italy

Company / Academic Institution

Università Ca' Foscari of Venice, Campus Scientificio, via Torino 155, 30170, Venezia Mestre, Italy

Cyclic peptides combine favourable properties of proteins (e.g., good binding affinity, target specificity) and small organic molecules (e.g., high stability, straightforward chemical synthesis, good diffusion properties), which make them suitable ligands for the development of therapeutics.

Towards this goal, our laboratory has recently developed a novel combinatorial platform to rapidly isolate and select genetically encoded disulfide-cyclised peptide binders against a wide range of protein targets. This platform allows generation and quantitative screening of >108 different yeast- encoded cyclic peptide ligands/hr. Isolated macrocyclic peptide ligands showed different amino acid sequences, topologies and ring size distribution. Most promising peptide sequences were chemically

synthesised using orthogonal cysteine protecting groups, cyclised, purified and characterised by LC-MS. For four out of the five protein targets screened, we isolated at least one cyclic peptide ligand with a binding affinity below 10 nM. Importantly, yeast-encoded cyclic peptides showed high specificity for the protein target they were selected for. Studies are ongoing to prove the efficacy and broad applicability of this technology to generate genetically encoded macrocyclic ligands against challenging protein targets and demonstrate their therapeutic uses in vivo.

P2: 03

Development of Membrane-Lytic Anticancer Peptides for the Treatment of Individual Cancer Types

Oluebube Nwajiaku & Martin Ulmschneider

Company / Academic Institution

Department of Chemistry, King’s College London, SE1 1DB, London, United Kingdom

Traditional cancer treatment is often hindered by significant challenges such as multi-drug resistance, lack of tumour selectivity, and severe side effects. In pursuing next-generation chemotherapeutics, membrane-perforating peptides have emerged as a promising solution. These anticancer peptides (ACPs) offer numerous advantages over conventional methods, including high activity, low immunogenicity, and excellent biocompatibility in vivo.  EEK¹, a recently developed 15-residue ACP, has broad-spectrum activity against different cancer types.  In vitro EEK dose-response measurements on a range of patient-derived cancer and healthy cells have shown that EEK has some selectivity towards the cancerous cells. The mechanism of peptide activity is the selective formation of large aqueous pores in cancer cell plasma membranes. Building upon these results, a new peptide library with greater selectivity is designed using both in silico and in vitro methods.

Reference: 

(1)       Chen, C. H.; Liu, Y.-H.; Eskandari, A.; Ghimire, J.; Lin, L. C.-W.; Fang, Z.-S.; Wimley, W. C.; Ulmschneider, J. P.; Suntharalingam, K.; Hu, C.-M. J.; Ulmschneider, M. B. Integrated Design of a Membrane-Lytic Peptide-Based Intravenous Nanotherapeutic Suppresses Triple-Negative Breast Cancer. Advanced Science 2022, 9 (13), 2105506. https://doi.org/10.1002/advs.202105506.

P2: 04

Generation of macrocyclic peptide inhibitors of a therapeutically relevant enzyme using yeast display

Giacomo Bettin1,7, Zhanna Romanyuk1,2, Paul Brear3, Sara Linciano1,2, Ylenia Mazzocato1,2, Sylvia Rothenberger5, Marko Hyvonen3 and Alessandro Angelini1,2,6,*

1 Arzanya S.r.l., Via Rezzonico 6, 35131 Padua, Italy

2 Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Via Torino 155, 30172 Mestre, Italy

3 Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, United Kingdom

5 Institute of Microbiology, University Hospital Center and University of Lausanne, Rue du Bugnon 48, 1011 Lausanne, Switzerland

6 European Centre for Living Technology (ECLT), Ca’ Bottacin, Dorsoduro 3911, Calle Crosera, 30123 Venice, Italy

7Department of Medicine, Universita` Degli Studi di Udine, P.le Kolbe 4, 33100 Udine, Italy

Company / Academic Institution

Department of Medicine, Universita` Degli Studi di Udine, P.le Kolbe 4, 33100 Udine, Italy

Macrocyclic peptides are becoming increasingly valuable molecular formats for drug development, positioning as a bridge between small-molecule drugs and larger biologics due to their favourable properties. Established in vitro display technologies, such as phage display and mRNA display, are potent tools for the discovery of these ligands. However, they rely on screening procedures which are often difficult to control. To overcome these limitations, we recently developed a strategy based on yeast display combined with FACS for the quantitative and real-time monitoring screening and characterization of macrocyclic peptide ligands. We recently applied this tool to rapidly isolate macrocyclic peptide inhibitors of a therapeutically relevant human protease. To evaluate the inhibitory activity of these macrocyclic peptides, we produced them using solid-phase peptide synthesis and an orthogonal cysteine approach. We then purified the peptides by reversed-phase high-performance liquid chromatography (RP-HPLC) and determined their molecular mass using high-resolution mass spectrometry (HRMS). The most potent inhibitor exhibited a KD value of 16.1 nM and inhibited the protease activity with an IC50 value of 7.5 nM. Additionally, X-ray crystal structure of the inhibitor in complex with the protease revealed optimal shape complementarity and extensive surface interaction, explaining its exquisite affinity and selectivity.

P2: 05

Evolving strategies for peptide-based APIs scalable manufacture: balancing speed, cost, and greenness

Ana Cruz, Ana Vicente, Maria Quental, Sandra Sanches, Nuno Lousa

Company / Academic Institution

Hovione Farmaciencia SA

The unprecedented surge in demand for complex peptide therapeutics, particularly evident in the booming GLP-1 agonist market, has highlighted critical bottlenecks in established peptide API production capacity. This escalating demand has outpaced traditional manufacturing capabilities, leading to significant supply chain vulnerabilities and material shortages. While solid-phase peptide synthesis (SPPS) remains a cornerstone due to its time-to-market, its inherent limitations in scalability, environmental impact and cost-effectiveness for multi-ton commercial production are increasingly apparent.

To address these challenges, pharmaceutical companies are actively exploring alternative strategies. Liquid-phase peptide synthesis (LPPS) technologies, including innovative approaches such as membrane-enhanced peptide synthesis (MEPS) and chemoenzymatic peptide synthesis (CEPS), are gaining significant traction. Recent advancements in MEPS demonstrate efficient purification and reduced solvent usage, while CEPS offers highly selective and environmentally favourable routes for complex sequences and longer peptides. Unlike SPPS, which often necessitates dedicated infrastructure, LPPS methods can leverage existing multi-purpose CDMO assets, offering a compelling pathway for supply chain de-risking and substantial cost reduction at commercial scale. Moreover, according to USP–NF/PF guidelines, differences between LPPS and SPPS significantly affect the impurity profiles of the final drug substance, with LPPS offering greater predictability and control through defined reaction steps and intermediate purification.

Ultimately, the future of large-scale peptide manufacturing will likely converge on a hybrid approach between SPPS and LPPS. This strategy combines the strengths of various synthetic methodologies, where shorter, well-characterized peptide fragments are efficiently produced using optimized SPPS or LPPS, followed by highly efficient fragment ligation using both conventional chemical coupling reagents and enzymatic methods. This emerging approach promises to achieve a critical balance between rapid development timelines, commercial scalability and cost-effectiveness, ensuring sustainable supply of next-generation peptide therapeutics. Herein we propose to compare and reflect on different manufacturing strategies comprising pure SPPS vs hybrid approaches, considering cycle time, cost effectiveness and greenness score.

P2: 06

Serving synthesis and post-conjugation of Oligonucleotides

Prabhakar Uttamrao Pawar, Shahadat Ahmed

Company / Academic Institution

Aurigene Pharmaceutical Services Ltd.

Oligonucleotides are emerging as an attractive modality to treat rare genetic

disorders. They are poised to become mainstream in drug discovery, once they are

amenable to a wider range of targets. Specificity, stability and toxicity are few

challenges that needs immediate attention. To address target specificity and

stability issues, oligonucleotides need to be functionalized with target specific

conjugates1 (GalNAc/ peptides/Lipids/Chelators). But incorporation of these

conjugates via amidites, on solid phase, are associated with challenges due to

stability and poor coupling efficiency. Therefore, post-synthetic conjugation, using

bio-orthogonal reaction, offers an attractive alternative.

Aurigene has developed in house methods to design, synthesis, and

characterization of customized oligonucleotides with various functional groups for

different applications. We support early discovery programs by producing

oligonucleotides at different scales with modifications of choice at terminal or

internal positions in bio-burden free environment. Our state-of-the-art facility,

coupled with extensive experience in biological evaluations of ASOs, makes

Aurigene a preferred partner in early discovery stage for a wide range of small

biotech and large pharma companies.

P2: 07

Design and synthesis of miniprotein as inhibitors of CD47/SIRPα interaction.

Paulina Fortuna, Mariusz Fleszar, Małgorzata Krzystek-Korpacka, Iwona Bednarz-Misa, Wioleta Szewczak, Karolina Mosna, Łukasz Berlicki

Company / Academic Institution

Omics Research Center, Wroclaw Medical University, Wroclaw, Poland

The CD47 protein is one of the most attractive regulators of innate immune checkpoints due to its disruption of macrophage and other antitumor cell activation pathways. Binding of CD47 to the signal regulatory protein α (SIRPα) has been shown to inhibit an effective antitumor immune response in many types of cancer. Therefore, inhibition of interactions between these two proteins plays a significant role in cancer immunotherapy by limiting the escape of cancer cells from immune control.

The design of CD47/SIRPα interaction inhibitors was based on the selection of a known miniprotein. After an in-depth analysis of the known structures of various mini-proteins, the MvaT protein was selected. It has been shown that the MvaT protein, it can fold into a stable tertiary structure consisting of two α-helices and three β-harmonics (PDB ID:2MXE). The interior of the structure forms a hydrophobic core. Due to its length (47amino acid residues) and defined, compact spatial structure, the C-terminal domain of the MvaT protein has been classified as an independent mini-protein. Its size allows for rational redesign, which means it can be a starting point in the design of protein-protein interaction inhibitors.

The design was started by analyzing the structure of MVaT miniprotein. It was indicated which side chains participate in forming the hydrophobic core, are located on the surface, or are at the core/surface interface. The resulting template is the input sequence to the PyRosetta software. Calculations were performed using the FastDesign protocol. 

An example of design is remodeling the MVaT miniprotein as a CD47 ligand. The surface of CD47 protein is curved, into which the structure of MVaT miniprotein consisting of two α-helices and three β-harmonics fits perfectly. In the obtained model, we observe that the molecule of the designed inhibitor fits well on the CD47 surface, where it interacts with SIRPα.

P2: 08

Synthesis of proline-rich cyclic peptoids with biological activities

V. Ferrara1, R. Mauro1, R. Schettini1, W. Cieślik2, P. Zioła3, A. Mrozek-Wilczkiewicz3, O. Janďourek4, M. Doležal4, G. Della Sala1, F. De Riccardis1, I. Izzo1

1 - University of Salerno, Department of Chemistry and Biology “A. Zambelli”, via Giovanni Paolo II, 132, 84084 Fisciano, Italy.

2 - University of Silesia, Institute of Chemistry, 75 Pułku Piechoty 1A,41-500 Chorzów, Poland.

3 - University of Silesia, Institute of Physics “A. Chełkowski”, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland.

4 - Charles University, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203, 50005 Hradec Králové, Czech Republic.

Company / Academic Institution

University of Salerno, Department of Chemistry and Biology “A. Zambelli”, via Giovanni Paolo II, 132, 84084 Fisciano, Italy.

