Search

We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor., National Institutes of Health (U.S.) (GM046059), National Institutes of Health (U.S.) (GM101762), MIT Faculty Start-up Fund, Singapore. Agency for Science, Technology and Research (National Science Scholarship), National Cancer Institute (U.S.) (P30-CA14051)

Singled out for special treatment: Naturally occurring glutathione S-transferase (GST) was used to catalyze an efficient “click” ligation between polypeptides with an N-terminal glutathione sequence and biomolecules or chemical probes containing perfluorinated aromatic groups (see scheme). The site-specific modification of one cysteine residue was possible in the presence of other unprotected cysteine residues and reactive functional groups., National Institutes of Health (U.S.) (GM101762), National Institutes of Health (U.S.) (Award GM46059), MIT Faculty Start-up Fund, Damon Runyon Cancer Research Foundation, Amgen Inc. (Summer Graduate Research Fellowship)

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., MIT Faculty Start-up Fund, Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund), Deshpande Center for Technological Innovation, Damon Runyon-Rachleff (Innovation Award), Sontag Foundation (Distinguished Scientist Award), C. P. Chu and Y. Lai Fellowship, Daniel S. Kemp Summer Fellowship, National Institute of General Medical Sciences (U.S.). Biotechnology Training Program (Grant 5T32GM008334-25), National Institutes of Health (U.S.) (Fellowship F32GM101762)

Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide library based on the S[subscript N]Ar 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 S[subscript N]Ar transformation allows for the rapid diversity-oriented synthesis of hybrid macrocyclic peptide libraries with varied chemical and structural complexities., National Institutes of Health (U.S.) (GM101762), National Institutes of Health (U.S.) (GM046059), MIT Faculty Start-up Fund, Sontag Foundation (Distinguished Scientist Award), Deshpande Center for Technological Innovation, Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund), Damon Runyon Cancer Research Foundation