Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions

Kim, Eun Sil; Yaylayan, Varoujan

doi:10.3390/app13137658

Open AccessArticle

Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions

by

Eun Sil Kim

and

Varoujan Yaylayan

^*

Department of Food Science & Agricultural Chemistry, McGill University, Lakeshore, QC H9X 3V9, Canada

^*

Author to whom correspondence should be addressed.

Appl. Sci. 2023, 13(13), 7658; https://doi.org/10.3390/app13137658

Submission received: 18 April 2023 / Revised: 8 May 2023 / Accepted: 21 June 2023 / Published: 28 June 2023

(This article belongs to the Special Issue Innovation in Microwave Technology—A Themed Issue in Memory of Prof. Dr. J. R. Jocelyn Paré, MSM, (1959–2022))

Download

Browse Figures

Review Reports Versions Notes

Abstract

:

Imine or Schiff base formation is considered as a key event in the catalytic mechanisms of many enzymes and in certain biological transformations, including glycation. In this process, less stable amino-acid-derived Schiff bases rearrange into more stable ketoamines or Amadori products. Schiff bases are also stipulated to be stabilized through complexation with metal ions, or through intramolecular cyclization to form more stable and reversible cyclic isomers, such as oxazolidin-5-ones. These intermediates can be easily detected relative to Schiff bases due to their higher stability. In this study, high-resolution mass spectrometry and isotope labeling techniques were used to identify labile imines as their oxazolidin-5-one derivatives in heated reaction systems of glucose/alanine/FeCl₂, including their ¹³C-labeled counterparts. The reaction mixtures were heated for 2h at 110 °C and were analyzed by high resolution qTOF/MS for the presence of masses corresponding to Schiff bases of α-alanine with short chain aldehydes that can be generated from glucose degradation and also for the incorporation of ¹³C-labeled atoms from ¹³C-3 alanine and ¹³C-U glucose. Analysis of the data has indicated that Schiff bases can indeed be detected in the form of oxazolidin-3-ones, when methanol is used as the solvent. Furthermore, it was discovered that metal-ion-stabilized Schiff bases, in addition to forming oxazolidin-3-ones, can also undergo aldol addition with short chain sugars and initiate oligomerization reactions, leading to the formation of dimeric or trimeric oxazolidin-3-one oligomers, as demonstrated by their characteristic MS/MS fragmentations.

Keywords:

Schiff bases; oxazolidine-5-one; glycation; isotope labeling; iron

Graphical Abstract

1. Introduction

The formation of advanced glycation end products (AGEs) in biological systems and the generation of flavors and colors during the thermal processing of foods, share a common origin in the Maillard reaction. This reaction is fundamentally a carbonyl-amine reaction between reducing sugars and amino acids leading to the formation of imines or Schiff bases [1] The process leads to protein cross-linking in biological systems with its inherent implications in cell pathology. At the same time, it can lead to the formation of thermally generated toxicants and heterocyclic aromatic compounds [2] associated with enhanced flavor and color in processed foods [3,4]. Upon the initiation of the Maillard reaction, amino acids and reducing sugars can form labile imines that are prone to hydrolysis, avoiding detection. However, their subsequent rearrangement into its more stable and irreversible isomer form the Amadori rearrangement products (keto-amines) [5,6], allowing for their facile detection. In fact, Schiff bases are presumed to be formed in processed food and biological tissues due to the detection of their more stable Amadori isomers; alternatively, their intramolecular cyclization [7] to form more stable but reversible oxazolidin-5-one isomers may also be used as indicators of imine formation [8,9]. In addition to promoting the stabilization of Schiff bases in dry systems, oxazolidin-5-ones can also undergo facile thermal decarboxylation at higher temperatures to form azomethine ylides [10,11,12]. These ylides subsequently degrade into isomeric imines and generate various aroma-active compounds [10]. Only few studies have been reported on monitoring the generation of oxazolidin-5-ones under thermal reaction conditions using Fourier–transform infrared (FTIR) spectroscopy or high-resolution mass spectrometry (HRMS) [8,9,10,13]. In the Maillard reaction mixtures containing metal ions, the increased content of Amadori products was attributed to the stabilization of their precursors, the Schiff bases, through metal ion complexation [14]. Here, we employed high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HR/ESI/qTOF/MS/MS) and isotope labeling techniques to study their formation and their further transformations into oligomeric forms in alanine/glucose/FeCl₂ model systems under Maillard reaction conditions (110 °C for 2 h).

2. Materials and Methods

2.1. Materials and Reagents

L-α-alanine (98%), D-glucose, paraformaldehyde, glycolaldehyde, acetaldehyde (99.5%), and iron(II) chloride (FeCl₂) (99%) were purchased from Sigma-Aldrich Chemical Co. (Oakville, ON, Canada). Alanine-3-¹³C (¹³CH₃CH(NH₂)CO₂H) (98%) and glucose-¹³C-U (¹³C₆H₁₂O₆) (99%) were purchased from Cambridge Isotope Laboratories (Andover, MI, USA). Liquid chromatography–mass spectrometry (LC–MS)-grade water and methanol (OmniSolv, >99%) were obtained from VWR International (Mississauga, ON, Canada).

