Synthesis of phosphine containing amino acids: utilization of peptide synthesis in ligand design

Combinatorial chemistry has recently burst on the scene as a valuable tool for the discovery of new drug candidates. The ability to synthesize hundreds of compounds for screening is a useful complement to rational drug design. There are many similarities between the design of new therapeutic agents and the development of new asymmetric ligands, the most important of which is the limitation of a rational design strategy. For this reason a program was begun that would allow the use of combinatorial technology in the development of new ligands for transition metal catalyzed asymmetric reactions. Because of the large number of catalytic reactions they are involved in the system was based around phosphine ligands. This paper reports the synthesis of phosphine derivatives of alanine, proline, and the aromatic amino acids tyrosine and hydroxyphenylglycine. Examples of the use of these amino acids in the synthesis of peptides possessing helical and beta-turn secondary structures are presented. Metal complexes of these peptide-based ligands are used in hydrogenation and alkylation reactions.     

Design and selection of ligands for affinity chromatography

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.     

Design and DNA-binding of metallo-supramolecular cylinders conjugated to peptides

Di-nuclear metallo-supramolecular “cylinders”, based on bis-pyridylimine ligands, are end-functionalised with short peptides. The design and synthesis of one tetra-cationic triple-stranded (iron(II)) and three di-cationic double-stranded (copper(I) or silver(I)) cylinder-peptide conjugates are described. DNA-binding experiments, using circular and linear dichroism spectroscopies, confirm the binding and indicate that the iron(II) complexes cause DNA to bend or coil. Artificial nuclease activity by the copper(I) complexes is demonstrated by gel electrophoresis studies.     

Recent advances in the synthesis of endocannabinoid related ligands

The chemical strategies used for the synthesis of various ligands related to the endocannabinoid system namely anandamide (AEA), 2-arachidonylglycerol (2-Ara-Gl), CB1/(vanilloid receptors) VR1, anandamide membrane transporter (AMT) and fatty acid amide hydrolase (FAAH) are described in this review. In general, the chemical synthesis of analogs with changes in the head group of AEA was quite straightforward involving the conversion of an acid to an amide or an ester. Analogs which had modifications in the end pentyl chain were more difficult to synthesize and required multistep synthetic sequences to prepare the target compounds. A facile total synthesis of 2-Ara-Gl was reported and an HPLC procedure for its identification and quantification was developed, but because of the instability of 2-Ara-Gl another synthesis was developed so that it can be stored as the more stable phenylboronate ester. Similarly the chemical synthesis of various ligands in the remaining areas of CB1/VR1, AMT and FAAH are described. A summary of the present state of knowledge about the SAR in each area is presented to help in the design and synthesis of novel ligands for the future.     

The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E- selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT- VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.     

Free energy calculations on the binding of novel thiolactomycin derivatives to E. coli fatty acid synthase I

Finding novel antibiotics to combat the rise of drug resistance in harmful bacteria is of enormous importance for human health. Computational drug design can be employed to aid synthetic chemists in the search for new potent inhibitors. In recent years, molecular dynamics based free energy calculations have emerged as a useful tool to accurately calculate receptor binding affinities of novel or modified ligands. While being significantly more demanding in computational resources than simpler docking algorithms, they can be employed to obtain reliable estimates of the effect individual functional groups have on protein-ligand complex binding constants. Beta-ketoacyl [acyl carrier protein] synthase I, KAS I, facilitates a critical chain elongation step in the fatty acid synthesis pathway. Since the bacterial type II lipid synthesis system is fundamentally different from the mammalian type I multi-enzyme complex, this enzyme represents a promising target for the design of specific antibiotics. In this work, we study the binding of several recently synthesized derivatives of the natural KAS I inhibitor thiolactomycin in detail based on atomistic modeling. From extensive thermodynamic integration calculations the effect of changing functional groups on the thiolactone scaffold was determined. Four ligand modifications were predicted to show improved binding to the E. coli enzyme, pointing the way towards the design of thiolactomycin derivatives with binding constants in the nanomolar range.     

