BACKGROUND OF THE INVENTION Modified human hemoglobin is a central component of most materials that have been developed as potential blood substitutes. Cross- linking, either by chemical modification or genetic engineering, is necessary to stabilize the tetrameric protein and counteract its tendency to dissociate into a (3 sub-units. As blood substitutes, cross-linked hemoglobins have to be tolerated in large quantities. Hemoglobin-based oxygen carriers (HBOCs) also provide a possible means of delivery of therapeutically active substances to sites of the body, since HBOCs are a circulatory, biocompatible medium which perfuse tissues and organs of the body. Their globin chains have many chemical sites theoretically capable of bonding to a therapeutic substance to act as a carrier therefor. Any such chemical bonding will, however, effect modification of the globin chains, and care must be taken to ensure that the modification does not have an adverse effect on other properties of the HBOC.

The functionality introduced by cross-linking hemoglobin also has the potential for creating sites for bioconjugation.

BRIEF REFERENCE TO THE PRIOR ART U. S. Patent 5. 399. 671 Kluger and Song, issued March 21, 1995, teaches cross-linking of hemoglobin with a trifunctional reagent in which only two of the functionalities react with the hemoglobin to effect cross-linking, leaving one functionality on the cross-linker residue available for reaction with an exogenous nucleophile.

International Publication W093/08842 (PCT/US92/09713) Somatogen Inc. (Anderson et al.) describes methods and compositions for delivering drugs to the body by binding them to hemoglobin, utilizing cysteine units of the native globin chains of hemoglobin ("internal sites") as the binding sites.

Many natural proteins contain internal disulfide bonds, naturally formed and imparting stability to the protein. These are formed between cysteine units of the peptide chains, and may stabilize the protein in a naturally folded condition. Albumin, for example, has cysteine residues which participate in disulfide bonds in naturally folded albumm Hemoglobin, although containing one or two cysteine residues in the globin chains of its sub-units, does not naturally include disulfide cross- links to confer stability on the tetramer.

SUMMARY OF THE INVENTION It is an object of the present invention to provide novel reagents for modifying proteins such as hemoglobin.

It is a further object to provide novel modified hemoglobin capable of bioconjugation to biochemically active compounds.

It is a further object of the invention to provide tetrameric hemoglobin stabilized by disulfide cross-links.

The present invention provides, from one aspect, modifying reagents for proteins such as hemoglobin, containing disulfide bonds. The reagents have functionalities which react site-specifically with globin chains of the hemoglobin either to effect cross-linking, while leaving the disulfide bonds intact, as part of the cross-link group, or to provide novel complexes having side chains containing disulfide groups. In either case, the products can be reacted with reducing agents to cleave the disulfide bond and to provide modified hemoglobin products with terminal sulfhydryl groups, available for conjugation to therapeutically active compounds, e. g. biomolecules. In addition, the products with terminal sulfhydryl groups can be oxidized to re-form the disulfide bonds and hence produce cross-linked hemoglobin.

The modified reagents of the present invention are derivatives of cystine, in which the free amino groups are derivatized with bulky protectant groups such as carbobenzoxy, and in which the carboxylic acid groups are esterified with amino-reactive leaving groups, such as methylphosphate and electronegatively substituted phenyl groups. The reagents correspond to the general formula A: where R represents an amino-reactive leaving group selected from lower alkyl phosphate and electronegatively substituted phenyl, and R' represents a bulky, N-protectant group such as carbobenzoxy, benzenesulfonyl, toluenesulfonyl, t-butyloxycarbonyl, fluorene- methoxycarbonyl, etc., as commonly used in protein chemistry. The reagents have a sufficiently short chemical chain length between respective amino-reactive leaving groups that they do not effect cross-linking between different hemoglobin tetramers to form 128 kd products (intermolecular cross-linking), only reaction with different ß-sub-units of the same tetramer.

