Food Chemistry 158 (2014) 340–344
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ZnCl2-mediated practical protocol for the synthesis of Amadori ketoses Nanishankar V. Harohally ⇑, Sudhanva M. Srinivas, Sushma Umesh Food Safety and Analytical Quality Control Laboratory, CSIR-CFTRI, KRS Road, Mysore 570020, Karnataka, India
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 6 February 2014 Accepted 20 February 2014 Available online 5 March 2014 Keywords: Amadori rearrangement Zinc ACE
a b s t r a c t An efficient and practical protocol for the synthesis of Amadori ketoses N-(1-deoxy-D-fructose-1-yl) amino acid (amino acid = L-valine (1), L-leucine (2), L-isoleucine (3), L-tryptophan (4), L-phenylalanine (5), L-arginine (6) has been accomplished by employing ZnCl2 as a catalyst. The developed method circumvents protection and deprotection steps as well as tedious ion-exchange and column chromatographic techniques. The accomplished Amadori ketoses showed moderate to weak angiotensin I converting enzyme (ACE) inhibitory activity. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Amadori rearrangement is a landmark reaction of carbohydrate chemistry reported a century ago (Maillard, 1912; Hodge 1955; Wrodnigg & Eder, 2001). It has attracted pivotal importance in the context of Maillard chemistry and glycation (Finot 2005; Hodge 1953; Monnier et al., 2008; Zhang, Ames, Smith, Baynes, & Metz, 2009). Amadori rearrangement allows the formation of 1-aminodeoxyketoses by reaction of aldose with amine/amino acids, without affecting the other functional groups. Amadori rearrangement comprises several steps wherein each step is reversible and product formation is mainly driven by the nature of the carbohydrate, amine/amino acids, as well as reaction parameters including temperature and duration. Historically, acetic acid has been extensively employed as a Lewis acid catalyst and also to reduce the pH of the reaction mixture. However, acetic acid as a Lewis acid catalyst is not successful in furnishing a high yield and also necessitates employment of tedious ion-exchange chromatography to achieve acceptable purity. Alternate synthetic approaches for accomplishing Amadori products have been intensely pursued but require multistep reactions with unavoidable protection and deprotection steps (Iwamoto, Kan, Katsumura, & Ohfune, 1996; Kojic-prodic et al., 1995; Turner et al., 1999). We recently reported an improved method for arginine and lysinederived simple Amadori and Heyns products, however, it is not applicable to numerous substrates, as it requires the employment of amino acid hydrochloride instead of amino acid and also stoichiometric amount of zinc powder is utilized to achieve the products
⇑ Corresponding author. Tel.: +91 821 2514972; fax: +91 821 2412064. E-mail address: [email protected] (N.V. Harohally). http://dx.doi.org/10.1016/j.foodchem.2014.02.094 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
(Srinivas & Harohally, 2012). With this backdrop, we envisioned to develop a catalytic method applicable to amino acids and also employing ZnCl2 as a newer Lewis acid catalyst. We present herein an improved method for synthesis of Amadori ketoses mediated by ZnCl2 catalyst, avoiding protection and deprotection steps as well as separation techniques to achieve pure compounds. 2. Materials and methods Hippuryl-histidyl-leucine, hippuric acid, sodium tetraborate, and Triton X-100 were procured from Sigma–Aldrich (St. Louis, MO). L-tryptophan, L-leucine, L-isoleucine, L-valine, and L-phenylalanine were procured from Loba chemie. Glacial acetic acid, D-glucose and were purchased from Merck India. Pyridine, zinc chloride and benzenesulfonyl chloride were from Spectrochem Pvt. Ltd. NMR spectra were recorded on a Bruker Avance instrument (400 MHz for 1H and 100 MHz for 13C{H} experiments). Proton and carbon chemical shifts are given relative to internal HOD signal (4.79 ppm) and external standard tetramethylsilane for D2O solutions. NMR spectral assignments were based on 1H, 13 C, HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond correlation), DEPT (distortionless enhancement by polarisation transfer) experiments. Mass spectra were recorded on a Q-TOF ULTIMA instrument (Waters Corporation, Milford, MA) in the ESI positive ion mode. L-arginine
2.1. N-(1-Deoxy-D-fructose-1-yl)valine (1) In an Erlenmeyer flask containing a magnetic stir bar, L-valine (1.17 g, 10 mmol) was dissolved in a mixture of solvent consisting of pyridine (30 mL) and glacial acetic acid (30 mL) for 1–1.5 h. Zinc
N.V. Harohally et al. / Food Chemistry 158 (2014) 340–344
