Macrocyclic Peptide Synthesis Service

JPT Peptide Technologies is a high-throughput peptide synthesis company producing up to 1 million peptides per year. The company combines 20 years of expertise with extensive manufacturing capabilities, allowing it to offer everything from small-scale to large-scale peptide syntheses. Besides macrocyclic peptides we offer many other peptide modifications and post-translational modifications as well as peptide analysis services.

Overview of Macrocyclic Peptides

Understanding the Role of Cyclic Peptides in Nature and Therapeutics

JPT mascot with bicycle- macrocyclic peptides

Cyclic peptides occur naturally in animal toxins (chlorotoxin, conotoxin …), as antimicrobial peptides (e.g. defensins) or as hormones (e.g. oxytocin). In most cases, natural cyclization is achieved by the formation of a single or multiple disulfide bridges. Disulfide-bridged peptides are known to be highly resistant to protease degradation and to have high binding selectivity for their target. Given the advantages of cyclic peptides, scientists have worked extensively to use cyclic peptides in the development of therapeutic applications. Several methods of cyclization have been developed for this purpose. Other important recent developments in cyclic peptide drug discovery include innovative screening and structure prediction technologies.

Macrocyclic Peptide Structures

1. Disulfide Bridge Peptides

Disulfide cyclization, typically between cysteine residues, is the most prevalent form of peptide cyclization found in nature. These Cys-Cys-cyclized peptides are particularly appealing for drug discovery, serving as both drug candidates and as components of screening libraries. JPT excels in synthesizing hundreds of thousands of disulfide-bridged peptides with unmatched speed and cost-effectiveness.

2. Thioether Cyclization Peptides

Thioether cyclization offers the dual benefits of peptide rigidity and enhanced target binding, using reagents like chloroacetic acid and 1,4-Bis(bromomethyl)benzene. These reagents selectively react with cysteine residues, making thioether-cyclized peptides a reliable choice for various applications.

3. Amide Bond / Lactam Bridge Cyclized Peptides

Cyclization of peptides via amide bond formation can be performed at different positions within the peptide, resulting in head-to-tail, side-chain to side-chain, or side-chain to side-chain cyclized peptides. This type of cyclization is very popular because the chemistry is robust and the amides formed resemble the natural peptide backbone. An example is the recently approved Bremelanotide, which is a 7-meric peptide made by side-chain to side-chain cyclization.

4. Stapled Peptides

Peptides can be cyclized by the ring-closing metathesis (RCM) reaction, which, by using the right building blocks and positioning them within the peptide, allows an alpha-helix to be stabilized. The concept was introduced about 15 years ago and has since yielded several promising drug candidates.

stapled peptides as macrocyclic peptides

5. Bicyclic Peptides

Bicyclic peptides feature two chemically constrained cycles, typically achieved using trivalent reagents like TBMB (CAS 18226-42-1) or TATA (CAS 959-52-4). This structure offers significant advantages, including the ability to simultaneously target two receptors, making bicyclic peptides a valuable tool in drug design.

bicyclic-peptides by thioether cyclization

6. Click-Chemistry Cyclization

Peptide cyclization by click chemistry is popular because it results in a stable bond and can be conveniently performed on peptides that are unprotected on their side chain. Therefore, such peptides are widely used, especially in screening libraries. JPT can routinely synthesize large numbers of such peptides.

click chemistry cyclized peptide by triazole cyclization

Pharmacokinetics of Macrocyclic Peptides

The use of peptides as therapeutics has long been discouraged due to their pharmacokinetic properties. The typical size of peptides used for therapeutic applications is in the range of 1000-4000 g/mol, which is outside of Lipinski’s rule of five (i.e. not suitable for absorption) and at the same time too small to avoid renal clearance. Peptides are also naturally sensitive to proteases. Thus, oral administration has long been considered impossible, while parenteral administration has required frequent injections due to the limited half-life. However, the potential of peptides and their chemical manufacturability make them an invaluable modality as therapeutics – so scientists are actively working to develop new technologies to overcome these limitations. For example, conjugating a lipid chain can increase albumin binding, which reduces renal clearance. These few modifications may improve the half-life from minutes to a week. Peptide cyclization has been found to be a another promising improvement for oral administration due to appropriate absorption and stability properties. Peptides such as cyclosporins or more recently MK-0616 are examples for oral cyclic peptides. Many more are under development for various applications.