The invention also provides a process of modifying hemoglobin which comprises reacting hemoglobin, in its deoxy state, with a modifying reagent of formula A give above. The acylation reaction occurs at amino acid residues in the diphosphoglycerate (DPG) binding site of the hemoglobin, specifically at the e-amino groups of lys-82 in the P-subunits, and produces some cross-linked product of the general formula B: along with modified products having disulfide linkages in side groups, of general formula C: In the general formulae, the symbol represents tetrameric hemoglobin. The group-Lys-ß-82-NH represents the lysine residues at position 82 on the respective (3-globin chains of hemoglobin with linkage to the side groups through the e-amino group thereof.

Another aspect of the invention is the modified hemoglobins so produced, which are novel products, represented by formulae B, and C given above.

A further aspect is the process of preparing modified hemoglobin having side chains with terminal sulfhydryl groups bonded to (3-lys-82 and/or (3'-lys-82, which comprises subjecting modified hemoglobins of formulae B and/or C, to reduction so as to effect reductive cleavage of the disulfide bond. The modified hemoglobin so formed, of general formula D: is then reactable with any of a wide variety of biochemically active compounds, to form a conjugate administrable to a patient as a means for delivering the biochemically active compound to the patient.

Yet another aspect of the present invention is a process for preparing cross-linked tetrameric hemoglobin having cross-links between respective lysine-82 groups of the (3-globin chains thereof, which comprises oxidizing the product of formula D given above, to re-form the disulfide bond. The cross-linked product so formed, which is the product of formula B given above, also constitutes an aspect of the present invention. This product B is a hemoglobin tetramer stabilized with disulfide cross-links to inhibit dissociation into dimeric sub-units. The disulfide cross-link resembles the natural disulfide cross-link present in many other naturally occurring proteins, and effecting a degree of conformational stability thereon.

The reagents of the present invention do not react with the N- terminal amino groups (Val-1) as do reagents that are bis methyl phosphates derived from simple dicarboxylic acids. It is likely that the bulk of the N-protected amino group in the reagents produces additional selectivity based on steric effects.

X-ray crystallographic structures of deoxy hemoglobin reveal that the P-Lys-82 s-amino groups are outside the protein at the interface between the DPG-binding site and the surrounding solution. By contrast, the a-amino group of the N-terminal (3-Val-1 is further within the DPG- binding site. Modeling of the reagent into the structure of deoxy hemoglobin suggests that the reagents bind to the DPG site at the solution interface, limiting reaction to the lysine residues. The steric bulk of two Cbz amino-protecting groups in the reagents provides selectivity beyond that found with acyl phosphate monoesters toward amino groups in hemoglobin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an anionic exchange HPLC chromatogram of the reaction mixture resulting from Example 2 below, run under conditions which do not dissociate uncross-linked hemoglobin tetramers; Figure 2 is an HPLC chromatogram of the separated globin chains from the reaction mixture in Example 2 below; Figure 3 is a chromatogram of separated globin chains from Example 3 below, taken at an early stage of the reaction; and Figure 4 is an HPLC chromatogram of the products of Example 4 below, taken at an early stage of the reaction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The preferred cross-linking reagent according to the present invention is N, N'-bis-carbobenzoxy-cysteinyl-bis- (methylphosphate), sodium salt of formula I: This new reagent is referred to hereinafter as"ZCMP". It be prepared from the reaction of the bis-acid chloride of N-protected cystine with dimethylphosphate followed by O-demethylation with methyl iodide in acetone. ZCMP is an example of a compound of general formula A above in which R represents methylphosphate and R'represents carbobenzoxy.

Other preferred reagents according to the invention are those in which R is an electronegatively substituted phenyl group of general formula where Z represents one or more independently selected electronegative groups having positive Hammet sigma values, and n is an integer from 1-5.

Examples of suitable groups Z are acetamido (at the ortho or meta positions), acetoxy, acetyl, carbomethoxy, carboxy, halo, cyano, ethoxy (at the ortho or meta positions), hydroxy (at the ortho or meta positions), nitro, phenyl, trifluoromethyl and trimethylammonio. Preferred as substituent group Z are carboxyl, phosphonate, sulfate, sulfate, phosphonate and halogen. Two or three such substituents are preferred, and especially cases where one of the substituents is carboxyl. When only one such substituent is present, it is preferred that it is located in the position ortho to the ester linkage to the aromatic nucleus. Most preferred for group R according to this embodiment is dibromosalicylate.