chloride (25 mg, 0.183 mmol) was added to the reaction mixture and subsequently D-glucose (1.80 g, 10 mmol) was dropped into the reaction mixture and stirring was carried out for 5 days at room temperature (30 °C). The reaction mixture was filtered and the filtrate was concentrated under vacuum to about 3–4 mL. Acetone was added (100 mL) to get a cream-coloured solid which was allowed to settle. Acetone was removed using a cannula under nitrogen pressure without disturbing the product. Subsequently, addition of acetone (50 mL), stirring, and further removal was carried out three or four times, followed by diethyl ether (50 mL), to remove completely pyridine. Finally, the residual solvents were removed under vacuum to get an off-white solid. Yield = 1.745 g, 62%. ESI-MS positive ion mode (M + H)+ m/z 280.1780, Exact mass (M + H)+ 280.1396. NMR data of b-pyranose form: 1.07 (d, 3H, J = 6.8 Hz, H3C(10)), 1.00 (d, 3H, J = 6.8 Hz, H3C(9)), 2.27 (m, 1H, HC(8)), 3.62 (d, 1H, J = 4.2 Hz, HC(7)), 3.30 (m, 2H, H2C(6)), 3.73 (d, 1H, J = 12.4 Hz, HC(5)), 4.00 (d, 1H, J = 12.4 Hz, HC(5)), 3.97 (m, 1H, HC(4)), 3.86 (dd, 1H, J = 3.2 Hz, J = 9.8 Hz, HC(3)), 3.73 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 172.33 (C-11), 17.04 (C-10), 18.60 (C-9), 29.16 (C-8), 69.18 (C-7), 53.48 (C-6), 64.00 (C-5), 69.02 (C-4), 69.4 7(C-3), 70.27 (C-2), 95.40 (C-1). 2.2. N-(1-Deoxy-D-fructose-1-yl)leucine (2) This compound was prepared utilising L-leucine (1.32 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol) following a similar procedure to that for compound 1. Yield = 1.91 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 294.1808, exact mass (M + H)+ 294.1552. NMR data of b-pyranose form: 0.94 (t, 3H, H3C(11)), 0.94 (t, 3H, H3C(10)), 1.68 (m, 1H, HC(9)), 1.68 (m, 2H, H2C(8)), 3.60 (m, 2H, HC(7)), 3.14 (d, 1H, J = 13 Hz, HC(6)), 3.20 (d, 1H, J = 3 Hz, HC(6)), 3.73 (d, 1H, J = 12.9 Hz, HC(5)), 4.00 (d, 1H, J = 12.9 Hz, HC(5)), 3.98 (m, 1H, HC(4)), 3.86 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz, HC(3)), 3.74 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 175.98 (C-12), 22.11 (C-11), 21.46 (C-10), 24.64 (C-9), 39.87 (C-8), 62.46 (C-7), 52.49 (C-6), 63.99 (C-5), 69.01 (C-4), 69.46 (C-3), 69.81 (C-2), 95.86 (C-1). 2.3. N-(1-deoxy-D-fructose-1-yl)isoleucine (3) Synthesis of this compound was performed similar to compound 1, utilising L-isoleucine (1.33 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 1.824 g, 62%. ESI-MS positive ion mode (M + H)+ m/z 293.9759, exact mass (M + H)+ 294.1552. NMR data of b-pyranose form: 0.94 (d, 3H, H3C(11)), 0.96 (t, 3H, H3C(10)), 1.54 (m, 2H, HC(9)), 1.34 (m, 2H, H2C(8)), 1.98 (m, 1H, HC(7)), 3.28 (d, 2H, J = 13 Hz, H2C(6)), 3.75 (d, 1H, J = 12.7 Hz, HC(5)), 4.03 (d, 1H, J = 12.7 Hz, HC(5)),4.00 (m, 1H, HC(4)), 3.88 (dd, 1H, J = 2.7 Hz, J = 9.9 Hz, HC(3)), 3.75 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 171.65 (C-12), 12.19 (C-11), 8.59 (C-10), 21.96 (C-9), 33.39 (C-8), 61.41 (C-7), 57.07 (C-6), 60.88 (C-5), 65.08 (C-4), 66.71 (C-3), 67.20 (C-2), 95.58 (C-1). 2.4. N-(1-Deoxy-D-fructose-1-yl)tryptophan (4) Synthesis of this compound was accomplished similar to compound 1, utilising L-tryptophan (2.05 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 2.38 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 367.0481, exact mass (M)+ 367.1505. NMR data of b-pyranose form: 7.49 (d, 1H, HC(15)), 7.69 (m, 1H, HC(14)), 7.16 (m, 1H, HC(13)) 7.24 (m, 1H, HC(12)), 7.30 (s, 1H, HC(9)), 3.36 (dd, 1H, J = 15.3 Hz, J = 7.0 Hz, HC(8)), 3.47 (dd, 1H, J = 15.4 Hz, J = 4.7 Hz, HC(8)), 4.04 (d, 1H, HC(7)), 3.14 (d, 1H, J = 12.8 Hz, HC(6)), 3.28 (d, 1H, J = 12.8 Hz, HC(6)), 3.58 (d, 1H, J = 12.7 Hz, HC(5)), 3.84 (d,
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1H, J = 12.7 Hz, HC(5)), 3.96 (m, 1H, HC(4)), 3.76 (dd, 1H, J = 9.8 Hz, J = 3.3 Hz, HC(3)), 3.62 (d, 1H, J = 9.8 Hz, HC(2)); 13C NMR dC 173.03 (C-17), 136.32 (C-16), 111.90 (C-15), 118.45 (C14), 119.27 (C-13), 122.29 (C-12), 126.59 (C-11), 107.05 (C-10), 125.02 (C-9), 25.65 (C-8), 63.07 (C-7), 52.65 (C6), 63.73 (C-5), 68.75 (C-4), 69.22 (C-3), 70.23 (C-2), 95.03 (C-1). 2.5. N-(1-deoxy-D-fructose-1-yl)phenylalanine (5) Synthesis of this compound was accomplished similar to compound 1, utilising L-phenylalanine (1.65 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 2.115 g, 64%. ESI-MS positive ion mode (M)+ m/z 328.1609, exact mass (M + H)+ 328.1396. NMR data of b-pyranose form: 7.31 (d, 2H, HC(14)), 7.41 (m, 1H, HC(13)) 7.36 (m, 1H, HC(12)), 7.41 (t, 1H, HC(11)), 7.31 (d, 3H, HC(10)), 3.23 (d, 2H, J = 6.5 Hz, H2C(8)), 3.99 (m, 1H, HC(7)), 3.23 (d, 1H, J = 12.8 Hz, HC(6)), 3.30 (d, 1H, J = 12.8 Hz, HC(6)), 3.70 (d, 1H, J = 12.6 Hz, HC(5)), 3.95 (d, 1H, J = 12.6 Hz, HC(5)), 3.96 (m, 1H, HC(4)), 3.85 (dd, 1H, J = 3.1 Hz, J = 10.0 Hz, HC(3)), 3.70 (d, 1H, J = 9.9 Hz, HC(2); 13C NMR dC 172.61 (C-15), 129.56 (C-14), 129.30 (C-13), 127.94 (C-12), 129.30 (C-11), 129.56 (C-10), 134.95 (C-9), 35.83 (C-8), 64.26 (C-7), 53.11 (C-6), 64.06 (C-5), 69.05 (C-4), 69.50 (C3), 70.44 (C-2), 95.31 (C-1). 2.6. N-(1-deoxy-D-fructose-1-yl)arginine (6) Synthesis of this compound was accomplished similar to compound 1 utilising L-arginine (1.74 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol) Yield = 2.195 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 337.1723, exact mass (M + H)+ 337.1743. NMR data of b-pyranose form: 1.56 (m, 2H, H2C(9)), 1.56 (m, 2H, H2C(8)), 3.15 (t, 2H, J = 6.4 Hz, H2C(10)), 3.09 (t, 1H, J = 5.9 Hz, H2C(7)), 3.70 (d, 1H, J = 10.1 Hz, H2C(2)), 3.62 (dd, 1H, J = 1.8 Hz, J = 12.8 Hz, HC(5)), 3.96 (d, 1H, J = 12.8 Hz, HC(5)), 3.92 (d, 1H, J = 1.8 Hz, HC(4)), 2.72 (bs, 2H, HC(6)), 3.81 (dd, 1H, J = 3.5 Hz, J = 9.8 Hz, HC(3)); 13 C NMR dC 184.78 (C-12), 159.43 (C-11), 43.61 (C-10), 27.34 (C9), 32.48 (C-8), 66.52 (C-7), 55.51 (C-6), 65.95 (C-5), 71.85 (C-4), 72.41 (C-3), 71.94 (C-2), 100.44 (C-1). 