Molecularly imprinted polymer catalysis of a Diels-Alder reaction

A series of synthetic polymers were designed and synthesized for enhancing the rate of the Diels-Alder cycloaddition reaction of 1,3-butadiene carbamic acid benzyl ester (1) and N,N-dimethyl acrylamide (2), to yield the corresponding endo- (3) and exo- (4) reaction products. Putative transition state analogues (TSAs) for the endo- (5) and exo- (6) reaction pathways were used as templates for the synthesis of molecularly imprinted methacrylic acid (MAA)–divinylbenzene (DVB) copolymers. The polymer system utilized was selected based upon a series of ¹H NMR studies of complex formation between template and a functional monomer analogue (K_d (app) ≈ 70 mM, d₈-toluene, 293 K). Batch binding studies revealed that the imprinted polymers were selective for the TSA corresponding to the template used in the polymer synthesis. Studies on the influence of the polymers on the catalysis of the reaction of 1 and 2 demonstrated a 20-fold enhancement of the rate of the reaction relative to the solution reaction. A surprising temperature dependence of the reaction of 1 and 2 in the presence of the polymers was observed, which provides support for the role of template-functional monomer complexes in the catalysis of the Diels-Alder reaction.     

72/74As-labeling of HPMA based polymers for long-term in vivo PET imaging

In the context of molecular imaging, various polymers based on the clinically approved N-(2-hydroxypropyl)-methacrylamide (HPMA) have been radio-labeled using longer-living positron emitters ⁷²As t_1/2 = 26 h or ⁷⁴As t_1/2 = 17.8 d. This approach may lead to non-invasive determination of the long-term in vivo fate of polymers by PET (positron emission tomography). Presumably, the radio label itself will not strongly influence the polymer structure due to the fact that the used nuclide binds to already existing thiol moieties within the polymer structure. Thus, the use of additional charges or bulky groups can be avoided.     

Synthesis,crystal structure and fluorescent characterization of a novel lanthanide tetraphosphonate with a layered structure

Hydrothermal reactions of Gd(III) nitrate with N,N,N′,N′-tetramethylenephosphono-1,4-diaminobutane, (H₂O₃PCH₂)₂N–(CH₂)₄–N(CH₂PO₃H₂)₂ (H₈L), afforded a novel Gd(III) phosphonate, namely, Gd[(O₃PCH₂)(HO₃PCH₂)N(H)(CH₂)₄N(H)(CH₂PO₃H)₂] · 2H₂O, [Gd(H₅L)] · 2H₂O. Its structure was established by a single-crystal X-ray diffraction study. In this compound, the Gd(III) ion is coordinated by eight phoshonate oxygen atoms from five different phosphonate groups, which belong to five different phosphonic ligands. Each Gd atom is connected with its neighboring Gd atoms by two phosphonate oxygens, forming a gadolinium phosphonate slab along the a-axis. Such slabs are bridged by tetraphosphonate H₅L anions, resulting in a 〈0 1 1〉 layer with the butane groups toward the interlayer space. These layers are further interlinked by strong hydrogen bonds formed by uncoordinated phosphonate oxygens into a 3D supermolecular structure. Luminescent studies indicate that this compound exhibits a broad blue fluorescent emission band at 441 nm.     

Interactions of bovine serum albumin with biological buffers,TES, TAPS,and TAPSO in aqueous solutions

The thermal stability of bovine serum albumin (BSA) in the aqueous solutions containing the biological buffers, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), N-[tris(hydroxylmethyl)methyl]-3-aminopropanesulfonic acid (TAPS), and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), was studied by using dynamic light scattering (DLS) at various temperatures and concentration ranges of buffers. It is found that the increase of the buffer concentration enhanced the thermal stability of protein BSA, and the stabilization tendency follows the order of TAPSO > TES ≈ TAPS. In this study, we have also investigated the interactions of BSA with TES, TAPS, and TAPSO by using various techniques, such as UV–vis absorption, fluorescence, and molecular docking. It is revealed that the main interactions between the studied buffers and the peptide moieties of proteins are electrostatic including hydrogen bonds. The results obtained from this series of studies confirmed that the biological buffers, TES, TAPS, and TAPSO can serve as good stabilizers for the globular protein BSA, in the aqueous solutions.     

Cyanoguanidine-based lactam derivatives as a novel class of orally bioavailable factor Xa inhibitors

The N,N′-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K_i = 6.5 nM, EC_2xPT = 32 μM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand–protein interactions are discussed.     

Small molecule antagonists of the gonadotropin-releasing hormone (GnRH) receptor: Structure–activity relationships of small heterocyclic groups appended to the 2-phenyl-4-piperazinyl-benzimidazole template

A previous report described the serum LH suppression pharmacology of the 2-phenyl-4-piperazinyl-benzimidazole N-ethyluracil GnRH receptor antagonist 1 following oral administration in rats. A series of small heterocycles were appended to the 2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole template in place of the N-ethyluracil. Two imidazole analogues, 32 and 41, were shown to possess substantial in vitro potency at the target receptor (hGnRH IC₅₀ = 7 and 18 nM, respectively) and aqueous solubility (55 and 100 μg/mL at pH 7.4, respectively). Both compounds had high oral bioavailability in rats and 32 was further examined in an orchidectomized rat model for serum LH suppression based on increased volume of distribution over 41. Serum LH levels trended lower in orchidectomized rats following oral administration of 32.     

