Human Taste and Umami Receptor Responses to Chemosensorica

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Human Taste and Umami Receptor Responses to Chemosensorica Generated by Maillard-type N2‑Alkyl- and N2‑Arylthiomethylation of Guanosine 5′-Monophosphates Barbara Suess,† Anne Brockhoff,‡ Andreas Degenhardt,† Sylvia Billmayer,† Wolfgang Meyerhof,‡ and Thomas Hofmann*,† †

Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Straße 34, D-85354 Freising-Weihenstephan, Germany ‡ Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany ABSTRACT: Structural modification of the exocyclic amino function of guanosine 5′-monophosphate (5′-GMP) by Maillardtype reactions with reducing carbohydrates was recently found to increase the umami-enhancing activity of the nucleotide upon S-N2-1-carboxyalkylation and S-N2-(1-alkylamino)carbonylalkylation, respectively. Since the presence of sulfur atoms in synthetic N2-alkylated nucleotides was reported to be beneficial for sensory activity, a versatile Maillard-type modification of 5′-GMP upon reaction with glycine’s Strecker aldehyde formaldehyde and organic thiols was performed in the present study. A series of N2(alkylthiomethyl)guanosine and N2-(arylthiomethyl)guanosine 5′-monophosphates was generated and the compounds were evaluated to what extent they enhance the umami response to monosodium L-glutamate in vivo by a paired-choice comparison test using trained human volunteers and in vitro by means of cell-based umami taste receptor assay. Associated with a high umami-enhancing activity (β-value 5.1), N2-(propylthiomethyl)guanosine 5′-monophosphate could be generated when 5′-GMP reacted with glucose, glycine, and the onion-derived odorant 1-propanethiol, thus opening a valuable avenue to produce highpotency umami-enhancing chemosensorica from food-derived natural products by kitchen-type chemistry. KEYWORDS: guanosine 5′-monophosphate, taste enhancer, umami, chemosensorica, Maillard reaction

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umami taste of L-glutamate (β-value ≤0.1).6 These findings suggest an electron-withdrawing group at position 6 of the purine ring, as well as the lack of a hydroxyl group at position 2 of the carbohydrate moiety, plays a crucial role in nucleotide/ taste receptor interaction. To gain more insight into the umami taste enhancement mechanism, a series of studies focused on the synthetic structural modification of nucleotide scaffolds. For example, the transformation of nucleotide 3 into 2-mercaptoinosine derivatives led to highly potent umami taste enhancers, such as 2-methylthioinosine 5′-monophosphate (4) with a β-value of 8.2 (Figure 1).6−8 Whereas replacement of the sulfur atom with oxygen (5) decreased the β-value of nucleotide 5 to 3.5,6 substitution of the mercapto group with a furfurylthio ether in nucleotide 6 and an allylthio ether moiety in 7 revealed strongly improved chemosensorica with high β-values of 17.3 (6) and 9.2 (7), respectively.7,8 Interestingly, substitution of a methylene group by a sulfur atom in synthetic N2-alkylated 5′-GMP derivatives also led to more potent umami enhancers,9 whereas further oxidation of the sulfur atom to the corresponding sulfoxide drastically diminished the sensory activity.10

INTRODUCTION Originating from the Japanese word “umai” (savory, delicious), the umami taste was reported for the first time by Ikeda in 1908 to be imparted by L-glutamic acid, 1 (Figure 1), identified in seaweed.1 Despite these early findings, the umami taste was not accepted as the fifth basic taste quality until the discovery of the T1R1/T1R3 umami taste receptor almost 100 years later.2,3 Intriguingly, the well-known taste synergism between monosodium L-glutamate (MSG) and ribonucleotides such as, for example, guanosine 5′-monosphosphate (2, 5′-GMP) and inosine 5′-monosphosphate (3, IMP), could be validated by demonstrating the enhanced response of the heterodimeric umami receptor to monosodium L-glutamate in the presence of small amounts of 2 or 3.4 The purine nucleotides were found to operate as allosteric umami taste enhancers by stabilizing the closed form of the venus fly trap binding site in the receptor’s T1R1 subunit when activated by L-glutamate.5 For reliable sensory evaluation of the potency of umami taste modulators, the so-called β-value was introduced in 1971.6 Binary mixtures containing constant levels of MSG (1) and increasing concentrations of IMP (3) served as references to determine the β-values, representing the potency of a test compound to enhance the umami taste of MSG solutions in relation to 3 as the reference.6 For 5′-GMP (2), a β-value of 2.3 was determined, based on the observation that nucleotide 2 was 2.3-fold more potent than nucleotide 3 to enhance the umami taste of amino acid 1. In contrast, adenosine 5′-monophosphate and deoxyribose nucleotides were found to hardly modify the © XXXX American Chemical Society

Received: September 28, 2014 Revised: November 5, 2014 Accepted: November 6, 2014

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dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of umami-tasting and umami-modulating molecules: monosodium L-glutamate (1), guanosine 5′-monophosphate (2), inosine 5′-monophosphate (3), 2-methylthioinosine 5′-monophosphate (4), 2-methoxyinosine 5′-monophosphate (5), 2-allylthioinosine 5′monophosphate (6), 2-furfurylthioinosine 5′-monophosphate (7), N2-(3-methylthiopropyl)guanosine 5′-monophosphate (8), N2-lactoylguanosine 5′-monophosphate (9), (S)-N2-(1-carboxyethyl)guanosine 5′-monophosphate (10), (S)-N2-{[1-(N-butylamino)carbonyl]ethyl}guanosine 5′monophosphate (11), and (S)-N2-{[1-(N-isobutylamino)carbonyl]ethyl}guanosine 5′-monophosphate (12).

carboxyethyl)guanosine 5′-phosphate (10) was isolated from yeast extracts.11,12 Accompanied by its corresponding (R)configured isomer, the latter nucleotide has been reported to be generated by Maillard-type reactions of nucleotide 2 with dihydroxyacetone, glyceraldehyde, and hexoses, respectively.12 Intriguingly, only the (S)-configured nucleotide 10 showed a high β-value of 7.0 while the corresponding (R)-isomer showed only marginal sensory activity,12 again demonstrating that small structural changes impact on the umami taste-enhancing activity of this molecule. To further widen the portfolio of

Despite these achievements in the synthesis of umami tasteenhancing nucleotides, the growing aversion of alienated consumers toward nonnatural chemicals added to foods is creating an increasing request for flavor molecules of biological origin or made by “greener” or “kitchen-type” chemistry.4 This inspired initiatives targeting the discovery of previously unknown, naturally occurring chemosensorica in processed food. Among those sharing a guanosine 5′-monophosphate scaffold, N2-lactoylguanosine 5′-monophosphate, 9 (Figure 1), has been identified in fermented tuna fish, while (S)-N2-(1B

dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. N2-Alkyl- and N2-arylthiomethylation of guanosine 5′-monophosphates by Maillard-type reaction of guanosine 5′-monophosphate (2), formaldehyde, and organic thiols.

Figure 3. Chemical structures of N2-(alkylthiomethyl)guanosine 5′-monophosphates 13−20 and N2-(arylthiomethyl)guanosine 5′-monophosphates 21−25.

