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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
ii
Synthesis of a Novel Acyl Phosphate Cross-linker and its
Modification of Hemoglobin
Elizabeth Wilson
Master of Science
Department of Chemistry University of Toronto
2012
Abstract
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.
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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.
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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 ..................................................................... 12
Purification of hemoglobin tetramers ............................................................................ 14
Purpose of thesis............................................................................................................ 15
Acyl phosphate cross-linking reagents .......................................................................... 16
Chapter 2: Results and Discussion ................................................................................ 18
Preparation of acyl phosphate cross-linker ................................................................... 18
Reaction of cross-linker with hemoglobin .................................................................... 22
Gel Filtration ................................................................................................................. 25
Heat treatment ............................................................................................................... 27
v
Bis-tetramer formation .................................................................................................. 28
Optimization of Heat Treatment Conditions ................................................................. 31
Chapter 3: Experimental ................................................................................................ 34
General methods ............................................................................................................ 34
Materials ........................................................................................................................ 34
Synthesis of acyl phosphate cross-linker 5 ................................................................... 35
Cross-linking with hemoglobin ..................................................................................... 39
HPLC analysis of modified hemoglobin ....................................................................... 40
Heat treatment of modified hemoglobin solutions ........................................................ 40
Coupling of tetramers – CuAAC “click” chemistry...................................................... 41
Chapter 4: Conclusions and Future Work ................................................................... 42
Conclusions ................................................................................................................... 42
Symmetrically vs. unsymmetrically cross-linked hemoglobin ..................................... 42
Optimization of purification of Hb-N3 .......................................................................... 43
Synthesis of alkyne cross-linker .................................................................................... 44
vi
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. ........................................................ 3
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. .............................................................................................. 3
Figure 3. Cross-linking hemoglobin prevents the dissociation of the tetramer into dimers
outside of the red blood cell. ............................................................................................... 5
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 ................................................................................................................................ 5
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. .............................................. 6
Figure 7. One step bis-tetramer formation with acyl phosphate linker. ............................. 8
Figure 8. Hydrolysis at an acyl directing group can prevent bis-tetramer formation. ....... 8
Figure 9. Acyl salicylate-based cross-linker 1 with free azide group. .............................. 10
Figure 10. Bisalkyne linkers used in attempted coupling of two equivalents Hb-N3. ...... 11
Figure 11. Trimesoyl tris(3,5-dibromosalicylate) (TTDS)................................................ 12
Figure 12. Target compound 5, an acyl phosphate monoester analogue of 1. .................. 16
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
vii
Figure 14. Overlaid spectra show that the reaction at pH 9.0 gave the best yield. ........... 24
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
Figure 16. Superdex gel filtration chromatogram of native Hb. ....................................... 26
Figure 17. Superdex gel filtration chromatogram of crude reaction mixture of Hb with
reagent 5 at pH 9.0. ........................................................................................................... 26
Figure 18. Heat treated crude reaction material from above. ............................................ 28
Figure 19. Size exclusion HPLC of click reaction, overlaid with native Hb. .................. 30
Figure 20. C4 reverse-phase HPLC chromatogram of heat treated Hb-N3 ....................... 30
Figure 21. Products in solution after heat treatment. Some of the original Hb-N3
remained (left) and some species where the azide was not stable to heat treatment and
decomposed to release nitrogen gas (right)....................................................................... 31
Figure 22. All possible species formed from unpurified CuAAC starting material.
Unspecified subunits may be either α- or β-subunits. ....................................................... 31
Figure 23. Superdex gel filtration chromatogram of click reaction before purification. .. 32
Figure 24. Superdex gel filtration chromatogram of after heat treatment. ........................ 33
Figure 25. Overlaid chromatograms of CuAAC solution before and after heat treatment.
