Asymmetric synthesis of N-protected chiral 1-aminoalkylphosphonic acids and synthesis of side chain-functionalized depsiphosphonopeptides.

Optically active 1-aminoalkylphosphonic acid derivatives were synthesized in moderate yields and optical purities via Mannich-type reactions of benzyl carbamate, aldehydes, and optically pure chlorophosphites. Side chain-functionalized depsiphosphonopeptides were also prepared in satisfactory yields directly from one-pot reactions of benzyl carbamate, aldehydes, and diethyl (R,R)-2-chloro-1,3,2-dioxaphospholane-4,5-dicarboxylate.     

Inhibition of acetylcholinesterase by derivatives of 1,3,2-dioxaphosphorinane 2-oxide.

Cholinesterases are inhibited by 2-fluoro-1,3,2-dioxaphosphorinane 2-oxide by a mechanism that involves a slow association step followed by a very slow phosphorylation step. No phosphorylation step was observed for the interaction between acetylcholinesterase and 2-S-[2'-(NN-diethylamino)ethyl]thio-1,3,2-dioxaphosphorinane 2-oxide.     

Synthesis of Some Nucleoside Cyclic Ethylene Phosphothionates

Abstract

This communication describes the synthesis of 5′-deoxy-5′-chloro-3′-(2-thio-1,3,2-dioxaphosphorinanyl)thymidine, N⁴,2′,3′-triacetyl-5′-(2-thio-1,3,2-dioxaphosphorinanyl)-1-β-D-arabinosyl-cytosine and N⁴-acetyl-5′-(2-thio-1,3,2-dioxaphosphorinanyl)-1-β-D-arabinosylcytosine.     

Formation of an unstable covalent intermediate during the inhibition of electric-eel acetylcholinesterase with 1,3,2-dioxaphosphorinane 2-oxides.

The kinetics of interaction of eel acetylcholinesterase (EC 3.1.1.7) with 1,3,2-dioxaphosphorinane 2-oxides were investigated. It was demonstrated that the rate of spontaneous re-activation as well as the re-activation profile in the presence of 2-pyridine aldoxime methiodide of the inhibited enzyme are irrespective of the leaving group of three inhibitors and exhibit the same values. The dissociation constant of the corresponding Michaelis complex was evaluated by two independent methods and the results were found to be in close agreement. It was shown that the active site is essential for interaction between the enzyme and the various dioxaphosphorinanes. The mixed anhydride of diethyl phosphoric acid and 2-hydroxy-1,3,2-dioxaphosphorinane 2-oxide behaves exactly as would be predicted from a typical diethyl phosphate inhibitor. Enxyme that was incubated with the cyclic acid or the corresponding methyl ester recovered immediately upon extensive dilution. Inhibition of enzyme in the presence of high concentratasions of the corresponding 2-chloro and 2-fluoro derivatives decreased the regeneration rates as well as the maximal amount of the re-activated enzyme. This observation could not be explained in terms of a classical aging process. On the basis of the kinetics observations it is suggested that an unstable covalent phospho-enzyme intermediate is formed during the reaction between acetylcholinesterase and 1,3,2-dioxaphosphorinane 2-oxides.     

Synthesis and characterization of amphiphilic polyphosphates with hydrophilic graft chains and cholesteryl groups as nanocarriers

