Synthesis of a Novel Acyl Phosphate Cross-linker and its Modification of Hemoglobin

Transcription

1 Synthesis of a Novel Acyl Phosphate Cross-linker and its Modification of Hemoglobin by Elizabeth Wilson A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Chemistry University of Toronto Copyright by Elizabeth Wilson 2012

2 Synthesis of a Novel Acyl Phosphate Cross-linker and its Modification of Hemoglobin Elizabeth Wilson Abstract Master of Science Department of Chemistry University of Toronto 2012 Hemoglobin-based oxygen carriers (HBOCs) are of great interest for their potential as a safer alternative to blood transfusions. To overcome the vasoactivity associated with small HBOCs, our group is interested in connecting two hemoglobin tetramers together, forming bis-tetramers. Bis-tetramers have previously been synthesized by our group, but yield and purity of the resulting solutions have been low and hindered their usefulness for trials. A new cross-linker was designed in an attempt to improve yield. This thesis describes the synthesis of an acyl phosphate cross-linker N-[bis(sodium methyl phosphate)isophthalyl]-4-azidomethylbenzoate (5), its modification of hemoglobin and subsequent purification attempts of the resulting solution. Cross-linker 5 was found to be selective to β-β-crosslinking and produced singly modified subunits as byproducts. Attempts to purify the resulting reaction mixture by heating resulted in the decomposition of the azide group on the cross-linker, which was critical for the coupling step. Efforts to overcome this problem were unsuccessful. ii

3 Acknowledgements First and foremost I would like to thank my supervisor, Dr. Ron Kluger, for providing invaluable guidance, support and encouragement throughout the duration of my thesis. I would like to thank the entire Kluger group, both past and present members: Dr. Ying Yang, Dr. Raj Dhiman, Sohyoung Her, Adelle Vandersteen, Yi Han, Liliana Guevara Opinska, Graeme Howe, Erika Siren, Aizhou Wang and Brian De La Franier for all of their help and input, as well as their company. I would like to thank Chung-Woo Fung, for all of his technical assistance, and the University of Toronto NMR and AIMS staff. Finally I would like to thank my friends and family for all of their encouragement and support. iii

4 Table of Contents Abstract... ii Acknowledgements... iii Table of Contents... iv List of Figures... vi List of Schemes... vii List of Abbreviations... ix Chapter 1: Introduction... 1 Blood substitutes... 1 Hemoglobin... 2 Diphosphoglycerate binding site: a target for cross-linking... 4 Problems in clinical trials... 7 One step bis-tetramers: tetrakis-acylating linkers... 8 Using click chemistry to couple tetramers... 9 Improved yield of bis-tetramer formation Purification of hemoglobin tetramers Purpose of thesis Acyl phosphate cross-linking reagents Chapter 2: Results and Discussion Preparation of acyl phosphate cross-linker Reaction of cross-linker with hemoglobin Gel Filtration Heat treatment iv

5 Bis-tetramer formation Optimization of Heat Treatment Conditions Chapter 3: Experimental General methods Materials Synthesis of acyl phosphate cross-linker Cross-linking with hemoglobin HPLC analysis of modified hemoglobin Heat treatment of modified hemoglobin solutions Coupling of tetramers CuAAC click chemistry Chapter 4: Conclusions and Future Work Conclusions Symmetrically vs. unsymmetrically cross-linked hemoglobin Optimization of purification of Hb-N Synthesis of alkyne cross-linker v

6 List of Figures Figure 1. Adult human tetrameric hemoglobin: α-subunits are shown in red; β-subunits are shown in blue. Heme groups are shown in green Figure 2. An allosteric regulator in the human circulatory system, 2,3-diphosphoglycerate (DPG) electrostatically binds to hemoglobin to stabilize the low oxygen affinity conformation of hemoglobin Figure 3. Cross-linking hemoglobin prevents the dissociation of the tetramer into dimers outside of the red blood cell Figure 4. Binding site of 2,3-diphosphoglycerate. Emphasized are β-lys82/β -lys82 and β- val1/β -val1, which contain free amino groups that are most readily acylated by crosslinkers Figure 5 Two classes of reagents used to cross-link hemoglobin: acyl salicylates (left) acyl phosphates (right)... 5 Figure 6. Proposed mechanism of acylation of hemoglobin Figure 7. One step bis-tetramer formation with acyl phosphate linker Figure 8. Hydrolysis at an acyl directing group can prevent bis-tetramer formation Figure 9. Acyl salicylate-based cross-linker 1 with free azide group Figure 10. Bisalkyne linkers used in attempted coupling of two equivalents Hb-N Figure 11. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) Figure 12. Target compound 5, an acyl phosphate monoester analogue of Figure 13. C-4 reverse-phase HPLC chromatograms of the reaction mixture of Hb with reagent 5 at ph 7.4 (top left), at ph 8.0 (top right), ph 9.0 (bottom left) and unmodified Hb (bottom right) vi

7 Figure 14. Overlaid spectra show that the reaction at ph 9.0 gave the best yield Figure 15. Species formed after reaction of 5 with hemoglobin. Singly modified β- subunits (left), singly modified α-subunits (middle, and β-β-cross-linked subunits were found (right) Figure 16. Superdex gel filtration chromatogram of native Hb Figure 17. Superdex gel filtration chromatogram of crude reaction mixture of Hb with reagent 5 at ph Figure 18. Heat treated crude reaction material from above Figure 19. Size exclusion HPLC of click reaction, overlaid with native Hb Figure 20. C4 reverse-phase HPLC chromatogram of heat treated Hb-N Figure 21. Products in solution after heat treatment. Some of the original Hb-N 3 remained (left) and some species where the azide was not stable to heat treatment and decomposed to release nitrogen gas (right) Figure 22. All possible species formed from unpurified CuAAC starting material. Unspecified subunits may be either α- or β-subunits Figure 23. Superdex gel filtration chromatogram of click reaction before purification Figure 24. Superdex gel filtration chromatogram of after heat treatment Figure 25. Overlaid chromatograms of CuAAC solution before and after heat treatment List of Schemes Scheme 1. Coupling of two Hb-N 3 tetramers together using bisalkyne linker 3. The product of the first click reaction brings the previously insoluble linker in to the aqueous phase. The second click reaction is expected to be faster vii

