Modification of Oligopeptides on Aspartic Acid or Lysine Residues by Solid-Phase Synthesis through On-Resin Side-Chain Conjugation

Abstract

On-resin side-chain conjugations of various moieties to oligo­peptides were performed through an orthogonal protecting protocol using side-chain-protecting groups for aspartic acid or lysine that could be selectively removed on-resin. Various types of modification, such as PEGylation, biotinylation, glycosylation, or fluorophore-labeling of peptides, were realized by using this strategy. The formation of ester, amide, hydrazide, and thiourea bonds was accomplished through the on-resin conjugation. Our work provides an improved and convenient solid-phase synthetic protocol for the modification of oligopeptides on their aspartic acid or lysine residues. This is a universal and practical method that is expected to increase the potential application of peptide-related drugs.

Key words

oligopeptides - solid-phase synthesis - protecting group - PEGylation - biotinylation - glycosylation

• References and Notes

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• 21 Modification of Thymopentin on the Asp Residue (TA_{1 – 6}); General Procedure Fully-protected thymopentin attached to a resin was synthesized by standard SPPS. Fmoc-protected amino acid (0.2 mmol) was anchored to Wang resin (0.05 mmol) by using EDC/HOBt (0.2 mmol) in DMF (4 mL), and the Fmoc group was removed by treatment with 20 vol% piperidine in DMF (5 mL). To remove the OAll group, the resin was treated with Pd(PPh₃)₄ (0.2 mmol) and PhSiH₃ (0.5 mmol) in CH₂Cl₂ (4 mL). The mixture was shaken at r.t. for 12 h. The solvent was then removed by filtration, and the resin was washed with CH₂Cl₂ (3 × 5 mL) and DMF (3 × 5 mL). Side-chain modifications were performed by using various modifiers under the appropriate conditions (Scheme 1). For TA₁ , MPEG₆-OH (0.2 mmol), HOBt (0.2 mmol), DIC (0.2 mmol), DMAP (0.1 mmol) were dissolved in DMF (4 mL). For TA₂ –TA₆ : R–H (0.2 mmol), HOBt (0.2 mmol), HBTU (0.2 mmol), and DIPEA (0.2 mmol) were dissolved in DMF (4 mL) [R–H = e.g., MPEG₆-NH₂, glucosamine·HCl, 2-(biotinamido)ethylamine, biotin hydrazide, or dansylhydrazine]. For all reactions, the reaction mixture was shaken at r.t. for 24 h. The solvent was removed by filtration and the resin was washed with CH₂Cl₂ (3 × 5 mL) and DMF (3 × 5 mL). The resin was then suspended in 20% piperidine/DMF for 30 min to remove the Fmoc group. The peptide was cleaved from the resin with 50 vol% TFA in CH₂Cl₂ (5 mL) for 2 h. The residue was dissolved in H₂O then freeze-dried to give the solid. The crude product was finally purified by preparative RP-HPLC. TA₁ : white solid; yield: 13 mg (26%); analytical HPLC: t_R  = 13.8 min, purity 98%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₃H₇₅N₉O₁₅: 479.7770; found: 479.7806. TA₂ : white solid; yield: 22 mg (44%); analytical HPLC: t_R  = 14.7 min, purity 99%; HR-ESI-MS: m/z [M + H]⁺ calcd for C₄₃H₇₆N₁₀O₁₄: 957.5615; found: 957.5636. TA₃ : white solid; yield: 16 mg (38%); analytical HPLC: t_R  = 8.4 min, purity 99%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₃₆H₆₀N₁₀O₁₃: 421.2243; found: 421.2239. TA₄ : white solid; yield: 15 mg (31%); analytical HPLC: t_R  = 13.7 min, purity 99%; HR-ESI-MS: m/z [M + H]⁺ calcd for C₄₂H₆₉N₁₃O₁₀S: 948.5084; found: 948.5087. TA₅ : white solid; yield: 12 mg (26%); analytical HPLC: t_R  = 12.9 min, purity 98%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₀H₆₅N₁₃O₁₀S: 460.7428; found: 460.7495. TA₆ : white solid; yield: 20 mg (42%); analytical HPLC: t_R  = 15.2 min, purity 99%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₂H₆₂N₁₂O₁₀S: 464.2289; found: 464.2310.
