Synthesis of Peptide-Peptide Nucleic Acid Conjugates

Peptide nucleic acids (PNAs), developed by Peter Nielsen in the early 90’s, constitute synthetic analogues of DNA and RNA, where the phosphodiester backbone of these natural oligonucleotides (ON) is replaced by a pseudopeptide structure. PNAs hybridize with complementary nucleic acid segments with higher affinity and specificity than natural ON, interfering with higher efficiency with target genes. Moreover, the unnatural backbone of PNAs makes them highly resistant to enzymatic degradation.

These characteristics make them valuable tools for various applications, from diagnostics to therapeutics. Limitations to their versatility include poor water solubility and low nuclear targeting capability. However, these have been successfully circumvented by the introduction of chemical modifications to PNA sequences, such as cell-penetrating peptides, which have been proven to be highly valuable in drug delivery and gene therapy. Just as for peptides, PNA can be obtained through standard SPPS procedures.

View this recording to hear a brief introduction of these ON analogues and address the synthesis of a target peptide-PNA sequence, particularly highlighting the differences between manual and automated approaches, using Symphony® X peptide synthesizer from Gyros Protein Technologies, and the challenges we faced during this process.

Dr. Luísa Aguiar,
Post-Doctoral researcher at Paula Gomes Lab, University of Porto


Development, optimization and scale up of therapeutic peptides: approaches to solid phase synthesis and purification

Recorded presentation from TIDES Digital Week

The application and reliance on Solid Phase Peptide Synthesis (SPPS) has increased dramatically over recent years with the rise of peptides in diagnostic and therapeutic research. With this rapid increase in development activity, automated synthesis methods have had to adapt to be compatible with the wide range of coupling chemistries, reaction conditions and synthesis monitoring available to the practicing chemist. The subsequent purification of the synthesized peptides in that process demands rapid scale-up what is easily achieved using the Peptide Easy Clean (PEC) technology. The need for parallel, simultaneous reaction screening to optimize SPPS and purification conditions has never been greater. Here we describe the automated screening of multiple reaction conditions for the synthesis of a selection of therapeutic peptides, enabling the optimum synthetic conditions to be selected for each target, followed by the scale-up of the synthesis on a pilot-scale instrument. The rapid purification from mg to gram-scale using the PEC technology is show-cased on Liraglutide

Andrew Kennedy, Ph.D.
Global Product Manager (Peptides Business)
Gyros Protein Technologies

Dr. Robert Zitterbart
Co-Founder, Head of R&D
Belyntic GmbH, Berlin


Peptide Therapeutics: Discovery, Development and Manufacture

Recorded Webinar 

Peptide-based therapeutics are increasingly being seen as the future for many drug development programs, and these compounds can be found in many clinical therapeutic areas such as cancer vaccines, personalized medicines, and treatments for metabolic and cardiovascular diseases. But how do researchers in academia and industry chose a target peptide for a particular therapy? What challenges do they face when chemically synthesizing peptides as part of process development, or manufacture? Join this panel discussion where selected experts in the field will discuss these topics and answer your questions! 


  • Learn from experts in the field of peptide drug development, discovery and chemical synthesis. 

  • Understand what factors influence target selection. 

  • Learn how to overcome solid phase peptide synthesis challenges such as increasing purity and yield of the target product, and reducing impurities and side-products. Learn how to optimize your peptide synthesis with a focus on scale-up and manufacture. 

How can we improve the purification process? This webinar will look at current techniques in purification, including Peptide Easy-Clean (PEC) Technology. 


Bigger, Better, Faster: New Tools for Chemical Protein Synthesis

Recorded Webinar 

The Kay lab at the University of Utah is focused on D-peptide inhibitor development, which requires the chemical synthesis of mirror-image protein targets. This webinar will describe the use of recently developed chemical and computational tools to accelerate chemical protein synthesis of large proteins. Specific topics will include recently developed next-generation “helping hand” traceless linkers to improve peptide solubility, the use of traceless templating to accelerate ligations, and our automated ligator program (Aligator) for prediction of the optimal synthetic routes.


  • “Helping hand” traceless linkers are a valuable tool for solubilizing difficult peptide segments.
  • Templated ligations greatly accelerate ligations and can rescue otherwise suboptimal ligation junctions.
  • Computational simulation of potential chemical protein synthesis routes can be used to predict optimal synthesis strategies.

