Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

28Coronavirus disease 19 is an emerging global health crisis. With over 200,000 29 confirmed cases to date, this pandemic continues to expand, spurring research to discover 30 vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It 31 initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the 32 receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding 33 to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, 34 presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive 35 solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact 36interface. Using molecular dynamics simulations based on the recently solved ACE2 and SARS-37CoV-2-RBD co-crystal structure, we observed that the ACE2 peptidase domain (PD) α1 helix is 38 important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we 39 chemically synthesized a 23-mer peptide fragment of the ACE2 PD α1 helix composed entirely of 40 proteinogenic amino acids. Chemical synthesis of this human derived sequence was complete in 41 1.5 hours and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer 42 interferometry revealed that this peptide specifically associates with the SARS-CoV-2-RBD with 43 low nanomolar affinity. This peptide binder to SARS-CoV-2-RBD provides new avenues for 44 COVID-19 treatment and diagnostic modalities by blocking the SARS-CoV-2 spike protein 45 interaction with ACE2 and thus precluding virus entry into human cells. 46 47 Key words ： SARS-CoV-2, peptide binder, protein-protein interaction inhibitor, coronavirus, 48 COVID-19, rapid response, peptide therapeutic, MD simulation, automated flow peptide synthesis 49 50 51 3

The synthesis, crystal structure, and electronic properties of perfluoro-isopropyl-substituted perfluorophthalocyanine bearing a copper atom in the central cavity (F(64)PcCu) are reported. While most halogenated phthalocyanines do not exhibit long-term order sufficient to form large single crystals, this is not the case for F(64)PcCu. Its crystal structure was determined by X-ray analysis and linked to the electronic properties determined by electron paramagnetic resonance (EPR). The findings are corroborated by density functional theory (DFT) computations, which agree well with the experiment. X-band continuous-wave EPR spectra of undiluted F(64)PcCu powder, indicate the existence of isolated metal centers. The electron-withdrawing effect of the perfluoroalkyl (R(f)) groups significantly enhances the complexes solubility in organic solvents like alcohols, including via their axial coordination. This coordination is confirmed by X-band (1)H HYSCORE experiments and is also seen in the solid state via the X-ray structure. Detailed X-band CW-EPR, X-band Davies and Mims ENDOR, and W-band electron spin-echo-detected EPR studies of F(64)PcCu in ethanol allow the determination of the principal g values and the hyperfine couplings of the metal, nitrogen, and fluorine nuclei. Comparison of the g and metal hyperfine values of F(64)PcCu and other PcCu complexes in different matrices reveals a dominant effect of the matrix on these EPR parameters, while variations in the ring substituents have only a secondary effect. The relatively strong axial coordination occurs despite the diminished covalency of the C-N bonds and potentially weakening Jahn-Teller effects. Surprisingly, natural abundance (13)C HYSCORE signals could be observed for a frozen ethanol solution of F(64)PcCu. The (13)C nuclei contributing to the HYSCORE spectra could be identified as the pyrrole carbons by means of DFT. Finally, (19)F ENDOR and easily observable paramagnetic NMR were found to relate well to the DFT computations, revealing negligible isotropic hyperfine (Fermi contact) contributions. The single-site isolation in solution and solid state and the relatively strong coordination of axial ligands, both attributed to the introduction of R(f) groups, are features important for materials and catalyst design.

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 show how machine learning enables de novo design of abiotic nuclear-targeting miniproteins to traffic antisense oligomers to the nucleus of cells. We combined high-throughput experimentation with a directed evolution-inspired deep learning approach in which the molecular structures of natural and unnatural residues are represented as topological fingerprints. The model is able to predict activities beyond the training dataset, and simultaneously deciphers and visualizes sequence-activity predictions. The predicted miniproteins, termed "Mach", reach 10 kDa average mass, are more effective than any previously known variant in cells, and can also deliver proteins into the cytosol. The Mach miniproteins are nontoxic and efficiently deliver antisense cargo in mice. These results demonstrate that deep learning can decipher design principles to generate highly active biomolecules that are unlikely to be discovered by empirical approaches.

To elucidate the factors that impart selectivity for nitroxyl (HNO) over nitric oxide (NO), thiols, and H2S in metal-based fluorescent probes, we investigated five Cu(II)-cyclam (14-N4) derivatives. Upon exposure to NO gas at pH 7, no changes occur in the UV-vis spectra of any of the complexes. Addition of Angeli's salt to generate HNO promotes reduction of Cu(II) only in the case of [Cu(II)(14-N4-Ts)(OTf)2], which has the most positive reduction potential of the series. To gain insight into the observed reactivity, we prepared the Cu(II) complex of the mixed thia/aza 14-N2S2 ligand. [Cu(II)(14-N2S2)(OTf)2] reacts reversibly with HNO at pH 7, although nonselectively over thiols and H2S. The recurrent sensing of HNO uncovered with the study of Cu(II) azamacrocyclic complexes is a remarkable feature that opens the door for the design of a new generation of metal-based probes.