Amino Acid Moiety Research Articles

A classical peptide coupling approach for the total synthesis of simnotrelvir

The total synthesis of an oral small-molecule antiviral agent; a structure based developmental drug simnotrelvir has been accomplished. The synthesis involves readily accessible commercial amino acids L-tert-leucine, 4-hydroxy-l-proline and l-glutamic acid that are easily converted to the key intermediates and are coupled together following classical peptide coupling technique using HATU and NMM. One of the crucial step in the synthesis involved a one pot Boc-deprotection and trifluoroacetylation of tert-leucine amino acid moiety mediated by ZnCl2.

Gold nanoclusters stabilized with dopa-containing ligands: Catalyst-indicator integrated probe for tumor cell screening

Elevated hydrogen peroxide (H2O2) levels not only inflict cellular damage but also serve as a harbinger for various diseases. Tumor cells, in particular, often exhibit an abundance of H2O2. Hence, the detection of this pivotal molecule assumes paramount importance in monitoring physiological states and expediting cancer diagnosis. To this end, we have ingeniously devised an enzyme-free and monomeric system for intracellular H2O2 detection. Our astute selection of dopa-containing peptidomimetics, replete with ortho-bisphenol and amino acid moieties, has engendered the synthesis of distinctive fluorescent gold nanoclusters (AuNCs). These nanoclusters not only function as a peroxidase-like catalyst, catalyzing the decomposition of H2O2 into hydroxyl radicals (·OH), but also serve as an indicator, with their fluorescence quenched in response to varying H2O2 concentrations. Experimental results evince that our GDpE-AuNCs exhibit remarkable sensitivity, boasting a detection limit of 0.49 μM and a linear range of 5–1000 μM. Moreover, the amalgamation of catalyst and indicator within a single structure, facilitating efficient cellular uptake, engenders intracellular H2O2 detection and discernment of tumor cells. This pioneering approach bequeaths a valuable assay probe for monitoring physiological states and ushering in early disease diagnosis.

(Invited) Tuning Chirality of Graphene Quantum Dots to Empower Medical Imaging and Theranostic Applications

Chiral nanostructures from metals and semiconductor, including graphene-based materials, are increasingly recognized for their potential in polarization-sensitive optoelectronics. Particularly, graphene quantum dots (GQDs) modified with L/D amino acids exhibit unique chiral properties due to edge-specific interactions, offering new possibilities in biomedicine. Our study delves into the interaction of these chiral nanomaterials with biological systems, revealing significant implications for their application in biology and medicine. We discovered that attaching L/D amino acid moieties to GQDs induces nanoscale chirality by helical buckling, influenced by the chiral interactions at the graphene edges. The chirality of GQDs affects their interaction at the nanoscale with cells and tissues. Molecular dynamics simulations show that D-GQDs are more likely to accumulate within cellular membranes compared to L-GQDs. Additionally, experiments on chiral GQD permeation through lipid membranes of extracellular vesicles demonstrated that optimizing chirality via ligand tuning and GQD size can significantly enhance their permeation efficiency into lipid bilayers, exceeding 80%. Leveraging this enhanced transmembrane transport, chiral GQDs have been applied in imaging and efficient drug loading for therapeutic extracellular vesicles. Furthermore, we explored how GQD chirality affects transport in tumor-like cellular spheroids. The results indicate that L-GQDs exhibit a 1.7 times higher apparent diffusion coefficient than D-GQDs, enhancing their transport into these spheroids. In summary, our research underscores the importance of controlling and engineering GQD chirality. This approach not only enhances the transport of drug carriers through biological barriers but also opens new avenues for advanced imaging and theranostic applications.

In vitro and in silico approach towards antimicrobial and antioxidant behaviour of water-soluble chitosan dialdehyde biopolymers

Chitosan dialdehyde (ChDA) was prepared from a three-step process initiated by thermal organic acid hydrolysis, periodate oxidization, and precipitation from native chitosan (NCh). The developed ChDA resulted in an aldehydic content of about 82 % with increased solubility (89 %) and maximum yield (97 %). The functional alteration of the aldehydic (-CHO) group in ChDA was established using vibrational stretching at 1744 cm−1. The increase in the zone of inhibition of ChDA compared to NCh has confirmed the inherent antimicrobial effect against bacterial and fungal species. ChDA showed better antioxidant activity of about 97.4 % (DPPH) and 31.1 % (ABTS) compared to NCh, measuring 45.3 % (DPPH) and 15.9 % (ABTS), respectively. The novel insilico predictions of the ChDA's biocidal activity were confirmed through molecular docking studies. The amino acid moiety such as ARG 110 (A), ASN 206 (A), SER 208 (A), THR 117 (B), ASN 118 (B), and LYS 198 (B) residues of 7B53 peptide from E. coli represents the binding pockets responsible for interaction with aldehyde group of ChDA. Whereas PHE 115 (E), ALA 127 (H), TYR 119 (C), GLN 125 (H), ASN 175 (E), ARG 116 (E), LYS 101 (H), and LYS 129 (H) of 1IYL A peptide from Candida albicans makes possible for binding with ChDA. Hence, the synergistic effect of ChDA as a biocidal compound is found to be plausible in the drug delivery system for therapeutic applications.

Bioinspired Hg2+-sensing fluorogenic probe based on amino acid–functionalized rhodamine

Inspired by the suitability of rhodamine spirolactam switching for superresolution imaging and fluorescent probe development, we herein synthesized rhodamine-based probes with sulfur-bearing amine and amino acid moieties and screened their abilities to respond to Hg2+ in the presence of other metal ions. The probe based on S-methylcysteine methyl ester–substituted rhodamine 6G (Rho6G-6) was identified as optimal because of its straightforward synthesis and rapid, selective, and sensitive (detection limit = 8 × 10–9 M) response to Hg2+ manifested by absorption spectrum and emission color changes. The complexation of Hg2+ by Rho6G-6 was examined by absorbance and fluorescence analyses, electrospray ionization–mass spectrometry, and 1H nuclear magnetic resonance (NMR) titration experiments. Furthermore, the practical applicability of the above probe was demonstrated by naked-eye colorimetric Hg2+ detection using paper test strips and the fluorescence-based qualitative mapping of the Hg2+ distribution in living HeLa cells and the organs of live mice. Thus, unlike other probe design strategies, the use of flexible S-methyl amino acids for Hg2+ binding provides new opportunities and strategies for photopromoting the ring switching of the rhodamine spirothiolactam.

Identifying novel small molecule integrin agonists to mimic the favourable effects of the RGD domain of IGFBP-1 to treat cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation (BHF) 4-Year PhD Programme Background Insulin-like growth factor binding protein (IGFBP)-1 has previously been shown to be associated with reduced type 2 diabetes (T2D) progression and preferable cardiovascular profiles in patients. Our laboratory previously demonstrated that IGFBP-1 is an insulin sensitiser in mouse models, in addition to having favourable vascular effects, independently of IGFs. IGFBP-1 has an Arg-Gly-Asp (RGD) domain which is thought to facilitate the direct interaction with the heterodimeric cell-surface receptor, integrin α5β1, inducing downstream cell signalling. We then showed the positive effects of IGFBP-1 could be mimicked with an RGD-containing linear peptide, GRGDTP, however this peptide is not a suitable therapeutic as it is rapidly metabolised in vivo. To our knowledge, RGD-based therapeutics have not been explored for cardiometabolic disease. Purpose There are currently few therapeutics which have been specifically designed to address both insulin resistance and the high risk of macrovascular disease in T2D. Therefore, exploring RGD-based integrin agonists will be essential in developing new insulin sensitising therapeutics which prevent cardiovascular complications. Methods The ROCS high speed ligand-based virtual screening tool was used to screen commercially available small molecule libraries mimicking the chemical structure of the RGD sequence of IGFBP-1. Additionally, we performed in silico modelling to predict the binding interactions of these molecules with the integrin α5β1 headpiece to select candidates. We then used a LIVE/DEAD cell viability assay to initially profile any cytotoxic effects of our selected compounds in integrin-expressing human umbilical vein endothelial cells (HUVECs) and C2C12 skeletal muscle myotubes. We further assessed the effects of compounds on relevant HUVEC functions using a high-throughput wound closure assay. Results The structure of the RGD domain of IGFBP-1 can be mimicked with small molecules containing amino acid moieties. These molecules are predicted to bind with micromolar affinity to the integrin, promoting beneficial interactions for integrin agonism. We selected 17 structurally diverse small molecules with the best predicted binding interactions. Initial in vitro profiling demonstrates that the molecules are not cytotoxic. In preliminary analyses, three of the selected compounds show a trend towards inhibition of HUVEC migration and proliferation, while six show a trend towards increased migration and proliferation. Conclusion The RGD domain of IGFBP-1 can be mimicked with novel small molecules, of which we have selected 17 ligands. Our compounds do not affect cell viability in relevant cell types, so can be further profiled. Our initial findings indicate that the compounds have different functional effects on HUVECs which allows us to prioritise hits having favourable effects in the context of T2D, such as enhancing angiogenic properties of endothelial cells.

Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2

ASCT2 (alanine serine cysteine transporter 2), a member of the SLC1 (solute carrier 1) family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anti-cancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor Lc-BPE with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that, despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The pre-incubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

Design, synthesis, and evaluation of novel arecoline-linked amino acid derivatives for insecticidal and antifungal activities

A series of arecoline derivatives with amino acid moieties were designed and synthesised using an acylamide condensation strategy, taking arecoline as the foundational structure. The insecticidal efficacy of these compounds against Aphis craccivora and Tetranychus cinnabarinus was evaluated. Notably, derivatives 3h and 3i demonstrated superior insecticidal activity compared with arecoline. Additionally, 3h and 3i showed good fungicidal effectiveness against two types of plant fungi. Moreover, molecular docking analyses suggested that 3h and 3i could affect the nervous systems of A. craccivora and T. cinnabarinus by binding to neuronal nicotinic acetylcholine receptors. These findings suggest that compounds 3h and 3i represent promising leads for further development in insecticide and fungicide research.

Design, synthesis and bioactivity of a new class of antifungal amino acid-directed phthalide compounds.

Peanut southern blight disease, caused by Sclerotium rolfsii, is a destructive soil-borne fungal disease. The current control measures, which mainly employ succinate dehydrogenase inhibitors, are prone to resistance and toxicity to non-target organisms. As a result, it is necessary to explore the potential of eco-friendly fungicides for this disease. Fourteen novel phthalide compounds incorporating amino acid moieties were designed and synthesized. The in vitro activity of analog A1 [half maximal effective concentration (EC50) = 332.21 mg L-1] was slightly lower than that of polyoxin (EC50 = 284.32 mg L-1). It was observed that on the seventh day, the curative activity of A1 at a concentration of 600.00 mg L-1 was 57.75%, while the curative activity of polyoxin at a concentration of 300.00 mg L-1 was 42.55%. These results suggested that our compound exhibited in vivo activity. Peanut plants treated with A1 showed significant agronomic improvements compared to the untreated control. Several compounds in this series exhibited superior root absorption and conduction in comparison to the endothermic fungicide thifluzamide. The growth promotion and absorption-conduction experiments demonstrated the reason for the superior in vivo activity of the target compound. Cytotoxic assays have demonstrated that this series of targeted compounds exhibit low toxicity levels toward human lo2 liver cells. Our results provide a new strategy for the design and synthesis of novel green compounds. Furthermore, the target compound A1 can serve as a lead for further development of green fungicides. © 2024 Society of Chemical Industry.

A Sequence-Defined ABC Dendritic Macromolecule with Amino Acid Peripheral Functionality via Iterative Chemoselective Reactions.

Chemoselective reactions allow near-precision control over the polymer composition and topology to create sequence-controlled polymers with similar secondary and tertiary structures to those found in proteins. Dendrimers are recognized as well-defined macromolecules with the potential to mimic protein surface functionality due to the large number of functional groups available at its periphery with the internal structure acting as the support scaffold. Transitioning from using small-molecule dendrimers to dendritic macromolecules will not only allow retention of the high peripheral functionality but also provide an internal scaffold with a desired polymer composition within each generational layer. Here, we exemplify a systematic approach to creating a dendritic macromolecule with the placement of different polymer building blocks in precise locations within the internal structure and the placement of three different amino acid moieties clustered at the periphery. The synthesis of this ABC dendritic macromolecule was accomplished through iterative chemoselective reactions.

A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDTGo, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDTGo-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDTGo can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDTGo reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

Stereodivergent 1,5-Conjugate Addition with Iminoesters via Pd/Cu Dual Catalysis

AbstractThe first stereodivergent umpolung 1,5-conjugate addition reaction via synergistic Pd/Cu catalysts is reported. By dictating the absolute configuration of each chiral ligand ligated to corresponding metals, all four stereoisomers of the products are easily achieved. A series of amino acid moieties are introduced to the γ-position of the conjugated esters in high yields with excellent diastereoselectivities and enantioselectivities. The downstream transformations highlight the synthetic value of the method.

