Improved Mobility-Stretchability Properties of Diketopyrrolopyrrole-Based Conjugated Polymers with Diastereomeric Conjugation Break Spacers.

Stretchable conjugated polymers with conjugation break spacers (CBSs) synthesized via random terpolymerization have gained considerable attention because of their efficacy in modulating mobility and stretchability. This study incorporates a series of dianhydrohexitol diastereomers of isosorbide (ISB) and isomannide (IMN) units into the diketopyrrolopyrrole-based backbone as CBSs. It is found that the distorted CBS (IMN) improves the mobility-stretchability properties of the polymer with a highly coplanar backbone, whereas the extended CBS (ISB) enhances those of the polymer with a noncoplanar backbone. Additionally, the different configurations of ISB and IMN sufficiently affect the solid-state packing, aggregation capabilities, crystallographic parameters, and mobility-stretchability properties of the polymer. The IMN-based polymers exhibit the highest mobility of 1.69 cm2 V-1 s-1 and crystallinity retentions of (85.7, 78.6)% under 20% and 60% strains, outperforming their ISB-based or unmodified counterparts. The improvement is correlated with a robust aggregation capability. Furthermore, the CBS content affects aggregation behavior, notably affecting mobility. This result indicates that incorporating CBSs into the polymer can enhance backbone flexibility via movement and rotation of the CBS without affecting the crystalline regions.

Differential performance regarding the relationship of C3-epi-25(OH)D3 levels and %C3-epi-25(OH)D3 with common pediatric diseases: a case control study.

BACKGROUND: Recently, the C3-epimer of 25-hydroxyvitamin D [C3-epi-25(OH)D] has become a topic of interest among 25-hydroxyvitamin D [25(OH)D] metabolites. Although it can lead to an overestimation of vitamin D storage, its relationship with disease occurrence remains controversial, possibly related to the great extent of tracking of 25(OH)D by C3-epi-25(OH)D over time. This study aimed to investigate the differential performance of C3-epi-25(OH)D3 and its percentage [%C3-epi-25(OH)D3] with respect to 20 common paediatric diseases. METHODS: This study involved 805 healthy children and adolescents and 2962 patients with common paediatric diseases. We investigated sex, age, and seasonal differences in C3-epi-25(OH)D3 and %C3-epi-25(OH)D3 levels; their variations on 20 common paediatric diseases; and their degree of correlation with 25(OH)D3 levels and various diseases. RESULTS: Among the healthy underage participants, C3-epi-25(OH)D3 and %C3-epi-25(OH)D3 changed similarly, with no sex differences. Moreover, their levels were higher in the infant period than in the other periods (t = 5.329-5.833, t = 4.640-5.711, all Padj < 0.001), and in spring and summer than in autumn and winter (t = 3.495-6.061, t = 3.495-5.658, all Padj < 0.01). Under healthy and disease conditions, C3-epi-25(OH)D3 was positively correlated with 25(OH)D3 (ρ = 0.318 ~ 0.678, all P < 0.017), whereas %C3-epi-25(OH)D3 was not, except in patients with nephrotic syndrome (ρ=-0.393, P = 0.001). Before and after adjusting for 25(OH)D3, the relationship of C3-epi-25(OH)D3 with the diseases was notably different. However, it was almost consistent for %C3-epi-25(OH)D3. Our results indicated that %C3-epi-25(OH)D3 was associated with short stature, nephrotic syndrome, lymphocytic leukaemia, rickets, paediatric malnutrition, and hypovitaminosis D (OR = 0.80 ~ 1.21, all P < 0.05). CONCLUSIONS: The %C3-epi-25(OH)D3 can correct the properties of C3-epi-25(OH)D3 to better track 25(OH)D3 and may be more suitable for exploring its pathological relevance. Further detailed studies of each disease should be conducted.

Biotransformation of docosahexaenoic acid into 10R,17S-dihydroxydocosahexaenoic acid as protectin DX 10-epimer by serial reactions of arachidonate 8R- and 15S-lipoxygenases.