........................................................................................................................................... 33
List of Schemes
Scheme 1. Coupling of two Hb-N3 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 ............................................ 10
viii
Scheme 2. Reaction between 1 and Hb to form Hb-N3.17 ................................................. 11
Scheme 3. Reaction between Hb and TTDS, followed by installation of azide moiety.17 13
Scheme 4. Synthesis of acyl phosphate cross-linker 5. ..................................................... 18
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. ........... 21
Scheme 6. Reaction of 5 with Hb. Side products from are shown in Figure 16. ............. 22
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). Scheme is originally from Gourianov’s Ph.D. dissertation.15 .............. 25
Scheme 8. Coupling reaction. ........................................................................................... 28
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) SOCl2, THF iv)
KOH (in 1:1 THF:MeOH) deprotection v) SOCl2, sodium dimethyl phosphate, THF vi)
NaI, acetone. ...................................................................................................................... 45
ix
List of Abbreviations
kDA kiloDalton
DMSO Dimethylsulfoxide
DPG 2,3-diphosphoglycerate
ESI Electrospray Ionization
Hb Hemoglobin
MS Mass Spectrometry
NMR Nuclear Magnetic Resonance
RBC Red Blood Cell
SDS-PAGE Sodium Dodecyl Sulphate – Polyacrylomide Gel
Electrophoresis
TFA Trifluoroacetic Acid
THF Tetrahydrofuran
Tris Tris(hydroxylmethyl)aminomethane
HBOC Hemoglobin-based oxygen carrier
P50 Oxygen pressure at which Hb is half saturated
n50 Hill’s coefficient of cooperativity at half saturation
NO Nitric oxide
HbCO Carbonmonoxyhemoglobin
DeoxyHb Deoxygenated hemoglobin
oxyHb Oxygenated hemoglobin
metHb Methemoglobin
UV-Vis Ultraviolet-visible
x
Bis-Tris Bis-(2-hydroy-ethyl)-amino-tris(hydroxymethyl)-methane
MOPS 3-(N-morpholino)praopanesulfonic acid
α-α-Hb α-α-cross-linked hemoglobin
β-β-Hb β-β-cross-linked hemoglobin
BT-Hb Bis-tetramers of hemoglobin
DCM Dichloromethane
DMF Dimethylformamide
EtOAc Ethyl acetate
MeOH Methanol
NaI Sodium iodide
DCC N,N’-Dicyclohexylcarbodiimide
D2O Deuterated water
CDCl3 Deuterated chloroform
ºC Degrees Celsius
h Hours
TLC Thin layer chromatography
MW Molecular weight
CuAAC Copper-catalyzed azide-alkyne cycloaddition
ppm Parts per million
Hz Hertz
MALDI-MS Matrix-assisted laser desorption/ionization mass
spectrometry
1
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, 538-543.
2 U.S. Food and Drug Administration. Blood Substitutes: Working to Fulfill a Dream.
http://blogs.fda.gov/fdavoice/index.php/2012/06/blood-substitutes-working-to-fulfill-a-dream/ (accessed
August 7, 2012).
2
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
because of complications that arise when hemoglobin is outside of a red blood cell.1, 3, 4
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, 389–399 4 Kluger, R.; Foot, J. S.; Vandersteen, A.A. Chem. Commun. 2010, 46, 1194-1202. 5 Perutz, M.F.; Rossmann, M.G.; Cullis, A.F.; Muirhead, H.; Will, G.; North, A.C.T. Nature. 1960, 185,
416–422.
6 Nobelprize.org. The Nobel Prize in Chemistry 1962.
Http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/ (accessed August 22nd, 2012)
3
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
4
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, 813-828.
8 Amberson, W.R.; Jennings, J.J.; Rhode, C.N. J. Appl. Physiol. 1949, 1, 469-489.
5
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).
6
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, 214-219.
7
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, 1843-1853.
11 Nobelprize.org. The Nobel Prize in Physiologoy or Medicine 1998. Http:
//www.nobelprize.org/nobel_prizes/medicinelaureate/1998/ (accessed August 14th, 2012)
12 Jia, L.; Bonaventura, C.; Stamler, J.S. Nature, 1996, 380, 221-226.
13 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, 1843-1853.
8
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, 11912-11919.
15 Gourianov, N. PhD Dissertation, University of Toronto, 2006.
9
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 cross-
linked 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, 2933-2945.
17 Foot, J.S.; Lui, F.E.; Kluger, R. Chem. Comm. 2009, 47, 7315-7317.
10
Scheme 1. Coupling of two Hb-N3 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-N3, 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.
11
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 cross-
linked product (Scheme 2).
Scheme 2. Reaction between 1 and Hb to form Hb-N3.17
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-cross-
linked (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-N3.
