3.2.2. Modification of the Peptides with Linker Structures to Obtain 10–21

General procedure for the synthesis of NHS-PEG_n peptides (GP2): All steps were carried out under an N₂ atmosphere. A total of 1.0 equiv. of the respective peptide was added to a solution of 1.0 equiv. bis-NHS-ester and 0.5–1.0 equiv. DIPEA in dry DMF. Subsequently, the reaction mixture was stirred for 5–40 min at room temperature, while reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in 5 min). After the removal of the solvent under reduced pressure, the corresponding NHS-PEG_n-peptide was obtained after purification via semipreparative HPLC. In addition to the respective target product, small amounts of the hydrolyzed compound HO-PEG_n-peptide and the dimer peptide-PEG_n-peptide were isolated (for analytical data, see Supplementary Materials Figures S65–S80).

NHS-PEG₁-c(RGDfK) (10): According to GP2, NHS-PEG₁-NHS (3.3 mg, 8.3 μmol, 1.0 equiv.), DIPEA (1.4 μL, 1.1 mg, 8.3 μmol, 1.0 equiv.) and c(RGDfK) (5.0 mg, 8.3 μmol, 1.0 equiv.) were reacted in 5 mL dry DMF for 20 min. After purification by HPLC (semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, t_R = 7.01 min), 10 was isolated as a colorless solid (3.0 mg, 3.4 μmol, yield: 41%, purity: >93%). MS (MALDI) m/z calculated for C₃₉H₅₆N₁₀O₁₄ [M]⁺: 888.40, found: 888.26; HR-ESI-MS m/z calculated for C₃₉H₅₇N₁₀O₁₄ [M + H]⁺: 889.4050, found: 889.4038.

NHS-PEG₃-c(RGDfK) (11): According to GP2, NHS-PEG₃-NHS (12.1 mg, 24.8 μmol, 1.0 equiv.), DIPEA (4.2 μL, 3.2 mg, 24.8 μmol, 1.0 equiv.) and c(RGDfK) (15.0 mg, 24.8 μmol, 1.0 equiv.) were reacted in 8 mL dry DMF for 15 min. After purification by HPLC (semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, t_R = 4.84 min), 11 was isolated as a colorless solid (7.0 mg, 7.2 μmol, yield: 29%, purity: >89%). MS (MALDI) m/z calculated for C₄₃H₆₄N₁₀O₁₆ [M]⁺: 976.45, found: 976.68; HR-ESI-MS m/z calculated for C₄₃H₆₅N₁₀O₁₆ [M + H]⁺: 977.4575, found: 977.4567.

NHS-PEG₅-c(RGDfK) (12): According to GP2, NHS-PEG₅-NHS (9.6 mg, 16.6 μmol, 1.0 equiv.), DIPEA (2.8 μL, 2.2 mg, 16.6 μmol, 1.0 equiv.) and c(RGDfK) (10.0 mg, 16.6 μmol, 1.0 equiv.) were reacted in 6 mL dry DMF for 30 min. After purification by HPLC (semipreparative, 15–30% MeCN + 0.1% TFA in 10 min, t_R = 4.44 min), 12 was isolated as a colorless solid (6.1 mg, 5.7 μmol, yield: 35%, purity: >91%). MS (MALDI) m/z calculated for C₄₇H₇₃N₁₀O₁₈ [M + H]⁺: 1065.51, found: 1065.22; HR-ESI-MS m/z calculated for C₄₇H₇₃N₁₀O₈ [M + H]⁺: 1065.5099, found: 1065.5099.

NHS-PEG₈-c(RGDfK) (13): According to GP2, NHS-PEG₈-NHS (13.5 mg, 19.1 μmol, 1.0 equiv.), DIPEA (3.3 μL, 2.5 mg, 19.1 μmol, 1.0 equiv.) and c(RGDfK) (11.5 mg, 19.1 μmol, 1.0 equiv.) were reacted in 6 mL dry DMF for 40 min. After purification by HPLC (semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, t_R = 7.01 min), 13 was isolated as a colorless solid (7.3 mg, 6.1 μmol, yield: 32%, purity: >82%). MS (MALDI) m/z calculated for C₅₃H₈₅N₁₀O₂₁ [M + H]⁺: 1197.59, found: 1197.34; HR-ESI-MS m/z calculated for C₅₃H₈₄N₁₀O₂₁ [M + H]⁺: 1197.5885, found: 1197.5892.

