Conjugating Hemoglobin and Albumin by Strain-Promoted Azide-Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted azide-alkyne cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each b-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from the unreacted hemoglobin. The oxygen binding properties of this two-protein conjugate demonstrate an oxygen affinity and binding cooperativity suitable as an acellular oxygen carrier.

Efficient conversion of hemoglobin to a non-vasoactive oxygen carrier by site-specific cross-linking with azido acyl methyl phosphates followed by bio-orthogonal CuAAC with a bis-alkyne.

While cross-linked hemoglobin tetramers are functional acellular oxygen carriers, their ability to scavenge endogenous nitric oxide (NO) by endothelial pore penetration results in adverse cardiovascular effects. Animal studies established that cross-linked human hemoglobins, chemically joined into a double protein, avoid NO scavenging, presumably due to their larger size preventing penetration into endothelial regions that produce NO. In the present report, we utilize azide-containing acyl phosphate reagents to form cross-linked hemoglobins then bio-orthogonally click-couple them with a bis-alkyne (CuAAC). The production of these larger oxygen-carrying hemoglobin conjugates is obtained in high yields through subunit-specific cross-linking between each ßLys82 ε-amino group. The methyl phosphate leaving groups provide electrostatically induced ß-subunit site-selectivity, producing azido-cross-linked hemoglobin that undergoes highly efficient CuAAC compared with previous cross-linkers. The acyl phosphates also efficiently cross-link both T-state and R-state hemoglobin. The resulting bis- and tris-tetrameric hemoglobin conjugates exhibit oxygen affinity and cooperativity that are comparable to those of the native protein. The hemoglobin derivatives from the process we describe can function as sources of oxygen in biomedical applications, such as in ex-vivo donor organ perfusion.

Proton Transfer via π-Interactions from Pyridine Provides a Facilitated Route for Transfer of CO2 in Its Complex with a Carbanion.

Aromatic π-interactions have been recognized as enhancing enzymatic catalytic processes, providing an efficient route to overcome entropic barriers. A nonenzymic analogue, a complex of protonated pyridine and a phenyl substituent in a thiamin conjugate, facilitates the departure of CO2 by protonation of a vicinal carbanion in a reactive complex. To evaluate the efficiency of the catalytic pathway from the π-associated proton donor, a system was assessed that produced measurable competition through the rates of formation of alternative products resulting from the same thiamin-derived carbanion. The barriers to competing pathways from the decarboxylation of p-(bromomethyl)-mandelylthiamin in the presence and absence of protonated pyridine were determined, establishing the efficiency of the vicinal proton transfer between π-associated species. The formation of the complex of CO2 and the co-formed carbanion also addresses the mechanism of the uncatalyzed exchange of 13CO2 into carboxyl groups discovered by Lundgren. Finally, microscopic reversibility implicates pyridine as a vicinal Brønsted base in thiamin-aldehyde adducts, producing carbanions that could incorporate dissolved CO2 into carboxyl groups.

Efficient formation of hemoglobin bis-tetramers via selective acetylation of α-subunit amino groups by methyl acetyl phosphate.

Chemical cross-linking of human adult hemoglobin (Hb) prevents dissociation of the tetrameric (αß)2 protein into its constituent non-functional αß dimers when present outside red cells, providing the possibility of being an acellular oxygen carrier in circulation. However, studies of cross-linked Hb (xlHb) in circulation established effects consistent with scavenging of endogenous nitric oxide, leading to hypertension. Bis-tetramers, composed of coupled Hb tetramers, are sufficiently large to avoid penetration of endothelia, thereby blocking access to endogenous nitric oxide. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) joins two azide-functionalized xlHbs to each end of a bis-alkyne to form bis-tetramers. The process critically depends on formation of a cross-link between lysyl amino groups of the ß-subunits while avoiding reactions with amino groups in the α-subunits. Highly selective acetylation of α-subunit amino groups with methyl acetyl phosphate (MAP) effectively directs subsequent cross-linking to the ß-subunits. This outcome leads to efficient production of hemoglobin bis-tetramers by CuAAC.

Rates of competing fluoride elimination and iodination from a thiamin-derived Breslow intermediate.