8 Scheme 2. Reaction between 1 and Hb to form Hb-N Scheme 3. Reaction between Hb and TTDS, followed by installation of azide moiety Scheme 4. Synthesis of acyl phosphate cross-linker Scheme 5. Materials to the left of the arrow cannot practically be separated, thus any acyl phosphate groups are cleaved by base-catalyzed hydrolysis to regenerate Scheme 6. Reaction of 5 with Hb. Side products from are shown in Figure Scheme 7. Dissociating conditions causes native Hb to split into α-β-dimers (top). Crosslinked hemoglobin, whether a tetramer or a bis-tetramer, does not dissociate into αβdimers (bottom). Scheme is originally from Gourianov s Ph.D. dissertation Scheme 8. Coupling reaction Scheme 9. Proposed synthetic scheme for cross-linker containing an alkyne functional group. i) DCC coupling ii) KOH (in 1:1 THF:MeOH) deprotection iii) SOCl 2, THF iv) KOH (in 1:1 THF:MeOH) deprotection v) SOCl 2, sodium dimethyl phosphate, THF vi) NaI, acetone viii

9 List of Abbreviations kda DMSO DPG ESI Hb MS NMR RBC SDS-PAGE kilodalton Dimethylsulfoxide 2,3-diphosphoglycerate Electrospray Ionization Hemoglobin Mass Spectrometry Nuclear Magnetic Resonance Red Blood Cell Sodium Dodecyl Sulphate Polyacrylomide Gel Electrophoresis TFA THF Tris HBOC P 50 n 50 NO HbCO DeoxyHb oxyhb methb UV-Vis Trifluoroacetic Acid Tetrahydrofuran Tris(hydroxylmethyl)aminomethane Hemoglobin-based oxygen carrier Oxygen pressure at which Hb is half saturated Hill s coefficient of cooperativity at half saturation Nitric oxide Carbonmonoxyhemoglobin Deoxygenated hemoglobin Oxygenated hemoglobin Methemoglobin Ultraviolet-visible ix

10 Bis-Tris MOPS α-α-hb β-β-hb BT-Hb DCM DMF EtOAc MeOH NaI DCC D 2 O CDCl 3 Bis-(2-hydroy-ethyl)-amino-tris(hydroxymethyl)-methane 3-(N-morpholino)praopanesulfonic acid α-α-cross-linked hemoglobin β-β-cross-linked hemoglobin Bis-tetramers of hemoglobin Dichloromethane Dimethylformamide Ethyl acetate Methanol Sodium iodide N,N -Dicyclohexylcarbodiimide Deuterated water Deuterated chloroform ºC Degrees Celsius h TLC MW CuAAC ppm Hz MALDI-MS Hours Thin layer chromatography Molecular weight Copper-catalyzed azide-alkyne cycloaddition Parts per million Hertz Matrix-assisted laser desorption/ionization mass spectrometry x

11 Chapter 1: Introduction Blood substitutes Interest in developing artificial blood has been around for years, but was renewed in the early 1980s when HIV and the transmission of viral containments through blood (transfusions) were discovered. 1 Transfusing donated blood is a common low-risk medical procedure in many countries, but it can be high-risk in less technologically advanced countries. Concerns arise from type-matching, stringent storage requirements and the increasing unmet demand for blood donations. The shelf life of donated blood is fairly short (42 days) and it must be kept refrigerated. Furthermore, donated blood typically requires 24 hours for the allosteric effector 2,3-diphosphoglycerate to be sufficiently depleted for the blood to be ready for use. Due to these restraints transfusions are somewhat limited in situations which required immediate usage, such as emergency medical procedures. Currently there is no FDA approved blood substitute for use in humans. 2 Ideally, researchers hope to create a blood substitute that would be universal to all blood types, have practical storage requirements and be free from contaminations. The primary purpose of modern transfusions is to ensure sufficient oxygen is delivered to tissues when significant blood has been lost. Tissues require oxygen for cell 1 Kluger, R. Curr. Opin. Chem. Bio. 2010, 14, U.S. Food and Drug Administration. Blood Substitutes: Working to Fulfill a Dream. (accessed August 7, 2012). 1

12 metabolism and without sufficient supply tissue damage will result. Blood had many functions, but the goal of most blood substitutes is solely to transport oxygen. There are two major approaches to developing a blood substitute: perfluorocarbon based oxygen carriers (PFCBOCs) and hemoglobin based oxygen carriers (HBOCs). Perfluorocarbon solutions are synthetic solutions of fluorocarbons (hydrocarbon compounds that have fluorine atoms in the place of hydrogen atoms) in which gases such as oxygen are extremely soluble. Hemoglobin based oxygen carriers (HBOCs) use hemoglobin, the metalloprotein that carries oxygen, to effectively deliver oxygen throughout the bloodstream. Hemoglobin is naturally found in red blood cells, but is extracted and purified for use in HBOCs. Modification of hemoglobin is necessary 1, 3, 4 because of complications that arise when hemoglobin is outside of a red blood cell. Hemoglobin The structure of hemoglobin was first elucidated in 1952 by Max Perutz using X-ray crystallography. 5 For this, Perutz shared the 1962 Nobel Prize of Chemistry with John Kendrew for their studies of the structures of globular proteins. 6 Human hemoglobin is a tetrameric protein, comprised of two α-subunits and two β-subunits (Figure 1). 3 Lowe, K.C. Tissue Eng, 2003, 9, Kluger, R.; Foot, J. S.; Vandersteen, A.A. Chem. Commun. 2010, 46, Perutz, M.F.; Rossmann, M.G.; Cullis, A.F.; Muirhead, H.; Will, G.; North, A.C.T. Nature. 1960, 185, Nobelprize.org. The Nobel Prize in Chemistry (accessed August 22 nd, 2012) 2

