Enzymatic release and macrolactonization of cryptophycins from a safety-catch solid support
Enzymatic release and macrolactonization of cryptophycins from a safety-catch solid support
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DOI：
10.1002/anie.200703665
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
--
作者：
Seufert, Wolfgang;Beck, Zachary Q.;Sherman, David H.
许多具有药理学重要性的天然产物由大环结构组成，例如聚酮抗生素红霉素、非核糖体肽环孢菌素和念珠藻素（cryptophycins）、缩肽家族和有效的抗肿瘤剂。在自然界中，这些大环化合物通过由聚酮化合物（PK）脱氢酶、非核糖体肽（NRP）合成酶和杂合NRP/PK合成酶组成的模块化酶促“组装线”合成。[1]在生物合成过程中，中间体通过硫酯与酶结合，并在最后一步中通过整合的C-末端硫酯酶（TE）结构域环化。NRP和PK生物合成与固相合成方法的类比启发我们开发用于念珠藻素和潜在类似物的固相化学酶促合成方法。以前成功的策略，合成和酶催化的树脂环化的肽涉及底物的酯或硫酯键的方式结合到固体支持物。[2]此外，最近的报告固相合成的线性聚酮化合物在不同的反应条件下，鼓励我们设计一个化学酶的树脂大环化策略，使用一个强大的连接器，是稳定的大多数化学合成条件。[3]为了促进大环化合物的大文库的合成，我们还需要适合于化合物在树脂上的直接释放和环化的方法。该模型研究描述了三个念珠藻素类似物的固相合成和树脂上环化。念珠藻素是一类大环缩肽，于20世纪90年代首次从念珠藻属ATCC 53789和念珠藻属GSV 224中分离得到。[4]这些天然产物的治疗潜力来自其有效和高度选择性的细胞毒性，即使是对多药耐药肿瘤细胞系。其生物学特性引起了人们对其大规模分离、全合成和修饰的极大兴趣。[5]目前，已经描述了超过25种天然存在的念珠藻素和数百种合成类似物。其中几种类似物已被确定为正在考虑进行临床评价的高级抗癌治疗先导药物。[6]大多数天然念珠藻素由四种羟基或氨基酸（分别为单元A-D）组成：δ羟基苯基辛烯酸、3-氯-O-甲基-d-酪氨酸、（R）-α-甲基-β-丙氨酸（或β-丙氨酸）和l-亮氨酸（图1）。[7]最近，从蓝细菌Nostoc sp. ATCC 53789和Nostoc sp. GSV 224中表征了负责产生念珠藻素的基因簇。[8]此外，参与其生物合成的特定酶已经异源表达、纯化和表征，包括念珠藻素硫酯酶（CrpTE），其负责线性中间体的大环内酯化。[9]我们的合成方法采用了改性的肯纳的安全捕获磺酰胺接头，这是在埃尔曼实验室开发的，因为它在合成过程中的稳定性。随后的安全锁扣接头的N-烷基化导致不稳定的酰胺键，其可以通过与硫醇、醇和胺的亲核攻击而被置换，以分别形成硫酯、酯和酰胺。[10]由于活化的酰基磺酰胺的不稳定性，我们设想CrpTE的亲核丝氨酸可以在合成后直接从固体载体上置换念珠藻素。采用具有低取代水平的聚（乙二醇）聚（N，N-二甲基丙烯酰胺）（PEGA）树脂，使得Crp TE能够充分接触固体支持物结合的基质。[11]为了测试酶固相的多功能性。
Many natural products of pharmacological importance consist of macrocyclic structures, for example the polyketide antibiotic erythromycin, the non-ribosomal peptide cyclosporine, and the cryptophycins, a family of depsipeptides and potent antitumor agents. In nature, these macrocyclic compounds are synthesized by modular enzymatic “assembly lines” consisting of polyketide (PK) synthases, non-ribosomal peptide (NRP) synthetases, and hybrid NRP/PK synthetases.[1] During biosynthesis the intermediates are bound to the enzymes by a thioester, and in the final step cyclized by an integrated C-terminal thioesterase (TE) domain. The analogy of NRP and PK biosynthesis to solid-phase synthetic methodology inspired us to develop a solid-phase chemoenzymatic synthetic approach for cryptophycins and potential analogues. Previous successful strategies for the synthesis and enzyme-catalyzed on-resin cyclization of peptides involved substrates bound by means of an ester or thioester linkage to a solid support.[2] In addition, recent reports of solid-phase synthesis of linear polyketides under diverse reaction conditions encouraged us to design a chemoenzymatic on-resin macrocyclization strategy using a robust linker that is stable to most chemical synthesis conditions.[3] To facilitate the synthesis of large libraries of macrocyclic compounds we also required a method suitable for the direct release and cyclization of compounds on-resin. This model study describes the solid-phase synthesis and on-resin cyclization of three cryptophycin analogues. Cryptophycins, a class of macrocyclic depsipeptides, were first isolated in the 1990s from Nostoc sp. ATCC53789 and Nostoc sp. GSV 224.[4] The therapeutic potential of these natural products arises from their potent and highly selective cytotoxicity even for multi-drug-resistant tumor cell lines. The biological properties have generated significant interest in their large-scale isolation, total synthesis, and modification.[5] Currently, more than 25 naturally occurring cryptophycins and several hundred synthetic analogues have been described. Several of these analogues have been identified as advanced anticancer therapeutic leads that are being considered for clinical evaluation.[6] Most natural cryptophycins consist of four hydroxy or amino acids (units A–D, respectively): δhydroxy phenyloctenoic acid, 3-chloro-O-methyl-d-tyrosine,(R)-α-methyl-β-alanine (or β-alanine), and l-leucic acid (Figure 1).[7]Recently, the gene cluster responsible for production of cryptophycins was characterized from the cyanobacteria Nostoc sp. ATCC53789 and Nostoc sp. GSV 224.[8] Furthermore, specific enzymes involved in its biosynthesis have been heterologously expressed, purified, and characterized including the cryptophycin thioesterase (CrpTE), which is responsible for the macrolactonization of the linear intermediate.[9] Our synthetic approach employed the modified Kenner s safety-catch sulfonamide linker, which was developed in the Ellman laboratory, because of its stability during synthesis. Subsequent N-alkylation of the safety-catch linker results in a labile amide bond that can be displaced by nucleophilic attack with thiols, alcohols, and amines to form thioesters, esters, and amides, respectively.[10] Because of the lability of the activated acylsulfonamide, we envisioned that the nucleophilic serine of CrpTE could directly displace cryptophycin from the solid support after synthesis. A poly (ethylene glycol) poly (N, N-dimethylacrylamide)(PEGA) resin with low levels of substitution was employed so that Crp TE has adequate access to the solid-support-bound substrate.[11] To test the versatility of an enzymatic solid-phase …