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Antibody drug conjugates are an exciting therapeutic modality that combines the targeting specificity of antibodies with potent cytotoxins to selectively kill cancer cells. The targeting component improves efficacy and protects non-target cells from the harmful effects of the payload. To date 15 ADCs have been approved by regulatory agencies for commercial use and shown to be valuable tools in the treatment of cancer. The assembly of an ADC requires the chemical ligation of a linker-payload to an antibody. Conventional conjugation methods targeting accessible lysines and cysteines have produced all the ADCs currently on the market. While successful, technologies aiming to improve the homogeneity and stability of ADCs are being developed and tested. Here we provide a review of developing methods for ADC construction. These include enzymatic methods, oligosaccharide remodelling, and technologies using genetic code expansion techniques. The virtues and limitations of each technology are discussed. Emerging conjugation technologies are being applied to produce new formats of ADCs with enhanced functionality including bispecific ADCs, dual-payload ADCs, and nanoparticles for targeted drug delivery. The benefits of these novel formats are highlighted. [ABSTRACT FROM AUTHOR]

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide–protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy–entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands. [ABSTRACT FROM AUTHOR]

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain.

Using 3,4-dihalo-2(5H)-furanones and easily available hemostatic drugs, such as tranexamic acid (TA), 4-aminomethylbenzoic acid (ABA), aminocaproic acid (AA) as starting materials, serial multi-functional molecules 2(5H)-furanonyl amino acids are designed by the combination of different pharmacophores, and successfully synthesized by a transition metal-free Michael addition–elimination reaction. The reaction is carried out under mild conditions with ethanol-dichloromethane as solvent and only stirring at room temperature for 24 h, and the yield can be up to 91%. All products are well characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), high-resolution mass spectra (HRMS). Ten typical target compounds among them are selected out for the experiments of hemostasis performance by the evaluation of in vitro clot formation model and liver hemorrhage model. The test results show that, their hemostasis effect is better than the original drugs. Especially the target compound G, a TA derivative from 5-borneoloxy-3,4-dibromo-2(5H)-furanone, has the best hemostasis effect among all the tested compounds. These obtained target molecules are expected to be used as multi-functional hemostatic drugs. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide–protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy–entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating N ε - (o -azidobenzyloxycarbonyl)- l -lysine (o -Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o -Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype. • A designer amino acid was site-specifically introduced into the human λ light chain. • The bio-orthogonal azido chemistry was used for site-specific chemical conjugation. • Multiple positions useful for the conjugation were identified in the λ light chain. • Bispecific Fab dimers made of the κ and λ isotypes showed antagonistic activity. [ABSTRACT FROM AUTHOR]

The ability to genetically encode non-natural amino acids (nnAAs) into proteins offers an expanded tool set for protein engineering. nnAAs containing unique functional moieties have enabled the study of post-translational modifications, protein interactions, and protein folding. In addition, nnAAs have been developed that enable a variety of biorthogonal conjugation chemistries that allow precise and efficient protein conjugations. These are being studied to create the next generation of antibody-drug conjugates with improved efficacy, potency, and stability for the treatment of cancer. However, the efficiency of nnAA incorporation, and the productive yields of cell-based expression systems, have limited the utility and widespread use of this technology. We developed a process to isolate stable cell lines expressing a pyrrolysyl-tRNA synthetase/tRNApyl pair capable of efficient nnAA incorporation. Two different platform cell lines generated by these methods were used to produce IgG-expressing cell lines with normalized antibody titers of 3 g/L using continuous perfusion. We show that the antibodies produced by these platform cells contain the nnAA functionality that enables facile conjugations. Characterization of these highly active and robust platform hosts identified key parameters that affect nnAA incorporation efficiency. These highly efficient host platforms may help overcome the expression challenges that have impeded the developability of this technology for manufacturing proteins with nnAAs and represents an important step in expanding its utility.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli ( E. coli ) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli . Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Introduction: Antibody drug conjugates (ADCs) have emerged as a potent tool in cancer treatment, where cytotoxic drugs are linked to antibodies targeting specific antigens. While conventional ADC synthesis methods have seen success as commercials therapeutics, there is a growing interest in next-generation ADCs, looking at homogeneity of the drug-to-antibody ratio., Areas Covered: The article provides a high-level overview for achieving said homogeneity by site-directed conjugations via encompassing engineered amino acids, enzyme-mediated strategies, peptide sequences, affinity peptides, and beyond. As the field rapidly evolves with multiple ADCs in clinical trials and the advent of biosimilars, the article explores the benefits and challenges in both conventional and non-platform ADC technologies., Expert Opinion: The choice of site selection approach must be based on multiple criteria as discussed in this report. Two ADCs made from conjugation to engineered cysteines have been approved by regulatory agencies which have contributed to the excitement in this space. For the others, though successful as proof-of-concept, the true test of merit will be determined as these technologies advance into the clinic. The promise of improving the therapeutics index and decreasing toxicities will continue to drive progress in this area.

