Prolonged infective SARS-CoV-2 omicron variant shedding in a patient with diffuse large B cell lymphoma successfully cleared after three courses of remdesivir.

We report a case of prolonged shedding of the infective SARS-CoV-2 omicron variant BA.1.1.2 in a 79-year-old male patient with diffuse large B-cell lymphoma, after receiving chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was admitted to our hospital in late March 2022 for the sixth course of R-CHOP chemotherapy. Initially, the patient tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using an in-hospital loop-mediated amplification assay with a nasopharyngeal swab, both on the day of admission and three days later. However, the patient developed fever and was diagnosed with coronavirus disease (COVID-19) six days after admission and was suspected to have contracted the infection in the ward. Viral shedding continued for more than three months, with confirmed viral infectivity. As compared to the original Wuhan-Hu-1/2019 strain, amino acid substitutions including S36 N in non-structural protein (NSP)2, S148P, S1265del and L1266I in NSP3, G105D in NSP4, G496S, A831V, or V987F in spike protein, and I45T in open-reading frame (ORF)9b were randomly detected in isolated viruses. Although the patient had received two doses of the BNT162b2 vaccine approximately six months earlier and the third dose on day 127 after the infection, both serum anti-spike and anti-nuclear protein IgG and IgM tests were negative at day 92, 114, and 149 after the infection. The patient finally cleared the virus after the third course of remdesivir and did not have further recurrence.

Development of a highly sensitive in vitro system to detect and discriminate between vitamin D receptor agonists and antagonists based on split-luciferase technique.

Split-luciferase techniques are widely used to detect protein-protein interaction and bioactive small molecules including some hormones and vitamins. Previously, we successfully expressed chimeric proteins of luciferase and the ligand binding domain (LBD) of the vitamin D receptor (VDR), LucC-LBD-LucN in COS-7 cells. The LucC-LBD-LucN biosensor was named split-luciferase vitamin D biosensor (SLDB). This biosensor can detect and discriminate between VDR agonists and antagonists in mammalian cells. In this study, we established an in vitro screening system for VDR ligands using the SLDB proteins expressed in Escherichia coli (E. coli) cells. Our in vitro screening system using cell lysate of recombinant E. coli cells could be completed within 30min, and its activity was unchanged after 10 freeze-thaw cycles. This highly sensitive and convenient system would be quite useful to screen VDR ligands with therapeutic potential for various bone-related diseases, age-related cognitive disorders, cancer, and immune disorders. In addition, our system might be applicable to diagnostic measurement of serum concentrations of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3.

Nickel-catalyzed hydrocarboxylation of ynamides with CO2 and H2O: observation of unexpected regioselectivity.

We describe the nickel-catalyzed hydrocarboxylation of ynamides with CO2 and H2O to afford a variety of α-amino-α,ß-unsaturated esters with high regioselectivities. The selective α-carboxylation of ynamides with this catalytic protocol is unexpected in view of the electronic bias of ynamides and is in sharp contrast to our previous study in which a stoichiometric amount of Ni(0) was used to form a ß-carboxylated product exclusively. We revealed that this unexpected C-C bond formation was induced by the combination of Zn and MgBr2.

Enantioselective synthesis of ß-amino acid derivatives via nickel-promoted regioselective carboxylation of ynamides and rhodium-catalyzed asymmetric hydrogenation.

We succeeded in the development of a new method for enantioselective synthesis of α-substituted-ß-amino acid derivatives. Thus, nickel(0)-promoted carboxylation of ynamide gave the α-substituted-ß-aminoacrylate derivative in a highly regioselective manner. Then, rhodium-catalyzed asymmetric hydrogenation of the α-substituted ß-aminoacrylate produced the corresponding α-substituted ß-amino acid derivative as an optically active form.

"Inverse" thermoresponse: heat-induced double-helix formation of an ethynylhelicene oligomer with tri(ethylene glycol) termini.

