Synthesis of 1β‐hydroxydeoxycholic acid in H‐2 and unlabeled forms

1β–hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1‐step oxidation from deoxycholic acid and [D₄]deoxycholic acid, respectively.     

Synthesis and characterization of human α‐defensins 4‐6

Abstract: Human α‐defensins are small, Cys‐rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high‐quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human α‐defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N‐diisopropylethylamine (DIEA) in situ neutralization/2‐(1 H‐benzotriazolyl)‐1,1,3,3‐tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid‐phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10–16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony‐counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.     

Synthesis and characterization of [vinylidene‐3H] (−)‐α‐kainic acid at high specific activity

[Vinylidene‐³H] (?)‐α‐Kainic acid was prepared by means of a Wittig reaction with a protected ketone precursor and has proved useful as a tool to study neuroexcitatory amino acid receptors. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis and characterization of tritium labeled N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide

N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide is a potent C‐C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography–mass spectrometry, time‐of‐flight mass spectrometry, and ³H‐NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

New insights into the synthesis and characterization of 2‐methoxy‐3‐alkylpyrazines and their deuterated isotopologues

A previously described synthetic route for preparation of 2‐methoxy‐3‐alkylprazines (MPs) based on condensation of glyoxal with an α‐amino acid amide, followed by methylation with iodomethane yields 3‐alkyl‐1‐methyl‐1H‐pyrazin‐2‐ones (N‐methyl derivatives), rather than the designated 2‐methoxy‐3‐alkylpyrazines (O‐methyl derivatives). Despite similar nuclear magnetic resonance and mass spectral properties, gas chromatographic (GC) retention indices differ significantly, indicating chemical difference. With the example of 3‐sec‐butyl‐1‐methyl‐1H‐pyrazin‐2‐one and its 3‐sec‐butyl‐1‐[²H₃]methyl‐1H‐pyrazin‐2‐one isotopologue, the position of the methyl group introduced could be assigned unambiguously, using heteronuclear multiple bond correlation (HMBC) NMR experiments. For future characterization, the spectroscopic (NMR, EI⁺MS) as well as GC retention index data on two stationary phases of the most aroma relevant MPs and their deuterated isotopologues are summarized. Copyright © 2011 John Wiley & Sons, Ltd.     

重组人尿苷二磷酸葡糖醛酸转移酶2B7三个功能性突变体的构建、表达及活性比较

目的构建人尿苷二磷酸葡糖醛酸转移酶(UGT)2B7重组酶的3个功能性突变体UGT2B7*71S(A71S,211G>T),UGT2B7*2(H268Y,802C>T)和UGT2B7*5(D398N,1192G>A),以进一步研究该酶野生型和其突变体在对底物作用过程中的功能差异。方法应用细菌/杆状病毒系统,将构建的pFastBac-UGT2B7*71S,pFastBac-UGT2B7*2和pFastBac-UGT2B7*5重组质粒转化E.coli DH10Bac大肠杆菌,通过转座作用获得各自的重组粘粒(bac-mid),然后将其转染草地夜蛾(Sf)9细胞后,产生重组杆状病毒。这些病毒再感染Sf9细胞,即可获得野生型UGT2B7*1及其突变体的重组酶。野生型及突变体酶的活性以7-羟基-4-三氟甲基香豆素(7-HFC)为底物,用荧光法测定并分析比较。结果利用杆状病毒/昆虫细胞系统,成功地构建人UGT2B7重组酶的3个功能性突变体。UGT2B7*1对7-HFC的Km值为(0.331±0.018)mmol·L-1,Vmax值为(2.14±0.04)μmol·min-1·g-1蛋白;UGT2B7*71S对7-HFC的Km值为(0.260±0.026)mmol·L-1,Vmax值为(1.36±0.05)μmol·min-1·g-1蛋白;UGT2B7*2对7-HFC的Km值为(0.53±0.06)mmol·L-1,Vmax值为(9.5±0.5)μmol·min-1·g-1蛋白;UGT2B7*5对7-HFC的Km值为(0.59±0.05)mmol·L-1,Vmax值为(7.52±0.28)μmol·min-1·g-1蛋白。结论应用细菌/杆状病毒系统,成功构建了UGT2B7的3个功能性突变体,这些突变体可进一步用于对其他底物的代谢活性比较。     

Synthesis of [13C4]‐labeled 2′‐deoxymugineic acid

The phytosiderophore 2′‐deoxymugineic acid (DMA) is exuded via the root system by all grasses (including important crop plants like rice, wheat and barley) to mobilize Fe(III) from soil and improve plant Fe nutrition, crucial for high crop yields. Elucidation of the biogeochemistry of 2′‐deoxymugineic acid in the rhizosphere requires its quantification in minute amounts. To this end, ¹³C₄‐DMA was synthesized for the first time, from cheap isotopically labeled starting materials. The synthetic route utilizes l ‐allyl(¹³C₂)glycine and l ‐(2‐¹³C)azetidine (¹³C)carboxylic acid as versatile labeled building blocks. The title compound was recently used as an internal standard for analysis of soil and plant samples allowing the first accurate quantification of DMA in these matrices by means of LC‐MS/MS. It is furthermore used in tracer experiments investigating biodegradation of DMA in soil.     

