Novel Maillard Pigment, Furpenthiazinate, Having Furan and

3 days ago - ACS Journals. ACS eBooks; C&EN Global Enterprise ..... By continuing to use the site, you are accepting our use of cookies. Read the ACS ...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/JAFC

Novel Maillard Pigment, Furpenthiazinate, Having Furan and Cyclopentathiazine Rings Formed by Acid Hydrolysis of Protein in the Presence of Xylose or by Reaction between Cysteine and Furfural under Strongly Acidic Conditions Kyoko Noda,† Ruriko Masuzaki,† Yuka Terauchi,† Shinji Yamada,‡ and Masatsune Murata*,† Department of Nutrition and Food Science and ‡Department of Chemistry, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan

Downloaded via UNIV OF SOUTH DAKOTA on October 25, 2018 at 03:08:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

ABSTRACT: A novel Maillard pigment having partial structures of furan and cyclopentathiazine, named furpenthiazinate, was isolated and identified. Although this pigment was found in an acid hydrolysate of a Maillard reaction solution between soy protein and xylose, the same pigment was also formed by the Maillard reaction under strongly acidic conditions between soy protein and xylose and cysteine and furfural. The structure of its reduced form by NaBH4 was determined by MS, NMR, and Xray analysis and identified as 7-(2-furanyl)-2,3,4,4a,5,6-hexahydrocyclopenta[b][1,4]thiazin-4-ium-3-carboxylate, indicating that the chemical structure of furpenthiazinate is 7-(2-furanyl)-2,3,5,6-tetrahydrocyclopenta[b][1,4]thiazine-3-carboxylic acid. Furpenthiazinate showed an absorption maximum at 400 nm and strong yellow color under acidic and neutral conditions. The color contribution of furpenthiazinate was estimated to be more than 60% in a reaction solution prepared from cysteine and furfural. KEYWORDS: Maillard reaction, soy protein, furfural, cyclopentathiazine, cysteine, xylose, acid hydrolysis

■

INTRODUCTION The Maillard reaction between an amino group of amino acids, peptides, and proteins and a carbonyl group of reducing sugars and their degradation products causes nonenzymatic browning. As results of this reaction, various pigments and flavor compounds are formed, which significantly affect the quality of food. Although melanoidins are the major brown pigments formed by the Maillard reaction, identification of their structure is theoretically difficult because they are heterogeneous polymers and have no definite repeated units.1,2 On the other hand, it is possible to identify a low molecular weight pigment formed by the Maillard reaction when enough pure material is obtained. Thus far, several colored compounds have been isolated and identified from model reaction systems containing some kinds of amino acids and sugars or their degradation products. For example, Hofmann reported yellow pigments formed from proline and furfural3 or alanine and xylose.4 Hayase et al.5 and Shirahashi et al.6 reported blue and red pigments formed from glycine and xylose. Our group also reported some yellow pigments such as furpipate,7,8 dilysyldipyrrolones,9−112,4dihydroxy-2,5-dimethyl-3(2H)-thiophenone,12,13 pyrrolothiazolate,14,15 and pyrrolooxazolates.16 Although there was only a small amount of these pigments in foods,12,13 it is important to analyze these pigments. We must acknowledge that various kinds of compounds or chromophores contribute to the overall color cumulatively even though the individual contribution of a pigment to the total color is low. Dilysyldipyrrolones (Figure 1) mentioned above are formed by the Maillard reaction between L-lysine and D-xylose, and its pyrrolyl-methylidene-pyrrolone ring is composed from amino groups of two molecules of lysine and two molecules of © XXXX American Chemical Society

Figure 1. Chemical structures of dilysyldipyrrolones A, B, and C.

degradation products of pentose. Among these dilysyldipyrrolones, dipyrrolone B (DPL B) is formed by the reaction between ε-amino groups of two molecules of lysine residues and two molecules of degradation products of pentose, indicating that α-amino groups are not involved in the formation of DPL B. This suggests that DPL B is formed by a reaction between lysine residues of proteins and pentoses. Then we examined an acid hydrolysate of a reaction mixture of soy protein and xylose using HPLC equipped with diode array detection (DAD). Unfortunately, DPL B was not stable under the acid hydrolysis condition and could not be detected in the hydrolysate, but instead, a unique peak (furpenthiazinate) showing an absorption maximum at 400 nm was detected. In this study, we showed the isolation and identification of this novel pigment, named furpenthiazinate, having furan and cyclopentathiazine rings. Received: October 1, 2018 Revised: October 10, 2018 Accepted: October 12, 2018

A

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. NMR Data of Furpenthiazinatea 2 3 4a 5 or 6c 7 7a 8 2′ 3′ 4′ 5′

δC (ppm)

DEPT 135

δH (ppm)

