ABSTRACT
This article reviews the analytical tool chest used for characterizing alkoxylates and their associated copolymer mixtures. Specific emphasis will be placed upon the use of mass spectrometry-based techniques as rapid characterization tools for optimizing reaction processes in an industrial R&D setting. An initial tutorial will cover the use of matrix-assisted laser desorption/ionization-mass spectrometry and tandem mass spectrometry fragmentation for detailed component analysis (e.g., polyol and isocyanate) of a model polyurethane-based foam. Next, this critical feedback information will be used with the guidance of mass spectrometry to initiate the development of a new, more efficient, tris(pentafluorophenyl)borane (FAB) catalyst-based alkoxylation process for generating the next generation of glycerin-initiated poly(propylene oxide)-co-poly(ethylene oxide) copolymers. Examples will be provided for each step in the FAB-based optimization process that were required to generate the final product. Following this example, two-dimensional liquid chromatography, supercritical fluid chromatography, and ion mobility separations, along with their coupling to mass spectrometry, will be reviewed for their efficiency in characterizing and quantitating the components within these complex polyether polyol mixtures.
ABSTRACT
Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.
Subject(s)
Glucuronates/pharmacology , Heparin Cofactor II/agonists , Heparitin Sulfate/pharmacology , Drug Discovery , Glucuronates/chemistry , Heparin Cofactor II/metabolism , Heparitin Sulfate/chemistry , Humans , Protein Binding , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Minimalist structures, H and I, were designed as protein interface mimics. Attributes of these chemotypes are (i) greater rigidity than conventional peptides, (ii) chiral and nonplanar heterocyclic backbones that are less prone to the hydrophobic aggregation effects, and (iii) potential to be prepared with a variety of side chains corresponding to natural amino acids. Intermediates, however, in the oligo(pyrrolidinone-piperidine)s H syntheses were vulnerable to epimerization. The origins of this epimerization were determined, then the study was focused on oligo(piperidinone-piperidine) compounds I. Mimics I were prepared via iterative couplings; a penta(piperidinone-piperidine) was prepared in this way. A series of lower homologues of this pentamer were crystallized and studied (single crystal X-ray), and four of them were used in a circular dichroism (CD) study. Thus, an estimate of 36 Å for the N-to-C distance of a typical conformation of the penta(piperidinone-piperidine) was made. CD spectra of four progressively longer oligomers allowed assignment of elipticity changes around 300 nm that can be attributed to increased conformational ordering of the longer oligomers in solution.
Subject(s)
Piperidines/chemistry , Molecular Structure , Piperidines/chemical synthesis , Protein BindingABSTRACT
Oligo-pyrrolinone-pyrrolidines (generic structure 1) have the potential to interfere with protein-protein interactions (PPIs), but to reduce this to practice it is necessary to be able to synthesize these structures with a variety of different side chains corresponding to genetically encoded proteins. This paper describes expansion of the synthetic scope of 1, the difficulties encountered in this process, particularly issues with epimerization and slow coupling rates, and methods to overcome them. Finally, spectroscopic and physicochemical properties as well as proteolytic stabilities of molecules in this series were measured; these data highlight the suitability of oligo-pyrrolinone-pyrrolidines for the development of pharmacological probes or pharmaceutical leads.
Subject(s)
Proteins/chemistry , Pyrrolidines/chemistry , Caco-2 Cells , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Structure , Protein Binding , Pyrrolidines/chemical synthesisABSTRACT
Small molecule probes that selectively perturb protein-protein interactions (PPIs) are pivotal to biomedical science, but their discovery is challenging. We hypothesized that conformational resemblance of semirigid scaffolds expressing amino acid side-chains to PPI-interface regions could guide this process. Consequently, a data mining algorithm was developed to sample huge numbers of PPIs to find ones that match preferred conformers of a selected semirigid scaffold. Conformations of one such chemotype (1aaa; all methyl side-chains) matched several biomedically significant PPIs, including the dimerization interface of HIV-1 protease. On the basis of these observations, four molecules 1 with side-chains corresponding to the matching HIV-1 dimerization interface regions were prepared; all four inhibited HIV-1 protease via perturbation of dimerization. These data indicate this approach may inspire design of small molecule interface probes to perturb PPIs.