2.2. Sample Preparation

The test model systems were prepared in 1:1:0.5 molar ratios by adding glucose (18 mg), alanine (9 mg), and FeCl₂ (6.4 mg) to methanol or water (1 mL) and heating in a stainless-steel reactor at 110 °C for 2 h, followed by evaporation of the solvent for 30 min at 110 °C. The control model system was prepared by heating glucose (18 mg) and alanine (9 mg) in methanol or water at 110 °C for 2 h in the absence of metal ions. All samples were analyzed in at least two replicates, as shown in Table 1.

2.3. Spiking Experiments with Selected Strecker Aldehydes

Experiments with selected Strecker aldehydes (formaldehyde, acetaldehyde, and glycolaldehyde) were conducted using excess (~1:10 w/w) aldehydes relative to alanine and glucose in the presence and absence of FeCl₂. The relative intensities of the oxazolidin-5-one derivatives were measured using ESI/qTOF/MS spectral peaks. These experiments were performed to confirm the identity of observed oxazolidin-5-one derivatives generated in the reaction mixtures from aforementioned aldehydes by observing the increases in their relative intensities (See Table 1 and Figure 1).

2.4. ESI/qTOF/MS Analysis

The samples were dissolved in liquid chromatography (LC) grade methanol at a concentration of 1 mg/mL. The samples were diluted 10-fold in 10% methanol prior to analysis using ESI/qTOF/MS in the positive-ion mode. The ESI/qTOF/MS system comprised a Bruker Maxis Impact quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in the positive-ion mode. Samples (1 μL) were injected directly into the ESI/qTOF/MS system. Instrument calibration was performed by using sodium formate clusters. The electrospray interface settings were as follows: nebulizer pressure, 0.6 bar (60,000 Pa); drying gas, 4 L/min; temperature, 180 °C and capillary voltage, 4500 V. The scan range was 70–1000. Molecular formulas were assigned to all observed peaks based on their exact m/z values using “ChemCalc” software (available online, Institute of Chemical Sciences and Engineering, Lausanne, Switzerland) [15]. ESI/qTOF/MS/MS was carried out in multiple reaction monitoring mode for the ions at m/z 227 and 328 using collision energy of 10 eV.

2.5. Structural Elucidation and Isotope Labeling Studies

Evidence for the proposed structures was provided through high-resolution ESI/qTOF/MS analysis of their elemental composition and isotope-labeling studies using [¹³C-U] glucose and [¹³C-3] alanine. The proposed structures are based on well-known Maillard reaction degradation products.

2.6. Criteria Applied for Tentative Identification of the Listed Ions

Most selected ions were detected in three different reaction systems: (1) un-labeled, (2) only glucose-labeled, and (3) only alanine-labeled systems. Each one was analyzed in at least two replicates. Ions with an error in their elemental composition higher than 10 ppm in only one of the above reaction systems were included if the corresponding ions in the remaining two reaction systems displayed errors with less than eight ppm.

3. Results and Discussion

In the presence of amino acids, reducing sugars undergo well-characterized degradation reactions, such as dehydrations, retro-aldol and redox reactions, to generate smaller and reactive sugar-derived carbonyl compounds critical for the formation of AGEs, aromatics, and toxicants in food [16,17,18,19,20]. In this study we took advantage of the Maillard reaction between α-alanine and glucose to demonstrate that the Schiff bases of the carbonyl compounds formed in this reaction system can be trapped as their oxazolidin-5-one derivatives in the presence of alanine.

3.1. Detection of Schiff Bases of Alanine with Sugar-Derived Aldehydes through Isotope Labeling

The α-alanine/glucose reaction mixtures heated for 2 h at 110 °C were analyzed by high resolution qTOF/MS for the presence of masses corresponding to Schiff bases of α-alanine with short chain aldehydes that can be generated from glucose degradation [14]. Peaks corresponding to adducts of formaldehyde, acetaldehyde and glycolaldehyde were observed only in methanol solutions and in the presence of FeCl₂, as listed in Table 2. The last two aldehydes have been previously reported to be generated also from formaldehyde alone in the presence of metal ions through the formose reaction (Butlerov reaction) [21,22,23]. The Schiff bases of α-alanine formed with formaldehyde (m/z 102), acetaldehyde (m/z 116), and glycolaldehyde (m/z 132) were observed only in the presence of FeCl₂ except the adduct formed with formaldehyde (Table 2). According to the HRMS data, the ion at m/z 102 was the most prominent. As shown in Table 2, spiking the reaction mixtures with excess formaldehyde increased the intensities of all the detected Schiff bases by more than four-fold. Furthermore, the reaction mixtures were also analyzed using isotope labeling techniques with [¹³C-U]glucose or [¹³C-3]alanine, targeting ions incorporating one C-3 atom of alanine and up to two sugar carbon atoms, depending on the type of aldehyde moieties involved in the Schiff base formation. The extracted ions from the HRMS data satisfying these requirements and exhibiting highly accurate elemental composition of the Schiff bases (shown in their isomeric oxazolidin-5-one forms) and their isotopically labeled counterparts using [¹³C-U]glucose or [¹³C-3]alanine are shown in Table 2. The isotope labeling data together with the spiking experiments and high-resolution MS data provided sufficient evidence for the identity of these ions. The fact that Schiff bases can be hydrolyzed during the reaction and are not stable enough to survive the reaction conditions lead us to conclude that the observed ions with a Schiff base composition represented their oxazolidin-5-one isomers [10]. When the reactions were carried out in water or without FeCl₂, the intensities of the proposed oxazolidin-5-one were significantly decreased or disappeared altogether (Table 2).

