Β-amino Acid Research Articles

Enhancing a Sphaerobacter thermophilus ω-transaminase for kinetic resolution of β- and γ-amino acids

Sphaerobacter thermophilus synthesizes an ω-transaminase (ω-TA) that allows the production of enantiomerically pure β-amino acids. To obtain ω-TA variants with a higher activity and more favorable properties for industrial use, we modified critical amino acid residues either in the catalytic center or in a previously proposed signature motif critical for aromatic β-amino acid ω-TAs. Seventeen different variants of this enzyme were generated and their activity was examined with four β-amino acids and one γ-amino acid, and compared with the wildtype’s activity. Among all variants, seven showed up to ninefold higher activity with at least one of the tested substrates. For most of these seven variants, the temperature optimum was even lower as in the wild type enzyme, with keeping a high temperature stability, making them more valuable for industrial purposes. Our results indicate that for the production of enantiomerically pure β-amino acids replacement of critical amino acid residues in the proposed signature motif of ω-TAs is a more effective strategy than modifying their catalytic center. Another finding was, that the proposed motif is not only suitable for aromatic amino acid ω-TAs, because some of the variants have a higher activity with β-alanine or β-leucine than with aromatic β-amino acids.Graphical

Taurine Protects Doxorubicin-Induced Hepatotoxicity via Its Membrane-Stabilizing Effect in Rats.

Doxorubicin (dox) is a chemotherapeutic agent widely used against various tumors. However, the clinical use of this agent is limited due to various organ toxicities. Taurine is an intracellular free β-amino acid with antioxidant properties. The present study investigated the protective mechanism of taurine on dox-induced hepatotoxicity. In total, 31 male Sprague-Dawley rats were used in the study. The control group received intraperitoneal (i.p.) 0.9% NaCl alone for 14 days; the taurine (Tau) group received i.p. taurine 150 mg/kg body weight/day for 14 days; the dox group received dox on days 12, 13, and 14 at a cumulative dose of 25 mg/kg body weight/3 days; and the tau+dox group received taurine and dox together at the same dose and through the same route. On day 15, biochemical evaluations were performed on blood samples taken from the left ventricle followed by histological examinations on liver samples. Dox was found to increase liver function enzymes and tissue protein carbonyl levels, causing congestion and tissue damage, thereby leading to dysfunction. Tau was found to histologically preserve the liver morphology without showing any corrective effect on oxidative stress parameters. These findings suggest that the membrane-stabilizing effect of taurine may be more effective than its radical scavenging activity in preventing dox-induced toxicity. Taurine can prevent doxorubicin-induced hepatotoxicity through non-antioxidant pathways.

Exploring a β-Amino Acid with a Seven-Membered Ring Constraint as a Foldamer Building Block for Nontraditional Helices.

We explored trans- and cis-2-aminocycloheptanecarboxylic acid (ACHpC) as potential building blocks for helical foldamers. trans-ACHpC does not show sufficient folding propensity in unnatural peptides. cis-ACHpC promotes nontraditional helices of two unnatural peptide backbones: the 11/9-helix for 1:1 α/β-peptides and the 12/10-helix for β-peptides with interconvertible handedness. The two opposite-handed 12/10-helices rapidly interconvert in solution by pseudorotation of the two twist chair forms of the cycloheptane moiety in each cis-ACHpC residue.

Application of Metathesis Protocols to the Stereocontrolled Synthesis of Some Functionalized β-Amino Esters and Azaheterocycles.

In this account our aim was to give an insight into the application of metathesis protocols (ROM, RCM, RCEYM, CM, RRM) for the synthesis of various azaheterocyclic frameworks. Due to the high biological potential and importance in peptide chemistry and drug design of β-amino acids our intention is to give a highlight on the synthetic procedures and transformation of these class of compounds with the above-mentioned metathesis strategies with emphasis on selectivity, stereocontrol, substrate-directing effect or functional group tolerance.

α-(Aminomethyl)acrylates as acceptors in radical-polar crossover 1,4-additions of dialkylzincs: insights into enolate formation and trapping.

We demonstrate that α-(aminomethyl)acrylates are suitable acceptors for 1,4-additions of dialkylzincs in aerobic conditions. The air-promoted radical-polar crossover process involves the 1,4-addition of an alkyl radical followed by homolytic substitution at the zinc atom of dialkylzinc. Coordination of the nitrogen atom to zinc enables this SH2 process which represents a rare example of alkylzinc-group transfer to a tertiary α-carbonyl radical. The zinc enolate thus formed readily undergoes β-fragmentation unless it is trapped by electrophiles in situ. Enolates of substrates having free N-H bonds undergo protodemetalation to provide ultimately the 1,4-addition adduct. In the presence of carbonyl acceptors, aldol condensation occurs providing overall a tandem 1,4-addition-aldol process. When a tert-butanesulfinyl moiety is present on the nitrogen atom, these electrophilic substitution reactions occur with good levels of chiral induction, paving the way to enantioenriched β2-amino acids and β2,2-amino acids.