26.5 57.5 182.9 29.8 157.7 119.1 173.6 150.2 121.6 114.8 149.7

− +

a 3.31, b 3.36 4.68

H, dd (J = 5.3, 13.7 Hz), H, dd (J = 3.0,7 13.7 Hz) H, t-like (J = 4.3 Hz)

−

3.25

2H, s

+ + +

7.35 6.79 7.92

connectivityb

H, d (J = 3.7 Hz) H, dd (J = 1.3, 3.7 Hz) H, d-like (J = 1.3 Hz)

H(3) H(2a), H(2b) H(3), H(5 or 6) H(5 or 6) H(2a), H(2b), H(5 or 6) H(2a), H(2b), H(3) H(5 or 6), H(3′), H(4′), H(5′) H(4′), H(5′) H(3′), H(5′) H(3′), H(4′)

a

Furpenthiazinate was dissolved in D2O. bThe connectivity was established by 1H−1HCOSY, HSQC, and HMBC experiments. Refer to C in Figure 6 for numbering. cNeither 5 nor 6 was not observed because of tautomerism.

■

preparative HPLC system under the following conditions: pump, L6000 (Hitachi, Tokyo); column, Mightysil RP-18 (20 mm i.d. × 250 mm, Kanto Kagaku); eluent, MeOH:0.1% HCOOH−water = 20:80 (v/v); flow rate, 9.99 mL/min; detector, L-4200 (Hitachi); detection wavelength, 400 nm. A peak at a retention time of about 25 min was collected and concentrated in vacuo, which was subjected to a second preparative HPLC system under the following conditions: column, TSK-gel G2500PW (25 mm i.d. × 300 mm, Tosoh, Tokyo); eluent, MeOH:water (30:70, v/v); flow rate, 6.0 mL/min; detection wavelength, 400 nm. A peak at a retention time of about 37 min was collected and concentrated in vacuo. A yellow powder of furpenthiazinate (ca. 7 mg, 0.04% yield from xylose (mol/mol)) was obtained. Isolation of Furenthiazinate from a Solution Prepared from Cysteine and Furfural under Strongly Acidic Conditions. A Maillard reaction solution (500 mL) containing 0.1% L-cysteine, 1.3% furfural, and 2 M HCl was refluxed for 2 h. The solution was cooled and applied to a column of DIAION HP20. After washing it with water until the pH of the eluate became about 5, furpenthiazinate was eluted with MeOH:water = 1:1 (v/v). Fractions containing the pigment were collected, concentrated in vacuo, and applied to a column of Chromatorex ODS, which was successively developed with aqueous MeOH (MeOH:water = 1:9, 2:8, and 5:5 v/v). Each fraction was analyzed with DAD-HPLC. Fractions containing furpenthiazinate (MeOH:water = 2:8) were concentrated in vacuo. A dark green powder of furpenthiazinate (ca. 60 mg, 0.4% yield from furfural (mol/ mol)) was obtained. Isolation of Reduced Furpenthiazinate. Furpenthiazinate (6 mg) was dissolved in distilled water (5 mL), to which NaBH4 (1 mg) was added. The solution was left for 1 h at room temperature before being applied to a column of DIAION HP20. After washing it with water until the pH of the eluate became about 7, reduced furpenthiazinate was eluted with MeOH. The eluate was concentrated in vacuo and then subjected to a preparative HPLC system under the following conditions: column, YMC Pack R&D ODS-A (20 mm i.d. × 250 mm, YMC, Kyoto); eluent, MeOH:0.1% HCOOH/water (25:75, v/v); flow rate, 9.99 mL/min; detection wavelength, 280 nm. A peak at a retention time of about 54 min was collected and concentrated in vacuo. A white powder of reduced furpenthiazinate (ca. 1 mg, 17% yield from furpenthiazinate (mol/mol)) was obtained. The powder was dissolved in a small amount of MeOH and placed at room temperature. A colorless prism crystal of reduced furpenthiazinate was obtained. Instrumental Analyses. Spectroscopic measurements were done by using the following instruments: spectrophotometer (Multispec1500, Shimadzu, Kyoto), spectrofluorophotometer (RF-540, Shimadzu), and NMR (Bruker Avance III 600, Bruker Biospin, Karlsruhe, Germany). LC/MS Analyses. The molecular weights of isolated furpenthiazinate and its reduced form were analyzed using a mass spectrometer (MS, Triple TOF 4600, AB Sciex, Foster City, CA) coupled with

