Identification of the electrophoretic peaks of the phenylthiohydantoin derivatives of amino acids

Two techniques for identifying the peaks of phenylthiohydantoin (PTH) amino acids separated under the conditions of micellar electrokinetic chromatography were compared. The first technique is linear regression analysis, in which the retention time of an amino acid is a function of the retention times of two retention-time standards. The second technique takes into account hydrophobicity constants logD′, which were calculated using the ACD/LC Simulator 8.0 program package from ACDLabs (Canada). These constants provide an opportunity to calculate the relative migration times of PTH amino acids taking into account the velocity of the electroosmotic flow. The first technique allows us to identify the electrophoretic peaks of all 16 amino acids separated; the second procedure allows us to predict the elution order of the electrophoretic peaks; the use of a correlation equation gives better results.     

G-蛋白偶联受体GPR120分子模建研究

新的长链脂肪酸受体G-蛋白偶联受体120 (G-protein-coupled receptor120, GPR120)是2型糖尿病的潜在治疗靶标. 由于其晶体结构迄今尚未获得, 成为基于结构的新药设计的瓶颈. 首先, 以人体β2肾上腺能素受体(human β2 adrenergic receptor, β2AR)晶体结构为模板, 通过同源模建方法构建GPR120三维结构, 对整个体系进行包膜的分子动力学模拟. 然后采用分子对接技术模建了GPR120的小分子激动剂GW9508与GPR120的相互作用模型, 发现了受体分子识别的关键性残基, 为开展定点突变实验提供了指导意义. 所建模型为研究受体与配体作用提供了合理的初始结构, 此方法也适用于其他G蛋白偶联受体的分子模建.     

Reprogramming the genetic code: from triplet to quadruplet codes

The genetic code of cells is near-universally triplet, and since many ribosomal mutations are lethal, changing the cellular ribosome to read nontriplet codes is challenging. Herein we review work on the incorporation of unnatural amino acids into proteins in response to quadruplet codons, and the creation of an orthogonal translation system in the cell that uses an evolved orthogonal ribosome to efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons. Using this system multiple distinct unnatural amino acids have been incorporated and used to genetically program emergent properties into recombinant proteins. Extension of approaches to incorporate multiple unnatural amino acids may allow the combinatorial biosynthesis of materials and therapeutics, and drive investigations into whether life with additional genetically encoded polymers can evolve to perform functions that natural biological systems cannot.     

Chiral Amino Acids‐Derived Catalysts and Ligands

Transforming amino acids into novel catalysts and ligands is a remarkable subset of new catalyst development in order to imitate enzymatic efficiencies. Their ability to perform a variety of asymmetric catalytic reactions is complimented by their ready availability, rich transformations, stability and easy procedure. Herein, we focused on describing our endeavor of developing new catalysts and ligands from primary and secondary amino acids. It includes C₂‐symmetric N,N'‐dioxides, guanidine‐amides, bispidine‐based diamines, and other organic salts. The account covered a brief introduction about their discovery, representative applications and related mechanisms.     

Homology modeling of 5-lipoxygenase and hints for better inhibitor design

Lipoxygenases (LOXs) are a group of enzymes involved in the oxygenation of polyunsaturated fatty acids. Among these 5-lipoxygenase (5-LOX) is the key enzyme leading to the formation of pharmacologically important leukotrienes and lipoxins, the mediators of inflammatory and allergic disorders. In view of close functional similarity to mammalian lipoxygenase, potato 5-LOX is used extensively. In this study, the homology modeling technique has been used to construct the structure of potato 5-LOX. The amino acid sequence identity between the target protein and sequence of template protein 1NO3 (soybean LOX-3) searched from NCBI protein BLAST was 63%. Based on the template structure, the protein model was constructed by using the Homology program in InsightII. The protein model was briefly refined by energy minimization steps and validated using Profile-3D, ERRAT and PROCHECK. The results showed that 99.3% of the amino acids were in allowed regions of Ramachandran plot, suggesting that the model is accurate and its stereochemical quality good. Like all LOXs, 5-LOX also has a two-domain structure, the small N-terminal beta-barrel domain and a larger catalytic domain containing a single atom of non-heme iron coordinating with His525, His530, His716 and Ile864. Asn720 is present in the fifth coordination position of iron. The sixth coordination position faces the open cavity occupied here by the ligands which are docked. Our model of the enzyme is further validated by examining the interactions of earlier reported inhibitors and by energy minimization studies which were carried out using molecular mechanics calculations. Four ligands, nordihydroguaiaretic acid (NDGA) having IC(50) of 1.5 microM and analogs of benzyl propargyl ethers having IC(50) values of 760 microM, 45 microM, and no inhibition respectively were selected for our docking and energy minimization studies. Our results correlated well with the experimental data reported earlier, which proved the quality of the model. This model generated can be further used for the design and development of more potent 5-LOX inhibitors.     

Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.     

Generate: a program for 3-D structure generation and conformational analysis of peptides and peptidomimetics

The program Generate, aimed at generating 3-D structures for peptides and peptidomimetics, is presented. The algorithm is based on a build-up procedure, using a library of conformations of amino acid residues. This library is built from conformational analysis of amino acids placed in a di- or tripeptide environment to mimic the surroundings of the amino acid in a true peptide, considering different positions of the residue in the peptide chain (peptidyl fragment, NH(+)(3)-terminus or COO(-)-terminus). Cis-trans isomerism in the amide bonds is taken into account by construction of rotamer libraries for different isomers. Water solvation is included through the GB/SA model. New amino acid residues can easily be added to the libraries, making it possible to generate conformations of peptidomimetics.     

SVM and SVR-based MHC-binding prediction using a mathematical presentation of peptide sequences

At present, there are a number of methods for the prediction of T-cell epitopes and major histocompatibility complex (MHC)-binding peptides. Despite numerous methods for predicting T-cell epitopes, there still exist limitations that affect the reliability of prevailing methods. For this reason, the development of models with high accuracy are crucial. An accurate prediction of the peptides that bind to specific major histocompatibility complex class I and II (MHC-I and MHC-II) molecules is important for an understanding of the functioning of the immune system and the development of peptide-based vaccines. Peptide binding is the most selective step in identifying T-cell epitopes. In this paper, we present a new approach to predicting MHC-binding ligands that takes into account new weighting schemes for position-based amino acid frequencies, BLOSUM and VOGG substitution of amino acids, and the physicochemical and molecular properties of amino acids. We have made models for quantitatively and qualitatively predicting MHC-binding ligands. Our models are based on two machine learning methods support vector machine (SVM) and support vector regression (SVR), where our models have used for feature selection, several different encoding and weighting schemes for peptides. The resulting models showed comparable, and in some cases better, performance than the best existing predictors. The obtained results indicate that the physicochemical and molecular properties of amino acids (AA) contribute significantly to the peptide-binding affinity.     

Development and Molecular Understanding of a Pd-Catalyzed Cyanation of Aryl Boronic Acids Enabled by High-Throughput Experimentation and Data Analysis

A synthetic method for the palladium-catalyzed cyanation of aryl boronic acids using bench stable and non-toxic N-cyanosuccinimide has been developed. High-throughput experimentation facilitated the screen of 90 different ligands and the resultant statistical data analysis identified that ligand σ-donation, π-acidity and sterics are key drivers that govern yield. Categorization into three ligand groups – monophosphines, bisphosphines and miscellaneous – was performed before the analysis. For the monophosphines, the yield of the reaction increases for strong σ-donating, weak π-accepting ligands, with flexible pendant substituents. For the bisphosphines, the yield predominantly correlates with ligand lability. The applicability of the designed reaction to a wider substrate scope was investigated, showing good functional group tolerance in particular with boronic acids bearing electron-withdrawing substituents. This work outlines the development of a novel reaction, coupled with a fast and efficient workflow to gain understanding of the optimal ligand properties for the design of improved palladium cross-coupling catalysts.     

Advances in the investigation of dioxouranium(VI) complexes of interest for natural fluids

