Unnatural Amino Acids Research Articles

Scalable synthesis of sulfonium and selenonium peptides for selective methyllysine reader crosslinking

Lysine methylation readers are an important class of proteins that bind site-specifically methylated proteins for downstream regulation. Chemical probes that crosslink lysine methylation readers are highly desired to investigate the proteins from cellular samples. We recently reported NleS+me2 (norleucine-ε-dimethylsulfonium) as dimethyllysine mimic selectively crosslinks corresponding methyllysine readers. Although the sulfonium tools exhibited great promise, the synthetic availability may limit broad applications. In order to incorporate the unnatural amino acid, l-Fmoc-NleSme-OH (Fmoc: fluorenyl methoxycarbonyl) was synthesized as an important building block for solid-phase peptide synthesis (SPPS) in the previous synthetic route. It took six steps with resolution of racemic mixture and radical mediated thiol-ene reaction. As a result, the synthesis was not scalable with only 4.4 % overall yield. Here we report a much-improved synthesis method so that diverse NleS+me2 peptides could be readily prepared. In addition, the new method enables preparation of selenonium peptides for methyllysine reader crosslinking. We thus believe this synthetic method will be widely used to prepare sulfonium and selenonium probes for site-selective crosslinking.

Reaching New Heights in Genetic Code Manipulation with High Throughput Screening.

The chemical and physical properties of proteins are limited by the 20 canonical amino acids. Genetic code manipulation allows for the incorporation of noncanonical amino acids (ncAAs) that enhance or alter protein functionality. This review explores advances in the three main strategies for introducing ncAAs into biosynthesized proteins, focusing on the role of high throughput screening in these advancements. The first section discusses engineering aminoacyl-tRNA synthetases (aaRSs) and tRNAs, emphasizing how novel selection methods improve characteristics including ncAA incorporation efficiency and selectivity. The second section examines high-throughput techniques for improving protein translation machinery, enabling accommodation of alternative genetic codes. This includes opportunities to enhance ncAA incorporation through engineering cellular components unrelated to translation. The final section highlights various discovery platforms for high-throughput screening of ncAA-containing proteins, showcasing innovative binding ligands and enzymes that are challenging to create with only canonical amino acids. These advances have led to promising drug leads and biocatalysts. Overall, the ability to discover unexpected functionalities through high-throughput methods significantly influences ncAA incorporation and its applications. Future innovations in experimental techniques, along with advancements in computational protein design and machine learning, are poised to further elevate this field.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins.

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Palladium‐Catalyzed Directing‐Group‐Mediated γ‐C(sp3)−H Glycosylation for Synthesis of C‐Alkyl Glycosides

AbstractC‐alkyl glycosides play a crucial role in various bioactive compounds. However, the synthesis of C‐alkyl glycosides poses significant challenges, particularly through C(sp3)−H glycosylation. Here, we report a set of reactions for constructing C‐alkyl glycosides through directing‐group‐mediated functionalization of unactivated γ‐C(sp3)−H bonds under mild conditions. These reactions not only achieve high regioselectivity and stereoselectivity in glycosylation, but also exhibit a wide substrate scope. They are compatible with both arene and alkane substrates, as well as natural and unnatural amino acid substrates. Mechanistic studies have shown that the directing‐group 8‐aminoquinoline (AQ) and picolinamide (PA) may affect the chirality of the β‐carbon of L‐valine through a sterically favorable trans‐palladacycle intermediate, resulting in (R) or (S) configuration of glycosylated amino acid, respectively. These reactions are promising to provide a convenient and powerful tool for constructing C‐alkyl glycosides and carbohydrate‐based drugs in the future.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins

AbstractSulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Voltage-clamp fluorometry for advancing mechanistic understanding of ion channel mechanisms with a focus on acid-sensing ion channels.

Voltage-clamp fluorometry (VCF) has revolutionized the study of ion channels by combining electrophysiology with fluorescence spectroscopy. VCF allows ion channel researchers to link dynamic structural changes, measured in real time, to function. Acid-sensing ion channels (ASICs) are Na+-permeable non-voltage-gated ion channels of the central and peripheral nervous system. They function as pH sensors, triggering neuronal excitation when pH decreases. Animal studies have shown the importance of ASICs for pain and fear sensation, learning, and neurodegeneration following ischaemic stroke. This review explores the technical bases and various developments of VCF, including fluorescence resonance energy transfer and the use of unnatural fluorescent amino acids. We provide an overview of VCF applications with a focus on ASICs, detailing how VCF has unveiled proton-induced conformational changes in key regions such as the acid pocket, wrist, and pore, crucial for understanding transitions between closed, open, and desensitized states.

Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR.

In-cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in-cell. However, the need to spin label proteins ex-situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in-cell spin labeling. We quantify the amount of Cu(II)-NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat-shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)-NTA into E. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community.

Protoporphyrin IX-Dependent Antiviral Effects of 5-Aminolevulinic Acid against Feline Coronavirus Type II.