The hemoglobin which is used in the process of the present invention is preferably human hemoglobin, although the invention is applicable to other types of hemoglobin also, such as bovine hemoglobin and porcine hemoglobin and other animal hemoglobins. The source of the hemoglobin is normally red blood cells, although hemoglobin obtained by genetic engineering, recombinant techniques is also useful.

The cross-linked hemoglobin so formed, with the disulfide bonds intact, can be safely administered to human patients as a hemoglobin-based oxygen carrier.

Alternatively and preferably, however, the cross-linked hemoglobin is subjected to reductive cleavage so as to cleave the disulfide bonds, and form terminal sulfhydryl groups on the side chains bonded to a specific site of a specific globin chain. The cleaved product is then reacted with a biochemical molecule, utilizing the terminal sulfhydryl group, and administered to the body to act as a delivery medium for delivery of the biochemically active compound to the body.

The attachment of the biochemically active compound to the modified hemoglobin according to the present invention utilizes sulfhydryl bonds, located at the terminus of side chains remote from the point of attachment to the globin chains of the hemoglobin, and hence available for ready reaction with biochemically active compounds. The preferred linkage is a disulfide link, formed from the sulfhydryl group of the hemoglobin side chain and a sulfhydryl group of the biochemically active compound, or a sulfhydryl group of a linker or spacer molecule to which the biochemically active compound is in turn linked. A disulfide bond is slowly reduced by reducing agents present in human serum, so that a biochemically active compound so linked to the hemoglobin is slowly released into the blood stream. Reagents and conditions for formation of disulfide bonds are well known in the art. Release of the biochemically active molecule from the hemoglobin, in order for it to exhibit its biochemical activity, is not necessary, in many cases. The conjugate with hemoglobin may exhibit the biochemical activity of the conjugant.

Biochemically active molecules which include sulfhydryl groups in their native structure include a wide variety of proteinaceous compounds which incorporate cysteine residues. These include atrial natriurectic factor (ANF), antithrombotic peptides such as RGDW; antiproliferatives or antimetastics such as RGD polymers or analogues thereof, and GRDGDS or analogues thereof ; antihypertensives-renin inhibitors such as Boc- HPFHL-CH (OH)-CH2-VIH or analogues thereof ; human growth hormone releasing factor analogues; anorexigenics; vasoconstrictors such as arginine vasopressin; vasodilators such as angiotensin converting enzyme inhibitors; and anti-AIDS drugs such as HIV protease inhibitors.

Other sulfhydryl group containing therapeutically active compounds which can be conjugated to the modified hemoglobin of the invention include captopril (1- (3-mercapto-2-methyl-1-oxopropyl)-L- proline).

The reactions of ZCMP with hemoglobin follow the patterns of other anionic electrophiles. The reagent reacts efficiently only with the deoxy form. Its acylating reaction occurs at residues in the DPG binding site at the e-amino groups of Lys-82 in the ß subunits.

The reaction of ZCMP with carbonmonoxy hemoglobin gives only a very small amount of product under the conditions that extensively modify the deoxy form. The product that does form is not cross-linked.

This result is consistent with the conformational change of hemoglobin in going from the R state to the T state. When deoxy hemoglobin binds to ligands such as oxygen and carbon monoxide, the extensive quaternary structural change leads to contraction of the DPG binding site. As a consequence, the DPG binding site is not large enough to be accessed by such a bulky reagent.

The disulfides of the two D-chain-modified hemoglobins, compounds B and C above, show different reactivity with mild reducing agents such as P-mercaptoethanol. With this reagent, the linear disulfide compound C is reduced to form compounds with terminal sulfhydryl groups (compound D) whereas the cross-linked disulfide B remains largely unaffected. The reaction mechanism and accessibility of the S-S bonds of modified hemoglobins explains this difference. Reduction of a disulfide by thiol reagents proceeds in two steps. A mixed disulfide of the cleaved disulfide and mercaptoethanol is an intermediate. In the thiol-disulfide exchange process, a large excess of the reagent is required to drive the reaction to completion.