Protectins, 10,17-dihydroxydocosahexaenoic acids (10,17-DiHDHAs), are belonged to specialized pro-resolving mediators (SPMs). Protectins are generated by polymorphonuclear leukocytes in humans and resolve inflammation and infection in trace amounts. However, the quantitative production of protectin DX 10-epimer (10-epi-PDX, 10R,17S-4Z,7Z,11E,13Z,15E,19Z-DiHDHA) has been not attempted to date. In this study, 10-epi-PDX was quantitatively produced from docosahexaenoic acid (DHA) by serial whole-cell biotransformation of Escherichia coli expressing arachidonate (ARA) 8R-lipoxygenase (8R-LOX) from the coral Plexaura homomalla and E. coli expressing ARA 15S-LOX from the bacterium Archangium violaceum. The optimal bioconversion conditions to produce 10R-hydroxydocosahexaenoic acid (10R-HDHA) and 10-epi-PDX were pH 8.0, 30 °C, 2.0 mM DHA, and 4.0 g/L cells; and pH 8.5, 20 °C, 1.4 mM 10R-HDHA, and 1.0 g/L cells, respectively. Under these optimized conditions, 2.0 mM (657 mg/L) DHA was converted into 1.2 mM (433 mg/L) 10-epi-PDX via 1.4 mM (482 mg/L) 10R-HDHA by the serial whole-cell biotransformation within 90 min, with a molar conversion of 60% and volumetric productivity of 0.8 mM/h (288 mg/L/h). To the best of our knowledge, this is the first quantitative production of 10-epi-PDX. Our results contribute to the efficient biocatalytic synthesis of SPMs.

Stereochemistry and antimalarial activity of C-10 carba analogues of artemisinin.

Artemisinin is an endoperoxide bond-containing sesquiterpene lactone showing potent antimalarial effect as well as antitumor and antivirus activities. Inspired by this unique pharmacorphore, researchers around the world developed numerous Artemisinin derivatives. Among these derivatives, the C-10 carba analogues of artemisinin are frequently reported. However, the stereochemistry of C-10 carba analogues of artemisinin is overlooked and the corresponding mixture of stereoisomers are used. Herein, we reported for the first time stereochemistry and antimalarial activity of C-10 carba analogues of artemisinin. We employed two approaches to obtain the pure isomer of C-10 carba analogues and presented an interesting observation about their antimalarial activities. The minor isomer with large-sized substitute and S configuration at C-10 position had much lower antimalarial effect than the major isomer with R configuration. The study will shed light on the development of effective antimalarial drugs based on ART.

Dynamics of the vitamin D C3-epimer levels in preterm infants.

OBJECTIVES: The primary objective was to determine levels of C3-epi-25(OH)D in very low birth weight infants. The secondary objective was to evaluate the possible influence of preterm birth, intrauterine growth restriction (IUGR), and season of birth on the production of C3-epimers. METHODS: A total of 127 infants with birth weight less than 1,500 g met the inclusion criteria of the study. We examined 25-hydroxyvitamin-D [25(OH)D] levels and C3-epi-25(OH)D in maternal serum before labor, and in cord blood and infants' serum on days 14 and 28, and at discharge. RESULTS: The mean levels (±SD) of C3-epi-25(OH)D of the cord, on day 14, on day 28, and at discharge were 2.2 (2.9), 7.7 (5.5), 11.7 (7.6) and 14.9 (11.7) nmol/L respectively. The proportion of total 25(OH)D as the C3-epimer was 6.9% (cord), 16.3% (day 14), 22.4% (day 28) and 23.3% (discharge). A statistically significant correlation between 25(OH)D and C3-epi-25(OH)D can be demonstrated from birth. The severity of immaturity and IUGR did not affect the production of C3-epimers. In summer/autumn vs. winter/spring, the mean (SD) percentage of total 25(OH)D as the C3-epimer significantly differs only in maternal serum samples and umbilical cord samples (p value <0.001). CONCLUSIONS: The production of C3-epi-25(OH)D is functional even in the most immature newborns, has fetal origins, and is largely dependent on circulating 25(OH)D. At the end of the first month of life, C3-epimers make up more than 20% of 25(OH)D.

