Racemization-free and scalable amidation of l-proline in organic media using ammonia and a biocatalyst only

Julia Pitzer, Kerstin Steiner, Christian Schmid, Viktor K. Schein, Christoph Prause, Claudia Kniely, Michaela Reif, Martina Geier, Elena Pietrich, Tamara Reiter, Philipp Selig, Clemens Stückler, Peter Pöchlauer, Georg Steinkellner, Karl Gruber, Helmut Schwab, Anton Glieder*, Wolfgang Kroutil

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Efficient amide formation is of high importance for the chemical and pharmaceutical industry. The direct biocatalytic one-pot transformation of acids into amides without substrate activation is a highly desirable but highly challenging reaction; this is why in general the acid is activated using additional reagents before amide formation occurs. In particular, amidation of α-amino acids is challenging and in general requires protection strategies for the amino functionality. A further challenge is the low solubility of the unprotected amino acids in organic solvents. Furthermore, the amidation process is prone to racemisation as observed for the acyl chloride derivative. These three challenges may be addressed using biocatalysis. Here the enzyme catalyzed, racemization-free amidation of unprotected l-proline with ammonia in an organic solvent is described. Comprehensive reaction, solvent and enzyme engineering allowed obtaining high l-prolinamide concentrations. For instance at 145 mM substrate concentration, 80% conversion was achieved employing an immobilized CalB variant and ammonia in 2-methyl-2-butanol at 70 °C. A two-fold increase in l-prolinamide formation was achieved employing the immobilized and engineered enzyme variant CalBopt-24 T245S compared to wild type CalB. In contrast to chemical processes, racemization, halogenated solvents and waste are avoided/minimized and atom efficiency is significantly improved from 45.5% to 86.4%. The excellent optical purity of the obtained product (ee >99%) and the stability of immobilized CalB pave the way for an innovative industrial process to produce l-prolinamide, a key intermediate in drug synthesis.

Original languageEnglish
Pages (from-to)5171-5180
Number of pages10
JournalGreen Chemistry
Issue number13
Publication statusPublished - 25 May 2022

ASJC Scopus subject areas

  • Environmental Chemistry
  • Pollution


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