Transaminase and Norcoclaurine Synthase One‐Pot Cascades Towards (1S)‐Tetrahydroisoquinolines

TL;DR

Researchers developed a one-pot enzyme cascade to efficiently produce biologically active THIQs from amines with high enantiomeric purity.

Contribution

A TAm-NCS cascade was developed for in situ aldehyde generation and THIQ synthesis with improved substrate tolerance.

Findings

The cascade produced (1S)-6,7-dihydroxy-THIQs with up to >99% enantiomeric excess and 83% yield.

The method works with diverse amines, both nonfunctionalized and functionalized.

A tyrosinase extension enabled the synthesis of 3-hydroxymethylene substituted THIQs.

Abstract

Tetrahydroisoquinolines (THIQs) are an important group of alkaloids, and many of these natural products and non‐natural analogues are biologically active. For this reason, concise stereochemical synthetic routes are of significant interest. One strategy has been to use the Pictet–Spenglerase enzyme norcoclaurine synthase (NCS) with an arylethylamine and aldehyde in a single step reaction, alternatively enzyme cascades have been developed. The use of a transaminase (TAm) potentially provides efficient access to the aldehyde component required in NCS cascades, but currently suffers from limited substrate tolerance, in part due to potential complexities with the formation of mixed products. Here we present the development of a TAm‐NCS cascade using structurally diverse primary amines to form (1S)‐6,7‐dihydroxy‐THIQs via the generation of, in particular, aliphatic aldehyde intermediates in situ. Application of the one‐pot cascade with amines successfully generated the corresponding products in up to >99% enantiomeric excess and 83% isolated yields. The cascade was also extended with a tyrosinase to produce a 3‐hydroxymethylene substituted THIQ.

A TAm‐NCS cascade towards THIQs has been established, starting from amines with the in situ production of aldehydes. The cascade was used with a variety of nonfunctionalized and functionalized amines, and reaction conditions modified to enhance formation of the desired cross‐products. This resulted in an efficient one‐pot approach to THIQs starting from readily available amines.© 2026 WILEY‐VCH GmbH

Figures

A comparison of previous work using TAms to provide precursors for NCS reactions and this work using one‐pot TAm‐NCS cascades to produce THIQs.

TAm‐NCS cascade towards (1S)‐6,7‐dihydroxy‐THIQs with nonfunctionalized amines. Reaction conditions: a: CvTAm CCL, b: pQR2189‐TAm CCL, c: desalted CCL CvTAm (blue text). Conversions (dopamine consumption) and relative yields of cross/self‐PSR products were determined by 1H NMR spectroscopy using maleic acid as the internal standard, or HPLC against product standards. For selected examples, ees were determined using CCL and desalted CvTAm, and some reactions were performed at a larger scale and products isolated. Ees were determined by chiral HPLC or the use of Marfey's reagent (see Supporting Information).

TAm‐NCS cascade towards (1S)‐6,7‐dihydroxy‐THIQs with functionalized amines. Reactions conditions: a: CvTAm CCL, b: pQR2189‐TAm CCL, c: desalted CCL CvTAm (in blue), d: pQR2208‐TAm. Conversions (dopamine consumption) and relative yields of cross/self‐PSR products were determined by 1H NMR spectroscopy using maleic acid as the internal standard or by HPLC analysis against product standards. For selected examples, ees were determined using CCL and desalted CvTAm, and some reactions were performed at a larger scale and products isolated. Ees were determined by chiral HPLC.

TYR‐TAm‐NCS cascades to give 7. Reaction conditions: 6 (10 mM), TYR lysates (10%, v/v), PLP (1 mM), DMSO (10% v/v), TAm lysates (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) in water or KPi buffer (50 mM, pH 7.5), 37°C, 18 h. aYields were calculated against product standards by HPLC analysis; diastereomeric ratios were determined by HPLC analysis and 1H NMR spectroscopy.

