Initial Protonation Research Articles

Computational Characterization of the Substrate-Binding Mode in Coproporphyrinogen III Oxidase

Oxygen-dependent coproporphyrinogen III oxidase catalyzes the sequential decarboxylation of the propionate substituents present on the A and B rings of coproporphyrinogen III in the heme biosynthetic pathway. Although extensive experimental investigation of this enzyme has already afforded many insights into its reaction mechanism, several key features (such as the substrate binding mode, the characterization of the active site, and the initial substrate protonation state) remain poorly described. The molecular dynamics simulations described in this paper enabled the determination of a very promising substrate binding mode and the extensive characterization of the enzyme active site. The proposed binding mode is fully consistent with the known selectivity of the active site toward substituted tetrapyrroles and explains the lack of activity of the H131A, R135A, D274A, and R275A mutants and the reasons behind the nonoccurrence of catalysis on the C and D rings of the tetrapyrrole. An important role in this binding mode is fulfilled by G276, as its carbonyl oxygen intervenes in the substrate anchoring by hydrogen bonding its ring D pyrrole NH group. The presence of this interaction (which is only possible with the protonated NH pyrrole group) and the absence of positively charged side chains close to the pyrrole nitrogen (which might stabilize the N-deprotonated pyrrole postulated in some mechanistic proposals) show that the pyrrole ring is very unlikely to undergo deprotonation during the catalytic cycle and allow the discrimination between the previously postulated mechanistic proposals.

Kinetic and Theoretical Studies on the Protonation of [Ni(2-SC6H4N){PhP(CH2CH2PPh2)2}]+: Nitrogen versus Sulfur as the Protonation Site

The complexes [Ni(4-Spy)(triphos)]BPh(4) and [Ni(2-Spy)(triphos)]BPh(4) {triphos = PhP(CH(2)CH(2)PPh(2))(2), 4-Spy = 4-pyridinethiolate, 2-Spy = 2-pyridinethiolate} have been prepared and characterized both spectroscopically and using X-ray crystallography. In both complexes the triphos is a tridentate ligand. However, [Ni(4-Spy)(triphos)](+) comprises a 4-coordinate, square-planar nickel with the 4-Spy ligand bound to the nickel through the sulfur while [Ni(2-Spy)(triphos)](+) contains a 5-coordinate, trigonal-bipyramidal nickel with a bidentate 2-Spy ligand bound to the nickel through both sulfur and nitrogen. The kinetics of the reactions of [Ni(4-Spy)(triphos)](+) and [Ni(2-Spy)(triphos)](+) with lutH(+) (lut = 2,6-dimethylpyridine) in MeCN have been studied using stopped-flow spectrophotometry, and the two complexes show very different reactivities. The reaction of [Ni(4-Spy)(triphos)](+) with lutH(+) is complete within the deadtime of the stopped-flow apparatus (2 ms) and corresponds to protonation of the nitrogen. However, upon mixing [Ni(2-Spy)(triphos)](+) and lutH(+) a reaction is observed (on the seconds time scale) to produce an equilibrium mixture. The mechanistic interpretation of the rate law has been aided by the application of MSINDO semiempirical and ADF calculations. The kinetics and calculations are consistent with the reaction between [Ni(2-Spy)(triphos)](+) and lutH(+) involving initial protonation of the sulfur followed by dissociation of the nitrogen and subsequent transfer of the proton from sulfur to nitrogen. The factors affecting the position of protonation and the coupling of the coordination state of the 2-pyridinethiolate ligand to the site of protonation are discussed.

ChemInform Abstract: Mechanism of Reaction of S,S-Diphenyl-S-methoxythiazyne with Thiols.

The reaction of S,S-diphenyl-S-methoxythiazyne with various thiols gave methyl sulfides. Kinetic investigations support a mechanism involving an initial protonation of the thiazyne nitrogen with thiol followed by a nucleophilic attack of thiolate anion on the methyl group.

ChemInform Abstract: Protonation of Monosubstituted Oxiranes: A Computational Molecular Orbital Study of Oxonium Ion versus Carbonium Ion Formation.

