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Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.

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Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the ⑀ amino groups of the two ␤ chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the ␣ subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the ⑀-amino groups of the two ␣ chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked ␤ chain species binds oxygen cooperatively with a high oxygen affinity (P 50 ‫؍‬ 4.8 torr at pH 7.4). The addition of the second cross-linking reduces the oxygen affinity to 15.9 torr, which compares with 13.0 torr for the singly cross-linked ␣ chain species. The doubly cross-linked hemoglobin retains significant cooperativity with a Hill coefficient of 2.3 compared with 3.0 for unmodified hemoglobin. Because some of the groups responsible for the Bohr effect are acylated, this doubly cross-linked hemoglobin exhibits 25% of the normal Bohr effect and less than 20% of the normal chloride effect. The use of two distinct cross-links within the same tetramer provides a material for physical and structural analysis as well as for further modifications for specific applications. The results indicate that the cross-link introducing the lowest oxygen affinity in the two singly cross-linked species appears to control the overall affinity in this doubly cross-linked species.

There are numerous reports of phosphonate analogues of biologically interesting compounds.1 A related class of analogues

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