Tables

TABLE 1. Initial development of the TAm‐NCS cascade between 1 and 2a to give 4a and 5.a

Transaminase	1	2a	3	Relative ratiob 4a:5
CvTAm	10 mM	7.5 mM	10 mM	1:3
CvTAm	10 mM	10 mM	10 mM	1:2
CvTAm	10 mM	20 mM	15 mM	1:1
pQR2189	10 mM	20 mM	15 mM	2:1
pQR2208	10 mM	20 mM	15 mM	3:1

Full text

Introduction

1

The application of biocatalytic synthetic strategies is of increasing importance due to the use of mild reaction conditions, environmentally friendly solvents, and high regio‐ and stereoselectivities that can be achieved. Moreover, the incorporation of enzymes into biocatalytic cascades avoids the isolation of intermediates and purification steps, which saves time, lowers the costs involved, and simplifies procedures [1]. This approach has been applied to several pharmaceutically important compounds such as islatravir [2] and MK1454 [3].

Tetrahydroisoquinolines (THIQs) are an important group of natural and synthetic alkaloids with a range of pharmaceutical applications such as the analgesics morphine and codeine, and vasodilator papaverine [4]. Other examples include ET‐743 an antitumour compound [5], and emetine which is used as an antiparasitic drug [6]. Several strategies for the asymmetric syntheses of natural and unnatural THIQs have been developed, among which the asymmetric Pictet–Spengler (PS) cyclization provides one of the most straightforward approaches by combining amine and aldehyde fragments [7]. Although (R)‐TRIP ((R)‐3,3′‐bis(2,4,6‐triisopropylphenyl)‐1,1′‐binaphthyl‐2,2′‐diyl hydrogenphosphate) [8, 9] and IDPi (imidodiphosphorimidate) [10] organocatalysts have been successfully applied in asymmetric PS reactions, N‐substituted substrates were required, adding additional steps. Biocatalytic routes to THIQs have proved to be particularly successful through application of the Pictet‐Spenglerase norcoclaurine synthase (NCS) in the key C—C bond forming step [11]. In biosynthetic pathways to benzylisoquinolines, NCS catalyzes the formation of (S)‐norcoclaurine from dopamine 1 and 4‐hydroxyphenylacetaldehyde [12, 13]. Recently, recombinant NCSs in Escherichia coli (E. coli) have been applied to synthesize a range of (S)‐THIQs starting from 1 and analogues, and aromatic and aliphatic aldehydes [14, 15, 16]. Notably, NCS has also been combined into cascades with other biocatalysts including tyrosinases, decarboxylases, carbonyl reductases and transaminases (TAms) to access dopamine and analogues [17, 18, 19, 20], and carbonyl reductases [21] or TAms [19, 20, 22, 23] to produce arylacetaldehydes and phenylpropanaldehydes (Scheme 1A). Sustainable access to other aldehydes for coupling to NCS would be valuable, as they are reactive compounds, traditional synthetic approaches use toxic reagents or solvents, and efficient in situ generation not requiring isolation would expand the structural diversity of alkaloids accessible. Indeed, TAms are a well‐established biocatalyst for amine synthesis, but by comparison have been used less frequently for aldehyde synthesis [24, 25]. Here, we present a TAm‐NCS cascade towards (1S)‐THIQs using a range of aliphatic and functionalized amines, highlighting the key challenges, and solutions identified to establish this strategy as a general approach (Scheme 1B). Moreover, the cascade was extended using a tyrosinase with tyrosinol to produce a 3‐hydroxymethylene substituted THIQ.

A comparison of previous work using TAms to provide precursors for NCS reactions and this work using one‐pot TAm‐NCS cascades to produce THIQs.

