(EN) This work aims at preparation, analysis and isolation of intermediates of biosynthetic pathways of 4-alkyl-L-proline derivatives for their structural elucidation. Compounds with incorporated 4-alkyl-L-proline derivatives include clinically used lincosamide antibiotic, lincomycin A, antitumor pyrrolobenzodiazepines and bacterial hormone hormaomycin. Detailed knowledge of biosynthetic pathways of these biological active substances can be used to prepare new, more efficient derivatives. The first part of this work focuses on yellow-coloured dicarboxylic intermediates 1 and 2 of the biosynthetic pathway of 4-propyl-L-proline - the precursor of lincomycin A. In the presence of the methylation agent, S-adenosyl-L-methionine, and LmbW C- -methyltransferase, 1 was partially converted into intermediate 2. Using ultra-high performance liquid chromatography, both intermediates were identified from absorption and mass spectrometry spectra. A semi-preparative chromatographic method for isolation of both intermediates was developed. Surprisingly, a significantly lower stability of 2 compared to intermediate 1 was observed in an in vitro enzymatic reaction mixture. The second part of the work focuses on 4-ethylidene-L-proline - the precursor of tomaymycin belonging to pyrrolobenzodiazepines. After...