MATERIALS AND METHODS

Chemicals. The following compounds were obtained commercially: D-xylose, L-asparagine, L-aspartic acid, L-cysteine, L-tryptophan, L -histidine, trifluoroacetic acid, 90% formic acid, and NaBH 4 (FUJIFILM Wako Pure Chemical, Osaka, Japan), HCl and MeOH (Kanto Chemical, Tokyo, Japan), furfural (Tokyo Chemical Industry, Tokyo), and D2O and dimethyl sulfoxide-d6 (Thermo Fisher Scientific, Waltham, Massachusetts). Soy protein was obtained from Fuji Oil (Izumisano, Japan). Acid Hydrolysis of a Solution of the Maillard Reaction between Soy Protein and Xylose. A solution of soy protein (7%) and xylose (0.5%) in distilled water (3 mL) was put into a test tube with a cap before being heated for 6 h at 95 °C. After cooling, 12 M HCl (3 mL) was added to the solution, and then the solution was heated again for 24 h at 110 °C. This reaction solution was cooled before being applied to DAD-HPLC analysis. Preparation of Model Solutions of the Maillard Reaction. An amino acid (1.5% L-asparagine, 0.9% L-aspartic acid, 0.1% Lcysteine, 0.1% L-tryptophan, or 0.2% L-histidine) and a sugar-related compound (2% D-xylose or 1.3% furfural) dissolved in water or 2 M HCl (6 mL) was put into a test tube with a cap before being heated for 1 h at 110 °C. After cooling, each reaction solution was subjected to DAD-HPLC analysis. HPLC Analyses. Each reaction solution was analyzed using a reversed-phase DAD-HPLC system under the following conditions: system, Agilent 1100 series (Palo Alto, CA); column, COSMOSIL 5C18-MSII (4.6 mm i. d. × 250 mm, Nacalai Tesque, Kyoto, Japan); eluent I for detection of DPL B, solution A (0.1% trifluoroacetic acid−water) and solution B (MeOH:0.1% trifluoroacetic acid−water = 70:30, v/v); eluent II for detection of furpenthiazinate, solution A (0.1% HCOOH−water, v/v) and solution B (MeOH:0.1% HCOOH−water = 70:30, v/v), 0−100% B (v/v) for 0−30 min with a linear gradient; flow rate, 1.0 mL/min; column temp., 50 °C; detection, 220−500 nm. DPL B and furpenthiazinate were detected at retention times of about 14 and 13 min, respectively, under these conditions. Isolation of Furpenthiazinate from a Solution Prepared from Soy Protein and Xylose under Strongly Acidic Conditions. A reaction solution (600 mL) containing 7% soy protein, 2% xylose, and 2 M HCl was refluxed for 30 h. The solution was cooled before being applied to a column of DIAION HP20 (Mitsubishi Chemical, Tokyo). After washing it with water until the pH of the eluate became about 5, furpenthiazinate was eluted with MeOH:water = 1:1 (v/v). Fractions containing furpenthiazinate were collected, concentrated in vacuo, and applied to a column of Chromatorex ODS (100−200 mesh; Fuji Sylysia Chemical, Kasugai, Japan), which was successively developed with aqueous MeOH (MeOH:water = 1:9, 2:8, 3:7, 4:6, and 5:5; v/v). Each fraction was checked for the existence of furpenthiazinate with DAD-HPLC under the condition mentioned above. Fractions containing the pigment (MeOH:water = 2:8) were concentrated in vacuo and subjected to a B

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry HPLC. The ionization mode was set to electrospray ionization (ESI +). HPLC conditions were as follows: system, Prominence (Shimadzu); column, Inertsil ODS-3 (2.1 mm i.d. × 150 mm, GL Science, Tokyo); eluent, solution A (0.1% HCOOH−water, v/v) and solution B (MeOH:0.1% HCOOH−water = 70:30, v/v), 0−100% B (v/v) for 0−30 min with a linear gradient; flow rate, 0.2 mL/min; column temp., 50 °C. Furpenthiazinate and its reduced form were detected at retention times of about 15 and 20 min, respectively, under this condition. X-ray Analysis of Reduced Furpenthiazinate. A colorless prism crystal of C12H13NO3S having approximate dimensions of 0.200 × 0.100 × 0.500 mm was mounted on a glass fiber. All measurements were made on a Rigaku R-AXIS RAPID diffractometer (Rigaku, Tokyo) using graphite-monochromated Cu Kα radiation. Indexing was performed from three oscillations that were exposed for 60 s, the crystal-to-detector distance being 127.40 mm. The structure was solved by direct method and refined by full-matrix least-squares on F2 using SHELXL-2014. Physicochemical Properties of Furpenthiazinate. UV λmax nm (ε): 400 (13 000) in 0.01 M HCl; 400 (13 000) in water; 340 (13 000) in 0.01 M NaOH. Fluorescence: λem nm 400, λex nm 480. MS(m/z): 250.0532 (M + H)+ calcd for C12H12NO3S, 250.0535. 1H and 13C NMR data are summarized in Table 1. Physicochemical Properties of Reduced Furpenthiazinate. Mp: 179 °C (dec). UV λmax nm: 300 in 0.01 M HCl, in water, and in 0.01 M NaOH. MS (m/z): 252.0705 (M + H)+ calcd for C12H14NO3S, 252.0691. 1H and 13C NMR data are summarized in Table 2.

solution was calculated as [furpenthiazinate concentration (mg/ mL)]/[its detection limit (mg/mL)]. The contribution of furpenthiazinate to total color (%) of the reaction solution was calculated by [color activity of furpenthiazinate in the reaction solution]/[detection limit of the reaction solution (dilution ratio)] × 100.