2.7. In vitro colorimetric assay of ACE inhibitor activity ACE was extracted from porcine kidney acetone powder using the procedure as reported earlier.12 ACE inhibitory activity was assayed by utilizing hippuryl-histidyl-leucine (HHL) as the substrate. The assay is carried out by monitoring the release of hippuric acid (HA) via hydrolysis of hippuryl-histidyl-leucine (HHL). The assay mixture comprised of 0.125 mL of 0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl, 0.05 mL of 5 mM HHL, and 0.025 mL of ACE enzyme extract (the enzyme extract incubated at 37 ± 2 °C with Amadori compound for 10 min). The reaction mixture which was incubated at 37 °C was arrested after 30 min by the addition of 0.2 mL of 1 M HCl. Then to this solution, 0.4 mL of pyridine were added and subsequently 0.2 mL of benzenesulfonyl chloride (BSC). This solution was mixed by inverting for 1 min and cooled on ice. The yellow colour developed was measured at 410 nm. One unit of ACE activity is defined as the amount of enzyme that releases 1 lmol of HA per min at 37 °C and pH 8.2. The IC50 value is defined as the quantity of the inhibitor required to decrease the ACE activity by 50%. The inhibition curves (residual activity) were plotted using a minimum of seven determinations for each inhibitor concentration, and IC50 values computed from the semi-logarithmic plots. A linear regression analysis was performed using Microsoft Excel. The IC50 values for Amadori
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compounds were determined in triplicates. The IC50 values are expressed as mean ± 2SD.
3. Results and discussion The aprotic solvent combination of pyridine and acetic acid was chosen for our efforts to accomplish Amadori ketoses, in view of its successful utilisation for the synthesis of Amadori and Heyns compounds (Keil, Mortensen, & Christophersen, 1985; Srinivas & Harohally, 2012). Kiel et al. have achieved the synthesis of Amadori compound 1 with a reaction duration of 4 days. Our initial efforts started with respect to optimisation of catalyst loading in the solvent combination of pyridine and acetic acid for the synthesis of 1 (Fig. 1). When we attempted the method developed by Keil et al. (1985) we observed that the reaction was not completed by 4 days (as indicated by NMR) and required 5 days of stirring. We then attempted the synthesis of 1 with ZnCl2 as a catalyst with loading of 2 mg (0.014 mmol) and reaction time consisting of 5 days, which displayed no yield enhancement for 1 (observed yield 29%). On further increase of ZnCl2 catalyst loading to 10 mg (0.073 mmol), we witnessed slight improvement (45% yield). More experiments led to the best yield of 62% for 25 mg (0.183 mmol) ZnCl2 catalyst (Table 1). With the best catalyst loading, we then tried to reduce the reaction duration from 5 days to a lesser period; however, this resulted in lower yield and necessitated chromatographic column separation. For the purpose of achieving higher yield and to circumvent tedious column separation, a reaction time of 5 days was employed. We were further interested in extending the optimised catalyst loading to different solvent systems including the classical methanol and acetic acid as well as a green solvent combination of aqueous acetic acid (Table 2). We screened methanol and acetic acid in the ratio (3:1) with 25 mg (0.183 mmol) as it represented the optimum catalyst loading. This solvent combination with ZnCl2 catalyst upon stirring for 5 days did not indicate any improvement with respect to yield (obtained yield 15%). Then, we switched to the green solvent combination consisting of water and acetic acid in the ratio (3:1). The ZnCl2 catalyst loading of 25 mg (0.183 mmol) with aqueous acetic acid solvent did not demonstrate any improvement in the yield (obtained yield 10%) and it necessitated
Table 1 Optimisation of catalyst loading. Compound
ZnCl2 amount
Yield (%)
1
2 mg (0.014 mmol) 10 mg (0.073 mmol) 25 mg (0.183 mmol)
29 45 62
Table 2 Optimisation of solvent system. Compound
Solvent system
Yield (%)
Separation technique
1
MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH
15 10 62 14 12 65 16 12 62 12 0 65 20 0 64 13 10 65
+ +
2
3
4
5
6
+ + – + + + + – + + + +
Reaction conditions:10 mmol of D-glucose + 10 mmol of amino acid with ZnCl2 (0.0183 mmol); MeOH–AcOH V/V ratio(3:1) and H2O–AcOH V/V ratio (3:1)
column chromatographic or ion-exchange chromatographic separation for achieving pure Amadori compound. Then, we screened all three solvent systems for the synthesis of various Amadori compounds (Table 2). The best solvent system in all cases was found to be pyridine-acetic acid with a yield of 62–65%. The yields of Amadori compounds were slightly lower compared to method reported by Srinivas and Harohally (2012), which could be attributed to longer reaction duration and also due to the nature of the catalyst. The method developed by Srinivas et al. utilises stoichiometric reagent zinc powder for deprotonation of amino acid hydrochloride
Fig. 1. Catalytic synthesis of Amadori compounds.