For the cysteinyl hemoglobin with no cross-links, one end of the reagent is attached to ßLys-82. The unreacted portion of the large, hydrophilic reagent is probably out of the DPG binding site. Therefore, the disulfide is accessible to 2-mercaptoethanol. On the other hand, if the P subunits of hemoglobin are cross-linked as the bis cysteinyl derivative, the S-S bond within the DPG cleft becomes less accessible to the thiol reagent. The DPG binding site protects the disulfide from reduction, and the reaction of that disulfide bond with 2-mercaptoethanol is blocked.

The S-S bond of the cross-linked hemoglobin is cleaved with more effective reducing reagents such as dithiothreitol (DTT). When dithiothreitol is used to reduce the S-S bond, the first step is also a thiol- disulfide exchange as with 2-mercaptoethanol. However, once the intermediate is formed, a second molecule of reagent does not participate in the reaction. Instead, an intramolecular reaction can occur, leading to the formation of the stable cyclic disulfide of DTT. The intramolecular process has a large entropic advantage in its favor. Since the cross-linked hemoglobin was reduced without the addition of denaturing reagents, the S-S bond is near the outside of the DPG cleft of the cross-linked hemoglobin and is exposed to solvent.

In any event, the end product of reduction of both compound B and compound C is a modified hemoglobin with terminal sulfhydryl (thiol) groups, of general formula D above.

Depending upon the choice of protectant group Rl in the modifying reagent, it may be desirable to remove it prior to administering products of the present invention to a patient. This will depend largely on toxicity considerations. Removal of the protectant groups can be accomplished by standard methods employed in peptide chemistry.

The oxidation of thiols to disulfides by oxygen normally proceeds only in the presence of catalytic quantities of metal ions such as iron and copper. The rate of the oxidation of thiols in the same molecule depends on the distance between the thiol groups. In the present case, it has been found that the modified hemoglobin with two free sulfhydryl groups attached at P-Lys-82 is oxidized spontaneously by atmospheric oxygen, quantitatively producing the cross-linked tetramer. It is possible that the iron of the heme facilitates the reaction, especially that which is present as Fe (III) met-hemoglobin. The sulfhydryl groups produced by reduction in the process of the present invention remain in close spatial proximity in order to form the disulfide bond between subunits by oxidation. Cleavage of the disulfide at each site, followed by oxidation permits spontaneous formation of the cross-link between dimers. This produces a much higher yield of cross-linked materials and provides an added benefit of the use of such reagents. Accordingly, a preferred process for making an HBOC according to the present invention comprises reacting human deoxy hemoglobin with a disulfide group containing protein modifying reagent as previously described, to form a mixture of cross-linked and non-cross-linked modified hemoglobin (products B and C) above, subjecting the mixture to reductive cleavage of the disulfide bonds to form a modified hemoglobin of general formula D, and then oxidizing this modified hemoglobin to form disulfide cross-linked hemoglobin. Such a process is capable of producing substantially quantitative yields of cross-linked hemoglobin.

The introduction of specific chemical alterations at defined sites within a protein is complementary to methods based on genetic engineering. The design of the modifying reagent ZCMP of the preferred embodiment of the invention permits site selection based on charge and steric factors. The fact that ZCMP is an amino acid derivative permits chemical techniques to be used that are extended from the chemistry of peptide formation. A wide variety of side chains, as well as longer peptides for conjugation, can be implemented in combination with the convenient methods developed for producing the mixed phosphate-amino acid anhydride.

The use of the disulfide-based reagent in the present study adds further possibilities for applications of cross-linking by converting sites that contain lysyl side chains into those connected as disulfides and also, by reduction, to sites for bioconjugation.

The invention is further described, for illustrative purposes, in the following specific examples.

EXPERIMENTAL PROCEDURES General Methods. Water was doubly distilled and deionized. All pH measurements were standardized against calibrated buffers using a combination glass electrode. Molecular mechanics was employed to obtain preferred conformations based on several initially estimated structures of bis (N-Cbz)-cysteinyl bis (methyl phosphate). The coordinates of deoxy hemoglobin of Fermi and Perutz (Re f. 1, infra) from the Brookhaven protein database were used for visualization of the structure of the protein.