Results and Discussion

2

Dopamine 1 and the functionalized amine methyl 4‐aminobutyrate 2a were used as model substrates for method development: the corresponding aldehyde of 2a had previously been successfully used with 1 [26]. Initially, Chromobacterium violaceum (Cv) TAm clarified cell lysates (CCL) and purified wild‐type Thalictrum flavum (Tf) NCS (N‐terminal 29 amino acid truncated, WT‐Δ29TfNCS) were used together in the same reaction vessel. In the presence of both biocatalysts, 1, 2a, the TAm amine acceptor sodium pyruvate 3 (which produces alanine (Ala)) and co‐factor pyridoxal phosphate (PLP), the enzymatic deamination and PS reaction led to the formation of the desired cross‐product 4a (Table 1, Supporting Information Figure S1) together with a self‐PS reaction product norlaudanosoline 5 [22]. THIQ 5 was formed from 1 and 3,4‐dihydroxyphenylacetaldehyde produced by the reaction of 1 with CvTAm. Clearly, this was one of the challenges to overcome, to enhance cross‐product formation rather than 5. Notably, the relative ratio of products depended on the comparative concentrations of 1, the amine 2a and amine acceptor 3: with the same concentrations of 1 and 3, and less 2a, the relative product ratio of 4a:5 was 1:3. However, on increasing the relative amounts of 2a and 3, 4a and 5 were formed in a 1:1 ratio. The control reaction with no NCS and only TAm also gave products 4a and 5 in lower quantities due to the background chemical PS reaction [26, 27], and this was explored further below.

In an attempt to decrease the formation of the side‐product 5, a two‐step one pot cascade was investigated with dopamine 1 added after the TAm reaction had commenced, but the ratio of 4a:5 did not improve (Supporting Information, Figure S2). Two other TAms were then explored, pQR2189‐TAm and pQR2208‐TAm [28]. Interestingly, both pQR2189‐TAm and pQR2208‐TAm gave higher comparative ratios of the desired cross‐product 4a compared to 5, than when using CvTAm, with relative ratios of 2:1 and 3:1 for 4a:5, respectively (Table 1, Supporting Information Figure S3 and LC‐MS Figure S4). This may reflect the relative rates of deamination by the TAms compared to NCS reaction rates. A trace amount of the cyclized lactam cross‐product formed from 4a was also detected by HPLC analysis [26].

To exemplify the methodology, the aromatic amine 2b was tested which was readily accepted in the cascade using CvTAm CCL to give (S)‐4b and the stereoselectivity determined as 95% enantiomeric excess (ee) by chiral HPLC (Supporting Information Figure S6) [20]. The major (S)‐stereoselectivity was in agreement with previous NCS studies. To further extend the substrates used, a range of nonfunctionalized amines 2c–2i were investigated (Scheme 2) and the stereoselectivities confirmed. Briefly, high conversions were achieved using linear chain alkyl amines, giving 72%, 78%, and 81% yields by ^1^H NMR spectroscopy of the corresponding THIQ products 4c–4e, with negligible self‐PS reaction product 5 (<5%) detected. The stereoselectivity in 4c when using 2c, was >90% ee (Scheme 2, Figure S7). An enzymatic scaled‐up reaction using amine 2d yielded 4d in 62% isolated yield after purification by an acid–base extraction method [26], and 90% ee, while 4e was formed in 95% ee (Figures S8 and S9). By adopting pQR2189‐TAm [28] in the cascade, moderate yields by ^1^H NMR spectroscopy of the desired cross‐products 4f (54%) and 4g (47%) were achieved with the short‐chain amines. Interestingly, 2‐cyclohexyl‐1‐ethylamine 2h and 2‐cyclopentyl‐1‐ethylamine 2i were also tolerated in the TAm‐NCS cascade using CvTAm. The yields by ^1^H NMR spectroscopy of 4h (84%) were more than twice that of 4i (37%), presumably reflecting the native reactivity of NCS with arylacetaldehydes. An enzymatic scaled‐up reaction (0.1 mmol scale) using 2h afforded 4h in 83% isolated yield and 89% ee, (determined via a derivatization method using Marfey's reagent, Scheme S1) [29, 30].

TAm‐NCS cascade towards (1S)‐6,7‐dihydroxy‐THIQs with nonfunctionalized amines. Reaction conditions: a: CvTAm CCL, b: pQR2189‐TAm CCL, c: desalted CCL CvTAm (blue text). Conversions (dopamine consumption) and relative yields of cross/self‐PSR products were determined by 1H NMR spectroscopy using maleic acid as the internal standard, or HPLC against product standards. For selected examples, ees were determined using CCL and desalted CvTAm, and some reactions were performed at a larger scale and products isolated. Ees were determined by chiral HPLC or the use of Marfey's reagent (see Supporting Information).

