Phenylalanine Analogues Research Articles

Reevaluating the Substrate Specificity of the L-Type Amino Acid Transporter (LAT1).

The L-type amino acid transporter 1 (LAT1, SLC7A5) transports essential amino acids across the blood-brain barrier (BBB) and into cancer cells. To utilize LAT1 for drug delivery, potent amino acid promoieties are desired, as prodrugs must compete with millimolar concentrations of endogenous amino acids. To better understand ligand-transporter interactions that could improve potency, we developed structural LAT1 models to guide the design of substituted analogues of phenylalanine and histidine. Furthermore, we evaluated the structure-activity relationship (SAR) for both enantiomers of naturally occurring LAT1 substrates. Analogues were tested in cis-inhibition and trans-stimulation cell assays to determine potency and uptake rate. Surprisingly, LAT1 can transport amino acid-like substrates with wide-ranging polarities including those containing ionizable substituents. Additionally, the rate of LAT1 transport was generally nonstereoselective even though enantiomers likely exhibit different binding modes. Our findings have broad implications to the development of new treatments for brain disorders and cancer.

Fluorinated phosphonate analogues of phenylalanine: Synthesis, X-ray and DFT studies

Due to their biological activity and structural analogy to corresponding α-amino acids, α-aminophosphonates and their fluorinated derivatives provide an important source for drug discovery. Therefore convenient access to this class of compounds is still desirable. Four series of novel phosphonate analogues of fluorinated phenylalanine containing variable number of fluorine atoms in different positions of the phenyl ring were synthesized and subjected to solid state characterization by single-crystal X-ray diffraction analysis, and to studies with the use of NMR, HRMS and DFT methods. Such an approach provided valuable information in regard to preferable conformation, hydrogen bonds and also weak intermolecular interactions present in the crystals investigated. As analogues of naturally occurring compounds, the obtained α-aminophosphonates have a big potential for biological activity. Formation of some indolinylphosphonates as minor products arisen from intramolecular SNAr reactions show that aminophosphonates exhibiting an electronically depleted aromatic group, and possessing a fluorine atom in ortho position of the phosphonoalkyl substituent may give an entrance to further derivatives that may exhibit entirely new properties.

Novel wine yeast with ARO4 and TYR1 mutations that overproduce 'floral' aroma compounds 2-phenylethanol and 2-phenylethyl acetate.

It is well established that the choice of yeast used to perform wine fermentation significantly influences the sensory attributes of wines; different yeast species and strains impart different profiles of esters, volatile fatty acids, higher alcohols, and volatile sulphur compounds. Indeed, choice of yeast remains one of the simplest means by which winemakers can modulate the sensory characteristics of wine. Consequently, there are more than 100 commercially available Saccharomyces cerevisiae wine yeast strains available, mostly derived by isolation from vineyards and successful fermentations. Nevertheless, some desirable characteristics such as 'rose' and 'floral' aromas in wine are not present amongst existing strains. Such aromas can be conferred from the higher alcohol 2-phenylethanol (2-PE) and its acetate ester, 2-phenylethyl acetate (2-PEA). These metabolites of the aromatic amino acid phenylalanine are present at concentrations below their aroma detection thresholds in many wines, so their contribution to wine style is often minimal. To increase the concentration of phenylalanine metabolites, natural and chemically mutagenised populations of a S. cerevisiae wine strain, AWRI796, were exposed to toxic analogues of phenylalanine. Resistant colonies were found to overproduce 2-PE and 2-PEA by up to 20-fold, which resulted in a significant increase in 'floral' aroma in pilot-scale white wines. Genome sequencing of these newly developed strains revealed mutations in two genes of the biosynthetic pathway of aromatic amino acids, ARO4 and TYR1, which were demonstrated to be responsible for the 2-PE overproduction phenotype.

Tailored Mutants of Phenylalanine Ammonia-Lyase from Petroselinum crispum for the Synthesis of Bulky l- and d-Arylalanines.