Michael S Kay MD/PhD
Biochemistry, University of Utah


SPPS Tips for Success: Optimizing Synthesis of SARS-CoV-2 Epitopes for GMP Manufacture

Recorded webinar

Therapeutic peptides have many advantages, including high activity, broad chemical and biological diversity, and low toxicity. These and the relative ease and low cost of peptide manufacture compared to protein-based biologicals has meant that therapeutic peptides are now being used to treat a range of conditions, including metabolic diseases, cancer, cardiovascular, and infectious diseases.

The speed and flexibility of peptide synthesis is a major advantage when handling rapidly evolving conditions, such as neoantigen peptide-based vaccines for the individualized immunotherapy of certain forms of cancer. The dynamics of neoantigen presentation by the tumor cells demands high peptide purity and yield, and also the ability to quickly synthesize many peptides in parallel for timely treatment. Such performance will be invaluable in fighting COVID-19.

This session in the “SPPS Tips for Success” webinar series will focus on the synthesis  of 24 SARS-CoV-2 peptides with therapeutic potential which represents a real-world example of synthetic challenges brought by a diverse range of sequences (based on published work by Grifoni and colleagues (1)). Other applications covered in this presentation include the synthesis of GLP-1 receptor agonists as an important treatment for type 2 diabetes, and the synthesis of neoantigen peptides for metastatic melanoma cell therapy development (2). In addition to application data, here we also discuss features of peptides synthesizers that are critical for cGMP and regulatory compliance when manufacturing peptide products for clinical use. Which hardware options are important? What software features should be included, such as designing for 21CFR Part 11 compliance? And what needs to be included in an IQ/OQ package for a peptide synthesizer instrument.

(1) Grifoni, A. et al. Cell Host & Microbe 27, 671–680 April 8, 2020
(2) Lu, Y et al. Clin Cancer Res. 2014 July 1; 20(13)


  • Understand how peptide-based epitopes and therapeutics are synthesized for COVID-19 and Neoantigen applications, as well as GLP-1 agonists therapeutics for type-2 diabetes treatments.

  • Learn how to optimize peptide syntheses through reagent and condition screening.

  • Understand which factors are important for the manufacture of clinical peptides under cGMP conditions.

Dr. Cyf Ramos-Colon, Senior Scientist,
Gyros Protein Technologies


The use of parallel peptide synthesizers in the manufacture of long and complex peptides and high throughput manufacture

Recorded Webinar from TIDES Digital Week

Reliance on Solid Phase Peptide Synthesis has increased dramatically over the decades to keep pace with the rise in the use of peptides in diagnostic and therapeutic research. In parallel, approaches to automated synthesis have evolved in order to be compatible with a diverse range of coupling chemistries, reaction conditions and synthesis monitoring. Automated peptide synthesises must be reliable, robust, fast, and easy to use.

Almac as a peptide CMO, has used parallel synthesisers for more than 15 years. In that time, we have developed methodology to manufacture very long and complex peptides, and we will present some examples in this filed. More recently, we have applied automated parallel synthesisers in a GMP setting in the manufacture of clinical grade peptides for use in vaccine products.

Alastair Hay, Account Manager
Peptides, Almac Group Ltd.


SPPS Tips for Success: Strategies for Minimizing Side-Reactions

Recorded Webinar 

As peptide chemists strive to achieve new methods and techniques for solid phase peptide synthesis (SPPS), strategies must also be implemented to help reduce the occurrence of unwanted side reactions that inevitability take place during the chemical synthesis process. Attend this free webinar to learn how to implement preventative measures to help reduce or eliminate unwanted side products from forming.

Lukasz Frankiewicz PhD, Senior Product Specialist
Gyros Protein Technologies


Venom Peptides: Rethinking Voltage-Gated Sodium Channel Inhibition

Recorded Webinar

In this webinar, delineating the mechanism of action behind venom peptide inhibition of voltage-gated sodium channels will be presented. This will include discussion on engineering peptides to achieve subtype specificity and complete inhibition of specific sodium channels to unlock the potential of potent venom peptides as therapeutic leads for the treatment of pain.

Christina I. Schroeder, Stadtman Investigator,
National Cancer Institute, National Institutes of Health


SPPS Tips for Success: Designing a Synthesis

Recorded Webinar

With the increasing chemical complexity of therapeutic peptides, how can you maximize the likelihood of success when developing a solid phase synthesis protocol to automate synthesis for a new peptide sequence? View this webinar to learn how to design SPPS synthesis protocols, analyze sequences and understand which factors influence decisions such as resin selection, choice of chemistry and coupling protocol.