13 Figure 1. Adult human tetrameric hemoglobin: α-subunits are shown in red; β-subunits are shown in blue. Heme groups are shown in green. Each subunit contains a non-covalently bound iron-heme group, which can reversibly bind one oxygen molecule at the iron center. Accordingly, at saturation one hemoglobin molecule can bind to four oxygen molecules. Oxygen is bound cooperatively - as one oxygen molecule binds to a free heme group, it induces discreet changes to the protein s conformation so that the next oxygen molecule to bind does so with more ease and so forth for each successive oxygen molecule. Cooperativity in binding allows hemoglobin to fluently pick up oxygen where it is in high concentration and release oxygen more readily where oxygen is required. Contained by the red blood cell, hemoglobin shuttles back and forth in the bloodstream between the lungs and oxygen-depleted tissues. Figure 2. An allosteric regulator in the human circulatory system, 2,3-diphosphoglycerate (DPG) electrostatically binds to hemoglobin to stabilize the low oxygen affinity conformation of hemoglobin. Assisting in this process is the allosteric regulator 2,3-diphosphoglycerate (DPG) (Figure 2). It electrostatically binds to a small cleft in hemoglobin, stabilizing hemoglobin in its low oxygen affinity state, assisting in offloading oxygen. DPG is found in high 3

14 concentration at oxygen-depleted tissues. Due to hemoglobin s natural efficiency at executing the complex process of delivering oxygen, it was a natural starting point for developing a blood substitute. Furthermore, purified hemoglobin lacks a blood type antigen and does not produce an immunological response, making it universal for all blood types. 7 The first major clinical testing with a hemoglobin-saline solution was discouraging as patients were found to have high renal toxicities. 8 Outside of the natural protective red blood cell environment, hemoglobin dissociates into α-β-dimers, losing functionality. These toxic byproducts are readily excreted. Different strategies to modifying hemoglobin revolve around preventing the dissociation of these dimers, while still retaining the HBOC`s oxygen-delivering capabilities. Besides being safe, blood substitute candidates must also be efficient at what they were originally intended for - delivering oxygen. When designing HBOCs, two qualities are looked at to ensure an effective oxygen deliverer: oxygen binding affinity and the cooperativity of oxygen binding. Discussion of these parameters is beyond the scope of this thesis as they are peripheral details to the project. For further information see reviews. 4 Diphosphoglycerate binding site: a target for cross-linking To prevent dissociation of the hemoglobin tetramer into α-β-dimers, reagents are designed that will bind to either both α- or β-subunits, cross-linking them together (Figure 3). Some of these cross-linking reagents have been designed to mimic the 7 Kim, H. W.; Greenburg, A.G. Artif. Organs, 2004, 28, Amberson, W.R.; Jennings, J.J.; Rhode, C.N. J. Appl. Physiol. 1949, 1,

15 Figure 3. Cross-linking hemoglobin prevents the dissociation of the tetramer into dimers outside of the red blood cell. regioselectivity of 2,3-diphosphoglycerate (DPG) and bind to a small cleft between the two β-subunits (Figure 44). 4 DPG is anionic in aqueous solution and is strongly attracted to the positively charged protonated amino groups located in this region. Figure 4. Binding site of 2,3-diphosphoglycerate. Emphasized are β-lys82/β -lys82 and β-val1/β -val1, which contain free amino groups that are most readily acylated by cross-linkers. 4 Cross-linking reagents contain functional groups that direct it to the DPG binding site and acylate amino groups located there, covalently linking the β-β-subunits together. Some reagents are not specific to the DPG binding site and are also able to acylate amino groups elsewhere in the protein. If the DPG is bound in its site on hemoglobin, reagents that would react in that site instead react at the other end, which is in the α-subunits. Acyl salicylates and acyl phosphates are anionic electrophiles and have been used to react selectively in the DPG-binding site (Figure 5). Figure 5 Two classes of reagents used to cross-link hemoglobin: acyl salicylates (left) acyl phosphates (right). 5

16 Acyl salicylates are able to react with hemoglobin at both the α-subunits and the β- subunits in the DPG-binding region. Kluger and coworkers probed the relationship between the substitution pattern on the phenyl ring of salicylate esters and the efficiency at acylating hemoglobin. 9 Results indicated that having a carboxyl group ortho to the phenolic ester is necessary to cross-link hemoglobin, due to interactions between the reagent and the surface of the protein at the active site, which are necessary to direct the reagent for acylation. 9 This supports a mechanism of acylation shown in Figure 6. 9 Figure 6. Proposed mechanism of acylation of hemoglobin. Acyl phosphates more closely resemble 2,3-diphosphoglycerate structurally. Like acyl salicylates, they are anionic and have a negative charge but the anion is part of the functional group itself in the acyl phosphate. This supports the idea that a negative charge is required to orient the reagent in the reaction site. 9 Acyl phosphate compounds as cross-linking reagents are discussed further in the introduction. Both acyl salicylates and acyl phosphates direct the reagent to the binding site of the protein where acylation occurs. As leaving groups that are not a part of the cross-linked product, analogous acyl salicylate and acyl phosphate reagents yield the same product after acylation. 9 Kluger, R.; De Stefano, V. J. Org. Chem. 2000, 65,

17 Problems in clinical trials Cross-linked hemoglobins that went to clinical trials induced blood pressure increases, heart attacks and deaths. The specific reasons are not certain but it is reasonable to assume that the materials scavenged nitric oxide, a signaling molecule for vasodilation found in the endothelium. 10 The 1998 Nobel Prize in Medicine was given jointly to Robert Furchgott, Louis Ignarro and Ferid Murad for their discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system, indicative of nitric oxide s acute importance to blood vessel relaxation. 11 Nitric oxide is able to bind at the heme groups (competing with oxygen) and at the thiol of cysteine. 12 It is believed that hemoglobin is able to scavenge nitric oxide from the endothelium because of its small size in comparison to red blood cells. In 2004 Vandegriff and coworkers reported a larger species, maleimide-polyethylene glycol-modified hemoglobin, which effectively delivered oxygen and did not induce high blood pressure when tested in swine. 13 However, there is no definitive data that increasing size alone is sufficient to make a safe HBOC. 10 Drobin, D.; Kjellstrom, B.T.; Malm, E.; Malavalli, A.; Loman, J.; Vandegriff, K.D.; Young, M.A.; Winslow, R.M. J. Appl. Physiol., 2004, 96, Nobelprize.org. The Nobel Prize in Physiologoy or Medicine Http: // (accessed August 14 th, 2012) 12 Jia, L.; Bonaventura, C.; Stamler, J.S. Nature, 1996, 380, Drobin, D.; Kjellstrom, B.T.; Malm, E.; Malavalli, A.; Lohman, J.; Vandegriff, K.D.; Young, M.A.; Winslow, R.M.; J. Appl. Physiol. 2004, 96,

