Side Chains Of Amino Acid Residues Research Articles

Evolutionary pathways for deep-sea adaptation in marine planktonic Actinobacteriota.

The deep ocean, one of the largest ecosystems on earth, is dominated by microorganisms that are keystones in the regulation of biogeochemical cycles. However, the evolutionary pathways underlying the specific adaptations required (e.g., high pressure and low temperature) by this unique niche remain understudied. Here, we analyzed the first representatives belonging to the order Acidimicrobiales, a group of marine planktonic Actinobacteriota, that specifically inhabits the aphotic zone of the oceanic water column (>200 m). Compared with their epipelagic counterparts, deep-sea representatives showed the same evolution in genome architecture with higher GC content, longer intergenic spaces as well as higher nitrogen (N-ARSC) and lower carbon (C-ARSC) content in encoded amino acid residue side chains consistent with the higher nitrogen concentration and lower carbon concentration in deep waters compared to the photic zone. Metagenomic recruitment showed distribution patterns that allowed the description of different ecogenomic units within the three deep water-associated genera defined by our phylogenomic analyses (UBA3125, S20-B6 and UBA9410). The entire genus UBA3125 was found exclusively associated with oxygen minimum zones linked to the acquisition of genes involved in denitrification. Genomospecies of genus S20-B6 recruited in samples from both mesopelagic (200-1,000 m) and bathypelagic (1000-4,000 m) zones, including polar regions. Diversity in the genus UBA9410 was higher, with genomospecies widely distributed in temperate zones, others in polar regions, and the only genomospecies associated with abyssal zones (>4,000 m). At the functional level, groups beyond the epipelagic zone have a more complex transcriptional regulation including in their genomes a unique WhiB paralog. In addition, they showed higher metabolic potential for organic carbon and carbohydrate degradation as well as the ability to accumulate glycogen as a source of carbon and energy. This could compensate for energy metabolism in the absence of rhodopsins, which is only present in genomes associated with the photic zone. The abundance in deep samples of cytochrome P450 monooxygenases associated with the genomes of this order suggests an important role in remineralization of recalcitrant compounds throughout the water column.

Entropy-Enthalpy Compensations Fold Proteins in Precise Ways.

Exploring the protein-folding problem has been a longstanding challenge in molecular biology and biophysics. Intramolecular hydrogen (H)-bonds play an extremely important role in stabilizing protein structures. To form these intramolecular H-bonds, nascent unfolded polypeptide chains need to escape from hydrogen bonding with surrounding polar water molecules under the solution conditions that require entropy-enthalpy compensations, according to the Gibbs free energy equation and the change in enthalpy. Here, by analyzing the spatial layout of the side-chains of amino acid residues in experimentally determined protein structures, we reveal a protein-folding mechanism based on the entropy-enthalpy compensations that initially driven by laterally hydrophobic collapse among the side-chains of adjacent residues in the sequences of unfolded protein chains. This hydrophobic collapse promotes the formation of the H-bonds within the polypeptide backbone structures through the entropy-enthalpy compensation mechanism, enabling secondary structures and tertiary structures to fold reproducibly following explicit physical folding codes and forces. The temperature dependence of protein folding is thus attributed to the environment dependence of the conformational Gibbs free energy equation. The folding codes and forces in the amino acid sequence that dictate the formation of β-strands and α-helices can be deciphered with great accuracy through evaluation of the hydrophobic interactions among neighboring side-chains of an unfolded polypeptide from a β-strand-like thermodynamic metastable state. The folding of protein quaternary structures is found to be guided by the entropy-enthalpy compensations in between the docking sites of protein subunits according to the Gibbs free energy equation that is verified by bioinformatics analyses of a dozen structures of dimers. Protein folding is therefore guided by multistage entropy-enthalpy compensations of the system of polypeptide chains and water molecules under the solution conditions.

Effect of roasting temperature on lipid and protein oxidation and amino acid residue side chain modification of beef patties.

