Liquid-phase Peptide Synthesis Research Articles

Scalable preparation of green C-terminal amidation peptide-synthesis TAGs and the optimized TAG-assisted liquid-phase synthesis of eptifibatide

Eptifibatide is a cycloheptapeptide with C-terminal amidation and head-to-tail disulfide bridging, which has been approved as an antithrombotic drug. Currently, both at the laboratory scale and industrial level, the synthesis of eptifibatide predominantly relies on solid-phase peptide synthesis (SPPS), which is not regarded as a green sustainable and cost-effective approach to prepare eptifibatide. Herein, we have investigated the strategy employing tag-assisted liquid-phase peptide synthesis (TAG-Assisted LPPS) technology that is scalable to multi-gram scale manufacturing for eptifibatide preparation. We successfully obtained soluble Rink-Amide TAGs by esterifying organophosphorus TAG-OH with Rink-Amide, thereby facilitating the transition from synthesizing C-terminal carboxyl peptides to C-terminal amidation peptides. The products obtained during the tag-assisted coupling and Fmoc deprotection of eptifibatide intermediates can be purified through precipitation processes, eliminating the need for complex chromatographic operations. Accordingly, we developed an optimized laboratory-scale process (300 mmol) for the preparation of TAG-Rink Amide TAG, which was subsequently upscaled successfully to a 50 mmol batch synthesis of eptifibatide. The subsequent work also explores the processes of peptide cleavage and disulfide bond oxidative cyclization in eptifibatide preparation.

Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis.

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Synthesis and anticancer evaluation of [d-Ala]-nocardiotide A.

Cancer is currently one of the biggest causes of death in the world. Like some microorganisms, cancer cells also develop resistance to various chemotherapy drugs and are termed multidrug resistant (MDR). In this regard, there is a need to develop new alternative anticancer agents. Anticancer peptides (ACPs) with high selectivity and high cell penetration ability are a promising candidate, as well as they are easy to modify. A cyclohexapeptide called nocardiotide A was isolated from the marine sponge Callyspongia sp., which is cytotoxic towards several cancer cells such as MM, 1S, HeLa, and CT26 cells. Previously, nocardiotide A was synthesized with a very low yield owing to its challenging cyclization process. In this study, we synthesized [d-Ala]-nocardiotide A as a derivative of nocardiotide A using a combination of solid phase peptide synthesis (SPPS) and liquid phase peptide synthesis (LPPS). The synthesis was carried out by selecting a d-alanine residue at the C-terminus to give a desired cyclic peptide product with a yield of 31% after purification. The purified [d-Ala]-nocardiotide A was characterized using HR-ToF MS and 1H and 13C-NMR spectroscopy to validate the desired product. The anticancer activity of the peptide was determined against HeLa cancer cell lines with an IC50 value of 52 μM compared to the parent nocardiotide A with an IC50 value of 59 μM. In the future, we aim to mutate various l-amino acids in nocardiotide A to d-amino acids to prepare nocardiotide A derivatives with a higher activity to kill cancer cells with higher membrane permeation. In addition, the mechanism of action of nocardiotide A and its derivatives will be evaluated.

Assessing the immunogenicity risk of salmon calcitonin peptide impurities using in silico and in vitro methods.

Advances in synthetic peptide synthesis have enabled rapid and cost-effective peptide drug manufacturing. For this reason, peptide drugs that were first produced using recombinant DNA (rDNA) technology are now being produced using solid- and liquid-phase peptide synthesis. While peptide synthesis has some advantages over rDNA expression methods, new peptide-related impurities that differ from the active pharmaceutical ingredient (API) may be generated during synthesis. These impurity byproducts of the original peptide sequence feature amino acid insertions, deletions, and side-chain modifications that may alter the immunogenicity risk profile of the drug product. Impurities resulting from synthesis have become the special focus of regulatory review and approval for human use, as outlined in the FDA's Center for Drug Evaluation and Research guidance document, "ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin," published in 2021. This case study illustrates how in silico and in vitro methods can be applied to assess the immunogenicity risk of impurities that may be present in synthetic generic versions of the salmon calcitonin (SCT) drug product. Sponsors of generic drug abbreviated new drug applications (ANDAs) should consider careful control of these impurities (for example, keeping the concentration of the immunogenic impurities below the cut-off recommended by FDA regulators). Twenty example SCT impurities were analyzed using in silico tools and assessed as having slightly more or less immunogenic risk potential relative to the SCT API peptide. Class II human leukocyte antigen (HLA)-binding assays provided independent confirmation that a 9-mer sequence present in the C-terminus of SCT binds promiscuously to multiple HLA DR alleles, while T-cell assays confirmed the expected T-cell responses to SCT and selected impurities. In silico analysis combined with in vitro assays that directly compare the API to each individual impurity peptide may be a useful approach for assessing the potential immunogenic risk posed by peptide impurities that are present in generic drug products.