Amino‐Acid Side‐Chain Nanoarchitectonics for Tuning the Chiroptical Properties and Supramolecular Structure of Pentameric Oligothiophenes

AbstractOligothiophenes with specific photophysical properties and molecular organization are of great interest, since this class of materials are used in organic electronics and bioelectronics, as well as biosensing. Herein, 8 different pentameric oligothiophenes, denoted proteophenes, with different amino acid substitution patterns at distinct positions along the thiophene backbone were investigated. Spectroscopic and microscopic studies of the ligands revealed the formation of optically active self‐assembled materials under acidic or basic conditions. The distinct photophysical characteristics, including induced circular dichroism, as well as the supramolecular structures of the assemblies deduced from light scattering and transmission electron microscopy, were highly influenced by the positioning of distinct amino acid moieties along the thiophene backbone. Proteophenes functionalized with only glutamate residues or these functionalities in combination with hydrophobic valine moieties formed fibrillar structures with excellent chiroptical properties under acidic conditions. In addition, the amino acid functionality at the β‐position of distinct thiophene moieties influenced the induced circular dichroism pattern observed from the proteophenes. Overall, the obtained results demonstrate how changes in the position of various amino acid functionalities, as well as the chemical nature of the amino acid side chain functionality greatly affect the optical properties as well as the architecture of the self‐assembled materials.

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

Precursor-directed production of water-soluble red Monascus pigments with high thermal stability via azaphilic addition reaction-based semi-synthesis

Red Monascus pigments (MPs) are a large group of polyketides from the fungus Monascus which have been widely used as food colorants. In this study, a variety of red MPs congeners were prepared to explore promising water-soluble candidates for application in liquid food formulations. The results showed that by combining the two-stage, low-pH fermentation strategy with a downstream purification step of fractional crystallization, precursors of red MPs, namely monascorubrin and rubropunctatin, were obtained with a purity of 91.9%. Then, via the azaphilic addition reaction, 18 types of red MPs congeners carrying different amino acid moieties (MPs-aa) were semi-synthesized. Compared to rubropunctamine and monascorubramine, the water solubility, pH and thermal stability of MPs-aa were improved greatly. MPs-His, MPs-Phe, MPs-Tyr and MPs-Trp were identified to be the most resistant to pasteurization. These findings provide water-soluble red MPs candidates with high thermal stability and an attractive approach for their large scale production.

Designing functionalized polymers through post-polymerization modification of side chain leucine-based polymer

The opportunity to modify the chemical functionality of the incorporated amino acid moiety in side chain amino acid-based polymers provides access to tailor their optical, mechanical, and biological properties. Methacrylate monomers from C-terminus modified amino acids and their subsequent polymerization leads to the formation of polymethacrylate polymers with pendant amine (-NH2) groups. To this end, the post-polymerization reactions on the amine groups offer a facile route to prepare functionalized side chain amino acid-based polymeric materials. Herein, we synthesized side chain L-leucine pendant homopolymer using reversible addition-fragmentation chain transfer (RAFT) polymerization of Boc-L-leucine methacryloyloxyethyl ester (Boc-leu-HEMA) and subsequently the Boc group is deprotected to prepare P(H3N+-leu-HEMA). Post-polymerization modifications on the pendant amine groups were performed to incorporate fluorescent probe, propargyl group, methacrylic moiety, hydrophobic fatty acid, anionic pendant, and hydrophilic polyethylene glycol (PEG) units; which demonstrates an efficient platform to build a library of functionalized polymers from a single polymeric backbone. We have studied the solvatochromic behavior of fluorescence probe bearing polymers using fluorescence spectroscopy. The ability of the pendent methacrylic unit containing polymers to undergo crosslinking and the mechanical properties of the synthesized gel are investigated by the rheological study.

Molecular docking approach on the binding stability of derivatives of phenolic acids (DPAs) with Human Serum Albumin (HSA): Hydrogen-bonding versus hydrophobic interactions or combined influences?

Molecular docking (Mol.Doc) techniques were employed to ascertain the binding affinity and energetics of hydroxy derivatives of benzoic and cinnamic acids extract from Psidium guajava L. with Human Serum Albumin (HSA). Caffeic acid (CA), Ferullic acid (FA), Sinapic acid (SA), Syringic acid (SyA) and Vanillic acid (VA) are the derivatives phenolic acids (DPA) employed in docking studies which acts as the guest molecule. Docking of various feasible conformers of DPA with HSA (host) was explored and these conformers were categorized based on the docking score which is correlated to the binding energy (BE) and the stability depends upon the molecular interactions. Among the phenolic acids, SA-HSA complex was energetically more favorable and feasible based on BE and the order of binding stability upon complex formation of various DPA-HSA follows the order SA > FA = CA > VA > SyA, though SA and SyA are structurally similar to each other, likewise FA and VA exhibit a similar structure. The stability upon complex formation is correlated to the docking of the guest molecule in the binding domains of HSA and several molecular interactions. Hydrogen-bonding (HB) interaction governs the stability of host-guest complex is established. Interestingly, the presence of multiple hydrophobic interactions (pi-pi, pi-alkyl, pi-cation or anion, pi-sigma and pi-amide) competes over HB interaction in several conformers resulting in a decrease in BE. We report that SA acts as an excellent site selective and site-specific ligand that prefers to dock in Sudlow binding site II comprising of sub domains IIIA and IIIB respectively. However, all other phenolic acids do not behave neither as site selective nor site specific ligand such that they prefer to reside both in site II and site III (non-Sudlow binding site) of HSA. We authenticate that all the DPA as well as the amino acid moieties in HSA act as HB donor as well as acceptor sites apart from several hydrophobic interactions. We further establish that all the DPA has the least probable affinity to reside in binding site I (warfarin binding site), whereas sub domain IIIA of site II is the most preferred site which is energetically most favoured among all the sub domains.