Thiolactones and Δ8,9-Pregnene Steroids from the Marine-Derived Fungus Meira sp. 1210CH-42 and Their α-Glucosidase Inhibitory Activity.

The fungal genus Meira was first reported in 2003 and has mostly been found on land. This is the first report of second metabolites from the marine-derived yeast-like fungus Meira sp. One new thiolactone (1), along with one revised thiolactone (2), two new Δ8,9-steroids (4, 5), and one known Δ8,9-steroid (3), were isolated from the Meira sp. 1210CH-42. Their structures were elucidated based on the comprehensive spectroscopic data analysis of 1D, 2D NMR, HR-ESIMS, ECD calculations, and the pyridine-induced deshielding effect. The structure of 5 was confirmed by oxidation of 4 to semisynthetic 5. In the α-glucosidase inhibition assay, compounds 2-4 showed potent in vitro inhibitory activity with IC50 values of 148.4, 279.7, and 86.0 µM, respectively. Compounds 2-4 exhibited superior activity as compared to acarbose (IC50 = 418.9 µM).

Chiral Separation, Configuration Confirmation and Bioactivity Determination of the Stereoisomers of Hesperidin and Narirutin in Citrus reticulata Blanco.

Hesperidin and narirutin are a class of flavanone glycosides, which are the main active constituents in Citrus reticulata Blanco. In the present study, a chiral HPLC-UV method with amylose tris(3,5-dimethylphenylcarbamate) as a stationary phase under a normal-phase mode was used to achieve the stereoselective separation of the C-2 diastereomers of hesperidin and narirutin simultaneously. The single epimer was then successfully prepared by applying semi-preparative chromatography, whose absolute configuration (R/S) was characterized by combining the experimental electronic circular dichroism (ECD) detection with time-dependent density functional theory (TDDFT) calculations. The epimer composition of these two chiral flavanone glycosides in Citrus reticulata Blanco was then determined, which was found to be slightly different in the herbs from different production regions. The anti-inflammatory activity of each prepared single epimer was further evaluated, and some differences between one pair of epimers of hesperidin and narirutin were observed, which suggested that the presence of different epimers should be considered in the quality evaluation and control of natural medicine.

[Applying Serum Vitamin D Metabolites in the Assessment of Renal Impairment in Diabetic Kidney Disease of Type 2 Diabetes Mellitus Patients: A Retrospective Study].

Objective: Total 25(OH)D (t-25[OH]D), a marker traditionally used in the assessment of vitamin D (VitD) in the human body, includes 25(OH)D 2, 25(OH)D 3, and C 3-epimers-25(OH)D 3(C 3-epi). In this study, we analyzed the relationship between serum VitD metabolites and renal impairment in patients with diabetic kidney disease (DKD). Methods: We covered, in the study, 339 subjects, including 114 otherwise healthy controls (HC), 74 type 2 diabetes mellitus (DM) patients with no glomerular filtration dysfunction, and 151 DKD patients. According to the results of combined evaluation of estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), the DKD patients were further divided into four subgroups, stage 2 subgroup of patients of DM combined with stage-2 chronic kidney disease (CKD2), stage 3 subgroup of patients of DM combined with CKD3, stage 4 subgroup of patients of DM combined with CKD4, and stage 5 subgroup of patients of DM combined with CKD5. The levels of 25(OH)D 2, 25(OH)D 3, and C 3-epi were measured by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the activity level of 25(OH) 3 (AVitD 3), t-25(OH)D concentration, 25(OH)D 2/25(OH)D 3 ratio, C 3-epi/t-25(OH)D ratio, and C 3-epi/AVitD 3 ratio were calculated. Results: The levels of 25(OH)D 3, t-25(OH)D, and AVitD 3 were lower in the DKD group than those in the DM and HC groups (all P<0.05). C 3-epi/t-25(OH)D ratio and C 3-epi/AVitD 3 ratio were higher in the DKD group than those in the HC group (all P<0.05). The levels of 25(OH)D 3, t-25(OH)D, AVitD 3, and C 3-epi were lower in the stage 5 subgroup than those in the stage 2 and stage 3 subgroups (all P<0.05). The levels of 25(OH)D 3, t-25(OH)D, and C 3-epi were lower in the stage 4 subgroup than those in the stage 3 subgroup (all P<0.05). The 25(OH)D 3, t-25(OH)D, and AVitD 3 levels were lower in the stage 4 subgroup than those in the stage 2 subgroup (all P<0.05). Conclusions: UPLC-MS/MS can be used to perform accurate evaluation of VitD nutritional status in DKD patients. DKD patients have decreased levels of serum t-25(OH)D, 25(OH)D 3, and AVitD 3, all of which progressively decrease along with the rise in CKD staging. The trend of C 3-epi and 25(OH)D 3 changes were not consistent.