Fast Solution-Phase and Liquid-Phase Peptide Syntheses (SolPSS and LPPS) Mediated by Biomimetic Cyclic Propylphosphonic Anhydride (T3P®).

The growing applications of peptide-based therapeutics require the development of efficient protocols from the perspective of an industrial scale-up. T3P® (cyclic propylphosphonic anhydride) promotes amidation in the solution-phase through a biomimetic approach, similar to the activation of carboxylic moiety catalyzed by ATP-grasp enzymes in metabolic pathways. The T3P® induced coupling reaction was applied in this study to the solution-phase peptide synthesis (SolPPS). Peptide bond formation occurred in a few minutes with high efficiency and no epimerization, generating water-soluble by-products, both using N-Boc or N-Fmoc amino acids. The optimized protocol, which was successfully applied to the iterative synthesis of a pentapeptide, also allowed for a decrease in the solvent volume, thus improving process sustainability. The protocol was finally extended to the liquid-phase peptide synthesis (LPPS), where the isolation of the peptide was performed using precipitation, thus also showing the suitability of this coupling reagent to this emerging technique.

Efficient Solution Phase Synthesis of PPII Helix Mimicking Ena/VASP EVH1 Inhibitors from Proline‐Derived Modules (ProMs)

AbstractIn the search for efficient inhibitors for the enabled/vasodilator‐stimulated phosphoprotein homology 1 (EVH1) domain to reduce cell motility in metastatic cancer, we previously developed a toolkit of proline‐derived modules (ProMs), which mimic the PPII helix found in the natural −FPPPP− binding motif of EVH1. In this work, we describe the modular assembly of these ProM‐based pentapeptidic EVH1 ligands through liquid phase peptide synthesis. We initially used pentafluorophenyl (Pfp) active esters for amide bond formation and built up the growing peptide chain from the C‐ to the N‐terminus. Switching to HATU/DIPEA coupling conditions and changing the directionality of the synthesis from the N‐ to the C‐terminus afforded the target ligands with improved overall yields and purity. Employing a Fmoc‐protected (instead of the N‐acetylated) phenylalanine derivative as N‐terminal building block significantly reduced epimerization. In contrast to the originally used solid phase peptide synthesis (SPPS), the developed solution phase method allowed for a facile alteration of the C‐terminal ProM unit and the production of various pentapeptidic ligands in an efficient fashion even on a multigram scale.

Super silyl-based stable protecting groups for both the C- and N-terminals of peptides: applied as effective hydrophobic tags in liquid-phase peptide synthesis.

Tag-assisted liquid-phase peptide synthesis (LPPS) is one of the important processes in peptide synthesis in pharmaceutical discovery. Simple silyl groups have positive effects when incorporated in the tags due to their hydrophobic properties. Super silyl groups contain several simple silyl groups and play an important role in modern aldol reactions. In view of the unique structural architecture and hydrophobic properties of the super silyl groups, herein, two new types of stable super silyl-based groups (tris(trihexylsilyl)silyl group and propargyl super silyl group) were developed as hydrophobic tags to increase the solubility in organic solvents and the reactivity of peptides during LPPS. The tris(trihexylsilyl)silyl group can be installed at the C-terminal of the peptides in ester form and N-terminal in carbamate form for peptide synthesis and it is compatible with hydrogenation conditions (Cbz chemistry) and Fmoc-deprotection conditions (Fmoc chemistry). The propargyl super silyl group is acid-resistant, which is compatible with Boc chemistry. Both tags are complementary to each other. The preparation of these tags requires less steps than previously reported tags. Nelipepimut-S was synthesized successfully with different strategies using these two types of super silyl tags.

