Synthetic Proteins behind the Plasma Barrier: Molecular Spies.

There is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes.Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein's sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes.Unfortunately, the low cell permeability of proteins limits their applications mainly to in vitro settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid-liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries.In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells.

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).

Copper(II)-Catalyzed Nitroaldol (Henry) Reactions: Recent Developments.

Self-assembled copper(II) complexes are described as effective catalysts for nitroaldol (Henry) reactions on water. The protocol involves a heterogeneous process and the catalysts can be recovered and recycled without loss of activity. Further, C2-symmetric N,N'-substituted chiral copper(II) salan complexes are found to be more effective catalysts than chiral copper(II) salen complexes for reactions in homogeneous catalysis, with high enantioselectivities. The reactions involve bifunctional catalysis, bearing the properties of a Brønsted base, as well as a Lewis acid, to effect the reaction in the absence of external additives.

Copper(II)-Catalyzed Oxidative Cross-Coupling of Anilines, Primary Alkyl Amines, and Sodium Azide Using TBHP: A Route to 2-Substituted Benzimidazoles.

Copper(II)-catalyzed oxidative cross-coupling of anilines, primary alkyl amines, and sodium azide is described in the presence of TBHP at moderate temperature. This one-pot multicomponent protocol involves a domino C-H functionalization, transimination, ortho-selective amination, and a cyclization sequence. The broad substrate scope and functional group compatibility are the significant practical features. The protocol can be extended to the coupling of benzyl alcohols with moderate yields.

Copper(I)-catalyzed regioselective amination of N-aryl imines using TMSN3 and TBHP: a route to substituted benzimidazoles.

A novel and efficient copper-catalyzed amination of N-aryl imines is described. This one-pot, multicomponent reaction, in which imine acts as a directing group by chelating to the metal center, affords a potential route for the transformation of the commercial aryl amines, aldehydes, and azides into valuable benzimidazole structural units with wide substrate scope and diversity. The synthetic and mechanistic aspects are presented.

Room-Temperature Cu(II)-Catalyzed Chemo- and Regioselective Ortho-Nitration of Arenes via C-H Functionalization.

An efficient Cu-catalyzed chemo- and regioselective ortho-nitration of N,1-diaryl-5-aminotetrazoles and N,4-diaryl-3-amino-1,2,4-triazoles have been described with good functional group compatibility. The procedure features the use of operationally simple protocol utilizing the commercially available less toxic CuCl2·2H2O as catalyst and Fe(NO3)3·9H2O as nitration source at room temperature. Removal of the 5-aminotetrazole directing group has been demonstrated using base hydrolysis to afford substituted 2-nitroanilines.

Organocatalytic syntheses of benzoxazoles and benzothiazoles using aryl iodide and oxone via C-H functionalization and C-O/S bond formation.

An organocatalytic protocol for the syntheses of 2-substituted benzoxazoles and benzothiazoles is described from alkyl-/arylanilides and alkyl-/arylthioanilides using 1-iodo-4-nitrobenzene as catalyst and oxone as an inexpensive and environmentally safe terminal oxidant at room temperature in air via oxidative C-H functionalization and C-O/S bond formation. The procedure is simple and general and provides an effective route for the construction of functionalized 2-alkyl-/arylbenzoxazoles and 2-alkyl-/arylbenzothiazoles with moderate to high yields. The synthetic and mechanistic aspects have been described.

Room-temperature Cu(II)-catalyzed chemo- and regioselective ortho-nitration of arenes via C-H functionalization.

An efficient Cu-catalyzed chemo- and regioselective ortho-nitration of N,1-diaryl-5-aminotetrazoles and N,4-diaryl-3-amino-1,2,4-triazoles has been described with good functional group compatibility. The procedure features the use of an operationally simple protocol utilizing the commercially available less toxic CuCl2·2H2O as catalyst and Fe(NO3)3·9H2O as nitration source at room temperature. Removal of the 5-aminotetrazole directing group has been demonstrated using base hydrolysis to afford substituted 2-nitroanilines.

Pd(II)-catalyzed aminotetrazole-directed ortho-selective halogenation of arenes.

A Pd(II)-catalyzed ortho-selective halogenation of N-aryl ring of N,1-diaryl-1H-tetrazol-5-amine has been described employing N-halosuccinimide as a halogen source via C-H bond activation. The present work features 5-aminotetrazole, as a directing group, for the chemo- and regioselective C-H halogenation of arenes. The kinetic isotope study (kH/kD = 2.9) suggests that the cleavage of the C-H bond takes place in the rate-determining step. The scope and mechanism of the protocol have been demonstrated.

Copper(II)-mediated regioselective N-arylation of pyrroles, indoles, pyrazoles and carbazole via dehydrogenative coupling.

A copper(II)-mediated regioselective N-arylation of azoles has been developed using 8-aminoquinoline amide as a directing group. This reaction shows a broad substrate scope with different azoles such as pyrroles, indoles, pyrazoles and carbazole with good yields.

Iodobenzene catalyzed C-H amination of N-substituted amidines using m-chloroperbenzoic acid.

The oxidative C-H amination of N"-aryl-N'-tosyl/N'-methylsulfonylamidines and N,N'-bis(aryl)amidines has been accomplished using iodobenzene as a catalyst to furnish 1,2-disubstituted benzimidazoles in the presence of mCPBA as a terminal oxidant at room temperature. The reaction is general, and the target products can be obtained in moderate to high yields.