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation.

INTRODUCTION: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites-vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)2D (1α25(OH)2D2, and 1α25(OH)2D3)-including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. METHOD: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. RESULTS: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze-thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)2D, and 7αC4 between SDD and SDL group. CONCLUSIONS: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D's metabolites and their epimers.

Isolation of N-Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography.

N-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5'''R- and 5'''S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-EGCG-cThea and S-EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) and 2. N-hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v/v/v/v) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio KD values, selectivity factors α, and resolution factors RS. After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R-EGCG-cThea and 2.9 mg of S-EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R- and S-EGCG-cThea possessed in vitro inhibition effects on α-glucosidase with IC50 of 70.3 and 161.7 µM, respectively.

[A novel dammarane-type saponin from Gynostemma pentaphyllum and its neuroprotective effect].

One new and two known dammarane-type saponins were isolated from the leaves of Gynostemma pentaphyllum using various chromatographic methods. Their structures were identified by HR-ESI-MS,~( 1)H-NMR, ~(13)C-NMR, 2 D-NMR spectra as 2α,3ß,12ß,20,24(S)-tetrahdroxydammar-25-en-3-O-[ß-D-glucopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-ß-D-xylopyranosyl(1→6)-ß-D-glucopyranoside(1, a new compound, namely gypenoside J5) and 2α,3ß,12ß,20,24(R)-tetrahdroxydammar-25-en-3-O-[ß-D-glucopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-ß-D-xylopyranosyl(1→6)-ß-D-glucopyranoside(2) and 2α,3ß,12ß,20-tetrahydroxy-25-hydroperoxy-dammar-23-en-3-O-[ß-D-glucopyranosyl(1→2)][ß-D-glucopyranosyl]-20-O-[ß-D-xylopyranosyl(1→6)]-ß-D-glucopy-ranoside(3), respectively. Compounds 1 and 2 were a pair of C-24 epimers. All compounds showed weak cytotoxicity agxinst H1299, HepG2, PC-3, SH-SY5 Y cancer cell lines. However, they exerted protective effect against SH-SY5 Y cellular damage induced by H_2O_2 dose-dependently, of which compound 1 displayed the strongest antioxidant effect. The present study suggested that G. pentaphyllum has antioxidative potential and the saponins from G. pentaphyllum are considered as the active compounds with neuroprotecitve effect.

[Separation and structural elucidation of C25 epimers of furostanol saponin from aerial parts of Paris polyphylla var. chinensis].