• 22 Modification of Thymopentin on the Lys Residue (TL_{1 – 6}); General Procedure Fully-protected thymopentin attached to a resin was synthesized by standard SPPS. Fmoc-protected amino acid (0.2 mmol) was anchored to Wang resin (0.05 mmol) by using EDC/HOBt (0.2 mmol) or HBTU/HOBt/DIPEA (0.2 mmol) in DMF (4 mL), and the Fmoc group was removed by treatment with 20 vol% piperidine in DMF (5 mL). To remove the Mtt group, the resin was treated with 5 vol% TFA and 5 vol% TIS in CH₂Cl₂ (5 mL, 2 × 5 min). The resin was washed with DMF (3 × 5 mL), CH₂Cl₂ (3 × 5 mL), and DMF (3 × 5 mL). Side-chain modifications were performed by using various modifiers under the appropriate conditions (Scheme 1). For TL₁ –TL₂ : R′-OH (0.2 mmol), HOBt (0.2 mmol), HBTU (0.2 mmol), and DIPEA (0.2 mmol) were dissolved in DMF (4 mL), and the mixture was shaken at r.t. for 4 h. The solvent was removed by filtration, and the resin was washed with CH₂Cl₂ (3 × 5 mL) and DMF (3 × 5 mL). [R′-OH = MPEG₆-CH₂-COOH, MPEG₆-(CH₂)₅CO₂H]. For TL₃ –TL₆ : R′-OSu (0.2 mmol) or FITC (0.2 mmol) was dissolved in DMF (4 mL), then DIPEA (0.2 mmol) was added. (R′-OSu = for example, biotin-OSu, MCA-OSu, FAM-OSu). The mixture was shaken at r.t. for 8 h. The reaction using FITC was shielded from light. Then, the solvent was removed by filtration and the resin was washed with CH₂Cl₂ (3 × 5 mL) and DMF (3 × 5 mL). The resin was then suspended in 20% piperidine/DMF to remove the Fmoc group. The peptide was cleaved from the resin with 50 vol% TFA in CH₂Cl₂ (5 mL) for 2 h. The residue was dissolved in H₂O then freeze-dried to give a solid. The crude product was finally purified by preparative RP-HPLC TL₁ : white solid; yield: 30 mg (57%); analytical HPLC: t_R  = 15.9 min, purity 99%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₅H₇₇N₉O₁₇: 508.7791; found: 508.7812. TL₂ : white solid; yield: 35 mg (54%); analytical HPLC: t_R  = 17.8 min, purity 99%; HR-ESI-MS: m/z [M + H + Na]²⁺ calcd for C₄₉H₈₅N₉O₁₇: 547.8014; found: 547.7995. TL₃ : white solid; yield: 12 mg (26%); analytical HPLC: t_R  = 14.6 min, purity 98%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₀H₆₃N₁₁O₁₁S: 453.7287; found: 453.7282. TL₄ : white solid; yield: 25 mg (54%); analytical HPLC: t_R  = 17.9 min, purity 99%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₄₂H₅₇N₉O₁₃: 448.7111; found: 448.7115. TL₅ : yellow solid; yield: 27 mg (48%); analytical HPLC: t_R  = 19.4 min, purity 98%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₅₁H₅₉N₉O₁₅: 519.7143; found: 519.7118. TL₆ : yellow solid; yield: 22 mg (40%); analytical HPLC: t_R  = 20.1 min, purity 99%; HR-ESI-MS: m/z [M + 2H]²⁺ calcd for C₅₁H₆₀N₁₀O₁₄S: 535.2080; found: 535.2041.
• 23 Iacobazzi RM. Porcelli L. Lopedota AA. Laquintana V. Lopalco A. Cutrignelli A. Altamura E. Di Fonte R. Azzariti A. Franco M. Denora N. Int. J. Pharm. 2017; 528: 485
• 24 Liao X. Ning X. Wang Y. Shen H. Shenyang Yaoke Daxue Xuebao 2017; 34: 1044