5-Aminolevulinic acid (5-ALA), a non-proteinogenic amino acid, is an intermediate in the biosynthesis of heme and exerts antiviral effects against feline coronavirus (FCoV); however, the underlying mechanisms remain unclear. In the biosynthesis of heme, 5-ALA is condensed and converted to protoporphyrin IX (PpIX), which is then transformed into heme by the insertion of ferrous iron. Previous research has suggested that the metabolites generated during heme biosynthesis contribute to the antiviral effects of 5-ALA. Therefore, the present study investigated the in vitro mechanisms responsible for the antiviral effects of 5-ALA. The results obtained revealed that 5-ALA and PpIX both effectively reduced the viral titer in the supernatant of FCoV-infected fcwf-4 cells. Moreover, PpIX exerted virucidal effects against FCoV. We also confirmed that 5-ALA increased PpIX levels in cells. While hemin induced heme oxygenase-1 gene expression, it did not reduce the viral titer in the supernatant. Sodium ferrous citrate decreased PpIX levels and suppressed the antiviral effects of 5-ALA. Collectively, these results suggest that the antiviral effects of 5-ALA against FCoV are dependent on PpIX.

Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in Escherichia coli Cells.

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.

Potent Inhibition and Rapid Photoactivation of Endogenous Bruton's Tyrosine Kinase Activity in Native Cells via Opto-Covalent Modulators.

Naturally, kinases exert their activities in a highly regulated fashion. A number of ingenious approaches have been developed to artificially control kinase activity by external stimuli, such as the incorporation of unnatural amino acids or the fusion of additional protein domains; however, methods that directly modulate endogenous kinases in native cells are lacking. Herein, we present a facile and potent method that takes advantage of recent developments in targeted covalent inhibitors and rapid light-mediated uncaging chemistry. Using an important drug target, Bruton's tyrosine kinase (BTK), as an example, these opto-covalent modulators successfully blocked the activity of endogenous BTK in native cells after simple incubation and washout steps. However, upon a few minutes of light irradiation, BTK activity was cleanly restored, and could be blocked again by conventional inhibitors. Promisingly, this photoactivation strategy easily worked in human peripheral blood mononuclear cells (hPBMCs).

Biosynthesis of novel non-proteinogenic amino acids β-hydroxyenduracididine and β-methylphenylalanine in Escherichia coli.

Non-proteinogenic amino acids (npAAs) are valuable building blocks for the development of advanced pharmaceuticals and agrochemicals. The surge in interest in their synthesis is primarily due to the potential to enhance and diversify existing bioactive molecules. This can be achieved by altering these bioactive molecules to improve their effectiveness, reducing resistance compared to their natural counterparts or generating molecules with novel functions. Traditional production of npAAs in native hosts requires specialized conditions and complex cultivation media. Furthermore, these compounds are often found in organisms that challenge genetic manipulation. Thus, the recombinant production of these npAAs in a model organism like Escherichia coli paves the way for groundbreaking advancements in synthetic biology. Two synthetic operons, comprising of five heterologous proteins were genomically integrated into E. coli for the synthesis of npAAs β-methylphenylalanine (BmePhe), β-hydroxyenduracididine (BhEnd), and enduracididine (End). Proteomic and metabolomic analysis confirmed production of these compounds in E. coli for the first time. Interestingly, we discovered that the exogenous addition of pathway precursors to the E. coli system enhanced the yield of BmePhe by 2.5 times, whereas it concurrently attenuated the production of BhEnd and End, signifying a selective precursor-dependent yield enhancement. The synthetic biology landscape is broadened in this study by expanding the repertoire of amino acids beyond the conventional set of 22 standard proteinogenic amino acids. The biosynthesized npAAs, End, BhEnd, and BmePhe hold promise for engineering proteins with modified functions, integrating into novel metabolites and/or enhancing biological stability and activity. Additionally, these amino acids' biological production and subsequent purification present an alternative to traditional chemical synthesis methods, paving a direct pathway for pharmacological evaluation.

Modulating the pH dependent photophysical properties of green fluorescent protein.

The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNO2Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the pK a of the 3-nitrophenol side chain of mNO2Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the pK a of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.

Expanding the Genetic Code of Bioelectrocatalysis and Biomaterials.

Genetic code expansion is a promising genetic engineering technology that incorporates noncanonical amino acids into proteins alongside the natural set of 20 amino acids. This enables the precise encoding of non-natural chemical groups in proteins. This review focuses on the applications of genetic code expansion in bioelectrocatalysis and biomaterials. In bioelectrocatalysis, this technique enhances the efficiency and selectivity of bioelectrocatalysts for use in sensors, biofuel cells, and enzymatic electrodes. In biomaterials, incorporating non-natural chemical groups into protein-based polymers facilitates the modification, fine-tuning, or the engineering of new biomaterial properties. The review provides an overview of relevant technologies, discusses applications, and highlights achievements, challenges, and prospects in these fields.