Paris polyphylla var. chinensis(PPC) is used as one of the origin plants of Paridis Rhizoma described in the Chinese Pharmacopoeia(2020 edition). Its resources shortage makes the planting scale gradually expand, and plenty of aerial parts are abandoned because of not being effectively used. On the basis of previous research, this study separated steroidal saponins to further clarify the chemical composition of the aerial parts of PPC. As a result, three pairs of 25R or 25S epimers of furostanol saponins were obtained by various column chromatography techniques. Their structures were identified as neosolanigroside Y6(1), solanigroside Y6(2), neoprotogracillin(3), protogracillin(4), neoprotodioscin(5) and protodioscin(6) by spectral data combining with chemical transformation. Compound 1 is a new compound, and compounds 2, 3 and 5 are isolated from Paris plants for the first time. Compounds 4 and 6 are isolated from this plant for the first time. Previously, only several spirostanol glycosides with 25S configuration were isolated from Paris plants. Guided by mass spectrometry, the present study isolated the furostanol saponins with 25S configuration from this genus for the first time, which further enriches the chemical information of Paris genus and provides a reference for the isolation of similar compounds.

Structural and functional consequences of age-related isomerization in α-crystallins.

Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here, we examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity.

Epimers of Vitamin D: A Review.

In this review, we discuss the sources, formation, metabolism, function, biological activity, and potency of C3-epimers (epimers of vitamin D). We also determine the role of epimerase in vitamin D-binding protein (DBP) and vitamin D receptors (VDR) according to different subcellular localizations. The importance of C3 epimerization and the metabolic pathway of vitamin D at the hydroxyl group have recently been recognized. Here, the hydroxyl group at the C3 position is orientated differently from the alpha to beta orientation in space. However, the details of this epimerization pathway are not yet clearly understood. Even the gene encoding for the enzyme involved in epimerization has not yet been identified. Many published research articles have illustrated the biological activity of C3 epimeric metabolites using an in vitro model, but the studies on in vivo models are substantially inadequate. The metabolic stability of 3-epi-1α,25(OH)2D3 has been demonstrated to be higher than its primary metabolites. 3-epi-1 alpha, 25 dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3) is thought to have fewer calcemic effects than non-epimeric forms of vitamin D. Some researchers have observed a larger proportion of total vitamin D as C3-epimers in infants than in adults. Insufficient levels of vitamin D were found in mothers and their newborns when the epimers were not included in the measurement of vitamin D. Oral supplementation of vitamin D has also been found to potentially cause increased production of epimers in mice but not humans. Moreover, routine vitamin D blood tests for healthy adults will not be significantly affected by epimeric interference using LC-MS/MS assays. Recent genetic models also show that the genetic determinants and the potential factors of C3-epimers differ from those of non-C3-epimers.Most commercial immunoassays techniques can lead to inaccurate vitamin D results due to epimeric interference, especially in infants and pregnant women. It is also known that the LC-MS/MS technique can chromatographically separate epimeric and isobaric interference and detect vitamin D metabolites sensitively and accurately. Unfortunately, many labs around the world do not take into account the interference caused by epimers. In this review, various methods and techniques for the analysis of C3-epimers are also discussed. The authors believe that C3-epimers may have an important role to play in clinical research, and further research is warranted.

Anti-Angiogenic Properties of Ginsenoside Rg3.

Ginsenoside Rg3 (Rg3) is a member of the ginsenoside family of chemicals extracted from Panax ginseng. Like other ginsenosides, Rg3 has two epimers: 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3). Rg3 is an intriguing molecule due to its anti-cancer properties. One facet of the anti-cancer properties of Rg3 is the anti-angiogenic action. This review describes the controversies on the effects and effective dose range of Rg3, summarizes the evidence on the efficacy of Rg3 on angiogenesis, and raises the possibility that Rg3 is a prodrug.

