Yasuyoshi Miki

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3, 5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length) x 50 microns I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 micrograms AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5-100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 microgram AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.

Reaction of indole-2,3-dicarboxylic anhydride with (3-bromo-4-pyridyl)triisopropoxytitanium: Synthesis of ellipticine

Abstract N -Benzylindole-2,3-dicarboxylic anhydride ( 1 ) was reacted with (3-bromo-4-pyridyl)triisopropoxytitanium to give 2-(3-bromoisonicotinoyl)indole-3-carboxylic acid ( 2 ) as the sole product in high yield, which could be converted to ellipticine in six steps.

Intramolecular palladium-catalyzed cyclization of methyl 1-(2-bromobenzyl)indole-2-carboxylates: Synthesis of pratosine and hippadine

Abstract Palladium-catalyzed cyclization of methyl 1-(2-bromo-4,5-dimethoxy-benzyl)indole-2-carboxylate in the presence of potassium acetate in hot 1,4-dioxane gave a 7 H -pyrrolo[3,2,1- de ]phenanthridine derivative, which was converted to pratosine in four steps. In a similar manner, hippadine was also prepared.

In-capillary derivatization with 1-methoxycarbonylindolizine-3,5-dicarbaldehyde for high-performance capillary electrophoresis

A simple, selective and reproducible high-performance capillary electrophoresis (HPCE) assay with in-capillary derivatization using 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA) as a derivatization reagent for the amino group has been developed. The method involves two modes of in-capillary derivatization, namely, ‘on-line’ and ‘sandwich’. The on-line mode involves derivatization and separation in a capillary filled with a run buffer containing IDA. The sandwich mode refers to a method in which the front end of a separation capillary for HPCE is used as a derivatization chamber, in which a sample buffer and reagent buffer are introduced into the injection end of a capillary as a ‘sandwich’, i.e., reagent buffer–sample solution–reagent buffer. In both modes, each sample was subsequently derivatized, electromigrated and detected (409 nm) by flow in the capillary. The assay reproducibility (n= 5) on the on-line mode, sandwich mode and pre-column method at 1 mmol l–1 alanine (Ala) was 1.31, 12.26 and 3.72%, respectively. The detection limit (S/N = 3) obtained by both modes was 1 µmol l–1 of Ala, which is nearly the same level as that obtained by the pre-column method. Calibration curves, for both modes, were constructed over the concentration range 10 µmol l–1 to 10 mmol l–1 Ala. The pH of the run buffer was limited to 10 for the on-line mode; for the sandwich mode a wide range of pH could be used. The present method was applied to the determination of 16-component amino acid solution, 4-component polyamine (cadaverin, histamine, spermidine and tyramine) solution, and 4-component aminocyclitol antibiotic (amikacin, arbekacin, micronomicin and netilmicin) solution.

[Cu(OH)(TMEDA)]2Cl2-Catalyzed Regioselective 2-Arylation of 5-Substituted Tetrazoles with Boronic Acids under Mild Conditions

A mild and regioselective 2-arylation of 5-substituted tetrazoles is described. The reaction proceeds regioselectively with a variety of arylboronic acids in the presence of [Cu(OH)(TMEDA)]2Cl2 to afford 2,5-disubstituted tetrazoles. This is the first report of highly regioselective arylation of 5-alkyltetrazoles.

Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of sarpogrelate (SG), a 5-HT2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5×10-7 M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (TNO) level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. Meanwhile, mitochondrial Ca2+ uptake on acidification or Ca2+ content changes of the perfusate was suppressed by pre-treatment with SG or the KATP channel opener diazoxide, but not the KATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT2A receptor induced in ischemia–reperfusion plays an important role in the mitochondrial KATP channel of hearts in close relation with NO and active oxygen radicals.