Beef is rich in nutrients and is one of the most important ingredients in the world. But in the process of cooking and heating, the nutrients of beef will change to varying degrees. How temperature affects the oxidation of lipids and proteins in beef, and the modification of amino acid residues is unclear. This study intended to heat beef at different roasting temperatures (150 °C, 190 °C, 230 °C, 270 °C, 310 °C), measure parameter including colour, peroxide value (PV), thiobarbituric acid-reactive substances (TBARS), thiol and carbonyl content, protein solubility, tryptophan and Schiff base content, protein molecular weight distribution and modification of amino acid residues to discussed the effects of different temperatures on the lipid and protein oxidation of beef patties, as well as the modification of amino acid residues. The results showed that the values of L* and b* increased with the temperature increased, and the values of a* decreased. With the increase of temperature, the lipid oxidation indexes PV and TBARS, Schiff base and carbonyl content also increased, and the thiol content and protein solubility decreased significantly (p < 0.001). SDS-PAGE showed that the band of myosin heavy chain (MHC, 220 kDa) was significantly degraded, while the band of actin (42 kDa) was still clearly visible. The analysis of UPLC-MS/MS results found that the aromatic amino acid residues in all samples were oxidized to a certain extent, especially tryptophan. Other oxidative modifications, including α-amiooadipic acid (AAA), hydroxyethyl lysine (CEL) and malondialdehyde (MDA), were only present in roasted samples and not in raw meat. The results suggested that lipid oxidation and protein oxidation were closely related to colour parameters. The oxidation of proteins and lipids was aggravated at higher temperature. Amino acid side chains were also modified at high temperature, and this change was particularly evident in aromatic amino acids. These results provided new insights for the oxidation of proteins and lipids of beef and the modification level of amino acid residues under high temperature conditions, which will help us to improve the cooking quality of meat foods.

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications.

Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

Rhodium(I) and Iridium(I) N-Heterocyclic carbene complexes of imidazolium functionalized amino acids and peptides

The conjugation of organometallic complexes to peptides is generally achieved through covalent organic linkages of the metal’s ligand to the peptide. Examples of direct coordination to metal centers by amino acid side chain residues remain rare. In one such example, side chain methylation of the natural amino acid histidine (His) resulted in an imidazolium functionalized amino acid which was used for the synthesis of rhodium(I), iridium(I), iridium(III), palladium(II) and ruthenium(III) N-heterocyclic carbene (NHC) complexes of the single amino acid and peptides containing this amino acid. Here, we have synthesized two new, non-natural imidazolium functionalized amino acid derivatives, which were used for solid phase peptide synthesis and for the synthesis of [M(COD)(NHC)Cl] (COD = 1,5 cyclooctadiene) complexes of Rh(I) and Ir(I). In total, six new complexes of the single amino acids and four complexes where the amino acids are present in a peptide environment were synthesized. Their characterization provides convincing evidence of conversion of the imidazolium moiety to an NHC ligand and thus the presence of a direct metal-carbon bond between the metal center and the amino acid side chain. Therefore, our compounds represent unique examples of peptide-conjugated complexes that bear the potential to be used for the synthesis of N-heterocyclic carbene complexes conjugated to cancer cell targeting peptides.

Redox Proteomes in Human Physiology and Disease Mechanisms.

Redox proteomics is a field of proteomics that is concerned with the characterization of the oxidation state of proteins to gain information about their modulated structure, function, activity, and involvement in different physiological pathways. Oxidative modifications of proteins have been shown to be implicated in normal physiological processes of cells as well as in pathomechanisms leading to the development of cancer, diabetes, neurodegenerative diseases, and some rare hereditary metabolic diseases, like classic galactosemia. Reactive oxygen species generate a variety of reversible and irreversible modifications in amino acid residue side chains and within the protein backbone. These oxidative post-translational modifications (Ox-PTMs) can participate in the activation of signal transduction pathways and mediate the toxicity of harmful oxidants. Thus the application of advanced redox proteomics technologies is important for gaining insights into molecular mechanisms of diseases. Mass-spectrometry-based proteomics is one of the most powerful methods that can be used to give detailed qualitative and quantitative information on protein modifications and allows us to characterize redox proteomes associated with diseases. This Review illustrates the role and biological consequences of Ox-PTMs under basal and oxidative stress conditions by focusing on protein carbonylation and S-glutathionylation, two abundant modifications with an impact on cellular pathways that have been intensively studied during the past decade.

Strong Purifying Selection Is Associated with Genome Streamlining in Epipelagic Marinimicrobia.