Melting points were obtained in a calibrated oil bath apparatus. Proton NMR spectra were recorded at 200 MHz and 300 MHz with chemical shifts reported relative to TMS or DSS. 13C NMR spectra were recorded on the same instruments at 50 MHz and 75 MHz. Carbon chemical shifts were measured relative to the chloroform-d with TMS at 0. 31p NMR spectra were recorded at 120 MHz and chemical shifts are relative to 85% phosphoric acid in water. Infrared spectra were recorded on a FT-IR spectrometer in KBr pellets. Mass spectra were recorded by electron- impact (EI) or fast atom bombardment ionization (FAB). The Mass Spectroscopy Laboratory, Department of Medical Genetics, University of Toronto provided electrospray ionization mass spectra. Chemical modification of hemoglobin and analysis followed general procedures as reported for related materials. (Refs. 2 and 3, infra) Example 1-Synthesis of N, N'-bis-Cbz-cystein l bis (Sodium Methyl Phosphate) (ZCMP) N, N'-bis-Cbz-cysteinyl dichloride (1.0 g, 1.8 mmol) and sodium dimethyl phosphate (0.54g, 3.6 mmol; from trimethyl phosphate and one equivalent of sodium iodide in dry acetone) were suspended in 30 mL dry tetrahydrofuran at 0° C under nitrogen and stirred for 1 hr. The resulting precipitate of sodium chloride was removed by filtration.

The filtrate contained the reaction product N-Cbz-cysteinyl bis-dimethyl phosphate, 2 Sodium iodide (2.2 g, 14.6 mmol) was added to the filtrate to replace one methyl group at each phosphate with sodium.

The mixture was stirred at 5° C for 48 hr. Solvent was removed by rotary evaporation. The resulting solid was crystallized from acetone-ether, producing an off-white powder. This was collected and washed three times with acetonitrile. The powder was dried in vacuum to give 1.0 g (1.35 mmol, 74% yield) N-Cbz-cysteinyl bis (sodium methyl phosphate) (ZCMP) as a white solid, mp > 200 °C. IR (KBr) C=O 1675 cnf 1,1695 cmi'; 1H NMR (200 MHz, (s, 10H, 2C6H5), 4.88 (s, 4H, 20CH2Ph), 4.44 (m, 4H, NH and NCH), 3.45 (dd, 6H, JP-H = 11.5 Hz, 20CH3), 2.70-3.25 (m, 4H, SCH2); 13C NMR (121 MHz, D20) 8168.3 (d, JP-C = 9.7 Hz, P-O-C=O), 157.29 (C=O), 135.9,128.4,128.3,127.7, 67.1,53.9 (d, JP-C = 6.4 Hz, OCH3), 37.8; 31P NMR (121 MHz, D20) 8- 5.4 (q, JP-H = 11.3 Hz); FAB mass (-, glycerol) 717 (38.0, M-Na+).

Example 2-Preparation of deoxy hemoglobin and its reaction with ZCMP A solution of hemoglobin (80 mg/mL in 6 mL 0.1M pH 8 MOPS) in a 50-mL rb flask was immersed in an ice-water mixture. This was connected to a rotating reactor containing tubes for inflow and outflow of gasses. Humidified oxygen was then passed through the solution for one hr with the flask illuminated by a tungsten lamp. The resulting oxy hemoglobin was converted to the deoxy form by passing a stream of humidified nitrogen over the rotating solution for 3 hr at 37°C.

A solution of ZCMP was added over 15 min to a solution of hemoglobin in buffer (0.5 mM) so that the final conc. of ZCMP in the mixture was 0.7 mM and that of hemoglobin was 0.25 mM (0.1 M MOPS, pH 8.0). Other buffering agents of the kind known as Good buffers may be used in lieu of MOPS, e. g. sodium borate, but more chemically reactive conventional buffers such as Tris and bisTris will react adversely with ZCMP or other protein-modifying agents according to the invention.

