Β-amino Acid Research Articles

Molecular dynamics simulations of putative primitive proteins including β-aspartic acid residues

Abstract Aspartic acid (Asp) has 2 carboxyl groups, either of which can form an amide bond. However, among the present proteins, only one is involved in a peptide bond, whereas the other is regarded as “side chain,” with only α-Asp identified as a gene-encoded amino acid residue. In this study, molecular dynamics simulations of random peptides were performed to investigate why β-Asp, which may have been present in primitive proteins in the origin of proteins, was excluded from proteinogenic amino acids. [GADV]-peptides, constructed from Gly, Ala, Asp, and Val, were used as putative primitive proteins. Molecular dynamics simulations were performed when β-Asp was used instead of Asp in the [GADV]-peptides and when β-Asp was added to the [GADV]-peptide. The secondary structures of peptides containing β-Asp were less than [GADV]-peptides, and their ability to form a protein-like structure was inferior to that of peptides excluding β-Asp. Thus, the ability to form protein-like structures may have acted as a selection pressure, leading to the elimination of protein synthesis systems utilizing β-Asp from primitive life. When the [GADVB]-peptide, i.e. a β-amino acid β-Ala, was added to the [GADV] amino acid set, the ability to form secondary structures reduced. Therefore, β-amino acids that altered the main chain length were excluded from primitive life, despite β-Ala being relatively abundant on primitive Earth.

Novel settlement inhibition oligopeptides containing β-amino acids

Efficient syntheses of tripeptides containing β-amino acids and their settlement inhibition activities toward two main foulants, the barnacle Amphibalanus amphitrite and the blue mussel Mytilus galloprovincialis, are described.

Photoinduced Concerted Fragmentation of Alkyl N-Phthalimidoyl Oxalates: Mechanisms and Applications.

In this work, we used experiments and density functional theory calculations to investigate the mechanism and driving forces of the reductive fragmentation of NHPI esters. Mechanistic studies suggest that the fragmentation behavior of the NHPI ester is influenced not only by the electronic nature of the substituent group but also by the stability of the radical intermediate. To further investigate this transformation, we next examined the aminoalkoxycarbonylation of alkenes using alkyl N-phthalimidoyl oxalates. With this approach, a variety of β-amino acid derivatives with diverse structures can be obtained in moderate to high yields.

Stereoselective Divergent Synthesis of Chiral Pyrazolone Derivatives via Multicomponent Cascade Reactions by Isothiourea Catalysis.

Isothiourea-catalyzed multicomponent cascade reactions are challenging due to the existence of competitive side reactions between multiple reaction partners and intermediates. Herein, we report a practical and efficient protocol for the stereoselective divergent synthesis of pyrazolone-derived β-amino acid esters and β-lactams by isothiourea catalysis. Two distinct reaction pathways are identified, which are controlled by esterification or lactamization of the zwitterionic intermediate. The strategy is attractive because of the convenient one-step procedure, mild conditions, high stereoselectivity, and good functional-group tolerance.

Sequential Photocatalysis for Homologative Diversification of α-Amino Acids to β-Amino Acids Via Phosphonium Ylide Linchpin Strategy.

β-Amino acids serve as crucial building blocks for a broad range of biologically active molecules and peptides with potential as peptidomimetics. While numerous methods have been developed for the synthesis of β-amino acids, most of them require multistep preparation of specific reagents and substrates, which limits their synthetic practicality. In this regard, a homologative transformation of abundant and readily available α-amino acids would be an attractive approach for β-amino acid synthesis. Herein, we disclose the development of a sequential process to provide diverse β-amino acids from α-amino acid derivatives and commercially available phosphonium ylides via visible light photoredox catalysis. In this two-step protocol, phosphonium ylides function as a bifunctional linchpin: they act as a carbon nucleophile to forge a C-C bond in the first step and as a carbon-centered radical source for diverse modifications of the β-amino acid scaffold in the second step. The orthogonal activation of these reactivities under mild photocatalytic conditions enables a modular three-component assembly to access β-amino acids and dipeptides with high structural diversity.

Axially Chiral Phenanthroline Ligand-Enabled Pd-Catalyzed Asymmetric Amination and Alkylation of Aryl-Substituted Morita-Baylis-Hillman Adducts.

