Fourth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-4), www.mdpi.org/ecsoc-4.htm, September 1-30, 2000

[C0031]

S. Diem, B. Gutsche, M. Herderich*

Lehrstuhl für Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
Fax: +49-931-8885484, E-mail: [email protected]

Received: 14 August 2000 / Uploaded: 16 August

INTRODUCTION

Until recently, investigations on the glycosylation of amino acids during the Maillard reaction, on the formation of advanced glycation endproducts (AGE) and the postranslational modification of proteins mainly concentrated on the amino acids lysine, arginine, asparagine, serine and threonine whereas the significance of tryptophan remained largely unnoticed. While studying occurrence and relevance of novel tryptophan metabolites in biological systems, our attention on tryptophan glycosylation was attracted by reports on mannosylated tryptophan residues in proteins [1-6]. In this contribution, we describe the characterization and structure elucidation of novel tryptophan glycoconjugates resulting from the chemical condensation of tryptophan and aldohexoses and demonstrate their occurrence in food samples [7]. In addition, we report on the identification of tryptophan-N- and -C-glycoconjugates, namely N¹-(b-D-glucopyranosyl-⁴C₁)-L-tryptophan and 2-(a -manno-pyranosyl-¹C₄)-L-tryptophan, formed enzymatically as novel tryptophan metabolites in plants [8] and man [7].

CHEMICAL FORMATION OF TRYPTOPHAN GLYCOCONJUGATES

With regard to the reactivity of the indole moiety towards electrophilic attack, several reaction products of tryptophan with aldohexoses could be postulated (Figure 1).

Besides the well-known Amadori rearrangement product (ARP), we considered tryptophan-N-glycosides, -C-glycosyl derivatives and glyco-tetrahydro-b-carbolines as primary condensation products. To prove formation of these novel tryptophan glycoconjugates, we performed model reactions heating tryptophan with D-glucose, D-galactose and D-mannose to 80 �C at pH 2 for 12 days. HPLC-ESI-MS analysis of each model reaction yielded up to four tryptophan glycoconjugates from each of the aldohexoses with molecular ions m/z 367 [M+H]⁺ other than the established Amadori rearrangement product [9]. Subsequent isolation of the compounds by RP-18 column chromatography and characterization by tandem mass spectrometry combined with NMR spectroscopy including HH-COSY, HMBC and HMQC experiments led to identification of the postulated structures [7].

The product ion spectrum of the tryptophan-N-glycosides as obtained by low-energy CID of the molecular ion m/z 367 [M+H]⁺ is dominated by ions m/z 205 and m/z 188 corresponding to the loss of an intact anhydro-sugar moiety C₆H₁₀O₅ and subsequent loss of NH₃ (Figure 2A). This fragmentation pattern is characteristic for N-glycosidically linked sugars [10] whereas in product ion spectra of C-glycosyl derivatives and glyco-tetrahydro-b-carbolines the prominent ions m/z 247 and m/z 229 result from loss of a C₄H₈O₄ moiety and subsequent loss of H₂O (Figure 2B).

Differentiation between C-glycosyl derivatives and glyco-tetrahydro-b-carbolines was achieved by low energy CID of the deprotonated molecular ions m/z 365 [M-H]^-. Product ion spectra of C-glycosyl derivatives (Figure 3A) are dominated by typical loss of 120 amu (m/z 245) whereas the major product ion m/z 215 of glyco-tetrahydro-b-carbolines results from the loss of a C₅H₁₀O₅ moiety (Figure 3B).

These characteristic fragmentation patterns enabled us to differentiate tryptophan glycoconjugates in complex samples by HPLC-ESI-MS/MS according to their linkage type. In order to clarify the relevance of the newly identified tryptophan glycoconjugates, several food samples including seasoning sauces, alcoholic beverages and fruit products were analysed by means of HPLC-ESI-MS/MS. For these studies, limits of detection of about 100 ng/mL were achieved using selective reaction monitoring (SRM). A typical HPLC-MS/MS chromatogram of a soy sauce sample is shown in Figure 4.

Notably, most food samples analysed contained complex mixtures of tryptophan glycoconjugates with concentrations ranging from 100 ng/mL to more than 10 m g/mL. Both, product pattern and concentration demonstrate, that the chemical condensation reaction with aldohexoses can count responsible for modification of up to 30 % of available tryptophan in nutritionals samples.