An ethynylhelicene oligomer [(M)-d-4]-C12-TEG with six tri(ethylene glycol) (TEG) groups at the termini was synthesized, and double-helix formation was studied using CD, UV-Vis, vapor pressure osmometry, dynamic light scattering, and 1H NMR. [(M)-d-4]-C12-TEG reversibly changed its structure between a double helix and a random coil in response to heating and cooling in aromatic solvents, non-aromatic polar organic solvents, and aqueous solvent mixtures of acetone/water/triethylamine. Notably, [(M)-d-4]-C12-TEG in acetone/water/triethylamine (1/2/1) formed a double helix upon heating and disaggregated into random coils upon cooling. The double helix/random coil ratio sharply changed in response to temperature changes. This is an unprecedented "inverse" thermoresponse, which is opposite to the "ordinary" thermoresponse in molecular dimeric aggregate formation. This phenomenon was explained by the dehydration of the terminal TEG groups and the formation of condensed triethylamine domains upon heating.

Development of Novel Bioluminescent Sensor to Detect and Discriminate between Vitamin D Receptor Agonists and Antagonists in Living Cells.

Active forms of vitamin D regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via the vitamin D receptor (VDR). Many vitamin D analogs have been synthesized for clinical use in the treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. We have constructed two fusion proteins containing split-luciferase and the ligand binding domain (LBD) of the VDR designated as LucN-LBD-LucC and LucC-LBD-LucN. Remarkably, the LucC-LBD-LucN, which has the C-terminal domain of luciferase at the N-terminus of the fusion protein, was a significantly better biosensor than LucN-LBD-LucC. Addition of the VDR agonists to COS-7 cells expressing LucC-LBD-LucN dramatically reduced luciferase activity. In contrast, the VDR antagonist significantly increased the chimeric luciferase activity in a dose- and time-dependent manner. Our results on chimeric luciferases containing the LBDs of mutant VDRs derived from patients with vitamin D-dependent type II rickets indicated that our system could detect a conformational change of the LBD of the VDR likely based on a positional change of the helix 12, which occurs upon ligand binding. This novel system to detect and discriminate between VDR agonists and antagonists could be useful for the screening and identification of chemical compounds that bind to normal or mutant VDRs with high affinity.

Nickel-promoted highly regioselective carboxylation of aryl ynol ether and its application to the synthesis of chiral ß-aryloxypropionic acid derivatives.

Nickel(0)-promoted carboxylation of aryl ynol ether proceeded in a highly regioselective manner to produce α-substituted-ß-aryloxyacrylic acid derivatives. The α-substituted-ß-aryloxyacrylic acids were transformed into the corresponding ß-aryloxypropionic acid derivative as an optically active form via rhodium-catalyzed asymmetric hydrogenation.

Human cytochrome P450-dependent differential metabolism among three 2α-substituted-1α,25-dihydroxyvitamin D(3) analogs.

Our previous studies revealed that C2α-substituted-1α,25(OH)(2)D(3) analogs had unique biological activities. For example, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10), which has a high affinity for vitamin D receptor (VDR), is more bioavailable and more potent than 1α,25(OH)(2)D(3) in inhibiting cancer cell growth and invasion because of its weaker binding to vitamin D binding protein (DBP), and more resistance to CYP24A1-dependent metabolism. In this study, we examined the metabolism of MART-10 and two other 2α-substituted analogs, 2α-(3-hydroxypropoxy)-1α,25(OH)(2)D(3) (O2C3) and 2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (O1C3) by using human liver microsomes and human P450s. We demonstrated that O2C3 was converted to 1α,2α,25(OH)(3)D(3) in human liver microsomes, whereas both O1C3 and MART-10 were hardly metabolized. The metabolism of O2C3 was significantly inhibited by ketoconazole, and the recombinant human CYP3A4 converted O2C3 to 1α,2α,25(OH)(3)D(3), which suggests that CYP3A4 is responsible for the metabolism of O2C3 in human liver. The k(cat)/K(m) values of CYP3A4 for O1C3 and MART-10 are much smaller than that for O2C3. The k(cat)/K(m) values of human CYP24A1 for the three analogs are 1% (MART-10), 3% (O2C3), and 4% (O1C3) of that for 1α,25(OH)(2)D(3), indicating that MART-10 is the most resistant to CYP24A1 hydroxylation. On the other hand, 1α,2α,25(OH)(3)D(3), the metabolite of O2C3 by CYP3A4, was metabolized by CYP24A1 via multiple pathways similar to 1α,25(OH)(2)D(3), which suggests that O2C3 can be metabolized by two sequential hydroxylations, first by CYP3A4 and then by CYP24A1 in human body. These results suggest that modification at C-2α position and C-19 demethylenation markedly change metabolic profiles and biological activities of vitamin D analogs.

