The aim of this study was to determine whether the derivatives of ursodeoxycholic acid (UDCA) are useful compounds for clinical medicine. 1-1) A conjugate (5-ASA-UDCA monophosphate) of UDCA monophosphate with 5-aminosalicylic acid (5-ASA) was newly synthesized, and basic studies on this compound were carried out. This compound was efficiently deconjugated by cholylglycine hydrolase (CGH) to release 5-ASA, whereas it was completely resistant to deconjugation by pancreatic and intestinal mucosal enzymes. In animal experiments, the urinary excretion of N-acetyl-5-ASA (Ac-5ASA) was measured for 24 h following the oral administration of 20 mg of 5-ASA-UDCA monophosphate. Control rats excreted 276.3±89.0μg (mean±S.E.) of Ac-5ASA whereas rats with intestinal bacterial overgrowth excreted more (1224.1±231.5μg;p<0.01). These basic studies indicate that this compound is likely to offer a simple method for the evaluation of intestinal microorganisms without the use of radioisotopes or expensive, special apparatus. 1-2) The disulphate ester of ursodeoxycholyl-p-aminobenzoic acid (PABA-UDCA) was synthesized and compared with PABA-UDCA for its use in the detection of intestinal bacteria. This compound, PABA-UDCA disulphate, had characters in common with PABA-UDCA in that it was deconjugated by CGH to release free PABA. Further, in rat experiments the urinary excretion of PABA was measured for 6 h after oral administration of 15 mg PABA-UDCA disulphate. Ten control rats excreted 188.2±13.6μg (mean±S.E.) of PABA; 10 rats with an intestinal stagnantloop excreted more (530.1±30.1μg;p<0.001) : whereas 10 rats in each of three groups pretreated with oral administration of various antibiotics excreted less. PABA-UDCA disulphate is a single pass type substance in the gut and its oral administration test reflects the sum of the activities of bacteria in the small intestine and colon. From the results the obtained PABA-UDCA disulphate was considered a good material to detect intestinal bacteria. 2) A conjugate (Lys-UDCA) of UDCA with L-lysine was newly synthesized. In the incubation experiments with plasma, homogenates of the liver and small intestine, various pancreatic enzymes and CGH, Lys-UDCA was deconjugated by carboxypeptidases B and CGH. In the experiment using rodent everted gut sac, Lys-UDCA was actively absorbed from the terminal ileum. Lys-UDCA was recovered well in the bile after intravenous or intraileal administration of Lys-UDCA in biliary fistula rat. These data suggest that Lys-UDCA is a good prodrug of UDCA for intravenous administration. 3) A novel calcium-chelating agent, N''-ursodeoxycholyl-diethylenetriamine-N, N, N'-triacetic acid (UDCA-DTTA), was synthesized to study its ability to dissolve calcified gallstones. In the presence of the agent, sliced human gallstone with a composition of more than 50% calcium bilirubinate was thoroughly dissolved, indicating that calcium bilirubinate was dissolved from the gallstone. The ability to dissolve calcium was comparable to that of EDTA. However, the laminar structure of the sliced gallstone did not disappear in the presence of EDTA, whereas the structure disappeared in the presence of UDCA-DTTA. These results indicate that UDCA-DTTA is an interesting compound as a parent substance for developing a prodrug for an oral or intravenous agent to dissolve calcium-containing gallstones.
Marine bacteria are unique in the requirement for Na+ for optimal growth. Using a marine bacterium Vibrio alginolyticus, it was confirmed that Na+ is essential for the active uptake of all amino acids. Furthermore, the respiratory chain of V. alginolyticus was found to require Na+ for the maximum activity. The site of Na+-dependent activation is localized in the NADH-quinone reductase segment, where Na+ is extruded from the cells as a direct result of redox reaction. Thus, marine bacteria are able to directly generate sodium-motive force by respiratory chain activity. The sodium-motive force is directly coupled to the active uptake of nutrients and to the rotation of polar flagella. In addition to the energy coupling by proton circulation, marine bacteria are unique in utilizing Na+ circulation for the energy coupling. The latter mode of energy coupling is superior to proton circulation especially at alkaline and Na+-rich conditions. The respiration-coupled Na+ pump is widely distributed among Gram-negative marine and moderately halophilic bacteria. Recently, it was found that the same type of Na+ pump is distributed in the Gram-negative pathogenic bacteria. Since the presence of Na+ pump widens the adaptability of bacteria to grow at harsh environments, Na+ pump is likely to be helpful for the growth of pathogenic bacteria in the host cells to manifest their pathogenicity.