ENZYMATIC TRYPTOPHAN GLYCOSYLATION IN PLANTS

During HPLC-MS/MS analysis of food samples, the observation of one distinct tryptophan-N-glycoside in fruit syrup, which was sold as natural sweetener, attracted our attention. Consequently, we studied the occurrence of tryptophan glycoconjugates in plants and detected a single glycosylation product in numerous fruit extracts and commercially available fruit juices (Figure 5) in concentrations between 0.1 mg/L and > 10 mg/L (Table 1).

Table 1. Tryptophan-N-glucoside in fruits and fruit juices.

^anumber of samples analysed

The product ion spectrum obtained by low-energy CID of the molecular ion m/z 367 [M+H]⁺ is dominated by the fragment ions m/z 205 and m/z 188 corresponding to loss of an intact anhydro-sugar moiety C₆H₁₀O₅ and subsequent loss of NH₃. As mentioned above, this fragmentation pattern is characteristic for N-glycosidically linked sugars [10], an assignment that is further supported by the retention time of the compound during HPLC analysis. Yet, information about configuration and conformation of the sugar residue is not readily available from the product ion spectra. Motivated by the adequate concentration of tryptophan-N-glycoside in pear juice, we started the isolation from this material. HR-ESI-MS analysis confirmed the molecular formula C₁₇H₂₃N₂O₇ for the molecular ion m/z 367 [M+H]⁺. The structure of the purified compound was deduced by ¹H, HH-COSY and ¹³C NMR spectroscopy [8]. The results are in good accordance with those of the N-glucoside which was prepared chemically [7]. Thus, the identity of the isolated compound was unambiguously established as N¹-(b-D-glucopyranosyl-⁴C₁)-L-tryptophan (Figure 6).

Figure 6. N¹-(b-D-glucopyranosyl-⁴C₁)-L-tryptophan.

Previous investigation of reactions of tryptophan with aldohexoses had revealed that temperatures above 50 �C and acidic pH were a prerequisite for formation of tryptophan-N-glycosides. Furthermore, by the chemical condensation reaction C-glycosyl conjugates and glyco-tetrahydro-b-carbolines were always generated together with N-glycosides [7]. Therefore, occurrence of a single N-glucoside in fruits lacking any heat treatment strongly suggested the specific enzymatic biosynthesis in the plant. To obtain deeper insight into the origin of the novel glycoconjugate, we studied formation of deuterium-labeled tryptophan glycoderivatives. To select appropriate ions for these SRM experiments we performed model reactions, heating L-[indole-d₅]-tryptophan with D-glucose to 80 �C at pH 1 for 12 days. As expected, MS analysis of the model reactions yielded the d₄-C-glucosyl conjugate and d₄-gluco-tetrahydro-b-carbolines detected by their molecular ions m/z 371 [M+H]⁺ as result of the loss of the deuterium at indole C-2 during the condensation reaction. However, for the chemically prepared N-glucoside molecular ions m/z 371 [M+H]⁺ (d₄-tryptophan-N-glucoside) besides m/z 372 [M+H]⁺ (d₅-tryptophan-N-glucoside) were found. This can be explained by deuterium-exchange at C-2: Following reversible protonation of C-3 in strongly acidic medium, H/D-exchange can occur by migration of deuterium from C-2 to C-3 and subsequent loss of deuterium during deprotonation [11,12]. In the product ion spectrum of the d₅-N-glucoside the prominent ions m/z 210 and m/z 192 result from loss of an intact anhydro-sugar moiety C₆H₁₀O₅ and subsequent loss of NH₂D. Based on the results of the model experiments with d₅-tryptophan the following ions were selected for subsequent SRM experiments: m/z 367/188 for d₀-tryptophan-N-glucoside, m/z 372/192 for d₅-tryptophan-N-glucoside and m/z 371/251 for the d₄-tryptophan-C-glucosyl derivative and d₄-gluco-tetrahydro-b-carbolines.

