Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPA^UPO). 4-OH-CPA^UPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPA^UPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.

中文翻译：

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轮生马拉丝霉过氧化物酶合成环磷酰胺代谢物，用于人类癌细胞的毒理学研究。

环磷酰胺（CPA）代表了一种广泛使用的抗癌前药，可通过肝细胞色素P450（CYP）酶转化为主要代谢物4-羟基环磷酰胺（4-OH-CPA），然后非酶促生成生物活性代谢物磷酰胺芥菜和丙烯醛。使用人类药物代谢物作为真实标准来评估其毒性对于药物开发至关重要。然而，4-OH-CPA的化学合成是复杂的，并且仅导致低产率和不期望的副产物。在过去的几年中，真菌非特异性过氧化酶（UPO）已发展成为强大的生物催化剂。它们可以发挥与CYP酶相同的有机化合物和药物相同的选择性氧官能化作用，而过氧化氢被用作唯一的共底物。在这里Marasmius rotula（Mro UPO）采用简单的反应设计。根据所使用的条件，可以获得初级肝脏代谢物4-OH-CPA，其互变异构体醛磷酰胺（APA）和过氧化产物4-酮环磷酰胺（4-酮-CPA）。使用动力学控制的方法，分离出4-OH-CPA，产率为32％（纯度> 97.6％）。用Mro UPO（4-OH-CPA ^UPO）产生的纯化的4-OH-CPA处理两种人癌细胞系（HepG2和MCF-7 ）。用发光细胞活力测定法测定的4-OH-CPA ^UPO诱导的细胞毒性和用γH2AX灶形成形成的基因毒性与市售标准无显着差异。4-OH-CPA ^UPO的高收率 其生物学活性表明，UPOs可有效用于产生CYP特异性药物代谢物，以进行药理评估。