Formation of Volatile Pyrazinones in the Asparagine Maillard Reaction Systems and Novel Pyrazinone Formation Pathways in the Amidated-Alanine Maillard Reaction Systems.

Maillard reaction (MR) plays a pivotal role in the food flavor industry, including a cascade of reactions starting with the reaction between amino compounds and reducing sugars, and thus provides various colors and flavors. A new group of volatile compounds called pyrazinones found in MR are now getting more attention. In this study, eight volatile pyrazinones were found in the asparagine MR systems, in which 3,5-dimethyl- and 3,6-dimethyl-2(1H)-pyrazinones were reported for the first time. The major formation pathways were the reactions between asparagine and α-dicarbonyls, with decarboxylation as a critical step. Besides, novel alternative pathways involving alanine amidation and successive reactions with α-dicarbonyls were explored and successfully formed eight pyrazinones. The major differences between alanine-amidated pathways and decarboxylation pathways are the amidation step and absence of the decarboxylation step. For the alanine-amidated pathways, the higher the temperature, the better the amidation effect. The optimal amidation temperature was 200 °C in this study. The reaction between the alanine amide and α-dicarbonyls after amidation can happen at low temperatures, such as 35 and 50 °C, proposing the possibility of pyrazinone formation in real food systems. Further investigations should be conducted to investigate volatile pyrazinones in various food systems as well as the biological effects and kinetic formation differences of the volatile pyrazinones.

Reducing Acrylamide Formation Potential by Targeting Free Asparagine Accumulation in Seeds.

Acrylamide is a probable carcinogen in humans and is formed when reducing sugars react with free asparagine (Asn) during thermal processing of food. Although breeding for low reducing sugars worked well in potatoes, it is less successful in cereals. However, reducing free Asn in cereals has great potential for reducing acrylamide formation, despite the role that Asn plays in nitrogen transport and amino acid biosynthesis. In this perspective, we summarize the efforts aimed at reducing free Asn in cereal grains and discuss the potentials and challenges associated with targeting this essential amino acid, especially in a seed-specific manner.

Predicting deamidation and isomerization sites in therapeutic antibodies using structure-based in silico approaches.

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.

Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m/z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

Mitigation of acrylamide formation in wood oven baked pizza base using wholemeal and refined wheat flour with low free asparagine content: considerations on fibre intake and starch digestibility.

BACKGROUND: In wheat-derived bakery products, the quantity of free asparagine (fAsn) has been identified as a key factor in acrylamide (AA) formation. Based on this assumption, four varieties of common wheat (Triticum aestivum L.), Stromboli, Montecarlo, Sothys and Cosmic, selected for their different fAsn content inside the grain, were studied to evaluate their potential in the production of pizza with reduced AA levels. To this purpose, wholemeal and refined flours were obtained from each variety. RESULTS: The fAsn content ranged from 0.25 to 3.30 mmol kg-1, with higher values for wholemeal flours which also showed greater amount of ash, fibre and damaged starch than refined wheat flours. All types of flours were separately used to produce wood oven baked pizza base, according to the Traditional Speciality Guaranteed EU Regulation (97/2010). AA reduction in the range 47-68% was found for all the selected wheat cultivars, compared with a commercial flour, with significantly lower values registered when refined flour was used. Moreover, refined leavened dough samples showed decreased levels of fAsn and reducing sugars due to the fermentation activity of yeasts. Furthermore, it was confirmed that pizza made with wholemeal flours exhibited lower rapidly digestible starch (RDS) and rapidly available glucose (RAG) values compared to that prepared with the refined flour. CONCLUSION: This study clearly shows that a reduced asparagine content in wheat flour is a key factor in the mitigation of AA formation in pizza base. Unfortunately, at the same time, it is highlighted how it is necessary to sacrifice the beneficial effects of fibre intake, such as lowering the glycaemic index, in order to reduce AA. © 2024 Society of Chemical Industry.

Adding pulse flours to cereal-based snacks and bakery products: An overview of free asparagine quantification methods and mitigation strategies of acrylamide formation in foods.