Amphiphilic polyphosphate graft copolymers with varied densities of cholesteryl esters and hydrophilic graft chains were prepared, and the solution properties of the graft copolymers were evaluated. Polyphosphates were synthesized as backbones by ring-opening polymerization of 2-isopropyl-2-oxo-1,3,2-dioxaphospholane (IPP), 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl-2-bromoisobutyrate) (OPBB), and 2-choresteryl-2-oxo-1,3,2-dioxaphospholane (ChOP) using triisobutylaluminum as an initiator. Three types of polyphosphates (PIBr(x)Ch(y), x = number of OPBB units in a polymer; y = number of ChOP units in a polymer) such as PIBr4, PIBr6Ch1, and PIBr3Ch2 were obtained. The molecular weights of these polymers were 2.4 x 10(4), 2.4 x 10(4), and 2.6 x 10(4) g/mol, respectively. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was grafted from the OPBB sites in PIBr(x)Ch(y) via atom transfer radical polymerization (ATRP) in EtOH. In each polymer system, the molecular weight of the graft polymer was linear with conversion. Furthermore, the polymer radical concentration remained constant during polymerization; that is, the molecular weights of the graft chains were easily controllable with polymerization time. The solution properties of amphiphilic PIBr(x)Ch(y)-g-PMPCs were investigated by the methods of surface tension measurement, light scattering, and fluorescence probe. The transition point (cmc) of the surface tension of the PIBr(x)Ch(y)-g-PMPCs aqueous solution decreased with an increase in the number of ChOP units in a graft polymer. Particularly, PIBr3Ch2-g-PMPC14.9K formed nanosized associates (R(h) = 7.5 nm) with 2.2 molecules above 0.1 wt %. v79 cells were used to evaluate the cytotoxicity of the graft polymers, but no cytotoxicity was observed. The graft polymers containing cholesteryl groups effectively enhanced the solubility of paclitaxel in an aqueous solution.     

New androst-4-en-17-spiro-1,3,2-oxathiaphospholanes. Synthesis, assignment of absolute configuration and in vitro cytotoxic and antimicrobial activities

The reactions of 17α-hydroxyprogesterone with Lawesson's reagent (LR) in toluene, CH(2)Cl(2) and/or CCl(4) gave, depending on the duration of the reaction, two diastereoisomeric androst-4-en-17-spiro-1,3,2-oxathiaphospholane-2-sulfide pairs 2a,b and 3a,b in approximately 7:3 ratio, differing in configuration at the phosphorus atom. A parallel analysis of heteronuclear 2D (1)H-(13)C spectra (HSQC and HMBC) and homonuclear 2D spectra (NOESY) enabled complete (1)H and (13)C assignments of each isomer. Also, analysis of NOESY correlations provided evidence for the preferred conformation. X-ray analysis of 3a confirmed the structure and absolute configuration on phosphorus. A pathway for the formation of 1,3,2-oxathiaphospholane ring was proposed. Cytotoxic activity in vitro was tested against three tumor cell lines (human cervix carcinoma HeLa cells and two human breast carcinoma MDA-MB-361 and MDA-MB-453 cells). Compound 3a and mixture 3a,b showed a moderate activity against HeLa and MDA-MB-453 cell lines while against MDA-MB-361 cell line all tested compounds exerted very weak cytotoxic effect. Antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal cells, toxicity to brine shrimp Artemia salina, were evaluated. All tested compounds showed strong antifungal activity.     

A study of the reaction of 2-phenyl-1,3,2-benzodiazaborolidine with benzoyl chloride

Reaction of 2-phenyl-1,3,2-benzodiazaborolidine (2) with benzoyl chloride afforded N,N′-dibenzoyl-o-phenylenediamine (8) regardless of the amount of the acid chloride employed. Depending on the work-up procedure, the 2-phenylboron moiety was isolated as either phenylboronic acid or phenylboronic acid anhydride. In the absence of added bases, the reaction was observed to be susceptible to solvent assistance by ethers, presumably by coordination with the empty p-orbital of boron. In the presence of added amines, the extent of benzoylation was altered considerably. With pyridine as a co-reagent, small amounts of 1,3-dibenzoyl-2-phenyl-1,3,2,-benzodiazaborolidine mono- or dihydrocholoride were isolated though this compound readily underwent hydrolysis or oxidation upon standing. Interaction of 2 with sodium hydride in tetrahydrofuran caused ionization of only one of the two hydrogens bound to nitrogen even upon extended reflux. Benzoylation of the heterocycle under these latter conditions again afforded only the dibenzoylated compound 8.     