The buffered hemoglobin solution was kept at 37 °C. For reactions with deoxy hemoglobin, the reagent was degassed and was introduced under nitrogen. The reaction was carried out under flowing nitrogen. For reactions with carbonmonoxy hemoglobin, the reaction solution was saturated initially with carbon monoxide. After the ZCMP was added to the solution of hemoglobin, the reaction was continued for 2 hr. The flask was then disconnected from the rotating reactor and carbon monoxide was introduced. The solution was passed through a column of Sephadex G-25 equilibrated with 0.1 M pH 8 MOPS to remove excess reagent. The resulting material (ca. 20 mL) was collected in a vial.

Analysis of the resulting modified hemoglobin was done with a combination of reversed-phase HPLC and ion exchange HPLC following previously reported procedures. (2) Product Analysis Extent of cross-linking. A sample of the reaction product (1.0 mL) was passed through a gel filtration column of Sephadex-100 (superfine) that had been equilibrated with 1.0 M magnesium chloride.

Under these conditions hemoglobin that is not cross-linked dissociates into aß dimers. The dimers elute more slowly than do cross-linked tetrameric species. The extent of cross-linking of globin chains was also determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE) on Mini-Protean II Ready Gels (12% polyacrylamide, 0.375 M Tris-HCl, pH 8.8) as reported previously. (Ref.

4, infra) The hemoglobins and globins from HPLC preparations were denatured in 0.065 M Tris-HCl, pH 6.8,2% SDS, 10-v/v glycerol.

Approximately 20 Rg of protein was applied to each lane of the gel and processed at 200 V for 1 hr. Protein bands on the gel were stained with Coomassie brilliant blue R-250.

Analytical and preparative separation of globin chains.

Modified hemoglobins were separated as intact tetramers by anion exchange HPLC on a SynChropak AX300 column (250 x 4.6 mm) using a mixture of 0.025 M bis-tris and 0.025 M tris with gradients starting from pH 8.5 to pH 6.7. The effluent was monitored at 540 nm. Heme and globin chains were separated by reversed phase HPLC using 330 A pore C4 Vydac columns (250 x 4.6 mm for analytical and 250 x 12 mm for preparative). Developers contained 0.1 % trifluoroacetic acid and various gradients of acetonitrile starting at 20% and ending at 60%. The effluent was monitored at 220 nm. Globin chains were recovered from the effluent by lyophilization.

Digestion of globin chains. Globin chains were dissolved in 8 M urea to increase susceptibility to hydrolysis and kept at room temperature for 2-4 hr. The solution was then diluted to 2 M urea with 80 mM ammonium bicarbonate buffer at pH 8.5. Trypsin (2% of total protein) was added, and the solution was digested for 24 hr at room temperature.

The hydrolysate was heated in boiling water for 2 min, diluted to 1 M urea with 80 mM ammonium bicarbonate buffer, and further digested with endoproteinase Glu-C (1% of total protein) for another 72 hr at room temperature. The hydrolysates were filtered before injection into the HPLC.

Chromatography of peptides. Peptide fragments were separated by analytical HPLC using reversed phase C18 columns (3.9 x 300 mm). Developers consisted of 0.1% trifluoroacetic acid in water with gradients of acetonitrile from 0 to 100% over ca 2 hr. The HPLC effluent was monitored at 214 and 280 nm.

Combination of ZCMP with deoxy hemoglobin (3: 1 reagent: protein) in aqueous buffer as described above produced a set of modified hemoglobins. Under the same conditions, carbonmonoxy hemoglobin underwent very little reaction. The resulting proteins from reaction of deoxy hemoglobin and ZCMP were separated by anion-ion exchange HPLC (AX-300). Enzymic digestion and mass spectral analysis were used to determine the sites of the modifications resulting from reaction with ZCMP. The HPLC chromatogram of the reaction mixture containing intact tetramers is presented as Figure I of the accompanying drawings and shows that the solution contained only a small amount of unreacted hemoglobin (oc (xßß) along with two major peaks corresponding to modified tetramers.

Two minor products elute after the major constituents.

Further analysis of the components of the modified proteins by reversed- phase HPLC indicates that the two major products have native oc chains along with modified ß chains (determined by comparison with unmodified protein).