Highly enantioselective allylic amination and alkylation of racemic sterically hindered aryl-substituted Morita-Baylis-Hillman (MBH) adducts have been achieved by using an in situ formed Pd-catalyst from an axially chiral phenanthroline ligand. This dynamic kinetic asymmetric transformation (DYKAT) is compatible with cyclic and acyclic secondary amines, dialkyl malonates, β-keto esters, acetylacetone, and malononitrile, affording the corresponding chiral products, such as β-amino acid esters, in up to 95% yield and with up to a 99:1 enantiomeric ratio.

Synthesis of extended fluorinated tripeptides based on the tetrahydropyridazine scaffold.

The synthesis of tripeptides incorporating new fluorinated heterocyclic hydrazino acids, based on the tetrahydropyridazine scaffold is described. Starting from simple fluorinated hydrazones, these non-proteinogenic cyclic β-amino acids were easily prepared by a zinc-catalyzed aza-Barbier reaction followed by an intramolecular Michael addition. Preliminary conformational studies on tripeptides including this scaffold in the central position show an extended conformation in solution (NMR) and in the solid state (X-ray).

Synthesis of Amphiphilic Cationic Poly(β-amino acid) Derivatives and Their PEG Length Optimization for mRNA Transfection.

We synthesized a series of amphiphilic cationic poly(ethylene glycol)-b-poly(β-amino acid) derivatives with various lengths of PEG to investigate the effects of PEG lengths on mRNA transfection. The surface charges of the polyplexes with a DPOEG ≥ 4 gradually decreased as DPOEG increased. This indicated that hydrophilic PEG with a DPOEG ≥ 4 was exposed on the surface of the polyplexes, which was further confirmed using 1H NMR. Polyplexes with a DPOEG ≤ 4 exhibited mRNA transfection efficacy similar to that of homopolymers. However, the transfection efficacy of the polyplex with a DPOEG ≥ 12 markedly decreased, mainly because of the decreased cellular uptake and stability of the polyplexes against serum albumin. This indicated that the PEG length considerably affected the delivery efficacy of IVT mRNA. Our results provide useful information for the fundamental polymer design to optimize the PEG length of amphiphilic cationic polymers for systemic IVT mRNA delivery.

Dissociation Chemistry of Protonated α-, β- and Other Amino Acids Using Multiple Stage Tandem Mass Spectrometry.

To discriminate amino acid isomers by multiple stage tandem mass spectrometry (MSn), the fragmentation of protonated amino acids were investigated by MSn with collision-induced dissociation (CID) and density functional theory calculations. The CID of protonated α-amino acids results in a loss of 46 Da, corresponding to H2O and CO, and iminium ions appear as resultant fragments. The CID of protonated β-amino acids also produces iminium ions, but the corresponding loss is 60 Da instead of 46 Da. H2O loss initiates the fragmentation of protonated β-amino acids, producing protonated β-lactams as an intermediate. Subsequently, protonated β-lactams are easily converted to iminium ions and CH2CO. By contrast, H2O loss from the protonated forms of γ- and ε-amino acids provides protonated lactams with 5- and 7-membered rings, respectively. Protonated lactams with more than 5-membered rings provide stable fragments and do not undergo further degradation during CID. In addition, protonated forms of γ- and ε-amino acids undergo NH3 loss as a competitive fragmentation pathway of H2O loss, producing protonated lactones. Because the fragmentation of protonated amino acid by CID depends on the position of amino and carboxyl groups, the tandem mass spectrometry with CID can discriminate α-, β-, and other amino acids.

Mirror-Image Random Nonstandard Peptides Integrated Discovery (MI-RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic β-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

In vitro characterization of taurine transport using the human brain microvascular endothelial cell line as a human blood-brain barrier model

Taurine, a sulfur-containing β-amino acid, has various roles in the brain including cellular osmoregulation and neuroprotection. For adequate supply to the brain, taurine has to pass through the blood-brain barrier (BBB); however, the associated mechanism behind crossing the human BBB is not fully understood. Therefore, we characterized taurine transport in vitro using the human brain microvascular endothelial (hCMEC/D3) cell line, a model of human BBB function. [3H]Taurine uptake by hCMEC/D3 cells exhibited time-, as well as extracellular Na+- and Cl−-dependence. The uptake was saturable with a Km of 19 μM and was inhibited by GABA at an IC50 of 328 μM, which were similar to Km values of taurine transporter (TauT)-mediated transport of taurine and GABA, respectively, suggesting that TauT is a major contributor to taurine uptake. For distribution to the brain, taurine must undergo cellular efflux after uptake. Taurine efflux from hCMEC/D3 cells increased for at least 60 min, and monocarboxylate transporter 7 (MCT7)-targeted siRNA significantly reduced MCT7 mRNA levels and [3H]taurine efflux by 93% and 12%, respectively, suggesting that MCT7 partly contributes to taurine efflux from hCMEC/D3 cells. Taken together, these results suggest that TauT and MCT7 function cooperatively in the human BBB.

