Hi1a is a venom peptide isolated from the Australian funnel-web spider Hadronyche infensa, containing an intricate "double knot" tertiary structure, and is currently under investigation for the treatment of ischemic stroke. The recombinant expression and chemical synthesis of Hi1a remain a significant challenge due to laborious protocols, delaying progress in translation to the clinic. We describe the first single-shot chemical synthesis of Hi1a (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (<4.5 h total synthesis time) to milligram quantities of linear Hi1a (>10 mg). This work highlights the utility of AFPS as a technology that enables chemical production of biomolecules challenging to obtain by recombinant methods.

An expansion of the hexanucleotide (GGGGCC) repeat sequence in the chromosome 9 open frame 72 (c9orf72) is the most common genetic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutation leads to the production of toxic dipeptide repeat proteins (DPRs) that induce neurodegeneration. However, the fundamental physicochemical properties of DPRs remain largely unknown due to their limited availability. Here, we synthesized the c9orf72 DPRs poly-glycine-arginine (poly-GR), poly-proline-arginine (poly-PR), poly-glycine-proline (poly-GP), poly-proline-alanine (poly-PA), and poly-glycine-alanine (poly-GA) using automated fast-flow peptide synthesis (AFPS) and achieved single-domain chemical synthesis of proteins with up to 200 amino acids. Circular dichroism spectroscopy of the synthetic DPRs revealed that proline-containing poly-PR, poly-GP, and poly-PA could adopt polyproline II-like helical secondary structures. In addition, structural analysis by size-exclusion chromatography indicated that longer poly-GP and poly-PA might aggregate. Furthermore, cell viability assay showed that human neuroblastoma cells cultured with poly-GR and poly-PR with longer repeat length resulted in reduced cell viability, while poly-GP and poly-PA did not, thereby reproducing the cytotoxic property of endogenous DPRs. This research demonstrated the potential of AFPS to synthesize low-complexity peptides and proteins necessary for studying their pathogenic mechanisms and constructing disease models.

Information storage in DNA forms the fundamental basis of cellular life, but inherent limitations of information stability and density hamper innovative applications such as data storage or drug discovery. Here, we establish abiotic peptides for next-generation information storage and apply them for the encoding of diverse small molecule synthesis. The chemical stability of the peptide-based tag allows the use of palladium-mediated reactions to efficiently synthesize peptide-encoded libraries (PELs) with large chemical diversity and excellent purity. We demonstrate the successful application of PELs in drug discovery by affinity selection yielding small molecules with nanomolar affinity toward their target protein. Collectively, this work establishes abiotic peptides as carriers of information, leveraged herein for the encoding of small molecule synthesis.

Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are rapidly synthesized with automated flow technology.

The human CAMP gene product LL-37 is thought to exert direct antimicrobial activity against gram-negative bacteria via membrane disruption. In the course of structure-activity relationship studies of LL-37 aimed at developing peptidomimetic antibiotics, however, we incidentally noted mutations in LL-37 that globally inhibit membrane disruption in both mammalian and gram-negative bacterial cells. Despite their diminished capacity for membranolysis, these variants retained full antibacterial activity against gram-negative bacteria. While testing LL-37 and derivatives thereof against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis, we further noted unusually high rates of elevated minimum inhibitory concentrations for LL-37. Further evaluation of these clinical isolates revealed that they are fully permeabilized by LL-37 without being killed. Thus, we have identified variants of LL-37 that kill gram-negative bacteria without permeabilizing, and gram-negative bacteria that are permeabilized by LL-37 without being killed. This may suggest the existence of one or more mechanisms other than membrane disruption by which LL-37 can kill gram-negative bacteria, which may open up new avenues for antibiotic development based on the naturally evolved, non-membrane targets of human host defense peptides. ### Competing Interest Statement MGH has filed a provisional patent application on the results described in this manuscript. BLP is a co-founder and/or member of the scientific advisory board of several companies focusing on the development of protein and peptide therapeutics. National Institute of Allergy and Infectious Diseases, K08 AI166345, T32 AI007061, U19 AI142780 Cystic Fibrosis Foundation, ALBIN21Q0, ALBIN22A0-KB, ALBIN19F0