[Determination of 25-hydroxyl vitamin D in serum by isotope dilution ultra-fast liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a rapid and accurate method for determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3 in serum by isotope dilution ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS). METHODS: The serum sample was extracted by nhexane after methanol/acetonitrile precipitation protein, and then the extract was concentrated by nitrogen and volumed with the primary mobile phase. The chromatographic separation was carried out on a Phenomenex Kinetex F5 column(2. 1 mm × 150 mm, 1. 7 µm) by using 0. 1%(V/V) formic acid and 0. 1%(V/V) formic acid/methanol solution as the mobile phase with the gradient elution. Detection was performed in positive multi-reaction monitoring(MRM) mode with the isotope internal labeling method for quantification. RESULTS: The baseline separation was obtained within 6 min for the epimer 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3, and the accurate qualification was obtained for the simultaneous determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3. The three analytes showed good linear relationship within the range of 0. 5-50. 0 µg/L with a correlation coefficient r >0. 9995. The limits of detection(LODs) and the limits of quantitation(LOQs) of the method were 0. 15 µg/L and 0. 5 µg/L, respectively. The recoveries of the method were84. 3%-109. 0%(n = 11) at the three spiked levels of 1. 0, 10. 0 and 30. 0 µg/L, and the relative standard deviations(RSDs) were between 0. 8%-6. 8%. At the same time, the certified standard reference materials(SRM) of the America National Institute of Standards and Technology(NIST) Level 1, Level 2, Level 3 and Level 4(SRM 972 a)were used as the quality control samples for verification, the relative deviations of the measurement result were less than 5% compared with the reference values. CONCLUSION: The developed method has the characteristics of simplicity, rapidity, sensitivity and accuracy, and is suitable for the simultaneous rapid determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3 in serum.

The C-3α Epimer of 25-Hydroxycholecalciferol from Endogenous and Exogenous Sources Supports Normal Growth and Bone Mineral Density in Weanling Rats.

BACKGROUND: The C-3α epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] is elevated in infants. OBJECTIVES: We tested whether increasing cholecalciferol intake results in a dose-response in plasma 3-epi-25(OH)D3 We also examined bone and mineral metabolism in response to 3-epi-25(OH)D3 treatment. METHODS: Sprague Dawley rats (4 wk old) were randomly assigned (n = 6/group of each sex) to AIN-93G diets with cholecalciferol at 1 (control), 2, or 4 IU/g diet for objective 1 and to diets with 3-epi-25(OH)D3 at 0.5 or 1 IU/g diet or 25-hydroxycholecalciferol [25(OH)D3] at 0.5 IU/g diet for objective 2 for 8 wk. Measurements at weeks 0, 4, and 8 included body weight and length, plasma vitamin D metabolites, bone biomarkers, and bone mineral density determined by using dual-energy X-ray absorptiometry. Lumbar vertebra 3 (L3) geometry and volumetric bone mineral density (vBMD) were measured using microcomputed tomography. Differences between groups were identified for males and females separately. RESULTS: Weight and food intake were not different between groups. Elevated plasma 3-epi-25(OH)D3 was observed only in females in the 4 IU cholecalciferol/g diet group (mean ± SD: 24.7 ± 17.1 ng/mL), compared with the control group (5.3 ± 1.4 ng/mL; P = 0.001). By week 8, both male and female rats in the 3-epi-25(OH)D3 groups had >87% greater plasma 3-epi-25(OH)D3 concentrations relative to the 25(OH)D3 reference group (P < 0.0001). At week 8 in males only, parathyroid hormone was significantly lower (P = 0.019) in both 3-epi-25(OH)D3 groups than in the 25(OH)D3 group, and L3 total vBMD was higher (P = 0.004) in the 0.5 IU 3-epi-25(OH)D3 group than in the 25(OH)D3 group. CONCLUSIONS: Endogenously generated 3-epi-25(OH)D3 is more prominent in female than in male rats. Exogenous 3-epi-25(OH)D3 was as effective as 25(OH)D3 in supporting bone mineral accretion in both sexes. It thus appears that 3-epi-25(OH)D3 has biological activity and should be further explored.

Design, synthesis and structure-bactericidal activity relationships of novel 9-oxime ketolides and reductive epimers of acylides.