18 One step bis-tetramers: tetrakis-acylating linkers Previous efforts by our group increased the size of hemoglobin tetramers by joining two tetramers together to form bis-tetramers. The main advantage to this approach is that instead of adding on non-functional mass, size is increased with material that carries oxygen. Initially bis-tetramers were formed using tetrakis-acylating linkers, which would simultaneously cross-link hemoglobin tetramers and from the bis-tetramers (Figure 7). 14,15 Figure 7. One step bis-tetramer formation with acyl phosphate linker. Problematically, hydrolysis at the directing groups is a competitive reaction to acylation (Figure 8). Hydrolysis at any one of these activated esters prevents that group from acylating hemoglobin, preventing the formation of a bis-tetramer. Figure 8. Hydrolysis at an acyl directing group can prevent bis-tetramer formation. The solution becomes a mixture of products of varying size: bis-tetramers, a tetramer connected to a dimer, two dimers connected, etc. are possible species in the solution. 14 Lui, F.E.; Kluger, R. Biochemistry, 2009, 48, Gourianov, N. PhD Dissertation, University of Toronto,

19 The lower molecular weight species are undesirable in an HBOC solution because of their likely effect on blood pressure due to their extravasation from blood vessels. Separation of these species is impractical on a large scale and so research focused on synthesizing bis-tetramers in higher purity. Using click chemistry to couple tetramers Recently our group developed a new strategy to increase the yield of bis-tetramer by minimizing competitive hydrolysis reactions. In this approach, hemoglobin is first crosslinked and then tetramers are coupled together using a copper-catalyzed azide-alkyne cycloaddition (CuAAC), a reaction that is orthogonal to hydrolysis. This cycloaddition is aptly called a click reaction because it is highly selective, high yielding and useful for joining two molecules together without forming messy byproducts. Using an azide group on one molecule and an alkyne group on the other, the two molecules are coupled by formation of a triazole. 16 To connect two hemoglobin tetramers an azide group was inserted on the cross-link and a bisalkyne linker was used to couple to two tetramers by undergoing two separate CuAACs (Scheme 1). 17 Initially the bisalkyne linker is 16 Liang, L.; Astruc, D. Coordin. Chem. Rev. 2011, 255, Foot, J.S.; Lui, F.E.; Kluger, R. Chem. Comm. 2009, 47,

20 Scheme 1. Coupling of two Hb-N 3 tetramers together using bisalkyne linker 3. The product of the first click reaction brings the previously insoluble linker in to the aqueous phase. The second click reaction is expected to be faster. 17 insoluble in the aqueous phase (where the hemoglobin is dissolved), so the initial coupling occurs between phases. After the first coupling reaction, the partially coupled linker is pulled into the same phase by the hemoglobin to which it is now attached, where there is excess Hb-N 3, and the second coupling occurs much faster. Excess bisalkyne linker can be used to drive the reaction forward, since once in the aqueous phase, coupling is rapid and it becomes unlikely that a bisalkyne linker will only couple to one cross-linked tetramer. 17 Figure 9. Acyl salicylate-based cross-linker 1 with free azide group. 10

21 The cross-linker 1 with a free azide group (Figure 9) was synthesized in four steps with a yield of 36%. 17 Two equivalents of 1 reacted with hemoglobin to give the desired crosslinked product (Scheme 2). Scheme 2. Reaction between 1 and Hb to form Hb-N Analysis of the reaction mixture with HPLC using a C4 column under fully dissociating conditions showed that both α-α (α-lys99/α -lys99) and β-β (β-lys82/β -lys82) sites are cross-linked by 1. The remaining species in the mixture are unsymmetrical cross-linked tetramer (β-val1/β -lys82) and singly modified β-subunits. Overall the mixture is approximately 40:30:30 of β-β-cross-linked (symmetrical) : α-α-cross-linked : β-β-crosslinked (unsymmetrical). This solution was then taken and subjected to the CuAAC. Various bisalkyne linkers were tested for efficiency (Figure 10). The most efficient was also the most electron deficient, 3, giving bis-tetramer as 20-25% of the final composition, which corresponded to 100% of the β-β-cross-linked symmetrical species coupling. Bisalkyne linkers 2 and 4 gave substantially lower yields. Figure 10. Bisalkyne linkers used in attempted coupling of two equivalents Hb-N 3. 11

22 HPLC and SDS-PAGE were used to confirm the identity of the species formed. Further attempts to increase the yield by optimizing the reaction conditions of the coupling step were not successful. It is speculated that the α-α-cross-linked and the β-βunsymmetrically cross-linked material did not undergo the coupling reaction because the azide was enclosed in folds of the protein and was not accessible. Improved yield of bis-tetramer formation To improve upon the yield an alternative approach was devised which avoided making α-α-hb-n 3. A known reagent was used to make exclusively β-β-cross-linked hemoglobin, and the azide group was installed on the cross-link in a second step. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) (Figure 11) cross-links hemoglobin exclusively β-β in the DPG-binding site, acylating the ε-amino groups Lys-82 of each beta-subunit. 18 The double acylation of a hemoglobin tetramer using this reagent leaves Figure 11. Trimesoyl tris(3,5-dibromosalicylate) (TTDS). one acyl dibromosalicylate of the triester free. Aminolysis is used to install the azide group here (Scheme 3). Unfortunately, a side reaction is the hydrolysis which cleaves the acyl dibromosalicylate group, leaving a free hydroxyl group which is no longer useful. Optimal results were achieved when 2.0 equivalents of TTDS were reacted with deoxyhemoglobin at 37 ºC in MOPS (0.1 M, ph 8.5) for 3 hours, yielding 67% of the 18 Yang, Y.; Kluger, R. Chem. Commun. 2010, 46,