The effect of substituents on the nature of the more stable species formed in the initial protonation of oxiranes-whether oxonium or carbonium ions-has been investigated by computational molecular orbital techniques. The geometries, electronic energies, and the distributions of total atomic charge for oxirane, for monosubstituted fluoro-, hydroxy-, amino, methyl-, cyano, formyl-, formaldimino-, and vinyloxiranes, and for the corresponding protonated species have been calculated using the 6-31G* basis set with full geometry optimization

Nucleophilic Cycloaromatization of Ynamide-Terminated Enediynes

Introduction of a nitrogen atom at one of the acetylenic termini of 10-, 11-, 12-, and 13-membered benzannulated cyclic enediynes results in a complete suppression of the conventional radical Bergman reaction in favor of a polar cycloaromatization. The latter reaction is catalyzed by acids and proceeds via initial protonation of an ynamide fragment. The resulting ketenimmonium cation then cyclizes to produce naphthyl cation, which rapidly reacts with nucleophiles or undergoes Friedel-Crafts addition to aromatic compounds. In alcohols, addition of the nucleophilic solvent across the activated triple bond competes with the cyclization reaction. The ratio of cyclized to solvolysis products decreases with the increase in ring size.

The role of hydrogen-bonding interactions in acidic sugar reaction pathways

Previously, theoretical multiple sugar (β- d-xylose and β- d-glucose) reaction pathways were discovered that depended on the initial protonation site on the sugar molecules using Car–Parrinello-based molecular dynamics (CPMD) simulations [Qian, X. H.; Nimlos, M. R.; Davis, M.; Johnson, D. K.; Himmel, M. E. Carbohydr. Res. 2005, 340, 2319–2327]. In addition, simulation results showed that water molecules could participate in the sugar reactions, thus altering the reaction pathways. In the present study, the temperature and water density effects on the sugar degradation pathways were investigated with CPMD. We found that changes in both temperature and water density could profoundly affect the mechanisms and pathways. We attributed these effects to both the strength of hydrogen bonding and proton affinity of water.

Mechanistic studies on the cis to trans epimerization of trisubstituted 1,2,3,4-tetrahydro-beta-carbolines.

It is well-known that N(b)-benzyltryptophan alkyl esters undergo the Pictet-Spengler reaction with aldehydes to furnish both cis- and trans-1,2,3,4-tetrahydro-beta-carbolines, with the trans isomer predominating. Epimerization at C-1 took place under acidic conditions to produce, exclusively, the thermodynamically more stable trans diastereomer via internal asymmetric induction. Recent kinetic experiments provided insight into the cis to trans epimerization mechanism involved in the Pictet-Spengler reaction of 1,2,3-trisubstituted tetrahydro-beta-carbolines. Since the epimerization reaction had been shown to be sensitive to electronic effects at C-1, the rate data for a series of 1-phenyl-substituted 1,2,3,4-tetrahydro-beta-carbolines was investigated via a Hammett study. Analysis of the data supported the presence of a positively charged intermediate with a rho value of -1.4, although the existence of an iminium ion intermediate or a carbocationic intermediate could not be determined from this data alone. Analysis of the rate of epimerization demonstrated first-order kinetics with respect to TFA following the initial protonation of the substrate. This observation was consistent with the formation of a doubly protonated intermediate as the rate-determining step in the carbocation-mediated cis to trans epimerization process. In addition, the observed first-order rate dependence was inconsistent with the retro-Pictet-Spengler mechanism since protonation at the indole-2 position was not rate determining as demonstrated by kinetic isotope effects. Based on this kinetic data, the retro-Pictet-Spengler pathway was ruled out for the cis to trans epimerization of 1,2,3-trisubstituted 1,2,3,4-tetrahydro-beta-carbolines, while the olefinic mechanism had been ruled out by experiments carried out in TFA-d.