Development of a nitrogen-bound hydrophobic auxiliary: application to solid/hydrophobic-tag relay synthesis of calpinactam.

In the last couple of decades, technologies and strategies for peptide synthesis have advanced rapidly. Although solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS) have contributed significantly to the development of the field, there have been remaining challenges for C-terminal modifications of peptide compounds in SPPS and LPPS. Orthogonal to the current standard approach that relies on installation of a carrier molecule at the C-terminus of amino acids, we developed a new hydrophobic-tag carbonate reagent which facilitated robust preparation of nitrogen-tag-supported peptide compounds. This auxiliary was easily installed on a variety of amino acids including oligopeptides that have a broad range of noncanonical residues, allowing simple purification of the products by crystallization and filtration. We demonstrated a de novo solid/hydrophobic-tag relay synthesis (STRS) strategy using the nitrogen-bound auxiliary for total synthesis of calpinactam.

Liquid-Phase Peptide Synthesis (LPPS): A Third Wave for the Preparation of Peptides.

Since the last century, peptides have gained wide acceptance as drugs, with almost 100 already in the market and a large number in the pipeline. In this context, peptide synthesis has grown massively as a stringent field for pharmaceuticals around the globe. Three methodologies, namely, classical solution peptide synthesis (CSPS), solid-phase peptide synthesis (SPPS), and liquid-phase peptide synthesis (LPPS), have made significant contributions to the field. This review provides a comprehensive and integrated vision of LPPS as the third wave for peptide synthesis. LPPS combines the advantages of CSPS and SPPS, where peptide elongation is carried out in solution and the growing peptide chain is supported on a soluble tag, which confers characteristic properties. LPPS protocols allow the large-scale production of peptides and reduce the use of excess reagents and solvents, thus meeting the principles of green chemistry. In this review, tags associated with LPPS are broadly discussed under the following headings: polydisperse polyethylene glycol (PEG), membrane-enhanced peptide synthesis (MEPS), fluorous technology, ionic liquids (ILs), PolyCarbon, hydrophobic polymers, and group-assisted purification (GAP). It also highlights the signature accomplishments of LPPS tags and the limitations of the same.

Rapid and column-free syntheses of acyl fluorides and peptides using ex situ generated thionyl fluoride.

Thionyl fluoride (SOF2) was first isolated in 1896, but there have been less than 10 subsequent reports of its use as a reagent for organic synthesis. This is partly due to a lack of facile, lab-scale methods for its generation. Herein we report a novel protocol for the ex situ generation of SOF2 and subsequent demonstration of its ability to access both aliphatic and aromatic acyl fluorides in 55–98% isolated yields under mild conditions and short reaction times. We further demonstrate its aptitude in amino acid couplings, with a one-pot, column-free strategy that affords the corresponding dipeptides in 65–97% isolated yields with minimal to no epimerization. The broad scope allows for a wide range of protecting groups and both natural and unnatural amino acids. Finally, we demonstrated that this new method can be used in sequential liquid phase peptide synthesis (LPPS) to afford tri-, tetra-, penta-, and decapeptides in 14–88% yields without the need for column chromatography. We also demonstrated that this new method is amenable to solid phase peptide synthesis (SPPS), affording di- and pentapeptides in 80–98% yields.

Silicon-based hydrophobic tags applied in liquid-phase peptide synthesis: protected DRGN-1 and poly alanine chain synthesis.