Novel Vibrational Proteins.

Genetically encoded green fluorescent protein (GFP) and its brighter and redder variants have tremendously revolutionized modern molecular biology and life science by enabling direct visualization of gene regulated protein functions on microscopic and nanoscopic scales. However, the current fluorescent proteins (FPs) only emit a few colors with an emission width of about 30-50 nm. Here, we engineer novel vibrational proteins (VPs) that undergo much finer vibrational transitions and emit rather narrow vibrational spectra (0.1-0.3 nm, roughly 3-10 cm-1). In response to an amber stop codon (UAG), a terminal alkyne bearing an unnatural amino acid (UAA, pEtF) is directly incorporated in place of Tyr64 in the chromophore of pr-Kaede by genetic code expansion. Essentially, the UAA64 further conjugates into a large π system with the contiguous two editable amino acid residues (His63 and Gly65), resulting in a programmable Raman resonance shift of the embedded alkyne. In the proof-of-concept experiment, we constructed a series of novel pEtF-VP mutants and observed fine Raman shifts of the alkynyl group in different chromophores. The genetically encoded novel VPs, could potentially label tens of proteins in the future.

Photoinduced Radical Approach for Desulfurative Alkylation of Cysteine Derivatives to Make Unnatural Amino Acids.

Unnatural amino acids (UAAs) are highly valuable molecules in organic synthesis, pharmaceutical sciences, and material science. Herein, we present a photocatalytic radical approach for desulfurative alkylation of cysteine derivatives with arenethiol as the hydrogen atom transfer catalyst for making UAAs and peptides. The formate salt, acting as the hydrogen atom donor, in situ generates the highly reductive CO2 radical anion species, which is the key to unlocking the C-S bond cleavage process with a simple benzoyl protecting group. No photocatalyst is required for the radical initiation and propagation, which makes such a visible-light-induced process mild, efficient, and sustainable.

A conserved peptide-binding pocket in HyNaC/ASIC ion channels

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

A Telescoped Photo-/Organo-Catalyzed Cross Dehydrogenative Coupling (CDC) between Glycine Derivatives and Ketones to Afford Nonproteinogenic Amino Acids (NPAAs) Enabled by Heterogeneous Continuous Flow Catalysis

A Telescoped Photo-/Organo-Catalyzed Cross Dehydrogenative Coupling (CDC) between Glycine Derivatives and Ketones to Afford Nonproteinogenic Amino Acids (NPAAs) Enabled by Heterogeneous Continuous Flow Catalysis

Exploiting the chemical diversity space of phosphopeptide binding to nasopharyngeal carcinoma PLK1 PBD domain with unnatural amino acid building blocks by using QSAR-based genetic optimization

Exploiting the chemical diversity space of phosphopeptide binding to nasopharyngeal carcinoma PLK1 PBD domain with unnatural amino acid building blocks by using QSAR-based genetic optimization

Insight into Protein Engineering: From In silico Modelling to In vitro Synthesis.

Protein engineering alters the polypeptide chain to obtain a novel protein with improved functional properties. This field constantly evolves with advanced in silico tools and techniques to design novel proteins and peptides. Rational incorporating mutations, unnatural amino acids, and post-translational modifications increases the applications of engineered proteins and peptides. It aids in developing drugs with maximum efficacy and minimum side effects. Currently, the engineering of peptides is gaining attention due to their high stability, binding specificity, less immunogenic, and reduced toxicity properties. Engineered peptides are potent candidates for drug development due to their high specificity and low cost of production compared with other biologics, including proteins and antibodies. Therefore, understanding the current perception of designing and engineering peptides with the help of currently available in silico tools is crucial. This review extensively studies various in silico tools available for protein engineering in the prospect of designing peptides as therapeutics, followed by in vitro aspects. Moreover, a discussion on the chemical synthesis and purification of peptides, a case study, and challenges are also incorporated.

De Novo Biosynthesis of L-Azetidine-2-Carboxylic Acid in Escherichia coli Strains for Powdery Mildew Treatment.

L-Azetidine-2-carboxylic acid (L-Aze), a natural nonproteinogenic amino acid found widely in plants, has recently been identified as an environmentally friendly agent for controlling powdery mildew with low toxicity. In this study, a biological route for L-Aze production via the methionine salvage pathway (Yang Cycle) was first in silico designed for Escherichia coli. Subsequently, systematic engineering strategies were employed to enhance the production efficiency, including the enhancement of the 5-phosphoribosyl 1-pyrophosphate (PRPP) supply, construction of the ATP-adenine cycle, and engineering of the strain's resistance to L-Aze. The final strain produced L-Aze from glucose with a titer of 568.5 mg/L. The antifungal activity of the produced L-Aze in the fermentation broth was also confirmed for treating powdery mildew in cucurbits. This approach not only provides a sustainable and green route for pesticide production to control powdery mildew but also expands our understanding of the exogenous construction of the Yang Cycle in E. coli.