Bengt Fransson

Ion-pair chromatography of acidic drug metabolites and endogenic compounds.

Liquid-liquid chromatographic systems based on ion-pair partition with silica microparticles as the support for the stationary phase have been used for the separation of anionic compounds of biochemical and pharmacological interest. A high separating efficiency can be obtained with both aqueous and organic mobile phases and the retention is easily regulated by the nature and the concentration of the quaternary ammonium counter ion, present in the aqueous phase. The influence of the composition of the liquid phases on the selectivity and separating efficiency has been studied, as well as equilibration methods and the stability of the systems. Examples are given of separations of sulphonamides, barbiturates, glucuronic and sulphuric acid conjugates of steroidal compounds and phenols glycine conjugates of carboxylic acids (hippuric, nicotinuric and salicyluric acid) and anionic metabolites of biogenic amines (indoleacetic, benzoic, mandelic and phenylacetic acid derivatives).

Ion-pair chromatography in the low concentration range by use of highly absorbing counter ions

Abstract High-performance systems for ion-pair chromatography that permit the detection of down to 7 pmole (about 1 ng) of non-UV-absorbing samples have been constructed, the high detector response being obtained by using a stationary phase containing a counter ion of high absorbance. Amino acids, dipeptides and alkylamines are separated with naphthalene-2-sulphonate as counter ion. The systems have a high selectivity, and a change of the amino acid sequence if often sufficient for a complete chromatographic resolution of dipeptides. A comparison with batch extraction data showed that the retention of the cations, except the most hydrophobic, is due mainly to liquid-liquid distribution. The naphthalene-2-sulphonate systems have a very high stability under careful thermostatted conditions. Systems for the separation of anionic compounds with highly absorbing quaternary ammonium ions in the stationary phase are being investigated.

Separation of enantiomers of α-hydroxy acids by reversed-phase liquid chromatography after derivatization with 1-(9-fluorenyl)ethyl chloroformate

Abstract The application of the chiral reagent (+)-1-(9-fluorenyl)ethyl chloroformate, also known as (+)-FLEC and originally introduced for the separation of enantiomers of α-amino acids and the determination of their optical purity by HPLC, has been extended to enantioseparation within a set of α-hydroxy acids. Retention of the acidic diastereomeric carbonate derivatives thereby formed is conveniently regulated by the pH of the mobile phase and separation can generally be accomplished within about 30 min under isocratic conditions with tetrahydrofuran as organic modifier. Of six α-hydroxy acids so far examined five had separation factors of 1.24 or higher, whereas for malic acid it was in the range 1.10–1.15.

Synthesis of amino acid derivatives substituted in the backbone with stable isotopes for application in peptide synthesis

Starting from the corresponding precursors, 1 and 2, the two well known procedures for asymmetric synthesis of amino acids, those of Schollkopf and Oppolzer, were compared for the preparation of [1,2-13C2,15N]-labelled (substituted) Boc-leucine enantiomers. Although both gave the final products in comparable overall yields, analysis of optical purity by two independent chromatographic methods revealed that the two procedures differed considerably in this respect. The enantiomeric excess found was 97.2–97.4 and 99.7%, respectively. As a consequence, the latter method was preferred for the synthesis of a number of additional backbone-labelled Boc-derivatives of the three proteinogenic amino acids alanine, phenylalanine and tyrosine, including such deuteriated in the amino acid side-chain. These derivatives exemplify precursors suitable for chemical synthesis of specifically backbone-labelled peptides and should allow greater exploitation of the properties of the 13C and, especially, the 15N nuclei in structural studies.

Separation of basic, hydrophilic peptides by reversed-phase ion-pair chromatography: I. Analytical applications with particular reference to a class of serine peptide substrates of cyclic AMP-stimulated protein kinase

Abstract Basic, hydrophilic peptides exemplified by Arg-Arg-Ala-Ser-Val, a substrate of cyclic AMP-stimulated protein kinase, could be efficiently retarded by ion-pair formation with n -hexanesulphonic acid on a C 18 column in phosphate buffer systems with ethanol as organic modifier. Reversed-phase ion-pair chromatography appears to be an ideal method for separating peptides of this type. Highly efficient separations under isocratic conditions are reported for peptides composed of 5–9 amino acids, with up to three basic residues, most having sequences derived from the phosphorylatable site of pyruvate kinase.

Separation of basic, hydrophilic peptides by reversed-phase ion-pair chromatography. II. Analytical applications with particular reference to phosphoserine peptides.