12
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-β-
unsymmetrically 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-N3. 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, 7557-7559.
13
targeted cross-linked hemoglobin with the free ester intact (DBST-Hb), 27% of the cross-
linked 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-N3 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 (P50 = 6.0, the partial pressure of oxygen required to half saturate
hemoglobin, used to determine oxygen affinity, and a Hill coefficient of n50 = 2.2, used to
assess the cooperativity in binding) and were compared to that of native hemoglobin (P50
= 5.0, n50 = 3.0).14 Based upon these values, it appears that the species formed may be a
14
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 cross-
link 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
8912456 A1 19891228, 1989.
15
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, 283-290.
21 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, 2011.
22 Hollocher, T.C. J. Biol. Chem. 1966, 241, 1958-1968.
23 Alves, O.C.; Wajnberg, E. Int. J. Biol. Macromol. 1993, 15, 273-27.
24 Seto, Y.; Kataoka, M.; Tsuge, K. Forensic Sci. Int. 2001, 121, 144-150.
25 Abe, H. Ikebuchi, K.; Hirayama, J.; Fujihara, M.; Takeoka, S.; Sakai, H.; Tsuchuda, E.; Ikeda, H. Art.
Cells, Blood Subs. 2001, 29, 381-388.
26 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, 577–582.
16
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-cross-
linking 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-N3, 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
17
hydrolysis and are more difficult to synthesize, their selectivity for β-β-cross-linking has
potential applications in making bis-tetramers.
18
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 MgSO4, which
was removed by gravity filtration. Evaporation of the solvent yielded a white powder (7).
Compound 7 was synthesized in 79% yield.
19
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 SOCl2 (15 mL) under an inert nitrogen atmosphere. Excess SOCl2 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 H2SO4 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 NaHCO3. Distilled water was added to aid in dissolving
NaHCO3 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 MgSO4. 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
20
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 H2O (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 MgSO4, 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
ddH2O) 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 MgSO4. 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 SOCl2 (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
21
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 31P
NMR spectrum. In a pure batch of 5, only one peak is present (31P NMR (121MHz,
D2O): δ -9.78 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 MgSO4. The drying agent was removed by gravity filtration and
22
excess solvent was evaporated. Regeneration of compound 9 was confirmed using 1H
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
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.
Figure 13. C-4 reverse-p(top left), at pH 8.0 (top r
heme heme
α-subunit
α-subunit
β-subunit
β,β-cross-linked
singly modified
hase HPLC chromatograms of the reaction mixture of ight), pH 9.0 (bottom left) and unmodified Hb (bottom
singly modified
singly modifiedβ,β-cross-linked
Hb w righ
β,β-cross-linked
β-subunit
β-subunitβ-subunit
α-subunit
α-subunitheme
heme
23
ith reagent 5 at pH 7.4 t).
24
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).
25
The reaction at pH 9.0 showed the most β-β-cross-linked hemoglobin (Hb-N3) (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.15
26
The chromatogram of the reaction mixture under these conditions confirmed that cross-
linking 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 cross-
linked 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 9.0.
27
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 Fe2+ to Fe3+ 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-N3).
28
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 K3Fe(CN)6 and
KCN and were spectrophotometrically assayed to determine concentration.27 Coupling
of the two Hb-N3 bis-tetramers was performed according to literature procedures
(Scheme 8).17 To a solution of Hb-N3 (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 H2O, 2 µmol),
Scheme 8. Coupling reaction.
27 Rosti, D. Atti. Accad. Med. Lomb. 1968, 23, 340-345.
29
CuSO4 (50 µL of 20 mM in H2O, 1 µmol) and L-ascorbic acid (200 µL of 100 mM in
H2O, 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-N3 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 bis-
tetramer 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.
The heat-treated hemoglobin solution (before the CuAAC was performed) was analyzed
by C4 reverse-phase HPLC (Figure 20).