Development & validation of a simple, sensitive, robust, reproducible & economical spectrofluorimetric method for estimation of taurine in novel chewable gel

BackgroundTaurine, known as 2-aminoethanesulphonic acid, is a sulfur-containing β-amino acid not incorporated into proteins. However, it takes part in biochemical reactions of significant importance, such as conjugation with bile acids to form bile salts essential for fat absorption, cell membrane stabilization, antioxidation, detoxification, osmoregulation, neuromodulation, and brain and retinal development. ObjectiveTo increase its dissolution and absorption rate for early onset of action, we developed a medicated gel formulation of taurine. Chewable gels are suitable dosage forms for pediatric, geriatric, and dysphagic patients. The current research aims to develop a simple, sensitive, and reproducible spectrofluorometric method for determining taurine in chewable gel. MethodA spectrofluorimetric method was developed, and the determination of taurine was based on Hantzsch condensation synthesis, and the fluorescence intensity was measured at emission wavelength (λem) of 474 nm after excitation (λex) at 416 nm. The spectrofluorimetry method for taurine was validated according to the Q2B(R1) ICH guideline for method validation and validated for linearity, LOD, LOQ, interday and intraday precision, accuracy, and robustness. ResultsThe calibration curve was linear in a 1.5–50 µg/ml concentration range. The correlation coefficient (r2) was found to be 0.994. The % RSD values of intra-day precision lie between 0.11 and 0.09; for inter-day precision, it was found to be between 0.09 and 0.11; in both cases, it is less than 2 %. The percentage accuracy study was between 91.81 and 99.93 with a % RSD value of 0.078, less than 2 %. LOD & LOQ values have been mathematically calculated to 4.03 µg/ml and 12.23 µg/ml, respectively. The method was precise, accurate, and robust. ConclusionAll validation parameters were found to be within their acceptable limits. Thus, the developed spectrofluorimetric method of taurine is economical, fast, and robust. Further, this method was utilized to estimate taurine content uniformity in chewable gel for in-vitro release studies.

Antimicrobial activity of α/β hybrid peptides incorporating tBu-β3,3Ac6c against methicillin-resistant Staphylococcus aureus.

The incorporation of β-amino acids into peptides is a promising approach to develop proteolytically stable therapeutic agents. Short α/β hybrid peptidescontaining tBu-β3,3Ac6cː H2N-Lys-tBu-β3,3Ac6c-PEA, P1; H2N-Orn-tBu-β3,3Ac6c-PEA, P2; H2N-Arg-tBu-β3,3Ac6c-PEA, P3; LA-Lys-tBu-β3,3Ac6c-PEA, P4; LA-Orn-tBu-β3,3Ac6c-PEA, P5; LA-Arg-tBu-β3,3Ac6c-PEA, P6; LAu-Lys-tBu-β3,3Ac6c-PEA, P7; LAu-Orn-tBu-β3,3Ac6c-PEA, P8; and LAu-Arg-tBu-β3,3Ac6c-PEA, P9 were prepared. The antimicrobial efficacies of all the peptides were evaluated against ESKAPE pathogens, along with a small panel of multi-drug resistant (MDR) clinical isolates of S. aureus. Among all the peptides, P4, P6, and P7 showed significant efficacies against P. aeruginosa, S. aureus, and MRSA with an MIC value ranging from 6.25 to 12.5 μM. Further, in vitro, anti-staphylococcal assessment with their antimicrobial synergy of the peptides P4, P6, and P7 was carried out against MRSA, due to its better efficacy. The peptides P6 and P7 exhibited MRSA biofilm inhibition of 70% and 77%, respectively, at 4×MIC concentration. At its MIC concentration, about 19% hemolysis was observed for P4, P6, and P7.

Base-Promoted Aminoamidation of Cinnamoyl Chlorides with Aryl Amines: Access to β-Amino Amides.

Herein, a base-promoted strategy for the synthesis of β-amino acids derivatives from α,β-unsaturated acyl chlorides derivatives and aryl amines has been described. In the presence of triethylamine, a tandem Michael addition and nucleophilic substitution progress was generated. The current method features readily available raw materials, mild reaction conditions, high atom economy, and wide tolerance for the coupling partners.