Maillard-type modified derivatives of nucleotide 2, the N2-(1carboxyethyl)guanosine 5′-phosphate scaffold was further altered by amidation of the side chain’s carboxy group, thus leading to butylated (11) and isobutylated ribonucleotides (12) with an increased β-value of 7.6.13,14 These results were verified

both by human psychophysical as well as cell-based T1R1/ T1R3 receptor assays.14 Inspired by the reported beneficial effect of a sulfur atom in N2-alkylated ribonucleotides,9 the objective of the present study was to prepare sulfur-containing guanosine 5′-monophosphates C

dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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then increasing the content of eluent B to 45% within 8 min, to 80% in 20 min, and finally to 100% in 1 min. Elution with 100% solvent B was continued for 5 min. N2-[(2-Acetylamino-2-carboxyethyl)thiomethyl]guanosine 5′-monophosphate (14) was isolated by increasing the content of solvent B from 0% to 15% within 10 min, then to 30% within 20 min, and finally within 10 min to 100%, with continued elution with 100% organic solvent for 5 min. Fractions showing UV absorbance at 260 nm were collected individually with an automated fraction collector, freed from organic solvents in vacuum, and lyophilized three times. S-[1-(N2-Guanosinyl)methyl]glutathione 5′-Monophosphate (13). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 681.1357 ([M − H]−, meas); m/z 681.1340 ([M − H]−, calcd for C21H30N8O14PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 2.05 [q, 2H, J = 7.2, 14.3 Hz, H−C(8″)], 2.43 [t, 2H, J = 7.5 Hz, H−C(7″)], 2.95 [dd, 1H, J = 8.7, 14.3 Hz, H− C(5″A)], 3.15 [dd, 1H, J = 8.7, 14.3 Hz, H−C(5″B)], 3.80 [t, 1H, J = 6.3 Hz, H−C(9″)], 3.89 [s, 2H, H−C(2″)], 4.05 [m, 1H, H−C(5′A)], 4.13 [m, 1H, H−C(5′B)], 4.30 [m, 1H, H−C(4′)], 4.41 [t, 1H, J = 5.1 Hz, H−C(3′)], 4.51 [d, 1H, J = 14.2 Hz, H−C(1‴A)], 4.62 [dd, 1H, J = 5.7, 8.8 Hz, H−C(4″)], 4.70 [t, 1H, J = 7.8 Hz, H−C(2′)], 4.71 [d, 1H, J = 14.2 Hz, H−C(1‴B)], 6.02 [d, 1H, J = 4.3 Hz, H−C(1′)], 8.52 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 25.8 [CH2, C(8″)], 30.8 [CH2, C(7″)], 32.3 [CH2, C(5″)], 41.2 [CH2, C(2″)], 43.5 [CH2, C(1‴)], 53.1 [CH, C(4″)], 53.2 [CH, C(9″)], 64.0 [d, CH2, 2JC,P = 4.3 Hz, C(5′)], 69.8 [CH, C(3′)], 74.3 [CH, C(2′)], 83.8 [d, CH, 3JC,P = 8.7 Hz, C(4′)], 88.6 [CH, C(1′)], 112.4 [C, C(5)], 137.0 [CH, C(8)], 150.2 [C, C(4)], 152.7 [C, C(2)], 156.4 [C, C(6)], 172.6 [C, C(10″)], 172.8 [C, C(3″)], 173.0 [C, C(1″)], 174.8 [C, C(6″)]. N2-[(2-Acetylamino-2-carboxyethyl)thiomethyl]guanosine 5′Monophosphate (14). UV/vis (1% formic acid in water/methanol, 80/20 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 537.0809 ([M − H]−, meas); m/z 537.0805; ([M − H]−, calcd for C16H22N6O11PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 2.00 [s, 3H, H−C(5″)], 3.04 [dd, 1H, J = 8.1, 14.0 Hz, H−C(3″A)], 3.23 [dd, 1H, J = 4.5 Hz, 14.0 Hz, H−C(3″B)], 4.11 [m, 2H, H−C(5′)], 4.32 [m, 1H, H−C(4′)], 4.47 [dd, 1H, J = 4.5, 8.1 Hz, H−C(2″)], 4.51 [t, 1H, J = 4.9 Hz, H−C(3′)], 4.62 [d, 1H, J = 14.3 Hz, H− C(1‴A)], 4.66 [d, 1H, J = 14.3 Hz, H−C(1‴B)], 4.80 [t, 1H, J = 4.4 Hz, H−C(2′)], 6.04 [d, 1H, J = 3.7 Hz, H−C(1′)], 8.16 [s, 1H, H− C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 24.8 [C(5″)], 36.2 [CH2, C(3″)], 46.7 [CH2, C(1‴)], 57.3 [CH, C(2″)], 67.5 [d, CH2, 2JC,P = 5.2 Hz, C(5′)], 73.2 [CH, C(3′)], 76.7 [CH, C(2′)], 86.4 [d, CH, 3JC,P = 8.8 Hz, C(4′)], 90.7 [CH, C(1′)], 119.2 [C, C(5)], 140.9 [CH, C(8)], 154.2 [C, C(4)], 154.7 [C, C(2)], 161.9 [C, C(6)], 176.4 [C, C(4″)], 179.3 [C(1″)]. N2-(2-Acetylaminoethylthiomethyl)guanosine 5′-Monophosphate (15). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 493.0904 ([M − H]−, meas); m/z 493.0824 ([M − H]−, calcd for C15H23N6O9PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 1.94 [s, 3H, H−C(4″)], 2.86 [t, 2H, J = 6.6 Hz, H−C(2″)], 3.42 [dt, 2H, J = 1.7, 6.5 Hz, H−C(1″)], 4.10 [ddd, 2H, J = 3.6, 5.6, 11.6 Hz, H−C(5′A)], 4.18 [ddd, 2H, J = 3.6, 5.6, 11.6 Hz, H−C(5′B)], 4.33 [m, 1H, H−C(4′)], 4.49 [t, 1H, J = 4.9 Hz, H−C(3′)], 4.62 [d, 1H, J = 14.3 Hz, H−C(1‴A)], 4.68 [d, 1H, J = 14.2 Hz, H−C(1‴B)], 4.76 [t, 1H, J = 4.9 Hz, H−C(2′)], 6.07 [d, 1H, J = 4.6 Hz, H−C(1′)], 8.48 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 24.5 [C(4″)], 32.8 [CH2, C(2″)], 41.7 [CH2, C(1″)], 45.8 [CH2, C(1‴)], 66.9 [d, CH2, 2JC,P = 5.6 Hz, C(5′)], 72.6 [CH, C(3′)], 76.8 [CH, C(2′)], 86.3 [d, CH, 3JC,P = 8.5 Hz, C(4′)], 90.1 [CH, C(1′)], 116.0 [C, C(5)], 139.7 [CH, C(8)], 153.4 [C, C(4)], 155.1 [C, C(2)], 160.1 [C, C(6)], 176.9 [C, C(3″)]. N2-(2-Hydroxyethylthiomethyl)guanosine 5′-Monophosphate (16). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 452.0630 ([M − H]−, meas); m/z 452.0642 ([M − H]−, calcd for C13H19N5O9PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 2.91 [t, 2H, J = 6.2 Hz, H−C(1″)], 3.73 [t, 2H, J = 6.2 Hz, H−C(2″)], 4.13 [m, 1H, H−C(5′A)], 4.27 [m, 1H,

by means of Maillard-type reactions of nucleotide 2 with glycine’s Strecker aldehyde formaldehyde and organic thiols, as depicted in Figure 2, and to evaluate their umami tasteenhancing potential of these chemosensorica in vivo by means of human sensory studies and in vitro by means of T1R1/T1R3 taste receptor experiments.