Thermal processing techniques can lead to the formation of heat-induced toxic substances. Acrylamide is one contaminant that has received much scientific attention in recent years, and it is formed essentially during the Maillard reaction when foods rich in carbohydrates, particularly reducing sugars (glucose, fructose), and certain free amino acids, especially asparagine (ASN), are processed at high temperatures (>120°C). The highly variable free ASN concentration in raw materials makes it challenging for food businesses to keep acrylamide content below the European Commission benchmark levels, while avoiding flavor, color, and texture impacts on their products. Free ASN concentrations in crops are affected by environment, genotype, and soil fertilization, which can also influence protein content and amino acid composition. This review aims to provide an overview of free ASN and acrylamide quantification methods and mitigation strategies for acrylamide formation in foods, focusing on adding pulse flours to cereal-based snacks and bakery products. Overall, this review emphasizes the importance of these mitigation strategies in minimizing acrylamide formation in plant-based products and ensuring safer and healthier food options.

Novel synergistic cross-linking ameliorate ready-to-eat sea cucumber deterioration and its quantum chemical analysis.

Synergistic cross-linkers could improve the taste acceptability of ready-to-eat sea cucumber (RSC). Besides, the hardness of RSC was increased by 331.00% and 266.87% after synergistic cross-linking. Synergistic cross-linking treatment could ameliorate the non-enzymatic degradation of RSC collagen and polysaccharides. Gaussian calculations results showed that dipeptides containing asparagine residues may have different reaction pathways. The main cleavage pathways of CH3CO-Asn-Gly-NHCH3 (NG) might be water-assisted side chain cyclization, stepwise cyclamide hydrolysis via a Gemdiol Intermediate, deamination, and peptide bond breakage. The relative free energy of cyclamide hydrolysis process of NG was increased by 8.2 kcal/mol after synergistic cross-linking. The mass spectrometry results showed that typical peptides could cleavage at NG, CH3CO-Asn-Lys-NHCH3 (NK) and CH3CO-Asn-Leu-NHCH3 (NL) sites after heating, which justified the breakage pattern of peptides in Gaussian calculations. It can offer a comprehensive theoretical basis for the processing of the ready-to-eat sea cucumber with storage stability.

Dissecting dual specificity: Identifying key residues in L-asparaginase for enhanced acute lymphoid leukemia therapy and reduced adverse effects.

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.

Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form.

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 µg.mL-1 and 5.6 µg.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

Characterizing the formation of process contaminants during coffee roasting by multivariate statistical analysis.

Coffee is a relevant source of dietary exposure for neoformed furan, alkyl furans and acrylamide. In this study, different statistical methods (hierarchical cluster analysis, correlation analysis, partial least squares regression analysis) were used for characterizing the formation of these process contaminants in green coffee beans roasted under the same standardized conditions. The results displayed a strong correlation between sucrose levels and furans in relation to the other sugars analyzed, while acrylamide formation was strongly related to the free asparagine. The data suggest that a sufficiently large amino acid pool in green coffee favors Maillard-induced acrylamide formation from asparagine, while reactions amongst the carbonyl-containing sugar fragmentation products leading to furan formation are suppressed. If the pool of free amino acids is small, it is depleted faster during roasting, thus favoring the formation of furans by caramelization, basically a sugar degradation process in which reactive carbonyl substances are generated and react together.

Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation.

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

Influence of Deamidation on the Formation of Pyrazines and Proline-Specific Compounds in Maillard Reaction of Asparagine and Proline with Glucose.

Maillard reaction products obtained from the model system of binary amino acids (asparagine and proline) with glucose were first studied. GC-MS results showed that proline-specific aromatic compounds, 2,3-dihydro-1H-pyrrolizines and cyclopent[b]azepin-8(1H)-ones, were dominant among overall products, followed by pyrazines at different temperatures. Aspartic acid was first applied to model reactions as the precise control of asparagine deamidation, and lysine was further introduced into model systems for improving pyrazine formation. Quantitative results of model reaction products demonstrated that pyrazines were not significantly increased in deamidated states (Asn-Asp-Pro and Asp-Pro) while proline-specific compounds had a rapid enhancement at the same time. With excellent ability to form pyrazines, lysine did help to increase the formation of pyrazines, but still far fewer than pyrrolizines and azepines. It was assumed that proline would preferentially react with α-dicarbonyl compounds in Maillard reaction cascades with lower activation energies.