Determination of amino acids by pre-column fluorescence derivatization with 1-methoxycarbonylindolizine-3,5-dicarbaldehyde

Abstract The fluorescence intensity of 15 amino acids and ammonia as their 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA) derivatives was investigated over the pH range 2–8. At pH 2–6, most derivatives showed strong fluorescence at 482 nm (emission); the intensity decreased with increasing pH (pH 6–8) when exposed to 291 and 414 nm light. The time course of the derivatization of the amino acids and their retention behavior on an ODS column in high-performance liquid chromatography (HPLC) were also investigated. Derivatizatiion of all amino acids — except for histidine, threonine and ammonia — was completed within 15 min and was relatively stable for 2 h. Using reversed-phase HPLC equipped with a fluorescence detector ( λ ex = 414 nm, λ em = 482 nm), a 15-component amino acid and ammonia standard mixture was analyzed on an ODS column with a 60-min sample turnover using binary gradient elution with 20 m M ammonium dihydrogen phosphate (adjusted to pH 2.6 with phosphoric acid) in the presence of 10 m M 1-octanesulfonic acid as an ion-pairing reagent and acetonitrile. The precision of the method is less than 2% (peak area) and 0.5% (retention time) at the 100 μM (166 pmol/injection) level. Linearity was established over the concentration range 0.5–100 μM of each derivative. The detection limits range from 0.2 fmol for leucine, isoleucine and phenylalanine to 200 fmol for methionine; however, proline, tyrosine and tryptophan did not respond at all to this method. In order to determine the accuracy, the data on the composition of protein hydrolysates (soybean and human hair) obtained with the present method are compared with those obtained with an amino acid analyzer.

Synthesis of ellipticine by reaction of 1-(4-methoxybenzyl)indole-2,3-dicarboxylic anhydride with (3-bromo-4-pyridyl)triisopropoxytitanium

Reaction of 1-benzyl- and 1-(4-methoxybenzyl)indole-2,3-dicarboxylic anhydride with (3-bromo-4-pyridyl)triisopropoxytitanium gave the corresponding 2-acylindole-3-carboxylic acids as the sole product. Deprotection of the 1-(4-methoxybenzyl) group of the 2-acylindole-3-carboxylic acid was performed by treatment with perchloric acid in acetic acid to afford 2-(3-bromoisonicotinoyl)indole, which was converted to ellipticine.

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt‐based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine‐assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6‐trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′‐dimethoxydiphenylmethyl and 4‐methoxybenzyloxymethyl groups were demonstrated to be acid‐labile S‐protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave‐assisted SPPS performed at a high temperature.

1-Methoxycarbonylindolizine-3,5-dicarbaldehyde as a derivatization reagent for amino compounds in high-performance capillary electrophoresis

The indolizine derivative 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA) was synthesized from 2-(1,3-dioxolan-2-yl)pyridine in four steps. The reactivity of the reagent towards primary amines was investigated by using alanine (Ala) as a model compound. The reagent easily reacted with Ala in 20 mmol l–1 phosphate–borate buffer at pH 10 containing 25–50% v/v of ethanol in the dark at room temperature, and the reaction was completed within 15 min. The IDA derivative of alanine (IDA-Ala) showed a strong absorption at 280 nm (Iµ= 3.31 × 104 l mol–1 cm–1) and 409 nm (Iµ= 2.18 × 104 l mol–1 cm–1). The derivative also showed fluorescence at 482 nm when irradiated at 282 or 414 nm. These wavelengths did not overlap with those of IDA. IDA-Ala showed a 1.5 times stronger absorption than that produced by the ninhydrin method, and the fluorescence intensity of IDA-Ala was about three times that produced by o-phthalaldehyde-Ala. On analysis of IDA-Ala by high-performance capillary electrophoresis (HPCE) in the capillary zone electrophoresis (CZE) mode with detection at 280 nm, the calibration graph showed good linearity over the range 0.0172–21.5 µmol ml–1 of alanine. The detection limit for alanine was about 5 nmol ml–1. The relative standard deviations in the determination of IDA-Ala at 10.8 and 21.5 µmol ml–1 were less than 2%(n= 5). Application to the analysis of a mixture of amino acids by HPCE using the micellar electrokinetic chromatography mode is also described.