Genetically Encoded Photocaged Proteinogenic and Non-Proteinogenic Amino Acids.

Photocaged amino acids could be genetically encoded into proteins via genetic code expansion (GCE) and constitute unique tools for innovative protein engineering. There are a number of photocaged proteinogenic amino acids that allow strategic conversion of proteins into their photocaged variants, thus enabling spatiotemporal and non-invasive regulation of protein functions using light. Meanwhile, there are a hand of photocaged non-proteinogenic amino acids that address the challenges in directly encoding certain non-canonical amino acids (ncAAs) that structurally resemble proteinogenic ones or possess highly reactive functional groups. Herein, we would like to summarize the efforts in encoding photocaged proteinogenic and non-proteinogenic amino acids, hoping to draw more attention to this fruitful and exciting scientific campaign.

N-Glycan Engineering: Constructing the N-GlcNAc Stump.

N-Glycosylation is often essential for the structure and function of proteins. However, N-glycosylated proteins from natural sources exhibit considerable heterogeneity in the appended oligosaccharides, bringing daunting challenges to corresponding basic research and therapeutic applications. To address this issue, various synthetic, enzymatic, and chemoenzymatic approaches have been elegantly designed. Utilizing the endoglycosidase-catalyzed transglycosylation method, a single N-acetylglucosamine (N-GlcNAc, analogous to a tree stump) on proteins can be converted to various homogeneous N-glycosylated forms, thereby becoming the focus of research efforts. In this concept article, we briefly introduce the methods that allow the generation of N-GlcNAc and its close analogues on proteins and peptides and highlight the current challenges and opportunities the scientific community is facing.

Spontaneous formation of autocatalytic sets with self-replicating inorganic metal oxide clusters.

Here we show how a simple inorganic salt can spontaneously form autocatalytic sets of replicating inorganic molecules that work via molecular recognition based on the {PMo12} ≡ [PMo12O40]3- Keggin ion, and {Mo36} ≡ [H3Mo57M6(NO)6O183(H2O)18]22- cluster. These small clusters are able to catalyze their own formation via an autocatalytic network, which subsequently template the assembly of gigantic molybdenum-blue wheel {Mo154} ≡ [Mo154O462H14(H2O)70]14-, {Mo132} ≡ [MoVI72MoV60O372(CH3COO)30(H2O)72]42- ball-shaped species containing 154 and 132 molybdenum atoms, and a {PMo12}⊂{Mo124Ce4} ≡ [H16MoVI100MoV24Ce4O376(H2O)56 (PMoVI10MoV2O40)(C6H12N2O4S2)4]5- nanostructure. Kinetic investigations revealed key traits of autocatalytic systems including molecular recognition and kinetic saturation. A stochastic model confirms the presence of an autocatalytic network involving molecular recognition and assembly processes, where the larger clusters are the only products stabilized by the cycle, isolated due to a critical transition in the network.

Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid.

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.

Bioconjugation via Hetero-Selective Clamping of Two Different Amines with ortho-Phthalaldehyde.

Amino groups are common in both natural and synthetic compounds and offer a very attractive class of endogenous handles for bioconjugation. However, the ability to differentiate two types of amino groups and join them with high hetero-selectivity and efficiency in a complex setting remains elusive. Herein, we report a new method for bioconjugation via one-pot chemoselective clamping of two different amine nucleophiles using a simple ortho-phthalaldehyde (OPA) reagent. Various α-amino acids, aryl amines, and secondary amines can be crosslinked to the ϵ-amino side chain of lysine on peptides or proteins with high efficiency and hetero-selectivity. This method offers a simple and powerful means to crosslink small molecule drugs, imaging probes, peptides, proteins, carbohydrates, and even virus particles without any pre-functionalization.

Toward an Orthogonal Protein Lysine Acylation and Deacylation System.

Lysine acetylation is one of the most basic molecular mechanisms to mediate protein functions in living organisms, and its abnormal regulation has been linked to many diseases. The drug development associated to this process is of great significance but severely hindered by the complex interplay of lysine acetylation and deacetylation in thousands of proteins, and we reasoned that targeting a specific protein acetylation or deacetylation event instead of the related enzymes should be a feasible solution to this issue. Toward this goal, we devised an orthogonal lysine acylation and deacylation (OKAD) system, which potentially could precisely dissect the biological consequence of an individual acetylation or deacetylation event in living cells. The system includes a genetically encoded acylated lysine (PhOAcK) that is not a substrate of endogenous deacetylases, and an evolved sirtuin (CobB2/CobB3) that displays PhOAcK deacylase activities as well as reduced deacetylase activities. We believe the strategy introduced here holds potential for future in-depth biological applications.