23 targeted cross-linked hemoglobin with the free ester intact (DBST-Hb), 27% of the crosslinked hemoglobin with the free acid and a minor amount of other modified proteins. The azide group was then installed via aminolysis between the free ester and excess 4- azidomethyl-benzylamine at room temperature for 24 hours in 0.1 M MOPS (ph 8.0). Scheme 3. Reaction between Hb and TTDS, followed by installation of azide moiety. 18 The same amount of hydrolyzed cross-linked material was present before and after aminolysis, indicating that further hydrolysis was not an issue at this step. Following this the CuAAC could occur as before to link two tetramers together. The yield of this cycloaddition was improved by replacing copper powder with L-ascorbic acid as the reducing agent to form Cu(I) from Cu(II) and using 4.0 instead of 6.0 equivalents of the activating ligand (sodium 4,4 -(1,10-phenanthroline-4,7- diyl)dibenzenesulfonate)). All of the Hb-N 3 in solution was able to couple, assuring that this reaction is quantitative, resulting in the solution 50% bis-tetramer. Species were confirmed using HPLC and SDS-PAGE. The oxygen-binding properties of these species were determined (P 50 = 6.0, the partial pressure of oxygen required to half saturate hemoglobin, used to determine oxygen affinity, and a Hill coefficient of n 50 = 2.2, used to assess the cooperativity in binding) and were compared to that of native hemoglobin (P 50 = 5.0, n 50 = 3.0). 14 Based upon these values, it appears that the species formed may be a 13

24 suitable candidate as an efficient HBOC. While this method improved upon the yield from before, the bis-tetramer solution was still not sufficiently pure to be useful. Purification of hemoglobin tetramers As illustrated above, an obstacle to developing usable HBOCs is that modifying hemoglobin typically results in a mixture of modified proteins, of which the target modified protein is one of several. The relationship between lower molecular weight species and their toxicity has been well-documented, but while in solution they cloud the structure-to-efficacy relationship of larger bis-tetramer species, as well as being undesirable to have in the HBOC. Appropriate purification methods are required which are efficient, inexpensive and suitable for large scale production. While challenging, recent efforts have certainly expanded this field. Researchers have developed methods for separating cross-linked hemoglobin from uncross-linked hemoglobin by thermally denaturing the less stable uncross-linked hemoglobin. 19 Solutions that were a mixture of cross-linked and uncross-linked hemoglobin tetramers were heated to a temperature between 60 and 85 ºC for 1-6 hours, at varying ph (optimal ph 7.5). Uncross-linked tetramers were denatured and precipitated out of solution while the cross-linked tetramers were unaltered and remained dissolved in solution. The supernatant was collected, which contained pure cross-linked hemoglobin. 19 It is expected that cross-linked tetramers could withstand intensified heat because the crosslink between subunits is covalent and so even if the solution is heated to higher 19 Estep, T.N.; Walder, J.A.; Hai, T. Precipitation method of purifying crosslinked Hb. Int. Appl. WO A ,

25 temperature, these bonds are unlikely to break. 20 Controlled thermal denaturation of hemoglobin for purification has progressed and other variations in process have been developed. 21 When performing these procedures, it is necessary to saturate hemoglobin with carbon monoxide prior to heating to stabilize hemoglobin. OxyHb is much less thermally stable and when heated is converted to methb. 22 MetHb is readily denatured and precipitates via hemichrome formation. 23 Conversely, HbCO is much more stable to heat denaturation. 24 An advantage to using heat for purification is that it simultaneously performs another useful action: inactivating viral contaminants. 25 Heating a solution of hemoglobin at 74 ºC for 90 min was found to inactivate human immunodeficiency virus (HIV), 26 solving an original problem that motivated developing blood substitutes. Purpose of thesis The purpose of this project was to develop a method of making bis-tetramers in higher yield in and in sufficient quantities for their use in various trials to be practical. An acyl 20 Yang, T.; Olsen, K.W. Arch. Biochem. Biophys. 1988, 261, Wong, B.L.; Kwok, S.Y. Method for removing unmodified hemoglobin from cross-linked hemoglobin solutions including polymeric hemoglobin with a high temperature short time heat treatment apparatus. US Patent 8,084,581, December 27, Hollocher, T.C. J. Biol. Chem. 1966, 241, Alves, O.C.; Wajnberg, E. Int. J. Biol. Macromol. 1993, 15, Seto, Y.; Kataoka, M.; Tsuge, K. Forensic Sci. Int. 2001, 121, Abe, H. Ikebuchi, K.; Hirayama, J.; Fujihara, M.; Takeoka, S.; Sakai, H.; Tsuchuda, E.; Ikeda, H. Art. Cells, Blood Subs. 2001, 29, Azari M.; Ebeling, A.; Baker, R.; Burhop, K.; Camacho, T.; Estep, T.; Guzder, S.; Marshall, T.; Rohn, K.; Sarajari, R. Artif. Cell. Blood Subs. 1998, 25,