The role of phosphate in the action of thymidine phosphorylase inhibitors: Implications for the catalytic mechanism

The design and synthesis of 5-fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU), a potent inhibitor of thymidine phosphorylase (TP) with K_i-values of 11 nM (ecTP) and 17 nM (hTP), are described. Kinetic studies established that the type of inhibition of TP by AIFU is uncompetitive with respect to inorganic phosphate (or arsenate). The results obtained suggest that AIFU and other zwitterionic thymine analog inhibitors of TP act as transition state analogs, mimicking the anionic thymine leaving group, consistent with an S_N2-type catalytic mechanism, and anchored by their protonated side chains to the enzyme-bound phosphate by electrostatic and H-bonding interactions.     

A novel side-bridged hybrid phosphonate/acetate pendant cyclam: Synthesis,characterization, and 64Cu small animal PET imaging

Copper-64 (t_1/2 = 12.7 h; β⁺: 0.653 MeV, 17.4%; β^?: 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik–Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with ⁶⁴Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the ⁶⁴Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in ⁶⁴Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.     

Methyl-palladium(II) complexes of pyridine-bridged bis(nucleophilic heterocyclic carbene) ligands: Substituent effects on structure,stability, and catalytic performance

A series of discrete, mononuclear palladium(II)–methyl complexes, together with several palladium(II)–chloro analogues, of pyridine-functionalised bis-NHC ligands have been prepared via ligand transmetallation from the silver(I)-NHC complexes. The reported complexes comprise examples with both the methylene-bridged 2,6-bis[(3-R-imidazolin-2-yliden-1-yl)methyl]pyridine (_RC^∧N^∧C; R = Mes, dipp, ^tBu) and planar 2,6-bis(3-R-imidazolin-2-yliden-1-yl)pyridine (_RCNC; R = Mes, dipp) ligands and, when combined with the previously reported _MeC^∧N^∧C/_MeCNC examples, cover a broad spectrum of ligand substituent steric and electronic properties, including the bulky Mes and dipp groups frequently used in catalytic applications. The palladium(II) complexes have been characterised by a variety of methods, including single crystal X-ray crystallography, with the shielding of the Pd–Me groups in the proton NMR spectra of some of the N-aryl substituted examples correlated with the proximity of the aryl rings to the methyl group in the solid state structures. The [PdMe(_RC^∧N^∧C/_RCNC)]⁺ complexes undergo thermal degradation via reductive methyl-NHC coupling to give 2-methyl-3-R-imidazolium-1-yl species with relative stabilities in the order of [PdMe(_MesC^∧N^∧C)]BF₄ > [PdMe(_MeC^∧N^∧C)]BF₄ ≈ [PdMe(_MesCNC)]BF₄ > [PdMe(_MeCNC)]BF₄ > [PdMe(_tBuC^∧N^∧C)]BF₄ ≫ [PdMe(_tBuCNC)]BF₄ (not isolable). A comparison of the activity of the complexes as precatalysts in a model Heck coupling reaction shows greatest activity in those species bearing bulkier N-substituents, with complexes bearing _RC^∧N^∧C ligands generally more efficient precatalysts than those bearing _RCNC ligands.     

Anthranilate phosphoribosyltransferase: Binding determinants for 5′-phospho-alpha-d-ribosyl-1′-pyrophosphate (PRPP) and the implications for inhibitor design

Phosphoribosyltransferases (PRTs) bind 5′-phospho-α-d-ribosyl-1′-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a “bianthranilate”-like moiety are inhibitors with IC₅₀ values of 10–60 μM, and K_i values of 1.3–15 μM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops.     

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

BackgroundAnalysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.MethodsThe method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (¹³C₃-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n = 6), brain tumour (n = 2), leukaemia (n = 5), and Salla disease (n = 1).ResultsLimit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n = 10) and 10.4% (n = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n = 10) and 12.8% (n = 40) respectively. Tested patients showed values of TSA above established reference value.ConclusionThe validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.     

Design,synthesis and anti-P. falciparum activity of pyrazolopyridine–sulfonamide derivatives

Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to benzenesulfonamide moieties with different substituents in the 4-position were synthesized and assayed against Plasmodium falciparum. These ten compounds exhibited activity in vitro against the chloroquine-resistant clone W2 with IC₅₀ values ranging from 3.46 to 9.30 μM. The most active derivatives with substituent R₂ = Cl or CH₃ at the benzenesulfonamide moiety exhibited the lowest IC₅₀. Compounds with an R₁ = CO₂Et substituent at the 5-position of the 1H-pyrazolo[3,4-b]pyridine ring presented lower activity than those with a CN substituent. The 1H-pyrazolo[3,4-b]pyridine system appears to be promising for further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.     