C15-functionalized 16-ene-1α,25-dihydroxyvitamin D3 is a new vitamin D analog with unique biological properties.

The Δ(16) structure as a vitamin D analog enhanced vitamin D receptor (VDR) binding affinity and induced significant cell differentiation, whereas its relative calcemic activity was reduced compared to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)). Methodologies available to introduce a double bond at C16-C17 of the D-ring on the seco-steroidal skeleton were limited; therefore, a new synthetic strategy was developed to obtain not only the Δ(16) structure, but also a new C15-functional group. Since C15-functionalization was unprecedented in vitamin D analog studies, the hybrid structure of Δ(16) and the C15-OH group at the D-ring may provide important information on the structure-activity relationship with vitamin D analogs. The synthesized 16-ene-2α-methyl-1α,15α,25-trihydroxyvitamin D(3) showed almost 3-times higher VDR binding affinity and an equipotent level of osteocalcin promoter transactivation activity in human osteosarcoma cells as compared to 1α,25(OH)(2)D(3).

The difference between 14-epi-previtamin D3 and 14-epi-19-norprevitamin D3: their synthesis and binding affinity for human VDR.

The synthesis of 14-epi-1α,25(OH)(2)previtamin D(3), 14-epi-19-nor-1α,25(OH)(2)previtamin D(3), and their 2-substituted analogs is described. The vitamin D receptor (VDR) binding affinity was further evaluated and 2α-methyl substituted 14-epi-1α,25(OH)(2)previtamin D(3) had 17-fold more potent affinity than 14-epi-1α,25(OH)(2)previtamin D(3).In the comparison of these compounds, the effects of thermal equilibrium, with or without 19-carbon at the A-ring, and their CD-ring structures are discussed.

Absorption of 6-O-caffeoylsophorose and its metabolites in Sprague-Dawley rats detected by electrochemical detector-high-performance liquid chromatography and electrospray ionization-time-of-flight-mass spectrometry methods.

Absorption and metabolism of a natural compound, 6-O-caffeoylsophorose (CS) from acylated anthocyanins in a red vinegar fermented with purple sweet potato, were clarified. The absorption of CS and conjugated CS in blood from orally administrated Sprague-Dawley rats at a dose of 400 mg/kg was investigated by electrochemical detection-high performance liquid chromatography. As a result, CS was successfully detected in rat plasma (AUC(0-6h), 108.6 ± 8.1 nmol h/mL) and was found to be an intact absorbable polyphenol. In addition, half of the absorbed CS was detected as its conjugates (AUC(0-6h), 50.7 ± 5.7 nmol h/mL) as well as caffeic and ferulic acids from CS. By a time-of-flight-mass spectrometric analysis of CS-administered plasma sample, glucuronide and methylated conjugates of CS were identified, in addition to glucuronide, methylated, or sulfate conjugates of caffeic and ferulic acids. Consequently, CS was absorbed in intact form into rat blood and partly degraded to caffeic and ferulic acids or metabolized by glucuronidation, methylation, or sulfatation.

Regio- and stereoselective synthesis of 2-amino-1,3-diene derivatives by ruthenium-catalyzed coupling of ynamides and ethylene.

A ruthenium-catalyzed hydrovinylation-type cross-coupling of ynamides and ethylene proceeds via ruthenacyclopentene to give 2-aminobuta-1,3-diene derivatives in a highly regioselective manner. It was also demonstrated that 2-aminobuta-1,3-diene derivatives reacted with various dienophiles or singlet oxygen to give a cyclic enamide derivative.