NHS-PEG₁-GG-Nle-c(DHfRWK) (16): According to GP2, NHS-PEG₁-NHS (5.4 mg, 6.1 μmol, 1.0 equiv.), DIPEA (0.5 μL, 0.4 mg, 3.1 μmol, 0.5 equiv.) and 7 (6.7 mg, 6.1 μmol, 1.0 equiv.) were reacted in 5 mL dry DMF for 5 min. After purification by HPLC (semipreparative, 15–50% MeCN + 0.1% TFA in 10 min, t_R = 4.80 min), 16 was isolated as a colorless solid (3.6 mg, 2.6 μmol, yield: 43%, purity: >93%). MS (MALDI) m/z calculated for C₆₄H₈₉N₁₈O₁₇ [M + H]⁺: 1381.66, found: 1381.34; HR-ESI-MS m/z calculated for C₆₄H₈₉N₁₈O₁₇ [M + H]⁺: 1381.6648, found: 1381.6643.

NHS-PEG₃-GG-Nle-c(DHfRWK) (17): According to GP2, NHS-PEG₃-NHS (4.7 mg, 9.6 μmol, 1.0 equiv.), DIPEA (1.6 μL, 1.2 mg, 9.6 μmol, 1.0 equiv.) and 7 (10.5 mg, 9.6 μmol, 1.0 equiv.) were reacted in 6 mL dry DMF for 40 min. After purification by HPLC (semipreparative, 10–30% MeCN + 0.1% TFA in 10 min, t_R = 8.51 min), 17 was isolated as a colorless solid (6.9 mg, 4.7 μmol, yield: 49%, purity: >91%). MS (MALDI) m/z calculated for C₆₈H₉₇N₁₈O₁₉ [M + H]⁺: 1469.72, found: 1469.47; HR-ESI-MS m/z calculated for C₆₈H₉₈N₁₈O₁₉ [M + 2H]²⁺: 735.3622, found: 735.3619; HR-ESI-MS m/z calculated for C₆₈H₉₇N₁₈O₁₉ [M + H]⁺: 1469.7172, found: 1469.7154.

NHS-PEG₅-GG-Nle-c(DHfRWK) (18): According to GP2, NHS-PEG₅-NHS (5.3 mg, 9.1 μmol, 1.0 equiv.), DIPEA (1.6 μL, 1.2 mg, 9.1 μmol, 1.0 equiv.) and 7 (10.0 mg, 9.1 μmol, 1.0 equiv.) were reacted in 5 mL dry DMF for 30 min. After purification by HPLC (semipreparative, 10–50% MeCN + 0.1% TFA in 8 min, t_R = 5.86 min), 18 was isolated as a colorless solid (5.9 mg, 3.8 μmol, yield: 42%, purity: >90%). MS (MALDI) m/z calculated for C₇₂H₁₀₄N₁₈O₂₁ [M]⁺: 1556.73, found: 1556.24; HR-ESI-MS m/z calculated for C₇₂H₁₀₆N₁₈O₂₁ [M + 2H]²⁺: 779.3884, found: 779.3884.

NHS-PEG₈-GG-Nle-c(DHfRWK) (19): According to GP2, NHS-PEG₈-NHS (3.3 mg, 4.7 μmol, 1.0 equiv.), DIPEA (0.8 μL, 0.6 mg, 4.7 μmol, 1.0 equiv.) and 7 (5.1 mg, 4.7 μmol, 1.0 equiv.) were reacted in 3 mL dry DMF for 40 min. After purification by HPLC (semipreparative, 0–50% MeCN + 0.1% TFA in 8 min, t_R = 6.09 min), 19 was isolated as a colorless solid (3.3 mg, 2.0 μmol, yield: 42%, purity: >90%). MS (MALDI) m/z calculated for C₇₈H₁₁₇N₁₈O₂₄ [M + H]⁺: 1689.85, found: 1689.09; HR-ESI-MS m/z calculated for C₇₈H₁₁₈N₁₈O₂₄ [M + 2H]²⁺: 845.4278, found: 845.4275.