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and involves generation of in situ stoichiometric amount of zinc catalyst. The nature of zinc catalyst is not probed by Srinivas et al., whereas we employ ZnCl2 as a catalyst. The obtained compounds were characterised by combination of 1 H and 13C NMR and ESI-mass spectral techniques. Previous conformation studies based on 13C NMR spectroscopic investigations by Roper, Roper, and Heyns (1983) revealed that these Amadori ketoses exists in aqueous solution as an equilibrium of different conformers including major b-pyranose (61%), and minor a-furanose (16%), b-furanose (15%) and a-pyranose (6%). We observed similar ratios for all these compounds except for compound 6 which predominantly occurred in b-pyranose form with minor a-pyranose form. The plausible mechanism for formation of Amadori compounds is as shown in Fig. 2. The first step comprises the reaction of D-glucose with amino acid, resulting in the formation of Schiff base. The Schiff base then binds to Zn2+ forming a five-membered intermediate complex. Further, due to the exertion of electron pull by Zn2+, the imine bound to Zn2+ undergoes transformation to Zn–amine complex. Then, elimination of Zn2+, followed by thermodynamically favoured enol to keto transformation, leads to formation of open chain keto form of the Amadori product. Subsequent ring closure leads to the formation of the pyranose form of Amadori compound. Efforts were made to establish the mechanism by monitoring the progress of the reaction by NMR and IR; however, we could not get direct evidence for the depicted intermediates to support the proposed mechanism. The zinc complexes of amino/sugar-derived Schiff bases are well documented in the literature (Adam & Hall, 1982, Yong, Jiming, Changjian, & Yune, 1997; Sah, Rao, Wegelius, Kolehmainen, & Rissanen, 2001; Singhal, Ramanujam, Ravikumar, & Rao, 2006). Further it is established that amino/sugar-derived Schiff bases exist in moderately polar solvents (including DMSO) in the keto-amine form in slight excess compared to enol-imine form (Sah et al., 2001). In addition, it has been observed that metal complexes of amino/sugar-derived Schiff bases have lower stability constants in MeOH (Costamagna, Lillo, Matsuhiro, Nosedo, & Villagran, 2003). Based on these previous observations, we infer that zinc Schiff base complex is stabilised in the aprotic solvent combination pyridine-AcOH, compared to other solvent combination, which leads to Amadori ketoses formation with higher yield. The fact that we are able to isolate pure Amadori ketoses after work up (utilising acetone and ether) indicates that the Amadori ketoses are not forming any complexes with ZnCl2
Table 3 IC50 values of Amadori compounds.
a
Compound
IC50 values (lM)
1 2 3 4 5 6
1277 ± 28 1728 ± 48 1756 ± 40 1293 ± 24 1731 ± 30 1038 ± 36a
From Srinivas & Harohally, 2012.
(ESI-MS also supports this fact). The Amadori compounds were found to be stable at room temperature (27–28 °C) for at least 3 months as indicated by the NMR spectroscopy. The ACE inhibitor activity of compounds 1–5 were evaluated by employing a high throughput ACE assay based on colorimetry (Table 3) (Jimsheena & Gowda 2009, Mallikarjun Gouda, Gowda, Rao, & Prakash, 2006). Among the Amadori compounds, 2 and 3 showed similar activity against ACE enzyme due to their structural similarity. On the other hand, compounds 1 and 4 displayed slightly lower activity compared to compound 6, indicating similar donor abilities compared to that of arginine towards Zn2+ of ACE enzyme. Compounds 2, 3 and 5 displayed lower ACE inhibitory activity compared to other compounds, suggesting weaker interaction of these compounds with the active site Zn2+. Lower IC50 value for compound 4, compared to 5, could be attributed to better donor abilities of L-tryptophan compared to L-phenylalanine. The IC50 values of compounds 1, 3 and 5 are comparable to fructose esters of amino acids consisting of 1-O-L-valyl-D-fructose (IC50 = 2.8 ± 0.29 mM), 1-O-L-phenylalanyl-D-fructose (IC50 = 13.6 ± 1.43 mM) and 1-O-L-tryptophanyl-D-fructose (IC50 = 0.9 ± 0.0092 mM) (Lohith, Vijayakumar, Somashekar, Sivakumar, & Divakar, 2006). Compounds 1 and 5 show significantly lower IC50 values compared to 1-O-L-valyl-D-fructose and 1-O-L-phenylalanyl-D-fructose and this is explained by considering the availability of the free carboxyl group in 1 and 5 and its interaction at the active site of ACE. 4. Conclusion We have demonstrated a catalytic method for synthesis of Amadori ketoses derived from amino acids, utilising ZnCl2 as a catalyst. This work may lead to the extension of the method to Amadori ketoses of numerous reducing sugar and amino acid derivatives; in addition it may also facilitate the employment of Amadori rearrangement reaction as a key reaction for facile synthesis of glyconjugates incorporating peptides and protein with carbohydrates. Acknowledgements This work was supported from an in-house CSIR project. We acknowledge Lalitha R.Gowda for providing ACE enzyme extract. We acknowledge Prasanna Vasu for helping in acquisition of mass spectral data. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 02.094. References