Figure 20. C4 reverse-phase HPLC chromatogram of heat treated Hb-N3 Peaks were collected and analysed by ESI-MS. The expected peaks wer
subunits and cross-linked β-β-subunits. We also expected that peaks cor
unmodified β-subunits and singly modified subunits would not be presen
new peak appeared at 50-55 min. The molecular weight of the species o
the peak at 53 min corresponded to that of the target material (Hb-N3). H
molecular weight of the material forming the peak at 50 min was lower t
(MW = 32140 kDa). This agreed with the hypothesis that the azide grou
the heat treatment conditions and had decomposed, releasing nitrogen ga
behind an amine functional group (Figure 21).
heme
α-subunit
Figure 19. Size exclusion HPLC of click reaction, overlaid with native Hb.
Hb-NH2
Hb-N3
30
e unmodified α-
responding to
t. However, a
f the conent of
owever, the
han expected
p is not stable to
s and leaving
31
Figure 21. Products in solution after heat treatment. Some of the original Hb-N3 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-N3, 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
32
described. The CuAAC was performed again. As expected, it gave a variety of species
with varying molecular weights (Figure 23), seen as multiple peaks in the size exclusion
HPLC chromatogram. This heterogeneous solution was subjected to the heat treatment
conditions previously used.
Figure 23. Superdex gel filtration chromatogram of click reaction before purification.
The hemoglobin solution 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 then heated to
78 °C in a hot water bath and was stirred for 95 min. A red precipitate formed. The
reaction mixture was then centrifuged for 0.5 h and the solution was decanted. The
G200 size exclusion HPLC chromatogram of the resulting solution shows two peaks,
indicating that while lower molecular weight species did precipitate, as shown by the
disappearance of the lower molecular weight peak. Clearly two peaks still remain,
indicating that it is not a homogenous solution (Figure 24).
33
Figure 24. Superdex gel filtration chromatogram of after heat treatment.
Size exclusion chromatograms from before and after heat treatment are overlaid (Figure
25) for clarity. The retention times remain the same for the species. The species with the
lowest molecular weight did disappear after the solution was heated, which was
presumably uncross-linked material. The smaller peak on the left is presumed to be the
desired product.
Figure 25. Overlaid chromatograms of CuAAC solution before and after heat treatment. Therefore, performing the CuAAC first and then heating the solution under these
conditions was not effective for obtaining the pure bis-tetramer and new methods of
synthesizing bis-tetramer still require further development.
34
Chapter 3: Experimental
General methods Manipulations requiring dry reaction conditions were carried out on a standard
Schlenk line under nitrogen atmosphere unless otherwise stated. NMR spectra were
recorded in CDCl3, DMSO, or D2O at room temperature on a Varian Mercury 400 MHz
spectrometer or a Varian Mercury 300 MHz spectrometer. Chemical shifts are given in
ppm and coupling constants in Hz. Mass spectrometry data was collected by the
Advanced Instrumentation for Molecular Structure (AIMS) laboratory of the University
of Toronto.
Materials
All reagents and solvents were obtained from commercial resources. Adult human
hemoglobin used for all modifications was donated from Oxygenix, Inc. THF was dried
over metallic sodium and acetone was dried over anhydrous MgSO4 prior to use. Unless
otherwise stated, reagents were used without further purification. Solutions of human
hemoglobin A were obtained as a gift from Oxygenix, Inc. Aliquots of solutions of
hemoglobin were converted into cyanomethemoglobin by K3Fe(CN)6 and KCN and were
spectrophotometrically assayed to determine concentration.27
35
Synthesis of acyl phosphate cross-linker 5
Sodium dimethyl phosphate Sodium dimethyl phosphate was prepared according to literature procedures.28 Trimethyl
phosphate (5.8 mL, 0.05 mol) and NaI (9.0 g, 0.06 mol) were dissolved in 120 mL
acetone. The solution was refluxed at 60 ºC for 20 min and then cooled to room
temperature. It was stored at 4 ºC overnight. A white precipitate was collected by
vacuum filtration and washed with cold acetone. Yield: 4.58 g (62%). 1H NMR (D2O): δ
3.60 (d, 6H, -CH3, 3J31P,1H = 12 Hz) ppm. 31P NMR (D2O): δ 4.09 (s, 1P) ppm.