Erythromycin was long viewed as a bacteriostatic agent. The erythromycin derivatives, 9-oxime ketolides have a species-specific bactericidal profile. Among them, the 3'-allyl version of the 9-oxime ketolide 1 (Ar=3-quinolyl; 17a) is bactericidal against Streptococcus pneumoniae and Streptococcus pyogenes. In contrast, the 2-fluoro analogs of 1, 13a (Ar=6-quinolyl), 13b (Ar=3-quinolyl) and 24a (Ar=4-isoquinolyl), show bactericidal activities against S. pneumoniae, Staphylococcus aureus and Moraxella catarrhalis, while the 2-fluoro analogs 13c (Ar=3-aminopyridyl) and 24b (Ar=3-carbamoylpyridyl) are only bactericidal against S. pneumoniae and Haemophilus influenzae. Reduction of the ketolides led to novel epiacylides, the 3-O-epimers of the acylides. Alteration of linker length (30b vs. 30a), 2-fluorination (33 vs. 30a) and incorporation of additional spacers at the 9-oxime or 6-OH (35, 40 vs. 30a) did not restore the epiacylides back to be as active as the acylide 31. Molecular docking suggested that epimerization at the 3-position reshapes the orientation of the 3-O-sidechain and leads to considerably weaker binding with bacterial ribosomes.

A new pre-column derivatization for valienamine and beta-valienamine using o-phthalaldehyde to determine the epimeric purity by HPLC and application of this method to monitor enzymatic catalyzed synthesis of beta-valienamine.

Valienamine and ß-valienamine are representative C7 N aminocyclitols with significant glycosidase inhibition activity that have been developed as important precursors of drugs for diabetes and lysosomal storage diseases, respectively. The quantitative analysis of these chiral compounds is crucial for asymmetric in vitro biosynthetic processes for converting valienone into valienamine epimers using aminotransferase. Here, we developed an efficient and sensitive method for separation and quantitative analysis of chiral valienamine using reversed-phase high-performance liquid chromatography (HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of the analytes. The epimers were derivatized by OPA in borate buffer (pH 9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18 (5 µm, 4.6 × 150 mm) column, eluted with 22% acetonitrile at 30 °C for 18 min, and detected by a fluorescence detector using 445 nm emission and 340 nm excitation wavelengths. The average resolution of the epimers is 3.86, and the concentration linearity is in the range of 0.02-20 µg/ml. The method proved to be effective, sensitive, and reliable with good intra- and inter-day precision and accuracy, and successfully evaluated the enantiopreference and catalytic capability of the potential aminotransferases on an unnatural prochiral substrate, facilitating the design of an asymmetric biosynthetic route for optically pure valienamine and ß-valienamine.

In vitro stereospecific hydration activities of the 3-vinyl group of chlorophyll derivatives by BchF and BchV enzymes involved in bacteriochlorophyll c biosynthesis of green sulfur bacteria.

The photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum produces bacteriochlorophyll (BChl) c pigments bearing a chiral 1-hydroxyethyl group at the 3-position, which self-aggregate to construct main light-harvesting antenna complexes, chlorosomes. The secondary alcoholic hydroxy group is requisite for chlorosomal aggregation and biosynthesized by hydrating the 3-vinyl group of their precursors. Using recombinant proteins of Cba. tepidum BchF and BchV, we examined in vitro enzymatic hydration of some 3-vinyl-chlorophyll derivatives. Both the enzymes catalyzed stereoselective hydration of zinc 3-vinyl-8-ethyl-12-methyl-bacteriopheophorbide c or d to the zinc 31 R-bacteriopheophorbide c or d homolog, respectively, with a slight amount of the 31 S-epimric species. A similar R-stereoselectivity was observed in the BchF-hydration of zinc 3-vinyl-8-ethyl- and propyl-12-ethyl-bacteriopheophorbides c, while their BchV-hydration gave a relatively larger amount of the 31 S-epimers. The in vitro stereoselective hydration confirmed the in vivo production of the S-epimeric species by BchV. The enzymatic hydration for the above 8-propylated substrate proceeded more slowly than that for the 8-ethylated, and the 8-isobutylated substrate was no longer hydrated. Based on these results, biosynthetic pathways of BChl c homologs and epimers are proposed.