Figure 2 shows the results of HPLC analysis of the reaction mixture under conditions that separate the globin chains. Structural analysis of each component (from digestion, SDS-PAGE, and peptide analysis) indicates that there are two p-modificd tetrameric products.

Peaks 1 and 2 correspond to modified ß subunits. The first peak is material that is modified by reaction with ZCMP without cross-linking (the cysteinyl amide, compound corresponding to general formula C above).

The second contains the two subunits cross-linked as the cysteinyl bis amide (Compound B above). Enzymic digestion followed by peptide analysis reveals that in both peaks, modification is at the e-amino group of lysine-82 of the ß subunits. Two minor products, peaks 3 and 4, were shown by a similar analysis to be modified a subunits. Ion-spray mass spectral analysis (Ref. 5, infra) of material from reversed-phase HPLC gives the molecular weight of the cross-linked ß chains as 32208, consistent with two ß chains linked as the N, N'-CBZ-cysteinyl bis-amide.

The peaks from the material that is not cross-linked are consistent with a species in which an amide has formed from reaction of ZCMP with the lysyl-82 s-amino group while the second acyl group of the reagent has undergone hydrolysis (Table 1). TABLE 1-Ion-spray mass spectral parent peaks of modified globin chains obtained from reaction of deoxv hemoglobin with ZCMP GLOBIN CHAIN OF FOUND EXPECTED HEMOGLOBIN (Molecular weight) (Molecular weight) Unmodified ß-15868.80 50 15867 subunits Modified P-subunits 16359.01 50 16357 Cross-linked P-32207.78 i 1.87 32206 subunits Unmodified a-15127.24 + 1. 55 15126 subunits Modified a-subunits 15616.19 55 15616 Where a cross-linked hemoglobin of the kind of Compound B according to the invention is not to be subjected to reductive cleavage of its disulfide crosslinks but, rather, is to be used as tetrameric (e. g., as an HOBC), the material may be reacted further with an os cross-linker such as <BR> <BR> 3,5 DBSF. Since ZCMP reacts with hemoglobin primarily at the p chains and 3,5 DPSF leads to cross-linking at the a Lys 99 positions, the result is a doubly cross-linked (or doubly-modified) hemoglobin. As reported previously by Kluger, et al. (Ref. 3, infra), if the DPG site is blocked (as it would be, given a Cbz-cysteinyl cross-link between the pLys 82 residues) then 3,5 DBSF cross-links only at the aLys 99 residues. Unintended cleavage of the Cbz-cysteinyl link in such a doubly cross-linked hemoglobin would still leave a fumaryl link between the os chains to keep the tetramer together.

Example 3-Reduction of the disulfide of bis-cysteinyl deoxy hemoglobin with 2-mercaptoethanol.

2-Mercaptoethanol (0.018 g) was dissolved in 1 mL 0.1 M pH 8.0 MOPS. 0.1 mL of the solution was added to one mL of a solution of the modified hemoglobins from the reaction of deoxy hemoglobin with ZCMP. The reaction mixture was stirred at 5 °C. Samples were analyzed by reversed-phase HPLC.

Progress of the cleavage reaction was followed by reversed- phase HPLC. When the modified hemoglobins from the reaction of ZCMP with deoxy hemoglobin were treated with a 20 fold excess of 2- mercaptoethanol for 1 hr, the modified but not cross-linked hemoglobin was reduced to yield two products in approximately 1: 1 ratio (Figure 3 shows a chromatogram from analysis early in the course of the reaction).

As peak 1 disappeared, two new peaks (peak 5 and 6) appeared with shorter retention times. The structures of these products were established <BR> <BR> <BR> <BR> by electrospray ionization mass spectral analysis. Peak 5 is the reduced ß chain corresponding to the desired product that results from the cleavage of the disulfide bonds. The reaction of 2-mercaptoethanol with modified (but not cross-linked hemoglobin is summarized in Scheme 1. The formation of a mixed disulfide intermediate and cleavage. occurs only with material that is not cross-linked by 1. Peak 6 is the mixed disulfide intermediate (of mercaptoethanol and modified and hemoglobin), with a parent mass of 16178 + 2 (calcd 16180). Such a mixed disulfide is consistent with formation of intermediates by this reagent. (21,22). Thus, the possible products are reduced of cross-linked hemoglobin and two hemoglobins with mixed disulfides. However, the reaction of the disulfide of cross-linked hemoglobin with 2-mercaptoethanol proceeded very slowly under the same conditions. Peak 2, corresponding to cross-linked hemoglobin, did not decrease. The calculated mass of the modified (3 chain with a cleaved sidulfide, a cysteinyl amide of the P subunit of hemoglobin, is 16,105; found: 16106.71.4.

