Expedient Synthesis of Peptides Containing N ^ε-Carboxymethyllysine

Abstract

Accumulation of advanced glycation endproducts ­(AGEs) is responsible for the development and progress of diabetes- and age-related complications. Synthesis of specific chemical probes is key for the detailed understanding of biochemical properties of AGEs and their precise roles in the progression of disease. We herein report the expedient synthesis of such probes in the form of peptides site-specifically glycated by the major lysyl AGE, N ^ε-carboxymethyllysine (CML). The facile and economical incorporation of CML into peptide sequences by using the nosyl group has been achieved in a single step on resin. This new method is a substantial improvement over the existing syntheses of CML-containing peptides in that it does not require the use of expensive reagents or elaborate purification techniques. The impact of CML on the proteolytic stability of the host peptide has been investigated using trypsin digest studies.

Key words

peptides - solid-phase synthesis - protecting groups - advanced glycation endproducts - N ^ε-carboxymethyllysine

• References and Notes

• 1 Kamalov M, Harris PW. R, Cooper GJ. S, Brimble MA. Chem. Commun. 2014; 50: 4944
• 2 Dunn JA, Patrick JS, Thorpe SR, Baynes JW. Biochemistry (Moscow) 1989; 28: 9464
• 3 Rabbani N, Thornalley P. Amino Acids 2012; 42: 1087
• 4 Beisswenger P. Amino Acids 2012; 42: 1171
• 5 Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR. J. Biol. Chem. 1996; 271: 9982
• 6 Golub LM, Greenwald RA, Zebrowski EJ, Ramamurthy NS. Biochim. Biophys. Acta 1978; 534: 73
• 7 Hamlin CR, Kohn RR, Luschin JH. Diabetes 1975; 24: 902
• 8 Woods TM, Kamalov M, Harris PW, Cooper GJ, Brimble M. Org. Lett. 2012; 14: 5740
• 9 Gruber P, Hofmann T. J. Pept. Res. 2005; 66: 111
• 10 Xue J, Rai V, Singer D, Chabierski S, Xie J, Reverdatto S, Burz DS, Schmidt AM, Hoffmann R, Shekhtman A. Structure 2011; 19: 722
• 11 Demmer O, Dijkgraaf I, Schottelius M, Wester H.-J, Kessler H. Org. Lett. 2008; 10: 2015
• 12 Monfregola L, Leone M, Calce E, De Luca S. Org. Lett. 2012; 14: 1664
• 13 Fukuyama T, Jow C.-K, Cheung M. Tetrahedron Lett. 1995; 36: 6373
• 14 Chu M.-L, de Wet W, Bernard M, Ding J.-F, Morabito M, Myers J, Williams C, Ramirez F. Nature (London) 1984; 310: 337
• 15 Woodley DT, Yamauchi M, Wynn KC, Mechanic G, Briggaman RA. J. Invest. Dermatol. 1991; 97: 580
• 16 Paul RG, Bailey AJ. Int. J. Biochem. Cell Biol. 1996; 28: 1297
• 17 Peptide Synthesis: SPPS was performed via the Fmoc strategy on Rink Amide resin using a Biotage Alstra peptide synthesiser on 0.1-mmol scale. The Fmoc group was deprotected with 20% piperidine in DMF for 2 min + 3 min at 60 °C. The coupling step was performed with Fmoc-AA-OH (5 equiv) in DMF (0.2 M), HBTU in DMF (4.5 equiv, 0.45 M) and DIPEA (10 equiv) for 5 min at 75 °C. The final Fmoc group was removed and the amine was acetylated by Ac₂O in the presence of DIPEA at r.t. for 10 min. The peptides were released from resin with concomitant removal of the side-chain protecting groups by treatment with TFA–TIS–H₂O (38:1:1, 5 mL) at r.t. for 2 h. Peptides were precipitated with cold Et₂O, isolated by centrifugation, washed in cold Et₂O, dissolved in MeCN–H₂O (1:1) containing 0.1% TFA and lyophilised. The peptides were analysed for purity by LCMS using a Zorbax C3 column (3.5 μm; 3 × 150 mm; Agilent) at 0.3 mL/min using a linear gradient. The solvent system used was A (0.1% formic acid in H₂O) and B (0.1% formic acid in MeCN). Purification of crude peptides was performed by semipreparative HPLC using a Gemini C18 column (10 μm; 250 × 10 mm; Phenomenex) at 5 mL/min using a shallow linear gradient. The solvent system used was A (0.1% TFA in H₂O) and B (0.1% TFA in MeCN). The resulting purified peptides were analysed by the LCMS system used for crude peptide analysis. The purities were extrapolated from integrating the peaks corresponding to peptide 1 (13.1 min) and to peptide 2 (12.7 min).
• 18 Halpin DR, Lee JA, Wrenn SJ, Harbury PB. PLoS Biol. 2004; 2: e175
• 19 Alkylation Conditions: Resin-bound peptide 10 or 12 (0.1 mmol, 1 equiv) was swollen in CH₂Cl₂ (30 min), then in DMF (10 min), and drained. A solution of tert-butyl bromoacetate (70 μL, 0.5 mmol, 5 equiv) with DIPEA (175 μL, 1 mmol, 10 equiv) in DMF (5 mL) was added in one portion and the reaction mixture was shaken overnight at r.t. to afford peptides 11 or 13, respectively, which were drained and washed with DMF.
• 20 Ns Deprotection: Resin-bound peptide 8 or 11 (0.1 mmol, 1 equiv) was swollen in CH₂Cl₂ (30 min), then in DMF (10 min) and filtered. A solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (90 μL, 0.6 mmol, 6 equiv) and 2-mercaptoethanol (42 μL, 0.6 mmol, 6 equiv) in DMF (5 mL) was added in one portion and the mixture was shaken for 15 min, drained, and washed with DMF.
• 21 Trypsin Digest: Bovine trypsin (0.3 mg, type XI, 9090 units/mg, Sigma) was dissolved in H₂O (1 mL) and 3.3 μL (9 units) of this solution diluted to 1 mL using Tris buffer (pH 8.0) and incubated at 37 °C for 30 min. Substrate peptide (0.21 μmol) was added in one portion and 50 μL aliquots removed every minute, quenched with 1 M HCl (50 μL) and analysed by analytical reverse phase-HPLC using a Luna C18(2) column (3μ; 150 × 3 mm; Phenomenex) at 0.3 mL/min using linear gradient. The concentrations were extrapolated from integrating the peaks corresponding to peptide 1 (14.9 min) and to peptide 2 (15.0 min).