Cell life is regulated by exchange, across cell membranes, of Na+, K+, Ca2+ and Cl- ions, controlling different biological processes including metabolism, regulation of cellular pH and signalling pathways. [1] These ions can cross the membrane with two different mechanisms: ion channels or ion carriers. These present amphipathic structures, which can stabilize polar species within the hydrophobic lipid bilayer. [2]

Cyclic peptoids, cyclic oligomers of N-glycines, are known to efficiently complex metal cations and act as synthetic carriers [3] and the introduction of proline residues into the cyclic backbone enhances their complexation abilities. [4] Inclusion of the 4-hydroxyproline residue, gave the opportunity to further functionalize the macrocyclic skeleton by adding lipophilic appendages and to tune the ionophoric properties of the synthetic derivatives. [5]

In this contribution we describe the design and synthesis, using a mixed “submonomer/monomer” solid-phase approach, of a library of hexameric cyclic peptoids containing different substituted L-prolines and various aliphatic and aromatic side chains. In this communication, preliminary antifungal, antibacterial and antitumor biological tests will be also reported.

Keywords: cyclic peptoids, antimicrobial activity, prolines



References

[1] F. Ratjen, S.C. Bell, S.M. Rowe, C.H. Goss, A.L. Quittner, A. Bush, Nat. Rev. Dis. Primers, 2015, 1, 15010.

[2] Y. Zhao, H. Cho, L. Widanapathirana, S. Zhang, Acc. Chem. Res., 2013, 46, 2763.

[3] N. Maulucci, I. Izzo, G. Bifulco, A. Aliberti, C. De Cola, D. Comegna, C. Gaeta, A. Napolitano, C. Pizza, C. Tedesco, D. Flot, F. De Riccardis, Chem. Commun., 2008, 3927.

[4] Izzo, G. Ianniello, C. De Cola, B. Nardone, L. Erra, G. Vaughan, C. Tedesco, F. De Riccardis, Org. Lett., 2013, 15, 598.

[5] R. Schettini, C. Costabile, G. Della Sala, J. Buirey, M. Tosolini, P. Tecilla, M. C. Vaccaro, I. Bruno, F. De Riccardis, I. Izzo, Org. Biomol. Chem., 2018, 16, 6708.

P2: 09

Unraveling the functional implications of site-specific post-translational modifications on heat shock protein 27: a semisynthetic approach

Sabrina Helena Schmied, Christian F.W. Becker

Company / Academic Institution

University of Vienna, Austria (Institute of Biological Chemistry)

Posttranslational modifications, particularly phosphorylations, can have profound influence on proteins with severe implications for diverse pathological conditions such as cancer or neurodegenerative diseases.[1]

Our focus lies on Hsp27, a member of the small heat shock protein (sHsp) family, known for its role in cellular stress response and apoptosis regulation.[1,2] The N-terminal domain of Hsp27 undergoes dynamic modulation through serine phosphorylations at residues 15, 78 and 82, mediated by cellular stress-induced activation of e.g. the MAPK pathway. These modifications induce a shift in oligomerization dynamics, thereby impacting chaperone activity.[2]

Investigating the influence of these phosphorylations will contribute to our understanding of the relationship between site-specific modifications and their effect on functional dynamics of Hsp27. Existing studies, employing cell induction methods or site-specific mutagenesis, fall short in dissecting the nuanced impact of individual phospho-variants.[3] Therefore, our objective is to generate variants of Hsp27 phosphorylated at positions 15, 78, and 82, using a semi-synthetic approach, combining solid-phase peptide synthesis (SPPS), recombinant protein expression and chemoselective ligation approaches.

To this end, suitably functionalized peptide segments of Hsp27 have been obtained through both SPPS and expression. For the expressed peptide segments, a thiol protection strategy with a phenacyl group will be strategically applied to preserve the native cysteine in Hsp27 during ligation reactions, enabling a traceless semi-synthesis.

The overall strategic approach allows for the preparation of full-length, functional Hsp27 with phosphorylations at distinct sites through sequential ligation and subsequent desulfurization. Further, we aim to conduct biochemical assessments, including chaperone activity assays and Hsp90 interaction studies, to unveil the functional properties of the obtained phospho-variants.

References

[1] Mukherjee, S., Vogl, D. P. & Becker, C. F. W, ACS Chem Biol, 2023, 18, 1760–1771. 

[2] Jovcevski, B. et al., Chem Biol, 2015, 22, 186–195.

[3] Collier, M. P. et al., Sci Adv, 2019, 5, 8421–8443.

P2: 10

Title to follow...

Franck Mevellec, Simone Giovanni Sala

Company / Academic Institution

Asymchem

P2: 11

PEG Power: Enhancing peptide vaccines for pancreatic cancer

Yomkippur Perez, Omar Luna, Daniele Ferrari, Sana Sayedipour, Miriam Royo, Gerardo Acosta, Luis Cruz, Frauke Alves, Erik Agner, Magne Sydnes, and Fernando Albericio

Company / Academic Institution

University of Stavanger/Polypure AS

Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments in these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC), by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60−90%), we developed an effective method to enhance the peptide yield and streamline the processes of synthesis and purification. PEGylation significantly improved the solubility, facilitating the single-step purification of long hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.

P2: 12

Towards the total synthesis of the bacteriocin Nisin A on solid support

Ilia Perov1, Bethan Donnelly1,, Tyler Mallett1, John Vederas1

Company / Academic Institution

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.

P2: 13

ONE-POT SOLUTION-PHASE STRATEGY FOR THE SYNTHESIS OF BICYCLIC PEPTIDES USING THE T3P/[PD(PPH3)4] SYSTEM

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.

P2: 14

Lanthanide-Labeled Self-Assembling Peptides

Carlo Martin M. Ocampo, Diego B. Sarte, and Aaron Joseph L. Villaraza

Company / Academic Institution

University of the Philippines - Diliman

The coordination chemistry of lanthanide ions bound to self-assembling peptides is interesting from the perspective of supramolecular science as well the development of probes responsive to the local environment. In one example, a peptide which forms a triple coiled coil in the presence of a lanthanide cation was analyzed by helical wheel simulation to determine the positions at which substitution with norleucine will result in a more stable supramolecular structure. Indeed, the resulting structure was found to exhibit greater thermodynamic and thermal stability via Circular Dichroism (CD) titration (1). In another example, a pH-dependent self-assembling peptide derived from the beak of the giant squid Dosidicus gigas was labeled with the paramagnetic complex Gd[DO3A] and the NMR spin-lattice relaxivity (r1) measured at different pHs; it was found that the labeled bioconjugate exhibits no relaxivity in the assembled state, but possesses enhanced relaxivity at low pH in the disassembled state (2). These results are particularly useful in the context of the design of Gd-labeled Magnetic Resonance Imaging (MRI) contrast agents: while it is possible to design more stable peptide supramolecular structures, and while it is assumed that MRI contrast agents possessing large molecular weights would exhibit enhanced spin-lattice relaxivities, our results demonstrate that the solution environment can have a significant effect on relaxivity, and should therefore be taken into consideration. 



References:

(1) Sarte, D.B. and Villaraza, A.J.L., J. Pept. Sci., 2025, 31: e3665

(2) Ocampo, C.M.M. and Villaraza, A.J.L., Dalton Trans., 2024, 53, 14971

P2: 15

Chemically stable diazo peptides as cysteine protease inhibitors active in living cells

J. Wahl, Ahsanullah, H. Zupan, F. Gottschalk, M. Mieth, C. Arkona, A. Hocke, B. Keller, J. Rademann

Company / Academic Institution

Freie Universität Berlin

The diazo group is an interesting tool in medicinal chemistry. To combine its selective and versatile reactivity with the selectivity of peptide sequences tuned to a target opens a promising new class of enzyme inhibitors. So far C- or N-terminal diazo peptides have been investigated [1] but exhibit only limited stability especially against acidic conditions. We designed internal diazo peptides, stabilized by two adjacent electron withdrawing groups to enhance the stability of the diazo moiety. 

Starting from polymer supported triphenylphosphine internal diazo peptides were obtained by C-acylation of phosphorane ylides and subsequent cycloaddition-reversion mechanism with electron-deficient azides. This procedure is stable towards standard SPPS conditions and delivers internal diazo peptides as amides and ester. These peptides are stable between pH 1-14 (amide) and pH 1-8 (ester), even in 95% TFA or HF/pyridine, as well as against thiol nucleophiles. [2]

A diazo peptide with the sequence of the human caspase-3 (hC-3) substrate showed IC50-values in the nanomolar range with a time-dependent covalent inhibition mechanism and a cell permeable fluorescent diazo peptide was used to image apoptosis in living cells by labelling of active hC3-protease.

To increase the activity of these enzyme inhibitors towards other targets we are investigating the diazo peptide ester as prodrugs of the corresponding diazo methylketone (DMK). With this approach the very stable diazo peptide gets transformed to the less stable but more reactive DMK by ester hydrolysis and subsequent decarboxylation. 



[1] Challis, B.C.; Latif, F. Synthesis and Characterisation of some New Diazopeptides. J. Chem. Soc. Perkin Trans. 1, 1990, 1005-1009. DOI: 10.1039/P19900001005

[2] Wahl, J.; Ahsanullah; Zupan, H.; Gottschalk, F.; Nerlich, A.; Arkona, C.; Hocke, A. C.; Keller, B. G.; Rademann, J. Chemically Stable Diazo Peptides as Selective Probes of Cystein Proteases in Living Cells. Angew. Chem. Int. Ed. 2024, 63, e202411006

P2: 16

Semisynthesis of photo-crosslinking probes for elucidation of targets and therapeutic scope of human Trefoil Factor Family peptide 2 in chronic gut disorders

S.S. Denisov, M. Muttenthaler

Company / Academic Institution

The Institute of Biological Chemistry, University of Vienna, Vienna, Austria

Inflammatory bowel disease (IBD) is a chronic gastrointestinal condition which is characterized by the degradation of the defence mucosal barrier and gut epithelium. IBD manifesting in two forms – Crohn’s disease and ulcerative colitis – affects 6 – 8 million people worldwide. Considering the growing prevalence and significant burden on patients’ quality of life of IBD, the development of novel therapies is of utmost importance. Although the current treatment is mostly focused on inflammation suppression, therapeutics which promote epithelium healing without immunosuppression could be more beneficial as they would prevent undesirable effects such as infections. However, the lack of understanding of the underlying processes of mucosal healing and its molecular agents significantly impedes progress in this direction. The trefoil factor family peptides (TFFs) – TFF1, -2, and -3 - are essential players in epithelial protection and repair and are involved in mucosal restitution, cell junction modulation, apoptosis, angiogenesis, and inflammation. Their exact molecular mechanisms remain elusive, with a broad range of receptors and glycoproteins proposed as putative interaction partners. Here, we present the synthesis of photo-cross-linking probes for the structurally most complex human TFF – TFF2 – which comprises two trefoil domains supported by 7 disulfide bonds. Firstly, TFF2 was produced via recombinant expression, its oxidative refolding was optimised, and the obtained native TFF2 was characterised using CD and NMR spectroscopy. Then, molecular probes containing 4-benzoyl-L-phenylalanine (Bpa) as a photo-reactive moiety were produced via two-fragment expressed protein ligation, refolded, and studied to confirm their structural identity with the native TFF2. Native TFF2 and its probes will be further used in TFF2 target identification by GPCR receptor screening and photo-cross-linking coupled with MS analysis.

P2: 17

The Effect of Curvature in Curved Gradient Elution on the Separation Parameters of Peptides Using HPLC

Mohammadreza Taheri; Stephan Uebel

Company / Academic Institution

Max Planck Institute of Biochemistry, Planegg, Bavaria, Germany

Peptide separation and purification are crucial steps in many biotechnological and pharmaceutical applications. Linear and segmented gradient high-performance liquid chromatography (HPLC) are widely accepted as effective methods for this task. However, little research has been conducted on whether curved gradient elution (CGE) is an even more effective method. To study the effect of gradient elution curvature on peptide separation efficiency, we created curves of varying sharpness, considering a linear gradient to be a flat line with zero curvature. We used a homemade standard peptide mixture and two different peptide mixtures obtained from the digestion of bovine serum albumin as the analytes. The mobile phase consisted of a mixture of water and acetonitrile containing 2% phosphoric acid. We plotted the critical chromatography parameters versus curvature. The results demonstrate CGE's potential for peptide separation and show how curvature affects separation parameters. The results also suggest the possibility of improving chromatography parameters for peptide separation.

