Enzymatic Noncovalent Synthesis.

Enzymatic reactions and noncovalent (i.e., supramolecular) interactions are two fundamental nongenetic attributes of life. Enzymatic noncovalent synthesis (ENS) refers to a process where enzymatic reactions control intermolecular noncovalent interactions for spatial organization of higher-order molecular assemblies that exhibit emergent properties and functions. Like enzymatic covalent synthesis (ECS), in which an enzyme catalyzes the formation of covalent bonds to generate individual molecules, ENS is a unifying theme for understanding the functions, morphologies, and locations of molecular ensembles in cellular environments. This review intends to provide a summary of the works of ENS within the past decade and emphasize ENS for functions. After comparing ECS and ENS, we describe a few representative examples where nature uses ENS, as a rule of life, to create the ensembles of biomacromolecules for emergent properties/functions in a myriad of cellular processes. Then, we focus on ENS of man-made (synthetic) molecules in cell-free conditions, classified by the types of enzymes. After that, we introduce the exploration of ENS of man-made molecules in the context of cells by discussing intercellular, peri/intracellular, and subcellular ENS for cell morphogenesis, molecular imaging, cancer therapy, and other applications. Finally, we provide a perspective on the promises of ENS for developing molecular assemblies/processes for functions. This review aims to be an updated introduction for researchers who are interested in exploring noncovalent synthesis for developing molecular science and technologies to address societal needs.

Enzymatically Forming Intranuclear Peptide Assemblies for Selectively Killing Human Induced Pluripotent Stem Cells.

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Heterotypic Supramolecular Hydrogels Formed by Noncovalent Interactions in Inflammasomes.

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.

An Exploration of Multiple Component Peptide Assemblies by Enzyme-Instructed Self-Assembly.

Based on the motifs (RNISY (M) and DEEVELILGDT (D)) in the protein crystal structures of Merlin and CRL4DCAF-1, we phosphorylated the tyrosine residue in M and conjugated M to a self-assembling motif to produce a phosphopeptide (1P) and examined enzyme-instructed self-assembly (EISA) of 1P with and without the presence of D (4). Our results show that EISA of 1P forms a hydrogel at exceedingly low volume fraction (~ 0.03%) even with the presence of the hydrophilic peptide, 4. Unlike 1P, 2P (a diastereomer of 1P) or 3P (the enantiomer of 1P) forms a hydrogel via EISA when their concentration is four or three times that of 1P, respectively. Circular dichroism (CD) spectra show that increasing the concentration of the phosphopeptides lowers the CD signals of the mixtures, and the magnitudes of the CD signals depends on the interaction between M and D. This work contributes insight for understanding multi-component hydrogels formed by self-assembly that involves both specific intermolecular interaction and enzymatic reactions.

Enzyme-instructed self-assembly of the stereoisomers of pentapeptides to form biocompatible supramolecular hydrogels.

This article reports enzyme-instructed self-assembly (EISA) of stereoisomers of pentapeptides as a simple approach for generating biocompatible supramolecular hydrogels as potential soft bionanomaterials. Peptide-based supramolecular hydrogels are emerging as a new type of biomaterials. The use of tyrosine phosphate offers a trigger for enzymatic hydrogelation, and the incorporation of D-amino acids can increase the proteolytic stability of peptides. This work compared four phosphorpeptides that are stereoisomers in terms of rate of dephosphorylation, proteolytic stability, and cell compatibility. The results show that the naphthyl (Nap)-capped pentapeptides, containing the amino acid sequence of Phe-Phe-Gly-Glu-pTyr, are able to undergo EISA to form the hydrogels consisting the nanofibres of the dephosphorylated pentapeptides. The naphthyl-capped D-phosphopentpeptides, contrasting to a naphthyl-capped D-phosphotripeptide (Nap-D-Phe-D-Phe-D-pTyr), are largely cell compatible. This result, suggesting that the sequence of phophopeptides also dedicates the cell compatibility of the peptide assemblies resulted from EISA, provides useful insights for developing supramolecular hydrogels as potential biomaterials with tailored properties.

Enzymatic Noncovalent Synthesis of Supramolecular Soft Matter for Biomedical Applications.

Enzymatic noncovalent synthesis (ENS), a process that integrates enzymatic reactions and supramolecular (i.e., noncovalent) interactions for spatial organization of higher-order molecular assemblies, represents an emerging research area at the interface of physical and biological sciences. This review provides a few representative examples of ENS in the context of supramolecular soft matter. After a brief comparison of enzymatic covalent and noncovalent synthesis, we discuss ENS of man-made molecules for generating supramolecular nanostructures (e.g., supramolecular hydrogels) in cell-free conditions. Then, we introduce ENS in a cellular environment. To illustrate the unique merits for applications, we discuss intercellular, peri- or intracellular, and subcellular ENS for cell morphogenesis, molecular imaging, cancer therapy, and targeted delivery. Finally, we provide an outlook on the potential of ENS. We hope that this review offers a new perspective for scientists who develop supramolecular soft matter to address societal needs at various frontiers.