Fig. 2. Plausible mechanism for formation of Amadori compound.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
ZnCl2-mediated practical protocol for the synthesis of Amadori ketoses Nanishankar V. Harohally ⇑, Sudhanva M. Srinivas, Sushma Umesh Food Safety and Analytical Quality Control Laboratory, CSIR-CFTRI, KRS Road, Mysore 570020, Karnataka, India
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 6 February 2014 Accepted 20 February 2014 Available online 5 March 2014 Keywords: Amadori rearrangement Zinc ACE
a b s t r a c t An efficient and practical protocol for the synthesis of Amadori ketoses N-(1-deoxy-D-fructose-1-yl) amino acid (amino acid = L-valine (1), L-leucine (2), L-isoleucine (3), L-tryptophan (4), L-phenylalanine (5), L-arginine (6) has been accomplished by employing ZnCl2 as a catalyst. The developed method circumvents protection and deprotection steps as well as tedious ion-exchange and column chromatographic techniques. The accomplished Amadori ketoses showed moderate to weak angiotensin I converting enzyme (ACE) inhibitory activity. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Amadori rearrangement is a landmark reaction of carbohydrate chemistry reported a century ago (Maillard, 1912; Hodge 1955; Wrodnigg & Eder, 2001). It has attracted pivotal importance in the context of Maillard chemistry and glycation (Finot 2005; Hodge 1953; Monnier et al., 2008; Zhang, Ames, Smith, Baynes, & Metz, 2009). Amadori rearrangement allows the formation of 1-aminodeoxyketoses by reaction of aldose with amine/amino acids, without affecting the other functional groups. Amadori rearrangement comprises several steps wherein each step is reversible and product formation is mainly driven by the nature of the carbohydrate, amine/amino acids, as well as reaction parameters including temperature and duration. Historically, acetic acid has been extensively employed as a Lewis acid catalyst and also to reduce the pH of the reaction mixture. However, acetic acid as a Lewis acid catalyst is not successful in furnishing a high yield and also necessitates employment of tedious ion-exchange chromatography to achieve acceptable purity. Alternate synthetic approaches for accomplishing Amadori products have been intensely pursued but require multistep reactions with unavoidable protection and deprotection steps (Iwamoto, Kan, Katsumura, & Ohfune, 1996; Kojic-prodic et al., 1995; Turner et al., 1999). We recently reported an improved method for arginine and lysinederived simple Amadori and Heyns products, however, it is not applicable to numerous substrates, as it requires the employment of amino acid hydrochloride instead of amino acid and also stoichiometric amount of zinc powder is utilized to achieve the products
⇑ Corresponding author. Tel.: +91 821 2514972; fax: +91 821 2412064. E-mail address: [email protected] (N.V. Harohally). http://dx.doi.org/10.1016/j.foodchem.2014.02.094 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
(Srinivas & Harohally, 2012). With this backdrop, we envisioned to develop a catalytic method applicable to amino acids and also employing ZnCl2 as a newer Lewis acid catalyst. We present herein an improved method for synthesis of Amadori ketoses mediated by ZnCl2 catalyst, avoiding protection and deprotection steps as well as separation techniques to achieve pure compounds. 2. Materials and methods Hippuryl-histidyl-leucine, hippuric acid, sodium tetraborate, and Triton X-100 were procured from Sigma–Aldrich (St. Louis, MO). L-tryptophan, L-leucine, L-isoleucine, L-valine, and L-phenylalanine were procured from Loba chemie. Glacial acetic acid, D-glucose and were purchased from Merck India. Pyridine, zinc chloride and benzenesulfonyl chloride were from Spectrochem Pvt. Ltd. NMR spectra were recorded on a Bruker Avance instrument (400 MHz for 1H and 100 MHz for 13C{H} experiments). Proton and carbon chemical shifts are given relative to internal HOD signal (4.79 ppm) and external standard tetramethylsilane for D2O solutions. NMR spectral assignments were based on 1H, 13 C, HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond correlation), DEPT (distortionless enhancement by polarisation transfer) experiments. Mass spectra were recorded on a Q-TOF ULTIMA instrument (Waters Corporation, Milford, MA) in the ESI positive ion mode. L-arginine
2.1. N-(1-Deoxy-D-fructose-1-yl)valine (1) In an Erlenmeyer flask containing a magnetic stir bar, L-valine (1.17 g, 10 mmol) was dissolved in a mixture of solvent consisting of pyridine (30 mL) and glacial acetic acid (30 mL) for 1–1.5 h. Zinc
