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This Pub. has the Guinness…for longest synthetic peptide!

In the May installment of the BPS Journal Club, I’d like to highlight a recent paper from the Walczak group (in Boulder!) that addresses a beast of a target, a microtubule-associated protein: Tau.

A 441-amino acid tour de force of chemical synthesis! There are plenty of nuggets here for chemists and biologists alike. If you're looking to assemble large peptides, or are in search of point mutations without the limitations of post-translational tools using the native sequences, or just simply curious of the odyssey, this publication will be a treat.

Tau is a focus point in studying neurodegenerative diseases, as they are characterized by intraneuronal deposits of this protein, hence their collective name, tauopathies. Tau exists in several variants and is prone to a vast array of post-translational modifications (PTMs). Examining the role of PTMs in tauopathies is thus a hot topic but is a challenging one in the
absence of a fully synthetic route to the protein. Semi-syntheses and synthetic PTMs of the native variants are limited to specific sites and substitutions.

The authors here propose a route to the longest isoform of the six Tau, by fragment-based chemical assembly. A retrosynthetic analysis identified three key fragments of ca. 150 amino acids each, which were further dissected into subunits <50 AA in length, at strategic cysteines or alanines. The preparation of each fragment then unfolds as a who's who of modern
SPPS techniques to obtain the desired fragments: resin/linker selection, protecting group selection, hydrazide approaches to thioesters or their surrogates, all while keeping the more conventional stowaways, such as aspartimides, at bay. Some fragments took a few tries. Some did not cooperate at the ligation stage and required rework. But eventually, the team had all the ingredients for the final native chemical ligations, both with sulfur and selenium, and the adequate sprinkle of desulfurizations.

This monumental synthesis is a strong one-up on the already formidable GFP precursor synthesis (Sakakibara et al, 238 AAs), not that there should be a competition! It should pave the way for more accessible, on-demand PTMs of long peptides and small proteins for the benefit of biologists and chemists alike.

The paper is available at JACS: Chemical Synthesis of Microtubule-Associated Protein Tau (https://pubs.acs.org/doi/10.1021/jacs.3c07338).

The authors have deposited a ChemRxiv open-access version as well (https://chemrxiv.org/engage/chemrxiv/article-details/64a7639f9ea64cc167918c5d).

Alaric Desmarchelier, Ph.D.
Member, BPF Scientific Advisory Board
https://www.linkedin.com/in/alaric-desmarchelier/

Read previous editions of the BPF Journal Club series: www.boulderpeptide.org/journal-club

Can Solid Phase Peptide Synthesis Learn to Become Leaner?

For the April edition of the BPF Journal Club, I would like to introduce an extremely interesting communication by Collins and co-workers from CEM Corporation (https://doi.org/10.1038/s41467-023-44074-5). The subject is of particular interest to those involved in peptide chemistry, peptide drug discovery, and manufacturing. The work addresses important issues related to solid-phase peptide chemistry (SPPS), in the context of large-scale production.

Since its introduction in the early 1960’s SPPS has revolutionized peptide science, especially the discovery and development of peptide therapeutics. Recent years have witnessed the emergence of incretin-based diabetes/obesity therapeutics which, as a result of unprecedented demand, require large-scale manufacturing. While some, including liraglutide and semaglutide (both from Novo-Nordisk), can be made in part by recombinant expression, others, notably tirzepatide (Eli Lilly & Co.), rely entirely on chemical synthesis. Utilizing SPPS at scales of hundreds of kilograms or more highlights several inherent limitations of the SPPS method. One of these is the poor atom-efficiency resulting from the several-fold stoichiometric excess of Fmoc-protected amino acid and coupling reagent used in each step. The second source of inefficiency involves the high consumption of solvents such as N,N-dimethylformamide (DMF) or N-methyl-pyrrolidone (NMP) used in wash steps between couplings. This produces a large volume of waste and substantially increases disposal costs. Both factors contribute to an unfavorably high process mass intensity (PMI) score (https://doi.org/10.1021/acs.joc.3c01494). This concern is further exacerbated by a regulatory climate that has restricted the use of DMF in the European Union (https://doi.org/10.1002/cssc.202301639).

The authors present an effective solution to the above problem by introducing a novel SPPS protocol that utilizes microwave-assisted evaporation and nitrogen flushing of pyrrolidine following the Fmoc deprotection step. Pyrrolidine is used in place of piperidine due to the former’s lower boiling point (bp 87 degC for pyrrolidine vs. bp 106 degC for piperidine). This modification results in a reduction of solvent and base waste of around 80%. The method is optimized using the Jung Reddman sequence and validated for several peptides including liraglutide and semaglutide, as well as the proteins proinsulin and barstar. The possibility of epimerization was checked by comparing a liraglutide sample made by the new method with a commercial sample noting no significant differences. The new methodology holds promise for minimizing not only production costs but also environmental and regulatory concerns for future large-scale peptide projects.