P2: 18

New MHB-Br resin for SPPS synthesis of high purity and yield C-terminal acid protected and unprotected peptides

Simona Serban, Chaoyang Wang, Cheng Zhang, Alessandra Basso, Beatriz de la Torre, Fernando Albericio

Company / Academic Institution

Sunresin New Materials Ltd

Through continuous innovation in the field of solid-phase peptide synthesis (SPPS), a novel bromine-functionalized polystyrene/ DVB resin was designed to enhance the synthesis of C-terminal acid peptides, achieving high yields and low impurities. 

Currently, the industry relies on two widely accepted resins for synthesizing acid peptides: the Wang resin and the 2-CTC resin. Each of these resins has its own well-documented advantages and limitations, making the selection process crucial for specific applications. 

The case studies presented herein demonstrate the effectiveness of the MHB-Br (4-Methylbenzhydryl bromide) resin in addressing the challenges associated with the synthesis of acid peptides showing low DKP and high stability to peptide elongation. These studies highlight the resin’s ability to facilitate the production of both protected and unprotected acid peptides, showcasing its potential to improve efficiency and purity in peptide synthesis and consequently supporting the peptide manufacturers in achieving better output.

P2: 19

Accelerated synthesis of modified peptide

Roni Freund-Cohen, Prof. Mattan Hurevich*

Company / Academic Institution

The Hebrew university of jerusalem (HUJI)

Chemical modifications on proteins create dynamic biological systems. Post translational modifications (PTMs) induce local structural changes which translates to a versatility in proteins function. 

The type, number, position and combinations of the PTMs forms a regulatory cascade . Studying the role of each modification patterns requires accessibility to the different variations. 

Since isolating native proteins with specific PTMs is impractical, the development of strategies to synthesize peptides with defined modification patterns is a viable alternative. The need to access a large number of peptides with defined PTMs combinations requires rapid, robust and high-efficiency solid phase peptide synthesis (SPPS) strategy. 

We developed a new SPPS strategy that relies on fast-stirring to accelerate the preparation of PTM peptides libraries. The strategy relies on the incorporation of pre-modified amino acids and overcomes many synthetic hurdles associated with phospho- and glyco-peptide synthesis.

 I will demonstrate that the strategy can be used to prepare multiphosphorylated and glycosylated peptides libraries .

I will show that using the accelerated process common setbacks like elimination and racemization cane be supresses. I will illustrate that the strategy can be handy in the introduction of O-GlcNAc on peptides in a late-stage modification approach. Integration between the above strategies can provide an accessible and complementary ways to access a variety of PTM peptides.

 1) D. Grunhaus, E. Rossich Molina, R. Cohen, T. Stein, A. Friedler* and M. Hurevich*, Org. Proc. Res. Dev., 2022, 26, 2492–2497.

P2: 20

Advancing antifungal frontiers with cationic cyclic peptides

Julia H. Huang, Jun X. Ang, David A. Wells, Rachel M. Chen, William W. Hunt, Nicholas L. Massey, Kylie A. Agnew-Francis, James A. Fraser, Avril A. B. Robertson

Company / Academic Institution

The University of Queensland

Invasive fungal infections (IFIs) account for an estimated 6.5 million cases and 3.8 million deaths annually. In 2022, the World Health Organisation identified Candida albicans, Candida auris, and Cryptococcus neoformans as pathogens of critical concern. Candida bloodstream infections and invasive candidiasis affect approximately 1.6 million people yearly, resulting in close to one million deaths. Cryptococcal meningitis has a mortality rate of 76%, translating to 147 000 deaths annually. These figures underscore the urgent need for antifungal therapies.

Current treatments are limited, with three of four major drug classes targeting the fungal cell membrane. Due to the high genomic similarity between human and fungal cells, treatments are often toxic. The rise of multidrug-resistant (MDR) and pan-resistant fungal strains, such as C. auris, further restricts the utility of existing antifungals. In the US, over 90% of C. auris isolates are resistant to the frontline drug, fluconazole, 30% to amphotericin B, and 1% to the echinocandin class.

Cationic cyclic peptides (CCPs) and amphiphilic peptides offer a promising solution. These positively charged peptides interact with negatively charged phospholipids in the fungal cell membrane, allowing penetration of a lipophilic moiety that leads to fungicidal activity. ROB072, a novel CCP developed by the Robertson group, has shown potent efficacy against azole-resistant and azole-susceptible isolates of C. auris, as well as other pathogenic yeasts such as C. albicans and C. neoformans. Ongoing research focuses on optimising ROB072 through structure-activity relationship studies. ROB072’s potential to combat MDR and pan-resistant fungal pathogens marks a critical advancement in treating IFIs.

P2: 21

HARNESSING A BIS-ELECTROPHILIC BORONIC ACID LYNCHPIN FOR AZABOROL THIAZOLIDINE (ABT) GRAFTING IN CYCLIC PEPTIDES

Arnab Chowdhury, Basab Kanti Das, Saurav Chatterjee, Nitesh Mani Tripathi, Bibekananda Pati, Soumit Dutta and Anupam Bandyopadhyay *

Company / Academic Institution

INDIAN INSTITUTE OF TECHNOLOGY, ROPAR

Chemical modifications of native peptides have significantly advanced modern drug discovery in recent decades. On this front, the installation of multitasking molecular grafts onto macrocyclic peptides offers numerous opportunities in biomedical applications. In this study, we present a novel class of borono-cyclic peptides featuring an azaborolo thiazolidine (ABT) graft, assembled via a bis-electrophilic boronic acid lynchpin. This strategy exploits the substantial reactivity difference (>10³ M⁻¹ s⁻¹) between N-terminal and backbone cysteines to achieve rapid and highly regioselective macrocyclization (∼1 h) under physiological conditions. The resulting ABT-crosslinked peptides exhibit remarkable stability in endogenous environments while retaining the ability to revert to linear diazaborine peptides through α-nucleophile treatment. This versatile crosslinking approach was further used for regioselective bicyclizations, enabling the precise engineering of α-helical peptide structures for structural and functional studies. ABT-grafted peptides also demonstrated utility in biorthogonal conjugation, significantly enhancing intracellular delivery of bioactive molecules. Notably, an ABT-grafted apoptotic peptide (KLA) exhibited potent cancer cell-killing activity, facilitating efficient intracellular delivery and targeted apoptosis induction. Beyond cancer therapeutics, the stability and modularity of ABT-grafted peptides position them as candidates for drug delivery systems, protein-protein interaction modulators, and imaging probes in diagnostic applications. The mechanism of apoptosis induced by the ABT-grafted KLA peptide was investigated, which shed light on its therapeutic potential. These findings highlight ABT-grafted peptides' broad applicability in advancing peptide-based drug discovery, opening up new avenues for precision medicine, targeted therapy, and biomedical research.

Keywords: Azaborol Thiazolidine, Borono hetrocycle, Apoptosis, KLA peptide, Crosslinking

P2: 22

Novel Strategies for Peptide Cyclisation: Acrylamide-Modified Amino Acids Drive Cysteine-Directed Cyclisation

Christina Dumitriu Jackson, Peptide Discovery, Discovery Sciences, R&D, AstraZeneca, UK 

Hannah Bolt, Peptide Discovery, Discovery Sciences, R&D, AstraZeneca, UK 

Carole Urbach, Phage Display, Biologics Engineering, R&D, AstraZeneca, UK 

Elizabeth brown, Phage Display, Biologics Engineering, R&D, AstraZeneca, UK 

Company / Academic Institution

AstraZeneca / Manchester Metropolitan

Peptide macrocycles are a promising modality in drug discovery with varied applications from therapeutic inhibitors of PPI interactions to targeting ligands for the delivery of other therapeutic payloads. Macrocyclic peptides have unique features, and cyclisation is important for optimising their structural stability, biological activity, and pharmacokinetic properties. Using traditional hit-finding techniques like phage display, cyclic peptides can be formed via disulfide bridges between cysteine residues. In more recent years, novel cyclisation chemistries have been developed to broaden the diversity and functionality of cyclised peptides. This is significant since such modifications have the potential to extend chemical stability and half-life, a key factor in optimising peptides for therapeutic use.

This work investigates novel acrylamide-functionalised amino acid analogues as alternative cyclisation handles in peptide discovery. These acrylamide groups undergo selective Michael addition with the thiol side chain of cysteine residues to form stable thioether linkages. Prior to applying recombinant peptide display technologies, the chemical feasibility of this approach was evaluated. Reaction conditions were optimised for efficient and selective chemical cyclisation of peptides containing acrylamide building blocks, and the methods developed were shown to be applicable to peptides with varying sequences, lengths, and loop sizes.

Use of acrylamide-containing amino acids in peptide drug discovery has several potential applications. In addition to use as a novel cyclisation strategy, acrylamides can act as an electrophilic warhead, providing the opportunity for covalent peptide discovery. In this approach, a stable, covalent bond is formed with the target protein for a more potent and irreversible interaction. Breaking beyond the boundaries of conventional disulfide-based peptide cyclisation, this innovative peptide strategy expands the drug discovery toolbox, unlocking new possibilities for targeting the most challenging diseases and delivering next-generation therapies for patients who need them most.

P2: 23

Greening Peptide Purification Through Replacement of Acetonitrile with Ethanol

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

P2: 24

Ultra-Efficient Solid-Phase Peptide Synthesis of Pharmaceutical Peptides

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.

P2: 25

Novel bio-based solvents for solid-phase peptide synthesis

Vincent Freiburghaus, Tom Nelis, Jeremy Luterbacher, Nina Hartrampf

Company / Academic Institution

Universität Zürich

The increasing demand for therapeutic peptides and peptide-based drugs drives the urgent need for more sustainable synthetic methods. Solid-phase peptide synthesis (SPPS) is the method of choice to reliably synthesize peptides and small proteins. However, the current manufacturing processes compare unfavourably in terms of process mass intensity (PMI) to other modalities such as small molecules and biopharmaceuticals. The main contributor to the unfavourable PMI of SPPS is the excessive solvent usage. N,N-dimethylformamide (DMF), the most used solvent in SPPS, is classified as carcinogenic and reprotoxic. Consequently, the European Union has restricted DMF usage as of December 2023, highlighting the need for alternatives.

Here, we present the usage of novel, bio-based solvents for SPPS. To assess SPPS compatibility, solubilities of common SPPS reagents and waste products were tested, as were the resin swelling capabilities of the newly developed solvents. Next, coupling and Fmoc-deprotection kinetics were investigated. Finally, several peptides have been synthesized without the use of concerning solvents. Difficult peptides were synthesized and compared to conventional synthesis in DMF. While the challenge of extensive solvent usage is yet to be solved, we show that DMF can be replaced with bio-based solvents, which is a big leap towards more sustainable SPPS.

The structures of the solvents are omitted for confidentiality reasons but will be shown on the event.

P2: 26

Cyclic & linear peptide modulation via late-stage C-H functionalization

Dr. Ralph Kirk, Dr. Mohit Kapoor, Ms Heidi Kingdon Jones and Dr. Lalit Mehta

Company / Academic Institution

Concept Life Sciences

Late-stage functionalisation (LSF) of peptides represents a powerful synthetic strategy for modifying complex biomolecules with precision, versatility, and minimal changes to their native structure. This approach enables the direct modification of linear and cyclic peptides after their synthesis, offering a streamlined route to diversification for therapeutic development and drug discovery. At Concept Life Sciences, we have synthesised a 19-mer linear and cyclic peptide using orthogonal protecting groups to provide a final ‘C-O-N-C-E-P-T-L-I-F-E-S-C-I-E-N-C-E-S’ peptide in excellent purity. Selective deprotection of functional groups enable us to do the diversification at the late stage. Furthermore, we have studied the Kolbe type functionalisation of amino acids using Electrochemistry, where we successfully fluorinated the side chains under mild conditions to expand the chemical repertoire for LSF, allowing the introduction of functionalities ranging from fluorophores and reactive handles to pharmacophores and targeting motifs. Despite ongoing challenges in controlling regioselectivity, particularly in densely functionalised or sequence-sensitive contexts; LSF continues to evolve as a transformative tool in peptide science, opening new frontiers in drug discovery, chemical biology, and precision medicine.