The modified protein mixture from the reaction of ZCMP with deoxy hemoglobin followed by treatment with an excess of 2- mercaptoethanol for a few minutes was analyzed as follows. Mass spectra reveal that Peak 5 is the (3 chain corresponding to the product that results from the cleavage of the disulfide bonds derived from the protein modified by ZCMP.

SCHEME 1 Example 4-Reduction of modified hemoglobin with dithiothreitol and subsequent re-oxidation Dithiothreitol (DTT) (0.073 g, four equivalents) was dissolved in 1 mL 0.1 M pH 8.0 MOPS. 0.01 mL of the DTT solution was added to 1 mL of the solution of modified hemoglobins from the reaction of deoxy hemoglobin with ZCMP. The reaction mixture was stirred at 5 °C and the sample analyzed by reversed-phase HPLC. After 15 min, the product was passed through a column of Sephadex G-25 equilibrated with 0.1 M pH 8.0 MOPS to remove excess dithiothreitol. The sample was collected and analyzed again by reversed-phase HPLC.

Dithiothreitol (DTT) is a more effective reducing agent than 2-mercaptoethanol. In this experiment with DTT, all disulfide bonds were cleaved within 15 minutes. The reversed phase HPLC showed none of the original peaks derived from the (3-subunits. There was a single product peak at the position of the cysteinyl derivative of the (3 subunit. This is the same species generated in the reaction with 2-mercaptoethanol and with the same mass (16,105). The bis cysteinyl amide (aap-Lys-82-cys-S-S- cys-(Lys-82)-ß)(Lys-82)-ß) is cleaved at the disulfide.

Figure 4 shows the reversed-phase HPLC chromatogram of the products from an early stage in the reaction with DTT (showing some remaining unreacted P subunits are present). The peaks are numbered as in Figure 3. The bis sulfide now appears as peak 5 but, as required, the intermediate from 2-mercaptoethnaol (peak 6) is not present. The reaction pattern with DTT is summarized in Scheme 2.

SCHEME 2 Gel filtration chromatography was used to separate modified protein from excess DTT after reduction. The product in the presence of air and the absence of DTT is rapidly oxidized to produce once again aaß-Lys-82 (£-NH-cys-S-S-cys-(s-NH-Lys-82) ß quantitatively. HPLC reverse phase analysis after 15 minutes reveals the decrease of peak 5 (modified, not cross-linked) and a large increase in peak 2, that of the cross-linked species. Re-treatment of the protein solution with DTT once again gives the disulfide-cleaved products exclusively (HPLC analysis).

Therefore, the reduction-oxidation procedure is an efficient way to convert material that is not cross-linked into cross-linked material (Scheme 3).

Scheme 3 References (1) Fermi, G. and Perutz M. F. (1984) The crystal structure of human deoxyhaemoglobin at 1.74 Angstroms resolution. J. Mol.

Biol., 175,159-174.

(2) Jones, R. T. (1994) Structural characterization of modified hemoglobins. Methods in Enzymology, 231,322-343.

(3) Jones, R. T., Shih D. T., Fujita T. S., Song Y., Xiao H., Head C. and Kluger R. (1996) A doubly cross-linked human hemoglobin.

Effects of crosslinks between different subunits. J. Biol. Chem., 271,675-680.

(4) Kluger, R. and Song Y. (1994) Changing a protein into a generalized acylating reagent. J. Org. Chem., 59,733-736.

(5) Fenn, J. B., Mann M., Meng C. K., Wong S. F. and Whitehouse C. M. (1989) Electrospray ionization for mass spectrometry of large molecules. Science, 246,64-71.