Synthesis of Small Cyclic Peptides Containing N-Methyl and β-Amino Acids

Synthesis of Small Cyclic Peptides Containing N-Methyl and β-Amino Acids

Effects of Replacing a Central Glycine Residue in GLP-1 on Receptor Affinity and Signaling Profile.

Agonists of the glucagon-like peptide-1 receptor (GLP-1R) are used to treat diabetes and obesity. Cryo-EM structures indicate that GLP-1 is completely α-helical when bound to the GLP-1R. The mature form of this hormone, GLP-1(7-36), contains a glycine residue near the center (Gly22). Since glycine has the second-lowest α-helix propensity among the proteinogenic α-amino acid residues, and Gly22 does not appear to make direct contact with the receptor, we were motivated to explore the impact on agonist activity of altering the α-helix propensity at this position. We examined GLP-1 analogues in which Gly22 was replaced with L-Ala, D-Ala, or β-amino acid residues with varying helix propensities. The results suggest that the receptor is reasonably tolerant of variations in helix propensity, and that the functional receptor-agonist complex may comprise a conformational spectrum rather than a single fixed structure.

Determination of taurine in soft drinks by an ultrahigh-performance liquid chromatography-mass spectrometry method

Abstract Taurine (2-aminoethanesulfonic acid) is a free sulfur-containing β-amino acid widely distributed in many mammalians. Owing to the energizing effects, it is mostly used in soft drinks and supplements for athletes. Regular intake of soft drinks may lead to an overdose of caffeine, taurine, and guarana and loss of bone mass, overweight, hypertension, and in older age, osteoporosis and cardiovascular diseases. Therefore, it is essential to control the maximum amount of taurine consumed by humans in the food and beverages. Here, a fast, simple, accurate, and robust method based on ultrahigh-performance liquid chromatography hyphenated with mass spectrometry (UHPLC-MS) was successfully applied for the determination of taurine in selected soft drinks sold in Slovakia. The method was characterized by coefficient of determination higher than 0.99, and the predicted value of the limit of detection was 4.29 μmol/L. The analyzed levels of taurine in selected commercial drinks ranged from 2.8 to 3.78 mg/mL. The concentration in one brand of the investigated drinks was found to be extremely low (about 70%) compared to the declared content by the manufacturer.

Versatile Post-synthetic Modifications of Helical β-Peptide Foldamers Derived from a Thioether-Containing Cyclic β-Amino Acid.

We introduce a novel cyclic β-amino acid, trans-(3S,4R)-4-aminotetrahydrothiophene-3-carboxylic acid (ATTC), as a versatile building block for designing peptide foldamers with controlled secondary structures. We synthesized and characterized a series of β-peptide hexamers containing ATTC using various techniques, including X-ray crystallography, circular dichroism, and NMR spectroscopy. Our findings reveal that ATTC-containing foldamers can adopt 12-helical conformations similar to their isosteres and offer the possibility of fine-tuning their properties via post-synthetic modifications. In particular, chemoselective conjugation strategies demonstrate that ATTC provides unique post-synthetic modification opportunities, which expand their potential applications across diverse research areas. Collectively, our study highlights the versatility and utility of ATTC as an alternative to previously reported cyclic β-amino acid building blocks in both structural and functional aspects, paving the way for future research in the realm of peptide foldamers and beyond.

Synthesis, Characterization, and Antimicrobial Activity of Ultra-Short Cationic β-Peptides.

