Josefa Donoso

The pyridoxamine action on Amadori compounds: A reexamination of its scavenging capacity and chelating effect.

Amadori compounds act as precursors in the formation of advanced glycation end products (AGEs) by non-enzymatic protein glycation, which are involved in ensuing protein damage. Pyridoxamine is a potent drug against protein glycation, and can act on several pathways in the glycation process. Nevertheless, the pyridoxamine inhibition action on Amadori compounds oxidation is still unclear. In this work, we have studied the Schiff base formation between pyridoxamine and various Amadori models at pH 7.4 at 37 degrees C in the presence of NaCNBH(3). We detected an adduct formation, which suggests that pyridoxamine reacts with the carbonyl group in Amadori compounds. The significance of this mechanism is tested by comparison of the obtained kinetics rate constants with that obtained for 4-(aminomethyl)-pyridine, a structural analogue of pyridoxamine without post-Amadori action. We also study the chelating effect of pyridoxamine on metal ions. We have determined the complexation equilibrium constants between pyridoxamine, N-(1-deoxy-d-fructos-1-yl)-l-tryptophan, aminoguanidine, and ascorbic acid in the presence of Zn(2+). The results show that the strong stability of pyridoxamine complexes is the key in its post-Amadori inhibition action. On the other hand results explain the lack of inhibition of aminoguanidine (a glycation inhibitor) in the post-Amadori reactions.

DFT Studies on Schiff Base Formation of Vitamin B6 Analogues. Reaction between a Pyridoxamine-Analogue and Carbonyl Compounds

A comprehensive theoretical study based on density functional theory calculations (B3LYP and M06-2X functionals) of the formation of Schiff bases of pyridoxamine analogues with two different aldehydes was conducted. The reaction mechanism was found to involve two steps, namely: (1) formation of a carbinolamine and (2) dehydration of the carbinolamine to give the final imine. Also, consistent with available experimental evidence, the carbinolamine dehydration was the rate-determining step of the process determined by means of M06-2X functional. Using an appropriate solvation method and reactant conformation ensures that all proton transfers involved will be intramolecular, which substantially reduces energy barriers and facilitates reaction in all cases. The formation of a Schiff base between pyridoxal 5-phosphate (PLP) and an amine or amino acid requires the contribution of an external water molecule in order to facilitate proton transfers. On the other hand, the formation of a Schiff base between pyridoxamine 5-phosphate (PMP) and a carbonyl compound requires no external aid since the spatial arrangement of the functional groups in PMP ensures that all proton transfers will be intramolecular.

Mechanistic insights in glycation-induced protein aggregation.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

Theoretical studies on Schiff base formation of vitamin b6 analogues

A comprehensive study of Schiff base formation of vitamin b6 analogues in gas-phase and water solvation environment is carried out using semiempirical (PM3) quantum mechanics calculations. Vitamin b6 analogues for the gas-phase calculations include one auxiliary water molecule whilst water solvation has been taken into account by using two different models: continuum solvation model on the gas-phase optimized structures and supermolecular approach, in which a complete reoptimization of the gas-phase structures surrounded with explicit water molecules is performed. These calculations result in the description of the geometries of all the intermediates and transition structures along the reaction pathway, which can be divided in three parts: carbinolamine formation, dehydration and imine formation. The carbinolamine is the main intermediate and dehydration is the limiting step of the reaction, in accordance with experimental evidence. The details of the mechanism highlight the key role of internal hydrogen transfers and of the hydrogen bonds from water molecules of the solvation sphere for the occurrence of the reaction.

Kinetic study of the Schiff-base formation between glycine and pyridoxal 5′-phosphate (PLP), pyridoxal (PL), and 5′-deoxypyridoxal (DPL)

The formation of Schiff bases between pyridoxal 5′-phosphate (PLP), pyridoxal (PL), 5′-deoxypyridoxal (DPL), and glycine has been investigated as a function of proton concentration at 25 °C and 0.1 mol dm–3 ionic strength. Ionization constants as well as equilibrium and microscopic kinetic constants of the ionic species present in solution have been determined.The results show that all the protonatable groups of the aldehyde molecule are involved in the intramolecular acidic catalysis of the dehydration process undergone by the α-hydroxyamine intermediate. Results also reveal that the formation of Schiff base with pyridoxal is greatly affected by the formation of the cyclic hemiacetal of this aldehyde.

A comparative study of the chemical reactivity of pyridoxamine, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds.