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MATERIALS AND METHODS

Chemicals and Materials. The following chemicals were obtained commercially: dipotassium hydrogen phosphate, formic acid, glycine, mercaptoethanol, monosodium L-glutamate monohydrate, orthophosphoric acid, potassium dihydrogen phosphate, and 1-propanethiol (Merck KGaA, Darmstadt, Germany); formaldehyde, D-glucose, Lglutathione (reduced), and 2-propen-1-thiol (Fluka, Buchs, Switzerland); 2-furanmethanethiol, inosine 5′-monophosphate disodium salt, 2-mercaptophenol, 2-mercaptobenzoic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, 4-mercaptophenol, N-acetylcysteamine, and N-acetyl-L-cysteine (Sigma−Aldrich, Steinheim, Germany); guanosine 5′-monophosphate disodium salt and 2-phenethylthiol (Acros Organics, Geel, Belgium); and HPLC-grade solvents (J. T. Baker, Deventer, Netherlands). Deionized water used for chromatography was purified by means of a Milli-Q gradient A10 system (Millipore, Molsheim, France). Deuterated solvents and 3-trimethylsilyl-2,2,3,3d4-propionic acid sodium salt were supplied by Euriso-Top (Gif-SurYvette, France). For medium-pressure liquid chromatography, RP-18bulk material (LiChroprep RP-18, 25−40 μm, Merck KGaA, Darmstadt, Germany) was used. Sensory analyses were performed with Evian bottled water (Danone Waters Deutschland, Frankfurt, Germany). Preparation of N2-(Alkylthiomethyl)guanosine and N2(Arythiomethyl)guanosine 5′-Monophosphate Derivatives. Following a literature protocol15 with some modifications, formaldehyde (2 mmol; 50 μL of a 36% solution in water) was added to a solution of potassium phosphate buffer (40 mL, pH 7.0; 30 mmol/L) containing one of the mercaptans (15 mmol/L), namely, L-glutathione, N-acetyl-L-cysteine, N-acetylcysteamine, 2-mercaptophenol, 2- and 3mercaptopropionic acid, 1-propanethiol, 2-propen-1-thiol, 2-furanmethanethiol, 2-phenethylthiol, 2- and 4-mercaptophenol, and 2mercaptobenzoic acid, respectively. After the mixture was stirred for 4 h at 40 °C in a closed vessel, guanosine 5′-monophosphate (0.6 mmol) was added and stirring was continued at 40 °C for another 16 h. Subsequently, the reaction product was isolated by means of reversed-phase medium-pressure liquid chromatography (RP-MPLC) on a Buechi-Sepacore system (Flawil, Switzerland) equipped with a 150 × 40 mm column filled with 25−40 μm LiChroprep RP-18 material (Merck KGaA Darmstadt, Germany). The effluent was monitored at 260 nm, and chromatography was performed at a flow rate of 40 mL/min with 1% aqueous formic acid (solvent A) and methanol (solvent B) as eluents. S-[1-(N2-guanosinyl)methyl]glutathione 5′-monophosphate, 13 (Figure 3), N 2 -(2acetylaminoethylthiomethyl)guanosine 5′-monophosphate (15), and N2-(phenethylthiomethyl)guanosine 5′-monophosphate (22) were isolated by eluting with 100% solvent A for 2 min, then increasing solvent B from 0 to 40% within 3 min, and finally increasing to 100% solvent B within an additional 16 min. 100% organic solvent was maintained for an additional 5 min. For isolation of nucleotide derivatives N2-(2-hydroxyethylthiomethyl)guanosine 5′-monophosphate (16), N2-(2-carboxyethylthiomethyl)guanosine 5′-monophosphate (17), N2-(1-carboxyethylthiomethyl)guanosine 5′-monophosphate (18), N2-(furfurylthiomethyl)guanosine 5′-monophosphate (21), N2-(2-hydroxyphenylthiomethyl)guanosine 5′-monophosphate (23), N2-(4-hydroxyphenylthiomethyl)guanosine 5′-monophosphate (24), and N2-(4-carboxyphenylthiomethyl)guanosine 5′-monophosphate (25), elution was started isocratically with 100% solvent A for 5 min, and then the content of solvent B was linearly increased to 100% during 35 min and maintained for an additional 5 min. Purification of N2-(propylthiomethyl)guanosine 5′-monophosphate (19) and N2(prop-2-enylthiomethyl)guanosine 5′-monophosphate (20) was achieved by isocratic elution with 100% eluent A for 2 min and D

dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

N2-(Furfurylthiomethyl)guanosine 5′-Monophosphate (21). UV/ vis (1% formic acid in water/methanol, 50/50 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 488.0634 ([M − H]−, meas); m/z 488.0641 ([M − H]−, calcd for C16H19N5O9PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 3.97 [d, 2H, J = 2.0 Hz, H−C(1″)], 4.10 [m, 2H, H−C(5′)], 4.32 [m, 1H, H−C(4′)], 4.51 [t, 1H, J = 4.6 Hz, H− C(3′)], 4.65 [d, 1H, J = 14.3 Hz, H−C(1‴A)], 4.71 [d, 1H, J = 14.3 Hz, H−C(1‴B)], 4.81 [t, 1H, J = 5.5 Hz, H−C(2′)], 6.02 [d, 1H, J = 5.6 Hz, H−C(1′)], 6.26 [d, 1H, J = 3.3 Hz, H−C(5″)], 6.34 [dd, 1H, J = 1.9, 3.0 Hz, H−C(4″)], 7.41 [d, 1H, J = 1.8 Hz, H−C(3″)], 8.11 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 30.2 [CH2, C(1″)], 46.4 [CH2, C(1‴)], 67.5 [d, CH2, 2JC,P = 5.1 Hz, C(5′)], 73.3 [CH, C(3′)], 76.6 [CH, C(2′)], 86.6 [d, CH2, 3JC,P = 8.2 Hz, C(4′)] 90.1 [CH, C(1′)], 110.4 [CH, C(4″)], 113.5 [CH, C(5″)], 119.2 [C, C(5)], 140.9 [CH, C(8)], 145.5 [CH, C(3″)], 154.3 [C, C(4)], 154.62 [C, C(2″)/C(2)], 154.64 [C, C(2″)/C(2)], 161.9 [C, C(6)]. N2-(Phenethylthiomethyl)guanosine 5′-Monophosphate (22). UV/vis (1% formic acid in water/methanol, 40/60 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 512.1019 ([M − H]−, meas); m/z 512.1005 ([M − H]−, calcd for C19H23N5O8PS). 1H NMR (500 MHz, D2O/methanol-d4, COSY) δ (ppm) 2.88 [t, 2H, J = 7.0 Hz, H− C(2″)], 3.03 [t, 2H, J = 7.0 Hz, H−C(1″)], 4.10 [m, 2H, H−C(5′)], 4.26 [m, 1H, H−C(4′)], 4.47 [t, 1H, J = 5.2 Hz, H−C(3′)], 4.51 [d, 1H, J = 14.2 Hz, H−C(1‴A)], 4.56 [d, 1H, J = 14.2 Hz, H−C(1‴B)], 4.70 [d, 1H, J = 5.2 Hz, H−C(2′)], 5.95 [d, 1H, J = 5.1 Hz, H−C(1′)], 7.14 [t, 1H, J = 7.0 Hz, H−C(6″)], 7.16−7.25 [m, 4H, H− C(4″,5″,7″,8″)], 8.08 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O/ methanol-d4, HMBC, HSQC) δ (ppm) 35.1 [CH2, C(1″)], 38.3 [CH2, C(2″)], 46.3 [CH2, C(1‴)], 67.5 [d, CH2, 2JC,P = 4.8 Hz, C(5′)], 73.2 [CH, C(3′)], 76.7 [CH, C(2′)], 86.3 [d, CH, 3JC,P = 8.2 Hz, C(4′)], 90.5 [CH, C(1′)], 119.5 [C, C(5)], 129.2 [CH, C(6″)], 131.2/131.7 [CH, C(4″,5″,7″,8″)], 140.8 [C, C(3″)], 143.4 [C, C(8)], 154.2 [C, C(4)], 154.5 [C, C(2)], 161.8 [C, C(6)]. N2-(2-Hydroxyphenylthiomethyl)guanosine 5′-Monophosphate (23). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 500.0640 ([M − H]−, meas); m/z 500.0641 ([M − H]−, calcd for C17H19N5O9PS). 1H NMR (500 MHz, methanol-d4) δ (ppm) 4.15 [ddd, 1H, J = 3.2, 5.5, 11.8 Hz, H− C(5′A)], 4.25 [ddd, 1H, J = 2.8, 4.4, 11.8 Hz, H−C(5′B)], 4.39 [m, 1H, H−C(4′)], 4.48 [t, 1H, J = 5.2 Hz, H−C(3′)], 4.64 [t, 1H, J = 5.2 Hz, H−C(2′)], 4.76 [d, 1H, J = 13.4 Hz, H−C(1‴A)], 5.02 [d, 1H, J = 13.4 Hz, H−C(1‴B)], 5.88 [d, 1H, J = 4.0 Hz, H−C(1′)], 6.84 [td, 1H, J = 1.1, 7.5 Hz, H−C(5″)], 6.88 [dd, 1H, J = 1.0, 8.2 Hz, H− C(3″)], 7.18 [td, 1H, J = 1.6, 8.1 Hz, H−C(4″)], 7.51 [dd, 1H, J = 1.6, 7.7 Hz, H−C(6″)], 8.63 [s, 1H, H−C(8)]. 13C NMR (125 MHz, methanol-d4, HMBC) δ (ppm) 46.1 [CH2, C(1‴)], 63.8 [d, CH2, 2JC,P = 4.7 Hz, C(5′)], 69.4 [CH, C(3′)], 74.3 [CH, C(2′)], 83.5 [d, CH, 3 JC,P = 8.9 Hz, C(4′)], 88.7 [CH, C(1′)], 115.2 [CH, C(3″)], 117.2 [C, C(1″)], 117.8 [C, C(5)], 120.8 [CH, C(5″)], 131.2 [CH, C(4″)], 136.4 [C, C(2″)], 137.5 [CH, C(8)], 149.7 [C, C(2)], 153.0 [C, C(4)], 156.4 [C, C(6)], 157.0 [C, C(6″)]. N2-(4-Hydroxyphenylthiomethyl)guanosine 5′-Monophosphate (24). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 500.0640 ([M − H]−, meas); m/z 500.0641 ([M − H]−, calcd for C17H19N5O9PS). 1H NMR (500 MHz, DMSO-d6) δ (ppm) 3.93 [m, 1H, H−C(5′A)], 4.05 [m, 1H, H− C(5′B)], 4.07 [m, 1H, H−C(4′)], 4.15 [dd, J = 3.9, 4.9 Hz, H−C(3′)], 4.50 [t, J = 5.3 Hz, H−C(2′)], 4.62−4.78 [m, 2H, H−C(1‴)], 5.77 [d, J = 5.7 Hz, H−C(1′)], 6.75 [d, 2H, J = 8.7 Hz H−C(3″/5″)], 7.31 [d, 2H, J = 8.7 Hz, H−C(2″/6″)], 7.95 [s, 1H, H−C(8)]. 13C NMR (125 MHz, DMSO-d6, HMBC) δ (ppm) 47.7 [CH2, C(1‴)], 66.1 [d, CH2, 2 JC,P = 5.4 Hz, C(5′)], 71.0 [CH, C(3′)], 73.5 [CH, C(2′)], 83.4 [d, CH, 3JC,P = 7.8 Hz, C(4′)], 87.3 [CH, C(1′)], 116.7 [CH, C(3″)/ C(5″)], 117.8 [C, C(5)], 122.5 [C, C(1″)], 134.9 [CH, C(2″)/ C(6″)], 136.7 [CH, C(8)], 150.9 [C, C(4)], 151.9 [C, C(2)], 156.7 [C, C(6)], 157.9 [C, C(4″)]. N2-(4-Carboxyphenylthiomethyl)guanosine 5′-Monophosphate (25). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. LC-MS (ESI−) m/z (%) 528 (85, [M − H]−), 362 (75, [M −