26 phosphate analogue of 1 (Figure 12) was synthesized and used to cross-link hemoglobin. Since acyl phosphate cross-linkers have shown selectivity towards β-β- vs. α-α-crosslinking unlike acyl dibromosalicylate cross-linkers, we proposed that the reaction of 5 Figure 12. Target compound 5, an acyl phosphate monoester analogue of 1. with hemoglobin would produce β-β-hb-n 3, which then would undergo the CuAAC. Other considerations of the project involved the optimization of the reaction between 5 and hemoglobin and purification of the resulting reaction solution. Acyl phosphate cross-linking reagents Acyl phosphates can be formed in two steps. In the first, an acid chloride is coupled to sodium dimethyl phosphate to form a diester, which is extremely susceptible to hydrolysis. The ester is dissolved in dry acetone containing dissolved sodium iodide, which cleaves one methyl group on the phosphate to give the monoester product while methyl iodide distills off. The insoluble sodium phosphate salt product precipitates. This cleavage is selective and occurs only once, leaving the other methyl ester intact. Advantages to using acyl phosphates over acyl salicylates to cross-link hemoglobin are that they are more soluble in water and react with hemoglobin more quickly than acyl salicylates. While downsides to using acyl phosphates are that they are more prone to 16

27 hydrolysis and are more difficult to synthesize, their selectivity for β-β-cross-linking has potential applications in making bis-tetramers. 17

28 Chapter 2: Results and Discussion Preparation of acyl phosphate cross-linker Scheme 4. Synthesis of acyl phosphate cross-linker 5. The synthesis of 5 is shown in Scheme 4. The starting compound, 4-(bromomethyl) benzoic acid, is from commercial sources. The first step in synthesizing 5 is replacing the bromine in 4-(bromomethyl) benzoic acid (3.03 g, 14 mmol) with an azide by refluxing at 110 ºC under an inert atmosphere with 3.0 equivalents of sodium azide (2.75 g, 42 mmol) in dimethylformamide (75 ml). After 15 hours, the reaction was cooled to room temperature and 250 ml of water was added. A white precipitate was collected by extraction with 4 x 100 ml of ether. The ether solution was washed with a saturated solution of sodium chloride. The ether solution was dried over anhydrous MgSO 4, which was removed by gravity filtration. Evaporation of the solvent yielded a white powder (7). Compound 7 was synthesized in 79% yield. 18

29 Once the azide functional group was installed 4-(azidomethyl) benzoic acid (1.51 g, 8.5 mmol) was converted to the corresponding acid chloride by refluxing for 18 h in excess SOCl 2 (15 ml) under an inert nitrogen atmosphere. Excess SOCl 2 was evaporated yielding a dark yellow liquid. Due to the reactivity of acid chlorides, the product was immediately dissolved in freshly distilled THF under a nitrogen atmosphere and formed a brown solution. Initially the acid chloride of 7 was coupled to (unprotected) 5-aminoisophthalic acid but the corresponding product from this coupling (9) contained impurities. Compound 9 it was not sufficiently soluble in organic solvents to be purified using column chromatography and could not be crystallized. Therefore, methyl protecting groups were added to the carboxylic acid groups of 5-aminoisophthalic acid to change the solubility of the product from the coupling reaction, in order to make column chromatography possible. Thus a convergent step in this synthesis was the protection of 5-aminoisophthalic acid: To a stirred suspension of 5-aminoisophthalic acid (2.5 g, 13.8 mmol) in 75 ml of methanol, 7.5 ml of concentrated H 2 SO 4 was added drop wise to give a clear yellow solution. This solution was heated to reflux. After 24 hours, the solution cooled to room temperature and approximately two thirds of the solvent methanol was evaporated. The remaining solution was neutralized with approximately 12.5 g of NaHCO 3. Distilled water was added to aid in dissolving NaHCO 3 into solution, facilitating neutralization. The product was extracted with 3 x 50 ml DCM, which was washed with a saturated solution of sodium chloride and dried over anhydrous MgSO 4. The drying agent was removed by gravity filtration and the remaining solvent was evaporated to yield a pale pink powder in 98% yield. Thus the acid chloride of 7 was then coupled to methyl protected 5-aminoisophthalic acid by the 19

30 dropwise addition of the acid chloride in THF to a second solution of 5-aminoisophthalic acid dimethyl ester (1.78 g, 8.5 mmol) in dry THF that had been cooled to 0 C, under nitrogen. The reaction stirred for 4 hours as it warmed to room temperature and an orange precipitate formed. Distilled H 2 O (60 ml) was added and 8 was extracted with ethyl acetate. The ethyl acetate solution was washed with a saturated solution of sodium chloride and was dried over anhydrous MgSO 4, which was removed by gravity filtration. Excess solvent was evaporated, yielding an orange solid. Column chromatography with a 7:3 DCM:EtOAc solvent system was used to purify 8. The yield for this reaction was 47%. Compound 8 (0.66 g, 1.7 mmol) was added to a 40 ml solution of 1:1 THF:MeOH (20 ml each), forming an orange suspension. Potassium hydroxide (2.10 g in 4 ml of ddh 2 O) was added drop wise to cleave methyl protecting groups, yielding a clear orange solution. The reaction stirred for 2 hours and was monitored by TLC. After 2 hours, 2M HCl was added drop wise until the solution was strongly acidic (approximately ph 2, measured with ph paper), precipitating out an off-white solid (9). This was extracted with 3 x 20 ml of ethyl acetate. The solution was washed with a saturated solution of sodium chloride and dried over anhydrous MgSO 4. The drying agent was removed by gravity filtration and excess solvent was evaporated, yielding a light yellow powder (9) in 74% yield. Compound 9 (0.32 g, 0.94 mmol) was refluxed in excess SOCl 2 (15 ml) under an inert nitrogen atmosphere, converting its free carboxylic acid groups into acid chloride groups. This product reacted with 2.0 equivalents of sodium dimethyl phosphate to form acyl phosphate diester groups, which are exceptionally unstable to hydrolysis. Thus, for 20