Mechanism of action of 2-aryloxy-2-thio-1,3,2-oxazaphosphorinane pesticides

The interaction of 2-aryloxy-2-thio-1,3,2-oxazaphosphorinanes exhibiting nematocide, insecticide/acaricide, and synergetic activities with monoamine oxidases and the interaction of the corresponding oxones, 2-aryloxy-2-oxo-1,3,2-oxazaphosphorinanes, with various cholinesterases, carboxyl esterases, and monoamine oxidases were studied. We showed that the thioderivatives inhibited monoamine oxidases, whereas oxones, which are, as a rule, weak cholinesterase inhibitors, strongly inhibited carboxyl esterases of the American cockroach and were transformed with monoamine oxidases into the strong cholinesterase inhibitors, acyclic phosphamidates. This allowed us to explain the low toxicity of the thioderivatives, the high toxicity of the oxoderivatives, and the great difference in toxicities of thio- and oxocompounds in the 1,3,2-oxazaphosphorinane series. The capacity of thioderivatives to inhibit monoamine oxidases and of oxoderivatives and their further activation products to inhibit carboxyl esterases, i.e., both enzymes responsible for pyrethroid detoxication in insects, explains the synergetic activity of the 1,3,2-oxazaphosphorinane series.     

8-Phosphorus substituted isosteres of purine and deazapurines.

Synthesis of 8-phosphorus substituted isosteres of purine [pyrimidino (4,5-d)-1,3,2-diazaphosphole], 1-deazapurine [pyridino (2,3-d)-1,3,2-diazaphosphole] and 3-deazapurine [pyridino (4,5-d)-1,3,2-diazaphosphole] has been achieved by the reaction of equimolar amounts of triphenylphosphite and 4,5-diaminopyrimidine, 2,3-diaminopyridine and 3,4-diaminopyridine, respectively. These compounds hydrolyzed (cleavage of the phosphorus-nitrogen bounds) in aqueous solutions to provide the corresponding diaminopyrimidine or diaminopyridines. These three new basic ring systems constitute the first reported synthesis of purines in which ring carbon atom is substituted with a phosphorus atom. 8-Phosphorus substituted purine at a concentration of 4 X 10(-4)M caused a 50% inhibition in the growth of leukemia L1210 cells in culture. The biochemical rationale for the synthesis of these compounds is discussed.     

[Z]-4-alkylidene-1,3,2-benzodioxaphosphorinane 2-oxides from stereospecific cyclization of 2-alkylketophenyl phosphonates and phosphates

O,O-Di(2-alkylketophenyl) phenylphosphonates and O-(2-alkylketophenyl) O-O-diphenyl phosphates undergo facile cyclization at 70°C in acetonitrile containing K₂CO₃ to 2-phenyl- and 2-phenoxy-4-alkylidene-1,3,2-benzodioxaphosphorinane 2-oxides, respectively. The Z isomer is exclusively formed with higher alkylidene derivatives. Metabolically formed 4-alkylidene- and 4-methyl-1,3,2-benzodioxaphosphorinane 2-oxides may contribute to the biological activity of some 2-ethylphenylphosphorus compounds.     

First and unexpected synthesis of macrocyclic cyclophane-based unusual alpha-amino acid derivatives by phosphazene base without high dilution conditions

First synthesis of a macrocylic cyclophane-based unusual alpha-amino acid derivative 11 by coupling of ethyl isocyanoacetate with 1,2-bis(4-bromomethylphenyl)ethane under phase-transfer catalysis (PTC) conditions. Phosphazene base such as 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) is useful to improve the yield of cyclophane derivative without high dilution conditions.     

Design, synthesis and biological study of pinacolyl boronate-substituted stilbenes as novel lipogenic inhibitors

A pilot library of novel 4,4,5,5-tetramethyl-2-(4-substitutedstyrylphenyl)-1,3,2 dioxaborolane derivatives has been synthesized. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphenylphosphonium bromide 3 was treated with various aldehydes in the presence of 3 equiv of (t)BuONa in DMF, and stirred at room temperature for 4-6h to yield the corresponding boron-containing stilbene derivatives in 71-94% yields. Several of them, including BF102 and BF175, have the lipogenesis inhibitory effect by suppressing lipogenic gene expression in mammalian hepatocytes. Further, BF102 also inhibits cholesterol biosynthesis by suppressing HMG-CoA reductase gene expression in hepatocytes. Interestingly, our preliminary in vivo data suggests that BF102 has no significant toxicity in mice at the highest possible dose we can administered. Thus, BF102 is a potential lead for the next generation of lipid-lowering drugs.     