This article reviews the efficient methods for diastereoselective and enantioselective syntheses of α-substituted serines. A newly designed bislactim ether, ethyl (5S)- or (5R)-3, 6-diethoxy-2, 5-dihydro-5-isopropyl-2-pyrazinecarboxylate, was treated with base or Lewis acid-tertiary amine to generate an enolate or enaminate (imine anion, metalloenamine). Alkylation or aldol-type reaction with the resultant enolate or enaminate proceeded in a highly diastereoselective manner to give the corresponding alkylated or aldol products, respectively. Reduction of these products with diisobutylaluminum hydride (DIBAL) followed by hydrolysis with hydrochloric acid afforded the desirable α-substituted serines. The enantioselective aldol-type reaction of an achiral dislactim ether, ethyl 3, 6-diethoxy-2, 5-dihydro-2-pyrazinecarboxylate, was also investigated by employing Sn(OSO2CF3)2-triethylamine in the presence of an external chiral ligand, (-)-sparteine. Not only a stoichiometric amount, but also a catalytic amount of (-)-sparteine promoted the highly enantioselective aldol-type reactions. Interestingly, the stereoselective outcome of the Sn(II)-mediated reaction differed from that of the Mg(II)-mediated one in the aldol-type reaction of the bislactim ethers with aliphatic aldehydes. On the other hand, chemoenzymatic synthesis of enantiomerically pure α-substituted serines must also be a convenient and useful procedure. Porcine liver esterase (PLE) or rabbit liver esterase (RLE) catalyzed hydrolysis of the pro-S ester group of diethyl α-alkyl-α-(benzyloxycarbonylamino)malonates to afford (R)-ethyl α-alkyl-α-(benzyloxycarbonylamino)malonates each in excellent enantiomeric excess. Enantiodivergent reductions of these acid esters readily proceeded to furnish both the corresponding enantiomeric α-substituted serines. Finally, the application of these methodologies mentioned above to a total synthesis of ISP-I (a potent immunosuppressive principle in the Isaria sinclairii metabolite) is described. Asymmetric total synthesis of ISP-I has been achieved in a highly stereoselective manner by utilizing the Mg(II)-promoted aldol-type reaction with ethyl (5R)-3, 6-diethoxy-2, 5-dihydro-5-isopropyl-2-pyrazinecarboxylate and Schlosser modification of the Witting reaction.
Enzymes proceed the reaction with high regio- and stereoselectivity under mild conditions, i.e. in an aqueous medium at room temperature. However, enzymatic reactions that catalyze carbon-carbon bond formation have not been utilized in organic synthesis until recently. We had an interest in an aldolase-catalyzed reaction which proceed carbon-carbon bond formation referred to aldol condensation, by which many bioactive compounds have been rationally synthesized. On the other hand, recent biological studies on cell recognition (cell adhesion) have disclosed the important roles of oligosaccharides on cell surfaces, especially which include glucuronic acid, 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), and sialic acid in the structures e.g. sialyl Lewis X and endotoxins, in differentiation, induction, viral and bacterial infections, and immune response. As well as acidic oligosaccharides, basic ones have been utilized as practical medicines in the clinical level, like acarbose that acts as an amylase inhibitor. Based on these background, we embarked the synthesis of carbohydrate related compounds which can control the interaction between carbohydrates and carbohydrate recognition protein by the use of several aldolases. Azasugars, potent inhibitors toward glycosidases, were synthesized using fructose-1, 6-diphosphate (FDP)-aldolase and other dihdroxyacetonephosphate (DHAP)-dependent aldolases in the key step. Sialyl Lewis X mimetic, peptidic mimetic of RNA having anti-Vero toxin activity, mycestericin D, and aza-idulonic acid were prepared by taking advantage of L-threonine aldolase catalyzed reaction, which afford β-hydroxy-α-L-amino acids. A precursor of KDO, featured acidic sugar of endotoxins was provided by the reaction catalyzed with kynureninase, which generates β-anion of L-alanine in its active site during the metabolic reaction from kynurenine to anthranilic acid.