Marine microorganisms inhabiting nutrient-depleted waters play critical roles in global biogeochemical cycles due to their abundance and broad distribution. Many of these microbes share similar genomic features including small genome size, low % G + C content, short intergenic regions, and low nitrogen content in encoded amino acid residue side chains (N-ARSC), but the evolutionary drivers of these characteristics are unclear. Here, we compared the strength of purifying selection across the Marinimicrobia, a candidate phylum which encompasses a broad range of phylogenetic groups with disparate genomic features, by estimating the ratio of nonsynonymous and synonymous substitutions (dN/dS) in conserved marker genes. Our analysis reveals that epipelagic Marinimicrobia that exhibit features consistent with genome streamlining have significantly lower dN/dS values when compared with their mesopelagic counterparts. We also found a significant positive correlation between median dN/dS values and % G + C content, N-ARSC, and intergenic region length. We did not identify a significant correlation between dN/dS ratios and estimated genome size, suggesting the strength of selection is not a primary factor shaping genome size in this group. Our findings are generally consistent with genome streamlining theory, which postulates that many genomic features of abundant epipelagic bacteria are the result of adaptation to oligotrophic nutrient conditions. Our results are also in agreement with previous findings that genome streamlining is common in epipelagic waters, suggesting that microbes inhabiting this region of the ocean have been shaped by strong selection together with prevalent nutritional constraints characteristic of this environment.

EMap: A Web Application for Identifying and Visualizing Electron or Hole Hopping Pathways in Proteins

eMap is a web-based platform for identifying and visualizing electron or hole transfer pathways in proteins based on their crystal structures. The underlying model can be viewed as a coarse-grained version of the Pathways model, where each tunneling step between hopping sites represented by electron transfer active (ETA) moieties is described with one effective decay parameter that describes protein-mediated tunneling. ETA moieties include aromatic amino acid residue side chains and aromatic fragments of cofactors that are automatically detected, and, in addition, electron/hole residing sites that can be specified by the users. The software searches for the shortest paths connecting the user-specified electron/hole source to either all surface-exposed ETA residues or the user-specified target. The identified pathways are ranked according to their effective length. The pathways are visualized in 2D as a graph, in which each node represents an ETA site, and in 3D using available protein visualization tools. Here, we present the capability and user interface of eMap 1.0, which is available at https://emap.bu.edu .

Axial bonds at the T1 Cu site of Thermusthermophilus SG0.5JP17-16 laccase influence enzymatic properties.

Laccase is a multi‐copper oxidase which oxidizes substrate at the type 1 copper site, simultaneously coupling the reduction of dioxygen to water at the trinuclear copper center. In this study, we used site‐directed mutagenesis to study the effect of axial bonds between the metal and amino acid residue side chains in lacTT. Our kinetic and spectral data showed that the replacement of the axial residue with non‐coordinating residues resulted in higher efficiency (k cat/K m) and a lower Cu2+ population at the type 1 copper site, while substitution with strongly coordinating residues resulted in lower efficiency and a higher Cu2+ population, as compared with the wild‐type. The redox potentials of mutants with hydrophobic axial residues (Ala and Phe) were higher than that of the wild‐type. In conclusion, these insights into the catalytic mechanism of laccase may be of use in protein engineering to fine‐tune its enzymatic properties for industrial application.

Characterisation of sensor kinase by CD spectroscopy: golden rules and tips.

This is a review that describes the golden rules and tips on how to characterise the molecular interactions of membrane sensor kinase proteins with ligands using mainly circular dichroism (CD) spectroscopy. CD spectroscopy is essential for this task as any conformational change observed in the far-UV (secondary structures (α-helix, β-strands, poly-proline of type II, β-turns, irregular and folding) and near-UV regions [local environment of the aromatic side-chains of amino acid residues (Phe, Tyr and Trp) and ligands (drugs) and prosthetic groups (porphyrins, cofactors and coenzymes (FMN, FAD, NAD))] upon ligand addition to the protein can be used to determine qualitatively and quantitatively ligand-binding interactions. Advantages of using CD versus other techniques will be discussed. The difference CD spectra of the protein–ligand mixtures calculated subtracting the spectra of the ligand at various molar ratios can be used to determine the type of conformational changes induced by the ligand in terms of the estimated content of the various elements of protein secondary structure. The highly collimated microbeam and high photon flux of Diamond Light Source B23 beamline for synchrotron radiation circular dichroism (SRCD) enable the use of minimal amount of membrane proteins (7.5 µg for a 0.5 mg/ml solution) for high-throughput screening. Several examples of CD titrations of membrane proteins with a variety of ligands are described herein including the protocol tips that would guide the choice of the appropriate parameters to conduct these titrations by CD/SRCD in the best possible way.

