Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers.

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 to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

Xenoprotein engineering via synthetic libraries.

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

A fully automated flow-based approach for accelerated peptide synthesis.

Here we report a fully automated, flow-based approach to solid-phase polypeptide synthesis, with amide bond formation in 7 seconds and total synthesis times of 40 seconds per amino acid residue. Crude peptide purities and isolated yields were comparable to those for standard-batch solid-phase peptide synthesis. At full capacity, this approach can yield tens of thousands of individual 30-mer peptides per year.

d-Amino Acid Scan of Two Small Proteins.

A "D-scan" of two small proteins, the disulfide-rich Ecballium elaterium trypsin inhibitor II (EETI-II) and a minimized Z domain of protein A (Z33), is reported. For each protein, the stereochemistry of one amino acid at a time was inverted to generate a series of diastereomers. In much the same way an alanine scan determines necessary residues for protein function, the D-scan elucidated the critical stereocenters of the 30-residue EETI-II and the 33-residue Z33. The folding properties and activity of each variant were investigated. A total of 24 out of 30 EETI-II D-scan analogues folded to give a three-disulfide product. Of the 24 variants that folded, half were high-affinity trypsin inhibitors, and three were as active as the wild type (WT). Of these 12 active variants, most were substantially less stable to reduction than WT EETI-II (WT first reduction potential -270.0 ± 1.5 mV, WT second reduction potential -307.2 ± 1.1 mV). Similarly, ten Z33 analogues retained high binding affinity to IgG (KD < 250 nM, WT: 24 ± 1 nM) and 12 additional analogues had reduced but appreciable IgG binding affinity (KD between 250 nM and 2.5 µM). As with EETI-II, most Z33 analogues were substantially less stable than the WT (ΔG(H2O, 263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show that the D-scan is powerful new strategy for studying how the stereochemistry of amino acids affects the structure and function of proteins.

C-Terminal Modification of Fully Unprotected Peptide Hydrazides via in Situ Generation of Isocyanates.

A method for chemo- and regioselective conjugation of nucleophiles to fully unprotected peptides and proteins via in situ generation of C-terminal isocyanates is reported. Oxidation of C-terminal peptide hydrazides in aqueous media followed by Curtius rearrangement of acyl azides reliably generates isocyanates, which react with a variety of external nucleophiles, such as hydrazines, hydrazides, aromatic thiols, and hydroxylamines. Multiple peptides and a 53 kDa protein hydrazide were conjugated to different nucleophiles using this reaction.

A perfluoroaromatic abiotic analog of H2 relaxin enabled by rapid flow-based peptide synthesis.

H2 relaxin is a pleiotropic peptide hormone with clinical potential. Here we report on the reaction and use of hexafluorobenzene as an intramolecular disulfide replacement between Cys10 and Cys15 in the A-chain of H2 relaxin. Using flow-based Fmoc solid-phase peptide synthesis methodology we were able to obtain high-quality H2 relaxin fragments that were previously reported as challenging to synthesize. Subsequent native chemical ligation and oxidative folding enabled total synthesis of both wild type H2 relaxin and a C6F4 linked analog. Cell-based activity assays revealed modest activity for the C6F4 linked H2 relaxin analog, albeit 100-fold reduced relative to wild type. This work demonstrates how perfluoroarylation-cysteine SNAr chemistry may be a useful tool for the selective replacement of native disulfide bonds in proteins.

Flow-based enzymatic ligation by sortase A.

Sortase-mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase-mediated side reactions. A flow-based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations. The platform was tested with several reaction partners and used to generate a protein bioconjugate inaccessible by solution-phase batch sortagging.

Rapid flow-based peptide synthesis.

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.

Rapid total synthesis of DARPin pE59 and barnase.

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.

Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides.

Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide library based on the SNAr reaction between cysteine thiolates and a new generation of highly reactive perfluoroaromatic small molecule linkers. This strategy enabled us to simultaneously "scan" two cysteine residues positioned from i, i + 1 to i, i + 14 sites in a polypeptide, producing 98 macrocyclic products from reactions of 14 peptides with 7 linkers. A complementary reverse strategy was developed; cysteine residues within the polypeptide were first modified with non-bridging perfluoroaryl moieties and then commercially available dithiol linkers were used for macrocyclization. The highly convergent, site-independent, and modular nature of these two strategies coupled with the unique chemoselectivity of a SNAr transformation allows for the rapid diversity-oriented synthesis of hybrid macrocyclic peptide libraries with varied chemical and structural complexities.

Mood assessment via animated characters: a novel instrument to evaluate feelings in young children with anxiety disorders.

We evaluated a novel, computerized feelings assessment instrument (MAAC) in 54 children with anxiety disorders and 35 nonanxious children ages 5 to 11. They rated their feelings relative to 16 feeling animations. Ratings of feelings, order of feeling selection, and correlations with standardized anxiety measures were examined. Positive emotions were rated more highly and visited earlier by nonanxious children. Children with anxiety disorders explored fewer emotions. MAAC ratings on several positive emotions showed inverse correlations with state anxiety. Although needing further evaluation, MAAC may facilitate feelings assessment in young children and may distinguish children with anxiety disorders from nonanxious children.