Design and synthesis of 6-methyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid derivatives as PPARgamma activators

The design and synthesis of novel series of 6-methyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid (pyrimidone) derivatives that are high affinity ligands for peroxisome proliferators activated receptor gamma have been reported as a potential substitute of 2,4-thiazolidinedione head group. The FlexX docking and radioligand binding affinity of some promising compounds of this series is comparable to that of thiazolidinedione based antidiabetic drugs currently in clinical use.     

Theoretical study of tryptophan operon: Application in microbial technology

A model for the tryptophan operon is formulated based on the genetic and biophysical data available on the structure of the operon and the nature of interactions between the represser and its ligands. Studies have been done, on wild-type, superrepressing, and loose-binding strains to identify conditions at which the stability of the system changes (i.e., evolves to a stable synthesis or periodic synthesis with increasing amplitude). Also, the factors that increase the yield of tryptophan are studied and predictions made, based on the results, for obtaining overproducing strains of tryptophan that can be used for the industrial production of this useful amino acid.     

Antibodies and genetically engineered related molecules: production and purification

Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification.     

Combinatorial Chemistry of Nucleic Acids: SELEX

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40–100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.     

Convenient synthesis of maleimido-derivatized lanthanide(III) chelates and their use in mercapto group conjugation

Simple synthesis of luminescent europium(III) and terbium(III) chelates tethered to a maleimido function (7, 8) is described. The method is based on the following: (i) synthesis of protected ligands tethered to a maleimido function and their purification on silica gel; (ii) deprotection by acidolysis; (iii) conversion of the deprotected ligands to the corresponding lanthanide(III) chelates by passing them through a column of strong cation exchange resin loaded with the appropriate lanthanide(III) ions. According to this procedure, large quantities of mercapto-selective biomolecule-labeling reactants of high purity can be prepared.     

Rational design,synthesis, and verification of affinity ligands to a protein surface cleft

The structure-based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas alpha-amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty-three compounds were selected for synthesis and screened in solution for binding toward alpha-amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand-coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.     

Cannabinoid receptors in atherosclerosis

PURPOSE OF REVIEW: Recent findings suggesting that cannabinoid receptors are potential targets for the treatment of atherosclerosis are reviewed. RECENT FINDINGS: Cannabinoids, such as Delta9-tetrahydrocannabinol, the major psychoactive compound of marijuana, their synthetic analogs and endogenous cannabinoid ligands, produce their biological effects by interacting with specific receptors. In the apolipoprotein E knockout mouse model of atherosclerosis, Delta9-tetrahydrocannabinol was shown to inhibit disease progression through pleiotropic effects on inflammatory cells. Blocking of cannabinoid receptor CB2, the main cannabinoid receptor expressed on immune cells, abolished the observed effects. The development of novel cannabinoid receptor ligands that selectively target CB2 receptors or pharmacological modulation of the endocannabinoid system might offer novel therapeutic strategies in the treatment of atherosclerosis. Several reports demonstrating an implication of the endocannabinoid system in different inflammatory conditions support this hypothesis. SUMMARY: The immunomodulatory capacity of cannabinoids is now well established and suggests a broad therapeutic potential of cannabinoids for a variety of conditions, including atherosclerosis. New strategies based on nonpsychotropic cannabinoid receptor ligands or compounds modulating endocannabinoid synthesis or stability might solve the problem of the unwanted side effects associated with cannabinoid administration.     