5-amino-isophthalic acid dimethyl ester
To a stirred suspension of 5-aminoisophthalic acid (2.5 g, 13.8 mmol) in MeOH (75 mL),
concentrated H2SO4 (7.5 mL) was added drop wise, yielding a clear yellow solution. The
solution was heated to reflux. After 24 hours, the solution was cooled to room
temperature and approximately two thirds of the solvent MeOH was evaporated. The
remaining solution was neutralized with NaHCO3 (approximately 12.5 g). Doubly
distilled H2O was added as required to dissolve NaHCO3 into solution, facilitating
neutralization. The product was extracted with 3 x 50 mL DCM and then washed with a
saturated solution of sodium chloride and dried over anhydrous MgSO4. The reaction
mixture was gravity filtered and the remaining solvent was evaporated to yield a pale
pink powder. Yield: 2.82 g (98%). 1H NMR (400 MHz, CDCl3): δ 8.05 (1H, t, 4JH,H = 1
Hz, Ar-H), 7.52 (2H, d, 4JH,H = 1Hz, Ar-H), 3.92 (6H, s, -COOMe). 13C NMR (400
28 Zervas, L.; Dilaris, I. J. Am. Chem. Soc. 1954, 77, 5354-5357.
36
MHz, CDCl3): δ 166.84, 147.05, 131.84, 121.01, 120.10, 52.62 ppm. ESI-MS: expected
209.1, found 210.1 (base peak).
4-(azidomethyl)benzoic acid (7)
To a solution of 4-(bromomethyl)benzoic acid (3.03 g, 14 mmol) in DMF (75 mL), 3.0
eq. of NaN3 (2.75 g, 42 mmol) were added and the mixture was heated to 110 °C under
an inert N2 atmosphere. The reaction was stirred for 15 h and a white precipitate formed.
The reaction was cooled to room temperature and 250 mL of ddH2O was added. The
organic product was extracted with 4 x 100 mL ether, washed with a saturated solution of
sodium chloride and dried over anhydrous MgSO4. MgSO4 was removed by gravity
filtration and ether was evaporated to yield a white powder. Yield: 1.96 g (79%). 1H
NMR (400 MHz, DMSO-d6): δ 7.94 (d, 2H, Ar-H, 3JH,H = 9 Hz), 7.56 (d, 2H, Ar-H, 3JH,H
= 9 Hz), 4.76 (s, 2H, -CH2-) ppm. 13C NMR (100 MHz, CDCl3): δ 170.67, 141.17,
130.80, 129.76, 128.09, 54.43 ppm. EA: calculated C8H7N3O2, found C8H6N3O2.
5-(4-Azidomethyl-benzoylamino)-isophthalic acid dimethyl ester (8)
4-(azidomethyl)benzoic acid (1.51 g, 8.5 mmol) was added to 15 mL of SOCl2, under an
inert nitrogen atmosphere and was heated to reflux to give a clear yellow solution. After
18 hours, the solution was cooled to room temperature and excess solvent was
evaporated. The product was immediately dissolved in freshly distilled THF under an
inert nitrogen atmosphere to give a brown solution. It was added dropwise to a second
solution of 5-amino-isophthalic acid dimethyl ester (1.78 g, 8.5 mmol) in dry THF under
a nitrogen atmosphere cooled to 0 °C. The solution was stirred for 4 hours as it warmed
37
to room temperature, forming an orange precipitate. ddH2O (60 mL) was added and 8
was extracted with EtOAc, washed with a saturated solution of sodium chloride and dried
over anhydrous MgSO4. MgSO4 was removed by gravity filtration and the solvent was
evaporated, yielding an orange solid. The crude product was purified by column
chromatography, using a 7:3 DCM:EtOAc solvent system. Yield: 1.48 g (47%). 1H
NMR (400 MHz, DMSO-d6): δ 10.69 (s, 1H, -NH-) 8.73 (s, 2H, Ar-H), 8.22 (t, 1H, Ar-
H), 8.04 (d, 2H, Ar-H, 3JH,H = 8 Hz), 7.55 (d, 2H. Ar-H, 3JH,H = 8 Hz), 4.58 (s, 2H, -CH2-
), 3.91 (s, 6H, -COOCH3) ppm. 13C NMR (100 MHz, DMSO-d6): δ 166.18, 166.01,
140.82, 140.38, 134.49, 131.29, 129.01, 128.90, 125.36, 125.14, 53.74, 53.25 ppm. MS
(ESI): calculated 368.1, found 369.1 (base peak). EA: expected C18H16N4O5, found
C18H17N4O5.