Crystal Structure of the pH-Dependent Green Fluorescent Protein WasCFP with a Tryptophan-Based Chromophore at an Extremely Low pH of 2.0

The green fluorescent protein WasCFP with a tryptophan-based chromophore (Thr65-Trp66- Gly67) exhibits considerable pH-dependent changes of spectral properties related to the reversible processes of tryptophan ionization and protonation. The crystal structure of WasCFP at pH 10.0, 8.0, and 5.5 was determined in our earlier studies. The spatial structure of WasCFP at an extremely low pH of 2.0 has been determined by X-ray diffraction with a resolution of 1.3 A in the present study. Synchronous changes in the conformation of amino acid residue side chains in the environment of the chromophore and the related changes in the local hydrogen bond network involving the chromophore were shown to occur at sequential pH changes from 10.0 to 2.0. A quantum chemical study of the effect of interactions between the chromophore and the key amino acid residues from its immediate environment has been performed.

Decomposition of total solvation energy into core, side-chains and water contributions: Role of cross correlations and protein conformational fluctuations in dynamics of hydration layer

Dynamical coupling between water and amino acid side-chain residues in solvation dynamics is investigated by selecting residues often used as natural probes, namely tryptophan, tyrosine and histidine, located at different positions on protein surface. Such differently placed residues are found to exhibit different timescales of relaxation. The total solvation response measured by the probe is decomposed in terms of its interactions with (i) protein core, (ii) side-chain and (iii) water. Significant anti cross-correlation among these contributions are observed. When the motion of the protein side-chains is quenched, solvation either becomes faster or slower depending on the location of the probe.

Zinc complex of tryptophan appended 1,4,7,10-tetraazacyclododecane as potential anticancer agent: Synthesis and evaluation

With the rising incidences of cancer cases, the quest for new metal based anticancer drugs has led to extensive research in cancer biology. Zinc complexes of amino acid residue side chains are well recognized for hydrolysis of phosphodiester bond in DNA at faster rate. In the presented work, a Zn(II) complex of cyclen substituted with two l-tryptophan units, Zn(II)-Cyclen-(Trp)2 has been synthesized and evaluated for antiproliferative activity. Zn(II)-Cyclen-(Trp)2 was synthesized in ∼70% yield and its DNA binding potential was evaluated through QM/MM study which suggested good binding (G=−9.426) with B-DNA. The decrease in intensity of the positive and negative bands of CT-DNA at 278nm and 240nm, respectively demonstrated an effective unwinding of the DNA helix with loss of helicity. The complex was identified as an antiproliferative agent against U-87MG cells with 5 fold increase in apoptosis with respect to control (2h post incubation, IC50 25µM). Electrophoresis and comet assay studies exhibited an increase in DNA breakage after treatment with complex while caspase-3/β-actin cleavage established a caspase-3 dependent apoptosis pathway in U-87 MG cells after triggering DNA damage. In vivo tumor specificity of the developed ligand was validated after radiocomplexation with 99mTc (>98% radiochemical yield and specific activity of 2.56GBq/µmol). Avid tumor/muscle ratio of >6 was depicted in biodistribution and SPECT imaging studies in U-87 MG xenograft model nude mice.

High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions.

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.

Radical-driven processes within a peptidic sequence of type I collagen upon single-photon ionisation in the gas phase.

We report on an experimental single-photon absorption study on gas-phase protonated collagen peptides employing a combination of mass spectrometry and synchrotron radiation. Partial ion yields for the main photoabsorption products vary steadily with photon energy over the range from 14 to 545 eV. At low energy, non-dissociative photoionisation competes with neutral molecule loss from the precursor ion, whereas fragmentation of the peptide backbone dominates at soft X-ray energies. Neutral molecule losses from the ionised peptide are found to have low energy barriers and most likely involve amino-acid residue side-chains with radical character, in particular aspartic acid. A particularly interesting finding is photoinduced loss of proline hydroxylation. The loss of this typical collagen post-translational modification might play a destabilizing role in the collagen structure.

Probing the Secondary Structure of Membrane Peptides Using (2)H-Labeled d(10)-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy.