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chloride (25 mg, 0.183 mmol) was added to the reaction mixture and subsequently D-glucose (1.80 g, 10 mmol) was dropped into the reaction mixture and stirring was carried out for 5 days at room temperature (30 °C). The reaction mixture was filtered and the filtrate was concentrated under vacuum to about 3–4 mL. Acetone was added (100 mL) to get a cream-coloured solid which was allowed to settle. Acetone was removed using a cannula under nitrogen pressure without disturbing the product. Subsequently, addition of acetone (50 mL), stirring, and further removal was carried out three or four times, followed by diethyl ether (50 mL), to remove completely pyridine. Finally, the residual solvents were removed under vacuum to get an off-white solid. Yield = 1.745 g, 62%. ESI-MS positive ion mode (M + H)+ m/z 280.1780, Exact mass (M + H)+ 280.1396. NMR data of b-pyranose form: 1.07 (d, 3H, J = 6.8 Hz, H3C(10)), 1.00 (d, 3H, J = 6.8 Hz, H3C(9)), 2.27 (m, 1H, HC(8)), 3.62 (d, 1H, J = 4.2 Hz, HC(7)), 3.30 (m, 2H, H2C(6)), 3.73 (d, 1H, J = 12.4 Hz, HC(5)), 4.00 (d, 1H, J = 12.4 Hz, HC(5)), 3.97 (m, 1H, HC(4)), 3.86 (dd, 1H, J = 3.2 Hz, J = 9.8 Hz, HC(3)), 3.73 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 172.33 (C-11), 17.04 (C-10), 18.60 (C-9), 29.16 (C-8), 69.18 (C-7), 53.48 (C-6), 64.00 (C-5), 69.02 (C-4), 69.4 7(C-3), 70.27 (C-2), 95.40 (C-1). 2.2. N-(1-Deoxy-D-fructose-1-yl)leucine (2) This compound was prepared utilising L-leucine (1.32 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol) following a similar procedure to that for compound 1. Yield = 1.91 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 294.1808, exact mass (M + H)+ 294.1552. NMR data of b-pyranose form: 0.94 (t, 3H, H3C(11)), 0.94 (t, 3H, H3C(10)), 1.68 (m, 1H, HC(9)), 1.68 (m, 2H, H2C(8)), 3.60 (m, 2H, HC(7)), 3.14 (d, 1H, J = 13 Hz, HC(6)), 3.20 (d, 1H, J = 3 Hz, HC(6)), 3.73 (d, 1H, J = 12.9 Hz, HC(5)), 4.00 (d, 1H, J = 12.9 Hz, HC(5)), 3.98 (m, 1H, HC(4)), 3.86 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz, HC(3)), 3.74 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 175.98 (C-12), 22.11 (C-11), 21.46 (C-10), 24.64 (C-9), 39.87 (C-8), 62.46 (C-7), 52.49 (C-6), 63.99 (C-5), 69.01 (C-4), 69.46 (C-3), 69.81 (C-2), 95.86 (C-1). 2.3. N-(1-deoxy-D-fructose-1-yl)isoleucine (3) Synthesis of this compound was performed similar to compound 1, utilising L-isoleucine (1.33 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 1.824 g, 62%. ESI-MS positive ion mode (M + H)+ m/z 293.9759, exact mass (M + H)+ 294.1552. NMR data of b-pyranose form: 0.94 (d, 3H, H3C(11)), 0.96 (t, 3H, H3C(10)), 1.54 (m, 2H, HC(9)), 1.34 (m, 2H, H2C(8)), 1.98 (m, 1H, HC(7)), 3.28 (d, 2H, J = 13 Hz, H2C(6)), 3.75 (d, 1H, J = 12.7 Hz, HC(5)), 4.03 (d, 1H, J = 12.7 Hz, HC(5)),4.00 (m, 1H, HC(4)), 3.88 (dd, 1H, J = 2.7 Hz, J = 9.9 Hz, HC(3)), 3.75 (d, 2H, J = 10.0 Hz, HC(2)); 13C NMR dC 171.65 (C-12), 12.19 (C-11), 8.59 (C-10), 21.96 (C-9), 33.39 (C-8), 61.41 (C-7), 57.07 (C-6), 60.88 (C-5), 65.08 (C-4), 66.71 (C-3), 67.20 (C-2), 95.58 (C-1). 2.4. N-(1-Deoxy-D-fructose-1-yl)tryptophan (4) Synthesis of this compound was accomplished similar to compound 1, utilising L-tryptophan (2.05 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 2.38 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 367.0481, exact mass (M)+ 367.1505. NMR data of b-pyranose form: 7.49 (d, 1H, HC(15)), 7.69 (m, 1H, HC(14)), 7.16 (m, 1H, HC(13)) 7.24 (m, 1H, HC(12)), 7.30 (s, 1H, HC(9)), 3.36 (dd, 1H, J = 15.3 Hz, J = 7.0 Hz, HC(8)), 3.47 (dd, 1H, J = 15.4 Hz, J = 4.7 Hz, HC(8)), 4.04 (d, 1H, HC(7)), 3.14 (d, 1H, J = 12.8 Hz, HC(6)), 3.28 (d, 1H, J = 12.8 Hz, HC(6)), 3.58 (d, 1H, J = 12.7 Hz, HC(5)), 3.84 (d,
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1H, J = 12.7 Hz, HC(5)), 3.96 (m, 1H, HC(4)), 3.76 (dd, 1H, J = 9.8 Hz, J = 3.3 Hz, HC(3)), 3.62 (d, 1H, J = 9.8 Hz, HC(2)); 13C NMR dC 173.03 (C-17), 136.32 (C-16), 111.90 (C-15), 118.45 (C14), 119.27 (C-13), 122.29 (C-12), 126.59 (C-11), 107.05 (C-10), 125.02 (C-9), 25.65 (C-8), 63.07 (C-7), 52.65 (C6), 63.73 (C-5), 68.75 (C-4), 69.22 (C-3), 70.23 (C-2), 95.03 (C-1). 2.5. N-(1-deoxy-D-fructose-1-yl)phenylalanine (5) Synthesis of this compound was accomplished similar to compound 1, utilising L-phenylalanine (1.65 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol). Yield = 2.115 g, 64%. ESI-MS positive ion mode (M)+ m/z 328.1609, exact mass (M + H)+ 328.1396. NMR data of b-pyranose form: 7.31 (d, 2H, HC(14)), 7.41 (m, 1H, HC(13)) 7.36 (m, 1H, HC(12)), 7.41 (t, 1H, HC(11)), 7.31 (d, 3H, HC(10)), 3.23 (d, 2H, J = 6.5 Hz, H2C(8)), 3.99 (m, 1H, HC(7)), 3.23 (d, 1H, J = 12.8 Hz, HC(6)), 3.30 (d, 1H, J = 12.8 Hz, HC(6)), 3.70 (d, 1H, J = 12.6 Hz, HC(5)), 3.95 (d, 1H, J = 12.6 Hz, HC(5)), 3.96 (m, 1H, HC(4)), 3.85 (dd, 1H, J = 3.1 Hz, J = 10.0 Hz, HC(3)), 3.70 (d, 1H, J = 9.9 Hz, HC(2); 13C NMR dC 172.61 (C-15), 129.56 (C-14), 129.30 (C-13), 127.94 (C-12), 129.30 (C-11), 129.56 (C-10), 134.95 (C-9), 35.83 (C-8), 64.26 (C-7), 53.11 (C-6), 64.06 (C-5), 69.05 (C-4), 69.50 (C3), 70.44 (C-2), 95.31 (C-1). 2.6. N-(1-deoxy-D-fructose-1-yl)arginine (6) Synthesis of this compound was accomplished similar to compound 1 utilising L-arginine (1.74 g, 10 mmol), zinc chloride (25 mg, 0.183 mmol) and D-glucose (1.80 g, 10 mmol) Yield = 2.195 g, 65%. ESI-MS positive ion mode (M + H)+ m/z 337.1723, exact mass (M + H)+ 337.1743. NMR data of b-pyranose form: 1.56 (m, 2H, H2C(9)), 1.56 (m, 2H, H2C(8)), 3.15 (t, 2H, J = 6.4 Hz, H2C(10)), 3.09 (t, 1H, J = 5.9 Hz, H2C(7)), 3.70 (d, 1H, J = 10.1 Hz, H2C(2)), 3.62 (dd, 1H, J = 1.8 Hz, J = 12.8 Hz, HC(5)), 3.96 (d, 1H, J = 12.8 Hz, HC(5)), 3.92 (d, 1H, J = 1.8 Hz, HC(4)), 2.72 (bs, 2H, HC(6)), 3.81 (dd, 1H, J = 3.5 Hz, J = 9.8 Hz, HC(3)); 13 C NMR dC 184.78 (C-12), 159.43 (C-11), 43.61 (C-10), 27.34 (C9), 32.48 (C-8), 66.52 (C-7), 55.51 (C-6), 65.95 (C-5), 71.85 (C-4), 72.41 (C-3), 71.94 (C-2), 100.44 (C-1). 