A series of hydroxy-1,2,3,6-tetrahydrophosphinine oxides were prepared by the two-step ring enlargement of 1-substituted 3-phospholene 1-oxides via the corresponding dichlorocarbene adducts. The two diastereomers of the P-ethoxy-3-phosphabicyclo-[3.1.0]-hexane 3-oxides could be identified by single crystal X-ray analysis, hence the isomers could be characterized by NMR methods. Detailed examination of the crystal structures of the two isomers shows weak O···H and Cl···H interactions, which are different for the two isomers, in accord with the different arrangements of the molecules in the solid state. The less hindered hydroxy-tetrahydrophosphinine oxide isomers were selectively phosphinoylated and thiophosphinoylated. The cytotoxic effect of the P-heterocycles synthesized was tested on U266 myeloma cells and on A2058 melanoma cells. The results are promising, as the viability of the cells was decreased drastically at the higher 100 μM concentration, especially in respect of one hydroxy-tetrahydrophosphinine oxide and the two P-functionalized derivatives, independently of the substituent's nature.

α-Hydroxyphosphonates and α-hydroxyphosphine oxides are important intermediates for additional biologically active species. While (hydroxy(phenyl)methyl)(diphenyl)phosphine oxide did not even undergo chlorination with thionyl chloride, the reaction of diethyl chloro(phenyl)methylphosphonate with potassium diphenylphosphide followed by oxidation with hydrogen peroxide resulted in the expected bis(>P(O)-functionalized) product only as a minor component. The similar reaction of the analogous (methanesulfonyloxy)(phenyl)methyl derivative afforded a mixture of a diethyl (diethoxyphosphonyl)(phenyl)methyl phosphate, a diethyl (diphenylphosphinyl)(phenyl)methyl phosphate, and a ((diphenylphosphinyl)(phenyl)methyl) diphenylphosphinate. The formation of the unexpected products was explained assuming rearrangements and reversible formation of the hydroxymethylene-bis(>P(O)-functionalized) intermediate. Contrary to the earlier experiences, the reaction of ((methanesulfonyloxy)(phenyl)methyl)(diaryl)phosphine oxides with potassium diphenylphosphide followed by oxidation took place in a clear-cut manner providing, with one exception, the corresponding α-phosphinylated α-hydroxyphosphine oxides that could also be synthesized by direct phosphinylation of the starting α-hydroxyphosphine oxide. Cell viability assays performed on U266 myeloma cells revealed concentration-dependent antiproliferative effects of the synthesized compounds on U266 myeloma cells, highlighting the key role of the phosphinoyloxy moiety in the cytotoxic activity, which may be enhanced further by 4-methyl groups in the P-phenyl rings.

Two new families of alpha-aminophosphonates were prepared to widen the pool of these potentially biologically active species. alpha-Alkylamino- and alpha-amino-arylmethylphosphonates were added on the triple bond of dialkyl acetylenedicarboxylates to afford the corresponding N-alkenylated alpha-aminophosphonic derivatives. Performing the reactions of alpha-alkylamino-phosphonates at 26 degrees C in the presence of diazabicycloundecene (DBU) as the catalyst, the additions took place in a diastereoselective manner to afford one isomer that was the E-form according to NOESY and ROESY NMR measurements. In the absence of a catalyst, completion of the addition required a longer time. The similar reaction of alpha-aminophosphonates with an NH2 group, in the presence of DBU, led to a mixture of two diastereoisomers. However, upon stirring the components without a catalyst at 26 degrees C for 24 h, or irradiating the mixture at 100 degrees C for 2 h, the addition of alpha-aminophosphonates to acetylenes, in this occasion, afforded diastereoselectively the Z isomer, as proven by NOESY/ROESY experiments. The effect of the new products was tested for cytotoxic activity on a myeloma cell line. It was found that these derivatives showed no antiproliferative effect on myeloma cells. Thus, there is a need for additional investigations to explore their antiproliferative properties and their effects on tumor cell viability.