H−C(5′B)], 4.39 [m, 1H, H−C(4′)], 4.52 [t, J = 5.3 Hz, 1H, H− C(3′)], 4.59 [d, 1H, J = 14.3 Hz, H−C(1‴A)], 4.63 [d, 1H, J = 14.3 Hz, H−C(1‴B)], 4.78 [m, 1H, H−C(2′)], 6.05 [d, J = 3.5 Hz, 1H, H− C(1′)], 8.77 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HSQC, HMBC) δ (ppm) 32.9 [CH2, C(1″)], 43.2 [CH2, C(1‴)], 60.7 [CH2, C(2″)], 63.8 [d, CH2, 2JC,P = 4.8 Hz, C(5′)], 69.4 [CH, C(3′)], 74.4 [CH, C(2′)], 83.6 [d, CH, 3JC,P = 8.5 Hz, C(4′)], 89.3 [CH, C(1′)], 110.4 [C, C(5)], 136.2 [CH, C(8)], 149.7 [C, C(4)], 153.0 [C, C(2)], 156.0 [C, C(6)]. N2-(2-Carboxyethylthiomethyl)guanosine 5′-Monophosphate (17). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 480.0591 ([M − H]−, meas); m/z 480.0590 ([M − H]−, calcd for C14H19N5O10PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 2.63 [m, 2H, H−C(2″)], 2.84 [m, 2H, H−C(3″)], 4.03 [m, 2H, H−C(5′)], 4.24 [m, 1H H−C(4′)], 4.43 [t, 1H, J = 4.7 Hz, H−C(3′)], 4.57 [d, 1H, J = 14.0 Hz, H−C(1‴A)], 4.65 [d, 1H, J = 14.1 Hz, H−C(1‴B)], 4.70−4.77 [m, 1H, H−C(2′)], 5.93 [d, 2H, J = 4.2 Hz, H−C(1′)], 8.01 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 28.7 [CH2, C(3″)], 38.0 [CH2, C(2″)], 46.2 [CH2, C(1‴)], 67.5 [d, CH2, 2JC,P = 4.8 Hz, C(5′)], 73.2 [CH, C(3′)], 76.7 [CH, C(2′)], 86.6 [d, CH, 3JC,P = 7.5 Hz, C(4′)], 90.4 [CH, C(1′)], 119.3 [C, C(5)], 140.7 [CH, C(8)], 149.9 [C, C(4)], 154.5 [C, C(2)], 155.0 [C, C(6)], 180.4 [C, C(1″)]. N2-(1-Carboxyethylthiomethyl)guanosine 5′-Monophosphate (18). UV/vis (1% formic acid in water/methanol, 60/40 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 480.0591 ([M − H]−, meas); m/z 480.0590 ([M − H]−, calcd for C14H19N5O10PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 1.49 [d, 3H, J = 7.1 Hz, H−C(3″)], 3.83 [m, 1H, H−C(2″)], 4.14 [m, 1H, H−C(5′A)], 4.25 [m, 1H, H− C(5′B)], 4.38 [m, 1H, H−C(4′)], 4.51 [m, 1H, H−C(3′)], 4.71 [m, 1H, H−C(2′)], 4.73 [d, 1H, J = 13.3 Hz, H−C(1‴A)], 4.83 [d, 1H, J = 13.0 Hz, H−C(1‴B)], 6.14 [d, 2H, J = 4.2 Hz, H−C(1′)], 8.87 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 16.9 [CH3, C(3″)], 41.4 [CH, C(2″)], 43.1 [CH2, C(1‴)], 63.9 [d, CH2, 2 JC,P = 4.6 Hz, C(5′)], 69.5 [CH, C(3′)], 74.3 [CH, C(2′)], 83.6 [d, CH, 3JC,P = 8.1 Hz, C(4′)], 89.0 [CH, C(1′)], 111.3 [C, C(5)], 136.5 [CH, C(8)], 149.9 [C, C(4)], 152.7 [C, C(2)], 156.6 [C, C(6)], 177.9 [C, C(1″)]. N2-(Propylthiomethyl)guanosine 5′-Monophosphate (19). UV/ vis (1% formic acid in water/methanol, 40/60 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 450.0843 ([M − H]−, meas); m/z 450.0848 ([M − H]−, calcd for C14H21N5O8PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 0.95 [t, 3H, J = 7.4 Hz, H−C(3″)], 1.65 [m, 2H, H−C(2″)], 2.69 [t, 2H, J = 7.4 Hz, H−C(1″)], 4.07 [m, 2H, H− C(5′)], 4.31 [m, 1H, H−C(4′)], 4.48 [t, 1H, J = 4.4 Hz, H−C(3′)], 4.57 [d, 1H, J = 14.1 Hz, H−C(1‴A)], 4.67 [d, 1H, J = 14.2 Hz, H− C(1‴B)], 4.75 [t, 1H, J = 5.7 Hz, H−C(2′)], 5.96 [d, 1H, J = 5.3 Hz, H−C(1′)], 8.12 [s, 1H, H−C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 15.6 [CH3, C(3″)], 25.8 [CH2, C(2″)], 35.9 [CH2, C(1″)], 46.0 [CH2, C(1‴)], 67.6 [d, CH, 3JC,P = 4.7 Hz, C(5′)], 73.6 [CH, C(3′)], 76.6 [CH, C(2′)], 86.8 [d, CH2, 2JC,P = 8.4 Hz, C(4′)], 90.2 [CH, C(1′)], 119.3 [C, C(5)], 140.7 [CH, C(8)], 154.5 [C, C(4)], 154.9 [C, C(2)], 161.7 [C, C(6)]. N2-(Prop-2-enylthiomethyl)guanosine 5′-Monophosphate (20). UV/vis (1% formic acid in water/methanol, 40/60 v/v) λmax = 260 nm. UPLC-TOF-MS (ESI−) m/z 448.0684 ([M − H]−, meas); m/z 448.0692 ([M − H]−, calcd for C14H19N5O8PS). 1H NMR (500 MHz, D2O, COSY) δ (ppm) 3.34 [d, 2H, J = 8.1 Hz, H−C(1″)], 4.12 [ddd, 1H, J = 3.5, 5.2, 11.7 Hz, H−C(5′A)], 4.21 [ddd, 1H, J = 3.4, 4.2, 11.8 Hz, H−C(5′B)], 4.36 [m, 1H, H−C(4′)], 4.49 [t, 1H, J = 5.0 Hz, H− C(3′)], 4.58 [d, 1H, J = 14.2 Hz, H−C(1‴A)], 4.62 [d, 1H, J = 14.2 Hz, H−C(1‴B)], 4.78 [t, 1H, J = 4.4 Hz, H−C(2′)], 5.16 [d, 1H, J = 10.0 Hz, H−C(3″A)], 5.21 [d, 1H, J = 17.0 Hz, H−C(3″B)], 5.91 [m, 1H, H−C(2″)], 6.09 [d, 1H, J = 4.2 Hz, H−C(1′)], 8.66 [s, 1H, H− C(8)]. 13C NMR (125 MHz, D2O, HMBC, HSQC) δ (ppm) 36.4 [CH2, C(1″)], 45.5 [CH2, C(1‴)], 67.0 [d, CH2, 2JC,P = 5.1 Hz, C(5′)], 72.6 [CH, C(3′)], 77.2 [CH, C(2′)], 86.6 [d, CH, 3JC,P = 8.0 Hz, C(4′)], 91.9 [CH, C(1′)], 114.8 [C, C(5)], 120.5 [CH2, C(3″)], 137.4 [CH, C(2″)], 139.7 [C−H, C(8)], 153.1 [C, C(4)], 155.7 [C, C(2)], 159.5 [C, C(6)]. E

dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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C8H7O2S]−). MS/MS (ESI−) m/z (%) 528 (100, [M − H]−), 362 (10, [M − C8H7O2S]−). 1H NMR (500 MHz, DMSO-d6) δ (ppm) 3.94 [m, 1H, H−C(5′A)], 4.01 [m, 1H, H−C(5′B)], 4.08 [m, 1H, H− C(4′)], 4.15 [dd, J = 3.7, 4.8 Hz, H−C(3′)], 5.03 [m, 2H, H−C(1‴)], 5.78 [d, J = 5.5 Hz, H−C(1′)], 7.53 [t, 2H, J = 8.5 Hz, H−C(3″/ 5″)], 7.86 [d, 2H, J = 8.5 Hz, H−C(2″/6″)], 7.96 [s, 1H, H−C(8)]. 13C NMR (125 MHz, DMSO-d6, HMBC) δ (ppm) 44.1 [CH2, C(1‴)], 65.9 [d, CH2, 2JC,P = 5.1 Hz, C(5′)], 77.1 [CH, C(3′)], 73.9 [CH, C(2′)], 83.9 [d, CH, 3JC,P = 8.2 Hz, C(4′)], 87.6 [CH, C(1′)], 117.9 [C, C(5)], 128.0 [C, C(4″)], 128.3 [CH, C(3″)/C(5″)], 130.3 [CH, C(2″)/C(6″)], 136.9 [CH, C(8)], 142.1 [C, C(1″)], 150.8 [C, C(4)], 151.8 [C, C(2)], 157.0 [C, C(6)], 167.4 [C, C(7″)]. Hydrolytic Stability of Nucleotide 21 in Aqueous Solution. A solution of N2-(furfurylthiomethyl)guanosine 5′-monophosphate (21; 1.0 mg) in potassium phosphate buffer (1 mL, pH 4.0/6.0/8.0) was stored at 22 °C and analyzed by means of RP-HPLC with diode array detection (DAD) after 7 h. To evaluate the stability over a longer period of time, the samples dissolved at pH 6.0 and 8.0 were stored at room temperature and analyzed daily by RP-HPLC-DAD. Sensory Analyses. Precautions Taken for Sensory Analysis. Prior to sensory analysis, the isolated compounds were suspended in water and, after removal of volatiles in high vacuum ( 0.99. Medium-Pressure Liquid Chromatography. Medium-pressure liquid chromatography (MPLC) was performed on a Sepacore chromatography system (Buechi, Flawil, Switzerland) consisting of two C-605 pumps with C-615 pump manager, manual rheodyne injection port (20 mL loop), C-635 UV/vis detector, and C-660 fraction collector. Chromatography was performed at a flow rate of 40 mL/min on a self-packed 150 × 40 mm i.d. polypropylene cartridge filled with 25−40 μm LiChroprep RP-18 Material (Merck KGaA, Darmstadt, Germany). High-Performance Liquid Chromatography. Analytical HPLC was carried out on an HPLC apparatus (Jasco, Groß-Umstadt, Germany), consisting of two PU-2087 Plus pumps, M800 gradient mixer, DG-2080−53 degasser, auto sampler (AS-2055 Plus) and diode array detector (MD-2010 Plus). Chrompass software was used for data acquisition. With a flow rate of 0.8 mL/min, chromatography was performed on a 250 × 4.6 mm i.d., 5 μm, Microsorb-MW 100−5 C18 column (Varian, Darmstadt, Germany). The output was monitored at 220 and 260 nm; 1% aqueous formic acid (solvent A) and methanol (solvent B) were used as eluents. After sample injection (10 μL), isocratic elution with 100% solvent A was performed for 5 min. Subsequently, the content of organic solvent was linearly increased to 100% within 25 min, and 100% solvent B was maintained for an additional 5 min. High-Performance Liquid Chromatography/Tandem Mass Spectroscopy. For HPLC-ESI-MS/MS analysis, a Dionex UHPLC UltiMate 3000 HPLC system (Dionex, Idstein), consisting of HPG3400SD type binary pump, SRD-3400 type degasser, WSP-3000TSL type autosampler, and TCC-3000SD type column compartment and operated with the DC MS Link 2.8.0.2633 software, was linked to an API 4000 QTRAP mass spectrometer (AB Sciex Instruments, Darmstadt, Germany), which was equipped with an electrospray ionization (ESI) source and operated in negative ionization mode. Nitrogen served as nebulizer gas (50 psi), and heated at 400 °C as turbo gas for solvent drying (60 psi). Nitrogen also served as curtain (20 psi) and collision gas (4.5 × 10−5 Torr). For the detection of 19, the following transition reactions were optimized prior to analyses and monitored for a duration of 100 ms each. Declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) are given in parentheses: m/z 450 → 374 (DP −65 V, CE −24 V, CXP −11 V), m/z 450 → 161 (DP −65 V, CE −50 V, CXP −11 V), and m/z 450 → 374 (DP −65 V, CE −36 V, CXP −1 V). Both quadrupoles were set at unit resolution. For HPLC-ESI-MS/MS analysis, data acquisition and integration were carried out with Analyst 1.5.1 software (Applied Biosystems). After sample injection (5 μL), chromatographic separation was performed on a 150 × 2.0 mm i.d., 3 μm, Phenomenex Luna PFP column (Phenomenex, Aschaffenburg, Germany) by use of a linear binary gradient with a flow rate of 200 μL/min. Eluent A was 1% formic acid in acetonitrile, and eluent B was 1% formic acid in water. Chromatographic separation was performed as follows: After isocratic elution with 100% solvent B for 5 min, the content of solvent A increased to 50% within 10 min and then to 100% within 2 min. The organic solvent was finally maintained at 100% for an additional 5 min. With help of a Valco valve, the effluent was directed into the mass spectrometer during minutes 12 and 22 of each chromatographic run, while the remaining effluent was split into the waste. To determine the effect of matrix components on the LC-MS/MS analysis, 5 μL of analyte-free matrix solutions were injected into the LC-MS/MS system described above, but in addition, a constant flow (10 μL/min) of a solution of 19 (0.19 μmol/L) was introduced by means of a PHD 4400 Hpsi type syringe pump (Harvard Apparatus, Holliston, MA) connected to the solvent flow via a three-way valve. Ultraperformance Liquid Chromatography/Time-of-Flight Mass Spectrometry. Mass spectra of the compounds were measured on a Waters Synapt G2 HDMS mass spectrometer (Waters, Manchester, U.K.) coupled to an Acquity UPLC core system (Waters) consisting of a binary solvent manager, sample manager and column oven. The compounds were dissolved in 1 mL of methanol ,and

