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Expedient Synthesis of Peptides Containing N ε-Carboxymethyllysine
Received: 11 March 2014
Accepted: 09 May 2014
06 June 2014 (online)
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 wordspeptides - solid-phase synthesis - protecting groups - advanced glycation endproducts - N ε-carboxymethyllysine
References and Notes
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- 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).
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- 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).