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利用Fmoc固相合成策略,Wang树脂为载体,使用三苯甲基(Trt)和乙酰胺甲基(Acm)保护基的半胱氨酸合成了3条[4 Trt+2 Acm]和3条[2 Trt+4 Acm]利那洛肽的线性前体化合物.在此基础上,采用半选择性氧化策略合成含有三对二硫键的利那洛肽.首先使用含三氟乙酸(TFA)的裂解剂脱除线性前体肽中半胱氨酸的Trt保护基,并使用氯化血红素催化氧化半胱氨酸自由巯基形成二硫键.下一步使用Ph S(O)Ph/CH3Si Cl3试剂体系脱除剩余保护半胱氨酸的Acm保护基,并同时形成二硫键.使用这种策略,在6条线性前体肽中,有3条可以得到利那洛肽,转化率分别为71.9%、31.5%、81.4%.通过分析6条线性前体肽中二硫键形成的先后顺序对目标产物生成的影响,发现二硫键Cys5-Cys13的形成对利那洛肽的氧化折叠非常关键,在选择性氧化合成利那洛肽时应当优先形成这对二硫键.
Three [4 Trt + 2 Acm] and three [4 Trt + 2 Acm] were synthesized using the Fmoc solid phase synthesis strategy and Wang resin as the carrier using cysteines with trityl (Trt) and acetamidomethyl (Acm) 2 Trt + 4 Acm] linaclotide linear precursor compound.On the basis of this, a semi-selective oxidative strategy was used to synthesize linaclotide containing three pairs of disulfide bonds.Firstly, using trifluoroacetic acid (TFA) -containing Cleavage reagent removes the Trt protecting group of cysteine in the linear precursor peptide and catalyzes the oxidation of cysteine free thiols to form disulfide bonds using hemin.The next step is to use Ph S (O) Ph / CH3Si Cl3 reagent System removes the Acm protecting group remaining cysteine and simultaneously forms disulfide bonds.Using this strategy, 3 of the 6 linear precursor peptides give linaclotide with conversion rates of 71.9 %, 31.5% and 81.4% respectively.It is found that the formation of disulfide bond Cys5-Cys13 is very important for the oxidative folding of linaclotide by analyzing the influence of the order of formation of disulfide bonds in six linear precursor peptides on the target product , The pair of disulfide bonds should be preferentially formed upon selective oxidation of linaclotide.