■

RESULTS AND DISCUSSION Analysis of an Acid Hydrolysate of a Solution for the Maillard Reaction between Soy Protein and Xylose. As mentioned in the Introduction, first, we examined the presence of DPL B in a solution of the Maillard reaction between soy protein and xylose using acid hydrolysis. Unfortunately we could not detect DPL B in the hydrolysate, because the compound was completely decomposed under the condition (data not shown). Instead, we detected unexpectedly a unique peak (furpenthiazinate) in the hydrolysate on DAD-HPLC (Figure 2). This peak had a maximum absorption wavelength

Table 2. NMR Data of Reduced Furpenthiazinatea δC (ppm)

DEPT 135

δH (ppm)

2

27.2

−

a 2.85, b 3.20

3

58.4

+

3.54

4a

63.6

+

4.19

5

27.3

−

6

29.7

−

a 1.83, b 2.37 a 2.55, b 2.79

7

128.2

7a

121.4

8 2′

166.9 149.7

3′ 4′

111.7 109.9

+ +

6.61 6.60

5′

142.9

+

7.76

connectivityb H, dd (J = 11.8, 13.5 Hz), H, dd (J = 2.9, 13.5 Hz) H, dd (J = 2.9, 11.8 Hz) H, t-like (J = 8.3 Hz) H, m, H, m H, m, H, m

H, d (J = 3.4 Hz) H, dd-like (J = 1.6, 3.4 Hz) H, d-like (J = 1.6 Hz)

H(3)

H(2a), H(2b) H(5a), H(5b), H(6b) H(4a), H(6a), H(6b) H(5a), H(5b)

Figure 2. Typical chromatograms of reversed-phase HPLC of acid hydrolysates of reaction solutions prepared from soy protein and xylose (top: SP+Xyl) and soy protein (bottom: SP). Solution (3 mL) containing 7% soy protein (bottom) or 7% soy protein and 0.5% Dxylose (top) was heated for 6 h at 95 °C. After cooling, 12 M HCl (3 mL) was added to the solution, which was heated again for 24 h at 110 °C. Each reaction solution was analyzed with HPLC equipped with diode array detection.

H(5b), H(6a), H(6b) H(2b), H(4a), H(5b), H(6a), H(6b) H(3) H(3′), H(4spm′), H(5′) H(4′), H(5′) H(3′), H(5′)

at 400 nm and did not appear in a reaction solution without xylose. These results suggest that this peak was a Maillard pigment formed from protein and xylose. Therefore, we tried to isolate and identify it, which was later named furpenthiazinate. Isolation and Structural Analyses of Furpenthiazinate. Although we found a peak of a pigment in an acid hydrolysate of a Maillard reaction solution between soy protein and xylose, the same peak was also detected in a heated solution containing soy protein and xylose under acidic condition. This result suggested that the furpenthiazinate was formed during acid hydrolysis by the reaction of amino acids and xylose. Then we used a heated solution containing soy protein, xylose, and HCl as a starting material for isolation after ascertaining the formation of the same pigment under this condition using DAD-HPLC. This compound was adsorbed onto a synthetic adsorbent and ODS, although not being extracted with organic solvents. We used several chromato-

H(3′), H(4′)

a

Reduced furpenthiazinate was dissolved in dimethyl sulfoxide-d6. b The connectivity was established by 1H−1HCOSY, HSQC, and HMBC experiments. Refer to A in Figure 6 for numbering. Evaluation of Color Activity and Color Contribution of Furpenthiazinate. A reaction solution containing 0.1% L-cysteine and 1.3% furfural and 2 M HCl was heated for 1 h at 110 °C. The color activity of furpenthiazinate and its color contribution to the solution were estimated with the color dilution method.17 An aqueous solution of furpenthiazinate or the reaction solution was successively diluted with distilled water, and each aliquot (200 μL) was placed into a 96-well microplate. Each detection limit of furpenthiazinate and the reaction solution was visually estimated by the triangle test using 11 panelists. The color activity (unit) of furpenthiazinate in the reaction C