Expanding the Scope of Genetically Encoded Lysine Post-Translational Modifications with Lactylation, ß-Hydroxybutyrylation and Lipoylation.

Post-translational modifications (PTMs) occurring on lysine residues, especially diverse forms of acylations, have seen rapid growth over the past two decades. Among them, lactylation and ß-hydroxybutyrylation of lysine side-chains are newly identified histone marks and their implications in physiology and diseases have aroused broad research interest. Meanwhile, lysine lipoylation is highly conserved in diverse organisms and well known for its pivotal role in central metabolic pathways. Recent findings in the proteomic profiling of protein lipoylation have nonetheless suggested a pressing need for an extensive investigation. For both basic and applied research, it is necessary to prepare PTM-bearing proteins particularly in a site-specific manner. Herein, we use genetic code expansion to site-specifically generate these lysine PTMs, including lactylation, ß-hydroxybutyrylation and lipoylation in proteins in E. coli and mammalian cells. Notably, using strategies including activity-based selection, screening and rational design, unique pyrrolysyl-tRNA synthetase variants were successfully evolved for each of the three non-canonical amino acids, which enabled efficient production of recombinant proteins. Through encoding these ncAAs, we examined the deacylase activities of mammalian sirtuins to these modifications, and importantly we unfold the lipoamidase activity of several sirtuins.

Sirtuin-Derived Covalent Binder for the Selective Recognition of Protein Crotonylation.

Lysine crotonylation (Kcr) is increasingly recognized as a key protein post-translational modification. However, selective detection and enrichment of crotonylated proteins remains a challenging task. Herein we present a covalent binder for the selective recognition of protein crotonylation. Based on proximity-induced crosslinking, a bacterial sirtuin (CobB) was remodeled with genetically installed thiol-bearing noncanonical amino acids at the Kcr-interacting site, which subsequently could react with Kcr sites in a unique NAD+ -dependent manner. The covalent binder has been used to selectively recognize crotonylated proteins in extracted histone samples and in fixed cells.

Site-Specific Protein Modification with Reducing Carbohydrates.

Protein glycosylation plays critical roles in many biological processes. However, the fundamental study and application of glycobiology are hindered by the heterogeneousness of oligosaccharides in natural glycoproteins and the difficulty in constructing glycoproteins of human design. Herein, we describe a semisynthetic method to site-specifically modify proteins with reducing carbohydrates. The method involves the genetic incorporation of a side-chain-esterified aspartate, which was subsequently quantitatively converted into alanine-ß-hydrazide (Aßz), and chemoselective conjugation of Aßz with a range of readily available reducing carbohydrates. The resulting Aßz-linked GlcNAc is a close mimic of native N-GlcNAc and could be installed on various proteins, including IL-17A and RNase A. Notably, Aßz-linked GlcNAc on proteins reacted with biantennary oligosaccharide oxazoline derivatives through endoglycosidase-catalyzed transglycosylation reactions to enable the assembly of homogeneous glycans on proteins.

A General Supramolecular Approach to Regulate Protein Functions by Cucurbit[7]uril and Unnatural Amino Acid Recognition.

Regulation of specific protein function is of great importance for both research and therapeutic development. Many small or large molecules have been developed to control specific protein function, but there is a lack of a universal approach to regulate the function of any given protein. We report a general host-guest molecular recognition approach involving modification of the protein functional surfaces with genetically encoded unnatural amino acids bearing guest side chains that can be specifically recognized by cucurbit[7]uril. Using two enzymes and a cytokine as models, we showed that the activity of proteins bearing unnatural amino acid could be turned off by host molecule binding, which blocked its functional binding surface. Protein activity can be switched back by treatment with a competitive guest molecule. Our approach provides a general tool for reversibly regulating protein function through molecular recognition and can be expected to be valuable for studying protein functions.