4- and 3-Bromophenyl-diphenylphosphine oxides prepared by us in a selective P-C coupling reaction offered themselves as excellent new starting materials in the Suzuki-Miyaura C-C and in the Hirao P-C cross-coupling reactions, as well as in the Michaelis-Arbuzov reaction. Novel bis(>P(O))-functionalized aromatics are the valuable products of the two latter phosphinoylations. The three transformations outlined were performed by applying metal catalysts (mainly Pd, but Ni may also be suitable) under microwave irradiation. Twenty-three, mostly new, P-functionalized aromatics were made available and fully characterized. On the other hand, the effect of the substituents in the aromatic ring on the reactivity, as well as the mechanism and energetics of the reactions, was studied by density functional theory calculations at the B3LYP/6-31G(d,p) level of theory. As regards the C-C cross-coupling, the 4- and 3-Ph2P(O)-substitution had only a minor effect on the value of the activation energy. In the transition metal-catalyzed Arbuzov reaction, the initial oxidative addition step shows comparable activation barriers for all investigated systems. However, in the subsequent steps, including the elimination of MeBr and the reductive elimination step, both the intermediates and transition states exhibit much larger differences in relative Gibbs free energy, which probably causes the observed differences in reactivity of the 4- and 3-Ph2P(O)-substituted models, as compared to the unsubstituted case. The elimination of MeBr has the highest Gibbs free energy requirement during the process. This high energy barrier may be overcome by the beneficial effects of microwave irradiation. In the Hirao cross-coupling, the 4-Ph2P(O)-substitution resulted in a decrease in the barrier of the oxidative addition step, whereas both the 3-Ph2P(O)-substitution and the electron-sending 4-Me- and 4-MeO-substituents led to a slight increase in the barrier. The theoretical data were in accord with the experimental findings.

Potentially biologically active alpha-aminophosphonic derivatives were prepared by the Kabachnik-Fields condensation of alpha-amino-alpha-aryl-methylphosphonates, arylaldehydes, and diethyl phosphite to afford bis(alpha-aryl-methylphosphonoyl)-amines as a mixture of racemic and meso isomers. To go "green"-performing the transformations under microwave irradiation-there was no need for a catalyst. On the other hand, the phospha-Mannich reaction of alpha-amino-alpha-phenyl-methylphosphonate with arylaldehydes led to (alpha-aryl-methylphosphonoyl)-(alpha-phenyl-methylphosphonoyl)-amines as a mixture of SS/RR and SR/RS racemates. Moreover, the respective symmetric products with identical aryl groups were also present. The outcome was similar, when alpha-amino-alpha-aryl-methyl-phosphonates were condensed with benzaldehyde and diethyl phosphite. The products were analyzed by 1D and 2D NMR spectroscopy. The combined NMR analysis of the products confirmed their structure.

Background: Inflammatory signaling and oxidative stress machinery are interconnected and play roles in apoptosis, proliferation, redox state control, and the progression of many diseases, including cancer. The marine environment harbors a wealth of organisms that produce a wide variety of bioactive molecules with significant biological activities. Over the last decade, the advent of AI-driven approaches has enhanced the study and analysis of peptides, helping to reduce costly and time-consuming conventional laboratory testing, validation, and synthetic procedures. Methods: In this study, we predicted the antioxidative and anti-inflammatory activities of peptides isolated from proteomic data obtained from circulating cells and humoral components of the sea cucumber defense system using a bioinformatic workflow based on different artificial intelligence tools. Results: We identified 40 top-ranked peptides with antioxidative and anti-inflammatory activity and a sub-class of eight peptides shared by FreD domains. Molecular docking and molecular dynamics simulations showed that they have active binding sites for different key molecules involved in inflammatory and oxidative processes. Conclusions: The results showed that the peptides highlighted by our analysis workflow can be identified as potential molecules used as therapeutic strategies for diseases by targeting both inflammatory and oxidative processes.