The rationale behind the divalent metal ion catalysis of oxazolidin-5-one formation and the proposed reaction pathways are shown in Figure 2. According to Figure 2, the added metal ions can stabilize the Schiff bases through complexation (see also Table 3), reducing the electrophilic character of the imine carbon and extending their half-life in the reaction mixture, thus retarding solvolysis. Table 3 provides evidence for the formation of iron complexes of some of the alanine Schiff bases with highly accurate elemental composition and isotope label incorporation data. Only when these complexes are dissociated, intramolecular cyclization will form stable oxazolidin-5-ones as shown in Figure 2.

Further evidence for the cyclic nature of the proposed Schiff bases was provided by the observation of their interaction products with aldehydes, such as formaldehyde, and formation of their corresponding iminium ions, such as [M]⁺ 114.0549, as shown in Figure 2 and listed in Table 4. Furthermore, the solvent methanol can trap the iminium ions as shown in Figure 2, providing additional structural information on iminium ions. Similarly, 4-methyl-5-oxaoxazolidin-5-one (m/z 102), shown in Figure 3, can undergo Schiff base formation with other available aldehydes and generate iminium ions at M⁺ 128, 144, and 114 as shown in Figure 3 and listed in Table 4. The open form Schiff bases will not be able to form such iminium ions.

It can be concluded that the Schiff bases were detected mainly due their existence in the more stable oxazolidin-5-one form, otherwise, they would have rapidly undergone hydrolysis before being detected. The ions listed in Table 2 incorporated one ¹³C-3 alanine atom, indicating the presence of alanine and up to four carbon atoms from glucose-derived aldehydes. The incorporation of one carbon atom from glucose can indicate that a formaldehyde Schiff base adduct was formed; incorporation of two carbon atoms can indicate acetaldehyde or glycolaldehyde Schiff base formation, and incorporation of one, three or four carbon atoms can indicate multiple addition adducts, considering their HRMS data.

3.2. Detection of Iron Complexes

Schiff bases incorporating formaldehyde, acetaldehyde, and glycolaldehyde were also detected as their iron(II) complexes as [M]⁺ at m/z 155.9738 [C₄H₆FeNO₂]⁺, m/z 169.9895 [C₅H₈FeNO₂]⁺, and m/z 185.9848 [C₅H₈FeNO₃]⁺, respectively (see Table 3). Supporting evidence for their formation were provided by observing the incorporation of one C-3 atom from [¹³C-3] alanine and up to two carbon atoms from [¹³C-U] glucose, depending on the conjugated aldehyde type.

3.3. Spiking Experiments with Simple Aldehydes

To provide supporting evidence for the formation of Schiff bases with sugar-derived aldehydes, spiking experiments were performed by adding formaldehyde, acetaldehyde, and glycolaldehyde to the reaction mixtures. There were statistically significant increases in the relative intensities of the corresponding ions upon spiking with the appropriate aldehyde; these increases are shown in Table 2. In addition, spiking the model system (Ala/Glu/FeCl₂) with excess formaldehyde (see Table 2) lead to an increase in the formation of adducts incorporating not only formaldehyde (R = CH₂ in Figure 3), but also acetaldehyde (R = CHCH₃), and glycolaldehyde (R = CHCH₂OH). These aldehydes have been previously reported to be thermally generated from formaldehyde in the presence of metal ions through the formose reaction (Butlerov reaction) [21,22].

3.4. Further Reactions of Oxazolidin-5-Ones with Aldehydes

The initially formed oxazolidin-5-ones are secondary amines and can further undergo Schiff base formation with available aldehydes and form the corresponding iminium ions (see Figure 3). The iminium ions can be easily detected under positive ionization mode without protonation or sodiation (Table 4). The oxazolidin-5-ones and their derivatives are mainly observed in the presence of metal ions during the reaction. This observation suggests that metal ions influence the formation of oxazolidin-5-one due to their ability to stabilize the Schiff bases through complexation, as discussed above. The detailed analysis of the HRMS data suggested that oxazolidin-5-one moieties can undergo further reactions with available sugar-derived aldehydes in the reaction mixture containing metal ions to form oxazolidin-5-one iminium ions (Figure 3). These derivatives can be traced by observing the incorporation of one C-3 alanine atom and several carbon atoms from [¹³C-U] glucose depending on the aldehyde. Nine such ions representing oxazolidin-5-one derivatives were detected, Figure 3 depicts three such ions originating from the most abundant 4-methyl-1,3-oxazolidinone observed at [M + H]⁺ 102 and listed in Table 4. As shown in Figure 3, the initial oxazolidin-5-one adducts consisting of alanine and formaldehyde can undergo further reactions with other aldehydes to form Schiff bases as iminium ions. As mentioned previously, further confirmation of these structures was performed by observing the incorporation of one C-3 atom from [¹³C-3] alanine and up to four carbon atoms from [¹³C-U] depending on the type of aldehyde (Table 4).

