Inverting the Regioselectivity of the Berberine Bridge Enzyme by Employing Customized Fluorine‐Containing Substrates

Abstract

Fluorine is commonly applied in pharmaceuticals to block the degradation of bioactive compounds at a specific site of the molecule. Blocking of the reaction center of the enzyme-catalyzed ring closure of 1,2,3,4-tetrahydrobenzylisoquinolines by a fluoro moiety allowed redirecting the berberine bridge enzyme (BBE)-catalyzed transformation of these compounds to give the formation of an alternative regioisomeric product namely 11-hydroxy-functionalized tetrahydroprotoberberines instead of the commonly formed 9-hydroxy-functionalized products. Alternative strategies to change the regioselectivity of the enzyme, such as protein engineering, were not applicable in this special case due to missing substrate–enzyme interactions. Medium engineering, as another possible strategy, had clear influence on the regioselectivity of the reaction pathway, but did not lead to perfect selectivity. Thus, only substrate tuning by introducing a fluoro moiety at one potential reactive carbon center switched the reaction to the formation of exclusively one regioisomer with perfect enantioselectivity.

Flavoprotein oxidases: classification and applications

“…As some of the 11-hydroxy functionalised products present biological activity, such as (S)-isocoreximine, (S)-corytenchine and phellodendrine, reticuline oxidase and its mutant were employed in the oxidative coupling of reticuline analogues, but only traces of the non-expected regioisomers were found. Only substrate modification, by introducing fluoro substituents, led to the synthesis of the 11-hydroxy compounds as sole products with good yields and excellent selectivities (Resch et al 2012).…”

Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

“…For example, NCS has been utilized to produce a wide range of tetrahydroisoquinolines (THIQ) from simple synthetic precursors 5. Similarly the berberine bridge enzyme has also been used to produce non‐natural scoulerine analogues 6. Additionally, enzymes from BIA biosynthesis, have been combined with enzymes from other pathways to create novel products 7.…”

“…Such insights can guide the engineering of enzymes towards improved activity or more stringent substrate specificity, which could reduce kinetic bottle necks or side‐product formation in engineered microbial BIA producing strains 2, 3. Similarly, engineered variants of BIA enzymes, with specificity for non‐natural substrates, could open up new routes to alternative bioactive THIQ scaffolds 4, 5, 6, 7. In this paper we describe the first crystal structure of CNMT, an essential enzyme in the core pathway to BIAs, which introduces the amino methyl functionality that is often essential for the bioactivity of most of the medically important BIAs (Figures 1 & S1).…”

Biocatalyzed C−C Bond Formation for the Production of Alkaloids

“…A follow‐up study by Resch et al . demonstrated that the regioselectivity of the products formed as a consequence of BBE‐catalyzed cyclization is influenced by the substitution pattern of the substrate, the pH of the reaction medium and the type and amount of co‐solvents used.…”