Asymmetric Synthesis of (R)‐1‐Alkyl‐Substituted Tetrahydro‐ß‐carbolines Catalyzed by Strictosidine Synthases

Abstract Stereoselective methods for the synthesis of tetrahydro‐ß‐carbolines are of significant interest due to the broad spectrum of biological activity of the target molecules. In the plant kingdom, strictosidine synthases catalyze the C−C coupling through a Pictet–Spengler reaction of tryptamine and secologanin to exclusively form the (S)‐configured tetrahydro‐ß‐carboline (S)‐strictosidine. Investigating the biocatalytic Pictet–Spengler reaction of tryptamine with small‐molecular‐weight aliphatic aldehydes revealed that the strictosidine synthases give unexpectedly access to the (R)‐configured product. Developing an efficient expression method for the enzyme allowed the preparative transformation of various aldehydes, giving the products with up to >98 % ee. With this tool in hand, a chemoenzymatic two‐step synthesis of (R)‐harmicine was achieved, giving (R)‐harmicine in 67 % overall yield in optically pure form.

Abstract: Stereoselective methods for the synthesis of tetrahydro-ß-carbolines are of significant interest due to the broad spectrum of biological activity of the target molecules.I nt he plant kingdom, strictosidine synthases catalyze the CÀC coupling through aP ictet-Spengler reaction of tryptamine and secologanin to exclusively form the (S)-configured tetrahydro-ß-carboline (S)-strictosidine.I nvestigating the biocatalytic Pictet-Spengler reaction of tryptamine with small-molecular-weight aliphatic aldehydes revealed that the strictosidine synthases give unexpectedly access to the (R)-configured product. Developing an efficient expression method for the enzyme allowed the preparative transformation of various aldehydes,giving the products with up to > 98 %ee. With this tool in hand, ac hemoenzymatic two-step synthesis of (R)harmicine was achieved, giving (R)-harmicine in 67 %overall yield in optically pure form.
ThePictet-Spenglerreaction [1] allows the synthesis of alarge variety of heterocyclic compounds, [2] and various chemical methods have been established, including many stereoselective approaches. [2][3][4] It is catalysed in nature by substratepattern-specific enzymes. [5] Them ost prominent and best characterized members of these Pictet-Spenglerases are the norcoclaurine synthases [6] and the strictosidine synthases (STRs). [7] In the natural reaction, the STR (EC 4.3.3.2 according to the Enzyme Commission classification) condenses tryptamine (1)a nd secologanin to generate the (S)-configured 1,2,3,4tetrahydro-ß-carboline (S)-strictosidine (Scheme 1). The latter serves as ac entral precursor of naturally occurring, biologically active indole alkaloids,thus the enzyme is located in the biosynthetic pathways at the central branching point of more than 2000 monoterpenoid indole alkaloids in higher plants,i ncluding some possessing extraordinary medicinal and therapeutic value. [8] In general STRs have been reported to show an arrow substrate spectrum with respect to the aldehyde reactant, transforming only secologanin and close derivatives in most cases; [7,9] to theb est of our knowledge, only one report indicated that simple non-natural aliphatic aldehydes might also be accepted. [10] Therefore,o ur aim was to investigate the substrate tolerance of STRs for non-natural aldehydes.
Our research focused on four strictosidine synthases originating from Catharanthus roseus (CrSTR), [11] Ophiorrhiza pumila, (OpSTR), [6e, 12] and Rauvolfia serpentina (RsSTR), [13] as well as its V208A variant (RvSTR), [13e] which was described to accept an even broader spectrum of amines. We quickly noticed that asignificant limitation of using STRs is the low expression level in E. coli when using various described constructs,although the activity with secologanin is high (e.g.3 1Umg À1 for RsSTR). [14] Attempts at expressing native sequences of the STRs (Table S1 in the Supporting Information) using various expression plasmids as well as various E. coli hosts,c haperones,a nd expression conditions resulted in no visible overexpression bands detectable by SDS PAGE, although the enzyme preparations allowed us to run the transformation of the natural substrate to completion within 24 hours at 1-2 mm substrate concentration. [6b] Interestingly,h ighest activities were found when the STRs were expressed with an Nterminal His 6 -tag.However, the use of anon-natural aldehyde such as isovaleraldehyde 2a did not lead to clear product formation independent of the enzyme preparation applied. Additionally,t he application of refolding strategies to solubilize inclusion bodies,w hich were formed in significant amounts,were not successful. Thekey to success was finally to design E. coli optimized DNAs equences devoid of the inherent signal peptide [15,16] but including an N-terminal His 6tag, which were expressed using E. coli Shuffle T7LysY as the expression host for promoting disulfide bond formation, and performing the expression in TB medium by ac areful selection of the expression conditions.T his approach finally led to clear detectable enzyme expression and up to 100-fold improvement in activity per mg cell-free extract when employing the natural substrates ( Figure S1 and Table S6).