Cyf Ramos-Colon PhD
Senior Scientist
Gyros Protein Technologies


Sequence-Specific “Peptoids” for Antifouling, Antibacterial and Self-Assembly Applications

Recorded Webinar

In this webinar, sponsored in partnership with Xtalks K.H Aron Lau will highlight recent efforts in exploring solid-phase synthesized peptoids for applications beyond their conventional use in combinatorial drug discovery. 

K.H Aaron Lau, PhD
Senior Lecturer (Associate Professor),
Department of Pure and Applied Chemistry,
University of Strathclyde, Glasgow, Scotland, United Kingdom


Sustainability Considerations in Peptide Chemistry

Recorded Webinar

In this webinar, sponsored in partnership with Xtalks, Craig Jamieson will discuss current efforts in the peptide chemistry community towards pursuing more sustainable replacement solvents for peptide synthesis, as well as other considerations such as coupling chemistry.

Craig Jamieson, PhD
Senior Lecturer
University of Strathclyde, Glasgow, UK


Innovation vs Application: Maximizing Efficiency in Peptide Drug Discovery from a Chemistry Perspective

Recorded Webinar

This presentation will discuss the discovery process used in the development and selection of a Glucagon agonist, including SAR studies and the positive impacts of integrating innovative technology. Faced with limited time and resources, flexible instrumentation has been used to efficiently incorporate molecular improvements that are critical for overcoming the inherent challenges of peptide drug discovery, including optimizing physical properties and receptor selectivity.


William Blackwell III, 
Investigator, Intarcia Therapeutics

James Cain, Ph.D., 
Global Product Manager, Gyros Protein Technologies


Challenging SPPS: Difficult sequences and side reactions

Recorded Webinar

Peptide therapeutics have seen increased interest in the last decade, making peptide synthesis a critical step in the drug discovery and development process. One of the major drawbacks in peptide synthesis is that success largely depends on the peptide sequence being synthesized, so what do you do when you have a sequence with failed couplings at certain cycles? Do you extend the coupling time? Change reagents? Do multiple couplings? Here we’ll discuss our take on these questions using fast parallel synthesis optimization of a sterically hindered model peptide, Aib-ACP (VQ-Aib-Aib-IDYING-NH2) which saw improvements from 7.8% using HCTU to 91% using COMU and increasing the reaction temperature to 75 °C with short reaction times (2 x 3 min). What if your synthesis challenges aren’t difficult couplings but side reactions? We’ll discuss strategies for minimizing their occurrence during fast SPPS.

Cyf Ramos-Colón, Ph.D., Senior Scientist, Gyros Protein Technologies


Polar Hinges as Functionalized Conformational Constraints in (Bi)cyclic Peptides 

Recorded webinar

Protein-protein interactions (PPI’s) mediate interactions that are not only essential to homeostasis, but are also responsible for initiating and maintaining a range of physiological disorders. As a result, the inhibition of PPI's is of increasing importance to the field of drug discovery. Despite the progress that has been made in the field of PPI modulation, the development of molecules that can interfere with protein-protein interactions remains challenging. Since contact surfaces involved in PPI’s are normally large and flat, it is a serious challenge for the traditional “small-molecule” approach to effectively disrupt such a large interface. In order to address this issue, we wish to devise larger biomolecular constructs, such as cyclic peptides and peptidomimetics as PPI inhibitors. The Liskamp group has developed 'Polar hinges’ (see 'Polar Hinges as Functionalized Conformational Constraints in (Bi)cyclic Peptides, ChemBioChem, 2017, 18 (4), 387-395') as scaffolds for the cyclization of peptides, which in turn yielded bicyclic peptides and cyclized peptides with improved solubility and biological activity. During the course of this research, we noted that aqueous solubility of candidate PPI inhibitors is an absolute prerequisite not only to be able to handle and purify our target peptides; but this intrinsic property is also crucial for validation of biological activity.

Learning Objectives:

  • Challenges in the development of molecules that interfere with protein-protein interactions (PPI inhibitors)
  • Cyclic peptides and peptidomimetics as PPI inhibitors
  • Polar hinges as scaffolds for the cyclization of peptides to yield bicyclic peptides and cyclized peptides
  • Importance of aqueous solubility of candidate PPI inhibitors in the validation of biological activity

Dr Alex Hoose
Post-Doctoral Research Associate
University of Glasgow

Professor Rob Liskamp
Chair of Chemical Biology and Medicinal Chemistry & Guest Professor of Molecular Medicinal Chemistry at Utrecht University
University of Glasgow & Utrecht University (The Netherlands)


Strategies to Increase Robustness in the Optimization and CMC Production of Peptide Therapeutics with Parallel Peptide Synthesis

Recorded Webcast from TIDES  Digital Week

With the advancement of personalized medicine and newer disease targets, peptide therapeutic development has greatly expanded. Its potential to achieve increased receptor specificity and decreased toxicity satisfies efficacy and safety regulatory requirements. Technology advances in automated SPPS enable screening and CMC production of peptides with greater structural complexity allows for the design of more physiologically stable products with increased target specificity and membrane permeability. Structural modification strategies for peptide lead optimization can include cyclization, stapling, and PEGylation, which can be difficult to synthesize and purify, requiring more efficient synthesis protocols and automation to ensure reproducible and efficient manufacturability. 