Synthesis, Assembly, and Sizing of Neutral, Lanthanide Substituted Molybdenum Blue Wheels {Mo90Ln10}.

Polyoxometalate molybdenum blue (MB) complexes typically exist as discrete multianionic clusters and are composed of repeating Mo building units. MB wheels such as {Mo176} and {Mo154} are made from pentagon-centered {Mo8} building blocks joined by equal number of {Mo1} units as loin, and {Mo2} dimer units as skirt along the ring edge, with the ring sizes of the MB wheels modulated by the {Mo2} units. Herein we report a new class of contracted lanthanide-doped MB structures that have replaced all the {Mo2} units with lanthanide ions on the inner rim, giving the general formula {Mo90Ln10}. We show three examples of this new decameric {Mo90Ln10} (Ln = La, Ce, and Pr) framework synthesized by high temperature reduction and demonstrate that later Ln ions result in {Mo92Ln9} (Ln = Nd, Sm), conserving one {Mo2} linker unit in its structure, as a consequence of the lanthanide contraction. Remarkably the {Mo90Ln10} compounds are the first examples of charge-neutral molybdate wheels as confirmed by BVS, solubility experiments, and redox titrations. We detail our full synthetic optimization for the isolation of these clusters and complete characterization by X-ray, TGA, UV-vis, and ICP studies. Finally, we show that this fine-tuned self-assembly process can be utilized to selectively enrich Ln-MB wheels for effective separation of lanthanides.

Anisotropic Polyoxometalate Cages Assembled via Layers of Heteroanion Templates.

The synthesis of anisotropic redox-active polyoxometalates (POMs) that can switch between multiple states is critical for understanding the mechanism of assembly of structures with a high aspect ratio, as well as for their application in electronic devices. However, a synthetic methodology for the controlled growth of such clusters is lacking. Here we describe a strategy, using the heteroanion-directed assembly, to produce a family of 10 multi-layered, anisotropic POM cages templated by redox-active pyramidal heteroanions with the composition [W16Mo2O54(XO3)]n-, [W21Mo3O75/76(XO3)2]m-, and [W26Mo4O93(XO3)3]o- for the double, triple, and quadruple layered clusters, respectively. It was found that the introduction of reduced molybdate is essential for self-assembly and results in mixed-metal (W/Mo) and mixed-valence (WVI/MoV) POM cages, as confirmed by an array of analytical techniques. To probe the archetype in detail, a tetrabutyl ammonium (TBA) salt derivative of a fully oxidized two-layered cage is produced as a model structure to confirm that all the cages are a statistical mixture of isostructures with variable ratios of W/Mo. Finally, it was found that multilayered POM cages exhibit dipolar relaxations due to the presence of the mixed valence WVI/MoV metal centers, demonstrating their potential use for electronic materials.

Stereoselective Assembly of Gigantic Chiral Molybdenum Blue Wheels Using Lanthanide Ions and Amino Acids.

The synthesis of chiral polyoxometalates (POMs) is a challenge because of the difficulty to induce the formation of intrinsically chiral metal-oxo frameworks. Herein we report the stereoselective synthesis of a series of gigantic chiral Mo Blue (MB) POM clusters 1-5 that are formed by exploiting the synergy between coordinating lanthanides ions as symmetry breakers to produce MBs with chiral frameworks decorated with amino acids ligands; these promote the selective formation of enantiopure MBs. All the compounds share the same framework archetype, based on {Mo124Ce4}, which forms an intrinsically chiral Δ or Λ configurations, controlled by the configurations of functionalized chiral amino acids. The chirality and stability of 1-5 in solution are confirmed by circular dichroism, 1H NMR, and electrospray ion mobility-mass spectrometry studies. In addition, the framework of the {Mo124Ce4} MB not only behaves as a host able to trap a chiral {Mo8} cluster that is not accessible by traditional synthesis but also promotes the transformation of tryptophan to kynurenine in situ. This work demonstrates the potential and applicability of our synthetic strategy to produce gigantic chiral POM clusters capable of host-guest chemistry and selective synthetic transformations.

Supramolecular Coordination Cages for Asymmetric Catalysis.