Pyridoxamine (PM) has long been known to inhibit protein glycation via various mechanisms of action. One such mechanism involves the scavenging of carbonyl compounds with glycating ability. Despite the abundant literature on this topic, few quantitative kinetic studies on the processes involved have been reported. In this work, we conducted a comparative kinetic study under physiological pH and temperature conditions of the reactions of PM, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds (viz. aldehydes, α-oxoaldehydes and ketones). The microscopic formation rate constants for Schiff bases of PM and various carbonyl compounds, k1, are of the same order of magnitude as those for the Schiff bases of Ac-Phe-Lys. However, because PM exhibits a higher proportion of reactive form at physiological pH, its observed second-order rate constant is ca. five times greater than that for Ac-Phe-Lys. That could explain PM ability to compete with amino residues in protein glycation. On the other hand, the observed formation rate constant for thiohemiacetals is four orders of magnitude greater than the formation constants for the Schiff bases of PM, which excludes PM as a competitive inhibitor of Cys residues in protein glycation.

Theoretical calculations of β-lactam antibiotics: Part 2. AM1, MNDO and MINDO/3 calculations of some cephalosporins

Abstract The geometries of several cephalosporins were determined by the MINDO/3, MNDO and AM1 semiempirical calculation methods. The geometric parameters of the bicyclic system thus obtained (bond distances and angles, and dihedral angles ) were compared with crystallographic and literature data. Of the three methods used, MINDO/3 provided the best estimations of the geometric values, while MNDO reproduced the pyramidal character of the β-lactam nitrogen with the greatest accuracy and AM1 yielded an intermediate solution. All three methods were used to carry out a comprehensive conformational analysis of the bicyclic system. In this respect, MINDO/3 only detected one minimum, while both MNDO and AM1 yielded two minima, corresponding to two extreme situations (S1-up and C2-up).

Formation of Schiff bases of 5′-deoxypyridoxal and hexylamine in aqueous and non-aqueous media

We have studied the formation of Schiff bases of 5′-deoxypyridoxal (DPL) and hexylamine in various non-aqueous solvents (dioxane, pentanol/dioxane mixtures, pentanol, butan-2-ol, propanol, ethanol, methanol and ethane-1,2-diol) at 25 °C. The results are interpreted in terms of the tautomer forms of DPL present in the medium, and the rate constant of formation of the Schiff base has been obtained for the neutral species of DPL and the zwitterionic form (k1n= 1.7 × 102 dm3 mol–1 min–1 and k1z= 3.6 × 103 dm3 mol–1 min–1, respectively). We also determined the constant of the tautomeric equilibrium between the corresponding forms of the uncharged species of DPL in the different solvents. These results and those obtained in water/dioxane media indicate that the intramolecular acid catalysis involved in the formation of Schiff bases is also governed by the occurrence of a charged transition state.

Theoretical calculations ofβ-lactam antibiotics: Part 1. AM1, MNDO and MINDO/3 calculations of some penicillins

Abstract MINDO/3, MNDO and AM1 calculations have been carried out in order to calculate the geometry of some penicillins. Theoretical bond lengths, bond angles and dihedral angles have been checked with crystallographic data. Results show that MINDO/3 is the best method for predicting the geometry of the bicyclic system. However, it is not very good in predicting the conformations and pyramidality of the β-lactam nitrogen. In spite of its poor accuracy in predicting the geometry of the antibiotic, the MNDO method reproduces very well the pyramidality of the β-lactam nitrogen. The AM1 method predicts the geometry of the system quite well, except for the S-C and C7-N4 bond lengths which are underestimated and overestimated respectively. In addition, AM1 is very good at treating conformations of the thiazolidine ring.

Kinetic and thermodynamic parameters for Schiff's base formation between pyridoxal 5′-phosphate and n-hexylamine

The kinetic and thermodynamic parameters for the reaction between pyridoxal 5′-phosphate and n-hexylamine in aqueous medium as a function of pH, at constant ionic strength (0.1) and temperatures of 10, 15, 20, and 30 °C have been calculated. The variation observed in the overall kinetic formation and hydrolysis constants of the corresponding Schiffs base can be accounted for on the basis of the protonation and reactivity of the substrates present (pyridoxal 5′-phosphate, n-hexylamine, and Schiffs base) in the medium as a function of pH. The first protonation, both of pyridoxal 5′-phosphate and of the Schiffs base, modifies the energies of activation corresponding to the formation and hydrolysis processes, respectively. The less negative charge the pyridoxal 5′-phosphate molecule bears, the more negative the ΔH and ΔS values become.