Unnatural amino acid

Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis...

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18...

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid-phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared-labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions...

Chiral recognition of amino acids is very important in both chemical and life sciences. Although chiral recognition with luminescence has many advantages such as being inexpensive, it is usually slow and lacks generality as the recognition module relies on structural complementarity. Here, we show that one single molecular-solid sensor, L-phenylalanine derived benzamide, can manifest the structural difference between the natural, left-handed amino acid and its right-handed counterpart via the difference of room-temperature phosphorescence (RTP) irrespective of the specific chemical structure...

A method was developed for the enantioselective formal 1,2-diamination of disubstituted ketenes using iminosulfinamides as nitrogen sources. The protocol involves the addition of lithium iminosulfinamides to ketenes to form N -iminosulfinyl amide metalloenolates. These metalloenolates then undergo a [2,3]-sigmatropic rearrangement to yield unnatural α,α-disubstituted α-amino acid derivatives with high enantiopurity. The chirality present at the sulfur atom in the iminosulfinamides is effectively transferred to α carbon of the resulting products, facilitating the highly enantioselective amination of ketenes...

The photoredox electron donor-acceptor (EDA) complex-mediated radical coupling reaction has gained prominence in the field of organic synthesis, finding widespread application in two-component coupling reactions. However, EDA complex-promoted multi-component reactions are not well developed with only a limited number of examples have been reported. Herein, we report a photoinduced and EDA complex-promoted highly chemoselective three-component radical arylalkylation of [1.1.1]propellane, which allows the direct functionalization of C(sp3)-H with bicyclo[1...

Deciphering ubiquitin proteoform signaling and its role in disease has been a long-standing challenge in the field. The effects of ubiquitin modifications, its relation to ubiquitin-related machineries, and its signaling output has been particularly limited by its reconstitution and means of characterization. Advances in genetic code expansion have contributed towards addressing these challenges by precision incorporation of unnatural amino acids through site selective codon suppression. This review discusses recent advances in studying the 'writers', 'readers', and 'erasers' of the ubiquitin code using genetic code expansion...

Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH2 and Tyr-Yyy-Trp-Thr-NH2 , where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds...

Homochiral α-amino acids are widely used in pharmaceutical design as key subunits in chiral catalyst synthesis or as building blocks in synthetic biology. Many synthetic methods have been developed to access rare or unnatural variants by controlling the installation of the α-stereocentre. By contrast, and despite their importance, α-amino acids possessing β-stereocentres are much harder to synthesize. Here we demonstrate an iridium-catalysed protocol that allows the direct upconversion of simple alkenes and glycine derivatives to give β-substituted α-amino acids with exceptional levels of regio- and stereocontrol...

Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression...

The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2...

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. Here, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines, namely unnatural amino acids 1-3, could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR)...

Targeting immunosuppressive metastatic cancer cells is a key challenge in therapy. We recently have shown that a rigid-rod aromatic, pBP-NBD, that responds to enzymes and kill immunosuppressive metastatic osteosarcoma (mOS) and castration resistant prostate cancer (CRPC) cells in mimetic bone microenvironment. However, pBP-NBD demonstrated moderate efficacy against CRPC cells. To enhance activity, we incorporated the unnatural amino acid L- or D-4,4'-biphenylalanine (L- or D-BiP) into pBP-NBD, drastically increasing cellular uptake and CRPC inhibition...

An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion...

Using a commercially available potentiostat, the electrochemical synthesis of unnatural amino acids bearing heteroaromatics on the lateral chain has been accomplished. This strategy exploits the side-chain decarboxylative arylation of aspartic/glutamic acid, a reaction that becomes challenging with electron-rich coupling partners such as 5- and 6-membered heteroaromatics. These rings are underrepresented in unnatural amino acids, therefore allowing a wider exploration of the chemical space, given the abundance of the aryl bromides employable in this reaction...

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins...

The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques...

Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of ( R )- or ( S )-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2'-bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues...

BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry...

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide- and protein-binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non-proteinaceous ligand-binding protein, flavin-based fluorescent protein CagFbFP...