Rates of fluoride elimination and iodination of the Breslow intermediate (BI) derived from 2-(1-hydroxy-2,2,2-trifluoroethyl)-thiamin provide a quantitative assessment of competing reactions at C2α of the BI. The competition probes the intrinsic reactivity of this important class of intermediates. Fluoride elimination, which occurs upon formation of the BI, produces 2-(2',2'-difluoroacetyl)-thiamin, while the rate of iodination of the same BI provides a basis for estimating the rate of the competing protonation. The results provide rates for reactions of the BI, the Brønsted ß for its formation by deprotonation, and the pKA of the conjugate acid of the BI at C2α. Comparison with reactions of 3-fluoropyruvate with enzymes that promote the decarboxylation of pyruvate (via the adduct of thiamin diphosphate) indicates that spontaneous fluoride elimination (kel = 7.5 ± 0.3 s-1) from the enzymic BI has a lower barrier than the reaction pathway that is normally promoted by the enzymes.

Origin of Free Energy Barriers of Decarboxylation and the Reverse Process of CO2 Capture in Dimethylformamide and in Water.

In aqueous solution, biological decarboxylation reactions proceed irreversibly to completion, whereas the reverse carboxylation processes are typically powered by the hydrolysis of ATP. The exchange of the carboxylate of ring-substituted arylacetates with isotope-labeled CO2 in polar aprotic solvents reported recently suggests a dramatic change in the partition of reaction pathways. Yet, there is little experimental data pertinent to the kinetic barriers for protonation and thermodynamic data on CO2 capture by the carbanions of decarboxylation reactions. Employing a combined quantum mechanical and molecular mechanical simulation approach, we investigated the decarboxylation reactions of a series of organic carboxylate compounds in aqueous and in dimethylformamide solutions, revealing that the reverse carboxylation barriers in solution are fully induced by solvent effects. A linear Bell-Evans-Polanyi relationship was found between the rates of decarboxylation and the Gibbs energies of reaction, indicating diminishing recombination barriers in DMF. In contrast, protonation of the carbanions by the DMF solvent has large free energy barriers, rendering the competing exchange of isotope-labeled CO2 reversible in DMF. The finding of an intricate interplay of carbanion stability and solute-solvent interaction in decarboxylation and carboxylation could be useful to designing novel materials for CO2 capture.

Competing Protonation and Halide Elimination as a Probe of the Character of Thiamin-Derived Reactive Intermediates.

Decarboxylation reactions from comparable thiamin diphosphate- and thiamin-derived adducts of p-(halomethyl)benzoylformic acids in enzymic and non-enzymic reactions, respectively, reveal critical distinctions in otherwise similar Breslow intermediates. The ratio of protonation to chloride elimination from the Breslow intermediate is 102-fold greater in the enzymic process. This is consistent with a lower intrinsic barrier to proton transfer on the enzyme, implicating formation of a localized tetrahedral (sp3) carbanion that is formed as CO2 is produced. In contrast, slower protonation in solution of the decarboxylated intermediate is consistent with formation of a delocalized planar carbanionic enol/enamine. The proposed structural and reactive character of the enzymic Breslow intermediate is consistent with Warshel's general theory of enzymic catalysis, structural characterization of related intermediates, and the lower kinetic barrier in reactions that occur without changes in hybridization.

Cross-linked hemoglobin bis-tetramers from bioorthogonal coupling do not induce vasoconstriction in the circulation.

BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) are potential alternatives to red blood cells in transfusions. Clinical trials using early versions of HBOCs noted adverse effects that appeared to result from removal of the vasodilator nitric oxide (NO). Previous reports suggest that size-enlarged HBOCs may avoid NO-rich regions along the vasculature and therefore not cause vasoconstriction and hypertension. STUDY DESIGN AND METHODS: Hemoglobin (Hb) bis-tetramers (bis-tetramers of hemoglobin that are prepared using CuAAC chemistry [BT-Hb] and bis-tetramers of hemoglobin that are specifically acetylated and prepared using CuAAC chemistry [BT-acHb]) can be reliably produced by a bio-orthogonal cyclo-addition approach. We considered that an HBOC derived from chemical coupling of two Hbs would be sufficiently large to avoid NO scavenging and related side effects. The ability of intravenously infused BT-Hb and BT-acHb to remain in the circulation without causing hypertension were determined in wild-type (WT) and diabetic (db/db) mouse models. RESULTS: In WT mice, the coupled oxygen-carrying proteins retained their function over several hours after administration. No significant changes in systolic blood pressure from baseline were observed after intravenous infusion of BT-Hb or BT-acHb in awake WT and db/db mice. In contrast, infusion of native Hb or cross-linked Hb tetramers in both animal models induced systemic hypertension. CONCLUSION: The results of this study indicate that bis-tetrameric HBOCs derived from the bio-orthogonal cyclo-addition process are likely to overcome clinical issues that arise from NO scavenging by Hb derivatives.

