Note Glycation of a lysine-containing tetrapeptide by D-glucose and D-fructose—influence of different reaction conditions on the formation of Amadori/Heyns products Andreja Jakas * , Anja Katic ´ , Nina Bionda, Štefica Horvat Division of Organic Chemistry and Biochemistry, Rudjer Boškovic ´ Institute, PO Box 180, Bijenic ˇka 54, HR-10002 Zagreb, Croatia article info Article history: Received 18 March 2008 Received in revised form 27 June 2008 Accepted 2 July 2008 Available online 10 July 2008 Keywords: Amadori compound Fructose Glucose Glycation Heyns compound Maillard reaction abstract The site specificity, extent, and nature of modification of the tetrapeptide, Leu-Ser-Lys-Leu (1), incubated with D-glucose or D-fructose in methanol, or in phosphate buffer of pH 5.7, 7.4, and 8.0 were investigated. The generated mono- and di-glycated Amadori (1-deoxy-D-fructosyl derivatives) and Heyns rearrange- ment products (N-alkylated glucosamine/mannosamine derivatives) were isolated and characterized by NMR and mass spectrometry. The results identified the e-amino group of the Lys residue as the pre- ferential glycation site in tetrapeptide 1. Under all conditions investigated, glucose afforded higher yields of glycation products than fructose. In the reactions carried out in buffer, glycation at pH 7.4 and 8.0 was much faster than at pH 5.7. Ó 2008 Elsevier Ltd. All rights reserved. The Maillard reaction involves the carbonyl of a reducing carbo- hydrate in reaction with free amino groups in peptides/proteins resulting, in the first step, in reversible formation of a Schiff base which can then undergo irreversible Amadori or Heyns rearrange- ments. 1–3 Further reactions lead to the formation of advanced gly- cation end (AGE) products, assumed to be responsible for a number of pathophysiological syndromes accompanying diabetes, aging, endothelial dysfunction, and vascular diseases. 4–6 Although the Maillard reaction is not an enzymatic reaction, a certain degree of specificity at the glycation site was observed. This has been rationalized by selective effects of the microenvironment on the isomerization of the protein-bound sugar to a protein-bound ketose or aldose. 7–10 To gain more detailed insight into peptide/ protein glycation processes under physiological conditions, model systems using mixtures of sugars with peptides and proteins con- taining selected structural elements should be studied. While there has been enormous advance in understanding the general chemis- try and biochemistry of AGE formation, the glycation adducts themselves have only rarely been isolated and rigorously puri- fied. 1,3,11–13 In our previous studies, 14–16 we characterized the gly- cation products generated from endogenous opioid peptides (enkephalins), which did not contain lysine residues. Here, we focus on the lysine-containing peptide Leu-Ser-Lys-Leu (1) and its reaction with D-glucose and D-fructose. The tetrapeptide se- quence Leu-Ser-Lys-Leu occurs in the inactive form of the trans- forming growth factor b (TGF-b), as part of the latency-associated protein (LAP). 17 Peptide 1 contains two amino groups available for glycation: at the N-terminal leucine residue and at the lysine side chain. The aim of the research presented was (a) to isolate and to characterize the products formed by condensation of D-glu- cose or D-fructose with tetrapeptide 1, (b) to study the influence of pH and sugar concentration on the kinetics of glycation product formation, and (c) to compare the reactivities of glucose and fruc- tose in these reactions. The findings that excessive fructose con- sumption may have a major role in the present epidemic of metabolic syndrome and obesity, due to its ability to raise uric acid, 18,19 suggest the potential value of studies on lysine-contain- ing peptides to assess the physiological significance of their glycat- ed products. The strategy for the preparation of glucose-derived Amadori products 4 and 5 included two synthetic approaches (Scheme 1). The fully protected di-glycated compound 3 was prepared in 58% yield by reductive amination of aldoketose 2 20 with tetrapeptide 1 in the presence of sodium cyanoborohydride. Removal of the ace- tonide protecting groups from 3 with aq TFA (90%) and subsequent purification by semipreparative RP-HPLC furnished di-glycated Amadori compound 4 in 40% yield. The second route involved the reaction of glucose and tetrapeptide 1 in MeOH at 70 °C to afford, after initial Schiff base formation, followed by Amadori 0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2008.07.003 * Corresponding author. Tel.: +385 1 46 80 103; fax: +385 1 46 80 195. E-mail address: jakas@irb.hr (A. Jakas). Carbohydrate Research 343 (2008) 2475–2480 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres