Fully synthetic whole proteins in reach Solid-phase peptide synthesis of homogeneous peptides longer than about 50 amino acids has been a long-standing challenge because inefficient coupling and side reactions. Hartrampf et al. used an automated chemistry platform to optimize fast-flow were able produce fully single-domain (see the Perspective by Proulx). The targets included proinsulin enzymes such as barnase version HIV-1 protease containing multiple noncanonical acids. Refolded nearly...

Abstract Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates fill such a role, but their challenging synthesis limits widespread application. To rapidly prototype potential PMO drug candidates, we report fully automated flow-based oligonucleotide synthesizer. Our optimized platform reduces coupling times by...

Despite tremendous efforts to engineer translational machinery, replacing the encoded peptide backbone with new-to-nature structures remains a significant challenge. C, H, O, and N are elements of life, yet ribosomes capable forming only C–N bonds as amides, C–O esters, C–S thioesters. There is no current strategy site-selectively form C–C ketones embedded in backbones ribosomal products. As an alternative direct bond formation, here we report that peptides containing dehydrolactic acid...

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression 50% all human cancers. Still, targeting remains a challenge due to the lack small molecule binding pockets tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic target DNA-binding site. We combined automated flow peptide chemistry palladium(II) oxidative addition complexes (OACs)...

The natural product himastatin has an unusual homodimeric structure that presents a substantial synthetic challenge. We report the concise total synthesis of from readily accessible precursors, incorporating final-stage dimerization strategy was inspired by detailed consideration compound's biogenesis. Combining this approach with modular enabled expedient access to more than dozen designed derivatives himastatin, including probes provide insight into its antibiotic activity.

Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric complexes MYC/MAX and MAX/MAX activate or inhibit, respectively, gene upon binding to same enhancer box DNA. Targeting these cancer a long-standing challenge. Inspired by inhibitory activity dimer, we engineered covalently linked, synthetic homo- heterodimeric protein attenuate oncogenic MYC-driven transcription. We prepared covalent (∼20 kDa, 167–231 residues) single shot via parallel automated flow...

Antisense peptide nucleic acids (PNAs) have yet to translate the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument manufacture CPP-conjugated (PPNAs) in single shot. The machine is rapid, with each amide bond being formed 10 s. Anti-IVS2-654 PPNA...

Abstract Proteins and polypeptides containing extended backbone monomers embody highly desirable structures functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions from anything other than an α-amino acid or α-hydroxy unknown. The second challenge absence of orthogonal enzymes that acylate tRNA with monomers. Here we describe a general approach programmed cellular synthesis...

Therapeutic macromolecules such as proteins and oligonucleotides can be highly efficacious but are often limited to extracellular targets due the cell's impermeable membrane. Cell-penetrating peptides (CPPs) able deliver into cells, structure-activity relationships inconsistent literature reports make it difficult design effective CPPs for a given cargo. For example, polyarginine motifs common in CPPs, promoting cell uptake at expense of systemic toxicity. Machine learning may address this...

Despite tremendous efforts to engineer translational machinery, replacing the encoded peptide backbone with new-to-Nature structures remains a significant and largely unmet challenge. C, H, O, N are elements of life, yet ribosomes only capable forming C–N bonds as amides, C–O esters, C–S thioesters; there is no current strategy form C–C ketones embedded in ribosomal products. We discovered that peptides containing dehydrolactic acid motif rapidly isomerize generate backbone-embedded...

ABSTRACT We describe a new assay that reports directly on the acylation state of user-chosen tRNA in cells. call this 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies high-resolution mass spectrometry identification acyl-adenosine species released upon RNase A cleavage isolated cellular tRNA. Here we develop PARTI workflow and apply it to understand three recent observations related incorporation non-α-amino acid monomers into protein: (1) origins apparent selectivity...

Abstract We describe a new assay that reports directly on the acylation state of user-chosen transfer RNA (tRNA) in cells. call this 3-Prime Adenosine-Retaining Aminoacyl–tRNA Isolation (PARTI). It relies high-resolution mass spectrometry identification acyl-adenosine species released upon RNase A cleavage isolated cellular tRNA. Here we develop PARTI workflow and apply it to understand three recent observations related incorporation non-α-amino acid monomers into protein: (i) origins...