The remarkable efficiency with which enzymes catalyze small-molecule reactions has driven their widespread application in organic chemistry. Here, we employ automated fast-flow solid-phase synthesis to access catalytically active full-length enzymes without restrictions on the number and structure of noncanonical amino acids incorporated. We demonstrate the total syntheses of iron-dependent Bacillus subtilis myoglobin (BsMb) and sperm whale myoglobin (SwMb). The synthetic enzymes displayed excellent enantioselectivity and yield in carbene transfer reactions. Absolute control over enantioselectivity in styrene cyclopropanation was achieved using synthetic L- and D-BsMb mutants, which delivered each enantiomer of cyclopropane product in identical and opposite enantiomeric enrichment. BsMb mutants outfitted with noncanonical amino acids were used to facilitate detailed structure-activity relationship studies, revealing a previously unrecognized hydrogen-bonding interaction as the primary driver of enantioselectivity in styrene cyclopropanation. We anticipate that our approach will advance biocatalysis by providing reliable and rapid access to fully synthetic enzymes possessing noncanonical amino acids.

Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations but remain restricted by the lack of mass tags optimized for data-independent acquisition (DIA) workflows. Here, we present a systematic approach screening a library of 576 compounds that identifies several small molecules that, when conjugated to peptides, improve their detection and sequence identification by mass spectrometry. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows simultaneously increasing proteome coverage and sample throughput for plexDIA workflows without compromising quantitative accuracy. We demonstrate 240 samples-per-day with 9-plexDIA, while acquiring 28,359 protein data points in the same time label-free methods acquire 4,340. Our approach constitutes an expandable framework for designing mass tags to overcome existing limitations in multiplexed proteomics and provides plexDIA reagents capable of analyzing over 1,000 samples per day when using 10 minute runs. By facilitating higher throughput and improved identification, this innovation holds significant potential for accelerating proteomic studies across diverse biological and clinical applications. ### Competing Interest Statement H.S., M.Y., S.S., D.B.S., M.A., W.V., A.Y., M.A.L., B.P. and N.S. are listed as inventors on a patent application for the tags described in this paper.

The chemical synthesis of proteins (CSP) has been an essential tool in studying and understanding the role of these biological polymers and in enabling the discovery of novel classes of inhibitors. However, CSP with commercially available synthesizers is typically limited to producing polypeptides of about 50 to 70 amino acids in length. Consequently, a wide range of protein targets have been inaccessible using these technologies, or they require cumbersome synthesis and purification of multiple peptide fragments. In this report, we employed a powerful combination of automated fast-flow peptide synthesis (AFPS), native chemical ligation (NCL), and high-throughput evaluation of refolding conditions to achieve the first chemical synthesis of both the wild-type and mirror-image forms of functional full-length cyclophilin A, which plays a vital role in proline cis-trans isomerization and other important processes. Functional assays confirmed that the chemically synthesized proteins retained their biological properties.

Flow synthesis with building block mixtures permits the generation of hyperdiverse polyamide libraries on a timescale of < 1 hour per library. The semi-automated flow platform on which we have previously developed this technique, howev-er, lacks the efficiency required to make protein-length polyamide chains. To facilitate the direct engraftment of diversity regions into longer polyamide chains, we adapted mixture-based library synthesis to automated flow peptide synthesis (AFPS) systems previously optimized for the production of 100-200-mer proteins. Using this approach, we demonstrate the ability to generate a 101-mer protein library with a diversity approaching quintillions of members. AFPS thus permits the incorporation of hyperdiverse polyamide libraries into synthetic protein scaffolds.

Mammalian cell toxicity of human host defense peptides (HDPs) is a key factor limiting the development of new antibiotic classes based on these natural product templates. In recent work, we have reported both residues in the human cathelicidin LL-37 that reduce its toxicity and strategies for enhancing the potency of short derivatives of LL-37 through mimicry of the LL-37 N-terminus. Here we describe structure-activity relationship studies of one such short derivative, FF-14, in which structure and ultimately separation of hemolytic and antimicrobial functions are supported via the mimicry of Aib-rich peptaibiotic natural products. ### Competing Interest Statement MGH has filed a provisional patent application on the results described in this manuscript. BLP is a co-founder and/or member of the scientific advisory board of several companies focusing on the development of protein and peptide therapeutics. National Institute of Allergy and Infectious Diseases, K08 AI166345, T32 AI007061, U19 AI142780 Cystic Fibrosis Foundation, ALBIN21Q0, ALBIN22A0-KB, ALBIN19F0

