Hemoglobin-based oxygen carriers have been sought as stable, sterile alternatives to red cells in transfusions. Problems in clinical trials using cross-linked tetramers have led to proposals that larger assemblies of tetramers may alleviate some of the problems. A study of such assemblies requires materials with defined structures and physical properties. Evaluation of cross-linked bis-tetramers with inflexible linear links between the tetramers revealed that these have very low cooperativity in oxygen binding and would thus be inefficient as oxygen carriers. New, more flexible reagents were designed to cross-link and connect tetramers in two modes: with angular connectors that permit torsional movement (1-3) and with linear connectors that resemble previously studied systems (4-6). The resulting cross-linked bis-tetramers were produced in high yield and were isolated and characterized. Digest mapping showed that modifications were specifically introduced as expected at amino groups in the 2,3-bisphosphoglycerate binding sites within beta subunits. Circular dichroism showed that the secondary structure of the globin chains is maintained while the microenvironment of the hemes is altered. The bis-tetramers derived from 1-3 have oxygen affinity (P(50) = 3.6-4.7) and cooperativity (n(50) = 2.2-2.7) that appear to be suitable for efficient oxygen delivery to hypoxic regions along with increased mass that is expected to minimize extravasation.

Chemically connected protein arrays have significant diverse applications including the production of red cell substitutes, bioconjugate drug delivery, and protein therapies. In order to make materials of defined structure, there is a need for efficient and accessible reagents. While chemical cross-linking with a multi-subunit protein can be achieved in high yield, connecting proteins to one another in a dendritic assembly along with concurrent cross-linking has met with limited success. This has now been overcome through the design and implementation of a readily prepared reagent with added reaction sites that compensate for competing hydrolysis. N,N',N''-Tris[bis(sodium methyl phosphate)isophthalyl]-1,3,5-benzenetricarboxamide (1), a hexakis(methyl phosphate) isophthalyl trimesoyl tris-amide, was designed and synthesized in high yield in three stages from a reactive trimesoyl core. This material has three pairs of coplanar cross-linking reaction sites in a symmetrical array. The presence of three sets of sites greatly increases the probability that at least two sets will produce cross-links within hemoglobin tetramers (in competition with hydrolysis) and thereby connect two cross-linked tetramers at the same time. Reaction of 1 with deoxyhemoglobin at pH 8.5 gives a material that contains two cross-linked hemoglobin tetramers connected to one another and to a constituent alphabeta dimer. Products were characterized by SDS-PAGE, MS, enzyme digestion and HPLC. The isolated dendritic-hemoglobin with 2.5 tetrameric components has the same oxygen affinity as native hemoglobin (P50 = 5.0 torr) and retains cooperativity (n50 = 2.0). Analysis of circular dichroism spectra indicates that the assembly retains proper folding of the globin chains while the hemes are in an altered environment.