This review summarizes the development of deprotecting methodologies for peptides and their practical application to the synthesis of disulfide bond- or phosphoamino acid-containing peptides. Acidic deprotecting systems utilizing Brφnsted acid (HF, trifluoromethanesulfonic acid (TFMSA) and HBr etc.) have been used for the removal of protecting groups in peptide chemistry; however, these reagents are not always applicable to all of the peptides including cystine- or phosphoamino acid-containing peptides. Our attempt to utilize Lewis acid for the deprotective reaction resulted in the development of efficient and practical reagent systems (1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf)-sulfide in trifluoroacetic acid (TFA) and 1 M trimethylsilyl bromide (TMSBr)-sulfide in TFA) suitable for peptide synthesis. A new disulfide bond-forming reaction using Tl(OCOCF3)3 was developed for the synthesis of cystine peptides. The use of TMSOTf or TMSBr-mediated deprotecting system in conjunction with the disulfide bond-forming reaction utilizing Tl(III) provides a procedure for the practical synthesis of cystine peptides. A two-step deprotection method consisting of high acidic (1 M TMSOTf-thioanisole in TFA, m-cresol, ethanedithiol) and low acidic (high acidic system + dimethyl sulfide - TMSOTf) treatments was successfully applied to the deprotection of protected phosphopeptide with dimethylprotected phosphoamino acids. Furthermore, we synthesized phosphatase-resistant phosphoamino acid isosters bearing the substitution of a phosphate oxygen with a difluoromethylene. The syntheses of peptides possessing these nonhydrolyzable phosphoamino acids were achieved utilizing two-step deprotecting methodologies. Additionally, we demonstrated the usefulness of phosphatase-resistant phosphopeptides as biochemical tools for understanding signal transduction.
A highly efficient process of aerobic oxidative coupling of 2-naphthol derivatives catalyzed by Cu(OH)Cl·TMEDA has been developed. Enantioselective oxidative coupling of 2-naphthol derivatives was achieved by the use of a chiral catalyst prepared from proline-derived diamine and cuprous chloride, affording the corresponding BINOL derivatives in good enantioselectivities of up to 78% ee. A new catalytic, enantioselective allylation of aldehydes with allyltrichlorosilanes exploiting (S)-3, 3'-dimethyl-2, 2'-biquinoline N, N'-dioxide as a catalyst affords homoallylic alcohols in virtually complete diastereoselectivities and high enantioselectivities of up to 92% ee, wherein the use of diisopropylethylamine as an additive has proven to be crucial for the acceleration of the catalytic cycle. It is also noteworthy that the above finding represents the first successful example of asymmetric reactions utilizing amine N-oxide as a chiral catalyst.