A new method to detect related function among proteins independent of sequence and fold homology

A new method has been developed to detect functional relationships among proteins independent of a given sequence or fold homology. It is based on the idea that protein function is intimately related to the recognition and subsequent response to the binding of a substrate or an endogenous ligand in a well-characterized binding pocket. Thus, recognition of similar ligands, supposedly linked to similar function, requires conserved recognition features exposed in terms of common physicochemical interaction properties via the functional groups of the residues flanking a particular binding cavity. Following a technique commonly used in the comparison of small molecule ligands, generic pseudocenters coding for possible interaction properties were assigned for a large sample set of cavities extracted from the entire PDB and stored in the database Cavbase. Using a particular query cavity a series of related cavities of decreasing similarity is detected based on a clique detection algorithm. The detected similarity is ranked according to property-based surface patches shared in common by the different clique solutions. The approach either retrieves protein cavities accommodating the same (e.g. co-factors) or closely related ligands or it extracts proteins exhibiting similar function in terms of a related catalytic mechanism. Finally the new method has strong potential to suggest alternative molecular skeletons in de novo design. The retrieval of molecular building blocks accommodated in a particular sub-pocket that shares similarity with the pocket in a protein studied by drug design can inspire the discovery of novel ligands.     

Discovery of thioazepinone ligands for Src SH2: from non-specific to specific binding

The structure-based design and synthesis of new thioazepinones as ligands for Src SH2 protein is presented. From benzothioazepinones, ligands with somewhat unspecific binding properties, simpler thioazepinones were designed, the best ones demonstrated nanomolar affinity for Src SH2. A few of these new ligands were crystallized with the protein and demonstrated a specific binding mode with the protein.     

Synthesis of paramagnetic ligands that target the C-terminal binding site of Hsp90

Identification of a ligand binding site represents the starting point for a structure-based drug development program. Lack of a binding site hampers the development of improved ligands that modulate the protein of interest. In this letter, we describe the development of chemical tools that will allow for elucidation of the Hsp90 C-terminal ligand binding site. Our strategy is based on the preparation of paramagnetic analogs of KU-596, an investigational new drug that is currently undergoing clinical trials for the treatment of neuropathy and interacts with the Hsp90 C-terminal domain. In particular, we report the design and synthesis of three novel paramagnetic analogs of KU-596, which will be used to obtain long range distances for NMR structural studies of Hsp90 in complex with C-terminal ligands.     

Selectins and glycosyltransferases in leukocyte rolling in vivo

Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified - P-, E- and L-selectin - which recognize and bind to crucial carbohydrate determinants on selectin ligands. Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo conditions. Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level. However, high endothelial venule-expressed selectin ligands were instrumental in uncovering the crucial role of post-translational modifications for selectin ligand activity. Several glycosyltransferases, such as core 2 beta1,6-N-acetylglucosaminyltransferase-I, beta1,4-galactosyltransferases, alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins. In addition, modifications, such as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands.     

Metal ligands in micronutrient acquisition and homeostasis

Acquisition and homeostasis of micronutrients such as iron (Fe) and zinc (Zn) pose specific challenges. Poor solubility and high reactivity require controlled synthesis and supply of ligands to complex these metals extracellularly and intracellularly. Cytosolic labile pools represent only a minute fraction of the total cellular content. Several low‐molecular‐weight ligands are known in plants, including sulfur ligands (cysteine and peptides), nitrogen/oxygen ligands (S‐adenosyl‐l ‐methionine‐derived molecules and histidine), and oxygen ligands (phenolics and organic acids). Some ligands are secreted into the extracellular space and influence the phytoavailability of metal ions. A second principal function is the intracellular buffering of micronutrients as well as the facilitation of long‐distance transport in xylem and phloem. Furthermore, low‐molecular‐weight ligands are involved in the storage of metals, predominantly in vacuoles. A detailed molecular understanding is hampered by technical limitations, in particular the difficulty to detect and quantify cellular metal–ligand complexes. More, but still too little, is known about ligand synthesis and the transport across membranes, either with or without a complexed metal. Metal ligands have an immediate impact on human well‐being. Engineering metal ligand synthesis and distribution in crops has tremendous potential to improve the nutritional quality of food and to tackle major human health risks.