3.5. Formation of Oxazolidin-5-One Oligomers

The metal ion-stabilized Schiff bases, in addition to promoting oxazolidin-5-one formation, can also undergo aldol addition with short chain aldo-sugars, such as glycolaldehyde (Figure 4a), or keto-sugars, such as dihydroxyacetone (Figure 4b), and serve as molecular scaffolds for the generation of oligomeric structures through the intramolecular cyclization and formation of a putative oxazolidin-5-one with a carbonyl-containing side chains, such as the ion at [M + H]⁺ 144.0648 shown in Figure 4a. Subsequently, this carbonyl group can undergo nucleophilic addition with the most abundant 4-methyl-oxazolidin-3-one to form oxazolidine-5-one oligomers or react with alanine to form the ion at [M + H]⁺ 215.1032, as shown in Figure 4a. Similarly, the carbonyl group of the ion observed at [M + H]⁺ 174.0766 shown in Figure 4b can undergo nucleophilic addition with the most abundant 4-methyl-oxazolidin-3-one to form ions at [M + H]⁺ 257.1138 and 328.1491.

3.6. Generation of Dimeric and Trimeric Oxazolidin-5-Ones Observed at [M + H]⁺ 227.1016 and 328.1491

According to Figure 4a, oligomerization can be initiated by the aldol addition of glycolaldehyde to an formaldehyde-alanine Schiff base followed by the migration of the carbonyl double bond and intramolecular cyclization to form an oxazolidine-5-one moiety with a carbonyl-containing side chain after a dehydration step. The subsequent carbonyl amine reaction between 4-methyl-oxzolidin-5-one (the most abundant oxzolidin-5-one in the reaction mixture) and the newly formed carbonyl side chain can generate the observed ion at [M + H]⁺ 227.1016, which can be considered as dimeric oxazolidine-5-one. Alternatively, the newly formed carbonyl group can also react with free alanine and form a dimeric structure observed at [M + H]⁺ 215.1032. As expected, these ions incorporated the expected number of carbon atoms from [¹³C-U] glucose and C-3 atoms from [¹³C-3] alanine (see Table 5). A similar oligomerization reaction can be initiated by the aldol addition of dihydroxyacetone with formaldehyde-alanine Schiff base followed by the migration of the carbonyl bond and intramolecular cyclization to form an oxazolidine-5-one moiety with a carbonyl-containing side chain after a dehydration reaction. The carbonyl amine reaction between 4-methyl-oxzolidin-5-one (the most abundant oxzolidin-5-one in the reaction mixture) and the newly formed carbonyl side chain on structure at [M + H]⁺ 174.0766 can generate a new structure with two oxazolidinone moieties with a carbonyl side chain after dehydration step observed at [M + H]⁺ 257.1138. This newly formed carbonyl moiety can subsequently undergo Schiff base formation with alanine, be stabilized as trimeric oxazolidine-5-one, and be observed at [M + H]⁺ 328.1491. As expected, this ion incorporated five carbon atoms from [¹³C-U] glucose and three C-3 atoms from [¹³C-3] alanine. Table 5 shows isotope incorporation data, and the elemental composition of MS/MS fragments are shown in Figure 4a,b. One of the characteristic properties of oxazolidinones is their ability to undergo facile decarboxylation due to the formation of relatively stable azomethine ylides [24,25,26] and this property is well demonstrated in their MS/MS fragmentations discussed below.

3.7. MS/MS Fragmentations of Ions at m/z 227 and 328 under 10eV Energy

As shown in Figure 5, dimeric and trimeric oxazolidin-5-ones exhibit decarboxylation reactions characteristic of oxazolidine-5-ones during MS/MS fragmentation, forming C-protonated azomethine ylides [27] observed at M⁺ 284.1571. In the case of the ion at [M + H]⁺ 328 and in the case of the ion [M + H]⁺ 227.1016, only its fragmentation product at [M + H] 155.0808 was detected. This ion can undergo electrocyclic ring closure to generate the proposed azetine ring system. Interestingly, trimeric oxazolidine-5-one (Figure 5b) undergoes multiple decarboxylations consistent with the number of oxazolidinone moieties in the structure to generate the ion at [M + H]⁺ 139.1218 after the loss of methanimine molecule. Furthermore, the MS/MS spectrum also shows characteristic oxazolidinone fragment ions at [M + H]⁺ 116.0699 and 102.0543 with consistent elemental composition.

