Enhancing the hydrodynamic volume of Hb

描述(由申请人提供)： 聚乙二醇化已被证明可以降低Hb诱导的血管活性。我们最近开发了一种新的非常有效的聚乙二醇化化学。这种化学已经被用来产生一种携带六个拷贝的PEG-5000链/四聚体的HBA。这种Hb现在被测试为HBOC的Hb基氧载体，名称为Hemostat TM，具有许多理想的性质，不具有血管活性。目前还不清楚达到这些效果所需的最低限度的修饰，甚至还不清楚需要哪些PEGylated-HBA的工程性质。显然，聚乙二醇化的Hb的正性部分源于它们的综合性质。这个项目试图了解综合性质如何随着聚乙二醇化参数的系统调整以及其他尺寸增强策略的变化而变化。 聚乙二醇经表面修饰后，Hb的分子体积/分子质量比显著增加，Hb溶液的粘度和胶体渗透压也随之增加。项目1的总体目标是将聚乙二醇化引起的血红蛋白分子体积/分子质量比的增加作为聚乙二醇链的数目和/或大小、聚乙二醇化中心(S)和聚乙二醇化化学的函数，并相互关联 作为分子体积/分子质量比变化的函数的综合性质Hb的变化。为了实现这一目标，我们致力于设计和评估新的合成策略，通过三种不同的合成方法来生成均一的尺寸增强型HBS：1)用聚乙二醇改性，2)用葡聚糖修饰，3)用聚乙二醇基双功能试剂进行齐聚。新型单官能团聚乙二醇类试剂 将合成马来酰亚胺(目标为-SH)、芳基异硫氰酸酯(目标为a-氨基)和酰胺化化学(目标为β-和羧基)，以实现所需尺寸的聚乙二醇链的特定位置共价连接。同样的聚乙二醇试剂的双功能版本也将被合成用于四聚体间的交联剂。还将开发用于葡聚糖修饰Hb的新化学物质。合并的潜在优势 将研究新的表面修饰策略，通过定点突变Hb来引入新的活性官能团(活性赖氨酸的半胱氨酸)来产生均一产物。这些新的策略将促进蛋白质生物化学核心的努力，以生产材料，将聚乙二醇化的Hb的生化、综合和生物物理性质与血管活性的减少联系起来，并探索两者之间可能的相互作用 聚乙二醇化介导了Hb性质的改变，以及Hb的氧和NO反应性的基因工程调节。

DESCRIPTION (provided by applicant): PEGylation has been shown to reduce Hb-induced vasoactivity. We have recently developed a new and very efficient PEGylation chemistry. This chemistry has been used to generate a species of HbA carrying six copies of PEG-5000 strands/tetramer. This Hb, now being tested as an Hb based oxygen carrier of HBOC under the name Hemostat TM, is vaso-inactive with many of desirable properties. It is not clear either as to the minimum modification needed to achieve these effects or even which engineered properties of PEGylated-HbA are required. It is apparent that the positive properties of PEGylated Hb arise in part from their colligative properties. This project seeks to understand how colligative properties vary with systematic tuning of PEGylation parameters as well as other size enhancement strategies. The surface decoration of Hb with PEG increases its molecular volume/molecular mass ration significantly with a concomitant increase in viscosity and colloidal osmotic pressure of the Hb solution. The overall objective of Project 1 is to correlate the increase in the molecular volume/molecular mass ratio of Hb resulting from PEGylation as a function of number and/or size of PEG-chains, site(s) of PEGylation, and PEGylation chemistry and cross-correlate the changes in the colligative properties Hb as a function of changes in the molecular volume/molecular mass ratio. Towards this goal, we undertake to design, and evaluate new synthetic strategies to generate homogeneous size-enhanced Hbs by three different synthetic approaches: surface decoration with 1) PEG, of 2) dextran and 3) oligomerization using PEG-based bifunctional reagents. New classes of monofunctional PEG reagents based on maleimide (targeted to -SH) , aryl isothiocyanate (targeted to a-amino) and amidation chemistry (targeted to beta and carboxyls) will be synthesized to achieve site specific covalent attachment of desired size PEG chains. The bifunctional versions of the same PEG-reagents will also be synthesized for inter-tetrameric cross linking. New chemistries will also be developed for surface decoration of Hb with dextran. The potential advantages of combining new surface decoration strategies with site directed mutagenesis of Hb to introduce new reactive functional groups (Cys for reactive Lys) to generate homogeneous products will be investigated. These new strategies will facilitate the efforts of the Protein Biochemistry Core to produce materials to correlate the biochemical, colligative and biophysical properties of PEGylated Hb with the reduction in vasoactivity, and to explore the possible interplay between the PEGylation mediated changes in the properties of Hb and the genetic engineering modulation of the oxygen and NO reactivity of Hb.