Charge Dispersion and Its Effects on the Reactivity of Thiamin-Derived Breslow Intermediates.

The enzymic decarboxylation of 2-ketoacids proceeds via their C2-thiazolium adducts of thiamin diphosphate (ThDP). Loss of CO2 from these adducts leads to reactive species that are known as Breslow intermediates. The protein-bound adducts of the 2-ketoacids and ThDP are several orders of magnitude more reactive than the synthetic analogues in solution. Studies of enzymes are consistent with formulation of protein-bound Breslow intermediates with localized carbanionic character at the reactive C2α position, reflecting the charge-stabilized transition state that leads to this form. Our study reveals that nonenzymic decarboxylation of the related thiamin adducts proceeds to the alternative charge-dispersed enol form of the Breslow intermediate. These differences suggest that the greatly enhanced rate of decarboxylation of the precursors to Breslow intermediates in enzymes arises from maintenance of the carbanionic character at the position from which the carboxyl group departs, avoiding charge dispersion by stabilizing electrostatic interactions with the protein as formulated by Warshel. Applying Guthrie's "no-barrier" addition to Marcus theory also leads to the conclusion that maintaining the tetrahedral carbanion at C2α of the resulting adduct minimizes associated kinetic barriers by avoiding rehybridization as part of steps to and from the intermediate. Finally, maintenance of the reactive energetic carbanion agrees with the concepts of Albery and Knowles as the outcome of evolved enzymic processes.

Lead-Catalyzed Aqueous Benzoylation of Carbohydrates with an Acyl Phosphate Ester.

Biochemical systems utilize adenylates of amino acids to aminoacylate the 3'-terminal diols of tRNAs. The reactive acyl group of the biological acylation agent is a subset of the general class of acyl phosphate monoesters. Those compounds are relatively stable in aqueous solutions, and their alkyl esters are conveniently prepared. It has previously been shown that biomimetic reactions of acyl phosphate monoesters with diols and carbohydrates are promoted by lanthanide salts. However, they also promote hydrolysis of acyl phosphate reagents, and the overall yields are modest. An assessment of the catalytic potential of alternative Lewis acids reveals that lead ions may be more effective as catalysts than lanthanides. Treatment of carbohydrates with benzoyl methyl phosphate (BMP) and triethylamine in water with added lead nitrate produces monobenzoyl esters in up to 75% yield. This provides a water-compatible pathway for novel patterns of benzoylation of polyhydroxylic compounds.

Carbon Kinetic Isotope Effects and the Mechanisms of Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid and CO2 Incorporation into 1,3-Dimethoxybenzene.

The rate of decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in parallel to the extent that the carboxyl group acquires a second proton (1H+). However, the conjugate acid would resist C-C bond breaking as that would lead to formation of doubly protonated CO2. An alternative via formation of a higher-energy protonated phenyl tautomer (2H+) prior to C-C bond breaking would produce protonated CO2, an energetically inaccessible species that can be avoided by transfer of the carboxyl proton to water in the same step. Headspace sampling of CO2 that evolves in the acid-catalyzed process and analysis by GC-IRMS gives a smaller than expected value of 1.022 for the carbon kinetic isotope (CKIE), k12/k13. While this value establishes that C-C cleavage is part of the rate-determining process, intrinsic CKIEs for decarboxylation reactions are typically greater than 1.03. Computational analysis of the C-C bond cleavage from 2H+ gives an intrinsic CKIE of 1.051 and suggests two partially rate-determining steps in the decarboxylation of 1: transfer of the second carboxyl proton to the adjacent phenyl carbon and C-C cleavage in which the carboxyl proton is also transferred to water. Applying the principle of microscopic reversibility to fixation of CO2 in acidic solutions reveals the importance of proton transfers to both carbon and oxygen in the overall fixation process.

