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.

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.

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.

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.

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.

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.

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.

Examining the Mechanism of Phosphite Dehydrogenase with Quantum Mechanical/Molecular Mechanical Free Energy Simulations.

The projected decline of available phosphorus necessitates alternative methods to derive usable phosphate for fertilizer and other applications. Phosphite dehydrogenase oxidizes phosphite to phosphate with the cofactor NAD+ serving as the hydride acceptor. In addition to producing phosphate, this enzyme plays an important role in NADH cofactor regeneration processes. Mixed quantum mechanical/molecular mechanical free energy simulations were performed to elucidate the mechanism of this enzyme and to identify the protonation states of the substrate and product. Specifically, the finite temperature string method with umbrella sampling was used to generate the free energy surfaces and determine the minimum free energy paths for six different initial conditions that varied in the protonation state of the substrate and the position of the nucleophilic water molecule. In contrast to previous studies, the mechanism predicted by all six independent strings is a concerted but asynchronous dissociative mechanism in which hydride transfer from the phosphite substrate to NAD+ occurs prior to attack by the nucleophilic water molecule. His292 is identified as the most likely general base that deprotonates the attacking water molecule. However, Arg237 could also serve as this base if it were deprotonated and His292 were protonated prior to the main chemical transformation, although this scenario is less probable. The simulations indicate that the phosphite substrate is monoanionic in its active form and that the most likely product is dihydrogen phosphate. These mechanistic insights may be helpful for designing mutant enzymes or artificial constructs that convert phosphite to phosphate and NAD+ to NADH more effectively.

Highly Selective Fluorescent Sensing of Phosphite through Recovery of Poisoned Nickel Oxide Nanozyme.

Phosphorus is a key element responsible for eutrophication, and its measurement and speciation is a critical topic in analytical chemistry. Most research efforts have been devoted to detecting phosphate (P(V)), while few reports on phosphite (P(III)) are available, making it difficult for sensor-based understanding of the phosphorus cycle. This study presents a fluorescent "turn-on" sensor for quantitative and highly selective analysis of phosphite based on the different coordination strength of N and P lone-pair electrons toward nickel oxide (NiO). A few N-containing compounds (mainly Good's buffers) were screened as inhibitors for the oxidase-like activity of NiO nanoparticles for the oxidation of Amplex red (AR). HEPES was found to be most effective for inhibiting the formation of fluorescent resorufin, the oxidation product of AR. Among various phosphorus-, arsenic-, selenium-, and sulfur-containing species, along with various cations, phosphite was the only one that could restore the activity, likely due to its stronger affinity with the surface, and it is not an inhibitor. Under the optimum condition, the sensor detected P(III) up to 1 mM with a detection limit of 1.46 µM. The phosphite analysis with recovery rates ranged from 74.2 ± 2.6% to 107.5 ± 0.5% in real water and biological samples, suggesting the potential applicability of this sensor.

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.

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.

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.

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.

The Synthesis of α-Aminophosphonates via Enantioselective Organocatalytic Reaction of 1-(N-Acylamino)alkylphosphonium Salts with Dimethyl Phosphite.

α-Aminophosphonic acids are phosphorus analogues of α-amino acids. Compounds of this type find numerous applications in medicine and crop protection due to their unique biological activities. A crucial factor in these activities is the configuration of the stereoisomers. Only a few methods of stereoselective transformation of α-amino acids into their phosphorus analogues are known so far and all of them are based on asymmetric induction, thus involving the use of a chiral substrate. In contrast, we have focused our efforts on the development of an effective method for this type of transformation using a racemic mixture of starting N-protected α-amino acids and a chiral catalyst. Herein, a simple and efficient stereoselective organocatalytic α-amidoalkylation of dimethyl phosphite by 1-(N-acylamino)alkyltriphenylphosphonium salts to enantiomerically enriched α-aminophosphonates is reported. Using 5 mol% of chiral quinine- or hydroquinine-derived quaternary ammonium salts provides final products in very good yields up to 98% and with up to 92% ee. The starting phosphonium salts were easily obtained from α-amino acid derivatives by decarboxylative methoxylation and subsequent substitution with triphenylphosphonium tetrafluoroborate. The appropriate self-disproportionation of enantiomers (SDE) test for selected α-aminophosphonate derivatives via achiral flash chromatography was performed to confirm the reliability of the enantioselectivity results that were obtained.

Continuous Flow Esterification of a H-Phosphinic Acid, and Transesterification of H-phosphinates and H-Phosphonates under Microwave Conditions.

The microwave (MW)-assisted direct esterification of phenyl-H-phosphinic acid, transesterification of the alkyl phenyl-H-phosphinates so obtained, and the similar reaction of dibenzyl phosphite (DBP) were investigated in detail, and the batch accomplishments were translated into a continuous flow operation that, after optimization of the parameters, such as temperature and flow rate, proved to be more productive. Alcoholysis of DBP is a two-step process involving an intermediate phosphite with two different alkoxy groups. The latter species are of synthetic interest, as precursors for optically active reagents.

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.

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.

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase.

Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD+, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary 2H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step. The KIEs on the pre-exponential factor (AH/AD) and the activation energy (ΔEa) were 1.6 ± 0.1 and 0.21 ± 0.05 kcal/mol, respectively, suggesting that 17X-PTDH facilitates extensive tunneling of both isotopes via a Marcus-like model. Site-directed mutagenesis was used to evaluate the role of an active site threonine (Thr104) found on the back face of the nicotinamide in promoting the close packing of the substrates. In mutants with reduced steric bulk at this position, values of AH/AD and ΔEa fall within the range describing semiclassical "over the barrier" reactivity, suggesting that Thr104 acts as a steric backstop to promote tunneling in 17X-PTDH. Whereas hydrogen tunneling is now a widely appreciated feature of C-H activating enzymes, these observations with a P-H activating system are consistent with the proposal that tunneling is likely to be a common feature on all enzymes that catalyze hydrogen transfers.