Two new recyclable silicon-based hydrophobic tags were developed for installing them at the C-terminal of peptides to increase the solubility in organic solvents and the reactivity of the peptides during liquid-phase peptide synthesis (LPPS). They comprise a siloxy group containing tag and an arylsilyl group containing tag. The hydrophobicity of these tags is much greater than those of previously reported examples. The siloxy group containing tag is compatible with Fmoc-deprotection conditions (Fmoc-chemistry) and hydrogenation conditions (Cbz-chemistry), while the arylsilyl group containing one is resistant to Fmoc-deprotection conditions (Fmoc-chemistry) and Boc-deprotection conditions (Boc-chemistry). Using the siloxy group containing tag, protected DRGN-1, a peptide containing 14 amino acid residues was prepared successfully by using linear synthesis combined with one convergent synthesis step. Using the arylsilyl group containing tag, a poly alanine chain containing 7 alanine residues was synthesized. The arylsilyl group containing tag can also be installed at the N-terminal to elongate the peptides from the N-terminal to the C-terminal. The yields and the solubility of the products in organic solvents in each step were good to excellent with the assistance of these silicon-based tags.

Quick Access to High-Purity Peptide Drugs Bradykinin, Leuprolide Analogue, 2(PZ-128), and Rapastinel with Minimal Reagents

Peptide drugs bradykinin, a leuprolide analogue, 2(PZ-128), and rapastinel are synthesized in 56-77% yield using heating-assisted liquid-phase peptide synthesis on a soluble polynorbornene support. These drugs of commercial utility and complex structures are obtained in 2-5.5 h with no epimerization and >95% purity using only 1.2 equivalents of amino acids and coupling reagents. The peptide yield and purity are comparable or superior to the reported methods.

Cover Feature: Peptide Head‐to‐Tail Cyclization: A “Molecular Claw” Approach (22/2021)

The Cover Feature shows a schematic impression of the “molecular claw” approach that enables an effective head (C-terminus)-to-tail (N-terminus) cyclization of peptides. Tag-assisted liquid phase peptide synthesis in combination with a backbone amide linker strategy is generally applicable to the head-to-tail cyclization, leading to the efficient synthesis of the cyclic heptapeptide stellarin E. The key for successful cyclization is found to be the tagging position that enables the peptide to be grabbed at the center as in a claw-like machine. More information can be found in the Communication by K. Chiba et al.

Construction and Piezoelectric Properties of a Single-Peptide Nanotube Composed of Cyclic β-peptides with Helical Peptides on the Side Chains.

To develop nanopiezoelectronics, it is necessary to investigate the relationship between the sizes and piezoelectric properties of the material. Peptide nanotubes (PNTs) composed of cyclic β-peptides have been studied as leading candidates for nanopiezoelectric materials. The current drawback of PNTs is aggregation to form a PNT bundle structure due to strong dipole-dipole interactions between PNTs. Here, we report the construction and piezoelectric properties of single PNTs without nonspecific aggregation by side-chain modification of helical peptides. A cyclic tri-β-peptide with a helical peptide was prepared by multiple-step liquid-phase peptide synthesis and assembled into PNTs by the vapor diffusion method. These nanotubes were characterized by polarized light microscopy and Fourier transform infrared (FTIR) spectroscopy. Additionally, atomic force microscopy (AFM) topographic images showed nanotubes with a height of 4 nm, which corresponds to the diameter of a PNT on a gold-coated mica substrate, indicating that a single PNT was prepared successfully. The converted piezoelectric response of a single PNT was determined to be 1.39 ± 0.12 pm/V. This value was consistent with that of a PNT bundle, which reveals that the piezoelectricity of PNTs is induced by deformation of their cyclic skeletons and is independent of the bundled structure. This finding not only demonstrates a new molecular design strategy to construct these smallest piezoelectric biomaterials by controlling the supramolecular hierarchical structures but also provides insights into the correlation between molecular assembly morphology and size-dependent piezoelectric properties.

Liquid Phase Peptide Synthesis via One-Pot Nanostar Sieving (PEPSTAR).

Herein, a one‐pot liquid phase peptide synthesis featuring iterative addition of amino acids to a “nanostar” support, with organic solvent nanofiltration (OSN) for isolation of the growing peptide after each synthesis cycle is reported. A cycle consists of coupling, Fmoc removal, then sieving out of the reaction by‐products via nanofiltration in a reactor‐separator, or synthesizer apparatus where no phase or material transfers are required between cycles. The three‐armed and monodisperse nanostar facilitates both efficient nanofiltration and real‐time reaction monitoring of each process cycle. This enabled the synthesis of peptides more efficiently while retaining the full benefits of liquid phase synthesis. PEPSTAR was validated initially with the synthesis of enkephalin‐like model penta‐ and decapeptides, then octreotate amide and finally octreotate. The crude purities compared favorably to vendor produced samples from solid phase synthesis.