4. Conclusions

Schiff bases can be stabilized by various mechanisms, including Amadori rearrangement. In the case of amino-acid-derived Schiff bases, stabilization can be achieved either through complexation with metal ions, or through intramolecular cyclization and formation of cyclic isomer the oxazolidin-5-one. Unlike oxazolidin-5-ones, Schiff bases cannot be easily detected owing to their facile conversion into Amadori products, especially under acidic pH conditions. The metal-ion complexation of Schiff bases enhances their stability, increasing their half-life and allowing for the formation of more oxazolidin-5-one. The observed ions corresponding to Schiff base structures suggested the possible formation of oxazolidin-5-one in the Maillard reaction mixtures in the presence of divalent iron. This helped to identify the previously unknown role of metal ions in the stabilization of Schiff bases and formation of oligomeric structures. Among the oxazolidin-5-one derivatives, the ion at m/z 102.0548 [C₄H₈NO₂]⁺, associated with alanine and the formaldehyde adduct, showed relatively higher intensity than that of the other oxazolidin-5-one derivatives. Oxazolidin-5-ones, furthermore, can be used as indicators for both Schiff base and Strecker aldehyde formation in the Maillard reaction when divalent iron is used as a reactant.

Author Contributions

E.S.K.: data curation, formal analysis, methodology, validation, and writing—original draft. V.Y.: supervision, conceptualization, project administration, reviewing—editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and McGill University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors wish to thank Alexander Wahba for his contributions to the qTOF/MS experiments.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

Hellwig, M.; Henle, T. Baking, Ageing, Diabetes: A Short History of the Maillard Reaction. Angew. Chem. Int. Ed. 2014, 53, 10316–10329. [Google Scholar] [CrossRef] [PubMed]
Mortzfeld, F.B.; Hashem, C.; Vranková, K.; Winkler, M.; Rudroff, F. Pyrazines: Synthesis and Industrial Application of these Valuable Flavour and Frarance Compounds. Biotechnol. J. 2020, 15, 2000064. [Google Scholar] [CrossRef]
Frank, O.; Hofmann, T. Characterization of Key Chromospheres Formed by Nonenzymatic Browning of Hexoses and L-Alanine by using the Colour Activity Concept. J. Agric. Food Chem. 2000, 48, 6303–6311. [Google Scholar] [CrossRef] [PubMed]
Yaylayan, V. Acrylamide formation and its impact on the mechanism of the early Maillard reaction. J. Food Nutr. Res. 2009, 48, 1–7. [Google Scholar]
Hofmann, T. Application of Site Specific ¹³C Enrichment and ¹³C NMR Spectroscopy for the Elucidation of the Formation Pathway Leading to a Red ¹H-Pyrrol-3(2H)-one during the Maillard reaction of Furna-2-carboxaldehyde and L-Alanine. J. Agric. Food Chem. 1998, 46, 941–945. [Google Scholar] [CrossRef]
Rizzi, G.P. Chemical structure of coloured Maillard reaction products. Food Rev. Int. 1997, 13, 1–28. [Google Scholar] [CrossRef]
RajanBabu, T.V. Stereochemistry of intramolecular free-radical cyclization reaction. Accounts Chem. Res. 1991, 24, 139–145. [Google Scholar] [CrossRef]
Chu, F.L.; Yaylayan, V.A. Post-schiff chemistry of the Maillard reaction. Ann. N. Y. Acad. Sci. 2008, 1126, 30–37. [Google Scholar] [CrossRef]
Chu, F.L.; Yaylayan, V.A. FTIR monitoring of oxazolidinone-5-one formation and decomposition in a glycolaldehyde-phenylalanine model system by isotope labelling techniques. Carbohydr. Res. 2009, 344, 229–236. [Google Scholar] [CrossRef]
Xing, H.; Yaylayan, V. Insight into the mechnochemistry of the Maillard reaction: Degradation of Schiff bases via 5-oxazolidinone intermediate. Eur. Food Res. Technol. 2021, 247, 1095–1106. [Google Scholar] [CrossRef]
Manini, P.; D’Ischi, M.; Prota, G. An Unusual Decarboxylative Maillard Reaction between l-DOPA and d-Glucose under Biomimetic Conditions: Factors Governing Competition with Pictet−Spengler Condensation. J. Org. Chem. 2001, 66, 5048–5053. [Google Scholar] [CrossRef]
Manini, P.; Napolitano, A.; D’ischia, M. Reactions of d-glucose with phenolic amino acids: Further insights into the competition between Maillard and Pictet–Spengler condensation pathways. Carbohydr. Res. 2005, 340, 2719–2727. [Google Scholar] [CrossRef]
Shimasaki, C.; Hirata, F.; Ohta, H.; Tsukurimichi, E.; Yoshimura, T. Thermal behaior and mass spectrometry studies of 2-oxazolidinone derivatives. J. Anal. Appl. Pyrolysis 1993, 24, 291–300. [Google Scholar] [CrossRef]
Kim, E.S.; Yaylayan, V. Identification of the Maillard reaction intermediates as divalent iron complexes in alanine/glucose/FeCl₂ model system using ESI/qTOF/MS/MS and isotope labelling technique. Curr. Res. Food Sci. 2021, 4, 287–294. [Google Scholar] [CrossRef]
Patiny, L.; Borel, A. ChemCalc: A Building Block for Tomorrow’s Chemical Infrastructure. J. Chem. Inf. Model. 2013, 53, 1223–1228. [Google Scholar] [CrossRef] [Green Version]
Smuda, M.; Glomb, M.A. Fragmentation Pathways during Maillard-Induced Carbohydrate Degradation. J. Agric. Food Chem. 2013, 61, 10198–10208. [Google Scholar] [CrossRef]
Davídek, T.; Robert, F.; Devaud, S.; Vera, F.A.; Blank, I. Sugar Fragmentation in the Maillard Reaction Cascade: Formation of Short-Chain Carboxylic Acids, by a New Oxidative α-Dicarbonyl Cleavage Pathway. J. Agric. Food Chem. 2006, 54, 6677–6684. [Google Scholar] [CrossRef]
Davídek, T.; Robert, F.; Devaud, S.; Vera, F.A.; Blank, I. Sugar Fragmentation in the Maillard Reaction Cascade: Isotope Labeling Studies on the Formation of Acetic Acid by a Hydrolytic β-Dicarbonyl Cleavage Mechanism. J. Agric. Food Chem. 2006, 54, 6667–6676. [Google Scholar] [CrossRef]
Thornalley, P.J. Dicarbonyl Intermediates in the Maillard Reaction. Ann. N. Y. Acad. Sci. 2002, 1043, 111–117. [Google Scholar] [CrossRef]
Gensberger-Reigl, S.; Glomb, M.A.; Pischetsrieder, M. Analysis of Sugar Degradation Products with α-Dicarbonyl Structure in Carbonated Soft Drinks by UHPLC-DAD-MS/MS. J. Agric. Food Chem. 2013, 61, 10238–10245. [Google Scholar] [CrossRef]
Breslow, R. On the mechanism of the formose reaction. Tetrahedron Lett. 1959, 1, 22–26. [Google Scholar] [CrossRef]
Breslow, R.; Appayee, C. Deuterium Studies Reveal a New Mechanism for the Formose Reaction Involvoing Hydride Shifs. J. Am. Chem. Soc. 2014, 136, 3720–3723. [Google Scholar] [CrossRef]
Usui, T.; Yanagisawa, S.; Ohguchi, M.; Yoshino, M.; Kawabata, R.; Kishimoto, J.; Arai, Y.; Watanabe, H.; Hayase, F. Identification and determination of α-Dicarbonyl Compounds Formed in the Degradation of Sugars. Biosci. Biotechnol. Biochem. 2007, 71, 2465–2472. [Google Scholar] [CrossRef] [PubMed]
Tsuge, O.; Kanemasa, S.; Ohe, M.; Takenaka, S. Simple Generation of Nonstabilized Azomethine Ylides through Decarboxylative Condensation of α-Amino Acids with Carbonyl Compounds via 5-Oxazolidinone Intermediates. Bull. Chem. Soc. Jpn. 1987, 60, 4079–4089. [Google Scholar] [CrossRef] [Green Version]
Choury, M.; Lopes, A.B.; Blond, G.; Gulea, M. Synthesis of Medium-Sized Heterocycles by Transition-Metal-Catalyzed Intramolecular Cyclization. Molecules 2020, 25, 3147. [Google Scholar] [CrossRef]
Feather, M.S.; Mossine, V.V. Correlations between Structure and Reactivity of Amadori Compounds: The Reactivity of Acyclic Forms. In The Maillard Reaction in Foods and Medicine; Woodhead Publishing: Cambridge, UK, 2005; pp. 37–42. [Google Scholar] [CrossRef]
Schmeyers, J.; Toda, F.; Boy, J.; Kaupp, G. Quantitative solid–solid synthesis of azomethines. J. Chem. Soc. Perkin Trans. 1998, 2, 989–994. [Google Scholar] [CrossRef]