5-(4-Azidomethyl-benzoylamino)-isophthalic acid (9)
Compound 8 (0.66 g, 1.7 mmol) was added to a solution of 1:1 THF:MeOH (40 mL),
forming an orange suspension. A solution of KOH (2.10 g in 4 mL of ddH2O) was added
drop wise, to yield a clear orange solution. The reaction stirred for 2 hours and was
monitored by TLC. After 2 hours, 2 M HCl(aq) was added drop wise until the solution
was strongly acidic (pH 2). A white solid precipitated which was extracted with 3 x 21
mL EtOAc. The EtOAc solution was washed with a saturated aqueous solution of
sodium chloride and dried over anhydrous MgSO4. The reaction was filtered and the
solvent was evaporated, yielding a light yellow powder. Yield: 0.45 g (74%). 1H NMR
(400 MHz, DMSO-d6): δ 10.61 (s, 2H, Ar-COOH), 8.67 (d, 2H, Ar-H, 4JH,H = 4 Hz), 8.21
(t, 1H, Ar-H, 4JH,H = 4 Hz), 8.03 (d, 2H, Ar-H, 3JH,H = 8 Hz), 7.54 (d, 2H, Ar-H, 3JH,H = 8
38
Hz), 4.57 (s, 2H, -CH2-) ppm. 13C NMR (100 MHz, DMSO-d6): δ 167.19, 166.12,
140.48, 140.26, 134.67, 132.35, 129.00, 128.88, 125.39, 53.75 ppm. MS (ESI) m/z
calculated 340.1, found 341.1 (base peak). EA: expected C16H12N4O5, found
C16H13N4O5.
Synthesis of N-[bis(sodium methyl phosphate)isophthalyl]-4-azidomethylbenzoate (5) 5-(4-Azidomethyl-benzoylamino)-isophthalic acid 9 (0.32 g, 0.94 mmol) was dissolved
in excess SOCl2 (15 mL) under a nitrogen atmosphere and was refluxed for 18 hours.
The reaction was cooled to room temperature and excess SOCl2 was evaporated. The
resulting brown film was dissolved in freshly distilled THF (20 mL) and was added
dropwise to a second solution of sodium dimethyl phosphate (0.35 g, 2.4 mmol)
dissolved in freshly distilled THF (20 mL) at 0 ºC in a nitrogen atmosphere. The reaction
was stirred for 18 h and was allowed to warm to room temperature. The solution was
filtered on a dry sintered glass funnel containing Celite to remove the NaCl(s), a
byproduct. The filtrate was collected and excess THF was evaporated. The resulting
yellow oil was dissolved in freshly dried acetone (20 mL) and the reaction flask was
covered with aluminum foil to prevent light from reaching the reaction mixture. NaI
(0.37 g, 2.4 mmol) dissolved in a minimal amount of dry acetone was added drop wise
and the reaction was allowed to stir overnight for 18 h at room temperature. A yellow
precipitate was collected using vacuum filtration. It was washed with a minimum amount
of cold dry acetone, and dried for 20 min under reduced pressure. Yield: 0.40 g (75%).
31P NMR (121MHz, D2O): δ -9.78 ppm. MS (ESI) m/z calculated 572.0, found (M - 2Na)
527 (parent peak).
39
Regeneration of 5-(4-Azidomethyl-benzoylamino)-isophthalic acid A crude mixture of attempted synthesis of 5 (0.60 g) was dissolved in ddH2O, which was
then titrated to pH 10 using aqueous sodium hydroxide. The reaction mixture was stirred
for 3 hours at room temperature. It was extracted with EtOAc (3 x 40 mL) and washed
with a saturated solution of sodium chloride. The EtOAc solution was dried with
anhydrous MgSO4 and excess solvent was removed. Yield: 0.32 g. 1H NMR (400 MHz,
DMSO-d6): δ 10.61 (s, 2H, Ar-COOH), 8.67 (d, 2H, Ar-H, 4JH,H = 4 Hz), 8.21 (t, 1H, Ar-
H, 4JH,H = 4 Hz), 8.03 (d, 2H, Ar-H, 3JH,H = 8 Hz), 7.54 (d, 2H, Ar-H, 3JH,H = 8 Hz), 4.57
(s, 2H, -CH2-) ppm.