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

group). These results suggest a partial structure of CH2−CH− COOH, which reminded us of an amino acid. On the other hand, the carbon at δC 150.2 ppm was quaternary and those at δC 114.8, 121.6, and 149.7 ppm were methine groups. These methine protons (δH 7.35, 6.79, and 7.92 ppm) connecting, respectively, to the carbons at δC 121.6, 114.8, and 149.7 ppm were coupled with each other. Further the HMBC data showed that these protons were correlated with the quaternary carbon at δC 150.2 ppm. These results show that these four carbons form a furyl group. In this stage we could not specify a structure satisfying all of the data observed by instrumental analyses. However, the NMR data showed that furpenthiazinate had partial structures of an amino acid residue and a furyl group derived from xylose, which induced us to examine a more efficient preparation method for furpenthiazinate. Analyses of Model Maillard Reaction Solutions Prepared from Amino Acids and Sugar-Related Compounds. The NMR data suggested that fupenthiazinate was formed from an amino acid having a methylene group in the βcarbon position. Therefore, each solution containing xylose or furfural and an amino acid having a methylene group in the βcarbon position (asparagine, aspartic acid, cysteine, tryptophan, or histidine) was heated in the presence or absence of HCl and then examined whether fupenthiazinate was formed or not. As a result, the pigment was formed only in the reaction solution containing L-cysteine and D-xylose or furfural in the presence of HCl (Figure 4). These results suggested that this

graphic procedures such as a synthetic adsorbent, ODS, reversed-phase HPLC, and size-exclusion HPLC. As a result, a yellow powder showing a single peak in HPLC was obtained. Its melting or decomposition point could not be determined as it was not melted, and the decomposition temperature was not clearly observed due to its color. Figure 3 shows the UV−vis spectra of this pigment. This compound showed an absorption maximum at 400 nm in

Figure 3. UV−vis spectra of furpenthiazinate. Furpenthiazinate was dissolved in 0.01 M HCl, water, and 0.01 M NaOH at a concentration of 10 μg/mL.

water and under acidic condition and at 340 nm under alkaline condition. The yellow color of the solution disappeared in the alkaline region. This result suggested that this compound had an amine or imine derived from protein in its chromophore. MS analysis showed a parent peak at m/z 250.0538, but this molecular weight could not be explained by the combination of carbon, hydrogen, nitrogen, and oxygen and showed that its molecular formula was C12H12NO3S. The NMR data are summarized in Table 1. The 1H NMR data showed a singlet methylene peak (δH 3.25 ppm), three double doublet methine peaks (δH 3.31, 3.36, and 6.79 ppm), a triplet-like methine peak (δH 4.68 ppm), a doublet methine peak (δH 7.35 ppm), and a doublet-like methine peak (δH 7.92 ppm). The 1H−1H correlation spectroscopy (COSY) data showed that two double doublet methine peaks (δH 3.31 and 3.36 ppm) and a triplet-like methine peak (δH 4.68 ppm) were coupled with each other. A doublet methine peak (δH 7.35 ppm), a doublet-like methine peak (δH 7.92 ppm), and a double doublet methine peak (δH 6.79 ppm) were also coupled with each other. The 13C NMR data showed 11 kinds of carbons, among which the C−H bonds of six carbons were assigned by a heteronuclear single-quantum correlation (HSQC) analysis. The HSQC data showed that the other five carbons at δC 119.1, 150.2, 157.7 (olefinic carbons), 173.6 (a carboxy group), and 182.9 ppm (a carbonyl group) were quaternary. Among the six carbon-bearing protons, the carbon at δC 57.5 ppm was a methine group to which a proton at δH 4.68 ppm was bound and the carbon at δC 26.5 ppm was a methylene group to which two protons at δH 3.31 and 3.36 ppm were bound. The heteronuclear multiple bond correlation (HMBC) analysis showed that these protons at δH 3.31, 3.36, and 4.68 ppm were correlated with a carbon at δC 173.6 ppm (a carboxy

Figure 4. Typical chromatograms of reversed-phase HPLC of isolated furpenthiazinate from a reaction solution between soy protein and xylose (top) and a reaction solution between cysteine and furfural (bottom; Cys+furfural). Isolated furpenthiazinate (top) and a reaction solution prepared from L-cysteine and furfural in 2 M HCl (bottom) were analyzed with HPLC equipped with diode array detection.

compound was formed from the reaction between cysteine and furfural, respectively, derived from protein and xylose under strongly acidic conditions. We could not detect the pigment in any reaction solutions without addition of HCl. On the basis of these results, we purified furpenthiazinate again from the model Maillard reaction solution between Lcysteine and furfural using a similar procedure as mentioned already. As a result, we obtained 60 mg of a pure sample. The NMR and MS data of this sample coincided with those of furpenthiazinate described above. D

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 5. ESI-MS and MS/MS spectra of furpenthiazinate (A and C) and its reduced form (B and D).