The interactions of dioxouranium(VI) cation with different organic and inorganic ligands of environmental and biological interest were carefully examined with the aim to draw a chemical speciation picture of this ion in natural aquatic ecosystems and in biological fluids. Since UO₂²⁺ ion shows a significant tendency to hydrolyze, particular attention was paid in considering the hydrolysis species formation both in the presence and in absence of ligands. The results reported in the literature show that formation of the hydrolytic species assumes a great importance in the complexation models for all the UO₂²⁺-ligand systems considered. In particular, the following ligands have been taken into account: (i) hydroxyl, chloride, fluoride, sulfate, carbonate and phosphate, as inorganic ligands, and (ii) carboxylates (with particular reference to oxalate and citrate), amines, amino acids, poly(amino carboxylates) (complexones), nucleotides, phosphonates, mercapto compounds and sulfonates, as organic ligands. In order to elucidate the speciation of uranyl in the presence of dissolved natural organic matter, the interactions with humic and fulvic acids were also considered. The strength of interaction in all the systems considered was expressed in terms of stability constants of complex species and, if available, of the relative thermodynamic stability parameters. When possible, if data reported in the literature were sufficiently homogeneous, trends of stability were found for the different ligands of the same class and for ligands of different classes. Moreover, relationships were derived for poly-functional ligands, such as poly-carboxylate, poly-amine and poly(amino carboxylate) ones, useful to predict the stability constants as a function of the number of binding sites per molecule, considering also, as in the case of amino acids, the contribution of the single functional groups to the whole stability of uranyl species formed. In addition, using the stability data collected for the uranyl-ligand systems considered, the sequestering capacity of some classes of ligands towards uranyl was calculated in terms of pL_0.5, i.e., the ligand concentration useful to bind at least 50% of the cation. A comparison of pL_0.5 of the most important classes of ligands considered was made to point out the different effectiveness in the UO₂²⁺ sequestration by the different ligands which can be present in multi-component solutions as natural waters and biological fluids. Finally, some considerations are reported about the different experimental techniques employed to study the complex formation in solution.     

PdII‐Catalyzed Enantioselective C(sp3)−H Activation/Cross‐Coupling Reactions of Free Carboxylic Acids

Pd^II‐catalyzed enantioselective C(sp³)?H cross‐coupling of free carboxylic acids with organoborons has been realized using either mono‐protected amino acid (MPAA) ligands or mono‐protected aminoethyl amine (MPAAM) ligands. A diverse range of aryl‐ and vinyl‐boron reagents can be used as coupling partners to provide chiral carboxylic acids. This reaction provides an alternative approach to the enantioselective synthesis of cyclopropanecarboxylic acids and cyclobutanecarboxylic acids containing α‐chiral tertiary and quaternary stereocenters. The utility of this reaction was further demonstrated by converting the carboxylic acid into cyclopropyl amine without loss of optical activity.     

Fluorinated amino acids in protein design and engineering

Selective incorporation of unnatural amino acids into proteins is a powerful tool for illuminating the principles of protein design. In particular, fluorinated amino acids have recently emerged as valuable building blocks for designing hyperstable protein folds, as well as directing highly specific protein-protein interactions. We review the collagen mimetic and coiled coil peptide systems that exemplify generalizable paradigms for future design. The unique electronic and phase properties of fluorocarbons are discussed, and protein synthesis using unnatural amino acids is briefly reviewed.     

A comparative docking study and the design of potentially selective MMP inhibitors

As part of a program aimed at the design and synthesis of constrained MMP inhibitors, a survey of the reported X-ray and NMR structures of MMP/inhibitor complexes was performed, revealing mutations of key amino acids at different subsites between MMPs. A comparative study of fully automated docking programs AutoDock and DOCK in closely approximating the X-ray crystal structures of ten selected MMP inhibitors was performed. AutoDock proved to be highly reliable, efficient and predictive for a set of inhibitors with less than six atom types.     

Determination of the protein and free amino acid content in a sample using o-phthalaldehyde and N-acetyl-L-cysteine

A spectrophotometric method is proposed for determining the protein content in a sample after total acid hydrolysis. In the procedure, free amino acids are caused to react with o-phthalaldehyde and N-acetyl-L-cysteine at pH 9.5, using isoleucine as the reference compound. Correction factors are used to take into account the differences between the molar absorptivities of the amino acid isoindoles and the recoveries of the amino acids after the hydrolysis treatment. The limit of detection was in the range 40-50 micrograms of protein, and the recoveries were usually 101 +/- 3% with a coefficient of variation lower than 4%. The free amino acid content in a partially hydrolysed protein was also determined.     

Employing the structural diversity of nature: development of modular dipeptide-analogue ligands for ruthenium-catalyzed enantioselective transfer hydrogenation of ketones

A library of novel dipeptide-analogue ligands based on the combination of tert-butoxycarbonyl(N-Boc)-protected alpha-amino acids and chiral vicinal amino alcohols were prepared. These highly modular ligands were combined with [[RuCl(2)(p-cymene)](2)] and the resulting metal complexes were screened as catalysts for the enantioselective reduction of acetophenone under transfer hydrogenation conditions using 2-propanol as the hydrogen donor. Excellent enantioselectivity of 1-phenylethanol (up to 98 % ee) was achieved with several of the novel catalysts. Although most of the ligands contained two stereocenters, it was demonstrated that the absolute configuration of the product alcohol was determined by the configuration of the amino acid part of the ligand. Employing ligands based on L-amino acids generated S-configured products, and catalysts based on D-amino acids favored the formation of the R-configured alcohol. The combination N-Boc-L-alanine and (R)-phenylglycinol (Boc-L-Ab) or its enantiomer (N-Boc-D-alanine and (S)-phenylglycinol, Boc-D-Aa) proved to be the best ligands for the reduction process. Transfer hydrogenation of a number of aryl alkyl ketones were evaluated and excellent enantioselectivity, up to 96 % ee, was obtained.     