Preparative experiments including the alkylation of diphenylthiophosphinic acid with a series of C1-C4 haloalkenes at 75/115°C in the presence of triethylamine in toluene suggested chemoselective formation of the corresponding S- (thiolic) ester. No O-(thionic) ester was formed. For this, we wished to clarify the confusion regarding the outcome of such alkylations described in the literature. A few analogous derivatives were also synthesized and characterized for comparison. First in the literature, crystal structures of two thiolic esters were analyzed in detail. The alkylation led to non-centrosymmetric crystal structures. Moreover, the two isostructural thiolic compounds formed chiral crystals, a rather unusual feature of achiral molecules. Weak interactions such as C-H … O bridges and dispersion dominate and tailor the structure of the crystals. As regards the mechanism, three possible pathways were investigated at the B3LYP/6-31G(d,p)/PCM[toluene] level of theory. The activation enthalpy for the rate-determining step was significantly lower for the triethylamine-catalyzed route than for the base-free variation. Moreover, in the base-catalyzed protocol, the difference of 30.8 kJ mol-1 between the enthalpy of activation for the S and O-alkylations justified the S-selectivity. The pathway involving the anion from the thiophosphinic acid was found unfavorable and hence was excluded.

Background: It is known that the α-hydroxyphosphonates and their derivatives may have potential biological activity. Methods: Within the prominent class of α-hydroxyphosphonates, α-hydroxy-alkylphosphonates and their derivatives were prepared as new representatives in the hope of obtaining biologically active species. During our work the Pudovik reaction, acylation and phosphinoylation/phosphorylation methods were used. The new compounds were characterized by NMR and MS spectroscopy. The antiproliferative effects were tested on U266 (myeloma multiplex) and A2058 (melanoma) cells. Results: Ethyl methyl ketone-dialkyl phosphite and secondary phosphine oxide adducts were synthesized by the Pudovik reaction on the earlier analogy of acetaldehyde- and acetone adducts. The hydroxyphosphonates and hydroxyphosphine oxides were acylated and phosphinoylated/phosphorylated. Due to the steric hindrance in the case of the preparation of the acetone-and ethyl methyl ketone-diethyl phosphite adducts, a two-step procedure was elaborated that was also suitable for the thiophosphinoylation of the adducts. A part of the α-hydroxyphosphonates could be successfully methanesulfonylated. The new derivatives prepared were tested on myeloma and melanoma cells, and it was found that the antiproliferative activity is primarily driven by phosphinoylation, particularly by diphenylthiophosphinoylation. The most promising compound, the diphenylthiophosphinoylated hydroxyphosphine oxide, reduced the viability of the U266 cells to less than 20% after a treatment with 100 µM concentration in a long-term experiment. Conclusions: A subset of the synthesized α-hydroxyphosphonate derivatives exhibited cytotoxic activity, supporting further structural optimization to identify compounds with enhanced biological efficacy.

Background: Methylenebisphosphonic derivatives including hydroxy-methylenebisphosphonic species may be of potential biological activity, and a part of them is used in the treatment of bone diseases. Methods: Methylenebisphosphonates may be obtained by the Michaelis–Arbuzov reaction of suitably α-substituted methylphosphonates and trialkyl phosphites or phosphinous esters, while the hydroxy-methylene variations are prepared by the Pudovik reaction of α-oxophosphonates and different >P(O)H reagents, such as diethyl phosphite and diarylphosphine oxides. Results: After converting α-hydroxy-benzylphosphonates and -phosphine oxides to the α-halogeno- and α-sulfonyloxy derivatives, they were utilized in the Michaelis–Arbuzov reaction with trialkyl phosphites and ethyl diphenylphosphinite to afford the corresponding bisphosphonate, bis(phosphine oxide) and phosphonate–phosphine oxide derivatives. The Pudovik approach led to α-hydroxy-methylenebisphosphonic species and to their rearranged products. A part of the derivatives revealed a significant cytotoxic effect on pancreatic adenocarcinoma or multiple myeloma cells. Conclusions: The new families of compounds synthesized by our novel approaches may be of practical importance due to the significant cytotoxic activity on the cell cultures investigated. Compounds lacking hydroxy groups showed anti-myeloma activity or limited effect on pancreatic cancer (PANC-1) cells unless substituted with para-trifluoromethyl group. Hydroxy-containing bisphosphonates and their rearranged derivatives demonstrated varying effects depending on structural modifications. While myeloma (U266) cells indicated greater sensitivity overall, the most significant reductions in cell viability were observed in PANC-1 cancer cells, raising potential therapeutic applications of bisphosphonates beyond myeloma-associated bone disease, particularly for malignancies like pancreatic ductal adenocarcinoma.

