Enzymatic and Chemoenzymatic Three‐Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines

Abstract Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4‐trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step‐efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3‐hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet–Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)‐metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo‐ and biocatalysts for optimal results.


Transformation, cultivation and purification 1.1 Expression and purification of the norcoclaurine synthase from Thalictrum flavum variant (△29TfNCS-A79I) for reference compound synthesis (UCL London)
Expression of △29TfNCS-A79I [1] : A pJ411 plasmid containing a codon optimised Δ29TfNCS-A79I was purchased from DNA2.0 and E. coli BL21 (DE3) cells were transformed with these plasmids by a standard heat-shock protocol.

Expression and purification of enzymes for biotransformation (Forschungszentrum Jülich
GmbH)

Chemoenzymatic 3-step cascade reaction
Step 1 and 2 occurred as described above.
Step    Table 6 Retention times of the carboligation step compounds measured by HPLC IE-column gradient method (Table 5).

Peak identification
For the identification of known metabolites we used a baseline noise subtracted fragment pattern in comparison to our inhouse database JuPoD, the commercial database NIST11 (National Institute of Standards and Technology, USA) and the free available database GMD (MPI of Molecular Plant Physiology, Golm, Germany). [11], [12] Unknown peaks were identified by structural combination of elemental compositions and verified by virtual derivatisation and fragmentation of the predicted structures.

GC-TOF-MS data of the 3-step cascade reaction.
Figure 10 GC-TOF-MS data of the carboligation step (1-hydroxy-1-(3-hydroxyphenyl)propan-2-one is identified as main product of this step, glycerol is found in the reaction mixture, due to the use of not purged syringe filters).

Figure 11
GC-TOF-MS data of the transamination step, 2-amino-1-(3-hydroxyphenyl)propan-1-ol is identified as main product of this step, glycerol is found in the reaction mixture, due to the use of not purged syringe filters.

Figure 12
GC-TOF-MS data of the Pictet-Spengler condensation step (1-benzyl-3-methyl-1,2,3,4-tetrahydroisoquinoline-4,6diol is identified as main product of this step, glycerol is found in the reaction mixture due to the use of not purged syringe filters).
To (1R,2S)-metaraminal bitartrate (15.9 mg, 0.050 mmol; 10 mM) and sodium ascorbate  The product (injection volume 5 μL) was eluted with MeOH (containing 0.2% AcOH and 0.1% TEA) at 1 mL/min and the (1S,3S,4R)-product had a retention time (rt) of 8.5 min.                  The reaction mixture was then extracted with ethyl acetate (3 × 10 mL). The organic phases were combined and washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to obtain the crude product. Both the reaction mixture above and the crude product were analysed by 1 H NMR spectroscopy and NMR NOESY experiments. In addition a small amount of the ortho-product (termed here minor-S or R) was produced <4%.

Substrate screening for phosphate catalysed Pictet-Spengler reaction (step 3)
A substrate screen for most suitable substrates in terms of good conversion and highest stereoselectivity for the phosphate catalysed Pictet-Spengler reaction was performed.
Product peaks were tentatively assigned, as chemical standards were not available at the time.