Here we describe the process development and parallel optimization of SPPS relating to different biologically relevant peptides. Solid-support screening, reagent screening and temperature screening are demonstrated using an automated peptide synthesizer as part of the optimization process for difficult peptides such as Aib-Enkephalin (90% optimized crude purity vs 21% crude purity) and JR 10-mer (67% optimized crude purity vs 21% crude purity). In addition, the synthesis optimization in parallel of GLP-1 related peptides and dual GLP-1/glucagon receptor agonists will be shown, testing multiple resins and coupling reagents in search of optimal crude purities that may translate into ease of purification during the manufacturing process. Finally, the fully automated synthesis, from linear, on-resin cyclization, and resin cleavage, of NYAD-1 stapled peptide will be shared.

James Cain, Global Product Manager, Gyros Protein Technologies


Strategies for efficient, high throughput optimization of biopolymers and mimetic compounds

Peptide, peptidomimetic and oligonucleotide therapeutics are increasingly at the forefront of drug development programs for personalized medicine, cancer therapeutics, and genetic diseases among others. This has driven the search for faster and more efficient solid phase synthesis (SPS) protocols, making method development crucial in the discovery process towards scale-up. Automated synthesizers are part of the SPS toolbox that allows simultaneous optimization and high-throughput synthesis via parallel synthesis.

In this webinar, we describe the process development and parallel optimization of SPS of different biologically relevant compounds. Solid-support screening and reagent screening are demonstrated using automated peptide synthesis as part of the optimization process for peptide nucleic acids (PNA), peptoids, and cyclic peptides. For example, parallel synthesis condition scanning for the synthesis of SK-8mer PNA analog, H-Lys-AGTGGATC-Lys-NH2, led to an increase of 27% crude purity resulting in high purity product (73% crude pure) for biological analysis. Coupling reagents, resins and reaction time combinations for increased crude purity results will be discussed for biopolymers and mimetic compounds.

James Cain, Global Product Manager, Gyros Protein Technologies
Cyf Ramos-Colón, Senior Scientist, Peptide Applications, Gyros Protein Technologies


Cell Penetration Profiling using the Chloroalkane Penetration Assay for Peptides, Proteins and Nucleic Acids

Biotechnology promises the ability to control biology and disease with laser-like precision, but biomolecules typically have poor cell penetration and unpredictable subcellular localization. Hundreds of peptides, proteins and nucleic acids are being developed as diagnostics and therapies. For many of these, intracellular delivery remains the primary obstacle. Currently, there are no quantitative, high-throughput tools to measure how much of a biomolecule enters a cell and where it distributes within the cell. Importantly, most widely-used methods for measuring cell penetration cannot rule out effects of material at the cell surface or trapped in endosomes.

We have devised a chloroalkane penetration assay (CAPA) that exclusively measures penetration to the cytoplasm. CAPA uses a cell line with stably expressed HaloTag in the cytoplasm, and measures the extent of covalent reaction between this enzyme and a small chloroalkane tag appended to the molecule of interest.

Joshua Kritzer
Associate Professor
Department of Chemistry
Tufts University


Chemical, Computational, and Instrumental Tools for Challenging Peptide Syntheses

The Kay lab at the University of Utah is focused on D-peptide inhibitor development, which requires the chemical synthesis of mirror-image protein targets. Mirror-image proteins are not found in nature and are promising therapeutic agents due to their resistance to degradation by natural proteases. This webinar will describe the use of chemical, instrumental, and computational tools to overcome challenges associated with the chemical synthesis of large proteins via Fmoc solid-phase peptide synthesis and native chemical ligation. Specific examples will include the use of a reversible solubilizing tag (“helping hand”), pseudoproline dipeptides, UV monitoring, and an automated ligator program (“Aligator”) to predict the most efficient synthesis schemes.

Michael S Kay MD/PhD
Biochemistry, University of Utah