600 controller pump

The peptide-based traditional Maleimidocaproyl(Mc)-Val-Cit-PABC-PNP linker or the rebridging linker were reacted with monomethyl auristatin E (MMAE). In the construction of traditional linker-MMAE payload, 18.20 μmol potent molecule MMAE, 16.38 μmol Mc-Val-Cit-PABC-PNP, and 3.64 μmol hydroxybenzotriazole were dissolved and mixed in 500 μL dimethylformamide. Then 18.20 μmol pyridine was added to the mixture after 2 min, and 20 μmol trifluoroacetic acid (TFA) was added after 24 h. In the construction of rebridging linker-MMAE payload, 13.55 μmol N,N'-diisopropylcarbodiimide, 13.55 μmol N,N-diisopropylethylamine, and 33.85 μmol synthesized rebridging linker were mixed in 0.25 mL dichloromethane, followed by frequent mixing for 1 h at room temperature. Then 13.55 μmol MMAE was added and frequently mixed for additional 16 h. After linker-MMAE conjugates were synthesized, the solvents were removed by vacuum pump and the conjugates were purified by a Waters HPLC system equipped with 600 Controller/Pump and 996 PDA detector (Waters, Milford, MA). A reversed-phase C₁₈ column with 5 μm C18(2) 100 Å and 250 x 10 mm (Phenomenex Luna; Torrance, CA) was used with gradient elution buffer of Phase A (water+0.1% TFA) and Phase B (acetonitrile). The purified products were confirmed by Agilent 6500 Series Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA).

Optical rotations were measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. UV spectra were recorded on a Perkin Elmer Lambda 40 spectrophotometer. IR spectra were obtained on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on a δ (ppm) scale using TMS as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. High resolution ESI mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer. Column chromatography separations were performed with Kieselgel 60 (Merck, Kenilworth, USA) and Sephadex LH-20 (Pharmacia & Upjohn Company LLC, USA). HPLC separations were conducted using a Pharmacia LKB 2248 liquid chromatography pump equipped with a RI-102 Shodex refractive index detector, using a Supelcosil SPLC-Si 5 μm (250 × 10 mm i.d.; Supelco) column. HPLC gradient elution separations for qualitative analysis were conducted using a Waters 600 Controller pump equipped with a Waters 996 Photodiode Array detector, using Kromasil Silica 8 μm (250 × 10 mm i.d.; AkzoNobel) column. TLC was performed with Kieselgel 60 F₂₅₄ (Merck aluminum-backed plates) and spots were detected after spraying with 15 % H₂SO₄ in MeOH reagent and heating at 100 °C for 1 min.