Inhibitory behavior and adsorption of asparagine dipeptide amino acid on the Fe(111) surface.

CONTEXT: The inhibitory effect of asparagine (Asn) and its derivatives on iron (Fe) corrosion was studied by performing density functional theory (DFT) calculations. In this paper, the global and local reactivity descriptors of Asn in the protonated and neutral forms were evaluated. Also, the changes in reactivity were investigated when dipeptides were combined with Asn. Due to the increase in the reaction centers within their molecular structure, there was an enhancement in the inhibitory effect of these dipeptides. Moreover, the adsorption energies (Eads) and the adsorption configurations of Asn and small peptides (SPs) with most stability were determined on the surface of Fe(111). It was found that dipeptides had a chemical adsorption on these substrates. In the protonated forms, there was an enhancement in the absolute values of Eads between the inhibitors and the Fe(111) surfaces. Peptides were more likely to be adsorbed on the Fe surfaces, showing the great inhibitory effect of these moieties. The results of the current research demonstrate the possibility of utilizing SPs as efficient "green" corrosion inhibitors. METHODS: DFT computations were undertaken by employing the BIOVIA Material Studio with B3LYP-D3 functional and 6-31 + G* basis set. The theoretical evaluation of the inhibitory effect of asparagine (Asn) dipeptides, and the potential analysis of small peptides to protect against the corrosion of Fe, was done.

Direct deamidation analysis of intact adeno-associated virus serotype 9 capsid proteins using reversed-phase liquid chromatography.

Recombinant adeno-associated viral (AAV) vectors have taken center stage as gene delivery vehicles for gene therapy. Asparagine deamidation of AAV capsid proteins has been reported to reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins that is detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid proteins is comparable to the peptide mapping-based deamidation analysis and both methods are suitable for deamidation monitoring of AAV9 capsid proteins.

Computing the Differences between Asn-X and Gln-X Deamidation and Their Impact on Pharmaceutical and Physiological Proteins: A Theoretical Investigation Using Model Dipeptides.

Protein deamidation is a degradation mechanism that significantly impacts both pharmaceutical and physiological proteins. Deamidation impacts two amino acids, Asn and Gln, where the net neutral residues are converted into their acidic forms. While there are multiple similarities between the reaction mechanisms of the two residues, the impact of Gln deamidation has been noted to be most significant on physiological proteins while Asn deamidation has been linked to both pharmaceutical and physiological proteins. For this purpose, we sought to analyze the thermochemical and kinetic properties of the different reactions of Gln deamidation relative to Asn deamidation. In this study, we mapped the deamidation of Gln-X dipeptides into Glu-X dipeptides using density functional theory (DFT). Full network mapping facilitated the prediction of reaction selectivity between the two primary pathways, as well as between the two products of Gln-X deamidation as a function of solvent dielectric. To achieve this analysis, we studied a total of 77 dipeptide reactions per solvent dielectric (308 total reactions). Modeled at a neutral pH and using quantum chemical and statistical thermodynamic methods, we computed the following values: enthalpy of reaction (ΔHRXN), entropy (ΔSRXN), Gibbs free energy of reaction (ΔGRXN), activation energy (EA), and the Arrhenius preexponential factor (log(A)) for each dipeptide. Additionally, using chemical reaction principles, we generated a database of computed rate coefficients for all possible N-terminus Gln-X deamidation reactions at a neutral pH, predicted the most likely deamidation reaction mechanism for each dipeptide reaction, analyzed our results against our prior study on Asn-X deamidation, and matched our results against qualitative trends previously noted by experimental literature.

Study the interaction of amino acids, sugars, thermal treatment and cooking technique on the formation of acrylamide in potato models.