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing (2)H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was (2)H-labeled, such as (2)H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with (2)H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique (2)H-ESEEM patterns were observed with the (2)H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the (2)H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, (2)H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.

Correlated Conformational Motions of the KH Domains of Far Upstream Element Binding Protein Complexed with Single-Stranded DNA Oligomers

Single-stranded DNA binding (SSB) proteins bind with single-stranded DNA (ss-DNA) segments that are generated as intermediates during DNA metabolic processes. The primary function of an SSB protein is to protect the ss-DNA from being degraded so that other enzymes can effectively act on it. We have performed atomistic molecular dynamics simulations of the two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element (FUSE) binding protein (FBP) complexed with two ss-DNA oligomers in aqueous solutions. Attempts have been made to study the effects of complexation on the internal motions of the protein domains and the correlated dynamics of the amino acid residue side chains. In agreement with experiments, KH3 domain has been found to be relatively more flexible in the complexed state. The calculations reveal increased long-range anticorrelated motions among several amino acid residues in the complexed forms. Compared to the KH4 domain, noticeable increase in N-H dipole ordering on complexation has been observed for the KH3 domain. Importantly, it is demonstrated that the effects of the DNA strands on the side chain orientations of the arginine and lysine residues and their ordering and dynamics play critical roles in forming the complexes and their structural stability.

Effects of implicit solvent and relaxed amino acid side chains on the MP2 and DFT calculations of ligand–protein structure and electronic interaction energies of dopaminergic ligands in the SULT1A3 enzyme active site

The electronic interaction energies between eight dopaminergic ligands and resveratrol and the SULT1A3 active site have been calculated using MP2 and M062X with the 6-311+G∗ basis set. The SULT1A3 active site was isolated from the crystal structure with dopamine bound (PBD ID 2A3R). Structures for the nine ligands bound in the active site were obtained by three different optimization approaches with M062X/6-31G: by optimization in vacuo with rigid amino acid residue side chains (i.e. using the side chain structures from the crystal structure; the ligand and all protons were optimized), with implicit solvation by water with rigid amino acid residue side chains, and with implicit solvation by water and relaxed amino acid residue side chains (i.e. the ligand and all amino acid residue side chains were optimized). The calculations show that the use of implicit solvent and a relaxed active site offer considerable improvement in ligand interaction energies (more strongly binding), and that the solvated-relaxed model shows less variability in ligand interaction than the other two models. Finally, 6-carboxydopamine was shown to be the most strongly binding of the nine ligands, with an interaction energy over 100kcal/mol stronger than its nearest neighbor. Four of the eight non-dopamine ligands studied interact with SULT1A3 more strongly than the endogenous ligand, dopamine, in the solvated-relaxed model.

Molecular origin of the hydrolytic activity and fixed regioselectivity of a Zr(IV) -substituted polyoxotungstate as artificial protease.

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells-Dawson polyoxometalate (POM), K15 H[Zr(α2 -P2 W17 O61 )2 ]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×10(8) M(-1) , the three remaining sites are characterized by a lower global affinity constant of 7×10(5) M(-1) . The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10(8) M(-1) obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ]. In addition, Eu(III) luminescence experiments with an Eu(III) -substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu(III) due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ] have demonstrated regioselective cleavage of HSA at the Arg114Leu115, Ala257Asp258, Lys313Asp314 or Cys392Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K15 H[Zr(α2 -P2 W17 O61 )2 ].

Synthesis of novel cinchona-amino acid hybrid organocatalysts for asymmetric catalysis

Three novel subclasses of cinchonidine derivatives coupled to diverse amino acids were prepared in very good overall yield and tested in a benchmark organocatalytic aldol reaction, between acetone and aromatic aldehydes. These subclasses are a family of amino acid-cinchonidine (subclass A), N-formamides-cinchonidine (subclass B) and dipeptide-cinchonidine (subclass C) hybrids. Our main goal, besides obtaining very good yields and enantioselectivities, was to understand the influence of the amino acid side chain residues on the enantioselectivity of the asymmetric aldol reactions. Different amino acid tethered cinchonidine hybrids were compared and their catalytic behaviour was evaluated, allowing good enantioselectivities to be achieved, 92% ee in one case. Other reactions such as Biginelli, Michael addition and ketimine hydrosilylation reactions were screened with these ligands, but the outcome was less successful.