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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.

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.

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.

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.

In connection with work related to cAMP-dependent protein kinase, a tritiated substrate, Arg-Arg-Ala-Ser-Val-Ala, was prepared from the corresponding unlabeled peptide by solid-state catalytic isotope exchange. Varying amounts of side products with chromatographic properties very similar to those of the substrate were observed. Careful chromatographic comparison with a complete set of diastereomers eliminated racemization as the reason for this heterogeneity. Instead, the serine residue was identified as the major source of by-products, presumably due to primary elimination of water followed by saturation of the dehydroalanine residue, transforming it into a racemic alanine, thus giving rise to Arg-Arg-Ala-( d, l -Ala)-Val-Ala.

Refluxing solutions of monosaccharides, unprotected at the 6-position and carrying O-methyl, S-ethyl, O-acetyl and OH-groups at the anomeric center, in acetonitrile containing a catalytic amount of a Lewis acid (O.1-0.4 equiv.) yielded 1,6-anhydrohexopyranoses in good to high yields. Best results were obtained with methyl 2,3,4-tri-O-dideuteriobenzyl-alpha-D-glycosides (87-91%). Dideuteriobenzyl protective groups were used to facilitate NMR spectral interpretations.

The resolving power of liquid chromatography systems containing 1-pentane sulfonate, previously used to analyze basic hydrophilic peptides related to substrates of cAMP-dependent protein kinase, has been studied with respect to diastereomeric peptides. A set of peptides comprising RRASV and its five diastereomers containing one D-amino acid were used as model compounds. Complete resolution of all the peptides could be accomplished both with ethanol and acetonitrile as organic modifiers. The separation of the various peptides within the set turned out to be only modestly changed under the conditions investigated. These new results clearly demonstrate the potential of the chromatography systems studied.

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.

Using phosphoserine peptides as model substances, we have studied the separation of acidic peptides as substituted ammonium ion-pairs on a macroporous copoly(styrene-divinylbenzene) ( PLRP-S ) chromatographic column. Various hydrophobic amines were examined, of which long-chain N-methyl- and N,N-dimethyl- n -alkylamines proved to be the best with respect to selectivity and peak symmetry. Excellent separations were obtained in ammonium bicarbonate buffer (p H 7.5) with ethanol as organic modifier under isocratic conditions using the corresponding heptylto nonylamines.

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.

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).

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.

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.

Calreticulin is an intracellular protein known to be involved in calcium binding, but is also known to appear as an autoantigen in certain autoimmune diseases. The cDNA sequence is known but the protein has not yet been well characterized at the amino acid level. Owing to the possible involvement of this protein in autoimmune disease and with the aim of making monoclonal antibodies for use in assay development and immunohistochemistry, we have purified calreticulin using human placental material. Amino acid analysis of the purified protein confirmed the cDNA-derived composition, and only one discrepancy between the cDNA-predicted sequence and the amino acid sequence was found by peptide mapping and microsequencing. The protein contains one disulfide bridge and has one free SH group and the protein is neither glycosylated nor phosphorylated. Affinity chromatography of a placental protein extract on a column with immobilized calreticulin showed the existence of at least six proteins interacting with calreticulin. Using the purified calreticulin in Western blots, two out of eight patients with autoimmune disease diagnosed as having anti DNA antibodies in their serum were found also to contain autoantibodies to calreticulin in their serum.