Figure 1. ESI/qTOF/MS spectra: (A) Heated alanine and glucose in the absence of FeCl₂ (Ala/Glu); (B) heated alanine and glucose, and excess of formaldehyde in the absence of FeCl₂ (Ala/Glu); (C) heated alanine and glucose, and excess of three aldehydes (formaldehyde, acetaldehyde, and glycolaldehyde) in the absence of FeCl₂ (Ala/Glu); (D) heated alanine, and glucose in the presence of FeCl₂ (Ala/Glu/FeCl₂); (E) heated alanine, glucose, and excess of formaldehyde in the presence of FeCl₂ (Ala/Glu/FeCl₂); (F) heated alanine, glucose, and excess of acetaldehyde in the presence of FeCl₂ (Ala/Glu/FeCl₂); (G) heated alanine, glucose, and excess of acetaldehyde in the presence of FeCl₂ (Ala/Glu/FeCl₂); and (H) heated alanine, glucose, and excess of three aldehydes (formaldehyde, acetaldehyde, and glycolaldehyde) in the presence of FeCl₂ (Ala/Glu/FeCl₂).

Figure 2. Reaction of formaldehyde with alanine and the rationale behind divalent metal ion catalysis of oxazolidin-5-one formation (see also Figure 3 and Table 3).