The phorbol esters, such as phorbol 12- myristate 13-acetate (PMA), are known to be powerful tumor promoters and activators of protein kinase C (PKC). First discovered by Nishizuka et al., PKC is a phospholipid- and calcium-dependent serine/threonine kinase, phisiologically activated by 1, 2-diacyl-sn-glycerol (DAG). PKC is also known to be an important target for other structurally diverse tumor promoters such as ingenols, teleocidins, and aplysiatoxins. Structure-activity analyses of a variety of analogs of DAG and these tumor promoters have been carried out. Although many pharmacophore models have been proposed from molecular modeling, no information about specific amino acid residues that interact with these ligands is available. Moreover it has been shown that the biological activity of 11-demethyl-13-deoxyphorbol esters 1, which were synthesized by our group, was not fully consistent with the pharmacophore models so far. Thus, we are now interested in determining the importance of the 13-acetoxy group in phorgbol ester-PKC complexes. This has led us to design new photoaffinity probes 66 and 67 and to carry out previously unprecedented photoaffinity labeling of PKC. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking. Although the cross-linking yield is not very high, we suppose that determination of the cross-linking site can be realized by taking advantage of subpicomole order analysis by mass spectrometry and other methodologies to clarify the role of individual cysteine rich domein (CRD) in native PKC. We have also designed a new phorbol ester-phosphatidylserine hybrid molecule 69. Because phosphatidylserines in phospholipid membranes are known to have specific interactions with phorbol ester-PKC complexes, such a hybrid molecule can be expected to act as a specific inhibitor of PKC by preventing PKC from interacting with phospholipid membranes. The hybrid molecule was synthesized and preliminary biological activities were examined to inhibit PKC. A catalytic asymmetric synthesis of phorbol PMA is also currently under investigation. Progress is discussed.
Molecular mechanism of structure-protection of dipalmitoylphosphatidylcholine (DPPC) liposomes by sucrose adsorption on/in the liposome surface was studied by performing fluorescence decay, quartz-crystal microbalance (QCM), and infrared reflection-absorption (IRRA) spectroscopic measurements. Thermally hydrated DPPC monolayers were newly introduced in place of the conventional DPPC liposomes, since the hydrated monolayers were expected to have similar surface property to that of liposomes. Studies of the fluidity and permeability of membrane in the monolayers and liposomes suggested that the thermally hydrated DPPC monolayers could be used as a model membrane in place of DPPC liposomes. With the hydrated DPPC monolayers, the adsorption rate of sucrose molecules in the DPPC monolayer was investigated by a combined technique of QCM and IRRA by the use of Langmuir-Blodgett (LB) technique. It was found that the incorporation rate of sucrose into the monolayer in the liquid-expanded state was proportional to the bulk concentration of sucrose in the Langmuir trough. The linear relationship depicts the incorporation mechanism that sucrose molecules were deeply incorporated in the DPPC molecules, and the layer is formed as a single mingled layer. This molecular scheme readily supports the conventional hypotheses based upon water-replacement and vitrification mechanisms. The IRRA spectra were further studied using newly found property of principal component analysis (PCA). PCA was found to have great potential to detect minute bands hidden in dominantly large bands, after theoretical speculation and simulations. The new analytical technique was employed to search for an unknown minute spectrum in the collection of raw spectra. The calculated abstract spectra yielded by PCA strongly suggested that there remained minute quantity of water in the dried LB films. The water molecules were also found to bind to PO2 group in the head group moiety of DPPC. The mass change of adsorbed and incorporated sucrose molecules in the DPPC monolayer were readily distinguished by PCA without any probe molecule, and they were followed semi-quantitatively by calculation of scores.
Human myeloid leukemia K562 cells can be induced to differentiate to mature cells bidirectionary, i.e., hemin induces erythroid differentiation, while 12-O-tetradecanoylphorbol 13-acetate (TPA) induces differentiation to monocytes. The differentiation-inducing activity of various hemin-related compounds suggested certain structural requirements for the activity : 1) the iron moiety of hemin is not essential, and 2) the propionic acid side chains of hemin play an important role in the differntiation and induction. In addition, we have examined the influence of some bioresponsemodifying factors on hemin/protoporphyrin IX-induced differentiation of K562 cell line. Retinoids and tubulindisruptors, themselves did not induce differentiation, enhanced hemin/protoporphyrin IX-induced differentiation of K562 cells. We also examined the possible involvement of peripheral-type benzodiazepine receptor (PBR) in hemin/protoporphyrin IX-induced differentiation on K562 cell lines. The PBR specific ligands modified hemin-induced differentiation. These results suggest a requirement for retinoids (or retinoids-like cofactors) for hemin/protoporphyrin IX-induced differentiation of K562 cells and the involvement of PBR in erythroid differentiation of K562 cell line. Further we showed that TPA suppresses hemin-induduced erythroid differentiation of K562 cells, while retinoids augment it. TPA is a potent inducer of heme oxygenase (HO), which catabolizes heme to biliverdin. An HO inhibitor, tin protoporphyrin (SnPP), suppresses TPA-induced K562 cell differentiation to monocytes. It was also found that cotreatment of K562 cells with SnPP and TPA induces erythroid differentiation of K562 cells, though SnPP alone or TPA alone does not induce erythroid differentiation, suggesting a role of HO in the directional switch of differentiation.
