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

JPT mascot with bicycle- macrocyclic peptidesCyclic 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

Disulfide Bridge Peptides

Disulfide cyclization is the most common type pf peptide cyclization type found in nature. Cys-Cys-cyclized peptides are also highly attractive for drug discovery, either as drugs themselves (such as the recently approved Vosoritide or Linaclotide) or as readily available screening libraries. JPT can produce hundreds of thousands of disulfide-bridged peptides at unmatched speed and costs.
disulfide bridge macrocyclic peptides


Thioether Cyclization Peptides

Thioether cyclization is often used to combine the advantages of cyclic peptides (rigidity, availability, target binding) with robust cyclization chemistry. Typical reagents used are chloroacetic acid and 1,4-Bis(bromomethyl)benzene, both of which react selectively with cysteine residues under appropriate conditions.
thioether cyclized peptidesthioether macrocyclic peptides

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.
amide-bond-cyclized-peptides

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

Bicyclic Peptides

Peptides can be cyclized to form a chemically constrained structure consisting of two cycles. This is usually done by cyclizing the peptide with a trivalent reagent such as TBMB (CAS 18226-42-1) or TATA (CAS 959-52-4). This has several advantages, including the ability to target two receptors simultaneously.

bicyclic-peptides by thioether cyclization


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. Semaglutide, for example, is an engineered version of the natural peptide GLP-1 for the treatment of diabetes. Compared to GLP-1, some amino acids have been mutated to reduce protease degradation, and conjugation to a lipid chain increases albumin binding reduces clearance. These few modifications have improved 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.

Cyclic Peptide Applications 

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 RoseTTAFoldRFdiffusion, and I-TASSER. Several databases have been established for the design of peptides as protein-protein interaction (PPI) inhibitors, including PPI-HotSpotDBASEdbSKEMPI 2.0 and 2P2I.

Macrocyclic Peptides as Protein-Protein Interaction Inhibitors 

protein-protein interaction inhibitonMacrocyclic 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

Macrocyclic peptides are also excellent delivery platforms. Based on the same principle as antibody-drug conjugates, peptide-drug conjugates have been widely developed. They are easier to manufacture and have valuable targeting, binding and penetration properties. Cyclic peptides can be conjugated to cytotoxic payloads such as MMAE, to oligos (PNA, siRNA…) or to radiotherapeutic agents (68Ga-DOTA, 68Ga-NOTA…). Examples are the radiolabeled somatostatin analogues DOTA-TOC, DOTA-NOC and DOTA-TATE, which are widely used for cancer molecular imaging and/or targeted radionuclide therapy (TRT). 


Custom Synthesis of Macrocyclic Peptides

Cyclic Peptide Manufacturing Capabilities 

JPT Peptide Technologies offers unique high-throughput peptide synthesis capabilities for microgram and milligram scale synthesis perfectly suited for the production of custom linear or cyclized peptide libraries (disulfide bridges, thioether cyclization, amide cyclization…). With a wide range of peptide synthesizers, preparative HPLCs and respective analytical systems, JPT can ensure the production of simple to complex libraries in a short and reliable timeframe. The company can produce tens of thousands of purified peptides on a mg scale and 1 million crude peptides on a µg scale per year. Beyond lab scales, JPT can produce cyclic peptides at multigram scale for advanced candidates, including pre-GMP and clinical quality grade (line clearance, CMC files…). JPT is your partner of choice to manage individual projects up to annual contracts. 

Quality Management 

The production of large custom peptide libraries is a quality challenge to avoid any errors. Since 2004, JPT has developed a strong QMS (ISO9001 certified) including a very robust LIMS system. Each batch is fully automatically recorded, tracked and released by authorized managers. Peptides are controlled at various stages of the production up to final QC using high resolution LCMS systems. With in-depth analytical expertise and AI-assisted solutions, JPT ensures the best-in-class determination of peptide integrity and purity rate. Learn more about our Quality Mangement System.

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/ 

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