The development of new antibiotics is urgently required because of the rapidly growing resistance against conventional antibiotics. The antimicrobial peptides show potential as small antibiotic molecules. The stability of peptides is a primary concern for the use of peptides as drugs. Introducing β-amino acids into peptide sequences can be useful in preventing biological degradation by proteolytic enzymes. Herein, we describe the synthesis, characterization, and antimicrobial activity of ultra-short cationic β-peptides, LA-β3,3-Pip-β2,2-Ac6c-PEA, P1; LA-β3,3-Pip(G)-β2,2-Ac6c-PEA, P2; LAU-β3,3-Pip-β2,2-Ac6c-PEA, P3, and LAU-β3,3-Pip(G)-β2,2-Ac6c-PEA, P4. Peptides P1-P4 were evaluated against Gram-negative, Gram-positive, MRSA, and multi-drug resistant E. coli (MDR-E. coli). P3 exhibited the most potent antimicrobial activity against E. coli, S. epidermidis, S. aureus, K. pneumoniae, S. mutans, and E. faecalis, with MIC values 0.5, 2, 0.5, 1, 2, and 1 μg/mL, respectively. P3 exhibited time- and concentration-dependent bactericidal activities against E. coli, S. aureus, and E. faecalis with a killing rate of 1.6 logs/h. The treatment of E. coli with peptide P3 showed membrane disruption. In addition, P3 exhibited the inhibition of biofilm produced by E. coli, synergism with antibiotics (ciprofloxacin, streptomycin, and ampicillin), 100% cell viability against AML12, RAW 264.7, and HEK-293 cell lines at 1, and 10 μg/mL concentrations.

Position-specific carbon isotopes of Murchison amino acids elucidate extraterrestrial abiotic organic synthesis networks

The Murchison meteorite is a well-studied carbonaceous chondrite with relatively high concentrations of amino acids thought to be endogenous to the meteorite, in part because they are characterized by carbon isotope (δ13C) values higher than those typical of terrestrial amino acids. Past studies have proposed that extraterrestrial amino acids in the Murchison meteorite could have formed by Strecker synthesis (for α-amino acids), Michael addition (for β-amino acids), or reductive amination, but a lack of constraints have prevented confident discrimination among these possibilities, or assignment of specific formation pathways to each of several specific amino acids. Position-specific carbon isotope analysis differentiates amongst these mechanisms by relating molecular sites to isotopically distinct carbon sources and by constraining isotope effects associated with elementary chemical reactions. Prior measurements of the position-specific carbon isotopic composition of α-alanine from the Murchison CM chondrite demonstrated that alanine’s high δ13CVPDB value is attributable to the amine carbon (δ13CVPDB = +142 ± 20‰), consistent with Strecker synthesis drawing on 13C-rich carbonyl groups in precursors (Chimiak et al., 2021). Here, we measured the δ13C composition of fragment ions generated by electron impact ionization of derivatized ⍺-alanine, β-alanine, and aspartic acid from Murchison via gas chromatography-Fourier transform mass spectrometry. α-Alanine’s amine carbon yielded δ13CVPDB = +109 ± 21‰, which is consistent with the previously measured value and with formation from 13C-rich precursors. β-Alanine’s amine carbon presents a lower δ13CVPDB = +33 ± 24‰, which supports formation from 13C-rich precursors but potentially via a Michael addition mechanism rather than Strecker synthesis. Aspartic acid’s amine carbon has δ13CVPDB = −14 ± 5‰, suggesting synthesis from precursors distinct from those that generated the alanine isomers. These measurements indicate that Murchison amino acids are a mixture of compounds made from different synthesis mechanisms, though some subsets likely drew on the same substrates; this conclusion highlights the complexity of extraterrestrial organic synthesis networks and the potential of emerging methods of isotope ratio analysis to elucidate the details of those networks.

Screening and identification of genes involved in β-alanine biosynthesis in Bacillus subtilis

β-alanine is the only naturally occurring β-amino acid, which is widely used in medicine, food, and feed fields, and generally produced through synthetic biological methods based on engineered strains of Escherichia coli or Corynebacterium glutamicum. However, the β-alanine biosynthesis in Bacillus subtilis, a traditional industrial model microorganism of food safety grade, has not been thoroughly explored. In this study, the native l-aspartate-α-decarboxylase was overexpressed in B. subtilis 168 to obtain an increase of 842% in β-alanine production. A total of 16 single-gene knockout strains were constructed to block the competitive consumption pathways to identify a total of 6 genes (i.e., ptsG, fbp, ydaP, yhfS, mmgA, and pckA) involved in β-alanine synthesis, while the multigene knockout of these 6 genes obtained an increased β-alanine production by 40.1%. Ten single-gene suppression strains with the competitive metabolic pathways inhibited revealed that the inhibited expressions of genes glmS, accB, and accA enhanced the β-alanine production. The introduction of heterologous phosphoenolpyruvate carboxylase increased the β-alanine production by 81.7%, which was 17-fold higher than that of the original strain. This was the first study using multiple molecular strategies to investigate the biosynthetic pathway of β-alanine in B. subtilis and to identify the genetic factors limiting the excessive synthesis of β-alanine by microorganisms.

Comparative Study of Molecular Mechanics Force Fields for β-Peptidic Foldamers: Folding and Self-Association.