Subject(s)
Fluorescent Dyes/pharmacology , HIV Protease/chemistry , Small Molecule Libraries/pharmacology , Algorithms , Dimerization , Fluorescent Dyes/chemistry , HIV Protease/metabolism , Models, Molecular , Protein Binding/drug effects , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Peptidomimetics 1-3 were prepared from amino acid-derived tetramic acids 7 as the key starting materials. Calculations show that preferred conformations of 1 can align their side-chain vectors with amino acids in common secondary structures more effectively than conformations of 3. A good fit was found for a preferred conformation of 2 (an extended derivative of 1) with a sheet/ß-turn/sheet motif.
Subject(s)
Peptidomimetics/chemistry , Pyrroles/chemistry , Pyrrolidines/chemistry , Models, Molecular , Peptidomimetics/chemical synthesis , Protein Structure, Secondary , Pyrroles/chemical synthesis , Pyrrolidines/chemical synthesisABSTRACT
Dermatan sulfate, an important member of the glycosaminoglycan family, interacts with heparin cofactor II, a member of the serpin family of proteins, to modulate antithrombotic response. Yet, the nature of this interaction remains poorly understood at a molecular level. We report the genetic algorithm-based combinatorial virtual library screening study of a natural, high-affinity dermatan sulfate hexasaccharide with heparin cofactor II. Of the 192 topologies possible for the hexasaccharide, only 16 satisfied the "high-specificity" criteria used in computational study. Of these, 13 topologies were predicted to bind in the heparin-binding site of heparin cofactor II at a â¼60° angle to helix D, a novel binding mode. This new binding geometry satisfies all known solution and mutagenesis data and supports thrombin ternary complexation through a template mechanism. The study is expected to facilitate the design of allosteric agonists of heparin cofactor II as antithrombotic agents.
ABSTRACT
Affinity CE (ACE) was used to study interactions of small, highly sulfated, aromatic molecules with antithrombin (AT). The high charge density of the small molecules induces differential migration of the complex resulting in a versatile method of assessing binding affinities, nature of interactions and site of binding on the inhibitor. Scatchard analysis of the interaction of three tetrahydroisoquinoline-based polysulfated molecules with AT results in monophasic profiles with affinities in the range of 40-60 microM in 20 mM sodium phosphate buffer, pH 7.4. For a pentasulfated molecule, a biphasic profile with affinities of 4.7 and 30 microM was observed. Measurement of K(D) as a function of ionic strength of the medium indicated that ionic and non-ionic forces contribute 2.4 and 1.9 kcal/mol, respectively, at pH 7.4 and 100 mM NaCl. Competitive binding studies showed that the tetrahydroisoquinoline-based molecules do not compete with a high-affinity heparin pentasaccharide. In contrast, the affinity of these tetrahydroisoquinoline derivatives decreases dramatically in the presence of an extended heparin-binding site ligand. Overall, ACE analysis of small, sulfated aromatic molecules interacting with AT is relatively easy and obviates the need for an external signal, e.g. fluorescence, for monitoring the interaction. In addition to affording biochemical knowledge, the small sample requirement and fast analysis time of ACE could be particularly advantageous for high-throughput screening of potential anticoagulants.
Subject(s)
Antithrombins , Binding Sites , Electrophoresis, Capillary/methods , Sulfates/metabolism , Tetrahydroisoquinolines/metabolism , Antithrombins/chemistry , Antithrombins/metabolism , Binding, Competitive , Heparin/metabolism , Kinetics , Oligosaccharides/metabolism , Phosphates/chemistryABSTRACT
Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS(5) containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS(5) activated the inhibitor nearly 30-fold, nearly 2- to 3-fold higher than our first generation flavanoid-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.