Figure 3. Observed iminium ions formed between 4-methyl-1,3-oxazolidin-5-one and various aldehydes (see also Table 3).

Figure 4. Proposed formation pathways of oxazoldine-5-one oligomers observed at (a) m/z 227 and (b) m/z 328 detected in the Ala/Glu/FeCl₂ model system (see Table 5).

Figure 5. (a,b) MS/MS fragmentations of the ions observed at using 10 eV collision energy.

Table 1. Composition of the model systems ^a.

Model System
Control Model System	Control model systems contained no metal ions—Ala/Glu
Spiked Model System	Paraformaldehyde was added to an alanine and glucose and heated in the absence of metal ions—Ala/Glu Three reactive aldehydes (paraformaldehyde, acetaldehyde, and glycolaldehyde) were added to an alanine and glucose solution, followed by heating without FeCl₂—Ala/Glu Reactive aldehyde (paraformaldehyde: acetaldehyde: B, and glycolaldehyde: C) were added to an alanine and glucose solution, followed by heating in the presence of FeCl₂—Ala/Glu/FeCl₂, Ala/Glu/FeCl₂, and Ala/Glu/FeCl₂ Three reactive aldehydes (paraformaldehyde, acetaldehyde, and glycolaldehyde) were added to an alanine and glucose solution, followed by heated in the presence of FeCl₂
Test Model System	Alanine was added to a glucose solution and heated in the presence of FeCl₂—Ala/Glu/FeCl₂
Isotope-Labeled Model System	Alanine [¹³C-3] was added to a glucose solution and heated in the presence of FeCl₂-[¹³C-3] Ala/Glu/FeCl₂
Isotope-Labeled Model System	Alanine was added to a glucose [¹³C-U] solution and heated in the presence of FeCl₂—Ala/[¹³C-U] Glu/FeCl₂

^a All model systems were heated either in a methanol or water at 110 °C for 2 h using a sealed stainless-steel reactor and analyzed in at least over two replicates.

Table 2. Label incorporation and observed intensities of the ions corresponding to α-alanine-derived Schiff bases from formaldehyde, acetaldehyde and glycolaldehyde shown in their oxazolidin-5-one isomeric forms, in glucose/α-alanine reaction mixtures.

	[M + H]⁺ = 102.0548 C₄H₈NO₂ (−1.52 ppm)	[M + H]⁺ = 116.0706 C₅H₁₀NO₂ (−0.04 ppm)	[M + H]⁺ = 132.0656 C₅H₁₀NO₃ (0.61 ppm)
Label incorporation data
Glu [¹³C-U]/Ala/FeCl₂	C₃[¹³C]H₈NO₂ (−3.01 ppm)	C₃[¹³C]₂H₁₀NO₂ (−0.97 ppm)	C₃[¹³C]₂H₁₀NO₃ (0.96 ppm)
Ala [¹³C-3]/Glu/FeCl₂	C₃[¹³C]H₈NO₂ (−2.04 ppm)	C₄[¹³C]H₁₀NO₂ (−3.07ppm)	C₄[¹³C]H₉NaNO₃ (−3.35 ppm)
% intensities of the detected ions in the presence FeCl₂ and spiking with formaldehyde
Ala/Glu/MeOH	0.6	Not detected	Not detected
Ala/Glu/FeCl₂/MeOH	14.5 ± 0.98	6.43 ± 0.32	0.83 ± 0.12
Ala/Glu/FeCl₂/H₂O	0.0	0.0	0.0
Ala/Glu/FeCl₂/CH₂O/	62.35 ± 7.42	62.1 ± 6.36	3.5 ± 0.28

Table 3. Observed label incorporation of the iron complexes of alanine-derived Schiff bases of formaldehyde, acetaldehyde and glycolaldehyde.

	M⁺ = 155.9738 C₄H₆FeNO₂ (−2.83 ppm)	M⁺ = 169.9895 C₅H₈ FeNO₂ (−2.3 ppm)	M⁺ = 185.9848 C₅H₈FeNO₃ (−0.03 ppm)
Label incorporation data
Glu [¹³C-U]/Ala	C₃[¹³C]H₆FeNO₂ 156.9765 (−6.98 ppm) C₄H₆FeNO₂ 155.9742 (−0.26 ppm)	C₃[¹³C]₂H₈FeNO₂ 171.99 (nd) 170.99 (nd)	C₃[¹³C]₂H₈FeNO₃ 187.9913 (−1.15 ppm)
Ala [¹³C-3]/Glu	C₃[¹³C]H₆FeNO₂ 156.9731 (−28 ppm)	C₄[¹³C]H₈FeNO₂ 170.9924 (−4.95 ppm) C₃[¹³C]₂H₈FeNO₂ 171.99 (nd)	C₄[¹³C]H₈ FeNO₃ 186.98 (nd)

Table 4. Elemental composition and label incorporation in the ions corresponding to further reactions of 4-methyl-1,3-oxazolidin-5-one with formaldehyde, acetaldehyde and glycolaldehyde in in glucose/α-alanine reaction mixtures.