<p>Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - chain elongation complete hours. We demonstrate utility this approach by chemical synthesis nine different protein that represent enzymes, structural units, and regulatory factors. After purification folding,...

Phosphorodiamidate morpholino oligonucleotides (PMOs) make up a promising class of therapeutics for genetic disease. PMOs designed "exon skipping" must be internalized into cells, reach the nucleus, and act on pre-mRNA to mediate their effects. One tactic improving PMO delivery exon skipping is covalently conjugate cell-penetrating peptides (CPPs). Here, we report synthesis conjugated CPP chimeras, constructed by combining multiple CPPs one sequence. The chimeric synergistically improve...

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter cytosol and deliver otherwise nonpenetrant macromolecules such as proteins oligonucleotides. For example, recent clinical trials have shown that a CPP attached phosphorodiamidate morpholino oligomers (PMOs) resulted in higher muscle concentration, increased exon skipping, dystrophin production relative another study of PMO alone patients Duchenne muscular dystrophy. Therefore, effective design CPPs could help enhance...

Abstract Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols after capture. These are serine (seramox) or isoserine (isoseramox) can be incorporated into during solid‐phase peptide synthesis through reductive amination. Both quantitatively cleaved within minutes under NaIO 4...

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) reached the market after 30 years investigation due to challenges discovery new, more efficient sequences. Here, we demonstrate a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) discover from synthetic library capable delivering macromolecule cargo cytosol. This was inspired by recent vivo selection...

Significance Erythrocyte-bound antigens can drive immune tolerance in an antigen-specific fashion. Exploiting this phenomenon, we developed a general strategy to promote by engineering peptide and protein bind erythrocytes. Here, showed that fully d -chiral library be selected vivo for the de novo discovery of robust erythrocyte binder, which attached antigens. An administration engineered mitigated inflammatory responses, suggesting generalizability tolerance-induction validating our ligand...

Abstract There are more amino acid permutations within a 40-residue sequence than atoms on Earth. This vast chemical search space hinders the use of human learning to design functional polymers. Here we couple supervised and unsupervised deep with high-throughput experimentation drive high-activity, novel sequences reaching 10 kDa that deliver antisense oligonucleotides nucleus cells. The models, in which natural unnatural residues represented as topological fingerprints, decipher visualize...

Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - chain elongation complete hours. We demonstrate utility this approach by chemical synthesis nine different protein that represent enzymes, structural units, and regulatory factors. After purification folding, synthetic...

Peptide nucleic acids (PNAs) are charge-neutral oligonucleotides with emerging potential for treatment of genetic, acquired, and viral diseases, including COVID-19. Their challenging synthesis, however, limits their use rapid therapeutic intervention widespread application. Here, we report a highly efficient technology that utilizes fully automated fast-flow instrument to manufacture cell-penetrating peptide-conjugated PNAs (PPNAs) in single shot. The machine is rapid: each amide bond formed...

The antisense phosphorodiamidate morpholino oligomer (PMO) drugs Eteplirsen and Golodirsen are improving the lives of some Duchenne muscular dystrophy (DMD) patients, but treating all DMD subtypes would require development over 50 novel therapies. To rapidly prototype personalized PMO for diseases such as DMD, we designed a fully automated flow-based oligonucleotide synthesizer. Our optimized high temperature synthesis platform reduces coupling times by up to 22-fold compared previously...

<p>The antisense phosphorodiamidate morpholino oligomer (PMO) drugs Eteplirsen and Golodirsen are improving the lives of some Duchenne muscular dystrophy (DMD) patients, but treating all DMD subtypes would require development over 50 novel therapies. To rapidly prototype personalized PMO for diseases such as DMD, we designed a fully automated flow-based oligonucleotide synthesizer. Our optimized high temperature synthesis platform reduces coupling times by up to 22-fold compared...

Abstract Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) reached the market after 30 years investigation due to challenges discovery new, more efficient sequences. We developed a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) discover from synthetic library capable delivering macromolecule cargo cytosol. This was inspired by recent vivo selection...