2.7. In vitro colorimetric assay of ACE inhibitor activity ACE was extracted from porcine kidney acetone powder using the procedure as reported earlier.12 ACE inhibitory activity was assayed by utilizing hippuryl-histidyl-leucine (HHL) as the substrate. The assay is carried out by monitoring the release of hippuric acid (HA) via hydrolysis of hippuryl-histidyl-leucine (HHL). The assay mixture comprised of 0.125 mL of 0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl, 0.05 mL of 5 mM HHL, and 0.025 mL of ACE enzyme extract (the enzyme extract incubated at 37 ± 2 °C with Amadori compound for 10 min). The reaction mixture which was incubated at 37 °C was arrested after 30 min by the addition of 0.2 mL of 1 M HCl. Then to this solution, 0.4 mL of pyridine were added and subsequently 0.2 mL of benzenesulfonyl chloride (BSC). This solution was mixed by inverting for 1 min and cooled on ice. The yellow colour developed was measured at 410 nm. One unit of ACE activity is defined as the amount of enzyme that releases 1 lmol of HA per min at 37 °C and pH 8.2. The IC50 value is defined as the quantity of the inhibitor required to decrease the ACE activity by 50%. The inhibition curves (residual activity) were plotted using a minimum of seven determinations for each inhibitor concentration, and IC50 values computed from the semi-logarithmic plots. A linear regression analysis was performed using Microsoft Excel. The IC50 values for Amadori
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compounds were determined in triplicates. The IC50 values are expressed as mean ± 2SD.
3. Results and discussion The aprotic solvent combination of pyridine and acetic acid was chosen for our efforts to accomplish Amadori ketoses, in view of its successful utilisation for the synthesis of Amadori and Heyns compounds (Keil, Mortensen, & Christophersen, 1985; Srinivas & Harohally, 2012). Kiel et al. have achieved the synthesis of Amadori compound 1 with a reaction duration of 4 days. Our initial efforts started with respect to optimisation of catalyst loading in the solvent combination of pyridine and acetic acid for the synthesis of 1 (Fig. 1). When we attempted the method developed by Keil et al. (1985) we observed that the reaction was not completed by 4 days (as indicated by NMR) and required 5 days of stirring. We then attempted the synthesis of 1 with ZnCl2 as a catalyst with loading of 2 mg (0.014 mmol) and reaction time consisting of 5 days, which displayed no yield enhancement for 1 (observed yield 29%). On further increase of ZnCl2 catalyst loading to 10 mg (0.073 mmol), we witnessed slight improvement (45% yield). More experiments led to the best yield of 62% for 25 mg (0.183 mmol) ZnCl2 catalyst (Table 1). With the best catalyst loading, we then tried to reduce the reaction duration from 5 days to a lesser period; however, this resulted in lower yield and necessitated chromatographic column separation. For the purpose of achieving higher yield and to circumvent tedious column separation, a reaction time of 5 days was employed. We were further interested in extending the optimised catalyst loading to different solvent systems including the classical methanol and acetic acid as well as a green solvent combination of aqueous acetic acid (Table 2). We screened methanol and acetic acid in the ratio (3:1) with 25 mg (0.183 mmol) as it represented the optimum catalyst loading. This solvent combination with ZnCl2 catalyst upon stirring for 5 days did not indicate any improvement with respect to yield (obtained yield 15%). Then, we switched to the green solvent combination consisting of water and acetic acid in the ratio (3:1). The ZnCl2 catalyst loading of 25 mg (0.183 mmol) with aqueous acetic acid solvent did not demonstrate any improvement in the yield (obtained yield 10%) and it necessitated
Table 1 Optimisation of catalyst loading. Compound
ZnCl2 amount
Yield (%)
1
2 mg (0.014 mmol) 10 mg (0.073 mmol) 25 mg (0.183 mmol)
29 45 62
Table 2 Optimisation of solvent system. Compound
Solvent system
Yield (%)
Separation technique
1
MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH MeOH–AcOH H2O–AcOH Pyridine–AcOH
15 10 62 14 12 65 16 12 62 12 0 65 20 0 64 13 10 65
+ +
2
3
4
5
6
+ + – + + + + – + + + +
Reaction conditions:10 mmol of D-glucose + 10 mmol of amino acid with ZnCl2 (0.0183 mmol); MeOH–AcOH V/V ratio(3:1) and H2O–AcOH V/V ratio (3:1)
column chromatographic or ion-exchange chromatographic separation for achieving pure Amadori compound. Then, we screened all three solvent systems for the synthesis of various Amadori compounds (Table 2). The best solvent system in all cases was found to be pyridine-acetic acid with a yield of 62–65%. The yields of Amadori compounds were slightly lower compared to method reported by Srinivas and Harohally (2012), which could be attributed to longer reaction duration and also due to the nature of the catalyst. The method developed by Srinivas et al. utilises stoichiometric reagent zinc powder for deprotonation of amino acid hydrochloride
Fig. 1. Catalytic synthesis of Amadori compounds.