Synthesis and characterization of radiolabeled 17β‐estradiol conjugates

The use of radioactive tracers for environmental fate and transport studies of emerging contaminants, especially for those that are labile, offers convenience in tracking study compounds and their metabolites, and in calculating mass balances. The aim of this study was to synthesize radiolabeled glucuronide and sulfate conjugates of 17 β ‐estradiol (17 β ‐E2). The conjugates 17 β ‐[4‐¹⁴C]estradiol‐3‐glucuronide ([¹⁴C]17 β ‐E2‐3‐G) and 17 β ‐[4‐¹⁴C]estradiol‐17‐sulfate ([¹⁴C]17 β ‐E2‐17‐S) were synthesized utilizing immobilized enzyme and chemical syntheses, respectively. Microsomal proteins from the liver of a phenobarbital induced pig (Sus scrofa domestica) were harvested and used to glucuronidate [¹⁴C]17 β ‐E2. Synthesis of [¹⁴C]17 β ‐E2‐17‐S consisted of a three‐step chemical process – introducing a blocking group at the C‐3 position of [¹⁴C]17 β ‐E2, sulfation at C‐17 position, and subsequent deblocking to yield the desired synthetic product. Successful syntheses of [¹⁴C]17 β ‐E2‐3‐G and [¹⁴C]17 β ‐E2‐17‐S were achieved as verified by liquid chromatography, radiochemical analyses, quadrupole‐time‐of‐flight (QTOF) mass spectrometry, and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Radiochemical yields of 84 and 44% were achieved for 17 β ‐E2‐3‐G and 17 β ‐E2‐17‐S, respectively. Synthetic products were purified using high‐performance liquid chromatography and radiochemical purities of 98% or greater were obtained. Copyright © 2011 John Wiley & Sons, Ltd.     

HPLC测定注射用葡醛酸钠的含量及有关物质

目的　建立测定注射用葡醛酸钠中葡醛酸钠含量及其有关物质的检查方法。方法　采用HPLC法HypersilC1 8色谱柱,1 0mmol·L-1 磷酸盐缓冲液(6mmol·L-1 NaH2 PO4 4mmol·L-1 Na2 HPO4) -甲醇(98∶2 ,磷酸调pH 6 )为流动相,检测波长2 2 5nm ,流速0 .8ml·min-1 。结果　葡醛酸钠在0 .1～2mg·ml-1 范围内,峰面积与浓度线性关系良好(r=0 .9999) ,高、中、低3种浓度的平均加样回收率为1 0 0 .0 %～1 0 0 .3%,RSD为0 .4 4 %～0 .82 %,有关物质检查的限量为1 .0 %。结论　所用方法准确、简便、快速,适用于注射用葡醛酸钠的质量控制。     

Identification of the anabolic steroid 6β‐chlorotestosterone in a dietary supplement

Capsules that were labeled to be performance‐enhancing dietary supplements obtained during an investigation were found to contain an unrecognized steroid‐like substance. This compound was isolated by liquid chromatography (LC) fraction collection and characterized using several qualitative analytical techniques, including ultraviolet (UV) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐high resolution accurate mass‐mass spectrometry (LC–HRAM–MS), as well as ¹H, ¹³C, and two‐dimensional nuclear magnetic resonance (NMR) spectrometry. This multi‐technique analytical approach was used to identify the designer steroid as 6β‐chloro‐4‐androsten‐17β‐ol‐3‐one (6β‐chlorotestosterone), an analog of testosterone about which little has been published.     

Analysis of γβ, βγ, γγ, ββ multiple turns in proteins

Abstract: The number of γ‐turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic γ‐turns and 57% inverse γ‐turns are associated as multiple turns with either another γ‐turn or a β‐turn. We refer to these as multiple turns of the (γβ)_1,2,3 or (βγ)_1,2,3 type, depending upon whether the γ‐turn is before or after the β‐turn along the protein chain, respectively. However, for multiple turns involving only γ‐turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) β‐turns. Fifty‐eight per cent β‐turns are associated as multiple turns with another β‐turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual γ‐ or β‐turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side‐chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins ( http://www.cdfd.org.in/dsmp.html ).     