Structural forms of green fluorescent protein by quantum mechanics/molecular mechanics calculations

The molecular modeling of structural forms of the green fluorescent protein (GFP) with the Ser65Thr single-site mutation was performed by the quantum mechanics/molecular mechanics (QM/MM) method. Two model systems were constructed based on the crystallographic structure from the Protein Data Bank (PDB entry code 1EMA.) The model systems differ in the initial protonation state of the side chain of the amino acid residue Glu222 near the chromophore. The atomic coordinates of the protein macromolecule corresponding to the equilibrium geometric configurations were determined by total energy minimization using the QM/MM method within the density functional theory approximation PBE0/cc-pVDZ for the quantum subsystem that consists of the chromophore, a water molecule, and the side chains of Arg96, Glu222, and Ser205, and with the parameters of the AMBER force field for the molecular mechanics subsystem. In the analysis of the results, particular attention was given to the hydrogen bond redistribution in the chromophore-containing region of the protein caused by a change in the protonation state of the chromophore. The results obtained from the model containing the initially protonated side chain of Glu222 suggest a new interpretation of the photophysical processes in the green fluorescent protein.

Epoxide-Nucleophile interactions: Acid-catalyzed reaction of ethylene oxide with water

We have studied the acid-catalyzed ring-opening interaction of ethylene oxide with water, a nucleophile. Ethylene oxide was selected as an initial model for the metabolically produced epoxides that may be the ultimate carcinogenic forms of some of the polynuclear aromatic hydrocarbons and are believed to undergo ring-opening interactions with a nucleophilic site (or sites) on DNA. We used an ab initio scf procedure (GAUSSIAN 70) to compute structures, energies, and charge distributions for the reacting system at several possible intermediate stages and in the final state, ethylene glycol. The initial protonation (due to the acidic conditions) facilitates ring opening both by weakening the C—O bonds and also by changing the charges on the carbons from negative to positive. For this gaseous phase interaction, a cis mode of attack (the water approaching from the oxygen side of the C—C bond) was found to be preferred over a trans attack. This may be due to internal hydrogen bonding, which also appears to play an important role at some other stages of the reaction.

Protein Autoproteolysis: Conformational Strain Linked to the Rate of Peptide Cleavage by the pH Dependence of the N → O Acyl Shift Reaction

Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --> O or N --> S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.

Meso-alkylidenyl-thia(p-benzi)porphyrins and their unusual protonation selectivity

Meso-alkylidenyl-thia(p-benzi)porphyrin and its ring expanded analog containing exocyclic C-C double bonds at meso-positions, undergo initial protonation at the exocyclic alkylidene alpha-carbon.

Proton catalyzed hydrolytic deamination of cytosine: a computational study

Two pathways involving proton catalyzed hydrolytic deamination of cytosine (to uracil) are investigated at the PCM-corrected B3LYP/6-311G(d,p) level of theory, in the presence of an additional catalyzing water molecule. It is concluded that the pathway involving initial protonation at nitrogen in position 3 of the ring, followed by water addition at C4 and proton transfer to the amino group, is a likely route to hydrolytic deamination. The rate determining step is the addition of water to the cytosine, with a calculated free energy barrier in aqueous solution of ΔG≠=140 kJ/mol. The current mechanism provides a lower barrier to deamination than previous work based on OH− catalyzed reactions, and lies closer to the experimental barrier derived from rate constants (Ea = 117 ± 4 kJ/mol).

Halophenol Rearrangement in Lewis Acid-Catalyzed Friedel - Crafts Conditions: Evidence of Competitive Initial Protonation and Acylation

Halogen rearrangement was observed during the Lewis acid-catalyzed Friedel–Crafts reaction of phthalic anhydride with bromophenols or bromoanisole. Further investigation revealed that 2-, 3-, and 4-bromophenols undergo rearrangement into other isomers under these reaction conditions. Product distribution from these reactions suggested that halogen rearrangement takes place during the s-complex intermediate of the condensation step. Furthermore, iodophenol undergoes hydrodeiodination rapidly rather than rearrangement, whereas chlorophenol does not undergo rearrangement at all.

Synthesis and Unusual Reactivity of N-Tosyl-4,5-benzoazacyclodeca-2,6-diyne, Yneamino-Containing Enediyne

Substitution of the propargylic carbon for nitrogen in cyclic 10-membered ring enediynes results in at least 2 orders of magnitude enhancement of reactivity. The mechanism of cycloaromatization of aza-enediynes, however, switches from radical (Bergman) to polar, which proceeds by initial protonation of a reactive yneamine moiety. Ketenimmonium cation produced on this step undergoes intramolecular nucleophilic cyclization. Reaction is strongly catalyzed by acids and exhibits substantial kinetic isotope effect.