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and involves generation of in situ stoichiometric amount of zinc catalyst. The nature of zinc catalyst is not probed by Srinivas et al., whereas we employ ZnCl2 as a catalyst. The obtained compounds were characterised by combination of 1 H and 13C NMR and ESI-mass spectral techniques. Previous conformation studies based on 13C NMR spectroscopic investigations by Roper, Roper, and Heyns (1983) revealed that these Amadori ketoses exists in aqueous solution as an equilibrium of different conformers including major b-pyranose (61%), and minor a-furanose (16%), b-furanose (15%) and a-pyranose (6%). We observed similar ratios for all these compounds except for compound 6 which predominantly occurred in b-pyranose form with minor a-pyranose form. The plausible mechanism for formation of Amadori compounds is as shown in Fig. 2. The first step comprises the reaction of D-glucose with amino acid, resulting in the formation of Schiff base. The Schiff base then binds to Zn2+ forming a five-membered intermediate complex. Further, due to the exertion of electron pull by Zn2+, the imine bound to Zn2+ undergoes transformation to Zn–amine complex. Then, elimination of Zn2+, followed by thermodynamically favoured enol to keto transformation, leads to formation of open chain keto form of the Amadori product. Subsequent ring closure leads to the formation of the pyranose form of Amadori compound. Efforts were made to establish the mechanism by monitoring the progress of the reaction by NMR and IR; however, we could not get direct evidence for the depicted intermediates to support the proposed mechanism. The zinc complexes of amino/sugar-derived Schiff bases are well documented in the literature (Adam & Hall, 1982, Yong, Jiming, Changjian, & Yune, 1997; Sah, Rao, Wegelius, Kolehmainen, & Rissanen, 2001; Singhal, Ramanujam, Ravikumar, & Rao, 2006). Further it is established that amino/sugar-derived Schiff bases exist in moderately polar solvents (including DMSO) in the keto-amine form in slight excess compared to enol-imine form (Sah et al., 2001). In addition, it has been observed that metal complexes of amino/sugar-derived Schiff bases have lower stability constants in MeOH (Costamagna, Lillo, Matsuhiro, Nosedo, & Villagran, 2003). Based on these previous observations, we infer that zinc Schiff base complex is stabilised in the aprotic solvent combination pyridine-AcOH, compared to other solvent combination, which leads to Amadori ketoses formation with higher yield. The fact that we are able to isolate pure Amadori ketoses after work up (utilising acetone and ether) indicates that the Amadori ketoses are not forming any complexes with ZnCl2
Table 3 IC50 values of Amadori compounds.
a
Compound
IC50 values (lM)
1 2 3 4 5 6
1277 ± 28 1728 ± 48 1756 ± 40 1293 ± 24 1731 ± 30 1038 ± 36a
From Srinivas & Harohally, 2012.
(ESI-MS also supports this fact). The Amadori compounds were found to be stable at room temperature (27–28 °C) for at least 3 months as indicated by the NMR spectroscopy. The ACE inhibitor activity of compounds 1–5 were evaluated by employing a high throughput ACE assay based on colorimetry (Table 3) (Jimsheena & Gowda 2009, Mallikarjun Gouda, Gowda, Rao, & Prakash, 2006). Among the Amadori compounds, 2 and 3 showed similar activity against ACE enzyme due to their structural similarity. On the other hand, compounds 1 and 4 displayed slightly lower activity compared to compound 6, indicating similar donor abilities compared to that of arginine towards Zn2+ of ACE enzyme. Compounds 2, 3 and 5 displayed lower ACE inhibitory activity compared to other compounds, suggesting weaker interaction of these compounds with the active site Zn2+. Lower IC50 value for compound 4, compared to 5, could be attributed to better donor abilities of L-tryptophan compared to L-phenylalanine. The IC50 values of compounds 1, 3 and 5 are comparable to fructose esters of amino acids consisting of 1-O-L-valyl-D-fructose (IC50 = 2.8 ± 0.29 mM), 1-O-L-phenylalanyl-D-fructose (IC50 = 13.6 ± 1.43 mM) and 1-O-L-tryptophanyl-D-fructose (IC50 = 0.9 ± 0.0092 mM) (Lohith, Vijayakumar, Somashekar, Sivakumar, & Divakar, 2006). Compounds 1 and 5 show significantly lower IC50 values compared to 1-O-L-valyl-D-fructose and 1-O-L-phenylalanyl-D-fructose and this is explained by considering the availability of the free carboxyl group in 1 and 5 and its interaction at the active site of ACE. 4. Conclusion We have demonstrated a catalytic method for synthesis of Amadori ketoses derived from amino acids, utilising ZnCl2 as a catalyst. This work may lead to the extension of the method to Amadori ketoses of numerous reducing sugar and amino acid derivatives; in addition it may also facilitate the employment of Amadori rearrangement reaction as a key reaction for facile synthesis of glyconjugates incorporating peptides and protein with carbohydrates. Acknowledgements This work was supported from an in-house CSIR project. We acknowledge Lalitha R.Gowda for providing ACE enzyme extract. We acknowledge Prasanna Vasu for helping in acquisition of mass spectral data. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 02.094. References
Fig. 2. Plausible mechanism for formation of Amadori compound.
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