The holothurian immune system is characterized by complex defense mechanisms that act through humoral and cellular pathways. Coelomocites are the cellular component of coelomic fluid, and they are involved in host defense, stress response, wound healing, organ regeneration, and tissue homeostasis. The close phylogenetic relationship between Holothuria tubulosa and chordate phylum makes it a good model for studying the evolution of immune processes. To elucidate the immune landscape in H. tubulosa, we applied an approach combining proteomic analysis of coelomic fluid separated into cellular fraction and extracellular fraction and bioinformatics and in silico analyses. A Search Tool for the Retrieval of Interacting Genes/Protein analysis indicated a highly functional homology to the human protein of immune recognition factors, non-canonical immune-related proteins, signaling molecules, and effector protein, cytoskeleton, and actin remodeling, and provided the first evidence in invertebrate immune cells of an intracellular protein fraction linked to ancestral structure resembling primary cilium involved in cell signaling.

Insight into molecular mechanisms of drug delivery enables development of new approaches for targeted drugs transportation. Facilitation of the transmembrane drug transfer is particularly significant for sparingly lipid-soluble medicines, as well as for certain anticancer drugs with high toxicity. In the present combined mass spectrometry and quantum chemical study we investigate intermolecular interactions between drug delivery facilitating agent dimethyl sulfoxide (DMSO) and selected anticancer thioderivatives of nucleobases. DMSO is a well-known enhancer of transmembrane and transdermal drugs penetration. Formation of stable noncovalent complexes of DMSO with 6-thiopurine and 2-thioadenine in the polar solvent methanol is revealed by the electrospray ionization mass spectrometry (ESI MS) probing. Structures and interaction energies are calculated using the MP2/aug-cc-pVDZ method for the complexes of DMSO with the anticancer agents observed in the ESI MS experiments. We also estimate the influence of the solvents on molecular interactions in the complexes. The calculation results confirm stability of the noncovalent complexes and provide information regarding their equilibrium structures. We believe that the study outcomes on the formation of stable noncovalent intermolecular complexes between the anticancer thioderivatives of nucleobases and the delivery facilitating molecules such as DMSO contribute to the elucidation of molecular mechanisms providing the drug delivery process.

This work presents the synthesis of three little‐investigated types of asymmetric cinchona organocatalysts. The cinchona moiety is considered one of the privileged chiral skeletons, and one of its well‐known modifications involves attaching H‐bond donor catalyst moieties at the C9 position. In our study, we aimed to introduce different types of H‐bond donor functional groups, namely squaramide and thiourea, at the relatively unexplored 5’‐position of the quinoline ring. Our goal was to compare the performance of these novel catalysts with those that are already established. Our test reaction was a pharmaceutically relevant Michael addition reaction, ultimately producing an intermediate for baclofen. The results indicated that the new substitution at the 5’‐position did not yield significant advantages in terms of either yield (34%–97%) or enantiomeric excess (0%–18%). Additionally, we synthesised a unique type of asymmetric organocatalyst that incorporates both an H‐bond donor and an enamine catalyst moiety, allowing for two different activation mechanisms. This innovative catalyst was also used in the synthesis of warfarin, resulting in lower yields (4%–26%) but promising enantiomeric excess values (13%–60%).