P2: 27

Peptide modifications via C-terminal enamides

Daria K. Wanic, Thomas Wharton, Thomas E. Nielsen, David Hymel, Wouter F. J. Hogendorf, David R. Spring

Company / Academic Institution

University of Cambridge

Peptide therapeutics offer significant advantages over small molecules and biologics due to their high selectivity, efficacy, low toxicity, and ability to mimic endogenous protein fragments. Despite their potential, the clinical application of peptide drugs is limited by poor pharmacokinetic properties, such as proteolytic degradation and renal excretion, resulting in short half-lives and frequent dosing requirements. 

In this work, we explore new strategies for site-selective peptide modifications at the C-terminus through the photocatalytically-enabled formation and subsequent functionalisation of enamides. This approach aims to develop a versatile platform for late-stage installation of diverse chemical handles under mild conditions, without the need for protecting groups. Leveraging the unique reactivity of enamides, we demonstrate successful peptide modifications including hydrothiolation, hydroarylation, and Heck cross-coupling. These transformations expand the chemical space of peptide derivatives and may enhance their therapeutic properties.

P2: 28

ONE-POT SOLUTION-PHASE STRATEGY FOR THE SYNTHESIS OF BICYCLIC PEPTIDES USING THE T3P/[PD(PPH3)4] SYSTEM

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.

P3: 07

Expanding the Nociceptin/Orphanin FQ landscape: synthesis and pharmacological characterization of dimeric ligands with unexplored architecture

Giulio Meneguzzo, Valentina Albanese, Pietro Pola, Michela Argentieri, Girolamo Calò, Tiziano De Ventura, Alessia Frezza, Remo Guerrini, Davide Illuminati, Davide Malfacini, Erika Marzola, Erika Morrone, Delia Preti, Alessandra Rizzo, Chiara Sturaro, Chiara Ruzza, Salvatore Pacifico.

Company / Academic Institution

University of Ferrara (Italy)

The nociceptin/orphanin FQ (N/OFQ) peptide is the endogenous ligand of the NOP receptor, a member of the opioid receptor family that, despite sharing approximately 60% sequence homology with classical opioid receptors (mu, delta, kappa), displays distinct pharmacological properties and ligand selectivity. The NOP receptor is involved in multiple physiological functions including pain modulation, emotional regulation, stress response, reward processing, neuroendocrine control, and motor coordination. Owing to this broad functional spectrum, the N/OFQ–NOP system has emerged as a promising therapeutic target. Despite this, only three small-molecule NOP ligands—cebranopadol, sunobinop, and LY2940094—are currently in clinical development.

Beyond non-peptide agents, peptide-based ligands continue to attract interest due to their intrinsic receptor affinity and selectivity. However, native peptides suffer from limited in vivo stability, prompting structural optimization strategies. 

Among these, peptide dimerization has garnered attention for its potential to enhance metabolic stability and pharmacodynamic properties. In this work, we report the design, synthesis, and characterization of covalently linked N/OFQ homodimers via site-selective disulfide bridge formation. Cysteine residues were strategically introduced at distinct positions within the address domain to explore the impact of linker geometry on pharmacological activity.

All synthesized dimers were evaluated in vitro in recombinant cells expressing the NOP receptor using both calcium mobilization and BRET assays to monitor receptor–G protein coupling. Selected compounds displaying potent agonist activity were further assessed ex vivo in the mouse vas deferens (mVD) and in vivo for their ability to induce loss of righting reflex (RR), a surrogate marker of hypnotic activity associated with NOP receptor stimulation.

Our findings demonstrate that dimerization of N/OFQ via disulfide linkage is a viable strategy that preserves the pharmacodynamic properties and selectivity of the peptide in vitro, while significantly enhancing its potency and duration of action in vivo. These results support its use in the development of novel NOP-targeted therapeutics.

P3: 08

Title to follow...

Christophe Andre

Company / Academic Institution

Polypeptide Laboratories France SAS

P3: 09

Title to follow...

Francesco Terzani

Company / Academic Institution

Polypeptide Laboratories France SAS

P3: 10

Ecumicin-like compounds targeting the ClpC protease of Staphylococcus aureus as novel antibacterial agents and precursors for the development of BacPROTACs against Gram-positive pathogens

Maria Tsipa, Sabryna Junker, Violetta Krisilia, Rainer Kalscheuer, Tim Clausen, Markus Kaiser

Company / Academic Institution

University of Duisburg-Essen, Center of Medical Biotechnology, Faculty of Biology

Antibacterial resistance, often termed the “silent pandemic”, is a major global concern as emerging resistant microbes evade treatment by many established antibiotics that operate through a traditional mode of action. This escalating threat has driven the search for alternative, novel antimicrobial chemotherapeutic strategies. Previous research from our lab, along with recent studies conducted by our collaborators, has highlighted Targeted Protein Degradation (TPD) as a promising field of research against bacteria. This innovative approach uses a bacterial variant of the PROteolysis TArgeting Chimeras (PROTAC) concept, denoted as a BacPROTAC. In this study, we aimed to expand the applicability of first- and second-generation BacPROTACs by developing cell-permeable compounds that target the ClpCP protease in Gram-positive bacteria. These third-generation bifunctional molecules were based on Ecumicin, the natural macrocyclic tridecapeptide that targets ClpC1, and demonstrates potent in vitro and in vivo activity against Mycobacterium tuberculosis, with selectivity toward mammalian cells. In the quest to identify potent Ecumicin-like compounds with high cell permeability in Gram-positive bacteria, we streamlined the synthesis of various Ecumicin derivatives employing solid-phase peptide synthesis, solution-phase reactions, CuAAC click-chemistry approaches and MW-assisted protocols. We subsequently assessed their bioactivity against mycobacteria and Gram-positive strains and identified two Ecumicin-Propargylglycine derivatives as the most effective candidates against Staphylococcus aureus. Further evaluation of the bioactivity of such molecules may not only reveal more Ecumicin-like prospective antibacterial agents, but the investigation of their PEG-modified derivatives could pinpoint those compounds with suitable exit vectors for the future development of Ecumicin-based BacPROTACs (EBPs).

P3: 11

Nature-Inspired Protection: Engineering Complement-Resistance via Surface-Tethered M22 Peptidomimetics

Sofia Pinheiro, Alexander John Lander, Ekatarina Umnyakova, Peter Rüthemann & Daniel Ricklin

Company / Academic Institution

University of Basel

The complement system plays a crucial role in immune defense, contributing to both innate and adaptive

immunity. However, because complement can also harm healthy human cells, its activity is tightly

regulated by various membrane-bound and soluble factors. Complement activation can also trigger

unintended attacks on non-self surfaces, such as transplanted organs. Preventing complement-mediated

injury to cells—for instance, during transplant rejection—is a pressing clinical challenge, yet current

therapeutic options have shown limited success. This work aims to take inspiration from nature to develop

new strategies to protect biosurfaces from complement-mediated cell damage.

Several pathogens have been identified to recruit a potent soluble inhibitor of the Classical and Lectin

pathway of the Complement System named C4b Binding Protein (C4BP), bypassing thus the complement

system attack when entering the human body. [1] Streptoccocus pyogenes binds to C4BP via a hyper-

variable domain in its M protein (M22), evading complement-mediated opsonization and phagocytosis.

Previously, a synthetic 52 aminoacids homodimeric peptide derived from M22 (M22-N), has been shown

to bind to C4BP. [2] Therefore, we synthesized site specifically labelled M22-N to enable capture onto

artifical biosurfaces, mimicking the natural presentation on bacterial cells. Flow cytometry analysis

demonstrated that the M22-N-decorated surface can efficiently recruit C4BP from normal human serum,

and prevents complement mediated attack of the model biosurface. Furthermore, we also characterized

target interaction kinetics between immobilized M22-N and soluble C4BP. Based on these insights, our

goal is to utilize structure guided design to develop shorter, more stable M22 peptidomimetics, with

improved physicochemical properties suitable for surface coatings. Therefore, we aim to develop a new

artificial coating strategy for mitigating complement damage of biosurfaces, by mimicking mechanisms

employed in nature.

[1] Ermert D., Blom A.M. Immunol Lett. 2015;169:82–92.

[2] Engberg A. E., Sandholm K., Bexborn F., et al. Biomaterials. 2009;30(13):2653–2659

P3: 12

Advancing arginine building blocks: Novel protecting group approaches for solid-phase peptide synthesis

Philipp Fronik, Swarup De, Timo Nuijens, Srinivas Banala, Amelia Taylor-Young, Noortje Coenen, Astrid Knuhtsen, Paul Alsters, Marijn Rijkers, Peter Quaedflieg and Daniel Sejer Pedersen

Company / Academic Institution

Novo Nordisk

The commonly used Pbf (2,2,4,6,7-pentamethyldihydrobenzofuranyl) protecting group for arginine, while beneficial in certain respects, presents substantial drawbacks that can hinder efficient peptide synthesis. Specifically, the removal of the Pbf group is generally slower than that of other protecting groups, leading to prolonged overall reaction times. Additionally, the Pbf cation formed during deprotection can modify the peptide itself via sulfonylation, resulting in unwanted side products and often necessitating additional development with regard to the cleavage conditions used.

In response to these challenges, we present a novel, divergent route for the synthesis of arginine building blocks that address the limitations of Pbf. We have developed a series of arginine analogs featuring various protecting groups and studied their deprotection reaction times under standard conditions. Additionally, we are assessing their compatibility with conventional coupling reagents and their integration into a model peptide sequence.

Our findings aim to highlight the advantages of these alternative protecting groups, emphasizing enhancements in deprotection times and overall efficiency in peptide synthesis.

P3: 13

Influence of Onopordum platylepis Murb. as a vegetable coagulant on the production and bioactivity of peptides in Murcia al Vino cheese.

1. Gregorio Molina Valero (Molina-Valero, G.)

2. Cindy María Bande De León (Bande-De León, C.)

3. Adela Abellán Guillén (Abellán, A.)

4. Teresa Muela Clemente (Muela, T.)

5. Luis Tejada Portero (Tejada, L.)

Company / Academic Institution

UCAM

P3: 14

Production of Bioactive Peptides from Chlorella vulgaris Through Enzymatic Hydrolysis with Plant- and Animal-Derived Proteases

Teresa Muela Clemente, Alfonso Pérez Garrido, María Dolores Gil Martínez, Gregorio Molina Valero, Noelia Hernández Correas, Adela Abellán Guillén

Company / Academic Institution

UCAM

The valorization of sustainable protein sources is a key strategy in the development of functional ingredients. Microalgae such as Chlorella vulgaris are particularly interesting due to their high protein content and their potential to generate bioactive peptides through controlled hydrolysis processes.

In this study, the hydrolytic capacity of two enzymes—porcine pepsin and a proteolytic extract from Cynara scolymus (artichoke flower)—was evaluated for their ability to release bioactive peptides from crude biomass and protein extracts of two C. vulgaris strains ("Gold" and "Green").

Functional activities of the hydrolysates were assessed through in vitro assays and LC-MS analysis, allowing for the identification of distinct peptide and functional profiles. Quantified data were represented using a heatmap and a radar chart, revealing differentiated bioactive patterns depending on the enzyme, microalgae strain, and substrate used. 

These results highlight the value of plant-derived enzymes for the generation of peptides with health-related properties and position Chlorella vulgaris as a promising microalgal source for the development of functional ingredients within a sustainable approach.