Computer-assisted study and design of non-natural peptidomimetics is increasingly important in the development of novel constructs with widespread applicability. Among these methods, molecular dynamics can accurately describe monomeric as well as oligomeric states of these compounds. We studied seven different sequences composed of cyclic and acyclic β-amino acids, the closest homologues of natural peptides, and compared the performance on them of three force field families in which specific modifications were made to improve reproduction of β-peptide structures. Altogether 17 systems were simulated, each for 500 ns, testing multiple starting conformations and in three cases also oligomer formation and stability from eight β-peptide monomers. The results indicated that our recently developed CHARMM force field extension, based on torsional energy path matching of the β-peptide backbone against quantum-chemical calculations, performs best overall, reproducing the experimental structures accurately in all monomeric simulations and correctly describing all the oligomeric examples. The Amber and GROMOS force fields could only treat some of the seven peptides (four in each case) without further parametrization. Amber was able to reproduce the experimental secondary structure of those β-peptides which contained cyclic β-amino acids, while the GROMOS force field had the lowest performance in this sense. From the latter two, Amber was able to hold together already formed associates in the prepared state but was not able to yield spontaneous oligomer formation in the simulations.

Study on the construction technology of β-alanine synthesizing Escherichia coli based on cellulosome assembly.

Introduction: β-Alanine is the only β-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of β-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method. Methods: In this study, we constructed an Escherichia coli recombinant strain for efficient β-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome. Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of β-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the β-alanine content. The key cellulosome elements, dockerin (docA) and cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. β-alanine production reached 7.439mg/L and 25.87mg/L in the two engineered strains. The β-alanine content reached 755.465mg/L in a 5L fermenter. Discussion: The content of β-alanine synthesized by constructed β-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of β-alanine using a cellulosome multi-enzyme self-assembly system.

Microwave Assisted Aza-michael Additions Towards β-amino Acids

Background: β-amino acids are non-natural amino acids, where the amino group is attached to the β-carbon instead of the α-carbon. Peptides containing β-amino acids present with particular secondary structures and remarkable chemical and biological properties. β-peptides are used as peptidomimetics, based on the resistance to hydrolysis in vivo, with various important applications in the field of Medicine. Methods: Several synthetic methods have appeared in recent years, with the Aza- Michael conjugate addition reactions, being a very effective approach towards β-amino acids. Microwave irradiation mediated reactions have also attracted much interest since they significantly improve the reaction yields while reducing the reaction time and avoiding by-products formation. The aim of this project has been the development of a reliable and general synthetic methodology towards β2 and β3 amino acids with the use of conjugate additions of N-nucleophiles to substituted acrylate derivatives. The application of effective catalysts has been also examined here. Results: The results show that the acrylate precursors of β2 and β3 amino acids with side chains corresponding to Phe, Asp, Ile, Leu, Val, and Tyr have been synthesized successfully by using a one-pot Horner-Wadsworth-Emmons reaction, and they have been used for the addition of Nnucleophiles, with a study on conditions and yields optimization. Conclusion: With the main challenge for β-amino acid synthesis being that there is still no general method to synthesize different types of β-amino acids corresponding to all-natural α-amino acids, the proposed synthetic methodology may offer this possibility.

Probing Molecular Chirality on the Self-Assembly and Gelation of Naphthalimide-Conjugated Dipeptides.

In this work, 1,8-naphthalimide (NMI)-conjugated three hybrid dipeptides constituted of a β-amino acid and an α-amino acid have been designed, synthesized, and purified. Here, in the design, the chirality of the α-amino acid was varied to study the effect of molecular chirality on the supramolecular assembly. Self-assembly and gelation of three NMI conjugates were studied in mixed solvent systems [water and dimethyl sulphoxide (DMSO)]. Interestingly, chiral NMI derivatives [NMI-βAla-lVal-OMe (NLV) and NMI-βAla-dVal-OMe (NDV)] formed self-supported gels, while the achiral NMI derivative [NMI-βAla-Aib-OMe, (NAA)] failed to form any kind of gel at 1 mM concentration and in a mixed solvent (70% water in DMSO medium). Self-assembly processes were thoroughly investigated using UV-vis spectroscopy, nuclear magnetic resonance (NMR), fluorescence, and circular dichroism (CD) spectroscopy. A J-type molecular assembly was observed in the mixed solvent system. The CD study indicated the formation of chiral assembled structures for NLV and NDV, which were mirror images of one another, and the self-assembled state by NAA was CD-silent. The nanoscale morphology of the three derivatives was studied using scanning electron microscopy (SEM). In the case of NLV and NDV, left- and right-handed fibrilar morphologies were observed, respectively. In contrast, a flake-like morphology was noticed for NAA. The DFT study indicated that the chirality of the α-amino acid influenced the orientation of π-π stacking interactions of naphthalimide units in the self-assembled structure that in turn regulated the helicity. This is a unique work where molecular chirality controls the nanoscale assembly as well as the macroscopic self-assembled state.