Applications of Macrocyclic Peptides

Macrocyclic Peptide Design

Over the last decade, major advances in phage display technologies, mRNA display, and in silico drug design have allowed millions of new structures to be generated with unprecedented speed, providing access to a new chemical space faster than ever before. Several software tools have been developed for predicting the three dimensional space of peptides/proteins, including AlphaFold, which has received tremendous attention recently. Other prediction tools include RoseTTAFold, RFdiffusion, and I-TASSER. Several databases have been established for the design of peptides as protein-protein interaction (PPI) inhibitors, including PPI-HotSpotDB, ASEdb, SKEMPI 2.0 and 2P2I.

Macrocyclic Peptides as Protein-Protein Interaction Inhibitors

protein-protein interaction inhibiton

Macrocyclic peptides are most commonly used to inhibit intracellular protein-protein interactions (PPIs) or as peptide-drug conjugates for targeted therapies. Macrocyclic peptides are small structures that can penetrate cells and selectively bind to specific protein domains due to their complex tridimensional structure. As a result, they are valuable candidates for targeting hotspots and inhibiting intracellular protein-protein interactions, such as preventing a transcription factor from binding DNA. Examples of macrocyclic peptides targeting PPIs in cancer have been intensively reviewed, and include the approved drugs pasireotide and lanreotide as well as many macrocyclic peptides in clinical development, including inhibitors of integrins, CXCR4 and MDMX/MDM2.

Macrocyclic Peptides as Drug Carriers

Cyclic peptides are increasingly being used as drug delivery platforms, similar to antibody-drug conjugates but with easier manufacturability. These peptides can be conjugated to cytotoxic agents, oligonucleotides, or radiotherapeutic agents like 68Ga-DOTA and 68Ga-NOTA, enhancing their targeting and penetration capabilities. Radiolabeled somatostatin analogues such as DOTA-TOC, DOTA-NOC, and DOTA-TATE are prime examples of cyclic peptides used for cancer molecular imaging and targeted radionuclide therapy (TRT).

Custom Synthesis of Macrocyclic Peptides

Cyclic Peptide Manufacturing Capabilities

At JPT Peptide Technologies, we offer unparalleled high-throughput peptide synthesis capabilities, ideal for producing custom linear or cyclized peptide libraries on both microgram and milligram scales. With our extensive range of peptide synthesizers, preparative HPLCs, and analytical systems, we can deliver even the most complex peptide libraries quickly and reliably. Whether you need thousands of purified peptides at the milligram scale or millions of crude peptides at the microgram scale, JPT is your trusted partner for high-quality, custom cyclic peptide synthesis.

Quality Management

Ensuring Precision and Reliability in Peptide Production

Producing large custom peptide libraries requires stringent quality control to ensure accuracy and consistency. JPT has developed a robust Quality Management System since 2004, incorporating a highly reliable LIMS system. Every batch is meticulously recorded, tracked, and released by authorized managers. Our peptides undergo rigorous quality checks using high-resolution LCMS systems, ensuring the highest standards of peptide integrity and purity. Our AI-assisted solutions further enhance the accuracy of our analytical processes.

Incorporation of Non-natural Amino Acids & Conjugates

JPT is strongly committed to providing an infinite level of customization. With a flexible structure and broad expertise, JPT can adapt to any need including the incorporation of non-natural amino acids into peptide libraries. With a large number of amino acids in stock and a dedicated sourcing department (AEO certified to ensure fast customs clearance), JPT ensures rapid sourcing of non-natural AAs. The company routinely incorporates azido-AAs, N-Methyl AAs, homo AAs… and conjugates chelators such as DOTA (tetraxetan) / NOTA to create precursors for Gallium 68 radiolabeling.

Compound Management Service

JPT’s customization service also includes the creation of libraries tailored to the needs of compound management departments. Do you need specific tubes, racks, 2D barcodes? JPT can adapt to any need to make the compound management process smoother. The Fill & Finish department is equipped with automated pipetting robots, labelers, and barcode readers, and data management and packaging can be carried out in a cleanroom. Peptides can be supplied as powder or ready-to-use solutions. JPT can perform solubility screening studies and cytotoxicity studies on single peptides and peptide libraries.

Take a look at some of our partners and collaborators who have already benefited from our services!

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References

https://www.nature.com/articles/s41589-023-01496-y
https://www.nature.com/articles/s41392-022-00904-4
https://pubs.acs.org/doi/10.1021/jacs.8b13178
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199541/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8372203/