WO1999023065A1 - Hemoglobin with chemically introduced disulfide cross-links and preparation thereof - Google Patents
Hemoglobin with chemically introduced disulfide cross-links and preparation thereof Download PDFInfo
- Publication number
- WO1999023065A1 WO1999023065A1 PCT/CA1998/001020 CA9801020W WO9923065A1 WO 1999023065 A1 WO1999023065 A1 WO 1999023065A1 CA 9801020 W CA9801020 W CA 9801020W WO 9923065 A1 WO9923065 A1 WO 9923065A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hemoglobin
- cross
- disulfide
- linked
- group
- Prior art date
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- 238000003786 synthesis reaction Methods 0.000 description 1
- QIQCZROILFZKAT-UHFFFAOYSA-N tetracarbon dioxide Chemical group O=C=C=C=C=O QIQCZROILFZKAT-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000005147 toluenesulfonyl group Chemical group C=1(C(=CC=CC1)S(=O)(=O)*)C 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- WVLBCYQITXONBZ-UHFFFAOYSA-N trimethyl phosphate Chemical compound COP(=O)(OC)OC WVLBCYQITXONBZ-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/095—Compounds containing the structure P(=O)-O-acyl, P(=O)-O-heteroatom, P(=O)-O-CN
- C07F9/096—Compounds containing the structure P(=O)-O-C(=X)- (X = O, S, Se)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
- C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
- C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
- C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to protein modification, and to processes and reagents useful therein. It also relates to novel, modified proteins, and to reactions of them to form conjugates with various biochemically active molecules and groups. More specifically, the invention relates to reagents for modifying hemoglobin, including cross- linking of hemoglobin, modified hemoglobins produced using the reagents, and use of the modified hemoglobin for conjugation of biomolecules thereto.
- 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 ⁇ 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.
- cross-linking hemoglobin also has the potential for creating sites for bioconjugation.
- Somatogen Inc. 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.
- 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 albumin. Hemoglobin, although containing one or two cysteine residues in the globin chains of its sub-units, does not naturally include disulfide crosslinks to confer stability on the tetramer.
- 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.
- 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.
- 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:
- R represents an amino-reactive leaving group selected from lower alkyl phosphate and electronegatively substituted phenyl
- R 1 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 ⁇ -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 ⁇ -subunits, and produces some cross-linked product of the general formula B:
- DPG diphosphoglycerate
- the group -Lys- ⁇ -82-NH represents the lysine residues at position 82 on the respective ⁇ -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 ⁇ -lys-82 and/or ⁇ '-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:
- biochemically active compound 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 ⁇ -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 ⁇ -Lys-82 ⁇ -amino groups are outside the protein at the interface between the DPG-binding site and the surrounding solution .
- the ⁇ -amino group of the N-terminal ⁇ - 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 an ⁇ jno-protecting groups in the reagents provides selectivity beyond that found with acyl phosphate monoesters toward amino groups in hemoglobin.
- 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.
- Figure 4 is an HPLC chromatogram of the products of Example 4 below, taken at an early stage of the reaction.
- the preferred cross-linking reagent according to the present invention is N,N-bis-carbobenzoxy-cysteinyl-bis-(methylphosphate), sodium salt of formula I:
- ZCMP 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.
- R is an electronegatively substituted phenyl group of general formula
- Z represents one or more independently selected electronegative groups having positive Hammet sigma values, and n is an integer from 1-5.
- 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, sulfonate, 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.
- 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 te ⁇ ninus 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.
- 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)-CH 2 -VIH or analogues thereof
- human growth hormone releasing factor analogues anorexigenics
- vasoconstrictors such as arginine vasopressin
- vasodilators such as angiotensin converting enzyme inhibitors
- anti-AIDS drugs such as HIV protease inhibitors.
- sulfhydryl group containing therapeutically active compounds which can be conjugated to the modified hemoglobin of the invention include captopril (l-(3-mercapto-2-methyl-l-oxopropyl)-L- proline).
- mild reducing agents such as ⁇ -mercaptoethanol.
- 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.
- the cysteinyl hemoglobin with no cross-links one end of the reagent is attached to ⁇ Lys-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.
- 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).
- DTT dithiothreitol
- the first step is also a thiol- disulfide exchange as with 2-mercaptoethanol.
- 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.
- 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.
- protectant group R 1 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.
- the modified hemoglobin with two free sulfhydryl groups attached at ⁇ -Lys-82 is oxidized spontaneously by atmospheric oxygen, quantitatively producing the cross-linked tetramer.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 cone, of ZCMP in the mixture was 0.7 mM and that of hemoglobin was 0.25 mM (0.1 M MOPS, pH 8.0).
- 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.