Electroreductive Cross-Electrophile Coupling of Aziridines and Aryl Bromides.

Herein, a nickela-electrocatalyzed cross-electrophile coupling of readily available aryl aziridines and aryl bromides under mild and sustainable electrochemical conditions to access synthetic useful β-arylethylamines is developed. This protocol is characterized by its exquisite chemo- and regioselectivity, broad substrate scope and good functional group compatibility. Mechanistic studies confirmed that the regioselectivity and reactivity observed are a result of electro-induced ring-opening of aziridines under electroreductive conditions to generate a benzyl radical intermediate as the active species. Furthermore, this strategy also enables cross-coupling with CO2 to access β-amino acids under mild conditions.

Antimicrobial efficacy of cyclic α- and β-peptides incorporated in polyurethane coatings.

Microbial growth on surfaces poses health concerns and can accelerate the biodegradation of engineered materials and coatings. Cyclic peptides are promising agents to combat biofouling because they are more resistant to enzymatic degradation than their linear counterparts. They can also be designed to interact with extracellular targets and intracellular targets and/or self-assemble into transmembrane pores. Here, we determine the antimicrobial efficacy of two pore-forming cyclic peptides, α-K3W3 and β-K3W3, against bacterial and fungal liquid cultures and their capacity to inhibit biofilm formation on coated surfaces. These peptides display identical sequences, but the additional methylene group in the peptide backbone of β-amino acids results in a larger diameter and an enhancement in the dipole moment. In liquid cultures, β-K3W3 exhibited lower minimum inhibitory concentration values and greater microbicidal power in reducing the number of colony forming units (CFUs) when exposed to a gram-positive bacterium, Staphylococcus aureus, and two fungal strains, Naganishia albida and Papiliotrema laurentii. To evaluate the efficacy against the formation of fungal biofilms on painted surfaces, cyclic peptides were incorporated into polyester-based thermoplastic polyurethane. The formation of N. albida and P. laurentii microcolonies (105 per inoculation) for cells extracted from coatings containing either peptide could not be detected after a 7-day exposure. Moreover, very few CFUs (∼5) formed after 35 days of repeated depositions of freshly cultured P. laurentii every 7 days. In contrast, the number of CFUs for cells extracted from the coating without cyclic peptides was >8 log CFU.

Biopotential insights and structural chemistry of some zirconocene incorporated heterocyclic β-diketones and flexible N-protected α/β- amino acids

The interaction of zirconocene dichloride with the heterocyclic β-diketones (LH) and flexible N-protected α/ β-amino acids (AH) in the presence of triethylamine in 1:1:1:2 molar ratio in refluxing dry THF has been resulted in the formation of zirconocene complexes of the general formula (where R= -CH3, -CH2CH3, p-ClC6H4-, -C6H5 and CHR’ = -CH2CH2and R’ = CH(CH3)2). These newly generated zirconocene incorporated organic-inorganic hybrid products have been characterized by FTIR, NMR (1H and 13C) and mass spectrometry. Some of these complexes and their corresponding ligands have been tested for their antimicrobial activity to examine the biopotential activity-structure relationship.

Modular enantioselective access to β-amino amides by Brønsted acid-catalysed multicomponent reactions.

β-Amino acids are structural motifs widely found in therapeutic natural products, novel biomimetic polymers and peptidomimetics. As a convergent method, the synthesis of stereoenriched β-amino amides through the asymmetric Mannich reaction requires specialized amide substrates or a metal catalyst for enolate formation. By a redesign of the Ugi reaction, a conceptually different solution to prepare chiral β-amino amides was established using ambiphilic ynamides as two-carbon synthons. The modulation of ynamides or oxygen nucleophiles concisely furnished three classes of β-amino amides with generally good efficiency as well as excellent chemo- and stereo-control. The utility is verified in the preparation of over 100 desired products that bear one or two contiguous carbon stereocentres, including those that directly incorporate drug molecules. This advance also provides a synthetic shortcut to other valuable structures. The amino amides could be elaborated into β-amino acids, anti-vicinal diamines, γ-amino alcohols and β-lactams or undergo transamidation with amino acids and amine-containing pharmaceuticals.