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RESULTS AND DISCUSSION Recent investigations revealed that N2-1-carboxyalkylation of guanosine 5′-monophosphate by Maillard-type reactions with reducing carbohydrates increased the umami-enhancing potential of the nucleotide; for example, (S)-N 2 -(1carboxyethyl)guanosine 5′-phosphate (10) was isolated as a high-potency umami enhancer from yeast extracts.12,13 Furthermore, amidation of the side chain’s carboxy group induced a further increase of sensory activity of 10; for example, butylation revealed the high-potency umami enhancer 11.14 Since the presence of sulfur atoms in synthetic N2-alkylated nucleotides was described to be beneficial for sensory activity,6−10 the following experiments targeted the substitution of the exocyclic amino function of guanosine 5′-monophosphate (2) with formaldehyde, the Strecker aldehyde of glycine, and alkyl- and arylmercaptans, respectively, by means of Maillard-type reaction chemistry following the scheme depicted in Figure 2. Preparation of N2-(Alkylthiomethyl)guanosine and 2 N -(Arylthiomethyl)guanosine 5′-Monophosphates 13− 25. In a first set of experiments, the umami-enhancing guanosine 5′-monophosphate (2) was reacted for 16 h at 40 °C with a binary mixture of formaldehyde and the kokumiactive glutathione, which was preincubated for 4 h at the same temperature. Reaction product 13 was isolated by means of RPMPLC in a purity of >98% [1H NMR, HPLC with evaporative light-scattering detector (ELSD)] and its structure was determined by means of UPLC-TOF-MS and 1D/2D-NMR experiments. UPLC-TOF-MS revealed m/z 681.1357 as a pseudo-molecular ion ([M − H]−), which is in accordance with the calculated mass of 681.1340 (C21H30N8O14PS). COSY, HSQC, and HMBC spectra allowed us to assign the proton signals at 2.05, 2.43, and 3.80 ppm to H−C(8″), H−C(7″), and G

dx.doi.org/10.1021/jf504686s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 4. Excerpt of HMBC spectrum (500 MHz, D2O) of S-[1-(N2-guanosinyl)methyl]glutathione 5′-monophosphate (13) generated from guanosine 5′-monophosphate (2), formaldehyde, and glutathione.

H−C(9″) of the glutamyl residue. 1JC,H couplings observed in the HSQC spectrum identified carbon signals resonating at 25.8, 30.8, and 53.2 parts per million (ppm) to correspond to the carbon atoms C(8″), C(7″), and C(9″), respectively. The two quaternary carbon atoms C(10″) and C(6″) resonated at 172.6 and 174.8 ppm and showed the expected 2,3JC,H-couplings in the HMBC spectrum. The cysteine moiety showed proton signals at 2.95 [H−C(5″A)], 3.07 [H−C(5″B)], and 4.62 ppm [H−C(4″))] and the corresponding carbon resonances at 32.3 [C(5″)] and 53.1 ppm [C(4″)]. The signal at 172.8 ppm was assigned to the carbon atom C(3″), showing heteronuclear 2,3Jcoupling with the protons H−C(2″), H−C(4″), and H−C(5″). The singlet at 3.89 ppm in the 1H NMR spectrum and at 41.2 ppm in the 13C NMR spectrum were attributed to methylene group of the glycine moiety. In addition, the HMBC experiment led to the identification of the carbon signal at 173.0 ppm as the glycine carboxy carbon C(1″). Moreover, the singlet at 8.52 ppm in the 1H NMR spectrum and the signal at 137.0 ppm in the 13C NMR spectrum were assigned to position C(8) of the purine scaffold. Due to heteronuclear 3J- and 4Jcoupling detectable in the HMBC, the signals resonating at 112.4, 150.2, and 156.4 ppm were assigned as the quaternary carbon atoms C(5), C(4), and C(6) of the nucleotide moiety. The proton signals at 4.05, 4.13, 4.30, 4.41, 4.70, and 6.02 ppm in the 1H NMR spectrum and at 64.0, 83.8, 69.8, 74.3, and 88.6 ppm in the 13C NMR spectrum were attributed to the positions 5′A+B, 4′, 3′, 2′, and 1′ of the nucleotide’s ribose moiety. The presence of the phosphate group in the molecule was confirmed by coupling of the 31P nucleus with the carbon atoms C(5′) and C(4′); for example, coupling constants of 4.3 and 8.7 Hz were observed for 2J- and 3J-coupling of the phosphate group with C(5′) and C(4′), respectively. Two doublets recorded in the 1H NMR spectrum at 4.51 and 4.71 ppm were assigned as protons of the methylene group C(1‴),

and with help of the HSQC spectrum, the carbon atom resonating at 43.5 ppm was assigned to this methylene group. This carbon atom showed heteronuclear 3J-coupling with diastereotopic methylene protons H−C(5″A+B) of the cysteine residue. Connection to the nucleotide scaffold was verified by heteronuclear 3J-coupling of the quaternary carbon atom C(2) at 152.7 ppm to two proton signals of the methylene group H− C(1‴) (Figure 4). With all these spectroscopic data taken into consideration, the reaction product 13 was unequivocally identified as S-[1-(N2-guanosinyl)methyl]glutathione 5′-monophosphate (Figure 3). By a straightforward synthetic approach, a series of N2(alkylthiomethyl)guanosine and N2-(arylthiomethyl)guanosine 5′-monophosphates, 14−25, was successfully generated by reacting guanosine 5′-monophosphate (2) with formaldehyde and N-acetyl-L-cysteine, N-acetylcysteamine, 2-mercaptophenol, 2- and 3-mercaptopropionic acid, 1-propanethiol, 2-propen-1thiol, 2-furanmethanethiol, 2-phenethylthiol, 2- and 4-mercaptophenol, and 2-mercaptobenzoic acid, respectively (Figure 3). To investigate the hydrolytic stability of such N 2 (alkylthiomethyl)guanosine 5′-monophosphates in aqueous solution, solutions of 21 in phosphate buffer were kept at pH 4.0, 6.0, and 8.0 at room temperature and repeatedly analyzed by RP-HPLC-DAD. While the target nucleotide remained rather stable at pH 4.0 and 6.0, storage under alkaline conditions induced rapid degradation with a half-life time of 8 days (data not shown). Sensory Evaluation of N2-(Alkylthiomethyl)guanosine and N2-(Arylthiomethyl)guanosine 5′-Monophosphates. In order to study the umami taste-enhancing properties of ribonucleotides 13−25, the highly purified substances were presented to a panel of trained assessors in a series of forcedchoice comparison tests described earlier (Table 1).6,13 To evaluate the umami taste synergism between L-glutamic acid H

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sequently, the β-value was calculated as the ratio of the inosine 5′-monophosphate concentration perceived as isointense to test solution and the concentration of the target ribonucleotide applied in this sensory test. Reference nucleotide 2 was evaluated with a β-value of 2.4, thus being well in line with literature data.6,13 Sensory analysis of the 5′-GMP/glutathione conjugate 13 revealed umami enhancement with a β-value of 1.1, which is close to that of the reference inosine 5′-monophosphate, 3 (Table 1). To evaluate the influence of mercaptan structure on the taste-enhancing properties of the nucleotide conjugate, the tripeptide glutathione was substituted by N-acetylcysteine to give nucleotide 14. Sensory evaluation revealed a β-value of 2.3, thus demonstrating that ribonucleotide 14 possessed tastemodifying properties equivalent to its mother nucleotide 3. Removal of the carboxy group from the N-acetylcysteine moiety showed an additional increase of the umami-enhancing potential (β-value 2.8) of the N-acetylcysteamine conjugate 15, while substituting the acetylated amino by an hydroxyl group to give 16 did not alter the sensory properties of the target nucleotide. Removal of the N-acetylamino group from Nacetylcysteine revealed a slightly lower β-value of 2.5 for the corresponding 3-mercaptopropionic acid conjugate 17. Shifting the carboxyl group from the α- into the β-position induced an increase in sensory activity from a β-value of 2.5 (17) to 3.1 (18). Complete absence of all functional groups in the mercaptan structure revealed the 5′-GMP/propanethiol conjugate 19 with a remarkably higher taste-enhancing potential (β-value 5.1) than the molecules evaluated before. Substitution of the propyl residue in 19 by an allyl residue (20) slightly decreased the β-value of the nucleotide to 4.1. These data confirm earlier observations,13,14 that the incorporation of small and nonpolar N-substituents in 2 evoke more pronounced umami-enhancement activity than large and hydrophilic substituents. A second set of experiments focused on the activity of N2(arylthiomethyl)guanosine 5′-monophosphates, exhibiting an