A facile construction of bi- or tricyclic skeletons by nickel-catalyzed stereoselective cyclization of alkynylcycloalkanone.

A nickel-catalyzed intramolecular cyclization of alkynylalkanone in the presence of Et(3)SiH using an NHC ligand produced various carbo- and heterocycles in a stereoselective manner. The reaction would proceed via formation of oxanickelacycle followed by sigma-bond metathesis with silane to give a bi- or tricyclic compound.

Synthesis of 2alpha-substituted-14-epi-previtamin D3 and its genomic activity.

We synthesized and isolated 2 alpha-substituted analogs of 14-epi-previtamin D3 after thermal isomerization at 80 degrees C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2 alpha-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D3.

Nickel-catalyzed [2+2+2] cycloaddition of arynes and an unactivated alkene: synthesis of 9,10-dihydrophenanthrene derivatives.

A nickel-catalyzed [2+2+2] cycloaddition of two molecules of aryne and an alkene moiety in a alpha,omega-diene afforded 9,10-dihydrophenanthrene derivatives in good yields.

Nickel-catalyzed highly regioselective multicomponent coupling of ynamides, aldehydes, and silane: a new access to functionalized enamides.

A new method for preparation of functionalized enamides by a nickel-catalyzed multicomponent coupling of ynamides, aldehydes, and silane has been developed. The coupling reaction proceeded in the presence of a nickel-IMes catalyst to give the corresponding gamma-silyloxyenamide derivative, which has an allylic alcohol moiety in the molecule, in a highly stereoselective manner.

(2S,2'R)-analogue of LG190178 is a major active isomer.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of the skin, prostate, colon, and breast as well as leukemia. LG190178 is a novel non-secosteroidal ligand for VDR. We synthesized and evaluated stereoisomers of LG190178 and found that only an (2S,2'R)-analogue of LG190178 (YR301) had strong activity.

Nickel-catalyzed enantio- and diastereoselective three-component coupling of 1,3-dienes, aldehydes, and silanes using chiral N-heterocyclic carbenes as ligands.

Nickel(0)-catalyzed asymmetric three-component coupling of 1,3-dienes, aldehydes, and silanes has been realized utilizing a chiral N-heterocyclic carbene as a ligand. On the basis of the screening of various NHC precursors, an imidazolium salt having 1-(2,4,6-trimethylphenyl)propyl groups on the nitrogen was designed and synthesized. In this reaction, various coupling products were produced in good yields with high regio-, diastereo- (anti selective in the case of the internal 1,3-diene), and enantioselectivities (up to 97% ee).

Creative synthesis of novel vitamin D analogs for health and disease.

We report new analogs of 1alpha,25-dihydroxyvitamin D(3) (1) in three categories. First, design and synthesis of ligands for a mutant vitamin D receptor (VDR)(Arg274Leu), which possess proper functional groups at both C1alpha and C2alpha positions of 1 to study the biological activity of the mutant VDR. Among our synthetic analogs, 1alpha-methyl-2alpha-(3-hydroxypropyl)-25-hydroxyvitamin D(3) (8) showed 7.3-fold greater transcriptional activity for the VDR(Arg274Leu) than that of 1. Next, we examined the antiproliferative activity of 2-substituted 19-norvitamin D(3) analogs on an immortalized normal prostate cell line, PZ-HPV-7, and we found MART 10 (14) showed the activity even at very low concentration of 10(-10) to 10(-11)M. We also synthesized 25-hydroxy-19-norvitamin D(3) (13) using Julia-type olefination to connect between the C5 and C6 positions, effectively, to test it as a prohormone type agent for antiprostate diseases. Synthesized compound 13 showed potent antiproliferative activity in PZ-HPV-7, which has high 1alpha-hydroxylase activity. Finally, we describe design and synthesis of a new TEI-9647 analog, 2alpha-(3-hydroxypropoxy)-24-propyl-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (17), which showed the strongest VDR antagonism. Its IC(50) value is 7.4pM to inhibit differentiation of HL-60 cells induced by 10nM of 1.