Structural Analysis of Furpenthiazinate and Its Reduced Form. The MS data of furpenthiazinate showed its molecular weight and molecular formula to be 249 and C12H11NO3S, respectively, which indicated the existence of a sulfur derived of cysteine. Considering its molecular formula and the fact that furpenthiazinate had a furan ring and a carboxyl group, the carbon at δC 182.9 ppm we first considered to be a carbonyl group should be that of an imine group. Nevertheless, the molecular formula showing the existence of 12 carbons did not agree with the NMR data showing the existence of 11 carbons. These results suggested that furpenthiazinate had a hardly observable carbon showing tautomerism around an imine group. Therefore, furpenthiazinate was reduced by NaBH4, and its reduced form was analyzed after isolation. The MS data of reduced furpenthiazinate showed that its molecular weight and molecular formula were 251 and C12H13NO3S, respectively. This molecular formula showed two more hydrogens than that of furpenthiazinate. Figure 5 shows the MS and MS/MS spectra of furpenthiazinate (A and C) and its reduced form (B and D). The fragmentation patterns of the parent peaks are depicted according to the determined structures described later. The NMR data of reduced furpenthiazinate are summarized in Table 2. The 13C NMR data clearly showed 12 kinds of carbons corresponding to the results of MS, among which 10 carbons (δC 27.2, either 27.3 or 29.7, 58.4, 109.9, 111.7, 121.4, 128.2, 142.9, 149.7, and 166.9 ppm) were also detected in furpenthiazinate. The HSQC and HMBC data showed that an imine group of furpenthiazinate (δC 182.9 ppm) was reduced and detected as a methine group (δC 63.6 ppm) bearing a proton at δH 4.19 ppm and that a newly detected carbon (δC

27.3 or 29.7 ppm) was a methylene group bearing two protons at δH 1.83 and 2.37 ppm or δH 2.55 and 2.79 ppm, respectively. It seemed that either of the two carbons was observed at δC 29.8 ppm in furpenthiazinate and that the other carbon was not observed by tautomerism. The 1H NMR data showed 11 kinds of peaks, among which 6 peaks (δH 2.85, 3.20, 3.54, 6.60, 6.61, and 7.76 ppm) were also detected in furpenthiazinate. The protons of a methylene group in furpenthiazinate (δH 3.25 ppm) were not detected in its reduced form, while two kinds of methylene protons showing geminal coupling (δH 1.83 and 2.37 ppm and δH 2.55 and 2.79 ppm) appeared in reduced one. It seemed that either of the two methylene groups was observed at δH 3.25 ppm in furpenthiazinate and that the other methylene group was not observed by tautomerism. A newly detected triplet-like methine peak (δH 4.19 ppm) was formed by reduction of an imine group in furpenthiazinate. The 1H−1H COSY data showed that this newly detected triplet-like proton (δH 4.19 ppm) and two multiplet protons (δH 1.83 and 2.37 ppm) connecting to a carbon at δC 27.3 ppm were coupled with each other, and four multiplet protons (δH 1.83 and 2.37 ppm and δH 2.55 and 2.79 ppm) were also coupled with each other. These results suggested that three carbons (δC 27.3, 29.7, and 63.6 ppm) formed a partial structure of CH2−CH2−CH(NH) as shown at positions 6, 5, and 4a in A or B of Figure 6. The 1H−1H COSY data further showed that two protons (δH 2.85 and 3.20 ppm) of a methylene group (δC 27.2 ppm) and a proton (δH 3.54 ppm) of a methine group (δC 58.4 ppm) were coupled with each other. These data indicated a partial structure of S−CH2−CH(N)−COOH derived from cysteine. The NMR data also showed the existence of a furyl group (δC 149.7, 111.7, 109.9, and 142.9 ppm). The protons and carbons E

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Cambridge CB2 1EZ, UK [Fax: +44 1223 336 033, E-mail: [email protected]]. X-ray analysis of reduced furpenthiazinate showed its chemical structure to be 7-(2-furanyl)2,3,4,4a,5,6-hexahydrocyclopenta[b][1,4]thiazin-4-ium-3-carboxylate (Figure 7A). This structure was a zwitterion form of the structure shown in Figure 6A. Figure 7B shows the unit cell of the crystal of reduced furpenthiazinate. This unit cell was composed of two pairs of its enantiomers that are (3R,4aS) and (3S,4aR). In addition to this result, the fact that both furpenthiazinate and its reduced form did not show optical activity (data not shown), indicating that racemization occurred at the chiral carbon of L-cysteine under strongly acidic condition during the formation of furpenthiazinate, although we used L-cysteine as a start material. From the structure of reduced furpenthiazinate, the structure of furpenthiazinate was identified as 7-(2-furanyl)-2,3,5,6tetrahydrocyclopenta[b][1,4]thiazine-3-carboxylic acid (Figure 6C). This pigment is a novel cyclopentathiazine derivative having a furyl and a carboxyl group and then named furpenthiazinate. This structure suggests that furpenthiazinate has two kinds of tautomers or yields a dynamic equilibrium in an aqueous solution (Figure 8), indicating why the carbon and protons at position 5 in Figure 6C were not observed in NMR measurement.