A genetic algorithm for structure-based de novo design

Genetic algorithms have properties which make them attractive in de novo drug design. Like other de novo design programs, genetic algorithms require a method to reduce the enormous search space of possible compounds. Most often this is done using information from known ligands. We have developed the ADAPT program, a genetic algorithm which uses molecular interactions evaluated with docking calculations as a fitness function to reduce the search space. ADAPT does not require information about known ligands. The program takes an initial set of compounds and iteratively builds new compounds based on the fitness scores of the previous set of compounds. We describe the particulars of the ADAPT algorithm and its application to three well-studied target systems. We also show that the strategies of enhanced local sampling and re-introducing diversity to the compound population during the design cycle provide better results than conventional genetic algorithm protocols.     

Electrostatic complementarity between proteins and ligands. 2. Ligand moieties

Summary Drug design strategies consider factors governing intermolecular interactions to build up putative ligands. In many strategies, the ligand is constructed using fragments which are placed in the site sequentially. The optimization is then performed with each fragment. We would like to examine if this optimization strategy could generate ligands with optimal electrostatic interactions. The electrostatic complementarities between constituent moieties and the receptor site have been calculated. The whole-ligand complementarity does not appear to be the mathematical mean of the individual complementarities, nor have we found a simple relationship between the moiety and whole-ligand complementarities. The results demonstrate clearly that, using a simple model, it is very difficult to predict the electrostatic potential complementarity of the whole ligand from the complementarities of its constituent chemical moieties. This means that ligand design strategies must optimize the electrostatic complementarity globally, and not moiety by moiety.     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.     

Formation of low-symmetric 2D superlattices of gold nanoparticles through surface modification by acid-base interaction

In the present work, a novel method was developed for the fabrication of 2D superlattices with different symmetries. Same-surface amino-functionalized Au nanoparticles as building blocks were self-assembled to form different 2D superlattices using surface modification with organic acids. The 2D superlattices of quasi-honeycomb and square structures were obtained by neutralizing amino-functionalized Au nanoparticles with 1,3,5-tribenzenecarboxylic acid and acetic acid, respectively. These results strongly suggest that the different types of 2D or 3D superlattices can be constructed by simple addition of proper acid to nanoparticles functionalized with amino groups. This method will allow us to obtain various desired metal superlattices without fully synthesizing the ligands.     

Relationship between the ligand structure and the luminescent properties of water-soluble lanthanide complexes containing bis(bipyridine) anionic arms

A series of six new ligands (L(1)-L(6)) suitable for the formation of luminescent lanthanide complexes in water is described. Ligands L(1)-L(4) are constructed from two 6'-carboxy-6-methylene-2,2'-bipyridine chromophoric arms bonded to the amino function of a 2-aminomethylene-6-carboxy-pyridine (L(1)), an N,N-diacetate-ethylene diamine (L(2)), a serine (L(3)), or an aminomalonic acid (L(4)). For ligands L(5) and L(6), the linking amino function is provided by a glutamic acid, and the anionic functions at the 6'-position of the bipyridyl arms are made of the sodium salts of monoethylphosphonic ester (L(5)) and phosphonic acid (L(6)). The synthesis and characterisation of the ligands are described, together with the study of the formation of lanthanide complexes with europium and terbium. In the case of L(3), the europium complex obtained in acidic conditions was crystallised and the X-ray crystal structure is depicted. Photophysical properties of the complexes were studied by means of UV-visible absorption, and steady-state and time-resolved luminescence spectroscopy. Excited-state luminescence lifetimes of the complexes were determined in water and deuterated water to gain insight into the number of water molecules directly coordinated in the first coordination sphere of the complexes. The coordination behaviour of the series of ligands is questioned in the light of the spectroscopic data and discussed in terms of protection of the cation towards water molecules and their impact on the luminescence efficiency.