Tailored mutants of phenylalanine ammonia‐lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non‐natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)‐arylacrylates. The wild‐type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well‐studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4‐methoxyphenyl)‐, (napthalen‐2‐yl)‐, ([1,1′‐biphenyl]‐4‐yl)‐, (4′‐fluoro‐[1,1′‐biphenyl]‐4‐yl)‐, and (5‐phenylthiophene‐2‐yl)alanines.

Adhesion control of human umbilical vein endothelial cells using clickable poly(2-oxazoline)-grafted biosynthesized extracellular matrix protein

A bacterial expression host was used for in vivo incorporation of p-azidophenylalanine (p-N3Phe), a phenylalanine (Phe) analog, into an artificial extracellular matrix protein (aECM-CS5-ELF). The host harbors the mutant phenylalanyl-tRNA synthetase (PheRS) which has an enlarged binding pocket, where the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) was expressed under the control of T7 promoters. Biosynthesized aECM-CS5-ELF containing p-N3Phe (aECM-CS5-ELF-N3) was coupled with alkyne-containing poly(2-oxazoline) prepared via ring-opening polymerization of 2-oxazolines (2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, and 2-iso-propyl-2-oxazoline) using methyl triflate (TfOMe) and propiolic acid as the initiator and terminator, respectively. A copper-catalyzed alkyne-azide click reaction was used to graft the poly(2-oxazoline)s onto the aECM. Examination of the solubility in organic and aqueous media, and the lower critical transition temperature (LCST), revealed a dependence on the incorporation ratio of p-N3Phe and graft chain species; the LCST behavior was markedly altered when poly(2-oxazoline) moieties were present as side chains. Circular dichroism measurements indicate that grafting was not responsible for conformational changes, because the conformation was retained both at below and above the LCST. Specific adhesion and subsequent temperature-sensitive detachment of human umbilical vein endothelial cells (HUVECs) onto the cross-linked aECM-CS5-ELF-N3-graft-poly(2-oxazoline) surfaces was also demonstrated.

Destabilization of ROS metabolism in tomato roots as a phytotoxic effect of meta-tyrosine

meta-Tyrosine (m-Tyr) is a non-protein amino acid produced in both plants and animals. Primary mode of action of this phenylalanine analog is its incorporation into protein structure leading to formation of aberrant molecules. An increased level of m-Tyr in animal cells is detected under oxidative stress and during age-related processes characterized by overproduction of reactive oxygen species (ROS). The aim of this study was to link m-Tyr physiological action to disturbances in ROS metabolism in tomato (Solanum lycopersicum L.) seedlings roots. Treatment of tomato seedlings with m-Tyr (50 or 250 μM) for 24–72 h led to inhibition of root growth without a lethal effect. Toxicity of m-Tyr after 72 h was connected with an increase in hydrogen peroxide concentration in roots and ROS leakage into the surrounding medium. On the contrary, membrane permeability and lipid peroxidation in roots were the same as for the control. This was accompanied by a decrease in total antioxidant activity and an increased accumulation of phenolic compounds. Catalase (CAT) activity declined in roots exposed to 50 μM m-Tyr after 24 h while after 72 h activity of this enzyme was inhibited in both treated and non-treated samples. Activities of different superoxide dismutase (SOD) isoforms were similar in m-Tyr stressed roots and in the control. Prolonged culture resulted in decrease of transcript level of genes coding CAT and SOD with the exception of FeSOD. Moreover, m-Tyr increased the level of protein carbonyl groups indicating induction of oxidative stress as a non-direct mode of action.

A novel synthesis of chromone based unnatural $$\upalpha $$ α -amino acid derivatives

An efficient method for the preparation of chromone based $$\upalpha $$ -amino acid derivatives by alkylation of glycinate schiff base with 3-bromomethyl chromone as well as 2-bromomethyl chromone has been described. Using this method, 2-amino-3-(4-oxo-2-chromenyl)propanoic acid and 2-amino-3-(4-oxo-3-chromenyl)propanoic acid, two novel chromone-amino acid conjugates have been prepared. Furthermore, the separation of chromone amino acid enantiomers by chiral column chromatography was accomplished. Synopsis: An efficient method for the preparation of chromone based $$\upalpha $$ -amino acid derivatives by alkylation of glycinate schiff base with 3-bromomethyl chromone as well as 2-bromomethyl chromone has been described. These conjugates can be considered as analogues of phenyl alanine where phenyl group has been replaced by chromone moiety.

Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands.

A series of racemic unnatural amino acids was rationally designed on the basis of recently published X-ray structures of the GluA2 LBD with bound phenylalanine-based antagonists. Twelve new diaryl- or aryl/heteroaryl-substituted phenylalanine derivatives were synthesized and evaluated in vitro in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of4.6μm for native AMPA receptors and over fourfold lower affinity for kainic acid receptors. Furthermore, 7e was evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. Recently reported X-ray structures 5CBR and 5CBS, representing two distinct antagonist binding modes, were used as templates for molecular docking of the synthesized series. Binding data supported with molecular modeling confirmed that aryl/heteroaryl-substituted phenylalanine analogues effectively bind to AMPA receptors with low micromolar affinity and high selectivity over native NMDA and kainate receptors. These properties make 7e a promising lead for the further development of new AMPA receptor ligands.

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria

BackgroundLactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment.ResultsThe pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23.ConclusionsAs reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.

Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

Recombineering in Streptococcus mutans Using Direct Repeat-Mediated Cloning-Independent Markerless Mutagenesis (DR-CIMM).

Studies of the dental caries pathogen Streptococcus mutans have benefitted tremendously from its sophisticated genetic system. As part of our own efforts to further improve upon the S. mutans genetic toolbox, we previously reported the development of the first cloning-independent markerless mutagenesis (CIMM) system for S. mutans and illustrated how this approach could be adapted for use in many other organisms. The CIMM approach only requires overlap extension PCR (OE-PCR) protocols to assemble counterselectable allelic replacement mutagenesis constructs, and thus greatly increased the speed and efficiency with which markerless mutations could be introduced into S. mutans. Despite its utility, the system is still subject to a couple limitations. Firstly, CIMM requires negative selection with the conditionally toxic phenylalanine analog p-chlorophenylalanine (4-CP), which is efficient, but never perfect. Typically, 4-CP negative selection results in a small percentage of naturally resistant background colonies. Secondly, CIMM requires two transformation steps to create markerless mutants. This can be inherently problematic if the transformability of the strain is negatively impacted after the first transformation step, which is used to insert the counterselection cassette at the mutation site on the chromosome. In the current study, we develop a next-generation counterselection cassette that eliminates 4-CP background resistance and combine this with a new direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM) system to specifically address the limitations of the prior approach. DR-CIMM is even faster and more efficient than CIMM for the creation of all types of deletions, insertions, and point mutations and is similarly adaptable for use in a wide range of genetically tractable bacteria.

A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases.

Aromatic amino acid ammonia‐lyases and aromatic amino acid 2,3‐aminomutases contain the post‐translationally formed prosthetic 3,5‐dihydro‐4‐methylidene‐5H‐imidazol‐5‐one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α‐ or β‐amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β‐position inhibited phenylalanine ammonia‐lyases (PAL), representing MIO enzymes. X‐ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene‐substituted substrate analogue as being a mirror image relation to the natural l‐phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)‐ and (S)‐enantiomers is possible by a mirror image packing.

Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.

Human serum albumin as vehicle for the solubilization of perylene diimides in aqueous solutions

The chemical, physical and photophysical properties of perylene diimides have attracted substantial attention for the potential applications in diverse fields ranging from advanced materials to biomedical applications. Some applications require the diimides to be in aqueous environment where they tend to dissolve poorly. We investigated the use of human serum albumin as a vehicle to increase the aqueous exposure of monomeric perylene diimides. Since studies on the interactions of these compounds with protein is scarce we characterized the binding and the possible effects on the protein. In order to increase the affinity of the dyes to the protein we have used perylene diimides with substituents that replicate the side chains of natural amino acids. The results show that only the dyes containing the side chain of leucine and phenylalanine yield measurable binding. Only the phenylalanine analogue promotes energy transfer with the lone tryptophan residue of albumin indicating different binding modalities for the dyes. In addition, this analogue is the only one which shows photochemical activity that prompts its release from the protein upon laser irradiation.