Biotransformation of tryptamine with the aliphatic aldehyde isovaleraldehyde 2a led to ac lear detection of product for all four investigated STRs when using freeze dried cellfree extracts.O ptimization of the reaction conditions (pH, buffer salt, temperature,s ubstrate concentration) enabled successful transformation of tryptamine 1 (10 mm)a nd isovaleraldehyde 2a (50 mm)i nto the corresponding 1,2,3,4tetrahydro-ß-carboline 3a with 77 %c onversion when employing RsSTR within 20 hours (Scheme 2). It has to be noted that 2.5 U strictosidine were employed for these reactions, which equals ac atalyst loading of 0.32-6.9 mg of pure STR (applied as 5-65 mg CFE) per 500 mLr eaction volume.T he STRs showed significant lower activity for the transformation of the non-natural aldehydes in comparison to the natural substrates,w hich can probably be attributed to less/no interaction sites between the enzyme and aldehyde substrate. Analysis of the optical purity indicated an essentially optically pure product for RsSTR, RvSTR, and OpSTR (ee > 98 %, HPLC) while for CrSTR, an ee of 88 %was obtained (Table 1, entries [1][2][3][4]. Deducing the absolute configuration of the enzymatically formed product 3a by comparison of previously reported elution order on aC hiralpak IC column [17] unexpectedly led to the conclusion that the newly formed chiral centre has to be (R)-configured. This is surprising because the natural reaction leads to the (S)-configured product (S)-strictosidine and the (S)c onfiguration has been assumed for product 3a in ap revious study. [18] To verify the absolute configuration, as emi-preparative biocatalytic Pictet-Spengler reaction was performed using RsSTR and the optical rotation of the obtained product 3a (85.4 mg, 75 %i solated yield) was measured (> 98 % ee:[a] 20 D : + 63 (c 1.0, MeOH). Comparison with published optical rotation values at comparable conditions for the (S)-enantiomer [19] and two reports for the (R)enantiomer [19b,20] clearly support the idea that the obtained product is (R)-configured, thus (R)-3a was obtained.
In as imilar fashion, the non-branched linear low-molecular-weight aldehyde n-butanal 2bwas transformed giving the corresponding (R)-3b product with up to 91 % ee (Entry 6). Thes mallest possible aldehyde leading to ac hiral product, acetaldehyde 2c,gave (R)-2cwith aclear enantiomeric excess (43 %) when employing RvSTR, while the variant RsSTR led to racemic product (Entry 10 and 11). In all these cases,t he (R)-configured product was formed as verified by assigning the absolute configuration by comparison with published optical rotation values (see the Supporting Information). [21] In the case of hexanal 2d,the conversions were low (max 8% within 20 h), leading to the corresponding b-carboline 3d with up to 82 % ee when using RvSTR (entry 14). With all four enzymes investigated, the same enantiomer of the product was in excess.T op rove the absolute configuration of the obtained product 3d,t wo independent asymmetric syntheses of optically enriched material according to reported procedures were repeated to have material to identify the elution order of the enantiomers.Both methods,namely using ac hiral Brønsted acid [4a] or ac hiral auxiliary, [22] are reported to give the (S)-enantiomer.C omparison of the elution order of the enantiomers clearly proved that the four enzymes investigated led to the formation of the R-configured (R)-3d (see Figure S20). [23] To demonstrate the synthetic applicability of the biocatalytic Pictet-Spengler reaction with non-natural aliphatic aldehydes,the synthesis of the natural alkaloid (R)-harmicine [(R)-5]w as envisioned. (R)-5 has been isolated from the Malaysian plant Kopsia griffithii,w hich displays strong anti-Leishmania activity. [24] Recently antinociceptive properties have been assigned to 5. [25] Consequently,t he biocatalytic transformation of commercially available methyl 4-oxobutanoate 2e with tryptamine 1 was performed and led to the formation of the Pictet-Spengler product (R)-3e followed by spontaneous ring closure to give the lactam product (R)-4 Scheme 2. Strictosidine-synthase-catalyzedP ictet-Spengler reaction between tryptaminea nd non-naturala liphatic low-molecular-weight aldehydes.  Afew asymmetric synthetic routes to (R)-harmicine [(R)-5]h ave been reported. [26] To the best of our knowledge,t he approach presented here,which enables the synthesis of (R)-5 in optically pure form in just two steps from commercial substrates,i st he shortest and highest yielding sequence reported to date.
Although optimization of the expression conditions to produce sufficient enzyme enabled actual preparative transformations,f urther optimization of the biocatalysts,f or example,b ye nzyme engineering,i sr equired to reduce the amount of catalyst needed even further.
In conclusion, it has been shown that strictosidine synthases can be used for preparative transformations of low-molecular-weight aldehydes,leading unexpectedly to the corresponding (R)-configured products,w hich is counterintuitive since the natural transformation with the aldehyde secologanin leads to the (S)-configured product. Consequently,t he natural alkaloid (R)-harmicine was synthesized with the developed method in just two steps with 62 %yield of isolated product in optically pure form. Thep resented asymmetric biocatalytic method extends the biocatalytic toolbox for the preparation of chiral amines [27] through biocatalytic CÀCb ond formation. [28] Experimental Section Preparative example for the biocatalytic synthesis of the precursors (R)-4 for (R)-harmicine: Freeze dried cell free extracts of recombinant His 6 -RsSTR overexpressed in E. coli/Shuffle T7LysY (500 U strictosidine )w ere dissolved in aP IPES-tryptamine*HClb uffer system [50 mL, 50 mm PIPES,10mm tryptamine*HCl 1*HCl, pH 6.1] in 150 mL Erlenmeyerflasks without baffles.The reaction was started by the addition of methyl 4-oxobutanoate 2e (75 mg,0.33 mmol, final concentration:5 0mm). Them ixture was incubated for 48 ha t 470 rpm at 35 8 8C, then quenched by the addition of aq. NaOH (5 mL, 10 N) and extracted with ethyl acetate (3 100 mL). The combined organic phase was dried over Na 2 SO 4 and the solvent was evaporatedu nder reduced pressure.P roduct( R)-4 was obtained as aw hite solid (75.4 mg,6 7%).