Figure 6. Two possible structures of reduced furpenthiazinate (A and B) and its corresponding furpenhthiazinte (C and D), and their NMR data.

corresponding to these partial structures were also apparent in furpenthiazinate. Considering the results of HMBC of reduced furpenthiazinate, we combined these partial structures and two olefinic and quaternary carbons (δC 121.4 and 128.2 ppm in reduced furpenthiazinate). Figure 6 shows two structures of reduced furpenthiazinate that satisfy all of the data described thus far. Although structure B is highly strained and structure A seems to be more correct, structure B could not be denied completely. As a crystal of reduced furpenthiazinate could be obtained from methanol, the crystal was applied to X-ray analysis. X-ray crystal data were as follows: C12H13NO3S, Mr = 251.30, monoclinic, space group P21/c (no. 14), μ(Cu Kα) = 26.168 cm−1, T = 123 K, a = 10.7001(4) Å, b = 8.9053(3) Å, c = 11.6952(4) Å, β = 101.499(2)°, V = 1092.03(6) Å3, Z = 4, Dc = 1.528 g cm−3, and F(000) = 528. A total of 11 841 reflections were collected, with 1995 being unique (Rint = 0.0443). R1, R, and wR2 were, respectively, 0.0370[I > 2σ(I)], 0.0433 (all data), and 0.1051 (all data). Further details of the crystal structure investigation are deposited in the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1869962. Copies of the data can be obtained free of charge by an application to CCDC at 12 Union Road,

Figure 8. Tautomerism of furpenthiazinate.

Figure 9 shows dissociation forms of furpenthiazinate in water under acidic, neutral, and alkaline conditions. Because

Figure 9. Dissociation forms of furpenthiazinate in water under acidic, neutral, and alkaline conditions.

Figure 7. Structure of reduced furpenthiazinate determined by X-ray analysis (A) and the unit cell in its crystal (B). F

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 10. Formation scheme of furpenthiazinate. Furpenthiazinate was formed by an acid hydrolysis of soy protein in the presence of xylose or by a reaction between furfural and cysteine under strongly acidic conditions.

furfural under strongly acidic conditions. We will further examine the existence in foods, more detailed conditions for its formation, and the formation mechanism using various kinds of proteins and labeled precursors.

this compound forms immonium cation under neutral and acidic conditions with conjugation of the double bond, its solution showed a strong yellow color. On the other hand, under alkaline condition, furpenthiazinate does not form immonium cation and the yellow color of the aqueous disappeared. Although some cyclopentathiazine derivatives have been synthesized,18−20 furpenthiazinate was for the first time isolated and identified as a colored Maillard reaction product from either a mixture of proteins and furfural or that of cysteine and furfural. This compound was formed only under strongly acidic conditions. It might be formed during food processing under strongly acidic conditions. We aim to further examine its existence in hydrolysate products of foods or solutions of amino acid analysis of foods. Our results showed that thiazine and furan rings of this compound were derived from cysteine or cysteine residues of proteins and furfural or xylose, respectively (Figure 10). However, it is not clear from what four carbons of a cyclopentene is formed, although we speculate that these carbons are derived from decomposed furfural. To confirm this, further experiments in which 13C-labeled furfural and pentose are used will be needed. Color Contribution of Furpenthiazinate. As a powder of furpenthiazinate was yellow or dark green and its aqueous solution was yellow, its color contribution to a model Maillard reaction solution was estimated. The detection limit of the solution of furpenthiazinate visually estimated was about 5.7 μg/mL. A reaction solution prepared from cysteine and furfural contained about 0.72 mg/mL of furpenthiazinate, and the detection limit of this solution was 190-fold dilution. These results showed that furpenthiazinate contributed about 67% of the total color of the reaction solution (0.72 [mg/mL] /5.7 × 10−3 [mg/mL]/190 × 100 = 67.48), meaning that the color of the reaction solution mainly derived from this compound. This result was supported by the fact that furpenthiazinate was the major peak with detection at absorbance at 400 nm of this reaction solution and that any other distinct peaks were not detected (Figure 4). In conclusion, a novel cyclopentathiazine derivative was isolated and identified as a Maillard pigment prepared from either solution of soy protein and xylose or that of cysteine and

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +81-3-5978-5755; E-mail: [email protected]. jp. ORCID