31 this and the following reaction, it is very important that all materials are completely dry. Due to its instability, the product (10) was immediately dissolved in dry acetone and a methyl group on each phosphate was cleaved using sodium iodide to produce the final compound 5 in 75% yield. Purity was determined by the number of peaks in the 31 P NMR spectrum. In a pure batch of 5, only one peak is present ( 31 P NMR (121MHz, D 2 O): δ ppm). The final step proved somewhat problematic, because if any moisture was present the reaction yielded a mixture of 9 with one carboxylic acid converted to an acyl phosphate and the target compound 5. These materials could not be separated (purified) or used as a mixture to modify hemoglobin. A simple, effective reaction was used to regenerate the starting material 9, so synthesis of 5 could be reattempted without remaking starting material entirely from scratch and thus it conserved resources (Scheme 5). The impure reaction material (0.60 g) was dissolved in water and titrated to ph 10 using a solution of sodium hydroxide. Scheme 5. Materials to the left of the arrow cannot practically be separated, thus any acyl phosphate groups are cleaved by base-catalyzed hydrolysis to regenerate 9. The reaction mixture was stirred for 3 hours and the regenerated product 9 was extracted with ethyl acetate. It was washed with a saturated solution of sodium chloride and was dried over anhydrous MgSO 4. The drying agent was removed by gravity filtration and 21

32 excess solvent was evaporated. Regeneration of compound 9 was confirmed using 1 H NMR. Reaction of cross-linker with hemoglobin Purified adult human hemoglobin was used for all modifications. It was stored in carbon monoxide as the stable form carbonmonoxyhemoglobin (HbCO) at 4 ºC until ready for use. In the initial reaction between 5 and hemoglobin (Scheme 6), hemoglobin was transferred into a sodium borate buffer (0.05 M, ph 9.0) by eluting through a Sephadex G-25 gel-filtration column. HbCO was converted to oxygenated hemoglobin (oxyhb) by exposure to a tungsten irradiation lamp for 2 h at 0 C. OxyHb was converted to Scheme 6. Reaction of 5 with Hb. Side products from are shown in Figure 16. deoxyhemoglobin (deoxyhb) by passing a stream of nitrogen over the hemoglobin solution for 2 h at 37 C, while slowly rotating. Cross-linker 5 (2.0 equivalents) was added and the reaction was stirred for 3 h at 37 C under a constant stream of nitrogen (Scheme 6). After 3 h, the modified hemoglobin in solution was converted back to HbCO by passing a stream of carbon monoxide over the solution for 20 min. It was transferred to a MOPS buffer (0.1 M, ph 8.0) via a Sephadex G-25 gel-filtration column to remove any traces of unreacted organic material. The reaction mixture was pushed through a syringe-driven filter unit (Mandel, 0.45 µm PVDF) for further purification. The 22

33 reaction mixture was centrifuged to concentrate the sample. It was then stored under carbon monoxide at 4 C. For subsequent reactions between 5 and Hb at different ph levels, various buffers were used (at ph 7.4, using a 0.01 M phosphate buffer; at ph 8.0, MOPS 0.1 M) but the procedure remained the same. Analysis of the reaction was carried out by reverse-phase HPLC on a C4 column, under fully dissociating conditions. Comparison of the reaction chromatograms to the chromatogram of native hemoglobin shows modification of both the α- and β-subunits. To confirm assignments, peaks were collected and analyzed by ESI-MS. The highest yields achieved are shown in Figure 13, which are estimated from the areas under the peaks. heme β-subunit heme β-subunit α-subunit α-subunit singly modified singly modified β,β-cross-linked β,β-cross-linked heme heme β-subunit β-subunit α-subunit singly modified α-subunit β,β-cross-linked Figure 13. C-4 reverse-phase HPLC chromatograms of the reaction mixture of Hb with reagent 5 at ph 7.4 (top left), at ph 8.0 (top right), ph 9.0 (bottom left) and unmodified Hb (bottom right). 23

34 Figure 14. Overlaid spectra show that the reaction at ph 9.0 gave the best yield. Peaks analyzed by ESI-MS corresponded to unmodified α- and β-subunits, singly modified α- and β-subunits and cross-linked β-β-subunits. Both singly modified α- and β-subunits appeared as a single peak in the chromatogram. In singly modified species, one acyl phosphate group acylated an amino group of hemoglobin, while the other acyl phosphate arm was hydrolyzed making that site unavailable for further reaction with hemoglobin (Figure 15). The disappearance of most of the β-subunit peak, which is prominent in the chromatogram of native hemoglobin, indicates that the majority of β- subunits were modified. β-β-cross-linked subunits appeared as a peak at approximately 55 minutes. As expected, no α-α-cross-linked material was found. Because acyl phosphates are prone to hydrolysis, especially at higher ph, the reaction was tested at lower ph to see if that would minimize hydrolysis and the formation of singly modified species. Figure 15. Species formed after reaction of 5 with hemoglobin. Singly modified β-subunits (left), singly modified α-subunits (middle, and β-β-cross-linked subunits were found (right). 24

35 The reaction at ph 9.0 showed the most β-β-cross-linked hemoglobin (Hb-N 3 ) (Figure 14). This is assumed to be the result of a lower ph causing the amino groups to be more highly protonated, making them unavailable as nucleophiles for the acylation of reagent 5. Gel Filtration The size of species was confirmed using a G200 size exclusion HPLC column under partially dissociating conditions. The reaction mixture was eluted in a 37.5 mm Tris-HCl buffer containing 0.5 M magnesium chloride, at ph 7.4. Under these conditions, hemoglobin tetramers dissociate into α-β-dimers (32 kda). If hemoglobin is cross-linked, it will not dissociate under these conditions and it will remain a tetramer (64 kda) (Scheme 7). 15 Species of different sizes elute at different speeds and thus have different retention times on a HPLC chromatogram. Species with higher molecular weights elute at lower retention times than species with lower molecular weights. Elution of species was monitored at 280 and 414 nm. Peaks were assigned by comparison of retention times to native hemoglobin and species with known molecular weights. Scheme 7. Dissociating conditions causes native Hb to split into α-β-dimers (top). Cross-linked hemoglobin, whether a tetramer or a bis-tetramer, does not dissociate into αβ-dimers (bottom). The scheme is taken from Gourianov s Ph.D. dissertation

36 The chromatogram of the reaction mixture under these conditions confirmed that crosslinking occurred. Native hemoglobin appears as a single peak at approximately 43 minutes (Figure 16). The reaction between Hb and 5 (at ph 9.0) appears as a large peak with a shoulder coming off of the right side. The large peak near the origin is from crosslinked Hb and the shoulder is a combination of all of the uncross-linked material, singly modified and unmodified subunits (Figure 17) appearing at roughly the same retention time as native hemoglobin. Figure 16. Superdex gel filtration chromatogram of native Hb. Figure 17. Superdex gel filtration chromatogram of crude reaction mixture of Hb with reagent 5 at ph