This study unveiled the effect of the suspected precursors of acrylamide (asparagine, glutamine) combined/separated with different formulations of glucose, fructose, and sucrose. To better understand the interaction between acrylamide precursors, cooking technique (deep vs air frying), and temperature (170 °C vs 190 °C), seven potato models from starch, sugars, amino acids, water and hydrocolloids (alginate and agar) were formulated. In line with previous findings, the present results showed that asparagine, glucose and fructose played an important role in acrylamide formation in these synthetic potato models. Furthermore, glutamine and sodium alginate might have an inhibitory effect on acrylamide formation. A significant impact of frying technique was also revealed. On the other hand, GC-FID analysis detected acrylamide in only these three models, (glucose-fructose, sucrose and asparagine-glucose/fructose/sucrose models > LOD 333.33 µg.kg-1).

In Silico Prediction Method for Protein Asparagine Deamidation.

In silico prediction methods were developed to predict protein asparagine (Asn) deamidation. The method is based on understanding deamidation mechanism on structural level with machine learning. Our structure-based method is more accurate than the sequence-based method which is still widely used in protein engineering process. In addition, molecular dynamics simulation was applied to study the time occupancy of nucleophilic attack distance, which is hypothesized as the most important step toward the rate-limiting succinimide intermediate formation. A more accurate prediction method for distinguishing potentially liable amino acid residues would allow their elimination or reduction as early as possible in the drug discovery process. It is possible that such quantitative protein structure-property relationship tools can also be applied to other protein hotspot predictions.

Synthesis, Processing, and Function of N-Glycans in N-Glycoproteins.

Many membrane-resident and secreted proteins, including growth factors and their receptors are N-glycosylated. The initial N-glycan structure is synthesized in the endoplasmic reticulum (ER) as a branched structure on a lipid anchor (dolicholpyrophosphate) and then co-translationally, "en bloc" transferred and linked via N-acetylglucosamine to asparagine within a specific N-glycosylation acceptor sequence of the nascent recipient protein. In the ER and then the Golgi apparatus, the N-linked glycan structure is modified by hydrolytic removal of sugar residues ("trimming") followed by re-glycosylation with additional sugar residues ("processing") such as galactose, fucose or sialic acid to form complex N-glycoproteins. While the sequence of the reactions leading to biosynthesis, "en bloc" transfer and processing of N-glycans is well investigated, it is still not completely understood how N-glycans affect the biological fate and function of N-glycoproteins. This review will discuss the biology of N-glycoprotein synthesis, processing and function with specific reference to the physiology and pathophysiology of the immune and nervous system, as well as infectious diseases such as Covid-19.

Acceleration effect of galacturonic acid on acrylamide generation: evidence in model reaction systems.

BACKGROUND: Acrylamide (AA) is a potential carcinogen formed in food rich in carbohydrate during heating. Recently, AA has been found in several fruit products, such as prune juice, sugarcane molasses and canned black olives. This study focused on the role of galacturonic acid (GalA), the main acid hydrolysis product of fruit pectin, in AA formation in three model systems - asparagine (Asn)/glucose (Glc), Asn/GalA, and Asn/Glc/GalA - during heating under different pH values (pH 3.8-7.8), Glc concentration (0-0.1 mol L-1 ), molar ratio of substrates (Asn/Glc = 1:1, 0.025-0.5 mol L-1 ) and temperature (120-180 °C) for 30 min, respectively. RESULTS: The results suggested that the addition of 0.1 mol L-1 GalA strongly accelerated AA formation in a manner dependent on pH value and temperature (P < 0.05). AA concentration under different Glc concentration and molar ratio of substrates suggested that GalA was more reactive than Glc when reacted with Asn. Furthermore, the Amadori rearrangement product/Schiff base/oxazolidine-5-one were identified as the intermediates formed in the Asn/GalA model system using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry. CONCLUSION: The results suggested that Maillard reaction between Asn and GalA might contribute to AA formation. This study is significant in elucidating the contribution of interaction between components for AA formation in fruit products. © 2022 Society of Chemical Industry.

Elucidation of the Reaction Mechanism of Cavia porcellus l-Asparaginase: A QM/MM Study.

The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.