P3: 15

Synthesis of Bicycle® Peptides Using Minimal-Protection Solid Phase Peptide Synthesis (MP-SPPS)

Nisha Mistry and Georgina Davis

Company / Academic Institution

Bicycle Therapeutics

Following the recent restrictions placed on the use of N,N-dimethylformamide (DMF) in Europe, there is an increasing demand for more sustainable practices when it comes to peptide production, where DMF was the standard production solvent. Establishing alternative organic solvents or solvent mixtures for Solid-Phase Peptide Synthesis (SPPS) has been the primary focus of many groups so far.1,2 However, it is expected that similar restrictions will be placed on trifluoroacetic acid (TFA), the solvent widely used to globally deprotect and cleave peptides from their solid support during SPPS. This creates a need to apply green chemistry principles to this part of the process. Commonly, global deprotection requires large amounts of TFA to remove protecting groups, such as 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) and trityl (Trt), from the side chains of the amino acids. Building on the work by Yang et al., we replaced or, in some cases, eliminated the need for these protecting groups in an approach called Minimal-Protection Solid-Phase Peptide Synthesis (MP-SPPS).3 This allowed for a 46% reduction in the volume of TFA required for the cleavage step, as well as a reduction in the time required to perform this step. This approach has demonstrated its utility, as well as improving the low atom economy associated with SPPS, thereby further reinforcing green chemistry principles.

1. J. Lopez, S. Pletscher, A. Aemissegger, C. Bucher and F. Gallou, Organic Process Research & Development, 2018, 22, 494–503.

2. K. P. Nandhini, N. Cele, B. G. de la Torre and F. Albericio, Green Chemistry Letters and Reviews, 2024, 17. 

3. Y. Yang, L. Hansen and P. Ryberg, Organic Process Research & Development, 2022, 26, 1520–1530.

P3: 16

Dual mechanisms and potent activity and selectivity of CRDK-S16 and CRDK-S18G peptides against Mycobacterium tuberculosis

Cesar A. Roque-Borda, Laura M. D. G. Primo, Rafaela M. Pogianeli, Flávia A. Resende, Emma Dyhr, Emily Helgesen, James Booth, Henrik Franzyk, Paul R. Hansen, Beatriz G. de la Torre; Fernando Albericio; João Perdigão, Fernando R. Pavan.

Company / Academic Institution

1) São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, São Paulo – Brazil. 

2) University of Lisbon. Faculty of Pharmacy, iMed.ULisboa–Institute for Medicines Research, Lisbon - Portugal. 

3) University of Copenhagen, Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, Denmark. 

4) Research Center on Biotechnology – UNIARA, Araraquara, São Paulo – Brazil. 5) Department of Microbiology, Oslo University Hospital, Norway.

6) Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, South Africa.

This study represents a major advancement in antimicrobial peptide (AMP) development through the synthesis and evaluation of 60 novel analogs derived from B1CTcu5, leading to the identification of two highly potent candidates: CRDK-S16 and CRDK-S18G. Both peptides displayed antimicrobial efficacy comparable to rifampicin and isoniazid against Mycobacterium tuberculosis (Mtb), with minimum inhibitory concentrations (MIC) of 1.14 µg/mL and 1.2 µg/mL, respectively, including activity against clinical drug-resistant strains. Synthesized via Fmoc-based solid-phase peptide synthesis, the peptides were comprehensively assessed for safety, selectivity, and functional efficacy. No cytotoxicity, mutagenicity (Ames test), or hemolytic activity was observed. Both peptides achieved significant intracellular killing of Mtb in RAW and THP-1 macrophages, with concurrent downregulation of inflammatory gene markers. In vivo, treatment of infected Galleria mellonella larvae resulted in a >99% reduction in bacterial load and full restoration of tissue integrity, with no systemic toxicity. Confocal microscopy confirmed efficient intracellular delivery, while scanning electron microscopy revealed pronounced bacterial membrane deformation. Despite differing by a single serine position, molecular dynamics simulations indicated that CRDK-S16 interacts with mycolic acids, suggesting a lipid-targeting mechanism, whereas CRDK-S18G does not—yet both share comparable bactericidal potency. Additional evaluations showed selective inactivity against non-tuberculous bacteria, strong antioxidant activity, and efflux pump inhibition by S18G in resistant strains. Together, these findings highlight CRDK-S16 and CRDK-S18G as promising AMP candidates with complementary mechanisms of action, high specificity, immune modulation capabilities, and strong in vivo efficacy for tuberculosis therapy.

P3: 17

Targeted delivery of oligonucleotide therapeutics using Bicycle® peptide technology

Megan A. St. Denis

Company / Academic Institution

Bicycle Therapeutics

Bicycle® molecules are constrained bicyclic peptides that are highly effective at engaging target proteins selectively and with high affinity. In addition to their potential to act as potent pharmaceutical agents, they can act as delivery vehicles for targeted therapies. Utilisation of the Bicycle® peptide technology enables targeted delivery of therapeutics to skeletal muscle, cardiac muscle, and the central nervous system.

P3: 18

An mRNA-display-based approach for discovering peptides with exposed C-termini

Huawu Yin, Louise J. Walport

Company / Academic Institution

Imperial College London

Biological peptide display technologies, such as phage display, ribosome display and mRNA display are powerful methodologies to develop peptide ligands for targeting a wide range of proteins. Despite their remarkable success, a prevalent shortcoming is the concealment of the C-terminus of displayed peptides in most display approaches. This concealment hampers the exploration of peptides where their C-terminus is crucial for binding, localization, or substrate recognition, which are found in diverse proteins and remain underexplored. While the PIII of phage has been engineered to display peptides with an exposed C-terminus by incorporating a suitable linker, this has not been possible for mRNA display as the C-terminus of displayed peptide is ligated with an mRNA through a puromycin linker. However, mRNA display would allow screening of higher diversity libraries and of libraries containing greater chemical diversity through the application of genetic code reprogramming. Herein, by integrating the mRNA display, flexible in vitro translation (FIT) system, and site-specific cleavage method, we developed an approach for displaying peptides with exposed C-termini, then we used it to identify peptide substrates or modulators of diverse protein targets including OaAEP, KLHDC2, and PDZ domain. We anticipate that the newly developed display technology will complement current display techniques, particularly those with exposed N-termini or cyclic peptides, and provide a valuable tool to elucidate the C-terminal functions of underexplored proteins.

P3: 19

Discovery of Insecticidal Cyclotides from Allexis batangae and Allexis obanensis

Negin Khatibi, Olivier Ndogo Eteme, Conan K. Wang, Edward K. Gilding, Ernestine Nkwengoua Zondegoumba, Michelle L. Colgrave, Yen-Hua Huang and David J. Craik

Company / Academic Institution

The University of Queensland

Cyclotides represent the largest class of cyclic peptides unique to the plant kingdom. They are recognized for their crucial role in host defense against insects and fungal pathogens. Comprising 28 to 37 amino acids, cyclotides feature a unique head-to-tail cyclic structure and a knotted arrangement of three conserved disulfide bonds, forming a cyclic cystine knot (CCK) motif. This structural feature endangers cyclotides with exceptional stability, making them a promising group of molecules for a diverse array of agricultural and pharmaceutical applications. Cyclotides are found in five major plant families: Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae, and Fabaceae. Every examined species in Violaceae has been shown to produce cyclotides, yet many species from different clades within the Violaceae phylogenetic tree remain unexplored. 

In the current study, the presence of cyclotides in Allexis batangae and Allexis obanensis from the Allexis genus in the Violaceae family is investigated. Five bracelet cyclotides, including three novel cyclotides, were isolated and characterized using MS-MS and NMR spectroscopy. The cytotoxic activity of these cyclotides was evaluated against Spodoptera frugiperda (Sf9) insect cells, demonstrating the high potency of cyclotides as insecticidal agents. Additionally, the insecticidal properties of the discovered cyclotides were evaluated in a feeding study using the Helicoverpa armigera L3 larvae. The results showed that both the 80% plant extract and individual cyclotides inhibited larval growth. These findings highlight the potential of cyclotides as potent bioinsecticidal agents and suggest promising applications in agricultural pest management.

P3: 20

Application of Edman degradation for de novo analysis of disulfide bonds in proteins

Toni Kühl, Yomnah Y. Elsayed, Alexander Terekhov, Diana Imhof

Company / Academic Institution

Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute

University of Bonn, Bonn, Germany

Analysis of cysteines and identification of their connectivity in peptides and proteins can be a laborious endeavour especially when multiple disulfide bridges are present in a sequence. Structural analysis of such complex structures using one method only may not necessarily lead to an unambiguous result and is often time-consuming involving complex data sets. Recently, we re-introduced Edman degradation as a suitable technique to complement the analytical toolbox for such investigations and established relevant standards such as PTH-Cys(Cam) and PTH-Cys(Me) as well as workflows for the analysis of disulfide connectivities [1]. Recently, we have expanded our set of standards by two new PTH-amino acid derivatives i.e., diPTH-cystine and PTH-cysteine, which have never been established before. We solved a long-time myth whether or not cysteine can be detected in Edman degradation and which conditions are required to determine its occurrence during automated Edman sequencing. The new PTH-standards now allow for identification of specific cysteines that are still involved in disulfide bridges without any need of further sample treatment such as partial reduction [2]. This is due to the fact that a disulfide bridge protects the otherwise free thiol group of cysteine from β-elimination and degradation. This detection option perfectly matches our previous approaches focusing on (partially) reduced cysteines for disulfide bond elucidation [1].

Utilizing the new PTH-Cys-based compounds we are able to provide a workflow which combines Edman degradation with tandem mass spectrometry to facilitate standard analyses of cysteine-rich peptides and proteins as well as reliable structure elucidation.

[1] Elsayed, Y. Y., Kühl, T., Imhof, D. (2024) Edman degradation reveals unequivocal analysis of the disulfide-connectivity in peptides and proteins. Anal. Chem., 96, 10, 4057–4066.

[2] Kühl, T., Elsayed, Y. Y., Terekhov, A., Imhof, D. (2025) DiPTH-cystine and PTH-cysteine in disulfide bond analysis using automated Edman degradation. J. Pept. Sci., in revision.

P3: 21

A Novel C-terminal Fluorescent Linker for Solid Phase Peptide Synthesis.

Connor B. Smieja, Kellan Xin, Tianhui Tang and Alison N. Hulme

Company / Academic Institution

School of Chemistry, University of Edinburgh

In solid-phase peptide synthesis (SPPS), precise labelling of peptides is vital for biochemical applications but is often challenging requiring multiple optimisations. This study investigates C-terminal labelling as a solution when the N-terminal is essential for subsequent processes, such as in expressed protein ligation (EPL)[1] or sortase-mediated C-terminal protein labelling.[2] By labelling the peptide’s C-terminus during SPPS, this approach bypasses the challenges of quantitative side-chain labelling and minimises additional purification steps.

Our method uses a coumarin-loaded azobenzene moiety (COLAZ) as a linker. While azobenzene molecules are not widely used in the peptide field, they are valued for their chemical properties and stability. This pre-loaded SPPS linker remains non-fluorescent and stable under SPPS conditions. Once the peptide is fully synthesised and side chains are deprotected, the azo-benzene unit in COLAZ can be reduced under conditions that we have previously shown to be mild and biologically compatible,[3,4] to reveal the active C-terminal coumarin-labelled peptide. We demonstrated this capability with model peptides WHISKEY, VAMPYRE, and FANATICQ. These sequences cover most natural amino acids, encompass different charge states, and have varied hydrophobicity profiles; successfully synthesising and purifying these peptides demonstrates the wide substrate tolerance of the COLAZ linker in SPPS.

Utilising COLAZ as a pre-loaded SPPS linker eliminates the need for tuning labelling quantification, providing a consistent and reliable fluorescent output. This innovation offers a robust tool for tracking, visualisation, and functional analysis of fluorescently labelled peptides, significantly enhancing their utility in biomolecular research.

[1] K.A. Scheibner, Z. Zhang, P.A. Cole, Anal.Biochem, 2003, 317, 226-232.

