Synthesis and DNA binding affinity evaluation of novel gilvocarcin-C-glucosides

描述(申请人提供)：许多已知与双链DNA结合的天然产物具有两个关键的结构特征，允许与其细胞靶标紧密结合：插入DNA主干的平面芳香族苷元，以及参与与主要和/或次要凹槽的非共价相互作用的一个或多个碳水化合物部分。C-芳基糖苷是一种在芳香族插层剂和一个或多个碳水化合物残基之间含有碳-碳键的物质，因此可以被视为开发高亲和力DNA结合剂的有前途的候选者。虽然已有研究表明，与没有这种残基的嵌入剂相比，含连接糖基残基的芳香族嵌入剂具有更好的结合DNA的能力，但文献中没有系统地比较含有一个、两个或三个附加糖基的嵌入剂与DNA的结合亲和力。在拟议的研究中，我们将制备一系列Gilvocarcin M发色团的单、双和三C-芳基葡萄糖苷衍生物，并用荧光和紫外光谱技术评估它们与双链DNA的结合常数。C-芳基糖苷天然产物Gilvocarcin家族的成员是进行此类研究的理想模板，因为它们是已知的DNA结合剂，具有高抗肿瘤活性和低总体毒性。该合成计划要求保护的碳水化合物、萘和邻碘苯甲酸亚基的聚合组装，并将允许在七到九步中制备四个吉尔沃卡星C-葡萄糖苷衍生物。芳香族和碳水化合物之间关键的碳-碳键形成反应将涉及芳基金属(芳基锂或芳基铜酸盐)试剂与糖硫内酯的直接偶联，然后是中间体半硫缩醛的立体选择性自由基还原。拟议的结合研究测量了我们的gilvocarcin衍生物与小牛胸腺DNA、聚(da/dt)7pol(da/dt)和聚(dg/dc)7Poly(dg/dt)相互作用的强度，这不仅使我们能够评估附加的碳水化合物与gilvocarcin发色团的结合改变的程度，而且还使我们能够在碳水化合物在发色团上的附着位置(对应于放置在主要和/或次要沟槽中)与DNA结合亲和力之间建立关联；此外，从获得的数据中也可以看出我们衍生物的DNA序列偏好。由于假设C-芳基糖苷天然产物的DNA序列特异性取决于糖苷取代基的性质，这些研究将为最终制备含有多种不同碳水化合物的吉尔沃卡星衍生物铺平道路，这有助于建立设计依赖于序列的DNA结合配体的范例。这些分子如果能够选择性地与靶基因的启动子或编码区相互作用，最终可能被用于调节基因转录，并作为广泛疾病状态的治疗剂。 公共卫生相关性：在当前癌症治疗方法的背景下，开发高亲和力DNA结合分子是一个至关重要的目标；事实上，为了最大限度地发挥肿瘤细胞的特异性细胞毒性，药物分子必须能够与癌细胞内确定的独特靶点紧密结合。这项拟议的研究将涉及Gilvocarcins的一系列单、双和三C-芳基葡萄糖苷衍生物的合成和DNA结合亲和力评价，Gilvocarcins是一类无毒的抗肿瘤抗生素，被视为设计基因特异性细胞毒剂的模板。通过这项研究，我们希望建立小分子-DNA相互作用的强度与碳水化合物残基在芳香族插层剂周围的附着数量和位置之间的关联。

DESCRIPTION (provided by applicant): Numerous natural products that are known to bind duplex DNA possess two key structural characteristics that allow tight binding interactions with their cellular target: a planar aromatic aglycone that intercalates the backbone of DNA, and one or more carbohydrate moieties that participate in non-covalent interactions with the major and/or minor grooves. C-Aryl glycosides, substances containing carbon-carbon bonds between an aromatic intercalator and one or more carbohydrate residues, may thus be viewed as promising candidates for the development of high affinity DNA-binding agents. Although it has been shown that aromatic intercalators containing attached glycosyl residues have a superior ability to bind DNA in comparison to intercalators lacking such residues, there exist in the literature no systematic studies quantitatively comparing the DNA binding affinity of intercalators containing one, two, or three appended glycosyl units. In the proposed research, we will prepare a series of mono-, bis-, and tris-C-aryl glucoside derivatives of the gilvocarcin M chromophore and evaluate their association constants for duplex DNA by fluoresence and ultraviolet spectrocopic techniques. Members of the gilvocarcin family of C-aryl glycoside natural products are ideal templates for undertaking such a study since they are known DNA-binding agents that possess high antitumor activity with low overall toxicity. The synthesis plan calls for the convergent assembly of protected carbohydrate, naphthalene, and o-iodobenzoic acid subunits, and will allow the preparation of four gilvocarcin C-glucoside derivatives in seven to nine steps. The key carbon-carbon bond-forming reactions between aromatic and carbohydrate moieties will involve the direct coupling of arylmetal (aryllithium or arylcuprate) reagents and sugar thiolactones, followed by stereoselective radical reduction of the intermediate hemithioketal. The proposed binding studies measuring the strength of interaction of our gilvocarcin derivatives with calf-thymus DNA, poly(dA/dT)7poly(dA/dT), and poly(dG/dC)7poly(dG/dC) will not only allow us to assess the degree to which the attachment of additional carbohydrates to the gilvocarcin chromophore alters DNA binding affinity, but also will allow us to establish a correlation between the site of carbohydrate attachment on the chromophore (corresponding to placement in the major and/or minor grooves) and DNA binding affinity; furthermore, the DNA sequence preferences of our derivatives will also be apparent from the data obtained. Since it has been hypothesized that the DNA sequence specificity of C-aryl glycoside natural products depends on the nature of the glycosidic substituents, these studies will pave the way for the eventual preparation of gilvocarcin derivatives bearing a variety of different carbohydrate moieties useful in establishing paradigms for the design of sequence-dependent DNA-binding ligands. Such molecules, if capable of selectively interacting with the promoters or coding regions of target genes, may ultimately be used for the regulation of gene transcription and as therapeutic agents for a wide range of disease states. PUBLIC HEALTH RELEVANCE: The development of high-affinity DNA-binding molecules is a crucially important goal in the context of current approaches to cancer therapy; indeed, to maximize tumor cell-specific cytotoxicity, drug molecules must be able to bind tightly to unique targets identified within cancer cells. The proposed research will involve the synthesis and DNA binding-affinity evaluation of a series of mono-, bis-, and tris- C-aryl glucoside derivatives of the gilvocarcins, a class of non-toxic antitumor antibiotics viewed as templates for the design of gene-specific cytotoxic agents. Through this study we hope to establish a correlation between the strength of small molecule-DNA interactions and both the number and position of attachment of carbohydrate residues around an aromatic intercalator.