Distinct Mechanisms of Bridging-Oxo Exchange in Di-μ-O Dimanganese Complexes with and without Water-Binding Sites: Implications for Water Binding in the O2-Evolving Complex of Photosystem II

Isotopic exchange between oxygens of water and mu-O bridges in the di-mu-O dimanganese complexes, [(mes-terpy)2Mn2(III/IV)(mu-O)2(H2O)2](NO3)3 (1, mes-terpy = 4'-mesityl-2,2':6',2' '-terpyridine) and [(phen)4Mn2III/IV(mu-O)2](ClO4)3 (2, phen = 1,10-phenanthroline), has been investigated by a study of the kinetics of exchange. The data provide evidence for distinct mechanisms of exchange in 1 and 2 and suggest that these differences arise due to the presence and absence of terminal water-binding sites in 1 and 2, respectively. Exchange of oxygen atoms between water and mu-O bridges must involve the elementary steps of bridge protonation, deprotonation, opening, and closing. On the basis of the existing literature on these reactions in oxo-bridged metal complexes and our present data, we propose pathways of exchange in 1 and 2. The mechanism proposed for 1 involves an initial fast protonation of an oxo-bridge by water coordinated to Mn(IV), followed by a slow opening of the protonated bridge as proposed earlier for an analogous complex on the basis of DFT calculations. The mechanism proposed for 2 involves initial dissociation of phen, followed by coordination of water at the vacated sites, as observed for rearrangement of 2 to a trinuclear complex. The subsequent steps are proposed to be analogous to those for 1. Our results are discussed in the context of data on 18O-labeled water isotope exchange in photosystem II and provide support for the existence of fully protonated terminal waters bound to Mn in the O2-evolving complex of photosystem II.

A quantum chemical study of the mechanism of action of Vitamin K epoxide reductase (VKOR): II. Transition states

A reaction path including transition states is generated for the Silverman mechanism [R.B. Silverman, Chemical model studies for the mechanism of Vitamin K epoxide reductase, J. Am. Chem. Soc. 103 (1981) 5939–5941] of action for Vitamin K epoxide reductase (VKOR) using quantum mechanical methods (B3LYP/6-311G**). VKOR, an essential enzyme in mammalian systems, acts to convert Vitamin K epoxide, formed by Vitamin K carboxylase, to its (initial) quinone form for cellular reuse. This study elaborates on a prior work that focused on the thermodynamics of VKOR [D.W. Deerfield II, C.H. Davis, T. Wymore, D.W. Stafford, L.G. Pedersen, Int. J. Quant. Chem. 106 (2006) 2944–2952]. The geometries of proposed model intermediates and transition states in the mechanism are energy optimized. We find that once a key disulfide bond is broken, the reaction proceeds largely downhill. An important step in the conversion of the epoxide back to the quinone form involves initial protonation of the epoxide oxygen. We find that the source of this proton is likely a free mercapto group rather than a water molecule. The results are consistent with the current view that the widely used drug Warfarin likely acts by blocking binding of Vitamin K at the VKOR active site and thereby effectively blocking the initiating step. These results will be useful for designing more complete QM/MM studies of the enzymatic pathway once three-dimensional structural data is determined and available for VKOR.

Reactivation pathway of the hydrogenase H‐cluster: Density functional theory study

AbstractThis work puts forth a reaction pathway for the reactivation of exogenous ligand inhibited H‐cluster, the active site of Fe‐only hydrogenases. The H‐cluster is a dimetal complex, Fe–Fe, with the metal centers bridged by di(thiomethyl)amine. Exogenous ligands, H2O, and OH−, are bound to the distal iron (Fed). Density functional theory (DFT) calculations on the native and ruthenium‐modified H‐cluster have been performed using the B3LYP functional with 6‐31+G** and 6‐311+G** basis sets. We have ascertained that there is a thermodynamically favorable pathway for the reactivation of the OH− inhibited H‐cluster, which proceeds by an initial protonation of the Fed–OH− complex. The proposed reaction pathway has all its intermediate reactions ensue exothermically. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