To investigate formation of deuterium-labeled tryptophan glycoconjugates in pear fruits, we injected L-[indole-d₅]-tryptophan subepidermally into the fruits. After storage at room temperature for 2 to 15 days, the fruit extracts were prepared as usual and analysed by HPLC-MS/MS. These experiments exclusively proved formation of the corresponding d₅-tryptophan-N-glucoside, while the presence of any chemically formed d₄-C-glucosyl conjugate and d₄-gluco-tetrahydro-b-carboline could be excluded conclusively (Figure 7).

Figure 7. HPLC-MS/MS analysis of pear juice obtained after application of d₅-tryptophan. Mass chromatograms A: for d₀-tryptophan-N-glycoside. B: for d₄-C-glycosyl conjugate and d₄-glyco-tetrahydro-b-carbolines. C: for d₅-tryptophan-N-glycoside.

To rule-out non-enzymatic condensation of tryptophan and glucose during storage of fruits, we also incubated d₅-tryptophan in a model juice containing 2.2 % glucose, 6 % fructose and 1.1 % sucrose with a pH of 3.8 (adjusted with malic acid). The sugar content and pH value were adjusted according to data published for pear fruits [13]. Again, in " pear model juice" no formation of deuterium-labeled tryptophan glycoconjugates was observed. Finally, we compared the mass spectra of deuterated N-glucoside obtained from chemical condensation with that of the deuterated N-glucoside found in pear juice after application of d₅-tryptophan (Figure 8).

As mentioned above, the chemically formed N-glucoside yields the molecular ions m/z 371 [M+H]⁺ and m/z 372 [M+H]⁺ due to deuterium exchange. In contrast, the N-glucoside in pear juice exclusively shows the molecular ion m/z 372 [M+H]⁺ as result of enzymatic biosynthesis which yields d₅-tryptophan-N-glucoside from the labeled precursor in a highly specific manner. Taken together, our data convincingly demonstrate the enzymatic formation of the N-glucoside from the amino acid tryptophan in pear fruits.

As conclusion, this is the first report on naturally occurring N¹-(b-D-glucopyranosyl-⁴C₁)-L-tryptophan representing a novel intermediate of tryptophan metabolism in plants. Future research will establish the potential of this compound to serve as a marker substance for authenticity control and will provide a reliable analytical basis for the detection of adulteration and processing of fruit products. Furthermore, the identification of this particular tryptophan metabolite provides the means to study enzymatic N-glycosylation reactions in plants [14] in greater detail.

ENZYMATIC TRYPTOPHAN GLYCOSYLATION IN MAN

Following the detection of tryptophan glycoconjugates in food as reported above, we analysed human urine for the occurrence of these tryptophan derivatives. By applying HPLC-ESI-MS, unexpectedly a single tryptophan glycoconjugate was detected (Figure 9).

The product ion spectrum as obtained by low-energy CID of the protonated molecule (ESI positive mode) shows a fragmentation pattern dominated by the loss of 120 amu, corresponding to the cleavage of a C₄H₈O₄-moiety characteristic for C-glycosyl derivatives and glyco-tetrahydro-b-carbolines. Low energy CID of the deprotonated molecular ion (ESI negative mode) yields a product ion m/z 245 again resulting from the characteristic loss of a C₄H₈O₄-moiety (Figure 9). Thus, the compound in human urine convincingly proved to be a C-glycosyl derivative of tryptophan. To further characterize the sugar residue of the glycoconjugate, we verified the structure by ¹H- and ¹³C-NMR spectroscopy as well as by HH-COSY, HMQC and HMBC experiments. Furthermore, NOE experiments and acid hydrolysis followed by GC-MS analysis of MOA derivatives enabled us to determine configuration and conformation of the hexopyranosyl moiety. Finally, the human metabolite was identified as 2-(a -mannopyranosyl)-L-tryptophan preferentially adopting a ¹C₄-conformation (Figure 10) [7].