Shinji Yamada: 0000-0002-5945-5996 Masatsune Murata: 0000-0002-5422-9535 Funding

This study was supported by a Grant-in-Aid for Scientific Research (B) [grant nos. 26282016 and 17H01958] from the Japan Society for the Promotion of Science. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Hodge, J. E. Chemistry of browning reactions in model systems. J. Agric. Food Chem. 1953, 1, 928−943. (2) Wang, H. Y.; Qian, H.; Yao, W. R. Melanoidins produced by the Maillard reaction: Structure and biological activity. Food Chem. 2011, 128, 573−584. (3) Hofmann, T. Characterization of the chemical structure of novel colored Maillard reaction products from furan-2-carboxaldehyde and amino acids. J. Agric. Food Chem. 1998, 46, 932−940. (4) Hofmann, T. Identification of novel colored compounds containing pyrrole and pyrrolinone structures formed by Maillard reactions of pentoses and primary amino acids. J. Agric. Food Chem. 1998, 46, 3902−3911. (5) Hayase, F.; Takahashi, Y.; Tominaga, S.; Miura, M.; Gomyo, T.; Kato, H. Identification of blue pigment formed in a D-xylose-glycine reaction system. Biosci. Biotechnol. Biochem. 1999, 63, 1512−1514. (6) Shirahashi, Y.; Watanabe, H.; Hayase, F. Identification of red pigment formed in D-xylose-glycine reaction system. Biosci. Biotechnol. Biochem. 2009, 73, 2287−2292. (7) Murata, M.; Totsuka, H.; Ono, H. Browning of furfural and amino acids, and a novel yellow compound, furpipate, formed from lysine and furfural. Biosci. Biotechnol. Biochem. 2007, 71, 1717−1723. (8) Totsuka, H.; Tokuzen, K.; Ono, H.; Murata, M. A novel yellow compound and furpipate derivatives formed from furfural or 5G

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry hydroxymethylfurfural in the presence of lysine. Food Sci. Technol. Res. 2009, 15, 45−50. (9) Sakamoto, J.; Takenaka, M.; Ono, H.; Murata, M. Novel yellow compounds, dilysildipyrrolones A and B, formed from xylose and lysine by the Maillard reaction. Biosci. Biotechnol. Biochem. 2009, 73, 2065−2069. (10) Nomi, Y.; Sakamoto, J.; Takenaka, M.; Ono, H.; Murata, M. Conditions for the formation of dilysil-dipyrrolones A and B, and novel yellow dipyrrolone derivatives formed from xylose and amino acids in the presence of lysine. Biosci. Biotechnol. Biochem. 2011, 75, 221−226. (11) Nomi, Y.; Masuzaki, M.; Terasawa, R.; Takenaka, M.; Ono, H.; Otsuka, Y.; Murata, M. Formation mechanism and characterization of dilysyl-dipyrrolons, the Maillard-type yellow pigments. Food Funct. 2013, 4, 1067−1075. (12) Satoh, M.; Nomi, Y.; Yamada, S.; Takenaka, M.; Ono, H.; Murata, M. Identification of 2,4-dihydroxy-2,5-dimethyl-3(2H)thiophenone as a low molecular weight yellow pigment in soy sauce. Biosci. Biotechnol. Biochem. 2011, 75, 1240−1244. (13) Furusawa, R.; Goto, C.; Satoh, M.; Nomi, Y.; Murata, M. Formation and distribution of 2,4-dihydroxy-2,5-dimethyl-3(2H)thiophenone, a pigment, an aroma and a biological active compound formed by the Maillard reaction, in foods and beverages. Food Funct. 2013, 4, 1076−1081. (14) Noda, K.; Yamada, S.; Murata, M. Isolation, identification, and formation conditions of a novel Maillard yellowish pigment, pyrrolothiazolate. Biosci. Biotechnol. Biochem. 2015, 79, 1350−1355. (15) Noda, K.; Terasawa, N.; Murata, M. Formation scheme and antioxidant activity of a novel Maillard pigment, pyrrolothiazolate, formed from cysteine and glucose. Food Funct. 2016, 7, 2551−2556. (16) Noda, K.; Murata, M. Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine. Biosci. Biotechnol. Biochem. 2017, 81, 343−349. (17) Hofmann, T. Characterization of the most intense coloured compounds from Maillard reactions of pentoses by application of colour dilution analysis. Carbohydr. Res. 1998, 313, 203−213. (18) DeStevens, G.; Halamandaris, A.; Dorfman, L. Investigations in heterocycles. VI. 1,4-thiazin-3-ones and related compounds. J. Am. Chem. Soc. 1958, 80, 5198−5200. (19) Anderson, A. G., Jr; Labar, R. A.; Haddock, R. D. 2,3,4,5,6,7Hexahydrocyclopenta-1,4-thiazine and the 8,9-dihydro-, 4-acetyl and 1-oxo - 4-acetyl derivatives. Org. Prep. Proced. Int. 1989, 21, 653−655. (20) Brun, O.; Agramunt, J.; Raich, L.; Rovira, C.; Pedroso, E.; Grandas, A. Selective derivatization of N-Terminal Cysteines Using Cyclopentationes. Org. Lett. 2016, 18, 4836−4839.

H

DOI: 10.1021/acs.jafc.8b05302 J. Agric. Food Chem. XXXX, XXX, XXX−XXX