Synthesis of α-, β- and cyclic spaglumic acids

A short, one-pot synthesis of α- and β-spaglumic acids (N-acetyl-L-aspartyl-L-glutamic acids, NAAGA) has been developed based on ultrasound-promoted acetylation of aspartic acid, followed by dehydration, condensation with glutamic acid dibenzyl ester and hydrogenolysis. The α- and β-peptides were separated by anion-exchange chromatography. The α-peptide shows a remarkable tendency to cyclize during methylation with diazomethane and yields cyclic N-acetylaspartylglutamic acid dimethyl ester, which could be hydrolysed to the hitherto unreported diketopiperazine dicarboxylic acid, cyclic spaglumic acid (cyclic NAAGA).     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

Structural analysis of fertilinβ cyclic peptide mimics that are ligands for α6β1 integrin

Abstract: The NMR structural analysis of two fertilinβ mimics cyclo(EC²DC¹)YNH_2, 1 , and cyclo(D²EC²D¹C¹)YNH_2, 2 is described. Both of these mimics are moderate inhibitors of sperm?egg binding with IC₅₀ values of 500 µm in a mouse in vitro fertilization assay. For peptide 1 , the optimized conformations that best match the NMR data have a pseudo‐type II′β‐turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC²D¹C¹ sequence is in a nonclassical (type IV) β‐turn. For peptide 2 , the conformation that best matches the NMR data has two turns: a pseudo‐type II′β‐turn in the D²EC²D¹ sequence followed by a nonclassical β‐turn in the EC²D¹C¹ sequence. The Cβ?Cβ distance between E and D¹ in peptide 1 is 9.1 Å, in peptide 2 , it is 7.7 Å. Thus, one possibility for the high IC₅₀ values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the α₆β₁ integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm?egg binding.     

Synthesis of all‐trans‐[10′‐3H]‐8′‐apo‐β‐carotenoic acid

The enzyme, 15,15′‐β‐carotene dioxygenase (BCDOX), facilitates the oxidation of β‐carotene to yield retinal. This is a remarkable process in which one of 11 double bonds in β‐carotene is selectively oxidized. To further probe the mechanistic aspects of BCDOX, the synthesis of all‐trans‐[10′‐³H]‐8′‐apo‐β‐carotenoic acid is reported. This compound will be used as a photoaffinity labeling reagent to probe the β‐carotene binding pocket within BCDOX. The synthesis outlines a simple and efficient route for the incorporation of tritium at the 10′ olefinic carbon of 8′‐apo‐β‐carotenoic acid. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of β‐amino acid derivatives and their inhibitory profiles against some metabolic enzymes

Sulfamate and its derivatives have a range of biological activities. One‐pot cyclocondensation of alkenes ( 1a–i ) with chlorosulfonyl isocyanate generates β‐lactams. β‐Amino acid derivatives ( 2a–i ) from β‐lactams were synthesized. Then, these highly reactive compounds were opened with MeOH to produce the corresponding sulfamate derivatives in good yields. The inhibitory effects of the novel sulfamate derivatives were tested on human carbonic anhydrase I and II isoenzymes (hCA I and hCA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α‐glycosidase (α‐Gly). Novel sulfamate derivatives showed K_i values in the range of 23.81–42.97 nM against hCA I, 8.95–52.23 nM against hCA II, 8.10–45.51 nM against AChE, 23.16–81.84 nM against BChE, and 14.02–48.68 nM against α‐Gly. As a result, the novel sulfamate derivatives had potent inhibitory effects against both isoenzymes. Overall, due to the inhibitory effects of the novel sulfamate derivatives on the tested metabolic enzymes, they are promising drug candidates for the treatment of diseases like glaucoma, epilepsy, leukemia, Alzheimer's disease, and type 2 diabetes mellitus, which are associated with high enzymatic activity of the indicated metabolic enzymes.     

Cyp3a11‐mediated testosterone‐6β‐hydroxylation decreased,while UGT1a9‐mediated propofol O‐glucuronidation increased,in mice with diabetes mellitus

The db/db mouse is one of the most popular animal models for type 2 diabetes mellitus, but changes in the activities of important P450s and UGTs are still not completely clear. This study was designed to investigate the alterations of major hepatic cytochrome P450s and UDP‐glucuronyltransferase enzymes in db/db mice. Mouse liver microsomes (MLMs) were obtained from male db/db mice and their wild type littermates. After incubation of the substrates separately with MLMs, the samples were pooled and analysed by high‐throughput liquid chromatography–tandem mass spectrometry system for the simultaneous study of nine phase I metabolic reactions and three glucuronidation conjugation reactions to determine the activity of the metabolic enzymes. Compared with normal controls, the Cl_int estimate for testosterone‐6β‐hydroxylation was lower (46%) (p < 0.05), while the V_max and Cl_int estimates for propofol O‐glucuronidation were 5‐fold higher (p < 0.01) in the liver microsomes from db/db mice. There was no significant difference in phase I metabolic reactions of phenacetin‐O‐deethylation, coumarin‐7‐hydroxylation, bupropion‐hydroxylation, omeprazole‐5‐hydroxylation, dextromethorphan‐O‐demethylation, tolbutamide‐4‐hydroxylation, chlorzoxazone‐6‐hydroxylation and midazolam‐1‐hydroxylation and in glucuronidation reactions of estradiol 3‐O‐glucuronidation, and 3‐azido‐3‐deoxythymidine glucuronidation. The data suggest that, in db/db mice, the activity of Cyp3a11, catalysing testosterone‐6β‐hydroxylation, decreased, while the activity of UGT1a9, catalysing propofol O‐glucuronidation, increased. Copyright © 2016 John Wiley & Sons, Ltd.