Thus, the structure of the carbohydrate moiety is consistent with the protein-bound 2-(a -mannopyranosyl)-moiety in RNase [1-3]. Concerning formation of the novel tryptophan metabolite, one has to consider enzymatic glycosylation of free tryptophan as well as degradation of proteins containing mannosylated tryptophan residues. Interestingly, Hofsteenge and coworkers reported on a novel posttranslational modification of tryptophan residues in proteins identifying the C-glycosidic attachment of a -D-mannopyranose to the C-2 of the indole ring of Trp⁷ in human RNase [1-3]. This novel kind of protein glycosylation proved to be enzyme catalyzed employing dolichyl phosphate mannose as a precursor [4,5]. Recent studies revealed that the C-mannosylation of tryptophan residues is a rather common modification and occurs for example in four proteins of the human complement system: in the complete " membrane attack complex" 50 of the 113 tryptophan residues were found to undergo C-mannosylation [6]. Still, clarification of the mechanism of the biosynthesis of 2-(a -mannopyranosyl)-L-tryptophan as well as its role in human metabolism requires further study.

CHEMICAL VERSUS ENZYMATIC GLYCOSYLATION OF TRYPTOPHAN

Applying extensive HPLC-MS/MS analysis, our studies on glycosylation of tryptophan clearly demonstrate the reactivity of the indole moiety towards modification by carbohydrates. In plant-derived nutritional sources up to 30 % of the available amino acid can be converted into the respective glycoderivatives by non-selective chemical condensation reactions. Interestingly, in numerous fruits selective and enzyme-catalyzed glycosyl transfer was observed, yielding exclusively N¹-(b-D-glucopyranosyl-⁴C₁)-L-tryptophan. Likewise, in humans specific formation of the novel C-glycosyl derivative 2-(a -mannopyranosyl-¹C₄)-L-tryptophan underlines the relevance of specific glycosylation reactions in biological systems. As conclusion, structural identification of both metabolites in plants and humans stimulates further studies on biosynthesis and function of tryptophan glycoconjugates.

REFERENCES

1 Hofsteenge, J., Mueller, D. R., de Beer, T., Loeffler, A., Richter, W. J. and Vliegenthart, J. F. G. New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us. Biochem. 1994, 33, 13524-13530.

2 de Beer, T., Vliegenthart, J. F. G., Loeffler, A. and Hofsteenge, J. The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is a-mannopyranose. Biochem. 1995, 34, 11785-11789.

3 Loeffler, A., Doucey, M. - A., Jansson, A. M., Mueller, D. R., de Beer, T., Hess, D., Meldal, M., Richter, W. J., Vliegenthart, J. F. G. and Hofsteenge, J. Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms. Biochem. 1996, 35, 12005-12014.

4 Doucey, M.-A., Hess, D., Cacan, R. and Hofsteenge, J. Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor. Mol. Biol. Cell. 1998, 9, 291-300.

5 Krieg, J., Hartmann, S., Vicentini, A., Glaesner, W., Hess, D. and Hofsteenge, J. Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp. Mol. Biol. Cell 1998, 9, 301-309.

6 Hofsteenge J.; Blommer M.; Hess D.; Furmanek A.; Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues. J. Biol. Chem. 1999, 274, 32786-32794.

7 Gutsche, B.; Grun, C.; Scheutzow, D.; Herderich, M. Tryptophan glycoconjugates in food and human urine. Biochem. J. 1999, 343, 11-19.

8 Diem, S.; Bergmann, J.; Herderich, M. Tryptophan-N-glucoside in fruits and fruit juices. J. Agric. Food Chem. 2000, in press.

9 Yaylayan, V. and Forage, N. G. Determination of the kinetics and mechanism of decomposition of tryptophan amadori rearrangement product by RP-HPLC analysis. J. Agric. Food Chem. 1991, 39, 364-369.

10 Frear, D. S.; Swanson, H. R.; Mansager, E. R. Picloram metabolism in leafy spurge: Isolation and identification of glucose and gentiobiose conjugates. J. Agric. Food Chem. 1989, 37, 1408-1412.

11 Gilchchrist, T. L. Heterocyclenchemie. VCH, Weinheim, 1995, 239-241.

12 Jackson, A. H.; Smith, P. Electrophilic substitution in indoles III - Rearrangement of 3,3-dialkyl indolenines. Tetrahedron 1968, 24, 2227-2239.

13 Belitz, H.-D.; Grosch, W. Lehrbuch der Lebensmittelchemie. Springer, Berlin, 1992, 723-774.

14 Rayon, C.; Lerouge, P.; Faye, L. The protein N-glycosylation in plants. J. Exp. Botany 1998, 49, 1463-1472.

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