Conjugates of a glycoprotein or a glycan with a toxic payload
a technology of glycoprotein and toxic payload, which is applied in the direction of biochemistry apparatus and processes, pharmaceutical non-active ingredients, microorganisms, etc., can solve the problems of toxic payload molecule not always efficiently released from the protein and/or delivered, bulky conjugates or conjugates with suboptimal solubility may not be optimal in terms of e,
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example 1
Synthesis of Dolastatin Derivatives
[0686]Unless otherwise noted, materials were obtained from commercial suppliers in the highest purity grade available and used without further purifications. Reaction solvents were dried and distilled prior to use when necessary. All reactions containing moisture- or air-sensitive reagents were carried out under an argon atmosphere. Monomethylauristatin F (MMAF) and monomethyldolastatin 10 were purchased from Concortis (San Diego, Calif., USA). Sodium cyanoborohydride, sodium hydride (NaH), methanol, 4-bromo-1-butyne, 5-iodo-1-pentyne, 2-deoxy-D-glucose, 6-O-(β-D-galacto-pyranosyl)-D-galactose, diisopropylethylamine and 2,5-dihydroxybenzoic acid were purchased from Sigma-Aldrich. Dimethylsuphoxide (DMSO) and N,N-dimethylformamide (DMF) were purchased from VWR. 2-acetamido-2-deoxy-4-O-(β-D-galactopyranosyl)-D-glucose, N-{4-O-[4-O-(α-D-galactopyranosyl)β-D-galactopyranosyl]-D-glucose and 4-O-[3-O-(α-N-acetyl-neuraminyl)-β-D-galactopyranosyl]-D-glucos...
example 2
In Vitro Cytotoxicity of Dolastatin Derivatives
[0710]Human ovarian cancer cell line SKOV-3 was from the ATCC (Manassas, Va., USA). The cells were grown according to the manufacturer's recommendations. Log phase cultures were collected and 5000 cells / well were seeded onto 96-well plates and incubated for 24 h. Serial dilutions of test molecules from a stock solution of 100 μM in 10% DMSO were made in cell culture medium, added to cells (maximum concentration of dimethylsulphoxide was 1%) and cultures were incubated further for 96 h. Cell viability was evaluated using PrestoBlue cell viability reagent (Life Technologies, Carlsbad, Calif., USA) according to the manufacturer's instructions. Cells were incubated for 2 h, and dye reduction was measured by absorbance at 570 nm. The compounds were assayed 1-2 times in triplicate.
[0711]Results of an exemplary assay are shown in FIG. 1, in which compound numbering is according to Example 1. The results are expressed in Table 1 as IC50 values ...
example 3
Synthesis of CMP-9-deoxy-9-azido-NeuNAc
[0712]
[0713]5-acetamido-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-2-nonulosonic acid (2): To a solution containing 63 mg of 1 (0.2 mmol) in 5 ml dry MeOH (under argon) was added 127 mg AG 50W-×8 (2 weight equiv.) and the resulting mixture was stirred at 45° C. o / n. The mixture was then filtered and concentrated to give methyl N-acetyl neuraminate as a white solid (65 mg, quantitative). TLC: Rf=0.43 (DCM:MeOH 3:1)
[0714]157 mg of methyl N-acetyl neuraminate (0.49 mmol) was dissolved in 5 ml of dry pyridine (under argon) and the reaction mixture was cooled to 0° C. 135 mg TsCl (0.7 mmol, 1.4 equiv.) was added and the reaction mixture was slowly warmed to RT and left to stir o / n. After 23 hours 134 mg TsCl (0.7 mmol, 1.4 equiv.) was added to the reaction mixture and it was stirred for an additional 2 hours at RT. The mixture was then cooled to 0° C. and the reaction quenched with MeOH. The mixture was concentrated and the crude product was purifie...
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