Synthesis of bisalkyne linker (3) This compound was prepared according to literature procedures.17
Cross-linking with hemoglobin Purified adult human hemoglobin was used. It was stored as stable HbCO at 4 ºC. In
preparation for cross-linking, hemoglobin was transferred into the selected buffer by
eluting through a Sephadex G-25 gel-filtration column. HbCO was converted to oxyHb
by exposure to a tungsten irradiation lamp for 2 h at 0 °C. It was converted to deoxyHb
by passing a stream of nitrogen over it for 2 h at 37 °C. Two equivalents of reagent 5
were added and the reaction was stirred for 3 h at 37 °C under a constant stream of
nitrogen. 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 then
transferred to a MOPS buffer (0.1 M, pH 8.0) via a Sephadex G-25 gel-filtration column
40
to remove any traces of unreacted organic material. The reaction mixture was
centrifuged to concentrate the sample. It was then stored under carbon monoxide at 4 °C.
HPLC analysis of modified hemoglobin Two high-performance liquid chromatography methods were used to analyse the
modified hemoglobin products. Analytical reverse-phase HPLC with a 330 Å pore-size
C-4 Vydac column (4.6 x 250 nm) under fully dissociating conditions was used to help
identify which subunit was modified. Gradient acetonitrile (20-60%) and ddH2O with
0.1% v/v TFA as an ion-pairing agent eluted separated modified hemoglobin chains.
Peaks were assigned by comparison of retention times to native hemoglobin and known
species and by collection and analysis by ESI-MS. Elution was followed at 220 nm.
Size exclusion HPLC with a Superdex G-75 HR (10 x 300 nm) column was used to
confirm the size distribution of modified protein mixtures. It was useful in distinguishing
between uncross-linked tetramers, cross-linked tetramers and bis-tetramers. Partially
dissociating conditions (37.5 mM Tris-HCl, pH 7.4, 0.5 M magnesium chloride)
separated uncross-linked hemoglobin proteins into α-β-dimers. Peaks were monitored at
280 nm and 414 nm. Peaks were assigned by comparison of retention times to native
hemoglobin and of species with known molecular weights. The yields of products of
reaction corresponded to the area under the peaks in chromatograms.
Heat treatment of modified hemoglobin solutions Samples of modified hemoglobin in a sealed vial under carbon monoxide were
submerged in a hot water bath and stirred at 78 ºC for 95 minutes. Once removed from
heat, the hemoglobin solutions were centrifuged for 0.5 h and the supernatant was
41
decanted to remove the precipitated denatured protein chains. Hemoglobin solutions
were passed through a syringe filter unit (Mandel, 0.45 µm PVDF) for finer purification.
A stream of carbon monoxide was passed over samples and they were stored at 4 ºC.
Samples were analyzed using both analytical reverse-phase G4 and G200 size exclusion
HPLC to determine the effectiveness of the heat treatment.
Coupling of tetramers – CuAAC “click” chemistry
Hemoglobin solutions were transferred into a phosphate buffer (0.02 M, pH 7.4) by
eluting through a Sephadex G-25 gel-filtration column. Aliquots were converted into
cyanomethemoglobin by K3Fe(CN)6 and KCN and were spectrophotometrically assayed
to determine concentration.27 To a solution of Hb-N3 (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
H2O, 2 µmol), CuSO4 (50 µL of 20 mM in H2O, 1 µmol) and L-ascorbic acid (200 µL of
100 mM in H2O, 20 µmol) were added by micropipette. Excess carbon monoxide was
used to flush the system and the reaction was shaken for 4 hours. The reaction was then
purified by eluting it through a Sephadex G-25 gel-filtration column. It was then
concentrated on a centrifuge and stored under carbon monoxide at 4 ºC. Yield was
determined using size-exclusion HPLC.
42
Chapter 4: Conclusions and Future Work
Conclusions
A novel acyl phosphate hemoglobin cross-linker was synthesized and used to cross-
link hemoglobin. The solution from this reaction was analyzed to determine the
products. The target product, β-β-cross-linked hemoglobin, was formed. Singly
modified and unmodified subunits were byproducts. Purification using heat to denature
uncross-linked protein was used to isolate the desired product in the reaction mixture.