Graphical abstract Abstract Hypothesis Release of lipopolysaccharides (LPS) from bacteria into bloodstream may cause serious unwanted stimulation of the host immune system. P-113 is a clinically active histidine-rich antimicrobial peptide. Nal-P-113, a β-naphthylalanine-substituted P-113, is salt-resistant but has limited LPS neutralizing activity. We suspected the size and shape of the non-natural bulky amino acid may affect its LPS neutralizing activity. Herein, antimicrobial, LPS neutralizing, and antiproteolytic effects of phenylalanine- (Phe-P-113), β-naphthylalanine- (Nal-P-113), β-diphenylalanine- (Dip-P-113), and β-(4,4′-biphenyl)alanine- (Bip-P-113) substituted P-113 were studied. Experiments Structure-activity relationships of P-113, Phe-P-113, Nal-P-113, Dip-P-113, and Bip-P-113 were evaluated using antimicrobial activity assays, serum proteolytic assays, peptide-induced permeabilization of large unilamellar vesicles, zeta potential measurements, dynamic light scattering measurement of LPS aggregation, and Limulus amebocyte lysate assays for measuring LPS neutralization. In vitro and in vivo LPS neutralizing activities were further confirmed by LPS-induced inflammation inhibition in an endotoxemia mouse model. Findings Bip-P-113 and Dip-P-113 had the longest and widest non-nature amino acids, respectively. Bip-P-113 enhanced salt resistance, serum proteolytic stability, peptide-induced permeabilization, zeta potential measurements, LPS aggregation, and in vitro and in vivo LPS neutralizing activities. These results could help design novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

Non-natural antimicrobial peptides are ideal as next-generation antibiotics because of their ability to circumvent the problems of drug resistance and in vivo instability. We report novel all-α- and α,γ-mixed Tat peptide analogues as potential antibacterial and anti-TB agents. These peptides have broad spectrum antibacterial activities against Gram-positive (MICs 0.61 ± 0.03 to 1.35 ± 0.21 μM with the peptide γTatM4) and Gram-negative (MICs 0.71 ± 0.005 to 1.26 ± 0.02 μM with γTatM4) bacteria and are also effective against active and dormant forms of Mycobacterium tuberculosis, including strains that are resistant to rifampicin and isoniazid. The introduction of the non-natural amino acids of the study in the Tat peptide analogues results in increased resistance to degradation by proteolysis, significantly increasing their half-life. The peptides appear to inhibit bacteria by a membrane disruption mechanism, and have only a low cytotoxic effect on mammalian cells. [ABSTRACT FROM AUTHOR]

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer. [ABSTRACT FROM AUTHOR]

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8–13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr 11 in NT(8–13). Additionally, Arg 8 , Arg 9 , and Ile 12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity ( K i (NTS2): 29 nM, K i (NTS1): 35,000 nM) was observed for the peptide analog 17 R trans . [ABSTRACT FROM AUTHOR]

The hydrochloride of the racemic amino acid (2-(7-hydroxycoumarin-4-yl)ethyl)glycine, which can serve as a fluorescent probe in proteins, and two halogen derivatives of it, were synthesized by using a new synthetic protocol in five steps. It is less costly and relatively easy to prepare this kind of fluorescent amino acid with the new synthetic method. Furthermore, it can be applied to synthesize other derivatives of the coumarin amino acid with some specific properties.

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering. [ABSTRACT FROM AUTHOR]

Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties. Here we describe the process by which fluorescent protein structures are determined, starting from recombinant protein production to structure determination by molecular replacement. We also describe how to incorporate and determine the structures of proteins containing non-natural amino acids. Recent advances in protein engineering have led to reprogramming of the genetic code to allow incorporation of new chemistry at designed residue positions, with fluorescent proteins being at the forefront of structural studies in this area. The impact of such new chemistry on protein structure is still limited; the accumulation of more protein structures will undoubtedly improve our understanding and ability to engineer proteins with new chemical functionality., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The recent advances in the molecular engineering of aromatic ammonia lyases (ALs) and aminomutases (AMs) attracted increased interest towards their applications in the treatment of phenylketonuria and/or the synthesis of non-natural amino acids. Herein we describe the cloning, isolation and purification of recombinant L-phenylalanine ammonia-lyase from Rhodosporidium toruloides for future biocatalytic and therapeutic applications. [ABSTRACT FROM AUTHOR]

Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p -azido- l -phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety. [ABSTRACT FROM AUTHOR]

