Ulf Ragnarsson

Synthetic methodology for alkyl substituted hydrazines

Substituted hydrazines have found many technical and commercial applications and this is reflected in the immense number of such compounds synthesized to date. In this review an attempt has been made to supplement the existing comprehensive literature by presenting only a selection of synthetic methods, judged to be of particular principal or practical interest, together with some additional, recent approaches. The important steps in the development from simple substituted hydrazines to rational synthesis of multisubstituted derivatives are recapitulated.

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver

Synthetic peptides, representing part of the phosphorylatable site of rat liver pyruvate kinase, were phosphorylated by (32P)ATP and the catalytic subunit of cyclic AMP-stimulated protein kinase. The shortest peptide which could be significantly phosphorylated was Arg-Arg-Ala-Ser-Val, with an apparent Km of 0.08 mM. The apparent Km for Arg-Arg-Ala-Ser-Val-Ala was 0.02 mM and that for Leu-Arg-Arg-Ala-Ser-Val-Ala was less than 0.01 mM. Peptides in which threonine was substituted for serine, or leucine for the one or the other arginine of the pentapeptide were not detectably phosphorylated. Substitution of phenylalanine for valine increased, and substitution of lysine or glycine for valine considerably decreased the rate of phosphorylation.

A general procedure for analysis of proenkephalin B derived opioid peptides

Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10 000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW greater than 5000).

Opiate receptor affinity of peptides related to Leu-enkephalin.

Abstract Several analogs of Leu-enkephalin were synthesized by the standard solid phase procedure in order to investigate structural requirements for binding to opiate receptors. Decisive features for receptor interaction seem to be the presence and location of the aromatic side chains of the tyrosine and phenylalanine residues. The terminal amino and carboxyl groups do not contribute significantly to binding affinity.

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.

Amino acid sequence at the phosphorylated site of rat liver phenylalanine hydroxylase and phosphorylation of a corresponding synthetic peptide

Abstract Purified rat liver phenylalanine hydroxylase was phosphorylated by incubation with ( 32 P)ATP and the catalytic subunit of pig muscle cyclic AMP-stimulated protein kinase. After digestion of the phosphorylated phenylalanine hydroxylase with pepsin, one major ( 32 P)phosphopeptide with the amino acid sequence Ser-Arg-Lys-Leu-( 32 P)SerP-Asx-Phe-Gly-Glx-Glx was isolated. Two non-phosphorylated octapeptides, Ser-Arg-Lys-Leu-Ser-Asp-Phe-Gly and Ser-Arg-Lys-Leu-Ser-Asn-Phe-Gly, were synthesized and characterized as substrates of the protein kinase.

Isolation and structure analysis of bee venom mast cell degranulating peptide

The venom of Apis mellifera was processed by gel permeation chromatography on Sephadex G-50 and by reversed-phase HPLC. The initial gel permeation step was carried out in the presence of phosphate ions (0.5 M). Ion pair reagents were required to resolve the strongly basic peptides, secapin, mast cell degranulating (MCD-) peptide and apamin, by reversed-phase (RP) HPLC. Using this relatively simple procedure it is possible to isolate these peptides essentially free of melittin (less than 1 in 10(7)) and phospholipase (less than 1 in 10(5] in high yield. The CD spectrum and secondary structure analysis are reported for MCD-peptide and on this basis a solution structure is proposed for this toxin.

The structural requirements of substrates of cyclic AMP-dependent protein kinase

The primary structure required by substrates of cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been detailed by the aid of synthetic peptides, representing the phosphorylatable site of pyruvate kinase (EC 2.7.1.40) of rat and pig livers [ 1,2]. The minimum substrate emerging from these studies may be represented by the general sequence: Arg-Arg-X-Ser-X [3]. The substitution of either arginine residue with other ammo acids considerably increases the app. Km for cyclic AMP-dependent protein kinase [2]. The distance between the phosphorylatable serine residue and the block of 2 arginine residues is critical, as elucidated in [4] using synthetic dodecapeptides of the general sequence: (Gly),-Arg-Arg-(Gly)y-Ala-Ser-Leu-Gly and the peptide: It has been deduced from this and other examples that the required structure of substrates of cyclic AMPdependent protein kinase is met also by the general structure Lys-Arg-X-X-&r-X [3]. However, in experiments with synthetic peptides of the general structure (Gly),-Lys-Arg-(Gly)y-Ala-Ser-I_euGly, the spacing of the Lys-Arg-block from the phosphorylatable serine residue by 2 neutral residues gave extremely high Km-values [4]. Additional structural elements are apparently required to form the optimal environment of the phosphorylatable serine residue in the P-subunit of phosphorylase kinase.

A proposal for the structure of apamin

Apamin is an octadecapeptide neurotoxin present in bee venom. It acts both centrally and peripherally, binding specifically to a protein in rat forebrain with high affinity (Kd 10-l’ M) [l] and acting as a potent noncompetitive inhibitor of a-adrenergic and purinergic agonists [2]. The amino acid sequence (fig.1) has been independently determined by two groups [3,4] and the positions of the disulphide bridges have been established [5]. Synthetic apamin [6-81 possesses full biological activity and hence the information for the correct folding of the peptide chain is contained in that chain and not in an apamin precursor. As a result, apamin is a suitable candidate for secondary structure prediction. This paper proposes a structure for apamin on the basis of such a study. The structure is capable of explaining all the available physico-chemical data of apamin and its derivatives, including its high degree of stability. The proposed conformation will facilitate structureactivity studies with this class of molecule.

A comparative structural study of apamin and related bee venom peptides.

Secondary structure analysis of apamin, mast cell degranulating peptide, tertiapin and secapin has been attempted, based on parameters produced by Levitt (Biochemistry (1978) 17, 4277--4285). The structural model, recently advanced for apamin, based on Chou and Fasmans parameters was confirmed. The predicted structure for mast cell degranulating peptide is almost spherical with the eight positive centres evenly distributed over the surface. On the basis of this analysis and related spectroscopic evidence, it is suggested that these four peptides share a common folding pattern, which is centered on a beta-turn covalently linked to an alpha-helical segment by two disulphide links (one disulphide link in the case of secapin). It is further suggested that apamin, mast cell degranulating peptide and tertiapin form a single molecular class.