Martin Flegel

Large‐scale synthesis of peptides

Recent advances in the areas of formulation and delivery have rekindled the interest of the pharmaceutical community in peptides as drug candidates, which, in turn, has provided a challenge to the peptide industry to develop efficient methods for the manufacture of relatively complex peptides on scales of up to metric tons per year. This article focuses on chemical synthesis approaches for peptides, and presents an overview of the methods available and in use currently, together with a discussion of scale-up strategies. Examples of the different methods are discussed, together with solutions to some specific problems encountered during scale-up development. Finally, an overview is presented of issues common to all manufacturing methods, i.e., methods used for the large-scale purification and isolation of final bulk products and regulatory considerations to be addressed during scale-up of processes to commercial levels.

Continuous-flow solid-phase peptide synthesis

Abstract We describe a simple manually operated synthesizer for solid-phase peptide synthesis (SPPS). The synthesis is performed on standard polystyrene-based resin in a flow reactor under low-pressure conditions. The usefulness of the present configuration of SPPS is exemplified on the synthesis of two octapeptide and one decapeptide amides.

Chiral analysis of biogenic DL‐amino acids derivatized by urethane – protected α‐amino acid N‐carboxyanhydride using capillary zone electrophoresis and micellar electrokinetic chromatography

A new analytical method for enantioselective separation of DL‐amino acids derivatized by N‐fluorenylmethoxycarbonyl‐L‐alanyl N‐carboxyanhydride (FMOC‐L‐Ala‐NCA) using capillary electrophoresis was developed. Separation parameters, such as composition and pH of the background electrolyte, and concentration of γ‐cyclodextrin (in capillary zone electrophoresis) and sodium dodecyl sulfate (in micellar electrokinetic chromatography) were optimized. The separation method was validated and it suits well for purity analysis. Detection limit of the method was 0.2% of the minor enantiomer in the major one. The level of racemization in coupling during solid‐phase peptide synthesis was studied using capillary electrophoresis with γ‐cyclodextrin as a chiral selector. The anchorage of the first (C‐terminal) amino acid derivative to the solid supports bearing the hydroxylic groups is the key step of the synthesis affecting the extent of its racemization. FMOC‐L‐phenylalanine was chosen as the suitable model amino acid derivative making it possible to study the degree of racemization of N‐fluorenylmethoxycarbonyl‐L‐alanine‐L‐phenylalanine synthesized on different polymer resins, using the different condensation agents.

Galanin modulating effect on restraint stress-induced short- and long-term behavioral changes in Wistar rats.

The neuropeptide galanin has been recognized as a possible neurotransmitter/neuromodulator, and in addition has been implicated in anxiety- and depression-related behaviors. The present study demonstrates increased locomotion and rearing after galanin (0.3mg/kg) that was given intraperitoneally (i.p.) to intact Wistar rats which were tested 1h later in the open field (OF). These effects, which suggest an anxiolytic-like action, were blocked by i.p. administered peptidic galanin antagonist M40. Further, the locomotion increase caused by galanin and the inhibitory effect of M40 persisted for 48h without additional treatment. Rats exposed to restraint stress (lasting 60min) for three consecutive days and tested 1h after stress termination exhibited reduced locomotion and exploration in the OF. Galanin (0.3 and 1.0mg/kg) given immediately after each stress exposure prevented the decrease of locomotion and exploration induced by stress in all trials. When the test was repeated 6 days later without stress and galanin treatment the reduction of locomotion produced by stress persisted; the anti-stress behavioral effects of both galanin doses were also present. Testing performed on the 12th day after the last stress and galanin treatment with 0.3mg/kg revealed an increased locomotion compared with unstressed and stress-exposed rats. Our results demonstrate that behavioral effects of the peptide galanin are evident even after i.p. administration. These results also suggest that galanin elicits stress-modulatory action, and support the notion that the galaninergic system may serve as a drug target in stress-related conditions.

Electronic and Vibrational Optical Activity of Several Peptides Related to Neurohypophyseal Hormones: Disulfide Group Conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.

Pharmacology of oxytocin-(1-6)-hexapeptide amide, vasopressin-(1-6)-hexapeptide amide and their deamino analogues.

Abstract Oxytocin-(1 -6)-hexapeptide amide and vasopressin-(1 -6)-hexapeptide amide had lower activities than the parent hormones on the isolated rat uterus and in the antidiuretic assay in hydrated rats; none of the analogues had pressor activity. Deamination of both rings increased uterotonic activity. The uterotonic effect of the analogues was blocked by N-carbamoyl-2-O-methyloxytocin, a competitive inhibitor of oxytocin, which proved their action to be specific. Mg2+ did not potentiate the effect of the ring molecules on the isolated uterus.

Acidic Cleavage of the Side Chain Protecting Groups of Peptides Attached to a Modified Polystyrene Carrier: Synthesis of Peptide Generics

High or good yield is essential for peptide production. The side chain protecting groups are cleaved usually in solution, after peptide detachment from the solid carrier. This can, however, be accompanied by the loss of material and the decrease in yield. Such problems can be circumvented when the side chain deprotection steps are carried out on the peptide still bound to resin. Just before purification, the final product can be detached from resin. Analogues of Vasopressin and LH-RH were synthesized on hydroxyalkylated aminopolystyrene. Stability of polymeric like alkyl ester towards hard acid (TFMSA, HF) makes possible the assembling of peptides protected with Benzyl and Tosyl groups. All cleavage steps were carried out on the peptide bound to resin. In this way salts and scavengers can also be washed away prior to detachment of peptide from the resin. Ammonolysis in gas [1] or ethylaminolysis was employed as the detachment step. The crude peptide amides were obtained in the high yield and purity. Conditions for the complete cleavage of Tos group were developed. Moreover, the resin could be used again for the new peptide synthesis.