- the reagent was degassed and was introduced under nitrogen. The reaction was carried out under flowing nitrogen.
- 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.
- 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 momtored 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.
- 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 hydroly sates 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 momtored at 214 and 280 nm.
- 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 ⁇ -modified tetrameric products. Peaks 1 and 2 correspond to modified ⁇ 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 ⁇ -amino group of lysine-82 of the ⁇ subunits.
- 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 cross-linker such as 3,5 DBSF.
- an cross-linker such as 3,5 DBSF. Since ZCMP reacts with hemoglobin primarily at the ⁇ chains and 3,5 DPSF leads to cross-linking at the Lys 99 positions, the result is a doubly cross-linked (or doubly-modified) hemoglobin.
- 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.
- Peak 2 corresponding to cross-linked hemoglobin, did not decrease.
- DTT Dithiothreitol
- 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 is a more effective reducing agent than 2-mercaptoethanol.
- DTT Dithiothreitol
- the reversed phase HPLC showed none of the original peaks derived from the ⁇ -subunits.
- the bis cysteinyl amide ( ⁇ -Lys-82-cys-S-S- cys-(Lys-82)- ⁇ ) 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 ⁇ 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.
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0010464A GB2346144A (en) | 1997-11-03 | 1998-11-03 | Hemoglobin with chemically introduced disulfide cross-links and preparation thereof |
CA002308277A CA2308277A1 (en) | 1997-11-03 | 1998-11-03 | Hemoglobin with chemically introduced disulfide cross-links and preparation thereof |
AU10156/99A AU1015699A (en) | 1997-11-03 | 1998-11-03 | Hemoglobin with chemically introduced disulfide cross-links and preparation thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6412297P | 1997-11-03 | 1997-11-03 | |
US60/064,122 | 1997-11-03 |
Publications (1)
Publication Number | Publication Date |
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WO1999023065A1 true WO1999023065A1 (en) | 1999-05-14 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/CA1998/001020 WO1999023065A1 (en) | 1997-11-03 | 1998-11-03 | Hemoglobin with chemically introduced disulfide cross-links and preparation thereof |
Country Status (4)
Country | Link |
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AU (1) | AU1015699A (en) |
CA (1) | CA2308277A1 (en) |
GB (1) | GB2346144A (en) |
WO (1) | WO1999023065A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2382347A (en) * | 2001-10-26 | 2003-05-28 | Ezio Panzeri | Haemoglobin conjugates |
US7235639B2 (en) | 2003-04-23 | 2007-06-26 | Ezio Panzeri | Hemoglobin conjugates |
EP4312284A3 (en) * | 2022-07-27 | 2024-02-28 | Samsung SDI Co., Ltd. | Positive electrode, and rechargeable lithium battery including the same |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5399671A (en) * | 1992-11-18 | 1995-03-21 | Kluger; Ronald | Specifically crosslinked hemoglobin with free functionality |
-
1998
- 1998-11-03 CA CA002308277A patent/CA2308277A1/en not_active Abandoned
- 1998-11-03 AU AU10156/99A patent/AU1015699A/en not_active Abandoned
- 1998-11-03 GB GB0010464A patent/GB2346144A/en not_active Withdrawn
- 1998-11-03 WO PCT/CA1998/001020 patent/WO1999023065A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5399671A (en) * | 1992-11-18 | 1995-03-21 | Kluger; Ronald | Specifically crosslinked hemoglobin with free functionality |
Non-Patent Citations (1)
Title |
---|
KLUGER R ET AL: "Efficient Chemical Introduction of a Disulfide Cross-Link and Conjugation Site into Human Hemoglobin at.beta.-Lysine-82 Utilizing a Bifunctional Aminoacyl Phosphate", BIOCONJUGATE CHEM. (BCCHES,10431802);97; VOL.8 (6); PP.921-926, University of Toronto;Lash Miller Laboratories Department of Chemistry; Toronto; M5S 3H6; ON; Can. (CA), XP002094745 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2382347A (en) * | 2001-10-26 | 2003-05-28 | Ezio Panzeri | Haemoglobin conjugates |
GB2382347B (en) * | 2001-10-26 | 2005-04-06 | Ezio Panzeri | Hemoglobin conjugates |
US7235639B2 (en) | 2003-04-23 | 2007-06-26 | Ezio Panzeri | Hemoglobin conjugates |
EP4312284A3 (en) * | 2022-07-27 | 2024-02-28 | Samsung SDI Co., Ltd. | Positive electrode, and rechargeable lithium battery including the same |
Also Published As
Publication number | Publication date |
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GB2346144A (en) | 2000-08-02 |
GB0010464D0 (en) | 2000-06-14 |
CA2308277A1 (en) | 1999-05-14 |
AU1015699A (en) | 1999-05-24 |
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