Table 1. Umami-Enhancing Activity (β-Value) of Reference Nucleotides (2 and 3) and N2-(Methylene-S-alkyl/ aryl)guanosine 5′-Monophosphate Derivatives (13−25) nucleotide derivativea inosine 5′-monophosphate, disodium salt (3) guanosine 5′-monophosphate, disodium salt (2) S-[1-(N2-guanosinyl)methyl]glutathione 5′-monophosphate (13) N2-[(2-acetylamino-2-carboxyethyl)thiomethyl]guanosine 5′monophosphate (14) N2-(2-acetylaminoethylthiomethyl)guanosine 5′-monophosphate (15) N2-(2-hydroxyethylthiomethyl)guanosine 5′-monophosphate (16) N2-(2-carboxyethylthiomethyl)guanosine 5′-monophosphate (17) N2-(1-carboxyethylthiomethyl)guanosine 5′-monophosphate (18) N2-(propylthiomethyl)guanosine 5′-monophosphate (19) N2-(prop-2-enylthiomethyl)guanosine 5′-monophosphate (20) N2-(furfurylthiomethyl)guanosine 5′-monophosphate (21) N2-(phenethylthiomethyl)guanosine 5′-monophosphate (22) N2-(2-hydroxyphenylthiomethyl)guanosine 5′-monophosphate (23) N2-(4-hydroxyphenylthiomethyl)guanosine 5′-monophosphate (24) N2-(4-carboxyphenylthiomethyl)guanosine 5′-monophosphate (25)

βvalue 1.0 2.4 1.1 2.3 2.8 2.8 2.5 3.1 5.1 4.1 3.1 2.7 2.1 3.0 3.6

a

Chemical structures and numbering of the compounds refer to Figures 1 and 4.

and ribonucleotides, each target ribonucleotide (50 μmol/L) was dissolved in a matrix of monosodium glutamate (3 mmol/ L; pH 6). In five duo tests, panelists had to evaluate this solution for umami taste intensity in comparison with reference solutions containing increasing concentrations (in 30% steps) of inosine 5′-monophosphate (3) dissolved in the monosodium glutamate matrix. The percent of panelists judging the test solution to exhibit more intense umami taste was calculated and, after Probit transformation, related to the logarithm of the corresponding inosine 5′-monophosphate reference solutions. By means of linear regression, the concentration of inosine 5′monophosphate was calculated at which panelists were unable to distinguish between test and reference solutions. Sub-

Figure 5. Umami-enhancing activity of selected N2-(alkylthiomethyl)guanosine 5′-monophosphates (19, 20) and N2-(arylthiomethyl)guanosine 5′monophosphates (21, 22) assessed in umami receptor assay. (A) Calcium fluorescence traces from PEAK Rapid/Gα15 cells expressing the umami receptor heteromer hT1R1/rT1r3 (upper row) and control cells (lower row) to application (↑) of 19 (0.05 mmol/L), 20 (0.05 mol/L), 21 (0.05 mmol/L), 22 (0.05 mol/L), and 5′-IMP (3; 0.5 mmol/L), in the absence (gray) and presence (black) of L-glutamate (0.5 mM). Control cells were transfected with empty vector. Scale: y-axis, 1050 counts; x-axis, 2 min. (B) Fluorescence ratios of cells expressing hT1R1/rT1r3 to L-glutamate (0.5 mM) in the absence (white bar) and presence of 5′-IMP (3; 0.5 mM; light gray bar) and nucleotides 19−22 (each 0.05 mmol/L; dark gray bars) (N = 2). I

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cells upon coapplication of L-glutamate (0.5 mM) and the target compounds 19, 20, and 21 (0.05 mM), respectively, significantly exceeded those induced by L-glutamate (0.5 mM) and 5′-IMP (3), even though 3 was employed at a 10-fold higher concentration (Figure 5B). Interestingly, the rank order of the compound’s in vitro efficacy in modulating the T1R1/R3 receptor (19 > 20 = 21 > 22) resembles that of the human sensory data, although the differences were less pronounced at the cellular level. For example, 19 (β-value 5.1) showed almost twice the activity of 22 (β value 2.7) in the human sensory experiment, but the fluorescence ratio of 0.116 ± 0.017 measured for 19 elicited an only 27% higher signal than 22 (0.091 ± 0.010). These differences between human sensory and taste receptor data may be explained either by response differences of the cells expressing the hT1R1-rT1r3 receptor construct when compared to human umami taste receptor cells or by assuming that the umami-enhancing activity of the modified nucleotides is only partially mediated by the T1R1/R3 umami receptor. Maillard-type Generation of N2-(Propylthiomethyl)guanosine 5′-monophosphate (19). Apart from the formation of volatile odorants,16 the generation of taste modulators by the Maillard reaction gained increasing attention during recent decades.12−14,19−25 As the Strecker aldehyde formaldehyde is released from the amino acid glycine upon thermal processing in the presence of reducing carbohydrates, the following experiments were done to investigate the generation of target nucleotide 19 by food-related Maillardtype reactions involving glycine, glucose, and 1-propanethiol, known as a key odorant released from freshly cut onions.26 After thermal reaction in phosphate buffer (pH 7.0), the reaction mixture was analyzed by means of HPLC-MS/MS (ESI−) analysis in the multiple reaction monitoring (MRM) mode. As depicted in Figure 6, the target molecule 19, eluted

aromatic ring in the mercaptan moiety. Conjugates of guanosine 5′-monophosphate with the key food odorants 2furfurylthiol and 2-phenethylthiol,16 respectively, showed only slightly increased umami-enhancement activity with β-values of 3.1 (21) and 2.7 (22) when compared to the reference nucleotide 2 (Table 1). While N2-(2-hydroxyphenylthiomethyl)guanosine 5′-monophosphate (23) showed an even lower β-value of 2.1, shifting the hydroxy group at the phenyl ring from the ortho into the para position resulted in ribonucleotide 24, evaluated with an increased β-value of 3.0. However, replacement of the hydroxyl group in 24 by a carboxy function induced a further increase in the taste-enhancing potential of the target nucleotide 25 to reach a β-value of 3.6. Intriguingly, substitution of the exocyclic amino group of 3 with N2-(arylthiomethyl) groups followed a different structure− activity relationship than substitution with N2-(alkylthiomethyl) groups. The sensory activity of N2-(arylthiomethyl) derivatives seems to depend on the substitution pattern of the aromatic moiety rather than on the size of the substituent. Enhancing Effect of Nucleotides on Functionally Expressed hT1R1/rT1r3 Umami Receptor. Although the candidate umami taste receptors in humans were identified almost a decade ago,4 only a few studies have combined human psychophysical experiments with functional expression of T1 receptors so far.14,17 As the human sensory data obtained for N 2 -(alkylthiomethyl)guanosine and N 2 -(arylthiomethyl)guanosine 5′-monophosphates implied high chemoselectivity of the umami receptor binding site, the enhancing effect of the most efficient compounds 19−22 on the L-glutamate-induced response of functionally expressed T1R1/T1r3 umami receptor was investigated (Figure 5). In human embryonic kidney PEAKRapid cells that stably express the G protein subunit mGα15, activation of the heteromeric umami receptor combination of hT1R1 and rT1r3 is coupled to release of calcium from the endoplasmatic reticulum that can be detected by fluorescent dyes. Since Lglutamate and nucleotide enhancer are known to interact with the T1R1 subunit of the umami receptor heteromer, the functional expression system is likely to resemble the detection of umami compounds in human taste receptor cells.5,14,18 With regard to its sensitivity to L-glutamate, the pharmacological characteristics of our umami functional expression system were well in accordance with previously published in vitro data.5 Calcium traces of T1R1/T1r3-expressing cells and mocktransfected cells (control) upon bath application of N2(alkylthiomethyl)guanosine 5′-monophosphates (19 and 20; 0.05 mmol/L each), N2-(arylthiomethyl)guanosine 5′-monophosphates (21 and 22; 0.05 mmol/L each), and reference nucleotide 3 (0.5 mmol/L each) in the presence or absence of L-glutamic acid (0.5 mmol/L) are depicted in Figure 5A. In the absence of L-glutamate, none of the compounds elicited calcium fluorescence responses from T1R1/R3-expressing cells nor from mock-transfected cells (Figure 5A, gray traces). These data propose that the N2-(alkylthiomethyl)guanosine and N2(arylthiomethyl)guanosine 5′-monophosphates do not exhibit intrinsic umami taste activity. However, the receptor-dependent fluorescence signal to L-glutamate (0.5 mmol/L) was strongly enhanced by 0.05 mM 19, 20, 21, and 22 (Figure 5A, black traces). Each of the tested ribonucleotides 19−22 increased receptor responses in a more pronounced manner than 5′-IMP did (3), thus confirming the human sensory data shown above. The average normalized fluorescence ratios of T1R1/R3-expressing