A 2-iridafuran from reaction between a 1-iridaindene and methyl propiolate

The coordinatively unsaturated 1-iridaindene, Ir[C 8H 5(Ph-3)]Cl(PPh 3) 2 has a labile chloride ligand and is easily converted to the corresponding iodide, Ir[C 8H 5(Ph-3)]I(PPh 3) 2 ( 1) by reaction with NaI. When Ir[C 8H 5(Ph-3)]I(PPh 3) 2 ( 1) is treated with methyl propiolate a reactive five-coordinate complex with both a diphenylvinyl ligand from ring-opening of the 1-iridaindene, and a 3-methoxy-3-oxoprop-1-ynyl ligand from deprotonation of methyl propiolate, is first produced. Reaction of this complex with aqueous HCl generates the 2-iridafuran, Ir[OC 3H(CH CPh 2-3)(OMe-5)]ClI(PPh 3) 2 ( 2) probably from initial protonation at the β-carbon of the 3-methoxy-3-oxoprop-1-ynyl ligand to form a vinylidene ligand and subsequent migration of the diphenylvinyl ligand to the α-carbon of this ligand accompanied by oxygen coordination to iridium. Similar treatment of 1 with methyl propiolate followed by aqueous HI gives the corresponding complex, Ir[OC 3H(CH CPh 2-3)(OMe-5)]I 2(PPh 3) 2 ( 3). The X-ray crystal structures of 2 and 3 together with NMR spectroscopic data confirm the 2-metallafuran structures of these complexes.

Formation and Reactivity of Ir(III) Hydroxycarbonyl Complexes

Kinetic studies show that the reaction of [TpIr(CO)2] (1, Tp = hydrotris(pyrazolyl)borate) with water to give [TpIr(CO2H)(CO)H] (2) is second order (k = 1.65 x 10(-4) dm(3) mol(-1) s(-1), 25 degrees C, MeCN) with activation parameters DeltaH++= 46+/-2 kJ mol(-1) and DeltaS++ = -162+/-5 J K(-1) mol(-1). A kinetic isotope effect of k(H2O)/k(D2O) = 1.40 at 20 degrees C indicates that O-H/D bond cleavage is involved in the rate-determining step. Despite being more electron rich than 1, [Tp*Ir(CO)2] (1*, Tp* = hydrotris(3,5-dimethylpyrazolyl)borate) reacts rapidly with adventitious water to give [Tp*Ir(CO2H)(CO)H] (2*). A proposed mechanism consistent with the relative reactivity of 1 and 1* involves initial protonation of Ir(I) followed by nucleophilic attack on a carbonyl ligand. An X-ray crystal structure of 2* shows dimer formation via pairwise H-bonding interactions of hydroxycarbonyl ligands (r(O...O) 2.65 A). Complex 2* is thermally stable but (like 2) is amphoteric, undergoing dehydroxylation with acid to give [Tp*Ir(CO)2H]+ (3*) and decarboxylation with OH- to give [TpIr(CO)H2] (4*). Complex 2 undergoes thermal decarboxylation above ca. 50 degrees C to give [TpIr(CO)H2] (4) in a first-order process with activation parameters DeltaH++ = 115+/-4 kJ mol(-1) and DeltaS++ = 60+/-10 J K(-1) mol(-1).

Allenediazonium ions and their protonation chemistry: a DFT study

The parent allenediazonium monocation H2C[double bond]C[double bond]CH(N2+) and ten of its substituted derivatives XYC[double bond]C[double bond]C(Z)N2+ (with F, CF3, Me, OMe, and Me2N as substituents) were studied by DFT at the B3LYP/6-31++G** level. Except for the Me2N-substituted derivative that forms a monocation-N2 complex, structurally intact allenediazonium ions were obtained as minima in all cases. Protonation studies at various sites were performed on allenediazonium cations, and relative energies of the resulting minima were used to identify the energetically most favored dications. In the majority of cases, protonation at the central carbon of the allenic moiety (C2) is most favored, forming delocalized allyl cation-N2+ species. The same dication structure is formed via initial C3-protonation, followed by a formal hydride shift, in cases where a carbocation-stabilizing group is placed at C3. When a CF3 group is placed at C3, initial protonation at C1 resulted in a 1,3-fluorine shift, to generate a fluoroallyl cation linked to a CH2N2+ moiety. Structural features in the allenediazonium monocations and their protonated dications were examined, taking into account their geometrical features, computed charges, and the GIAO NMR shifts.