Highly conformationally-constrained novel α-amino acid derivatives ((2R/3S) and (2S/3R)-2-(tert-butoxycarbonylamino)-3-cyclohexyl-3-phenylpropanoic acids) have been synthesised with high stereoselectivity (> 90% de) and in 36-37% overall yields. In the synthesis, Evans' auxiliary (4(R/S)-4-phenyl-oxzaolidin-2-one) was used to control the stereoselectivity via the key reactions of asymmetric Michael addition, azidation and catalytic hydrogenolysis. [ABSTRACT FROM AUTHOR]

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]

Abstract: We synthesized novel photochromic non-natural amino acids based on diarylethene skeletons. The non-natural amino acids can be introduced at any positions in various types of peptides by solid/solution phase synthesis. In this study, linear and cyclic peptides including the diarylethene residues were prepared. Their peptides showed photochromism peculiar to original diarylethenes. [Copyright &y& Elsevier]

In this report, we describe dendritic peptides possessing central fluorescent amino acids with adjacent branched amino acids. These fluorescent-peptide dendrimers were prepared using (9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis and Fmoc-derivative fluorescent and branched amino acids. The branched amino acids featured multiple carboxylic acids in their side chains, making the fluorescent-peptide dendrimers highly water-soluble compared with the analogous peptides containing the fluorescent amino acids only. The branched amino acid units also improved the fluorescence intensity of the dendrimers. Based on high-pressure liquid chromatography and fluorescence spectroscopy results, we determined that the fluorescent groups were located in the core and that the carboxylic acids were located on the surface of the dendrimers. Fluorescence resonance energy transfer was achieved among the three proximal fluorescent groups in one of the fabricated fluorescent-peptide dendrimers. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 64-70, 2013. [ABSTRACT FROM AUTHOR]

Abstract: Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated k cat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4×10−5 s−1. Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450±20μg/ml CAT109pAzF and 428±2μg/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe. [Copyright &y& Elsevier]

Abstract: The genetic encoding of synthetic or “non-natural” amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG “stop” triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites. [Copyright &y& Elsevier]

Abstract: The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins. [Copyright &y& Elsevier]

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CHOH, NH, and CHO, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y and b ions in MS spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y and b ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y and b ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b ions are formed, presumably with stable oxazolone structures when the C-terminus of the b ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b ions. [ABSTRACT FROM AUTHOR]

A gram-scale synthesis of a novel densely functionalized and orthogonally protected β-homoproline was achieved from l-tartaric acid derived nitrone through a highly stereoselective 1,3-�dipolar cycloaddition to γ-crotonolactone as the key initial step, followed by appropriate elaboration of the adduct. [ABSTRACT FROM AUTHOR]

A variety of unique codons have been employed to expand the genetic code. The use of the opal (UGA) codon is promising, but insufficient information is available about the UGA suppression approach, which facilitates the incorporation of non-natural amino acids through suppression of the UGA codon. In this study, the UGA codon was used to incorporate 4-iodo-l-phenylalanine into position 32 of the Ras protein in an Escherichia coli cell-free translation system. The undesired incorporation of tryptophan in response to the UGA codon was completely repressed by the addition of indolmycin. The minor amount (3%) of contaminating 4-bromo-l-phenylalanine in the building block 4-iodo-l-phenylalanine led to the significant incorporation of 4-bromo-l-phenylalanine (21%), and this problem was solved by using a purified 4-iodo-l-phenylalanine sample. Optimization of the incubation time was also important, since the undesired incorporation of free phenylalanine increased during the cell-free translation reaction. The 4-iodo-l-phenylalanine residue can be used for the chemoselective modification of proteins. This method will contribute to advancements in protein engineering studies with non-natural amino acid substitutions. [ABSTRACT FROM PUBLISHER]

Abstract: Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the +1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous +1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region. [Copyright &y& Elsevier]

Abstract: Different types of cyclobutane-containing peptides (CBPs) were screened for the first time as ligands of metallocarboxypeptidases (MCPs). CBPs are conformationally constrained, low molecular-weight compounds which showed moderate yet selective inhibitory activity against mammalian MCPs. The most potent compound was a carboxypeptidase B inhibitor. Docked protein–ligand complexes indicated that CBPs may bind to the target proteases via electrostatic interactions and aromatic stacking to catalytically crucial residues and that the placement of functional groups seems to be assisted by the rigid CBP backbone. The easily obtainable CBPs may offer a valuable alternative in the design of novel inhibitors to disease-linked metallocarboxypeptidases like human plasma carboxypeptidase B. [Copyright &y& Elsevier]

Abstract: Incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 can competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia coli K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS–PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricolum Trp1 tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into proteins in response to the amber codon. [Copyright &y& Elsevier]