Subject(s)
Antithrombins/chemical synthesis , Drug Design , Tetrahydroisoquinolines/chemical synthesis , Antithrombins/chemistry , Antithrombins/pharmacology , Binding Sites , Factor Xa Inhibitors , Heparin/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Tetrahydroisoquinolines/chemistry , Tetrahydroisoquinolines/pharmacologyABSTRACT
Factor Xa and thrombin, two critical pro-coagulant enzymes of the clotting cascade, are the primary target of current anticoagulation research that aims to develop potent, orally bioavailable, synthetic small-molecule inhibitors. To determine structural features that might play important roles in factor Xa and thrombin recognition and oral bioavailability, quantitative structure-activity and structure-property analyses were performed on the factor Xa and thrombin inhibition data and Caco-2 cell-permeability data of 3-substituted pyrazole-5-carboxamides reported by Pinto et al. (J. Med. Chem. 2001, 44, 566). The factor Xa and thrombin inhibition potencies, and Caco-2 cell permeability of the 3-substituted pyrazole-5-carboxamides could be quantitatively described through molecular connectivity and atom level E-state indices. Different quantitative structure-activity and structure-property models were derived for each of the three biological properties. The models are statistically relevant with correlation coefficients of at least 0.9, and contain only two or three molecular descriptor variables. The study demonstrates the use of molecular connectivity and E-state indices in understanding factor Xa and thrombin inhibition. In addition, the models may be useful for predictive purposes in generating molecules with better potency, specificity, and oral bioavailability.
Subject(s)
Factor X/antagonists & inhibitors , Pyrazoles/chemistry , Thrombin/antagonists & inhibitors , Algorithms , Caco-2 Cells , Factor X/chemistry , Humans , Models, Chemical , Permeability , Pyrazoles/pharmacology , Quantitative Structure-Activity Relationship , Thrombin/chemistrySubject(s)
HIV-1/drug effects , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Lignin/chemistry , Lignin/pharmacology , Biopolymers/chemistry , Biopolymers/pharmacology , Cell Fusion , Cell Line , Dimerization , Heparitin Sulfate/chemistry , Humans , Models, Molecular , Molecular StructureABSTRACT
Sulfation of multiply hydroxylated small organic molecules is fraught with problems of poor yield, multitude of products and long reaction times. We have developed a rapid microwave-based method for synthesis of highly sulfated small organic molecules, which affords the per-sulfated product in moderate to excellent yields and high purity. The method is expected to be of value in the discovery of per-sulfated organic molecules as mimics of glycosaminoglycans, which are being increasingly recognized as modulators of key physiological functions.
ABSTRACT
Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.
Subject(s)
Blood Coagulation Factors/antagonists & inhibitors , Coumaric Acids/chemical synthesis , Coumaric Acids/pharmacology , Protease Inhibitors/chemical synthesis , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Anticoagulants/pharmacology , Blood Coagulation Tests , Combinatorial Chemistry Techniques , Coumaric Acids/chemistry , Dimerization , Drug Evaluation, Preclinical/methods , Humans , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
We describe a combinatorial virtual screening approach for predicting high specificity heparin/heparan sulfate sequences using the well-studied antithrombin-heparin interaction as a test case. Heparan sulfate hexasaccharides were simulated in the 'average backbone' conformation, wherein the inter-glycosidic bond angles were held constant at the mean of the known solution values, irrespective of their sequence. Molecular docking utilized GOLD with restrained inter-glycosidic torsions and intra-ring conformations, but flexible substituents at the 2-, 3-, and 6-positions and explicit incorporation of conformational variability of the iduronate residues. The approach reproduces the binding geometry of the sequence-specific heparin pentasaccharide to within 2.5 A. Screening of a combinatorial virtual library of 6,859 heparin hexasaccharides using a dual filter strategy, in which predicted antithrombin affinity was the first filter and self-consistency of docking was the second, resulted in only 10 sequences. Of these, nine were found to bind antithrombin in a manner identical to the natural pentasaccharide, while a novel hexasaccharide bound the inhibitor in a unique but dramatically different geometry and orientation. This work presents the first approach on combinatorial library screening for heparin/heparan sulfate GAGs to determine high specificity sequences and opens up huge opportunities to investigate numerous other physiologically relevant GAG-protein interactions.
Subject(s)
Combinatorial Chemistry Techniques , Heparin/chemistry , Heparitin Sulfate/chemistry , Models, Molecular , Antithrombins/chemistry , Carbohydrate Sequence , Databases, Factual , Molecular Sequence Data , Protein BindingABSTRACT
The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.