37 Heat treatment Uncross-linked hemoglobin can be denatured and precipitated out of solution by heating. 19 We expected this treatment could be used to separate the unmodified and singly modified subunits from the cross-linked tetramers. The hemoglobin solution, still in MOPS buffer (0.1 M, ph 8.5), was transferred to a vial containing a small stirring bar and was passed under a stream of carbon monoxide for 20 min. The solution was heated to 78 C in a hot water bath and stirred for 95 min. A red precipitate formed. The reaction mixture was centrifuged for 0.5 h and the supernatant was decanted. The solution was concentrated by centrifugation. UV spectroscopy monitored the reaction solution before and after heat treatment at 640 nm to check that methemoglobin (methb, hemoglobin where the iron in the heme group has been oxidized from Fe 2+ to Fe 3+ and can no longer bind oxygen) did not form as a result of the heat treatment. The purity of the remaining solution was confirmed using G200 size exclusion HPLC. In the HPLC chromatogram before heat treatment two merged peaks are seen (Figure 17) but after heat treatment one peak remains (Figure 18). The material in the lower molecular weight shoulder peak (corresponding to unmodified and singly modified hemoglobin) has been removed. Therefore, the reaction mixture after heat treatment was purified and of uniform species (Hb-N 3 ). 27

38 Figure 18. Heat treated crude reaction material from above. Bis-tetramer formation The purified hemoglobin solution was transferred into a phosphate buffer (0.02 M, ph 7.4) by eluting through a Sephadex G-25 gel-filtration column. Several aliquots of solutions of hemoglobin were converted into cyanomethemoglobin by K 3 Fe(CN) 6 and KCN and were spectrophotometrically assayed to determine concentration. 27 Coupling of the two Hb-N 3 bis-tetramers was performed according to literature procedures (Scheme 8). 17 To a solution of Hb-N 3 (1.9 ml, 0.5 µmol), bisalkyne linker 3 (50 µl of 0.1 M in DMSO, 5 µmol), the activating ligand (100 µl of 20 mm in H 2 O, 2 µmol), Scheme 8. Coupling reaction. 27 Rosti, D. Atti. Accad. Med. Lomb. 1968, 23,

39 CuSO 4 (50 µl of 20 mm in H 2 O, 1 µmol) and L-ascorbic acid (200 µl of 100 mm in H 2 O, 20 µmol) were added by micropipette. A stream of carbon monoxide was used to saturate the system and the reaction mixture was shaken for 4 hours on a Fisher Vortex Genie 2. The reaction was purified by eluting it through a Sephadex G-25 gel-filtration column, concentrated on a centrifuge and stored under carbon monoxide at 4 ºC. The yield was determined using G200 size-exclusion HPLC. Because only one peak was seen in the size exclusion chromatogram of the starting material, it was assumed that the starting material was purified and was a single species. Since this coupling reaction has previously been shown to couple quantitatively two β-β-hb-n 3 tetramers, 18 we expected to see one peak in the size exclusion chromatogram. Unexpectedly two peaks were present, indicating that despite reaction conditions being replicated, 100% of the bistetramer was not formed (Figure 19). The reaction chromatogram is overlaid with the chromatogram of native Hb as a reference for calibrating the species size. Based upon this comparison, it appears that a small amount of bis-tetramer was formed (seen at approximately 35 min) and cross-linked material (seen at 40 min) is still present. A small amount of impurity is seen at approximately 20 min. Based on these results, it appears that not all of the cross-linked material underwent the coupling reaction. It is likely therefore that the azide was not stable to the heating purification process. 29

40 Figure 19. Size exclusion HPLC of click reaction, overlaid with native Hb. The heat-treated hemoglobin solution (before the CuAAC was performed) was analyzed by C4 reverse-phase HPLC (Figure 20). heme α-subunit Hb-NH 2 Hb-N 3 Figure 20. C4 reverse-phase HPLC chromatogram of heat treated Hb-N 3 Peaks were collected and analysed by ESI-MS. The expected peaks were unmodified α- subunits and cross-linked β-β-subunits. We also expected that peaks corresponding to unmodified β-subunits and singly modified subunits would not be present. However, a new peak appeared at min. The molecular weight of the species of the conent of the peak at 53 min corresponded to that of the target material (Hb-N 3 ). However, the molecular weight of the material forming the peak at 50 min was lower than expected (MW = kda). This agreed with the hypothesis that the azide group is not stable to the heat treatment conditions and had decomposed, releasing nitrogen gas and leaving behind an amine functional group (Figure 21). 30

41 Figure 21. Products in solution after heat treatment. Some of the original Hb-N 3 remained (left) and some species where the azide was not stable to heat treatment and decomposed to release nitrogen gas (right). Optimization of Heat Treatment Conditions In an attempt to make a pure solution of bis-tetramer, the coupling reaction was performed before the heating process. The starting material (reaction solution of Hb and 5) was a mixture of Hb-N 3, unmodified Hb and singly modified α- and β-subunits (Figure 15). Thus, since all modified materials presumably contained a free azide group, a variety of species was expected: β-β-cross-linked bis-tetramer, β-β-cross-linked tetramer linked to a singly modified subunit, β-β-cross-linked tetramer with both opposite acyl phosphate groups cleaved by hydrolysis, two α-β-dimers linked or a singly modified subunit, where all other three acyl phosphate groups were cleaved by hydrolysis (Figure 22). Figure 22. All possible species formed from unpurified CuAAC starting material. Unspecified subunits may be either α- or β-subunits. Hemoglobin was transferred into a phosphate buffer (0.02 M, ph 7.4) by eluting through a Sephadex G-25 gel-filtration column. The concentration of hemoglobin in solution was determined by conversion of several aliquots to cyanomethemoglobin as previously 31