Abstract: We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNAPyl in Chinese hamster ovary cells. When tRNAPyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNAPyl pair facilitated the most efficient incorporation of a pyrrolysine analog, Nε -tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNAPyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to Nε -benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to Nε -acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells. [Copyright &y& Elsevier]

Abstract: A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination. [Copyright &y& Elsevier]

Abstract: Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins. [Copyright &y& Elsevier]

Abstract: Amino acid residues with aromatic side chains, such as Tyr and Phe, are known to play essential roles in forming and stabilizing the amyloid fibrils of pathogenic polypeptides by affecting their amyloid forming propensity. We have studied the amyloid-type aggregation of peptides containing non-natural amino acid derived from a core part of human pathogenic protein, tau. The hydrophobic nature of the biphenyl group and its intermolecular aromatic interactions strongly alter their amyloid formation properties. [Copyright &y& Elsevier]

Kinetic parameters of d-amino acid oxidase from R. gracilis (DAAO) towards d-2-naphthyl alanine (d-2-NAla) and of l-aspartate amino transferase (l-AAT) from Escherichia coli towards 2-naphthyl pyruvate (2-NPA) were measured. The two enzymes were then combined in a one-pot reaction in which DAAO was used to generate 2-NPA which was the substrate of l-AAT in the presence of cysteine sulphinic acid (CSA) as an amino donor. The combined reactions afforded enantiomerically pure l-2-NAla in almost quantitative yield. The extremely low water solubility of 2-NAla can be partially overcome by running the biotransformation in suspension with higher formal concentration. In these conditions multiple enzyme additions are required. [ABSTRACT FROM AUTHOR]

Abstract: The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein. [Copyright &y& Elsevier]

A series of ?-bromo-2(S)-methyl acids has been synthesized as precursors of novel arginine (Arg), lysine (Lys), and mercapto analogues. These intermediates contain a-methyl groups and are designed to mask the N-terminal amine when incorporated in pharmaceutically relevant peptides. [ABSTRACT FROM AUTHOR]

New bioinorganic complexes of non-natural amino acids (p-methyl-,p-trifluoromethyl-,p-bromo-phenylalanine) and divalent copper ion are synthesized. The crystal structure of the complex ofp-methylphenylalanine and copper belongs to monoclinic system and that ofp-trifluoromethyl- orp-bromo-phenylalanine and copper belongs to orthorhombic system. The infrared spectra can demonstrate the complex formation. The large difference of the magnetic susceptibilities and of the thermal decomposition processes of the complexes shows that the bonding or coordination structure in the complexes may be different. [ABSTRACT FROM AUTHOR]

Abstract: In vitro non-natural amino acid mutagenesis requires aminoacyl-charged suppressor transfer RNAs which read an internal stop codon. For the synthesis of aminoacyl-tRNAs loaded with non-natural amino acids, T4 RNA ligase is used to ligate a chemically synthesised aminoacyl-dinucleotide to a truncated 74mer tRNA(−CA) lacking the two 3′ end nucleotides. The 74mer tRNA(−CA) in turn is generated by run-off transcription from a linearised plasmid encoding the tRNA sequence under control of the T7 promoter. Transcripts with heterogeneous ends are commonly obtained, which interfere with subsequent reactions such as ligation or translation. Here we report an improved procedure for the generation and chromatographic purification of large amounts of homogeneous 3′ end tRNA(−CA) by hepatitis delta virus ribozyme cis-cleavage and the first application of this tRNA to in vitro non-natural amino acid mutagenesis. Stop codon suppression is increased compared to conventionally synthesised suppressor tRNA; 2.5μg of mutated protein was synthesised in a 50μl batch reaction. [Copyright &y& Elsevier]

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system. [Copyright &y& Elsevier]

Abstract: A series of non‐natural isosteric analogs of the cationic, ion‐pairing, natural amino acids arginine and lysine have been synthesized, characterized with regard to relevant physical parameters, and protected for routine inclusion in Merrifield solid‐phase synthesis. The design of these molecules is based on the concept of steric inhibition of solvation, in that judicious placement of alkyl groups can destabilize aqueous ion solvation and favor ion‐pairing [see Beeson & Dix (1993) J. Am. Chem. Soc.115, 10275]. When the residues are substituted for the natural amino acids in biologically active peptides, enhanced ion‐pairing of the peptides to their receptors to increase the peptides’ biological activities can result. The increased lipophilicity of the non‐natural residues can also improve pharmacokinetic parameters and agonist/antagonist behaviors of peptides. While the synthesis of the L‐series is described, theD‐isomers were also prepared using identical chemistry. [ABSTRACT FROM AUTHOR]