Modifying aroylhydrazone prochelators for hydrolytic stability and improved cytoprotection against oxidative stress

BSIH ((E)-N′-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) is a prodrug version of the metal chelator SIH ((E)-N′-(2-hydroxybenzylidene)isonicotinohydrazide) in which a boronate group prevents metal chelation until reaction with hydrogen peroxide releases SIH, which is then available for sequestering iron(III) and inhibiting iron-catalyzed oxidative damage. While BSIH has shown promise for conditionally targeting iron sequestration in cells under oxidative stress, the yield of SIH is limited by the fact that BSIH exists in cell culture media as an equilibrium mixture with its hydrolysis products isoniazid and 2-formylphenyl boronic acid. In the current study, several BSIH analogs were evaluated for their hydrolytic stability, reaction outcomes with H₂O₂, and prochelator-to-chelator conversion efficiency. Notably, the para-methoxy derivative (p-OMe)BSIH ((E)-N′-(5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) and the meta-, para-double substituted (MD)BSIH ((E)-N′-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxol-5-yl)methylene)isonicotinohydrazide) showed 1.3- and 1.9-fold improved hydrolytic stability compared to BSIH, respectively, leading to a 22 and 50% increase in chelator released. Moreover, both prochelators were found to protect retinal pigment epithelial cells stressed with either H₂O₂ or paraquat insult.     

On the synthesis of a 32P-labelled Edman reagent for the sensitive identification of amino-acid derivatives

A phosphorus-32 containing derivative of phenylisothiocyanate was prepared to increase the sensitivity of amino-acid sequence determination. The respective compound 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide showed about the same reactivity, stability, and polarity as the Edman reagent itself. A repetitive yield of 94% was obtained in the stepwise degradation of insulin B chain using a solid phase sequencer. The synthesis of this radioactive reagent was achieved within 5 h but with a specific activity of 1 Ci/mol. Eight amino acids were reacted with the 32P-labelled reagent and identified by autoradiography after two dimensional thin-layer chromatography.     

In silico and biological activity evaluation of quercetin-boron hybrid compounds,anti-quorum sensing effect as alternative potential against microbial resistance

Boronic acid compounds and the natural flavonoid compound quercetin were handled to synthesize two novel ligands encoded as B1(2,2′-(1,4-phenylenebis (benzo [1,3,2] dioxaborole-2,5-diyl)) bis (3,5,7-trihydroxy-4H- chromen-4-one) and B2(3.3.6. 3,5,7-trihydroxy-2-(2-(6-methoxypyridin-3-yl)benzo[d][1,3,2]dioxaborol-5-yl)− 4 H-chromene-4). Antioxidant activities of ligands were investigated by DPPH, ABTS and CUPRAC methods. Cholinesterase inhibition effects of ligands were determined by acetylcholinesterase and butyrylcholinesterase enzyme inhibition methods, cytotoxic effects of ligands were applied to healthy breast and colon cancer cell lines by MTT method, and urease and tyrosinase enzyme activities were determined. Antimicrobial properties of the compounds were analyzed by detecting their anti-QS potentials on Chromobacterium violaceum biosensor strain. Both compounds were found to have significant antioxidant effects compared to controls. It was determined that the compound B1 at 1–10 µg/mL was more active than the reference compounds (α-TOC and BHT). Moreover, the enzyme activity studies on ligands demonstrated that acetylchoinesterase and butyrylcholinesterase enzyme inhibitions were higher than the reference compounds. As expected, boron derivatives exhibited respectable activity against the biofilms of Escherichia coli (E. coli) and P. aeruginosa (P. aeruginosa). These results demonstrate the potential applicability of boron derivatives in the treatment of biofilm-associated infections and provide a practical strategy for the design of new boron-based antimicrobial materials. In silico molecular docking studies were performed on ligands to identify newly synthesized compounds. The binding parameter values and binding sites of the compounds were also determined. In conclusion, our studies showed that newly synthesized hybrid compounds could be solutions for antimicrobial resistance and enzyme-related disorders.     

Identification of an orthogonal peptide binding motif for biarsenical multiuse affinity probes

Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of coexpressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4',5'-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha-subunit prior to the labeling of the KA-tag sequence of the beta-subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.     

Structure of cyclic dihydroxyacetone phosphate dimethyl acetal, a cyclic DHAP precursor, in the crystalline state

The six-membered cyclic phosphate diester, 5,5-dimethoxy-2-oxo-1,3,2-dioxaphosphorinane-2-ol, the dimethyl acetal of cyclic dihydroxyacetone phosphate, (MeO)(2)cDHAP, was obtained by the isolation of an intermediate in the basic hydrolysis of the cyclic triester derivative. The compound had been isolated in the form of the crystalline cyclohexylammonium (cha) salts: (cha)[(MeO)(2)cDHAP].3H(2)O (5a) and (cha)[(MeO)(2)cDHAP].H(2)O (5b), which were then converted into the free acid: (H(5)O(2))[(MeO)(2)cDHAP] (5c) and then into a series of different salts: Na[(MeO)(2)cDHAP].2H(2)O (5d), K[(MeO)(2)cDHAP].1.5H(2)O (5e), K[(MeO)(2)cDHAP].0.5H(2)O (5e'), Ca[(MeO)(2)cDHAP](2).2H(2)O (5f), CaK[(MeO)(2)cDHAP](3).2H(2)O (5g) and NH(4)[(MeO)(2)cDHAP] (5h). The synthesis of the compounds, their crystallization and crystal structures determined by X-ray crystallography are described. The most interesting structural feature observed in 5a-g anions in the crystalline state is the chair conformation of the P/O/C/C/C/O 1,3,2-dioxaphosphorinane ring, which is generally not flattened, in contrast to the deformations often observed in the analogous aryl derivatives (also in (MeO)(2)cDHAP(Ph); Slepokura, K.; Lis, T. Acta Crystallogr. Sect. C2004, 60, o315-o317). However, the anion in crystal 5h is disordered and exists in two conformations, 76% of which is the skew, S, conformation, not observed so far in the compounds of related structure.     

The structure of the benzeneboronate of pentane-1,3,5-triol

The relative ease of formation of five-, six-, seven-, and eight-membered cyclic benzeneboronates is discussed. Pentane-1,3,5-triol forms, by interaction with benzene-boronic anhydride, exclusively DL-4-(2-hydroxyethyl)-2-phenyl-1,3,2-dioxaborinane. It is suggested that, in this compound, the oxygen atom of the hydroxyl group is intramolecularly co-ordinated with the boron atom.     

CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems

The cis/trans diastereoisomeric composition of hydroxyl radical adducts to chiral cyclic nitrones can be used to approach mechanisms of free radical formation in biological systems. Such determination is greatly simplified when both diastereoisomers have ESR spectra with at least two non-overlapping lines. To achieve this prerequisite, a series of DEPMPO-derived spin traps bearing one unsubstituted or alkyl-substituted 2-oxo-1,3,2-dioxaphosphorinane ring were synthesized and their structures were confirmed by X-ray diffraction, (1)H, (13)C and (31)P NMR. These CyDEPMPOs nitrones showed variable lipophilicities and LD(50) values on murine fibroblasts compatible with a safe use in biological spin trapping. All CyDEPMPOs formed persistent spin adducts with a series of free radicals, including superoxide and hydroxyl (i.e., CyDEPMPOs-OH) and the in vitro half-life times of these two latter were at least as extended as those of parent DEPMPO. Using four methods of CyDEPMPOs-OH formation, the cis-CyDEPMPOs-OH percentage was found significantly varied with substitution on the P-containing ring and, more interestingly, with the generating system.     

The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.

Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5'-end of synthetic oligonucleotides on solid supports to produce amino or thio-linked oligonucleotides. Following deprotection and purification, fluorescent dyes, biotin derivatives and poly-L-lysine-maleimide were separately attached to the functionalised oligonucleotides. Identical derivatized oligomers were obtained with cyanoethyl-N,N-diisopropylamidite chemistry and amidites (5a-e) of the respective alcohols.