	M⁺ = 114.0549 C₅H₈NO₂ (−0.48 ppm)	M⁺ = 128.0709 C₆H₁₀NO₂ (1.98 ppm)	M⁺ = 144.0655 C₆H₁₀NO₃ (−0.14 ppm)
Label incorporation data
Glu [¹³C-U]/Ala/FeCl₂	C₃[¹³C]₂H₈NO₂ (nd) C₄[¹³C]₁H₈NO₂ (nd) C₅H₈NO₂ (−5.74 ppm)	C₃[¹³C]₃H₁₀NO₂ 131.0778 (21ppm) C₄[¹³C]₂H₁₀NO₂ (nd)	C₃[¹³C]₃H₁₀NO₃ 147 (2.15 ppm) C₄[¹³C]₂H₁₀NO₃
Ala [¹³C-3]/Glu/FeCl₂	C₄[¹³C]H₈NO₂ 115.0573 (−8.78pm)	C₄[¹³C]₂H₁₀ NO₂ (nd) C₅[¹³C]₁H₁₀ NO₂ (nd) C₃[¹³C]₃H10 NO₂	C₅[¹³C]H₉NO₃ 145 (−5.22 ppm)

Table 5. Elemental composition and isotope incorporation in the proposed oxazolidine-5-one derivatives observed in alanine/glucose/FeCl₂ model system using methanol as solvent (see Figure 3, Figure 4 and Figure 5).

[M + X]⁺	Elemental Composition	Error ppm	Glu [¹³C-U]	Elemental Composition	Error ppm	Ala [¹³C-3]	Elemental Composition	Error ppm
102.0548	C₄H₈NO₂	−1.52	103.058	C₃[¹³C]H₈NO₂	−3.01	103.0581	C₃[¹³C]H₈NO₂	−2.04
114.0549	C₅H₈NO₂	−0.48	nd ^b			115.0573	C₄[¹³C]H₈NO₂	−8.78
116.0706	C₅H₁₀NO₂	−0.04	118.0772	C₃[¹³C]₂H₁₀NO₂	−0.97	117.0736	C₄[¹³C]₁H₁₀NO₂	−3.07
128.0709	C₆H₁₀NO₂	1.98	131.0778	C₃[¹³C]₃H₁₀NO₂	nd	167.0321	C₅[¹³C]H₉KNO₂	nd
132.0656	C₅H₁₀NO₃	0.61	134.0721	C₃[¹³C]₂H₁₀NO₃	−0.96	155.0503	C₄[¹³C]₁H₉NaNO₃	−3.35
142.0866	C₇H₁₂NO₂	2.43	146.097	C₃[¹³C]₄H₁₂NO₂	nd	143.088	C₆[¹³C]₁H₁₂NO₂	nd
144.0655	C₆H₁₀NO₃	3.2	147.0759	C₃[¹³C]₃H₁₀NO₃	2.15	183.0238	C₅[¹³C]H₉KNO₃	−5.22
155.9738	C₄H₆FeNO₂	−2.83	156.9765	C₃[¹³C]₁H₆FeNO₂	−6.98	156.9731	C₃[¹³C]₁H₆FeNO₂	nd
158.0815	C₇H₁₂NO₃	2.09	nd	nd	nd	159.0818	C₆[¹³C]H₁₂NO₃	nd
169.9895	C₅H₈FeNO₂	−2.3	171.99	nd	nd	170.9924	C₄[¹³C]H₈FeNO₂	−4.95
174.0766	C₇H₁₂NO₄	0.189	nd	nd	nd	175.0794	C₆[¹³C]H₁₂NO₄	−0.22
185.9848	C₅H₈FeNO₃	−0.03	187.9913	C₃[¹³C]₂H₈FeNO₃	−1.15	nd
227.1016	C₁₀H₁₄N₂O₄	−2.12	231.1147	C₆[¹³C]₄H₁₄N₂O₄	−5.85	nd
328.1491	C₁₄H₂₂N₃O₆	−3.69	333.1695	C₉[¹³C]₅H₂₂N₃O₆	7.25	nd
215.1032	C₉H₁₄N₂O₄	{1.084	218.1132	C₆[¹³C]₃H₁₄N₂O₄	−10.99	nd

^b nd: error higher than 10 ppm.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Share and Cite

MDPI and ACS Style

Kim, E.S.; Yaylayan, V. Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions. Appl. Sci. 2023, 13, 7658. https://doi.org/10.3390/app13137658

AMA Style

Kim ES, Yaylayan V. Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions. Applied Sciences. 2023; 13(13):7658. https://doi.org/10.3390/app13137658

Chicago/Turabian Style

Kim, Eun Sil, and Varoujan Yaylayan. 2023. "Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions" Applied Sciences 13, no. 13: 7658. https://doi.org/10.3390/app13137658

APA Style

Kim, E. S., & Yaylayan, V. (2023). Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions. Applied Sciences, 13(13), 7658. https://doi.org/10.3390/app13137658

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Menu

Amino-Acid-Derived Oxazolidin-5-Ones as Chemical Markers for Schiff Base Formation in Glycation Reactions

Abstract

1. Introduction