Advances in Transition-Metal Catalysis and Organocatalysis Approaches towards Asymmetric Synthesis of β-Amino Acid Derivatives

AbstractThe stereoselective synthesis of β-amino acids has attracted major attention among the synthetic community in recent years. This review provides an overview of the important advances in chiral β-amino acid synthesis over the past decade. It covers the development of enantioselective methods using transition-metal complexes or organocatalysts, mainly including catalytic asymmetric hydrogenation, the Mannich reaction, multicomponent reactions of diazo compounds, and conjugate addition. Additionally, the asymmetric synthesis of optically active β-amino acids by other approaches are also summarized.1 Introduction2 Strategies towards the Asymmetric Synthesis of β-Amino Acids2.1 Hydrogenation2.2 Mannich Reaction2.3 Conjugate Addition2.4 Multicomponent Reactions2.5 Miscellaneous2.5.1 Synthesis of β-Amino Acids from Chiral Amines2.5.2 Synthesis of β-Amino Acids from Isoxazolidinones2.5.3 Synthesis of β-Amino Acids by Other Methodologies3 Summary and Outlook

Hydroxylamines: From Synthetic Intermediates to Synthetic Targets.

ConspectusSynergy between the teaching and research activities of a University should be a source of new ideas, each informing the other. A classroom discussion gave rise to our concept of using hydroxylamines as a form of "tethered nitrogen" for alkaloid synthesis. The "tether" temporarily connects a nucleophilic nitrogen atom to the substrate, rendering an intermolecular reaction intramolecular, thus providing stereo- and regiochemical control for C-N bond formation. In the context of the synthesis of 1,3-amino alcohols, this necessitated the synthesis of isoxazolidines. This concept led to the exploration of methods for the synthesis of these heterocycles beyond the well-established 1,3-dipolar cycloadditions. The first two methods developed based upon this concept were palladium catalyzed cyclocarbonylation and silver catalyzed allene cyclization. These new methods were applied to two syntheses of sedamine and the synthesis of three Nuphar alkaloids. Intramolecular aza-Michael addition, combined with the use of cross-metathesis to generate the substrates, gave access to both isoxazolidines and tetrahydro-1,2-oxazines. This new method made possible a synthesis of monomorine with high stereochemical control. The extension to more complex alkaloids required the incorporation of additional chemistry. Combining allene cyclization with iminium ion chemistry allowed the extension to the more complex piperidine alkaloids porantheridine and sedinine. In these cases, successful stereocontrol relied on the ability to predict the conformation of the intermediates and the trajectory of the nucleophilic attack, which may be sterically or stereoelectronically controlled. Inspection of our collection of methods revealed that we had good methods for cis-3,5-disubstituted isoxazolidines and trans-3,6-disubstituted tetrahydro-1,2-oxazines. Inspired by earlier work involving iminium ions, methods were developed to provide the complementary trans-isoxazolidines and cis-oxazines, using an allylation reaction. This, combined with our interest in applications of hydroformylation, led to the synthesis of 5-hydroxysedamine. Venturing away from piperidines, this method was successfully applied to the synthesis of the β-amino acid antibiotic negamycin. Use of N,O-heterocycles as intermediates naturally sparked an interest in N,O-heterocycles as synthetic targets, as there are many natural products that contain the hydroxylamine moiety, often incorporated into an isoxazolidine or 1,2-oxazine ring. As the presence of the hydroxylamine moiety cannot be fully demonstrated using the usual spectroscopic methods, total synthesis can be employed to bridge this logical gap. A synthesis of raistrickindole A, utilizing an intramolecular Mitsunobu reaction of a hydroxamic acid to form a 1,2-oxazine, confirmed the structure of this diketopiperazine natural product. A synthesis of preisomide, using an aza-Michael addition to form the required 1,2-oxazine, also confirmed the structure of this natural product.

Combination of the amide-to-triazole substitution strategy with alternative structural modifications for the metabolic stabilization of tumor-targeting, radiolabeled peptides.