Figure 6. HPLC-MS/MS (ESI−) analysis of (A) purified sample of N2(propylthiomethyl)guanosine 5′-monophosphate (19) and (B) nucleotide 19 in a Maillard-type reaction mixture of guanosine 5′monophosphate (2), glucose, glycine, and 1-propanethiol. Signal intensity of the mass chromatograms is normalized.

after 14.1 min, was detected at the same retention time in the Maillard reaction system and was further verified by means of cochromatography. External quantitation of 19 by means of HPLC-MS/MS in reaction systems varying the molar carbohydrate/amino acid ratio between 1 and 1000 revealed that the generation of 19 went through a maximum of 6.5 μg/ J

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(4) Behrens, M.; Meyerhof, W.; Hellfritsch, C.; Hofmann, T. Sweet and umami taste: Natural products, their chemosensory targets, and beyond. Angew. Chem., Int. Ed. 2011, 50, 2220−2242. (5) Zhang, F.; Klebansky, B.; Fine, R. M.; Xu, H.; Pronin, A.; Liu, H.; Tachdijian, C.; Li, X. Molecular mechanism for the umami taste synergism. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 20930−20934. (6) Yamaguchi, S.; Yoshikawa, T.; Ikeda, S.; Ninomiya, T. Measurement of the relative taste intensity of some L-α-amino acids and 5′-nucleotides. J. Food Sci. 1971, 36, 846−849. (7) Imai, K.-I.; Marumoto, R.; Kobayashi, K.; Yoshioka, Y.; Toda, J.; Honjo, M. Synthesis of compounds related to inosine 5′-phosphate and their flavor enhancing activity. IV. 2-Substituted inosine 5′phosphates. Chem. Pharm. Bull. 1971, 19, 576−586. (8) Mizuta, E.; Toda, J.; Suzuki, N.; Sugibayashi, H.-M.; Imai, K.-I.; Nishikawa, M. Structure-activity relationship in the taste effect of ribonucleotide derivatives. Chem. Pharm. Bull. 1972, 20, 1114−1124. (9) Cairoli, P.; Pieraccini, S.; Sironi, M.; Morelli, C. F.; Speranza, G.; Manitto, P. Studies on umami taste. Synthesis of new guanosine 5′phosphate derivatives and their synergistic effect with monosodium glutamate. J. Agric. Food Chem. 2008, 56, 1043−1050. (10) Morelli, C. F.; Manitto, P.; Speranza, G. Study on umami taste: the MSG taste-enhancing activity of N2-alkyl and N2-alkanoyl-5′guanylic acids having a sulfoxide group inside the N2-substituent. Flavour Fragrance J. 2011, 26, 279−281. (11) De Rijke, E.; Ruisch, B.; Bakker, J.; Visser, J.; Leene, J.; Haiber, S.; de Klerk, A.; Winkel, C.; König, Th. LC-MS study to reduce ion suppression and to identify N-lactoylguanosine 5′-monophosphate in bonito: a new umami molecule? J. Agric. Food Chem. 2007, 55, 6417− 6423. (12) Festring, D.; Hofmann, T. Discovery of N2-(1-carboxyethyl)guanosine 5′-monophosphate as an umami-enhancing Maillardmodified nucleotide in yeast extracts. J. Agric. Food Chem. 2010, 58, 10614−10622. (13) Festring, D.; Hofmann, T. Systematic studies on the chemical structure and umami enhancing activity of Maillard-modified guanosine 5′-monophosphates. J. Agric. Food Chem. 2011, 59, 665− 676. (14) Festring, D.; Brockhoff, A.; Meyerhof, W.; Hofmann, T. Stereoselective synthesis of amides sharing the guanosine 5′monophosphate scaffold and umami enhancement studies using human sensory and hT1R1/rT1R3 receptor assays. J. Agric. Food Chem. 2011, 59, 8875−8885. (15) Lu, K.; Ye, W.; Gold, A.; Ball, L. M.; Swenberg, J. Formation of S-[1-(N2-Deoxyguanosinyl)methyl]glutathione between glutathione and DNA induced by formaldehyde. J. Am. Chem. Soc. 2009, 131, 3414−3415. (16) Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.; Hofmann, T. Nature’s chemical signatures in human olfaction: A foodborne perspective for future biotechnology. Angew. Chem., Int. Ed. 2014, 53, 7124−7143. (17) Dewis, M. L.; Phan, T.-H. T.; Ren, Z.; Meng, X.; Cui, M.; Mummalaneni, S.; Rhyu, M.-R.; DeSimone, J. A.; Lyall, V. N-Geranyl cyclopropyl-carboximide modulates salty and umami taste in humans and animal models. J. Neurophysiol. 2013, 109, 1078−1090. (18) Xu, H.; Staszewski, L.; Tang, H.; Adler, E.; Zoller, M.; Li, X. Different functional roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14258−14263. (19) Intelmann, D.; Batram, C.; Kuhn, Ch.; Haseleu, G.; Meyerhof, M.; Hofmann, T. Three TAS2R bitter taste receptors mediate the psychophysical responses to bitter compounds of hops (Humulus lupulus L.) and beer. Chem. Percept. 2009, 2, 118−132. (20) Ottinger, H.; Hofmann, T. Identification of the taste enhancer alapyridaine in beef broth and evaluation of its sensory impact by taste reconstitution experiments. J. Agric. Food Chem. 2003, 51, 6791−6796. (21) Ottinger, H.; Soldo, T.; Hofmann, T. Discovery and structure determination of a novel Maillard-derived sweetness enhancer by application of the comparative taste dilution analysis (cTDA). J. Agric. Food Chem. 2003, 51, 1035−1041.

100 mL at a glucose/glycine ratio of 10 (Figure 7). Furthermore, varying the amount of 1-propanethiol between

Figure 7. Influence of glucose (0.001−1.0 mmol/L) on the formation of N2-(propylthiomethyl)guanosine 5′-monophosphate (19) from 2 (25 mmol/L), glycine (1 mol/L), and 1-propanethiol (5 mmol/L) in potassium phosphate buffer (2 mol/L, pH 7).

5 and 50 mmol/L at a glucose/glycine ratio of 10 revealed almost the same concentration of 19 varying between 5.8 and 7.1 μg/100 mL (data not shown), thus demonstrating the presence of formaldehyde rather than the thiol to be a limiting factor in the generation of 19. In summary, structurally versatile N2-(alkylthiomethyl)guanosine and N2-(arylthiomethyl)guanosine 5′-monophosphates were preparatively generated and evaluated in their activity to enhance the umami taste of monosodium Lglutamate in vivo (human sensory experiments) and in vitro (T1R1/T1r3 taste receptor assay). The most active umami enhancer, N2-(propylthiomethyl)guanosine 5′-monophosphate (19), showing a β-value of 5.1, was generated by Maillard-type reactions from guanosine 5′-monophosphate (2), glucose, glycine, and 1-propanethiol. Future studies are needed to fully exploit the potential of the exocyclic amino group of guanosine 5′-monophosphate as an effective anchor group to chemically fix small and hydrophobic food-derived molecules. This strategy opens a valuable avenue to produce high-potential chemosensorica from food-derived natural products by kitchentype chemistry.

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AUTHOR INFORMATION

Corresponding Author

*Phone +49-8161/71-2902; fax +49-8161/71-2949; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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L

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