Hirofumi Hirata

SEPARATION OF SECONDARY ALCOHOL ENANTIOMERS USING SUPERCRITICAL FLUID CHROMATOGRAPHY

Abstract Secondary alcohol enantiomers were separated using pre-column derivatization with (R)-(−)-1-(1-naphthyl)ethyl isocyanate and supercritical fluid chromatography. The chromatographic properties of the derivatized alcohols on three kinds of archiral stationary phases were investigated in this study. The selectivity of the two enantiomers depended on the properties of the stationary phase more than those of the mobile phase. The resolution properties depended on the species of secondary alcohols. The enantiomers of secondary alcohols with a longer carbon chain were better resolved, and the resolution became poor as the hydroxyl group was located towards the center of the carbon chain.

Effect of Phospholipids on Carrier-Mediated Transport of Lanthanide Ions through Cellulose Triacetate Membranes

Abstract Fluxes of 14 kinds of lanthanide ions across cellulose triacetate membranes were determined by using mixtures of o-nitrophenyl n-octyl ether and synthetic phospholipids as plasticizers, and 4-benzoyl-3-methyl-1-phenyl-5-pyrazolone (BMPP) and hinokitiol (HIPT) as carriers. The phospholipids used were didecanoyl-, dilauroyl-, and dimyristoyl-l-α-phosphatidylcholines. The effect of these phospholipids on the flux was demonstrated. The flux using BMPP increased with an increase in ionic radius of the lanthanide and reached a maximum value at an ionic radius around that of dysprosium. Subsequently, it decreased and then increased again in the range of praseodymium to lanthanum. The variation in the flux decreased with an increase in alkyl chain length of the phospholipid. For HIPT, an appreciable increase in the flux was observed in the range of erbium to europium.

Chemistry of Succinimido Esters.XX

GC-MS analysis of N-arylcarbonyl-O, O-bis (TMS) derivatives (4) of synthetic dihydrosphingosines (1) was conducted to establish a convenien method for determining the erythro and threo isomers of (1) by mass spectrometry. The structures of fragment ions were determined by comparing the mass spectra of (4) with different substituents in the aryl group and carbon chain of (1). The structures were indicated based on differences in mass numbers of (4) -d18 and (4) h18 derivatives [N-arylcarbonyl-O, O-bis (TMS) -d18 and O, O-bis (TMS) -h18 derivatives of (1)]. One characteristic difference was relative intensity (RI) at m/z=M-103; the relative intensities of threo compounds (RI=30-70%) exceeded those of erythro isomers (RI<10%). The ratios, γ=RI (M-103) /RI (M-15), of threo compounds were greater than those of the erythro isomers. Values γ (threo) /γ (erythro) were from 3 to 7. From these results, it should be possible to determine the erythro and threo isomers of (1) by mass spectrometry in the form of (4).

Chemistry of Succinimido Esters. XIX

Chemical modification (N-arylacetylation) of amino groups in proteins by N-succinimidyl arylacetates (1a) was studied kinetically using bovine serum albumin (BSA) as the model protein in the range of 7.0≤pH≤9.0 at 20°C. N-Arylacetylation was competitive with the hydrolysis of (1a). Its rate was expressed as V= k2 [NH2 of BSA] [1a].The logarithm of the second order rate constant, k2, increased linearly with the relative acid strength of the corresponding benzoic acid derivatives, log (K/K0), for meta-substituted (1a). The slope was independent of pH. The rate of ortho-substituted (1a) was less than that expected from log (K/ K0) owing to steric hindrance of the substituent. pH profiles gave slopes of less than unity since the amino groups of BSA were not equivalent. The rate ratio, 10-3 k2/k1, where k1 is the first order rate constant for the hydrolysis of (1a), exceeded unity for all cases and was maximal at about pH 8.5. The reactivity of (1a) was compared with those of N-succinimidyl benzoates (1b) and N-acyloxysuccinimides (1c).Strong hydrophobic interaction between BSA and the imido ester was observed for acylation by (1c) with a long carbon chain. However, little or no significant rate acceleration could be observed for (1a) (except p-chlorosubstituent) or (1b) because of the weaker hydrophobicity of the arylacetyl and aryl groups compared with the long alkyl chain.Based on the present results, (1a) is concluded applicable for use as an N-arylacetylation reagent for the chemical modification of Lys and terminal amino groups in proteins. A possible mechanism is also proposed and discussed.

Chemistry of Succinimido Esters. XXI

A sensitive determination of sphingolipid bases (1) was made using synthetic Cn-dihyrosphingosine and naturally occurring C18- (1) aromatized with N-succinimidyl arylacetate (2a). The amino group in (1) easily reacted with (2a) to give the corresponding N-arylacetyl derivative (3a). The isomers of (1) were separated on a normal phase HPLC column in the form of (3a) which was detectable by the absorption in UV region. The isomer content of (1) as determined by the present method using (3a) having a different substituent showed agreement with that determined by GC method in the form of N-acetyl-O, O-bis (TMS) derivatives of (1) and the HPLC method in the form of N-arylcarbonyl derivative of (1), (3b).

Preparation of N'-Succinimidyl Carboxylates with the Use of N, N'-Disuccinimidyl Oxalate (DSO)

A method was devised for preparation of N-succinimidyl carboxylates (N-succinimidyl esters of fatty, benzoic and arylacetyl acids) using N, N-disuccinimidyl oxalate (DSO). Reaction of acids with DSO in the presence of pyridine in acetonitrile gave the corresponding esters in good yields. N-Hydroxysuccinimide which was formed during the reactions and pyridine in the reaction mixtures could be easily removed by treatment with water. This method was compared with those previously reported using N, N-dicyclohexylcarbodiimide (DCC) and N, N-disuccinimidyl carbonate (DSC).