Nonsymmetric α‐hydroxy‐ethylidenebisphosphonic (HEBP) derivatives were synthesized by the Pudovik reaction of dialkyl α‐oxo‐ethylphosphonates (DAOEPs) and P‐reagents, such as dialkyl phosphites, alkyl phenyl‐H‐phosphinates (APHPs) and diarylphosphine oxides to make available a new family of compounds with two different P‐functions. Performing the addition of APHPs to DAOEPs in the presence of 40% of the diethylamine catalyst, the primary adducts rearranged to the mixture of the corresponding phosphonoyl‐phosphonate and phosphoryl‐phosphinate. Acylation of the HEBPs led to the formation of the respective acylated derivatives. A part of the new P‐compounds was subjected to cytotoxic screening on myeloma and metastatic cell lines. Among these, two derivatives, dibutyl 1‐(butyl‐phenylphosphinoyl)‐1‐hydroxy‐ethylphosphonate 4a and tetrabutyl α‐acetyloxy‐ethylidenebisphosphonate 8c, demonstrated the strongest antiproliferative effects on the A2058 (metastatic melanoma) and U266 (multiple myeloma) cell lines, highlighting their potential as promising candidates for further therapeutic development. The bisphosphonic derivatives 4a and 8c reduced the viability of U266 cells by 94% and 91%, respectively

The outcome of the condensation of alpha-hydroxybenzyl-diphenylphosphine oxide (HBDPPO) with dialkyl phosphites and that of diethyl alpha-hydroxy-benzylphosphonate (HBP) with diphenylphosphine oxide producing, beyond the alpha-(phosphonoyloxi-)benzyl-diphenylphosphine oxides, also "crossed" condensed products suggested the reversible formation of HBDPPO and HBP, respectively, meaning that the starting adducts may decompose to benzaldehyde and the corresponding > P(O)H species. The products formed were identified by P-31 NMR spectroscopy, LC-MS and HRMS. [GRAPHICS]

Genetic alterations in key oncogenes have been frequently identified in lung adenocarcinoma (LUAD), including genes encoding epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK). In this pilot study, we aimed to characterize the differences in enriched biological pathways and phosphorylation events between LUAD tumors harboring EGFR, KRAS, or echinoderm microtubule-associated protein-like 4 (EML4)-ALK oncogenic alterations and triple wild-type LUAD tumors (WT, without EML4-ALK, KRAS, or EGFR alterations) by mass spectrometry (MS)-based quantitative proteomics and phosphoproteomics. We analyzed tumor regions of 82 formalin-fixed paraffin-embedded (FFPE) tissue sections with 6, 23, 31, and 22 samples from the EML4-ALK, EGFR, KRAS, and WT sample groups, respectively. A total of 1377 to 2189 proteins and 73 to 1781 phosphosites were quantified in these analyses. Based on the results, the samples clustered according to their genetic alteration type, and EGFR-mutated samples showed unique protein expression patterns. Membrane organization, vesicle organization, and vesicle-mediated transport Gene Ontology Biological Process (GOBP) terms were significantly downregulated in EGFR-mutated samples compared to the other sample groups. Changes in 36 proteins and 52 phosphosites were also identified as potentially specific to a given genetic alteration. Many of these proteins have previously been linked to EGFR or KRAS mutations [e.g., cathepsin L, stimulator of interferon genes protein (STING)], whereas several phosphoproteins are associated with RNA splicing [e.g., serine/arginine-rich splicing factor 1 (SRSF1), SRSF2, and SRSF7 proteins]. Kinase-substrate enrichment analysis indicated altered activities of 10 kinases, including mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs). For example, CDK2 activity was elevated in EML4-ALK samples compared to the other sample groups. Our results could provide significant insights into further studies that could contribute to developing improved diagnostic and therapeutic strategies for LUAD.

In order to make available new derivatives, diethyl α-amino-α-aryl-methylphosphonates were subjected to phosphorylation, phosphinoylation and even thiophosphinoylation by reaction with phosphoryl chlorides, diphenylphosphinoyl chloride, and with the mixture of diphenylchlorophosphine and elemental sulfur, respectively. The X-ray crystal structures of the diphenylphosphinoyl and the diphenylthiophosphinoyl derivatives revealed molecular and supramolecular similarities, as well as a few differences too. An essential conformation change, along with packing differences are attributable to a change of one heteroatom: an oxygen for a sulfur in one of the P=X function. The diethyl diethylphosphoryl-aminobenzylphosphonates showed the highest antiproliferative effects on multiple myeloma cells, while the thiophosphinoylated diethyl aminobenzylphosphonate was the most effective on pancreatic ductal adenocarcinoma cells.