Insulin is a key life-saving drug for patients with diabetes and is used clinically worldwide. To address the physicochemical challenges of insulin, such as low solubility and aggregation, glycosylated insulins have been chemically synthesized, exhibiting improved stability due to the hydration effect of glycans. In this work, we demonstrated the rapid synthesis of glycosylated insulins (glycoinsulins) using flow-based solid-phase peptide synthesis (SPPS). The insulin A-chain and glycosylated B-chain were synthesized by flow-based SPPS, with each elongation cycle completed in just 3 minutes. Through our investigations, the glycosylation step was successfully performed within 10 minutes under optimized flow-based conditions. Additionally, we examined the incorporation of dipeptide units (isoacyl dipeptide and pseudoproline) under flow conditions and demonstrated efficient peptide elongation by combining flow-based SPPS with these dipeptide units. The synthesized A- and B-chains were subsequently used for the stepwise formation of disulfide bond linkages. The resulting glycoinsulins exhibited comparable binding affinities to insulin receptors. These findings highlight a novel flow-based approach for the rapid synthesis of glycosylated peptide and protein drugs.

The synthesis and study of antibiotic natural products with unique structures and mechanisms of action represents a proven strategy to combat the public health crisis posed by antibiotic-resistant bacteria. The natural product himastatin is an antibiotic with an unusual homodimeric structure that presents a significant synthetic challenge. We report the concise total synthesis of himastatin by a newly developed final-stage dimerization strategy that was inspired by a detailed consideration of its biogenesis. Combining our bio-inspired dimerization approach with a modular synthesis enabled expedient access to a number of designed derivatives of himastatin, including synthetic probes that provide insight into its antibiotic activity.

Covalent peptides have found widespread applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and highly tunable affinity selection method to enrich covalent reactive peptides from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform enabled by tandem high resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfides to build reversible peptide-protein conjugates that can enrich crosslinked peptides that after reduction can be sequenced with MS/MS. Using this platform, we achieved maturation of covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The resulting peptides selectively covalently crosslink Cys58 of E6 with 96% yield in 4 hours at 37 °C and Cys113 of Pin1 with >99% yield in 1 hour at room temperature, respectively. This approach enables the identification of highly selective covalent peptide inhibitors for diverse molecular targets, introducing an applicable method to assist pre-clinical therapeutic development pipelines.

Host defense peptides (HDPs) have long been studied as templates for the development of new antibiotic classes, and human HDPs hold unique potential among possible templates due to the evolution of the template peptide as part of the broader human immune system. In this work, we describe the development of lead peptidomimetics based on the human HDP LL-37. Using automated flow peptide synthesis technology, we rapidly generated a series of peptides and peptidomimetics based on our prior structure-activity work in the full-length LL-37 template, which led to the discovery of a modified minimal unit of LL-37 in which potency is enhanced by appending an N-terminal biphenyl clamp from the native sequence to the previously described core activity region. Activity of this lead derivative, termed FF-14, was further improved through the application of D-amino acids and C-terminal amidation as well as selected N-lipidation moieties to the template sequence, providing the foundation for future iteration on a small, highly active derivative of LL-37. ### Competing Interest Statement MGH has filed a provisional patent application on the results described in this manuscript. BLP is a co-founder and/or member of the scientific advisory board of several companies focusing on the development of protein and peptide therapeutics. National Institute of Allergy and Infectious Diseases, K08 AI166345, T32 AI007061, U19 AI142780 Cystic Fibrosis Foundation, https://ror.org/00ax59295, ALBIN21Q0, ALBIN22A0-KB, ALBIN19F0

Despite their potential, the preparation of large synthetic macrocyclic libraries for ligand discovery and development has been limited. Here, we produce 100-million-membered macrocyclic libraries containing natural and nonnatural amino acids. Near-quantitative intramolecular disulfide formation is facilitated by rapid oxidation with iodine in solution. After use in affinity selection, treatment with dithiothreitol enables near-quantitative reduction, rendering linear peptide analogs for standard tandem mass spectrometry. We use these libraries to discover macrocyclic binders to cadherin-2 and anti-hemagglutinin antibody clone 12ca5. Structure-activity relationship studies of an initial cadherin-binding peptide [CBP; apparent dissociation constant (Kd) = 53 nanomolar] reveal residues responsible for driving affinity (hotspots) and mutation-tolerant residues (coldspots). Two original macrocyclic libraries are prepared in which these hotspots and coldspots are derivatized with nonnatural amino acids. Following discovery and validation, high-affinity ligands are discovered from the coldspot library, with NCBP-4 demonstrating improved affinity (Kd = 29 nanomolar). Overall, we expect that this work will improve the use of macrocyclic libraries in therapeutic peptide development.

