J. Chem. Soc., Perkin Trans. 2, 1997 1523 CD and FTIR spectroscopic studies of Amadori compounds related to the opioid peptides S ˇ tefica Horvat,* , a Andreja Jakas, a Elemér Vass, b János Samu b and Miklós Hollósi b a Department of Organic Chemistry and Biochemistry, Rud – er Bos ˇkovic ´ Institute, POB 1016, 10001 Z agreb, Croatia b Department of Organic Chemistry, Eötvös University Budapest, POB 32, H-1518 Budapest, Hungary Circular dichroism (CD ) and Fourier transform infrared (FTIR) spectroscopy have been used to investigate conformational effects of glycation on the secondary structure of opioid peptide Leu- enkephalin and on structurally related peptides in 2,2,2-trifluoroethanol (TFE) solution. CD spectral analysis of Leu-enkephalin-related Amadori compounds revealed that attachment of the protected or free sugar may influence not only the distribution of the backbone but also the side-chain conformation of the Tyr moiety. The amide I region of the FTIR spectra analysed by self-deconvolution and curve-fitting methods revealed that Leu-enkephalin is present as a mixture of -sheet and -turn conformers in TFE solution, while its methyl ester likely adopts a -turn conformation. FTIR spectroscopy has shown that no major spectral changes occur in the peptide part of glycated (Amadori) compounds as compared to parent peptides. The structurally related Tyr-Gly-Gly tripeptide derivatives contain amide I components at ca. 1630 and ca. 1645 cm -1 consistent with the presence of -turns with strong and weak 1 ← 3 H-bondings, respectively. The attachment of the protected or free sugar moiety to pentapeptides appears to destabilize -turns but not to affect H-bonded -turns. In the spectra of Amadori compounds containing a free sugar moiety, the component band at ca. 1730 cm -1 suggests the presence of the open-chain sugar form. Based on the studies presented herein, FTIR spectroscopy is shown to be a powerful tool for the structural analysis of glycated peptides, in particular for the detection of the keto form of the sugar and turn conformations of the peptide part of the molecule. Introduction The process involving the nonenzymatic modification of vari- ous proteins by physiological sugars (Maillard reaction) in vivo has been associated with the progression of many long term complications of diabetes, renal disorders and normal ageing. 1,2 This modification involves covalent binding of reducing sugars, such as glucose, to primary amino groups of proteins forming 1-amino-1-deoxy-ketose derivatives by Amadori rearrange- ment. Amadori adducts then slowly undergo further intra- molecular rearrangement to produce advanced glycation end products (AGEs) which are responsible for the modification of circulating and tissue proteins that lead to structural and functional changes. 3,4 The chemistry of Maillard transformations in biological systems is extremely complex and yet poorly understood; thus synthetic Amadori compounds are considered to be effective tools for investigating the impact of glycation on the biological, physical and chemical properties of protein fragments. Although many studies dealing with the synthesis and proper- ties of Amadori derivatives of amino acids have been pub- lished, 5 there are only a few reports referring to the synthesis of peptide-related Amadori compounds. 6–8 In an effort to better understand the role that Amadori com- pounds may play in biologically relevant interactions we have recently reported 9 the synthesis, analytical characterization and 13 C NMR analysis of Amadori compounds related to the endogenous opioid peptide Leu-enkephalin (Tyr-Gly-Gly- Phe-Leu), involved in numerous physiological functions. 10 It is reasonable to suppose that glycation of the N -terminal amino group of the tyrosine residue will affect the biological activity of Leu-enkephalin since it was shown that after N -alkylation of enkephalins, depending on substitution pattern, activity may be enhanced, reduced, eliminated or even reversed to give antagonists. 11 Although there have been many contributions in the last few years which demonstrate that nonenzymatic glycation leads to functional changes in the affectedproteins, 1–4 little is known of how the attached carbohydrate molecule influences the conformational properties of the N -alkylated protein. To address this question, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies were performed on Leu-enkephalin-related Amadori compounds. FTIR spectroscopy has been shown to be a sensitive tool for providing information on the secondary structure of proteins in solution. 12–14 This method has also been used for acquiring information about the structure of linear 15,16 and cyclic pep- tides. 17 An important advantage of FTIR spectroscopy is that it provides information on both the side-chain functionalities 18 and backbone conformation in terms of changes of pH, sol- vation or addition of metal ions, detergents etc. 14,19,20,23–25 This is the first report on the chiroptical and vibrational spectroscopic characterization of well-defined Amadori com- pounds related to biologically active opioid peptides. In order to detect differences caused by the presence of sugar, we investigated protected (3–5,9,10) and unprotected (6,7,11) N -glycated derivatives in comparison with the corresponding parent peptides (1,2,8). N ,N -Dialkylated Amadori compounds 4 and 10 were selected on the basis of the observation 21 that reactive aldehydes formed in vivo by oxidation and decom- position of Amadori products may further react with amino acid residues in proteins to form cross-linkages. Results and discussion CD studies The dependence on solvent, pH and cations of the CD spectra Published on 01 January 1997. Downloaded on 20/05/2015 07:37:04. View Article Online / Journal Homepage / Table of Contents for this issue