John P. Mayer, Ph.D.
Member, BPF Scientific Advisory Board
linkedin.com/in/john-mayer-4587556

Discover the Future of Vaccination: Introducing 'String-of-Beads' Vaccines. In this enlightening Nature Communications article, Dasari et al. unveil the revolutionary development of an Epstein-Barr virus (EBV) vaccine, employing a cutting-edge multi-epitope subunit approach (https://doi.org/10.1038/s41467-023-39770-1). This breakthrough paves the way for analogous advancements in infectious and cancer vaccine research, driven by substantial enhancements in the biochemical production of such sophisticated constructs. Mirroring the principles of mRNA vaccine strategies, these novel approaches promise superior performance through improved stability and enhanced delivery to MHC complexes, all while eliminating the reliance on mRNA-based biosynthesis within human cells. Check out how these innovations could redefine disease prevention and treatment.

Christian Schwarz, Ph.D.
Member, BPF Scientific Advisory Board
https://www.linkedin.com/in/christian-dr-schwarz-8a314b155

The February BPS Journal Club explores lasso peptides, a fascinating class of bioactive macrocyclic peptide natural products. These peptides have a unique structure with a threaded (rotaxane) motif, where the C-terminal tail is captured within a macrolactam through an isopeptide bond. The lasso fold provides pharmacokinetic advantages, including high proteolytic and metabolic stability, along with resistance to degradation. This defined structure can be utilized to optimize interactions with therapeutically relevant targets.

Chemical synthesis of lasso peptides poses a significant challenge, as the nonthreaded isomer (referred to as a tadpole isomer) is kinetically favored over forming the lariat knot-like fold. While companies like Lassogen are making significant progress with engineered therapeutic lasso peptides (Lassotides™), reliance on biosynthetic routes limits the incorporation of noncanonical amino acids and other structural investigations.

A team led by Professor Andrew G. Roberts at the University of Utah recently reported the solid-phase peptide synthesis of cyclic, cyclic-branched (tadpole), and linear isomers of sungsanpin and ulleungdin, lasso peptide natural products known to inhibit cell migration in models of metastasis (https://pubs.acs.org/doi/10.1021/acschembio.3c00525¹).  Prof. Roberts and collaborators discovered that the lasso motif itself was not critical for inhibition of cell migration by synthetically accessible analogs of these natural products.

In their publication, the Roberts group details a general approach to the SPPS and on-resin cyclization of lasso peptide isomers and highlights the use of Fmoc-Asp(CSY)−OH (CAS 2379679-90-8) to mask the carboxylic acid functionality of Asp in certain aspartimide prone sequences. So, while the class II lasso scaffold remains a synthetic challenge yet to be overcome, the Roberts group and collaborators remind us that promising hits can be obtained from evaluating structurally complex natural molecules and applying ingenuity in peptide design to discover chemically tractable leads.

1. Nonthreaded Isomers of Sungsanpin and Ulleungdin Lasso Peptides Inhibit H1299 Cancer Cell Migration; Lori Digal, Shiela C. Samson, Mark A. Stevens, Abhijit Ghorai, Hyungyu Kim, Marcus C. Mifflin, Keith R. Carney, David L. Williamson, Soohyun Um, Gabe Nagy, Dong-Chan Oh, Michelle C. Mendoza, and Andrew G. Roberts. ACS Chemical Biology 2024 19 (1), 81-88. DOI: 10.1021/acschembio.3c00525

Wendy Hartsock
Member, BPF Scientific Advisory Board
https://www.linkedin.com/in/wendy-hartsock-49938512/

With this post, the scientific advisory board of the Boulder Peptide Foundation aims to establish a new tradition: The BPF Journal Club. Monthly, we will feature a reference and commentary on an article within the peptide field that a board member deems significant or interesting for the peptide community. Considering the diverse interests on our board, we believe these articles will be compelling reads.

Today, I'd like to highlight an article that delineates a novel orthogonality in SPPS, DNPBS (https://doi.org/10.1038/s41467-023-41115-x). The authors introduced a new N-alpha protection (DNPBS, 2,4-dinitrobenzenesulfenyl) to peptide chemistry, which is orthogonal to Fmoc, Boc, and potentially Aloc and ivDde/DDe chemistries (although not explicitly tested). Post coupling via classical SPPS methods and activations, DNPBS can be eliminated by p-toluenethiol in pyridine (~12%). While this protective group isn't entirely new—previously used to protect the 5’-OH in oligonucleotide synthesis (for once oligos prove useful)—its application here is innovative.

The authors meticulously prepared the twenty DNPBS N-alpha protected amino acids (employing classical Fmoc side chain protections) and utilized these derivatives to synthesize test peptides via SPPS, achieving considerable success. An intriguing aspect is the absence of detectable racemization during the integration of DNPBS- His(Trt)-OH and DNPBS-Cys(Trt)-OH. Additionally, the deprotection of DNPBS-Asp(OtBu)-OH, while expected, proceeded without aspartimide formation.

It's unlikely that we'll discard all our Fmoc amino acids from the shelf, but it's probable that many will attempt to adapt these reagents for intricate peptide chemistry (think complex multicycle peptides—FOG, are you listening?), even though adequate protection for the corresponding carboxylic groups is yet to be established. Hopefully, someone will replace p-toluenethiol with a less pungent reagent. Although I still feel a nostalgic pang for my younger self—a Post Doc who relished spending late nights in the lab using Reagent K (despite preferring sleep), as the other labs on the floor didn't share my affinity for its scent.

Stay tuned for the February Journal Club article.

Matteo Villain

www.linkedin.com/in/matteo-villain