Pseudomonas putida as a platform for the synthesis of aromatic compounds.

Aromatic compounds such as l-phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by 'green' alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l-1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheAfbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l-1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l-1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.

Penicillin G acylase-mediated kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters

Extensive studies on the penicillin G acylase-mediated kinetic resolution of N-acylated 1-aminoalkylphosphonic and 1-aminoalkylphosphinic acids as well as their esters were carried out to recognise the relationships between the substrate structure, reaction conditions, and the enzymatic hydrolytic deacylation efficiency and stereoselectivity. Reactivity of 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters in the penicillin G acylase-mediated hydrolytic deacylation reaction depends strongly on the kind of their N-acyl group, with high preference to the hydrolytic splitting of the N-phenylacetyl moiety. The initial hydrolysis rates of 1-(N-phenylacetylamino)alkylphosphonic acid dimethyl esters 2 are mostly distinctly lower in comparison with the corresponding free acids 3 and rapidly decrease with the increasing steric effect of the substituent at the α-position. In contrary to the substituents at the α-carbon, bulky substituents at the phosphorus hinder the enzymatic hydrolysis to a much lesser degree. The penicillin G acylase-mediated stereospecific hydrolysis of N-acyl group of both racemic 1-(N-acylamino)alkylphosphonic acids 3 and their dimethyl esters 2 proved to be, in most cases, a highly effective method for the kinetic resolution of these compounds. High enzyme enantioselectivity E-values exceeding 100, or synthetically useful E-values exceeding 20 (in two cases) were obtained for the N-acylated phosphonic acid analogues of alanine, phenylalanine, valine, leucine, and asparagine, as well as for their dimethyl esters, with the exception of the dimethyl ester of phosphonic analogue of valine 2e, that E-value was low (E=1.2). Also for the N-acylated H-phosphinic acid analogues of alanine, as well as phenylphosphinic acid analogue of alanine, high enzyme enantioselectivity values exceeding 100 were obtained. In contrary, E-values for both diastereomers of ethyl ester of phenylphosphinic analogue of alanine 2k were low (E=7 and 13). For the all accomplished assignments penicillin G acylase exhibited stereochemical preference for the (R)-substrate.

LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs

The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described.

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Azide-Incorporated Clickable Silk Fibroin Materials with the Ability to Photopattern.

Incorporation of unnatural amino acids (UAAs) bearing bioorthogonal reactive groups into proteins could be a powerful tool for developing novel protein-based biomaterials with innovative and controlled performance. Bombyx mori silk fibroin is one of naturally derived protein materials extensively studied for biomaterials development due to its mechanical strength and biocompatibility. We recently reported the in vivo incorporation of UAAs, 4-substituted analogues of phenylalanine including 4-azidophenylalanine (AzPhe), into silk fibroin by expanding the repertoire of amino acids for protein biosynthesis in silk glands of B. mori using transgenic techniques. We demonstrated that azide groups in AzPhe incorporated into silk fibroin can be selectively modified by bioorthogonal azide-alkyne cycloaddition reactions (click chemistry). However, the incorporation of AzPhe into silk fibroin required a special feeding condition, which led to the limited production of silk fibroin. Here we report more efficient production of an AzPhe-incorporated silk fibroin (termed AzidoSilk) and its modification by click chemistry in varied material forms (thread, film, and porous sponge). Using this methodology, photolithographic micropatterning of fluorescent molecules directly onto silk fibroin film was achieved and should further expand the availability of silk-based biomaterials for cell culture substrates, drug delivery, tissue scaffolds, implantable devices, and so on.

Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.