General procedure for the synthesis of HO-DIG- and HO-Ox-EGEGE peptides (GP3): All steps were carried out under an N₂ atmosphere. In total, 1.0–10.0 equiv. of the respective peptide was added to a solution of 1.0–10.0 equiv. bis-NHS-ester and 1.0 equiv. DIPEA in dry DMF. Subsequently, the reaction mixture was stirred for 5–50 min at room temperature, while the reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in 5 min). After the removal of the solvent under reduced pressure, the corresponding HO-DIG- or HO-Ox-EGEGE peptides were obtained after semipreparative HPLC purification. In addition to the target products, small amounts of the dimers peptide-DIG/Ox-EGEGE-peptide were isolated (for analytical data, see Supplementary Materials Figures S81–S84).

HO-DIG-c(RGDfK) (14): According to GP3, NHS-DIG-NHS (8.2 mg, 24.9 μmol, 1.5 equiv.), c(RGDfK) (10.0 mg, 16.6 μmol, 1.0 equiv.) and DIPEA (2.8 μL, 2.1 mg, 16.6 μmol, 1.0 equiv.) were reacted in 11 mL dry DMF for 50 min. After purification by HPLC (semipreparative, 5–40% MeCN + 0.1% TFA in 10 min, t_R = 3.80 min), 14 was isolated as a colorless solid (4.4 mg, 6.1 μmol, yield: 37%, purity: >99%). MS (MALDI) m/z calculated for C₃₁H₄₅N₉O₁₁ [M]⁺: 719.32, found: 719.59; HR-ESI-MS m/z calculated for C₃₁H₄₆N₉O₁₁ [M + H]⁺: 720.3311, found: 720.3309.

HO-Ox-EGEGE-c(RGDfK) (15): According to GP3, NHS-Ox-NHS (17.9 mg, 63 μmol, 5.0 equiv.), 8 (13.9 mg, 12.6 μmol, 1.0 equiv.) and DIPEA (10.7 μL, 8.1 mg, 63 μmol, 5.0 equiv.) were reacted in 8 mL dry DMF for 5 min. After purification by HPLC (semipreparative, 5–20% MeCN + 0.1% TFA in 8 min, t_R = 4.29 min), 15 was isolated as a colorless solid (5.9 mg, 5.0 μmol, yield: 40%, purity: >99%). MS (MALDI) m/z calculated for C₄₈H₆₉N₁₄O₂₁ [M + H]⁺: 1177.48, found: 1177.02; HR-ESI-MS m/z calculated for C₄₈H₆₈N₁₄O₂₁ [M]⁺: 1176.4683, found: 1176.4913.

HO-DIG-GG-Nle-c(DHfRWK) (20): According to GP3, NHS-DIG-NHS (1.5 mg, 3.6 μmol, 1.0 equiv.), 7 (5.0 mg, 3.6 μmol, 1.0 equiv.) and DIPEA (0.6 μL, 0.5 mg, 3.6 μmol, 1.0 equiv.) were reacted in 2 mL dry DMF for 25 min. After purification by HPLC (semipreparative, 10–40% MeCN + 0.1% TFA in 10 min, t_R = 5.36 min), 20 was isolated as a colorless solid (2.7 mg, 2.2 μmol, yield: 61%, purity: >99%). MS (MALDI) m/z calculated for C₅₆H₇₈N₁₇O₁₄ [M + H]⁺: 1212.59, found: 1212.16; HR-ESI-MS m/z calculated for C₅₆H₇₉N₁₇O₁₄ [M + 2H]²⁺: 606.7991, found: 606.7991.

HO-Ox-EGEGE-GG-Nle-c(DHfRWK) (21): According to GP3, NHS-Ox-NHS (17.8 mg, 62.6 μmol, 10.0 equiv.), 9 (10.0 mg, 6.3 μmol, 1.0 equiv.) and DIPEA (10.7 μL, 8.1 mg, 62.6 μmol, 10.0 equiv.) were reacted in 5 mL dry DMF for 10 min. After purification by HPLC (semipreparative, 0–40% MeCN + 0.1% TFA in 8 min, t_R = 6.41 min), 21 was isolated as a colorless solid (4.5 mg, 2.7 μmol, yield: 44%, purity: >99%). MS (MALDI) m/z calculated for C₇₃H₁₀₀N₂₂O₂₄ [M]⁺: 1668.73, found: 1668.90; HR-ESI-MS m/z calculated for C₇₃H₁₀₂N₂₂O₂₄ [M + 2H]²⁺: 835.3713, found: 835.3716.