NOVEL ORGANIC REAGENTS FOR BIOMEDICAL APPLICATIONS

我们建议合成一系列多功能有机试剂（PORs），用于化学修饰无细胞的人血红蛋白，作为紧急输注中携带氧气的血液替代品。这些por是专门设计用于执行分子内和分子间血红蛋白交联的，具有内置的质量控制功能，可以同时产生交联和低聚血红蛋白产品，具有所需的分子量。目前，以血红蛋白为基础的血液替代品研究的紧迫问题之一是增加交联血红蛋白在血管内的保留时间，但目前采用的聚合方法大多产生随机的、异质的混合物，其中大多数存在自氧化、高氧亲和力和/或抗原性的问题。提出了一种控制寡聚化的方法，每次进行交联时产生所需分子量的特定产物。我们将纯化修饰的血红蛋白，并研究它们的物理、组成和功能特性，包括分子量、高铁血红蛋白水平、储存稳定性、氧亲和力（P50）、协同性（Hill n值）以及P50对pH（玻尔效应）和阴离子（氯化物和磷酸盐）的依赖性。我们还将通过胰消化和MALDI质谱分析的结合来阐明交联的位点。然后，我们将使用大鼠和家兔对最有希望的改性血红蛋白进行广泛的动物研究，以评估其血管内持久性、氧运输/递送和生理反应，包括血流动力学反应性和毒性。

We propose to synthesize a series of polyfunctional organic reagents (PORs) for chemically modifying cell-free human hemoglobins for use as oxygen-carrying blood substitutes in emergency transfusions. These PORs have been specifically designed to perform both infra- and intermolecular hemoglobin cross-linking, with a built-in quality control feature, to yield simultaneously cross-linked and oligomerized hemoglobin products with the desired molecular weights. One of the pressing issues in hemoglobin-based blood substitute research at the present time is to increase the intravascular retention time of the cross-linked hemoglobin, but the current methods employed for polymerization largely yield random, heterogeneous mixtures of products, most of which suffer from problems of auto-oxidation, high oxygen affinity and/or antigenicity. Proposed is a method of controlled oligomerization that yields a specific product of desired molecular weight each time the cross-linking is carried out. We will purify the modified hemoglobins, and investigate their physical, compositional, and functional properties, including molecular weight, methemoglobin levels, storage stability, oxygen affinity (P50), cooperativity (Hill n value), and dependence of P50 on pH (Bohr effect) as well as on anionic species (chloride and phosphate). We will also elucidate the sites of cross-links by a combination of tryptic digestion and MALDI MS analyses. We will then perform extensive animal studies, using rats and rabbits, on the most promising modified hemoglobins to evaluate their intravascular persistence, oxygen transport/delivery, and physiological responses, including hemodynamic reactivity and toxicity.