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 alkyne-azide 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 β-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 unreacted hemoglobin.

Proton Transfer via π-Interactions from Pyridine Provides a Facilitated Route for Transfer of CO 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 CO 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.

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

Chemical cross-linking of human adult hemoglobin (Hb) prevents dissociation of the tetrameric (αβ) protein into its constituent non-functional αβ 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.

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.

Origin of Free Energy Barriers of Decarboxylation and the Reverse Process of CO 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 CO 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 CO capture by the carbanions of decarboxylation reactions.

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 -(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 10-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 (sp) carbanion that is formed as CO is produced.

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.

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 CO 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.

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.

Carbon Kinetic Isotope Effects and the Mechanisms of Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid and CO 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 CO. An alternative via formation of a higher-energy protonated phenyl tautomer (2H) prior to C-C bond breaking would produce protonated CO, an energetically inaccessible species that can be avoided by transfer of the carboxyl proton to water in the same step.

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

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 CO 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.

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 CO from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed.

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.

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.

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.

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 (α2β2) 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.

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.

Subunit-directed click coupling via doubly cross-linked hemoglobin efficiently produces readily purified functional bis-tetrameric oxygen carriers.

While cross-linked hemoglobin (Hb) tetramers can deliver oxygen as a supplement to red cells, they also cause unacceptable increases in blood pressure, presumably from their penetration of the linings of blood vessels (endothelia) where the internal hemes bind endogenous nitric oxide (NO). This penetration would lower the local concentration of NO that normally induces vasodilation. Enlarging the effective size of the oxygen-carrying protein by coupling two Hbs can prevent their extravasation.

Lithium-stabilized nucleophilic addition of thiamin to a ketone provides an efficient route to mandelylthiamin, a critical pre-decarboxylation intermediate.

Mandelylthiamin (MTh) is an accurate model of the covalent intermediate derived from the condensation of thiamin diphosphate and benzoylformate in benzoylformate decarboxylase. The properties and catalytic susceptibilities of mandelylthiamin are the subjects of considerable interest. However, the existing synthesis gives only trace amounts of the precursor to MTh as it is conducted under reversible conditions.

Bioorthogonal phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC) coupling of selectively cross-linked superoxide dismutase dimers produces a fully active bis-dimer.

Superoxide dismutase (SOD) is a 32 kDa dimeric enzyme that actively removes a toxic oxygen species within red cells. The acellular protein itself does not survive circulation as it is filtered through the kidney. Conjugating the protein to another SOD should increase the size of the dual protein above the threshold for filtration by the kidney, making the material a potential therapeutic in circulation.

Decarboxylation without CO2: why bicarbonate forms directly as trichloroacetate is converted to chloroform.

Patterns in the observed catalysis of decarboxylation reactions required us to conclude that these reactions involve initial hydration of the carboxylate and subsequent loss of bicarbonate. This raises the important and general question of why CO2 is not formed directly. Reaction profiles for the direct decarboxylation of trichloroacetate were generated with DFT calculations and show no significant barrier to the recombination of the incipient trichloromethide and CO2.