Multifunctional hydrolytic catalysis: VI. Catalytic hydrolysis of p-nitrophenyl acetate by N-(4-imidazolylmethyl)benzohydroxamic acid

A bifunctional catalyst, N-(4-imidazolylmethyl)benzohydroxamic acid, was synthesized from benzohydroxamic acid and chloromethylimidazole, and used for the hydrolysis of p-nitrophenyl acetate. The reaction proceeded via the formation of the acetyl hydroxamate and its subsequent decomposition. The deacylation step was shown to be general base-catalyzed by the intramolecular imidazole group on the basis of the deuterium solvent kinetic isotope effect of 2.0. The efficiency of water attack on the acetyl hydroxamate was enhanced 130-fold by the imidazole group. The catalytic process is compared with the reactions of related monofunctional compounds, and finally its significance as a model of the charge relay system is discussed.     

Synthesis and structure–activity relationships of 4-fluorophenyl-imidazole p38α MAPK,CK1δ and JAK2 kinase inhibitors

The synthesis and structure–activity relationships of novel 4-(4′-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via ‘click’ reactions. The kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited p38α MAPK with IC₅₀ = 250 nM and 96 nM, respectively. Pyridine 3 gave CK1δ inhibition with IC₅₀ = 89 nM and pyridin-2-one 31 gave JAK2 inhibition with IC₅₀ = 62 nM.     

2-Dialkynyl derivatives of (N)-methanocarba nucleosides: ‘Clickable’ A3 adenosine receptor-selective agonists

We modified a series of (N)-methanocarba nucleoside 5′-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A₃ adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A₃AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A₃AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (K_i 6.5 nM), although some of the click products had K_i values in the range of 200–400 nM. Other potent, selective derivatives (K_i at A₃AR in nM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), α-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A₃AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A₃AR agonists to reporter groups of diverse structure and to carrier moieties.     

Structural and diffusion properties of formamide/water mixture interacting with TiO2 surface

The photoreaction and adsorption properties on surfaces, thermal decomposition, chemical transformation, and other properties of the formamide molecule are widely used to understand the origins of the formation of biological molecules (nucleosides, amino acids, DNA, monolayers, etc.) needed for life. The titanium oxide (TiO₂) surface can act both as a template on which the accumulation of adsorbed molecules like formamide occurs through the concentration effect, and as a catalytic material that lowers the activation energy needed for the formation of intermediate products. In this paper, a formamide–water solution interacting with TiO₂ (anatase) surface is simulated using the molecular dynamics method. The structural, diffusion and density properties of formamide–water mixture on TiO₂ are established for a wide temperature range from T = 250 K up to T = 400 K.     

Neocarzinostatin-based hybrid biocatalysts with a RNase like activity

A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N′(17′-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called ‘Trojan horse’ strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (k_cat/K_M = 13.6 M⁻¹ s⁻¹ at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the ‘Trojan horse’ strategy for obtaining better catalysts of selective reactions will be further studied.     

Indene-based frameworks targeting the 5-HT6 serotonin receptor: Ring constraint in indenylsulfonamides using cyclic amines and structurally abbreviated counterparts

Further studies in quest of 5-HT₆ serotonin receptor ligands led to the design and synthesis of a few selected examples of N-(inden-5-yl)sulfonamides with a ring-constrained aminoethyl side chain at the indene 3-position, some of which exhibited a high binding affinity, such as the pyrrolidine analogue 28 (K_i = 3 nM). Moreover, the structurally abbreviated N-(inden-5-yl)sulfonamides showed K_i values ?43 nM, which indicates that neither the N,N-aminoethyl nor the conformationally restricted aminoethyl side arm at the indene 3-position are required for binding. Selected compounds were then tested in a functional cAMP stimulation assay and found to act as 5-HT₆ antagonists, although with moderate potency at the micromolar level.     

α-Chymotrypsin stabilization by chemical conjugation with O-carboxymethyl-poly-β-cyclodextrin

Bovine pancreatic α-chymotrypsin was covalently modified with the O-carboxymethyl poly-β-cyclodextrin (M=1.3×10⁴, 40% COOH groups) using a water-soluble carbodiimide as a coupling agent. The conjugate prepared by this procedure retained high proteolytic and esterolytic activity and contained about 74% carbohydrate by weight of transformed protein. The optimum temperature for α-chymotrypsin was increased by 5 °C after this transformation. The thermostability of the polymer–enzyme adduct was also increased by 5 °C. The conjugate prepared was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 55 °C. Additionally, the modified enzyme was 11-fold more stable at pH 9.0. The direct influence of supramolecular interactions between the hydrophobic amino acid residues located at the surface of the protease and the attached polycyclodextrin moieties on α-chymotrypsin stabilization was demonstrated.     

Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO₂ hydration: PfCAdom showed a k_cat of 3.8 × 10⁵ s⁻¹ and k_cat/K_m of 7.2 × 10⁷ M⁻¹ × s⁻¹, whereas PfCA showed a lower activity compared to PfCAdom, with a k_cat of 1.4 × 10⁵ s⁻¹ and k_cat/K_m of 5.4 × 10⁶ M⁻¹ × s⁻¹. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed K_Is in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with K_Is in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.