Affinity selection-mass spectrometry (AS-MS) is a ligand discovery platform that relies upon mass spectrometry to identify molecules bound to a biomolecular target. When utilized with large peptide libraries (108 members), AS-MS sample complexity can surpass the sequencing capacity of modern mass spectrometers, resulting in incomplete data, identification of few target-specific ligands, and/or incomplete sequencing. To address this challenge, we introduce pyBinder to perform quantitation on AS-MS data to process primary MS1 data and develop two scores to rank the peptides from the integration of their peak area: target selectivity and concentration-dependent enrichment. We benchmark pyBinder utilizing AS-MS data developed against antihemagglutinin antibody 12ca5, revealing that peptides that contain a motif known for target-specific high-affinity binding are well characterized by these two scores. AS-MS data from a second protein target, WD Repeat Domain 5 (WDR5), is analyzed to confirm the two pyBinder scores reliably capture the target-specific motif-containing peptides. From the results delivered by pyBinder, a list of target-selective features is developed and fed back into subsequent MS experiments to facilitate expanded data generation and the targeted discovery of selective ligands. pyBinder analysis resulted in a 4-fold increase in motif-containing sequence identification for WDR5 (from 3 to 14 ligands discovered), showing the utility of the two scores. This work establishes an improved approach for AS-MS to enable discovery outcomes (i.e., more ligands identified), but also a way to compare AS-MS data across samples, protocols, and conditions broadly.

Hi1a is a venom peptide isolated from the Australian funnel-web spider Hadronyche infensa, containing an intricate “double knot” tertiary structure and is currently under investigation for the treatment of ischemic stroke. The recombinant expression and chemical synthesis of Hi1a remains a significant challenge due to poor yields and laborious protocols, delaying progress in the translation to the clinic. Herein, we describe the first single-shot chemical synthesis of the Hi1a peptide (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (4.3 h total synthesis time) to quantities of linear Hi1a (> 10 mg). Our robust protocol facilitated rapid and efficient synthesis of chemical analogues for structure-activity relationship studies, demonstrating that chemical modification of the N- or C-terminus of Hi1a does not significantly perturb binding to acid sensing ion channel 1a (ASIC1a). The synthesis of fluorescently labelled Hi1a permitted live cell imaging using in vitro confocal microscopy, and RNA sequencing demonstrated that Hi1a did not perturb the genetic state of human neurological tissue. This work highlights AFPS as a technology that might address manufacturing issues associated with peptide production.

Flow chemistry has revolutionized poly amide synthesis, allowing access to entire synthetic proteins in a matter of hours. In principle, the efficiency of similar flow-based methods should also allow rapid access to extremely large compound libraries for selection-based drug discovery. To determine whether flow chemistry could be used for polyamide library synthesis, we adapted a semi-automated flow platform to the task of making combinatorial libraries, including both canonical and noncanonical amino acid building blocks. Using this platform, we then demonstrate the ability to decrease the turnaround time for custom library synthesis from days to <1 h while accessing quintillion-member libraries with orders of magnitude more compounds than can be made with current technologies. Flow synthesis is thus a powerful approach for the rapid generation of hypervariable libraries for selection-based drug discovery.

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and nonviral vectors) or require external devices (e.g., electroporation), limiting therapeutic applications. To address these problems, we developed a protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen (PA) proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that an engineered Acr (LFN-Acr) binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells (e.g., immortalized cell lines, embryonic stem cells, and 3D cell cultures) at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

Flow chemistry has revolutionized polyamide synthesis, allowing access to entire synthetic proteins in a matter of hours. In principle, the efficiency of similar flow-based methods should also allow rapid access to extremely large compound libraries for selection-based drug discovery. To determine whether flow chemistry could be used for polyamide library synthesis, we adapted a semi-automated flow platform to the task of making combinatorial libraries including both canonical and noncanonical amino acid building blocks. Using this platform, we then demonstrate the ability to decrease the turnaround time for custom library synthesis from days-to-weeks to < 1 hour while accessing quintillion-member libraries with diversities orders of magnitude beyond those achievable with current technologies. Flow synthesis is thus a powerful approach for the rapid generation of hyperdiverse libraries for selection-based drug discovery.