Radiolabeled peptides play a key role in nuclear medicine to selectively deliver radionuclides to malignancies for diagnosis (imaging) and therapy. Yet, their efficiency is often compromised by low metabolic stability. The use of 1,4-disubstituted 1,2,3-triazoles (1,4-Tzs) as stable amide bond bioisosteres can increase the half-life of peptides in vivo while maintaining their biological properties. Previously, the amide-to-triazole substitution strategy was used for the stabilization of the pansomatostatin radioligand [111In]In-AT2S, resulting in the mono-triazolo-peptidomimetic [111In]In-XG1, a radiotracer with moderately enhanced stability in vivo and retained ability to bind multiple somatostatin receptor (SSTR) subtypes. However, inclusion of additional 1,4-Tz led to a loss of affinity towards SST2R, the receptor overexpressed by most SSTR-positive cancers. To enhance further the stability of [111In]In-XG1, alternative modifications at the enzymatically labile position Thr10-Phe11 were employed. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide conjugates were synthesized with a 1,4-Tz (Asn5-Ψ[Tz]-Phe6) and either a β-amino acid (β-Phe11), reduced amide bond (Thr10-Ψ[NH]-Phe11), or N-methylated amino acid (N-Me-Phe11). Two of the new peptidomimetics were more stable in blood plasma in vitro than [111In]In-XG1. Yet none of them retained high affinity towards SST2R. We demonstrate for the first time the combination of the amide-to-triazole substitution strategy with alternative stabilization methods to improve the metabolic stability of tumor-targeting peptides.

Peptide-Based Strategies: Combating Alzheimer's Amyloid β Aggregation through Ergonomic Design and Fibril Disruption.

Amyloidosis of amyloid-β (Aβ) triggers a cascade of events, leading to oxidative damage and neuronal death. Therefore, inhibiting Aβ amyloidosis or disrupting the matured fibrils is the primary target to combat progressive Alzheimer's disease (AD) pathogenesis. Here, we undertake optimization strategies to improve the antiamyloid efficiency of our previously reported NF11 (NAVRWSLMRPF) peptide. Among the series of peptides tested, nontoxic and serum-stable peptide 1 or P1 containing an anthranilic acid residue shows immense potential in not only inhibiting the Aβ42 amyloid formation but also disrupting the mature Aβ42 fibrils into nontoxic small molecular weight soluble species. Our studies provide high-resolution characterization of the peptide's mechanism of action. With a binding affinity within the micromolar range for both the monomer and aggregated Aβ42, this α/β hybrid peptide can efficiently modulate Aβ amyloidosis while facilitating the clearance of toxic aggregates and enforcing protection from apoptosis. Thus, our studies highlight that incorporating a β-amino acid not only imparts protection from proteolytic degradation and improved stability but also functions effectively as a β breaker, redirecting the aggregation kinetics toward off-pathway fibrillation.

Synergistic Enhancement of 5-Fluorouracil Chemotherapeutic Efficacy by Taurine in Colon Cancer Rat Model.

Colorectal cancer (CRC) is one of the top 10 most common cancers worldwide and caused approximately 10 million deaths in 2022. CRC mortality has increased by 10% since 2020 and 52.000 deaths will occur in 2024, highlighting the limitations of current treatments due to ineffectiveness, toxicity, or non-adherence. The widely used chemotherapeutic agent, 5-fluorouracil (5-FU), is associated with several adverse effects, including renal, cardiac, and hepatic toxicity; mucositis; and resistance. Taurine (TAU), an essential β-amino acid with potent antioxidant, antimutagenic, and anti-inflammatory properties, has demonstrated protective effects against tissue toxicity from chemotherapeutic agents like doxorubicin and cisplatin. Taurine deficiency is linked to aging and cancers such as breast and colon cancer. This study hypothesized that TAU may mitigate the adverse effects of 5-fluorouracil (5-FU). Carcinogenesis was chemically induced in rats using 1,2-dimethylhydrazine (DMH). Following five months of cancer progression, taurine (100 mg/kg) was administered orally for 8 days, and colon tissues were analyzed. The results showed 80% of adenocarcinoma (AC) in DMH-induced control animals. Notably, the efficacy of 5-FU showed 70% AC and TAU 50% while, in the 5-FU + TAU group, no adenocarcinoma was observed. No differences were observed in the inflammatory infiltrate or the expression of genes such as K-ras, p53, and Ki-67 among the cancer-induced groups whereas APC/β-catenin expression was increased in the 5FU + TAU-treated group. The mitotic index and dysplasia were increased in the induced 5-FU group and when associated with TAU, the levels returned to normal. These data suggest that 5-FU exhibits a synergic anticancer effect when combined with taurine.

Structural basis for sequence context-independent single-stranded DNA cytosine deamination by the bacterial toxin SsdA.

DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA represents the bacterial deaminase toxin family-2 (BaDTF2) and it deaminates ssDNA cytosines with little sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues of SsdA engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5' or 3' to the target cytosine are stacked linearly and make few sequence-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and may contribute to the stability of SsdA as a toxin. Structure- function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering.