Michael Addition Reaction between Dehydroalanines and Phosphites Enabled the Introduction of Phosphonates into Oligopeptides.

A method for introducing a range of phosphonates into oligopeptides through a Michael addition reaction between dehydroalanine and phosphite is presented. The method offers a mild, cheap, and straightforward approach to peptide phosphorylation that has potential applications in chemical biology and medicinal chemistry. Moreover, the introduction of a phosphonate group into short antibacterial peptides is described to demonstrate its utility, leading to the discovery of phosphonated antibacterial peptides with potent broad-spectrum antibacterial activity.

Contamination of dairy products with tris(2,4-di-tert-butylphenyl) phosphite and implications for human exposure.

Tris(2,4-di-tert-butylphenyl) phosphite (AO168), an organophosphite antioxidant, can be oxidized to tris(2,4-di-tert-butylphenyl) phosphate (AO168 = O) during the production, processing, and application of plastics. AO168 = O can be further transformed to bis(2,4-di-tert-butylphenyl) phosphate and 2,4-di-tert-butylphenol. Here, we discovered the contamination of AO168 and its transformation products in dairy products for the first time. More samples contained AO168 (mean concentration: 8.78 ng/g wet weight [ww]), bis(2,4-di-tert-butylphenyl) phosphate (mean:11.1 ng/g ww) and 2,4-di-tert-butylphenol (mean: 46.8 ng/g ww) than AO168 = O (mean: 40.2 ng/g ww). The concentrations of AO168 and its transformation products were significantly correlated, and differed with the packaging material and storage conditions of the product. Estimated daily intakes (EDIs) of AO168 and its transformation products were calculated. Although the overall dietary risks were below one, transformation products accounted for 96.7% of the total hazard quotients. The high-exposure EDIs of total AO168 were above the threshold of toxicological concern (300 ng/kg bw/day), and deserve continual monitoring.

An efficient synthesis of functionalized ß-amino- and ß-hydrazinoalkylphosphonates by three-component reaction between trialkyl phosphites, dialkyl acetylenedicarboxylates and aromatic amines or hydrazines.

Three-component reaction between trialkyl phosphites, dialkyl acetylenedicarboxylates and aromatic amines afforded ß-aminoalkylphosphonate derivatives. Similar reaction between trialkyl phosphites, dialkyl acetylenedicarboxylates and dinitrophenylhydrazine afforded ß-hydrazinooalkylphosphonate derivatives. This method includes both the C-N and C-P bond formation in a one pot and single synthetic step in neutral and simple reaction conditions. All reactions were conducted in CH2Cl2 as solvent at room temperature without using any catalyst, and the stable products were obtained in high yields. The structures of all products were proved by 1H, 13C and 31P NMR and IR spectral and elemental analysis data.

An efficient synthesis of phosphonated cyclopentenones by NaN3-catalyzed three-component reaction between trialkyl phosphites, ethyl arylmethylidenecyanoacetates and dialkyl acetylenedicarboxylates.

NaN3-catalysed three-component reaction between trialkyl phosphites, dialkyl acetylenedicarboxylates and ethyl arylmethylidenecyanoacetates afforded phosphonated cyclopentenone derivatives. The process involves one C-P and two C-C bond formations in one synthetic step. All reactions were conducted in acetone as solvent at room temperature and the products were obtained in high yields as stable solids. The products were isolated and purified by simple washing with water and diethyl ether without need to tedious chromatography methods. The structures of products were proved by 1H, 13C and 31P NMR and IR spectral and elemental analysis data.

Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy.

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

A concise access to bridged [2,2,1] bicyclic lactones with a quaternary stereocenter via stereospecific hydroformylation.

Chiral bridged [2,2,1] bicyclic lactones are privileged structural units in pharmaceutics and bioactive nature products. However, the synthetic methods for these compounds are rare. Here we report an efficient method for enantioselective construction of bridged [2,2,1] bicyclic lactones bearing a quaternary stereocenter via Rh-catalyzed asymmetric hydroformylation/intramolecular cyclization/pyridium chlorochromate (PCC) oxidation. By employing a hybrid phosphine-phosphite chiral ligand, a series of cyclopent-3-en-1-ols are transformed into corresponding γ-hydroxyl aldehydes with specific syn-selectivity. Then, hemiacetals form in situ and oxidation with PCC in one-pot affords bridged [2,2,1] bicyclic lactones in high yields and excellent enantiomeric excess. Replacing the hydroxyl group by an ester group, cyclopentanecarbaldehydes with a chiral all-carbon quaternary stereocenter in the γ-position can be generated efficiently.

Adenylate Kinase-Catalyzed Reaction of AMP in Pieces: Enzyme Activation for Phosphoryl Transfer to Phosphite Dianion.

The binding of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) to adenylate kinase (AdK) drives closure of lids over the substrate adenosyl groups. We test the hypothesis that this conformational change activates AdK for catalysis. The rate constants for Homo sapiens adenylate kinase 1 (HsAdK1)-catalyzed phosphoryl group transfer to AMP, kcat/Km = 7.0 × 106 M-1 s-1, and phosphite dianion, (kHPi)obs ≤1 × 10-4 M-1 s-1, show that the binding energy of the adenosyl group effects a ≥7.0 × 1010-fold rate acceleration of phosphoryl transfer from ATP. The third-order rate constant of kcat/KHPiKEA = 260 M-2 s-1 for 1-(ß-d-erythrofuranosyl)adenine (EA)-activated phosphoryl transfer to phosphite dianion was determined, and the isohypophosphate reaction product characterized by 31P NMR. The results demonstrate the following: (i) a ≥14.7 kcal/mol stabilization of the transition state for phosphoryl transfer by the adenosyl group of AMP and a ≥2.6 × 106-fold rate acceleration from the EA-driven conformational change and (ii) the recovery of ≥8.7 kcal/mol of this transition state stabilization for EA-activated phosphoryl transfer from ATP to phosphite.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Recoverable Phospha-Michael Additions Catalyzed by a 4-N,N-Dimethylaminopyridinium Saccharinate Salt or a Fluorous Long-Chained Pyridine: Two Types of Reusable Base Catalysts.

Phospha-Michael addition, which is the addition reaction of a phosphorus-based nucleophile to an acceptor-substituted unsaturated bond, certainly represents one of the most versatile and powerful tools for the formation of P-C bonds, since many different electrophiles and P nucleophiles can be combined with each other. This offers the possibility to access many diversely functionalized products. In this work, two kinds of basic pyridine-based organo-catalysts were used to efficiently catalyze phospha-Michael addition reactions, the 4-N,N-dimethylaminopyridinium saccharinate (DMAP·Hsac) salt and a fluorous long-chained pyridine (4-Rf-CH2OCH2-py, where Rf = C11F23). These catalysts have been synthesized and characterized by Lu's group. The phospha-Michael addition of diisopropyl, dimethyl or triethyl phosphites to α, ß-unsaturated malonates in the presence of those catalysts showed very good reactivity with high yield at 80-100 °C in 1-4.5 h with high catalytic recovery and reusability. With regard to significant catalytic recovery, sometimes more than eight cycles were observed for DMAP·Hsac adduct by using non-polar solvents (e.g., ether) to precipitate out the catalyst. In the case of the fluorous long-chained pyridine, the thermomorphic method was used to efficiently recover the catalyst for eight cycles in all the reactions. Thus, the easy separation of the catalysts from the products revealed the outstanding efficacy of our systems. To our knowledge, these are good examples of the application of recoverable organo-catalysts to the DMAP·Hsac adduct by using non-polar solvent and a fluorous long-chained pyridine under the thermomorphic mode in phospha-Michael addition reactions.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers ΔG⧧ for kcat/Km for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose 6-phosphate isomerase are reduced by 11-13 kcal/mol by interactions between the protein and the substrate phosphodianion. Between 4 and 6 kcal/mol of this dianion binding energy is expressed at the transition state for phosphite dianion activation of the respective enzyme-catalyzed reactions of truncated substrates d-xylonate or d-xylose. These and earlier results from studies on ß-phosphoglucomutase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase define a cluster of six enzymes that catalyze reactions in glycolysis or of glycolytic intermediates, and which utilize substrate dianion binding energy for enzyme activation. Dianion-driven conformational changes, which convert flexible open proteins to tight protein cages for the phosphorylated substrate, have been thoroughly documented for five of these six enzymes. The clustering of metabolic enzymes which couple phosphodianion-driven conformational changes to enzyme activation suggests that this catalytic motif has been widely propagated in the proteome.

Vibrational Properties and DFT Calculations of Perovskite-Type Methylhydrazinium Manganese Hypophosphite.

Recently discovered hybrid perovskites based on hypophosphite ligands are a promising class of compounds exhibiting unusual structural properties and providing opportunities for construction of novel functional materials. Here, we report for the first time the detailed studies of phonon properties of manganese hypophosphite templated with methylhydrazinium cations ([CH3NH2NH2][Mn(H2PO2)3]). Its room temperature vibrational spectra were recorded for both polycrystalline sample and a single crystal. The proposed assignment based on Density Functional Theory (DFT) calculations of the observed vibrational modes is also presented. It is worth noting this is first report on polarized Raman measurements in this class of hybrid perovskites.

Pillared-layered indium phosphites templated by amino acids: isoreticular structures, water stability, and fluorescence.

Two crystalline open-framework indium phosphites (denoted SCU-31 and SCU-32) were prepared using amino acids as structure-directing agents. They have isoreticular pillared-layered structures built up from 6 × 1 and 4 = 1 clusters. Notably, the two compounds show excellent water stability and exhibit blue fluorescence under UV light irradiation at room temperature. The proton-conducting behaviour of SCU-31 was also investigated.

Study on the Microwave-Assisted Batch and Continuous Flow Synthesis of N-Alkyl-Isoindolin-1-One-3-Phosphonates by a Special Kabachnik-Fields Condensation.

A simple and efficient microwave (MW)-assisted method was elaborated for the catalyst-free synthesis of isoindolin-1-one-3-phosphonates by the three-component condensation of 2-formylbenzoic acid, aliphatic primary amines and various dialkyl phosphites. The batch and the continuous flow reactions were optimized in respect of the temperature, the reaction time and the molar ratio of the starting materials. To evaluate the potential of MW irradiation, comparative thermal experiments were also carried out. In order to obtain "real time" information about the condensation, the special Kabachnik-Fields reaction of 2-formylbenzoic acid, butylamine and diethyl phosphite was monitored by in situ FT-IR spectroscopy. The novel title compounds could be prepared in high yields at low temperature under a short reaction time. A suitable method could also be developed for the preparation of the isoindolin-1-one-3-phosphonates at a "few g" scale by using a continuous flow MW reactor.

Solvent-free, under air selective synthesis of α-glycosides adopting glycosyl chlorides as donors.

α-Glycosides are highly relevant synthetic targets due to their abundance in natural oligosaccharides involved in many biological processes. Nevertheless their preparation is hampered by several issues, due to both the strictly anhydrous conditions typically required in glycosylation procedures and the non-trivial achievement of high α-stereoselectivity, one of the major challenges in oligosaccharide synthesis. In this paper we report a novel and efficient approach for the highly stereoselective synthesis of α-glycosides. This is based on the unprecedented solvent-free combination of triethylphosphite, tetrabutylammonium bromide and N,N-diisopropylethylamine for the activation of glycosyl chlorides under air. Despite the relative stability of glycosyl chlorides with respect to more reactive halide donors, the solvent-free procedure allowed a wide set of α-glycosides, including biorelevant fragments, to be obtained in much shorter times compared with similar glycosylation approaches in solution. The presented method features a wide target scope and functional group compatibility, also serving with partially disarmed substrates, and it does not require a high stoichiometric excess of reagents nor the preparation of expensive precursors. The solvent-free glycosylation can be even directly performed from 1-hydroxy sugars without purification of the in situ generated chloride, providing an especially useful opportunity in the case of highly reactive and labile glycosyl donors.

Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials.

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

The Staudinger Ligation.

While the Staudinger reaction has first been described a hundred years ago in 1919, the ligation reaction became one of the most important and efficient bioconjugation techniques in the 1990s and this century. It holds the crucial characteristics for bioorthogonal chemistry: biocompatibility, selectivity, and a rapid and high-yielding turnover for a wide variety of applications. In the past years, it has been used especially in chemical biology for peptide/protein synthesis, posttranslational modifications, and DNA labeling. Furthermore, it can be used for cell-surface engineering, development of microarrays, and drug delivery systems. However, it is also possible to use the reaction in synthetic chemistry for general formation of amide bonds. In this review, the three major types, traceless and nontraceless Staudinger Ligation as well as the Staudinger phosphite reaction, are described in detail. We will further illustrate each reaction mechanism and describe characteristic substrates, intermediates, and products. In addition, not only its advantages but also stereochemical aspects, scope, and limitations, in particular side reactions, are discussed. Finally, the method is compared to other bioorthogonal labeling methods.

Redox mechanism and stability of uranyl phosphites at mineral surfaces: Cooperative proton/electron transfer and high efficacy for Uranium(VI) reduction.

Uranium phosphites have recently emerged as promising materials to remediate radioactive contamination. In this study, the redox mechanisms of uranyl phosphites at mineral surfaces have been addressed by periodic DFT calculations with dispersion corrections. Different from other ligands, the phosphite anions (H2PO3-, HPO32-) are efficient reducing agents for uranyl reduction, and the redox reactions are divided into three steps, as isomerization between two phosphite anion isomers (Step 1), conformational transition (Step 2) and dissociation of the water molecule (Step 3). A second water molecule is critical to lower the activation barriers of Step 1, and all activation barriers are moderate so that the redox reactions occur favorably under normal conditions, which are further dramatically accelerated by the highly exergonic Step 3. Accordingly, formation of uranyl phosphites becomes an effective approach to manage uranium pollution. Moreover, the lower activation barriers for H2PO3- rather than HPO32- rationalize the superior reduction activities of uranyl phosphites and the enhanced stability of U(IV) products at lower pH conditions. Owing to the cooperative proton/electron transfer, the U(VI) reduction to U(IV) and P(III) oxidation to P(V) are completed within one step, with transition states being featured by the U(V) and P(IV) species.

Three-Component Reaction of Diamines with Triethyl Orthoformate and Diethyl Phosphite and Anti-Proliferative and Antiosteoporotic Activities of the Products.

A three-component reaction between diamines (diaminobenzenes, diaminocyclohexanes, and piperazines), triethyl orthoformate, and diethyl phosphite was studied in some detail. In the case of 1,3- and 1,4-diamines and piperazines, products of the substitution of two amino moieties-the corresponding tetraphosphonic acids-were obtained. In the cases of 1,2-diaminobenzene, 1,2-diaminocyclohexanes and 1,2-diaminocyclohexenes, only one amino group reacted. This is most likely the result of the formation of hydrogen bonding between the phosphonate oxygen and a hydrogen of the adjacent amino group, which caused a decrease in the reactivity of the amino group. Most of the obtained compounds inhibited the proliferation of RAW 264.7 macrophages, PC-3 human prostate cancer cells, and MCF-7 human breast cancer cells, with 1, trans-7, and 16 showing broad nonspecific activity, which makes these compounds especially interesting in the context of anti-osteolytic treatment and the blocking of interactions and mutual activation of osteoclasts and tumor metastatic cells. These compounds exhibit similar activity to zoledronic acid and higher activity than incadronic acid, which were used as controls. However, studies of sheep with induced osteoporosis carried out with compound trans-7 did not support this assumption.

Mechanochemical Synthesis of Short DNA Fragments.

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.

Asymmetric Transfer Hydrogenation of Arylketones Catalyzed by Enantiopure Ruthenium(II)/Pybox Complexes Containing Achiral Phosphonite and Phosphinite Ligands.

A family of complexes of the formula trans-[RuCl2(L)(R-pybox)] (R-pybox = (S,S)-iPr-pybox, (R,R)-Ph-pybox, L = monodentate phosphonite, PPh(OR)2, and phosphinite, L = PPh2(OR), ligands) were screened in the catalytic asymmetric transfer hydrogenation of acetophenone, observing a strong influence of the nature of both the R-pybox substituents and the L ligand in the process. The best results were obtained with complex trans-[RuCl2{PPh2(OEt)}{(R,R)-Ph-pybox}] (2c), which provided high conversion and enantioselectivity (up to 96% enantiomeric excess, e.e.) for the reduction of a variety of aromatic ketones, affording the (S)-benzylalcohols.