While material that was not cross-linked did precipitate out of solution, the azide group
on the cross-link partially decomposed. This lowered the yield of the subsequent CuAAC
coupling reaction to form bis-tetramer. In an attempt to improve bis-tetramer formation
yield, the cross-linked material underwent coupling first and the resulting mixture was
subjected to the heat purification process. However, this still gave a mixture of products.
While this project did not achieve its goal of improving the yield of bis-tetramer in
solution, the information gathered from it will be useful in guiding future research.
Symmetrically vs. unsymmetrically cross-linked hemoglobin While the decomposition of the azide would certainly have been responsible for the
failure of the coupling reaction, another possible reason for the low yield of the CuAAC
is that 5 did not cross-link hemoglobin symmetrically. In the previous work using a
CuAAC coupling to form bis-tetramers, it was found that the cross-linker 1 was able to
cross-link β-β-subunits both symmetrically (β-lys82/β’-lys82) and unsymmetrically (β-
val1/β’-lys82). However, only the symmetrically cross-linked hemoglobin was able to
undergo the coupling reaction. It was presumed that the azide group on the
43
unsymmetrically cross-linked hemoglobin was within the protein and was not accessible
for reaction. It may be necessary to determine which amino groups within the DPG-
binding site 5 acylates. This can be resolved by a tryptic peptide digest, where isolated β-
subunits of cross-linked hemoglobin and unmodified hemoglobin are digested with
trypsin into fragments and analyzed using MALDI-MS. Comparison of the two can
determine the location of the cross-link on the modified hemoglobin. If both
symmetrically and unsymmetrically cross-linked material was formed by the reaction of
Hb and 5, it may be necessary to design a cross-linker that either exclusively cross-links
symmetrically (β-lys82/β’-lys82), or one that, regardless of how it cross-links, the azide
will always be accessible. To achieve this, it might be necessary to extend the length of
the cross-linker between the directing groups and the free azide.
Optimization of purification of Hb-N3
Also worth looking into might be optimizing the purification process. It is worth
investigating whether it is possible to use 5 to cross-link hemoglobin and obtain pure Hb-
N3. Possible strategies to explore:
i. Heating just the azide linker, to determine its stability in relation to heat and time
and using this to optimize heat treatment of Hb-N3.
ii. Form the bis-tetramer with the CuAAC coupling first, then heat the resulting
mixture of species to an even higher temperature than attempted in this
project.
iii. Examine heat-treating at lower temperatures and varying time to denature the
uncross-linked globins, whist maintaining the azide functional groups
44
iv. An alternative method of denaturing globins in literature involves shocking the
system with heat - 3 minutes at 90 ºC – and seeing if this will also destroy the
azide.
Synthesis of alkyne cross-linker One potential direction to take the results of this thesis in is to develop a new acyl
phosphate linker, similar to 5 but with the azide functional group substituted for an
alkyne functional group. Presumably, despite this minor deviation the new acyl
phosphate cross-linker will have a similar reactivity with hemoglobin as 1. Ideally β-β-
Hb will remain the only cross-linked species, with similar byproducts. If this were the
case, the heat treatment conditions used to purify the solution from the cross-linking
reaction may be suitable to cleanly purify the reaction mixture of the new cross-linker
and hemoglobin. Alkynes are much more thermally stable than azides and would be
much less likely to degrade at 78 ºC. Thus, it may be possible to obtain a clean solution
of cross-linked hemoglobin tetramers with a free functional group on the cross-link that is
completely able to undergo a coupling reaction. A potential synthetic scheme has been
designed to create a new acyl phosphate cross-linker (Scheme 9).
45
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) SOCl2, THF iv) KOH (in 1:1 THF:MeOH) deprotection v) SOCl2, sodium dimethyl phosphate, THF vi) NaI, acetone.
If the cross-linker contained the alkyne group, then the linker must contain the azide
group. An azide would be attached on each end of the linker. Thus, using 5 as a
template, instead of simply substituting the alkyne groups for azide groups, it might be
worth extending the length of the cross-linker to increase the carbon to nitrogen ratio of
the resulting compound to increase its stability.