Abstract Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C 18 column, using phosphate buffers with pH 3.2–4.5, n -hexane sulfonic acid as counter ion, and ethanol as organic modifier.

Synthesis of 15N-labelled chiral Boc-amino acids from triflates: enantiomers of leucine and phenylalanine

An efficient synthesis of 15N-labelled chiral Boc-amino acids by triflate alkylation of di-tert-butyl [15N]imidodicarbonate is reported. Both enantiomers of Boc-leucine and -phenylalanine were synthesized from commercial α-amino acids of opposite configuration viaα-hydroxy carboxylic acids provided by diazotization, thus extending the scope of an earlier exploratory study. The high chiral purity of the final products was confirmed by HPLC. These labelled amino acid derivatives are suitable for direct application to the synthesis of labelled peptides.

Reversed-phase ion-pair chromatography of basic, hydrophobic peptides

The effect of alkane sulphonates, varying in size from ethane to n-pentane, on the retention of substance P to alkylsilane-bonded (C6, C8 and C18) microsilica columns with ethanol as orgnaic modifier has been studied. Applying the same counter-ion in constant concentration, for this hydrophobic peptide the three columns gave essentially the same degree of retention. The sulphonate of n-pentane and n-butane and, to some extent, n-propane could be exploited for regulation of the retention, however, ethane sulphonate proved inefficient for this purpose. The peak symmetry varied among the supports studied. Peaks with good symmetry were obtained only when fully capped reversed-phase supports were used. On the other hand, with partly capped supports the peak symmetry could be greatly improved by the addition of amines or quaternary ammonium compounds to the mobile phase. A high separation efficiency (h < 10) was obtained with the fully capped alkyl silane-bonded supports.

Direct synthesis of N-protected chiral amino acids from imidodicarbonates employing either Mitsunobu or triflate alkylation. Feasibility study using lactate with particular reference to 15N-labelling

Two novel approaches to N,N-diprotected chiral α-amino acid esters, based on selected imidodi-carbonates as amine synthons, have been explored. Thus, N-alkylation of these substrates was smoothly accomplished by the Gabriel or Mitsunobu methods as well as by the use of triflates. Ethyl (R,S)-2-bromopropionate underwent nucleophilic substitution when treated with the potassium salt of selected imidodicarbonates in dry dimethylformamide to furnish the corresponding fully blocked (R,S)-alanines in high yield. The chirality of ethyl (S)-lactate was largely conserved when it was condensed with free imidodicarbonates and tosylcarbamates under conventional Mitsunobu conditions. The yield of the corresponding N,N-di-protected ethyl (R)-alaninate was strongly dependent on the electron-withdrawing properties of the imidodicarbonate alkyl groups. Thus, Boc2NH gave < 5% of the product whereas Troc-NH-Z afforded the corresponding analogue in 83% yield under comparable conditions. On the other hand, triflates of various lactic acid esters reacted smoothly with the lithium salt of Boc2NH, also with clean inversion, as a result of which, after selective removal of two blocking groups, the N-protected alanine of opposite configuration could be isolated in high yield and excellent stereochemical purity. Both methods have been used for the synthesis of 15N-labelled N-protected (R)- and (S)-alanines, suitable for direct application to peptide synthesis.

Synthesis of substrates of cyclic AMP-dependent protein kinase and use of their protected precursors for the convenient preparation of phosphoserine peptides

The synthesis of protected hexa- to nona-peptide precursors of substrates of cyclic AMP-dependent protein kinase, based on a partial amino acid sequence from rat liver pyruvate kinase, as well as of related phosphoserine peptides has been explored. A convenient scheme has been developed which furnishes both N-terminally elongated peptides of variable lengths and intermediates suitable for chemical phosphorylation. The use of adamantyloxycarbonyl as a protecting group for the two important guanidine functions involved, gave rise to the highly lipophilic intermediates (9), (21), (22), (28), and (31), which could easily be purified. Treatment of these with anhydrous hydrogen fluoride [or trifluoroacetic acid in the case of compound (31)] afforded the pure substrate peptides (10), (23), (24), (29), and (32) in high overall yield. All of the free peptides obtained could be phosphorylated by cyclic AMP-dependent protein kinase at a significant rate. The chemical synthesis of two phosphoserine peptides (12) and (14) and their purification by preparative reversed-phase ion-pair chromatography are also reported.