The most commonly used formulations for dry powder inhalations are binary ordered mixes composed of micronized drugs and coarse carriers. An optimal dry powder aerosol formulation should possess an optimal inhalation property and a good flow property. These characteristics are especially important for a multidose dry powder inheler (DPI). In the present study, model powder blend were prepared consisting of synthesized sugar (different particle sized isomalt; IM-PF, IM-FS, IM-F) as a carrier and micronized salbutamol sulfate (SS). These ordered mixtures were aerosolized by the multidose JAGO DPI (SkyePharma AG) and in vitro deposition properties (fine particle fraction, FPF) were evaluated by a twin impinger (TI) at a flow rate of 60 l/min. The separation property between SS and carrier particles was investigated by the centrifuge method and air jet sieve (AJS) method. It was found that FPF decreased with increasing carrier particle size. However, a large carrier particle possesses a good flow property. Therefore, the effect of mixing of fine carrier particles (IM-PF) into the large carrier particles (IM-FS) on dry powder inhalation property was investigated. When the proportion of IM-PF (fine carrier) increase from 0% to 25% of the total carrier powder blend, the FPF also increases from 16.7% to 38.9%. It is concluded that the effect of mixing of fine carrier particles might be a suitable method for improving the dry powder inhalation properties.
Objectives : The purpose of this study was to develop, implement, and assess an estimation procedure for preventing adverse drug reaction by subjective symptoms (complaints) of patients. This time, we focused and studied on drug-induced extrapyramidal symptoms. Methods : We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information Systems) since 1987, and the case reports of adverse drug reaction accumulated in the CARPIS database to be total about 16000. We studied for 180 cases of drug-induced extrapyramidal symptoms cumulated in CARPIS database. The evaluation scores were created based on the subjective symptoms and backgrounds of the patients. We estimated 180 cases using these evaluation scores. Results : We could estimate 178 cases (98.9%) in 180 cases to be drug-induced extrapyramidal symptoms using these evaluation scores. The validity of these evaluation scores were sensitivity=98.9%, specificity=98.0% and predictive value of positive test=98.9%. The positive likelihood ratio was 49.5 and the negative likelihood ratio was 0.01. Conclusions : This study confirmed the validity of our evaluation scores. We reported the evaluation scores about drug-induced liver diseases before. Therefore, to apply these evaluation scores onto the clinical practice, we prepared an evaluation form for subjective symptoms and backgrounds of the patients with adverse drug reactions.
We purified an antibacterial protein named sarcotoxin III from the hemolymph of injured larvae of Sarcophaga peregrina (Baba K., et al., J. Biochem., 102, 69-74(1987)). In this report we determined the partial amino acid sequence of sarcotoxin III and tried to conduct the cloning of sarcotoxin III cDNA. As a result we obtained two clones coding a protein similar to sarcotoxin III in sequence. Their primary structures are highly homologous to N-terminal sequences of an antibacterial protein from Phormia, diptericin, and another 8 kD antibacterial protein purified from Sarcophaga peregrina. These results suggested that sarcotoxin III is one of the proteins forming a diptericin family in Sarcophaga peregrina as well as newly obtained clone-coded products and 8 kD protein.