In recent years, alternative enzymes with varied specificities have gained importance in MS-based bottom-up proteomics, offering orthogonal information about biological samples and advantages in certain applications. However, most mass spectrometric workflows are optimized for tryptic digests. This raises the questions of whether enzyme specificity impacts mass spectrometry and if current methods for nontryptic digests are suboptimal. The success of peptide and protein identifications relies on the information content of MS/MS spectra, influenced by collision energy in collision-induced dissociation. We investigated this by conducting LC-MS/MS measurements with different enzymes, including trypsin, Arg-C, Glu-C, Asp-N, and chymotrypsin, at varying collision energies. We analyzed peptide scores for thousands of peptides and determined optimal collision energy (CE) values. Our results showed a linear m/z dependence for all enzymes, with Glu-C, Asp-N, and chymotrypsin requiring significantly lower energies than trypsin and Arg-C. We proposed a tailored CE selection method for these alternative enzymes, applying ca. 20% lower energy compared to tryptic peptides. This would result in a 10-15 eV decrease on a Bruker QTof instrument and a 5-6 NCE% (normalized collision energy) difference on an Orbitrap. The optimized method improved bottom-up proteomics performance by 8-32%, as measured by peptide identification and sequence coverage. The different trends in fragmentation behavior were linked to the effects of C-terminal basic amino acids for Arg-C and trypsin, stabilizing y fragment ions. This optimized method boosts the performance and provides insight into the impact of enzyme specificity. Data sets are available in the MassIVE repository (MSV000095066).

A series of α-hydroxy-alkylphosphonates and α-hydroxy-alkylphosphine oxides were synthesized by the Pudovik reaction of acetaldehyde and acetone with dialkyl phosphites or diarylphosphine oxides. The additions were performed in three different ways: in liquid phase using triethylamine as the catalyst (1), on the surface of Al2O3/KF solid catalyst (2), or by a MW-assisted Na2CO3-catalyzed procedure (3). In most of the cases, our methods were more efficient and more robust than those applied in the literature. Two of the α-hydroxy-alkylphosphonates were subjected to single-crystal X-ray analysis, suggesting a dimeric and a chain supramolecular buildup in their respective crystals. Four α-hydroxy-alkylphosphonates and one α-hydroxy-ethylphosphine oxide were reacted with different acid chlorides to afford ten α-acyloxyphosphonates. Diethyl α-hydroxy-ethylphosphonate was transformed to the methanesulfonyloxy derivative that was useful in the Michaelis–Arbuzov reaction with triethyl phosphite and ethyl diphenylphosphinite to afford tetraethyl ethylidenebisphosphonate and diethyl α-(diphenylphosphinoyl)-ethylphosphonate, respectively. The α-hydroxyphosphonates and α-hydroxyphosphine oxides prepared were subjected to bioactivity studies, and the compounds tested exhibited limited cytotoxic effects on U266 cells with modest reductions in viability at a concentration of 100 μM.

A series of new dialkyl alpha-diethylphosphonoylethyl-alpha-hydroxy-ethylphosphonates were prepared using the Pudovik reaction of the corresponding gamma-oxophosphonate with dialkyl phosphites performed on the surface of Al2O3/KF. The adducts revealed unexpected reactivity in the attempted O-acylation reaction, and provided the corresponding 5-phosphonoyl-1,2-oxaphospholane 2-oxides. On treatment with Cs2CO3, instead of the expected rearrangement a cyclization reaction leading to the same ring products took place. Three of the phosphonoylethyl-alpha hydroxy-ethylphosphonates, along with two phosphonoylmethyl analogues revealed significant and selective anticancer effect on A431 cells, and occasionally, on PC-3 and MDA-MB 231 cells.