Inspired by the high efficiency and specificity of enzymes in living systems, the development of artificial catalysts intrinsic to the key features of enzyme has emerged as an active field. Recent advances in supramolecular chemistry have shown that supramolecular coordination cages, built from non-covalent coordination bonds, offer a diverse platform for enzyme mimics. Their inherent confined cavity, analogous to the binding pocket of an enzyme, and the facile tunability of building blocks are essential for substrate recognition, transition-state stabilization, and product release. In particular, the combination of chirality with supramolecular coordination cages will undoubtedly create an asymmetric microenvironment for promoting enantioselective transformation, thus providing not only a way to make synthetically useful asymmetric catalysts, but also a model to gain a better understanding for the fundamental principles of enzymatic catalysis in a chiral environment. The focus here is on recent progress of supramolecular coordination cages for asymmetric catalysis, and based on how supramolecular coordination cages function as reaction vessels, three approaches have been demonstrated. The aim of this review is to offer researchers general guidance and insight into the rational design of sophisticated cage containers for asymmetric catalysis.

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Rational design of a function-based selection method for genetically encoding acylated lysine derivatives.

A foundation of genetic code expansion is the evolution of orthogonal aminoacyl-tRNA synthetase to recognize a non-canonical amino acid, which typically involves read-through of amber stop codon in the genes used in selection. The process is time-consuming, labor-intensive, and susceptible to certain bias. As a complementation, herein, we rationally designed a function-based method to directed evolution of aminoacyl-tRNA synthetase for acylated lysine derivatives.

Recombinant thiopeptides containing noncanonical amino acids.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Ligand-Directed Template Assembly for the Construction of Gigantic Molybdenum Blue Wheels.

Template-mediated synthesis is a powerful approach to build a variety of functional materials and complex supramolecular systems. However, the systematic study of how templates structurally evolve from basic building blocks, and then affect the templated self-assembly, is critical to understanding and utilizing the underlying mechanism, to work towards designed assembly. Here we describe the templated self-assembly of a series of gigantic Mo Blue (MB) clusters 1-4 using l-ornithine as a structure-directing ligand. We show that by using l-ornithine as a structure director, we can form new template⊂host assemblies. Based on the structural relationship between encapsulated templates of {Mo8 } (1), {Mo17 } (2) and {Mo36 } (4), a pathway of the structural evolution of templates is proposed. This provides insight into how gigantic Mo Blue cluster rings form and could lead to full control over the designed assembly of gigantic Mo-blue rings.

Digital Control of Multistep Hydrothermal Synthesis by Using 3D Printed Reactionware for the Synthesis of Metal-Organic Frameworks.

Hydrothermal-synthesis-based reactions are normally single step owing to the difficulty of manipulating reaction mixtures at high temperatures and pressures. Herein we demonstrate a simple, cheap, and modular approach to the design reactors consisting of partitioned chambers, to achieve multi-step synthesis under hydrothermal conditions, in digitally defined reactionware produced by 3D printing. This approach increases the number of steps that can be performed sequentially and allows an increase in the options available for the control of hydrothermal reactions. The synthetic outcomes of the multi-stage reactions can be explored by varying reaction compositions, number of reagents, reaction steps, and reaction times, and these can be tagged to the digital blueprint. To demonstrate the potential of this approach a series of polyoxometalate (POM)-containing metal-organic frameworks (MOFs) unavailable by "one-pot" methods were prepared as well as a set of new MOFs.

Genetically Encoded Fluorescent Probe for Detecting Sirtuins in Living Cells.

Sirtuins are NAD+ dependent protein deacetylases, which are involved in many biological processes. We now report a novel genetically encoded fluorescent probe (EGFP-K85AcK) that responds to sirtuins in living cells. The probe design exploits a lysyl residue in EGFP that is essential for chromophore maturation, and is also an efficient deacetylation substrate for sirtuins. Analysis of activity in Escherichia coli ΔcobB revealed that the probe can respond to various human sirtuins, including SIRT1, SIRT2, SIRT3 and SIRT5. We also directly monitored SIRT1 and SIRT2 activity in HEK293T cells with an mCherry fusion of EGFP-K85AcK, and showed that this approach can be extended to other fluorescent proteins. Finally, we demonstrate that this approach can be used to examine the activity of sirtuins toward additional lysyl posttranslational modifications, and show that sirtuins can act as erasers of HibK modified proteins.