Determining Carbon Kinetic Isotope Effects Using Headspace Analysis of Evolved CO2.

Isotope ratio mass spectrometry (IRMS) provides accurate measurements of relative abundance of isotopes of heavy atoms for reactions that are subject to kinetic isotope effects (KIEs). The recent development of compound-specific isotope analysis (CSIA) allows the use of multiple time points that provide data for a rate plot as well as isotope ratios. Utilizing CSIA in enzymology presents opportunities for obtaining heavy atom KIEs in diverse areas.

The Need for an Alternative to Radicals as the Cause of Fragmentation of a Thiamin-Derived Breslow Intermediate.

Mandelylthiamin (1) is a conjugate of benzoylformate and thiamin that loses CO2 to form the classic Breslow intermediate (2), whose expected fate is formation of the thiamin conjugate of benzaldehyde (3). Surprisingly, it was observed that 2 decomposes to 4 and 5 and rearranges to 6 in competition with the expected protonation to give 3. Recent reports propose that the alternatives to protonation arise from homolysis followed by radical-centered processes. It is now found, instead, that the spectroscopic observations cited in support of the proposed radical pathways are likely to be the result of other events. An alternative explanation is that ionization of the enolic hydroxy group of 2 and resultant electronic reorganization leads to C-C bond cleavage and non-radical intermediates that readily form 4, 5, and 6.

The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation.

It has previously been established that the deprotonated amino substituent of the pyrimidine of thiamin diphosphate (ThDP) acts as an internal base to accept the C2H of the thiazolium in ThDP-dependent enzymes. The amino group has also been implicated in assisting the departure of the aldehydic product formed after loss of CO2 from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed. Oxythiamin contains a hydroxyl group in place of the pyrimidine amino group in thiamin, providing a basis for comparison of reactivity. Lactyl-oxythiamin (LOTh), the conjugate of pyruvic acid and oxythiamin was prepared by condensation of ethyl pyruvate and hydroxyl-protected oxythiamin followed by deprotection and acidic hydrolysis of the ethyl ester. The rate constants observed for the decarboxylation of LOTh in neutral and acidic solutions are about four times smaller than those for the corresponding compound that contains the amino group, lactylthiamin. The difference in reactivity is consistent with the amino group's participation in facilitating the decarboxylation step by allowing a competitive addition pathway that produces bicarbonate and has implications for the corresponding enzymic reaction.

Strain-promoted azide-alkyne cycloaddition for protein-protein coupling in the formation of a bis-hemoglobin as a copper-free oxygen carrier.

Conventional chemical approaches to protein-protein coupling present challenges due to the intrinsic competition between the desired interactions of reagents with groups of the protein as well as reactions with water. Biorthogonal Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC)-processes provide a basis to direct reactivity without functional group interference. However, the requirement for Cu(i) in CuAAC leads to complications that result from the metal ion's interactions with the protein. In principle, a similar but metal-free alternative approach to coupling could employ the reaction of an alkyne that is strained in combination with an azide (strain-promoted azide-alkyne cycloaddition, SPAAC). The method is exemplified by the combination of a cyclooctyne derivative of hemoglobin with an azide-modified hemoglobin. The bis-hemoglobin tetramer that is produced has properties consistent with those sought for use as a hemoglobin-based oxygen carrier (HBOC).

Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen Affinity in Hemoglobin Bis-Tetramers.

The vasoactivity of circulating cross-linked hemoglobin is consistent with the acellular protein penetrating the endothelial lining of blood vessels where hemoglobin can bind nitric oxide, the signal for relaxation of the muscles that surround blood vessels. In an important contrast, derivatives of bis-tetramers that are produced from hemoglobin by chemical coupling do not cause vasoconstriction in animal models. Presumably, they are unable to enter the endothelia where hemoglobin tetramers bind to nitric oxide. In addition, hemoglobin bis-tetramers can produce nitric oxide in circulation through their intrinsic nitrite reductase activity. Examination of this activity for hemoglobin-derived bis-tetramers that are acetylated at lysyl amino groups in their α subunits reveals enhanced activity (k = 2.21 M(-1) s(-1)) compared to that of nonacetylated bis-tetramers (k = 0.70 M(-1) s(-1)). Plots of nitrite reductase activities as a function of the corresponding oxygen affinities of certain allosteric-state-stabilized derivatives reveal a significant correlation, providing a basis for interpretation of the correlated functions.

How Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid Avoids Formation of Protonated CO2.

The decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in acidic solutions. The rate of reaction depends upon solution acidity in a manner that is consistent with the formation of the conjugate acid of 1 (RCO2H2(+)), with its higher energy ring-protonated tautomer allowing the requisite C-C bond cleavage. However, this would produce the conjugate acid of CO2, a species that would be too energetic to form. Considerations of mechanisms that fit the observed rate law were supplemented with DFT calculations. Those results indicate that the lowest energy pathway from the ring-protonated reactive intermediate involves early proton transfer from the carboxyl group to water along with C-C bond cleavage, producing 1,3-dimethoxybenzene and CO2 directly.

Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin-Avidin Interactions.

Hypertension resulting from vasoconstriction in clinical trials of cross-linked tetrameric (α2ß2) human hemoglobins implicates the extravasation of the hemoglobins into endothelia where they scavenge nitric oxide (NO), which is the signal for relaxation of the surrounding smooth muscle. Thus, we sought an efficient route to create a larger species that avoids extravasation while maintaining the oxygenation function of hemoglobin. Selectively formed cysteine-linked biotin conjugates of hemoglobin undergo self-assembly with avidin into a stable triple protein, hemoglobin-avidin-hemoglobin (HbAvHb), which binds and releases oxygen with moderate affinity and cooperativity. The triple protein is likely to be stabilized by interactions of each constituent hemoglobin (pI 6.9) with the oppositely charged avidin (pI 10.5) as well as the strong association of the biotin moieties on hemoglobin with avidin.

Decarboxylation, CO2 and the reversion problem.

Decarboxylation reactions occur rapidly in enzymes but usually are many orders of magnitude slower in solution, if the reaction occurs at all. Where the reaction produces a carbanion and CO2, we would expect that the high energy of the carbanion causes the transition state for C-C bond cleavage also to be high in energy. Since the energy of the carbanion is a thermodynamic property, an enzyme obviously cannot change that property. Yet, enzymes overcome the barrier to forming the carbanion. In thinking about decarboxylation, we had assumed that CO2 is well behaved and forms without its own barriers. However, we analyzed reactions in solution of compounds that resemble intermediates in enzymic reaction and found some of them to be subject to unexpected forms of catalysis. Those results caused us to discard the usual assumptions about CO2 and carbanions. We learned that CO2 can be a very reactive electrophile. In decarboxylation reactions, where CO2 forms in the same step as a carbanion, separation of the products might be the main problem preventing the forward reaction because the carbanion can add readily to CO2 in competition with their separation and solvation. The basicity of the carbanion also might be overestimated because when we see that the decarboxylation is slow, we assume that it is because the carbanion is high in energy. We found reactions where the carbanion is protonated internally; CO2 appears to be able to depart without reversion more rapidly. We tested these ideas using kinetic analysis of catalytic reactions, carbon kinetic isotope effects, and synthesis of predecarboxylation intermediates. In another case, we observed that the decarboxylation is subject to general base catalysis while producing a significant carbon kinetic isotope effect. This requires both a proton transfer from an intermediate and C-C bond-breaking in the rate-determining step. This would occur if the route involves the surprising initial addition of water to the carboxyl, with the cleavage step producing bicarbonate. Interestingly, some enzyme-catalyzed reactions also appear to produce intermediates formed by the initial addition of water or a nucleophile to the carboxyl or to nascent CO2. We conclude that decarboxylation is not necessarily a problem that results from the energy of the carbanionic products alone but from their formation in the presence of CO2. Catalysts that facilitate the separation of the species on either side of the C-C bond that cleaves could solve the problem using catalytic principles that we find in many enzymes that promote hydrolytic processes, suggesting linkages in catalysis through evolution of activity.