Liquid Phase Peptide Synthesis via One‐Pot Nanostar Sieving (PEPSTAR)

AbstractHerein, a one‐pot liquid phase peptide synthesis featuring iterative addition of amino acids to a “nanostar” support, with organic solvent nanofiltration (OSN) for isolation of the growing peptide after each synthesis cycle is reported. A cycle consists of coupling, Fmoc removal, then sieving out of the reaction by‐products via nanofiltration in a reactor‐separator, or synthesizer apparatus where no phase or material transfers are required between cycles. The three‐armed and monodisperse nanostar facilitates both efficient nanofiltration and real‐time reaction monitoring of each process cycle. This enabled the synthesis of peptides more efficiently while retaining the full benefits of liquid phase synthesis. PEPSTAR was validated initially with the synthesis of enkephalin‐like model penta‐ and decapeptides, then octreotate amide and finally octreotate. The crude purities compared favorably to vendor produced samples from solid phase synthesis.

Greener liquid-phase synthesis and the ACE inhibitory structure-activity relationship of an anti-SARS octapeptide.

A high-efficiency strategy for resin-free and large scale liquid phase synthesis of the anti-SARS octapeptide AVLQSGFR is described. Herein, tri(4'-diphenylphosphonyloxylbenzoyl phenyl)phosphate (TDPBP) derivatives were designed as C-terminal supports to aid octapeptide intermediate purification without the need for chromatographic separation. Furthermore, the ACE inhibitory structure-activity relationship (SAR) of the anti-SARS octapeptide and its alanine-scanning analogues was systematically studied by in vitro assay and 3D-QSAR via molecular docking. This paper provides a new strategy for the development of peptide-based drugs. Simultaneously, a study on the ACE inhibition and structure-activity relationship of the anti-SARS octapeptide also lays a foundation for further understanding how the anti-SARS octapeptide acts as an ACE inhibitor.

Improved Tag-Assisted Liquid-Phase Peptide Synthesis: Application to the Synthesis of the Bradykinin Receptor Antagonist Icatibant Acetate

A cost- and time-effective synthetic method is a prerequisite to producing therapeutic peptides on a commercial scale. Herein we demonstrate that a soluble-tag-assisted liquid-phase peptide synthes...

Gram-Scale Preparation of C-Terminal-Modified Enkephalin Analogues by Typical Liquid-Phase Peptide Synthesis.

This article describes the gram-scale liquid-phase peptide synthesis of C-terminal-modified enkephalin analogues that possess high analgesic efficacy in animals, high potency for mu and delta opioid receptors, and high metabolic stability and potential blood-brain barrier permeability. Despite the long cycle time and tedious purification steps, liquid-phase synthesis is still a preferred method for large-scale peptide synthesis due to its cost effectiveness (i.e., amount of amino acids and reagents required), easy detection, and isolation of impurities compared with solid-phase synthesis. A robust liquid-phase synthesis protocol is described, involving BOP-assisted coupling and Boc deprotection, which has been well established in the laboratory and is a useful synthetic protocol for cost-effective production of peptide drugs. © 2019 by John Wiley & Sons, Inc.

Synthesis of the Antimalarial Peptide Aldehyde, a Precursor of Kozupeptin A, Utilizing a Designed Hydrophobic Anchor Molecule.

This Letter describes an efficient method of synthesizing highly bioactive peptide aldehydes without any concern about epimerization by liquid-phase peptide synthesis through the use of newly designed hydrophobic anchor molecules. Peptide elongation reactions effectively proceeded in less polar solvents, and direct crystallization by the addition of polar solvents enabled easy purification. This method also represents a new concept for the efficient synthesis of peptide derivatives. The development of new antimalarial drug candidates will be accelerated using this methodology.