[2] C.P. Guimaraes, M.D. Witte, C.S Theile, G.Bozkurt, L. Kundrat, A.E.M. Blom, H.L. Ploegh, Nature Protocols , 2013, 8, 1787–1799.

[3] C. Allan, M. Kosar, C.V. Burr, C.L. Mackay, R.R. Duncan, A.N. Hulme, ChemBioChem, 2018, 19, 2443-2447.

[4] F. Landi, C.M. Johansson, D.J. Campopiano, A.N. Hulme, Org.Biomol.Chem., 2010, 8, 56-59.

P3: 22

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) analogues as PAC-1 receptor antagonists

Main author: Sidra Faryal a

Co-authors: Dr. Chris Coxon a, Dr. Graeme Cottrell b

a EaStChem School of Chemistry, University of Edinburgh, The King’s Buildings, EH9 3FJ, UK

b Reading School of Pharmacy, University of Reading, Hopkins Building, RG6 6UB, UK

Company / Academic Institution

University of Edinburgh

Migraine is the most disabling neurological disorder, affecting ~15% of the global population and posing significant societal and economic burdens.1 Neuropeptides such as calcitonin gene-related peptide (CGRP) are key players in migraine pathology. Antagonising the CGRP receptor in the peripheral nervous system is a validated treatment strategy, though it benefits only ~60% of patients. Recently, other neuropeptides have gained attention as potential therapeutic targets. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that activates the G-protein coupled receptor, PAC1R. A recent clinical trial showed a monoclonal antibody (mAbs) (AG09222) binds to PACAP and prevent the onset of migraine.2 However, the blockade of PAC1 receptor using mAbs (AMG301) did not provide the expected benefits.3 Therefore, the actual receptor which PACAP binds and triggers migraine is still unknown.

In this study, we systematically truncated amino acids from the N-terminus of PACAP using solid-phase peptide synthesis (SPPS) to assess antagonist potency against PAC1R. Approximately 40 truncated analogues, ranging from PACAP 6–38 to PACAP 28–38, were synthesized and purified via preparative HPLC, achieving high homogeneity as confirmed by analytical HPLC. To further understand binding contributions, computational (BUDE) and experimental alanine scanning mutagenesis was performed on PACAP 28–38. A FRET-based cellular assay was used to evaluate cAMP accumulation, confirming the antagonistic activity of the analogues. To enhance pharmacokinetic properties, hydrocarbon stapling was introduced at various positions based on computational modelling of binding interactions and steric hindrance. Circular dichroism (CD) confirmed improved helicity in stapled peptides. Future work will focus on sequential structural modifications to evaluate antagonism across the receptor family: PAC1, VPAC1, and VPAC2, thereby advancing structure–activity relationship (SAR) studies.

P3: 23

Developing and optimising cyclic peptide inhibitors against human neuraminidases

Emma Wadforth, Sabrina Mackinnon, Qiaochu Zhang, Tom McAllister, Frances Platt, Wyatt Yue, Akane Kawamura

Company / Academic Institution

Newcastle University, Newcastle Upon Tyne, UK

Neuraminidases (NEU), otherwise known as sialidases, are responsible for cleaving terminal sialic acid residues from glycoproteins. In humans, these exist as four isoenzymes (NEU1, NEU2, NEU3 and NEU4), with differing subcellular localisation and substrate specificity. Despite having overall sequence similarity of <40%, the highly conserved active sites make finding specific binders particularly difficult. While several viral NEU inhibitors used as influenza treatments exhibit weak inhibition against some human isoenzymes, selective chemical probes for human NEU are needed as starting points for drug discovery. Studies have confirmed the implications of this enzyme class in an array of human disorders, including cancer, lysosomal storage disorders and neurodegenerative diseases. Finding selective chemical inhibitors for these enzymes could enable potential new therapeutic strategies for targeting these pathologies. 

The isoenzyme NEU2 has a published crystal structure available, and can be readily expressed and purified in E.coli. Previously in the group, thousands of cyclic peptide hits against NEU2 were discovered through mRNA display selection technology. Several of the top-most abundant binders with good chemical diversity were successfully synthesised using Solid-Phase Peptide Synthesis (SPPS). To probe their function, Differential Scanning Fluorometry (DSF) and a fluorescence-based assay was used to screen the hit cyclic peptides, with greater thermal stabilisation and enzymatic inhibition reproducibly observed, providing promising starting points for hit discovery. Co-crystal structure of NEU2 complexed with a hit cyclic peptide N2CP2 drove the hit optimisation through structure-activity relationship (SAR) studies. In addition, click chemistry was performed on hit cyclic peptide variants to allow for the incorporation of fluorescent dyes for cellular imaging. Dosing of these peptide-dye conjugates in immunofluorescence studies gave promising initial signs of peptide internalisation. This work has helped identify some of the most potent and novel NEU2 inhibitors to date; with the potential to become insightful chemical probes or scaffolds for future drug design.

P3: 24

Targeting overexpressed receptors on cancer cells with TAT cell-penetrating peptide

Alina Asandei, Alina Petre, Corina Ciobanasu

Company / Academic Institution

Alexandru I. Cuza University of Iasi

Cancer remains a major global health challenge, where early detection is critical to improving patient outcomes. Peptides targeting receptors overexpressed on cancer cells offer a highly promising strategy for precise and sensitive tumor detection. These short biomolecules can be engineered to selectively recognize malignant cells with minimal off-target effects. Their small size ensures improved tissue penetration and low immunogenicity, making them highly promising candidates for clinical applications.

The arginine-glycine-aspartate (RGD) motif, present in extracellular matrix (ECM) proteins, exhibits high binding affinity toward integrin receptors such as ανβ3, frequently upregulated in tumor vasculature and cancer cells. Neuropilin-1 (NRP-1), a transmembrane glycoprotein that functions as a co-receptor for vascular endothelial growth factor (VEGF) is frequently overexpressed in malignant tissues, where it plays a critical role in tumor progression, angiogenesis, invasion, and metastasis. Here, we report a TAT-derived peptide exhibiting dual targeting of integrin ανβ3 and NRP-1. Utilizing single molecule and confocal microscopy combined with model membrane systems, we evaluated the binding affinity and membrane-penetrating capabilities of these peptides. The dual receptor specificity, combined with efficient membrane translocation, make them potential theranostic agents and incorporating these peptides into detection and delivery platforms offers a non-invasive, highly selective strategy for both early cancer diagnosis and targeted therapies.

References:

[1] Ciobanasu, C; Pernier, J and Le Clainche, C, Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes, 2024, ChemBiochem 25 (2).

Acknowledgment:

The present study was financially supported by a grant PN-IV-P1-PCE-2023-0678 (UEFISCDI-Romania).

P3: 25

Fatty C18-C20 Di-Acids for GLP-1-like Drugs via Metathesis Technology

Cengiz Azap, PhD

Company / Academic Institution

Apeiron Synthesis

Fatty C18-C20 Di-Acids for GLP-1-like Drugs via Metathesis Technology

Glucagon-like peptide-1 receptor agonists (GLP-1RAs), which mimic the hormone GLP-1 in the body have gained growing attention recently, displaying remarkable effect in the treatment of obesity and weight control.

Fatty C18-C20 di-acids side chains display a common feature of these drugs, extending the half-life of the drugs in the body.

Apeiron Synthesis offers efficient and robust ruthenium-based metathesis catalysts of which 1 kg produces 2,817 kg of the desired di-acid starting from 10-undecenoic acid.

P4: 01

M2e Antigen-Containing Peptidyl and Peptide Amphiphile Micelles as Universal Influenza Vaccines

Megan C. Schulte, Agustin T. Barcellona, Xiaofei Wang, Adam G. Schrum, and Bret D. Ulery

Company / Academic Institution

University of Missouri

A critical problem with existing influenza vaccines is the dependence on predictions of the predominant strain for a given flu season. However, inaccurate predictions reduce vaccine efficacy and increase disease burden. Significant research efforts are being made in developing universal vaccines that harness conserved epitopes on the virus to elicit an immune response against a broad spectrum of viral strains, thereby reducing reliance on accurate predictions. One such conserved region on the influenza virus is the extracellular domain of the M2 viral envelope protein. To take advantage of this epitope, while avoiding the traditional shortcomings of peptide vaccines (including protease degradation and weak immunogenicity), we leveraged the self-assembly of peptides into micelles. In this work, we studied two vaccine formulations containing the M2(2-16) conserved antigen: the unmodified M2(2-16) peptide and an M2(2-16)-containing peptide amphiphile (Palm2K-M2(2-16)-(KE)4). We found that both the unmodified M2(2-16) peptide and the Palm2K-M2(2-16)-(KE)4 peptide amphiphile self-assembled, with each formulation exhibiting different physical characteristics that correlated with differences in the immune responses mounted against each vaccine. While the M2(2-16) peptides formed peptidyl micelles (PMs) with strong IgG responses after a prime-booster regimen, the peptide amphiphile micelles (PAMs) elicited significant IgM and IgG responses after just one dose. This study provides a proof-of-concept in which PAMs could be useful in one-dose vaccines (like in areas with inconsistent access to medical care), while both PMs and PAMs show potential for prime-booster vaccine strategies.

P4: 02

Simplified catch-and-release protocol transforms difficult-to-dissolve peptides into routine purifications

Robert Zitterbart, Stephan Luedtke, Manoj Muthyala, Dominik Sarma

Company / Academic Institution

Gyros Protein Technologies

Peptide purification remains the primary bottleneck in peptide production, despite significant advances in chemical synthesis technology. While HPLC (High-pressure Liquid Chromatography) continues to dominate, complementary purification methods are urgently needed. 

We present PEC (PurePep® EasyClean) 2.0, an enhanced catch-and-release purification protocol that eliminates the precipitation step after TFA (trifluoroacetic acid) cleavage from the SPPS (solid-phase peptide synthesis) resin, thereby addressing critical workflow inefficiencies associated with traditional methods, particularly for peptides that are difficult to dissolve.

The original PEC protocol required ether precipitation post-cleavage from the SPPS resin, introducing ether-derived aldehyde and ketone contaminations that interfered with the catch-and-release linker chemistry.[1-2] Additionally, precipitation and redissolution steps presented significant challenges for hydrophobic peptides, resulting in material loss and extended processing times.

Building on novel TFA-stable purification beads and findings by Mthembu et al. [3] demonstrating superior performance of thiol-free cleavage cocktails in suppressing back-alkylation, we developed PEC 2.0. This innovation enables direct purification from the TFA cleavage cocktail, as the absence of thiol scavengers prevents interference with the aldehyde-functionalized purification resin. The protocol leverages the TFA cocktail as an optimal peptide solvent, eliminating dissolution challenges.

Our results demonstrate comparable or superior purities and yields compared to previous protocols, with exceptional performance for hydrophobic peptides. The streamlined workflow reduces processing time and standardizes procedures across diverse peptide sequences.

This novel catch-and-release methodology represents a significant advancement in peptide purification technology. By providing a robust alternative to conventional methods that bridges synthesis and purification workflows without intermediate precipitation, PEC 2.0 addresses longstanding challenges in peptide production. The simplified protocol enables researchers to tackle increasingly complex peptide targets while maintaining high throughput and scalability, ultimately accelerating peptide drug development and research applications.

[1] J. Pept. Sci. 2018, 15, e3136

[2] Chem. Sci. 2021, 12, 2389-2396

[3] Org. Process Res. Dev. 2025, 29, 3, 691–703

P4: 03

Synthetic Peptide Peptides as Potential Therapeutics for Alzheimer's Disease

Francesca Barrera, Helena Bland, Dr Louise Serpell, and Dr Cristina Pubill-Ulldemolins

Company / Academic Institution

University of Barcelona

Alzheimer’s disease (AD) is a global health concern, affecting 55 million people worldwide. With current treatments only symptomatic in nature there remains a need for a novel therapeutic that can both alleviate symptoms and prevent disease progression. Recent studies demonstrated that short leptin peptide sequences are able to restore cognition and prevent the detrimental effects of amyloid-β accumulation in laboratory AD models. These discoveries provide an exciting new opportunity to synthetically modulate these peptides, with the goal of developing orally available therapeutics for AD. 