Synthesis, characterization and differentiation of the structural isomers of valine and tert-leucine derived synthetic cannabinoids.

The identification of the synthetic cannabinoids receptor agonists (SCRAs) has always posed a great challenge to drug testing laboratories with slight structural modifications aimed at evading drug legislation. In addition, the most prevalent synthetic cannabinoids have valine and tert-leucine amino acid moieties where re-arrangement of the carbon chains can result in structural isomers that are very similar to the parent synthetic cannabinoids. This makes their analysis and identification challenging, and the problem is compounded with the difficulty in purchasing reference standards quickly and a lack of literature for comparison. Therefore, in this investigation, four series of synthetic cannabinoids (AB-PINACA, AB-CHMINACA, MMB-FUBINACA and 5-fluoro-MDMB-PINACA) and their alkyl chain structural isomers at the amino acid moieties were synthesized and characterized using various analytical techniques-gas chromatography-mass spectrometry (GC-MS), gas chromatography-infrared detection (GC-IRD) and nuclear magnetic resonance (NMR) spectroscopy to evaluate the ability of each analytical technique to differentiate the respective isomers for their identification. A total of 12 isomers were synthesized and analysed together with the four parent synthetic cannabinoids. NMR was able to differentiate between all the compounds, whereas GC-IRD was able to discern between most of the synthetic cannabinoids and their isomers. GC-MS had the least discriminating power and was not able to differentiate some of the compounds that has very similar mass spectra. The results from this work will be useful to other drug testing laboratories that are facing the identification of related synthetic cannabinoids.

Evaluation of mode of indoleamine 2,3-dioxygenase 1 inhibition by 4,7-dichloroquinolines

Herein, we report the development of synthetically simpler analogs of 4,7-dichloroquinoline (DCQ) as apo-indoleamine 2,3-dioxygenase 1(IDO1) protein targeting ligand to inhibit the activity of IDO1 enzyme. Derivatization of the DCQ moiety improved the IDO1 inhibitory activity both against purified enzyme and the cellular environment without any significant cytotoxicity, leading to the identification of piperazine containing DCQ derivative DCQ4 as a potent IDO1 inhibitor. A series of biophysical studies, including UV–Vis spectroscopy of the Soret band, docking, as well as protoporphyrin IX binding studies, suggested that the IDO1 inhibitory activity of potent compound could be due to its direct binding to apo-IDO1 protein and formation of DCQ4-heme complex. This simple strategy of developing apo-IDO1 targeting molecules having DCQ, piperazine, and amino acid moieties as potent IDO1 inhibitors could be useful in fighting against immune-related diseases.

Determination of A1 and A2 β-Casein in Milk Using Characteristic Thermolytic Peptides via Liquid Chromatography-Mass Spectrometry

β-casein, a protein in milk and dairy products, has two main variant forms termed as A1 and A2. A1 β-casein may have adverse effects on humans. The fact that there is only one amino acid variation at the 67th position between A1 and A2 β-casein makes it difficult to distinguish between them. In this study, a novel method using characteristic thermolytic peptides is developed for the determination of A1 and A2 β-casein in milk. Firstly, caseins extracted from milk samples are thermolytic digested at 60 °C without any denaturing reagents required for unfolding proteins, which simplifies the sample pretreatment procedure. The characteristic thermolytic peptides (i.e., fragments 66–76 and 59–76 for A1 and A2 β-casein, respectively) selected to specifically distinguish A1 and A2 β-casein only have eleven or eighteen amino acid moieties. Compared with tryptic characteristic peptides with a length of 49 amino acid moieties, these shorter thermolytic characteristic peptides are more suitable for LC-MS analysis. This novel method, with the advantages of high specificity, high sensitivity, and high efficiency, was successfully applied for the analysis of six milk samples collected from a local supermarket. After further investigation, it is found that this method would contribute to the development of A2 dairy products for a company and the quality inspection of A2 dairy products for a government.