In our multidisciplinary research group, we utilized our in-house CEM Liberty Blue Microwave Peptide Synthesizer to efficiently generate a series of leptin peptide analogues incorporating unnatural amino acids, based on patented sequences. Through rational design, supported by in silico modelling and biological assays, we evaluated whether modifications to the native peptides improved metabolic stability while preserving their neuro-enhancing properties. At the EPSC 2025, we will present our latest findings on the design, synthesis, and biological evaluation of these synthetic leptin peptides.

P4: 04

Cell-penetrating peptides modulating extracellular matrix components

Melina Ruppel, Anna Bornikoel, Beate Eckes, Thomas Krieg, Vivek Malhotra, Ishier Raote, Ines Neundorf

Company / Academic Institution

Department of Chemistry and Biochemistry, Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany

The extracellular matrix is a dynamic network of proteins and macromolecules that plays a vital role in regulating various cellular processes. The primary constituent of both the extracellular matrix and fibrotic scar tissue is fibrillar collagen. Excessive secretion of extracellular matrix (ECM) proteins, particularly collagen, contributes to fibrotic diseases such as scleroderma. Typical symptoms include skin thickening, impaired flexion of joints and fingers, internal organ dysfunction, and muscle weakness. Despite their prevalence and urgent requirement, antifibrotic therapies remain limited.

The Transport and Golgi organization 1 (TANGO1) protein family has emerged as a key organizer of ER exit sites (ERES), forming transient inter-organelle tunnels via their ER-luminal domain. The cytoplasmic region facilitates the efficient secretion of bulky cargoes such as collagen into the ECM. A crucial step in the secretory pathway involves the interaction of the coiled-coil 2 (CC2) domains of TANGO1 with its paralog cutaneous T cell lymphoma-associated antigen 5 (cTAGE5). This initiates the formation of coat protein complex II (COPII)-coated vesicles, transferring collagen and ECM components to the extracellular space. Due to the critical role of TANGO1 proteins in collagen trafficking, their functional inhibition offers a promising strategy to reduce collagen hypersecretion.

In this work, we aimed to develop peptide-based inhibitors that specifically target the TANGO1-cTAGE5 binding interface, disrupting the interaction between these two proteins, thereby reducing excessive deposition of collagen in the ECM. For this, peptide inhibitors were designed incorporating a peptide motif targeting the TANGO1 or cTAGE5 interaction and the cell-penetrating peptide sC18*. These peptides were tested regarding their cytotoxicity, cellular uptake, and inhibitory effect on collagen I secretion in dermal fibroblasts. 

Our results demonstrate that disrupting TANGO1-cTAGE5 interaction with peptide inhibitors could offer a novel therapeutic strategy to modulate ECM secretion in fibrotic disorders.

P4: 05

Targeting fibrin with EPep: A peptide probe for stroke research

Philipp Holz, Dmitrii Antuganov, Qinyu Wang, Lukas Vieth, Bernd Neumaier, Heike Endepols, Holger Grüll, Ines Neundorf.

Company / Academic Institution

Institute of Biochemistry, Department of Chemistry and Biochemistry, University of Cologne, 50674 Cologne.

Thrombosis, defined by the intravascular formation of fibrin-rich clots, is a major driver of stroke and other ischemic thrombi worldwide. It remains a leading contributor to global morbidity and mortality. Early detection and precise localization of fresh thrombi are crucial for timely intervention. Conventional imaging modalities such as CT or MRI are often limited in identifying small or evolving infarcts. Molecular imaging with fibrin-specific probes offers the sensitivity and specificity needed to visualize thrombus formation at the biochemical level. Our overarching goal was to develop a biocompatible PET/SPECT tracer capable of early thrombus detection, real-time monitoring of clot evolution, and longitudinal assessment of therapeutic outcomes in preclinical models. Based on the EP2104R peptide, we designed EPep: a cyclic, fibrin-targeting peptide probe incorporating unnatural amino acids and a stabilizing disulfide bridge. These structural features enhance binding affinity, proteolytic resistance, and in vivo stability. EPep was synthesized via a combination of automated and manual solid-phase peptide synthesis, ensuring sequence fidelity and high yields. For multimodal imaging, a NOTA chelator was appended to facilitate radiolabeling with 68Ga and Al18F under mild conditions. In vitro assays investigated hemolytic activity and stability in human serum. In vivo evaluation in a mouse model of arterial thrombosis revealed rapid and specific accumulation of radiolabeled EPep at clot sites, with high target-to-background ratios. Ongoing investigations extend these promising findings in other thrombosis models to further characterize probe pharmacokinetics and imaging performance.

Collectively, our data underscore the potential of EPep derivatives as high-performance imaging agents for early detection and longitudinal monitoring of thrombotic and ischemic pathologies, paving the way for future clinical translation and improved patient management.

P4: 06

Chemically Tuned Peptides as Synthetic Substrates of Farnesyltransferase: A New Strategy Against Oncogenic Ras

Martin Matijass, Merlin Klußmann, Ines Neundorf

Company / Academic Institution

Institute of Biochemistry, Department of Chemistry and Biochemistry, University of Cologne, Zuelpicher Strasse 42, 50674 Cologne

Protein prenylation, particularly farnesylation, plays a key role in regulating membrane localization and activity of oncogenic Ras proteins. Building on previous findings that CaaX-motif-conjugated cell-penetrating peptides (CPPs) can hijack this modification machinery, we further developed and functionally characterized bifunctional peptides targeting this pathway. We used the amphipathic CPP sC18*, covalently linked to C-terminal CaaX motifs derived from Ras proteins, to generate synthetic prenylation substrates. These peptides showed selective uptake in cancer cell lines and decreased cellular internalization upon cysteine-to-serine mutation, confirming the significance of the CaaX motif. Extending these findings, we now show that the lead construct CaaX-1 significantly reduces plasma membrane localization of endogenous KRas in PANC-1 cells, suggesting functional competition with native substrates at the level of the farnesyltransferase.

Western blot analyses further revealed that CaaX-1 modulates Ras signaling by altering key regulatory nodes. Notably, we observed an impact on both, tumor suppressor proteins as well as Ras activators, indicating a dual impact on Ras pathway dynamics. In parallel, we synthesized a conformationally stabilized variant of the CPP segment using a dibromoxylene-based crosslinker. As a next step, this stabilized scaffold will be equipped with a CaaX motif to generate stabilized bifunctional peptides. These constructs will be characterized with the goal to enhance intracellular activity and amplify the observed biological effects. 

Together, our results demonstrate that bifunctional CaaX-peptides not only interfere with prenylation-dependent localization of KRas but also impact Ras signaling networks in cancer cells. The introduction of structural constraints via chemical crosslinking represents a promising strategy to enhance intracellular peptide stability and activity. These findings position CaaX-peptides as valuable synthetic tools for probing and modulating prenylation-dependent oncogenic signaling in live cells.

P4: 07

Rational design and synthesis of MOp3-derived peptides with enhanced broad-spectrum antimicrobial activity

Moreno Bernal Rachel; González Rocha Gerardo; Weber Paixão Márcio; Jiménez Águila Claudio

Company / Academic Institution

University of Concepción, Chile

The growing emergence of antimicrobial resistance has intensified the search for novel therapeutic agents with news mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates due to their broad-spectrum activity and low likelihood of resistance development. However, natural AMPs often suffer from low metabolic stability, cytotoxicity, and poor bioavailability. In this study, we aimed to design, synthesize, and evaluate synthetic analogs inspired by the natural peptide MOp3, previously identified from Moringa oleifera seed protein hydrolysates, to improve antimicrobial activity and stability.

Initially, alanine-scanning mutagenesis was performed on MOp3 to identify residues critical for antimicrobial activity. Seven linear peptides were synthesized via solid-phase peptide synthesis (SPPS) and evaluated against Gram-positive and Gram-negative bacteria. Replacement of Gly¹ and Met⁶ significantly reduced activity (91% and 44% inhibition loss, respectively), indicating their essential role. Based on these results, eight new MOp3-derived analogs were designed by incorporating hydrophobic (e.g., Leu) and positively charged residues (e.g., Lys, Arg), as well as D-amino acids to enhance proteolytic resistance.

Characterization was carried out by analytical RP-HPLC and completed via mass spectrometry. Circular Dichroism spectroscopy indicated flexible secondary structures, predominantly random coil with partial α-helix and β-turn characteristics. Antimicrobial activity was assessed using broth microdilution against S. aureus, Kocuria spp, S. Typhimurium and E. coli. Notably, analogs 4 and 8 exhibited MIC values of 0.5 mg/mL against S. aureus and Kocuria spp., while analog 8 showed MIC values of 1 mg/mL against E. coli and S. Typhimurium, representing a fourfold improvement compared to the MIC values reported for the native MOp3 peptide.

These findings highlight the potential of rational peptide design to enhance AMP properties, supporting the development of effective alternatives to conventional antibiotics.

P4: 08

Desulfurative borylation enables efficient synthesis of boronoalanine

Kohei Yamada, Jeongsu Lee, Tomoko Sasaki, and Yasuchika Yamaguchi

Company / Academic Institution

Faculty of Pharmaceutical Sciences, Nagasaki International University

Native chemical ligation (NCL) is widely used in the synthesis of polypeptides. While NCL relies on the presence of cysteine at the ligation site, there are cases where cysteine is not required in the final peptide. Developing methods to convert these cysteine residues to natural or non-natural amino acid residues could greatly advance peptide drug discovery. We thought that the conversion of the C-S bond of a cysteine residue to a C-B bond would allow access to amino acid residues with arbitrary substituents via various boron transformations. Although several desulfurative borylation methods have been reported, many of them require multi-step procedures or specific conditions such as white light irradiation. To address these limitations, we developed a simple method that avoids such additional steps. In this presentation, we will discuss the reaction conditions, substrate scope, and proposed mechanism.

P4: 09

Development of a microfluidic platform towards high-throughput synthesis and screening of antimicrobial peptides

Amalie K. Gudum, Tomas Sneideris, Klavs Jermakovs, Lydia Good, David R. Spring, Tuomas Knowles

Company / Academic Institution

University of Cambridge

Antimicrobial resistance (AMR) poses an ever-growing threat to global health and the search for new antibiotics has been marked as a top priority by the World Health Organization (WHO). Antimicrobial peptides (AMPs) represent a promising field within antibiotics, exhibiting high activity, low cytotoxicity and increased resilience towards antimicrobial resistance. Recent research has suggested that certain antimicrobial peptides work intracellularly by forming condensates with biomolecules such as RNA and DNA through liquid-liquid phase separation. By sequestering RNA and DNA in condensates they will no longer be available for translation and transcription which ultimately leads to cell death. This poster will show the development of a large-scale synthesis and screening platform targeting the formation of RNA condensates towards the discovery of new antimicrobial peptides. The platform is based on droplet microfluidics, which enables the individual compartmentalisation of large-scale compound libraries within hours. The platform will support the synthesis of millions of peptides and enable high-throughput screening to identify phase separation-prone amino acid sequences. Furthermore, the platform will aid in elucidating trends linking amino acid sequences with other bactericidal mechanisms of action such as cell lysis and inhibition of expression.

P4: 10

Palladium-mediated S-arylation for late-stage peptide functionalisation and protein conjugation

Ross Ballantine, Mélanie Chenon, Massimiliano Beltramo, Vincent Aucagne and Samir Messaoudi

Company / Academic Institution

CNRS CBM

The chemoselective functionalisation of peptides and proteins through manipulation and modification of the integral components within a complex polyfunctional system represents a key area of research for the elucidation of biological mechanisms and generation of potential pharmaceuticals. 

Messaoudi and coll. recently reported the palladium catalysed Buchwald-Hartwig-Migita (BHM) coupling of thiosugars as well as aryl- and alkyl-thiols with various iodoarenes at room temperature. The coupling involves the use of the Pd G3 XantPhos as a precatalyst and exhibits a high functional group tolerance (1). The synthetic utility of this method was subsequently expanded by using semi-aqueous conditions to perform various conjugation reactions on unprotected para-iodophenylalanine (pIPhe) containing peptides, with various glycosyl thiols (2).

Herein, we report the extension of this BHM coupling beyond thioglycoconjugation. We applied these novel methodologies to the labelling of a biologically relevant peptide, kisspeptin-10, and evaluated the binding of the peptides to their receptor (hKISS1r) in vitro. To demonstrate the impressive functional group tolerance, we then applied the methodology to a mono-iodo-labelled SUMO-2 and showed site-selective labelling with high rates of conversion.

In conclusion, we have shown that the methodology previously used to obtain thioglycoconjugates has a much broader application as a tool for late-stage peptide and protein functionalisation. 

[1] A. Bruneau, M. Roche, A. Hamze, J.-D. Brion, M. Alami, S. Messaoudi, Chem. Eur. J., 2015, 21, 8375 – 8379. [2] D. Montoir, M. Amoura, Z. El Abidine Ababsa, T. M. Vishwanatha, E. Yen-Pon, V. Robert, M. Beltramo, V. Piller, M. Alami, V. Aucagne, S. Messaoudi, Chem. Sci., 2018, 9, 8753 – 8759.

P4: 11

Applicability of solvent-free, catalytic approaches to coupling and deprotection steps in peptide-, glycopeptide- and peptidomimetic synthesis

Serena Traboni, Fabiana Esposito, Emiliano Bedini, Alfonso Iadonisi

Company / Academic Institution

Department of Chemical Sciences, University of Naples Federico II, Via Cinthia 4, 80126 Naples, Italy

The need for more sustainable methods in organic synthesis warrants the search for greener and safer alternatives to classic organic solvents. In this frame, solvent-free protocols, featuring enhanced reaction efficiency and a low economic/environmental impact, have also been developed, although hardly applicable to complex compounds. [1] Our effort has been recently focused on original solvent-free procedures for various organic reactions, culminating with the first examples of solvent-free approaches for the construction of key natural structural motifs, encompassing glycosidic, ester and amide bonds. Herein presented there is the first solvent-free revisiting of the carbodiimide coupling chemistry and its versatile application to the synthesis of both ester and amide (including peptide) linkages. [2] It is also discussed our most recent finding introducing the first catalytic, solvent-free protocol for the cleavage of the popular 9‑fluorenylmethoxycarbonyl (Fmoc) protecting group, with selected examples of the one-pot construction of glyco-peptide and depsi-peptide model structures based on the merging of the abovementioned procedures.[3] These methodologies may offer convenient alternatives to routinely adopted protocols for various synthetic applications, also in the field of peptide synthesis, where the search for more efficient and sustainable procedures at different key stages such as coupling and deprotection reactions, is a fundamental goal.

[1] S. Zangade et al, A Review on Solvent-free Methods in Organic Synthesis. Curr. Org. Chem. 2019, 23, 2295.

[2] S. Traboni et al, A comprehensive solvent-free approach for the esterification and amidation of carboxylic acids

mediated by carbodiimides. Tetrahedron 2023, 133, 133291.

[3] S. Traboni et al, Catalytic Cleavage of the 9‑Fluorenylmethoxycarbonyl (Fmoc) Protecting Group under Neat

Conditions. Org. Lett. 2024, 26, 3284.

P4: 12

Advancing peptide synthesis: Continuous-flow SPPS on silica support

Beáta Mándityné Huszka1,2, Steeves Potvin3, Georges Thibaut Koumba3, Brunello Nardone3, Eric Beausoleil3, Francois Beland3, and István M. Mándity1,2,4

1HUN-REN Artificial Transporter Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, 1117 Budapest, Hungary
2Ladon Therapeutics Inc, 6726 Szeged, Hungary
3SiliCycle Inc. 2500 Parc-Technologique Blvd. Quebec City, QC, G1P 4S6, Canada
4Institute of Organic Chemistry, Faculty of Pharmacy, Semmelweis University, 1085 Budapest, Hungary

Company / Academic Institution

Semmelweis University Institute of Organic Chemistry

The synthesis of peptides is crucial due to their roles in peptide-based medicines and drug discovery. Since its introduction by Merrifield, peptide synthesis has primarily been carried out on solid supports, and solid-phase peptide synthesis (SPPS) has undergone significant advancements. However, traditional SPPS methods still require a large excess of amino acids, and the widespread use of green solvents remains a challenge.
Recently, continuous-flow (CF) techniques have gained prominence in synthetic methodologies. Here, we demonstrate that by applying CF technology and optimizing reaction parameters, the amino acid equivalents required for SPPS can be drastically reduced to approximately 1.5 equivalents. [1] Additionally, the use of propylene carbonate (PC), regarded as one of the greenest solvents, was implemented in SPPS, leading to successful scale-up. [2]
We present a novel approach to solid-phase peptide synthesis (SPPS) utilizing silica-based particles functionalized with Rink Amide linkers. These supports, such as SiliCycle’s SiliaSphere™ Amide particles, offer enhanced mechanical and thermal stability compared to conventional polystyrene resins, enabling seamless integration into continuous-flow synthesis platforms. Their defined porosity and narrow particle size distribution support efficient coupling, reduced backpressure, and improved compatibility with automation. Notably, this support performs well in green solvent like PC, contributing to more sustainable synthetic workflows. Standard TFA-based cleavage protocols remain applicable, and the typical loading range (0.2–0.6 mmol/g) supports scalable peptide production. This technology provides a promising foundation for environmentally conscious and flow-compatible peptide manufacturing.
Complete reaction parameter optimization was performed followed by the synthesis of some α-peptides and foldamers.

References
[1] Mándity, I. M.; Ötvös, S. B.; Fülöp, F. ChemSusChem 2014, 7, 3172.
[2] Varró, N.; Mándityné Huszka, B.; Erdei, E.; Mándoki, A.; Mándity, I. M. Chemistry‐Methods, 2025, 2500010.

P4: 13

Potential therapeutic of peptide-ionic liquid conjugates

Ana Gomes a,* , Ermelindo C. Leal b,c,d,*, Jessica da Silva b,c,e, Inês Teixeira b,c, Ricardo Ferraz a,f,g, Daniela Calheiros b,c,h, Teresa Gonçalves b,c,i, Eugénia Carvalho b,c,d, Paula Gomes a 

a LAQV-REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal
b CNC-UC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
c CIBB – Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal
d Institute of Interdisciplinary Research, University of Coimbra, Coimbra, Portugal
e University of Coimbra, Institute of Interdisciplinary Research, Doctoral Program in Experimental Biology and Biomedicine (PDBEB), Coimbra, Portugal
f Center for Translational Health and Medical Biotechnology Research (TBIO)/Health Research Network (RISE-Health), ESS, Polytechnic of Porto, Porto, Portugal
g Polytech Institute of Porto, School of Health, Chemical & Biomolecular Sciences, Porto, Portugal
h Faculty of Medicine of the University of Coimbra, Doctoral Program in Health Sciences (PDDHS), Coimbra, Portugal
i Faculty of Medicine of the University of Coimbra, Coimbra, Portugal

Company / Academic Institution

LAQV-REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto

Chronic wounds, such as diabetic foot ulcers, present significant therapeutic challenges due to their impaired healing and high susceptibility to infection. Our research focuses on the design and development of peptide-based conjugates for topical application in the treatment of such complex skin conditions. We have advanced peptide-ionic liquid conjugates (PILCs) as promising active pharmaceutical ingredients. These conjugates combine a short cosmeceutical peptide known for its collagen-promoting properties, with an ionic liquid moiety that possesses inherent antimicrobial activity and could act as a dermal permeation enhancer.
Encouraged by our previous in vitro findings, demonstrating antibacterial, antifungal, and collagen-inducing properties,1 we selected the most potent PILC candidate for an in vivo evaluation in a streptozotocin (STZ)-induced type 1 diabetic mouse model.2 The results obtained so far indicate significant potential for promoting wound healing in this context. This presentation will highlight the in vivo therapeutic outcomes. 

References 
1. Gomes, A.; Bessa Lucinda, J.; Fernandes, I.; Aguiar, L.; Ferraz, R.; Monteiro, C.; Martins, M. C. L.; Mateus, N.; Gameiro, P.; Teixeira, C.; Gomes, P., Boosting Cosmeceutical Peptides: Coupling Imidazolium-Based Ionic Liquids to Pentapeptide-4 Originates New Leads with Antimicrobial and Collagenesis-Inducing Activities. Microbiology Spectrum 2022, 10 (4), e02291-21.
2. Gomes, A.; Leal, E. C.; Da Silva, J.; Teixeira, I.; Ferraz, R.; Calheiros, D.; Gonçalves, T.; Carvalho, E.; Gomes, P., Enhancement of wound healing in diabetic mice by topical use of a peptide-ionic liquid conjugate. The International Journal of Biochemistry & Cell Biology 2025, 180, 106753.


Acknowledgements:
AG thanks PT national funds (FCT/MECI, Fundação para a Ciência e Tecnologia and Ministério da Educação, Ciência e Inovação) for AGs contract 2022.08044.CEECIND/CP1724/CT0004 (DOI: https://doi.org/10.54499/2022.08044.CEECIND/CP1724/CT0004) and for the exploratory project reference 2023.12331.PEX, (DOI https://doi.org/10.54499/2023.12331.PEX). 

P4: 14

Title to follow...

Zeynep Kanlidere

Company / Academic Institution

ACIBADEM UNIVERSITY, Istanbul, Turkey

P4: 15

Green Peptide Synthesis Powered by Electrochemical Amino Deprotection

Esteban Suárez-Picado and Lara R. Malins

Company / Academic Institution

Australian National University

Peptide synthesis technologies are in high-demand for the efficient production of complex bioactive peptides, which are p romising alternatives to small-molecule drugs.Traditional synthetic methods, including liquid-phase and solid-phase synthesis, rely on protecting groups to precisely control the chemo- and regioselectivity of reactions. However, selecting appropriate orthogonal protecting groups can be challenging, especially for increasingly large and complex peptides. Orthogonal protecting groups that are compatible with Fmoc-based SPPS and amenable to selective deprotection are particularly attractive. While advancements have been made, traditional deprotection methods often involve excess reagents or harsh conditions, limiting the sustainability and efficiency of peptide synthesis.
To address this challenge, electrochemically sensitive protecting groups (e-PGs) stand as a milder alternative. In this study, we explore the application of an electrochemically cleavable amine protecting group in Fmoc-SPPS. We designed a model peptide containing five distinct protecting groups: Fmoc, Alloc, Boc, Dde, and one of two electroactive 1,3-dithiane-based groups—2-(hydroxymethyl)-1,3-dithiane (Dmoc) and dimethyl-1,3-dithian-2-ylmethyl (dM-Dmoc). This design enabled investigation of the selective electrochemical deprotection of the 1,3-dithiane-based groups (Dmoc and dM-Dmoc). By varying electrochemical conditions, we optimized the orthogonal deprotection of Dmoc using a Ni/Ni electrode pair in MeCN/NaAc, achieving isolated yields of 57-62%, while leaving all other protecting groups intact. This approach was successfully applied to complex bioactive peptides, demonstrating its effectiveness. However, deprotection of the more sterically hindered dM-Dmoc group proved more challenging, highlighting the need for further optimization.
Overall, our research shows that electrochemistry is a promising tool for efficient peptide synthesis, offering an alternative to traditional methods. This approach aligns with the principles of green chemistry by reducing the need for harsh chemicals and minimizing waste, contributing to more sustainable and environmentally-friendly processes for peptide synthesis.