Background PS reactions can occur in the presence of phosphate [27], so it was determined whether endogenous phosphates could significantly affect the stereoselectivities in cascades. To explore this for selected substrates, reactions using desalted CCL CvTAm were performed and compared to when using CCL (Scheme 2). The cascades with amine 2b afforded 4b in 98% ee with desalted CCL TAm, compared to an 95% ee in CCL TAm. For other substrates, ees improved slightly such as 4c–4e (92%–95% ee). This highlighted that endogenous phosphates in the TAm CCL only had a small effect on the stereoselectivities of the products. Ees for 4f, 4g, and 4i were then also determined using desalted TAms as >99%, 88%, and 70%, respectively (Figures S10, S11, and S12).

The TAm‐NCS cascade was then applied to functionalized amines (Scheme 3). First, 2a and the longer chain analogue 2j [31] were compared giving 4a in 57% and 4j in 73% HPLC yields (against product standards) using pQR‐2208 TAm, which had given the highest selectivities for 4a (Table 1), although 5%–10% 5 was formed (Figure S5). The higher yield of 4j was consistent with our previous kinetic study that the longer chain aldehyde formed from 2j reacted more rapidly with NCS**,** due to reduced steric interactions in the active site which had been explored using in silico modelling [26]. Amine 2k was then examined, containing an endocyclic C=C bond, giving the desired cross‐PSR product 4k in 31% HPLC yield and ees of 85% in CCL TAm and >95% in desalted CCL TAm. In addition, amine 2l, containing a terminal alkene, was used in the cascade to generate 4l in 72% yield by HPLC (against product standards), with a 92% ee in CCL TAm and 97% ee when desalted CCL CvTAm was applied.

TAm‐NCS cascade towards (1S)‐6,7‐dihydroxy‐THIQs with functionalized amines. Reactions conditions: a: CvTAm CCL, b: pQR2189‐TAm CCL, c: desalted CCL CvTAm (in blue), d: pQR2208‐TAm. Conversions (dopamine consumption) and relative yields of cross/self‐PSR products were determined by 1H NMR spectroscopy using maleic acid as the internal standard or by HPLC analysis against product standards. For selected examples, ees were determined using CCL and desalted CvTAm, and some reactions were performed at a larger scale and products isolated. Ees were determined by chiral HPLC.

Cascades with amino alcohol 2m and pQR2189‐TAm then gave THIQ 4m in 46% yield by ^1^H NMR spectroscopy, although around 20% of the self‐product 5 was also observed, most likely reflecting that the intermediate aldehyde produced by 2m can reversibly cyclize in situ to form a hemiacetal, reducing availabilities. Linear aliphatic diamines 2n, 2o were also tested, and 39% and 31% yields by ^1^H NMR spectroscopy of the THIQs 4n, 4o were observed, respectively. An enzymatic scaled‐up reaction with 2o was also carried out with CCL CvTAm and 4o was isolated in 36% yield after a 4 h reaction. Stereoselectivities were investigated with TAm CCL giving 4n in 62% ee in desalted CCL TAm (15% ee nondesalted). This may reflect the more rapid background PS reaction in the presence of endogenous phosphates and highly soluble aldehydes.

To further extend the cascade, R‐tyrosinol 6 was synthesised (see Supporting Information) [32]. A new cascade was then established using a tyrosinase from Candidatus nitrosopumilus (CnTYR) [19, 20], together with TfNCS and CvTAm in a one pot reaction. The tyrosinase was employed to introduce an ortho‐hydroxyl group to produce the catechol 1b from 6. Sodium ascorbate was added to suppress over‐oxidation of the catechol. The cascade with 6 and 2l then gave 7 in 80% yield (by HPLC against product standards) (Scheme 4). No self‐product was formed from the reaction of CvTAm and the ketone formed from 1b, as NCS‐mediated reactions with ketones are much slower for steric reasons. The cascade gave 7 in a 93:7 diastereomeric ratio (d.r.) with desalted CvTAm lysates, or a 90:10 d.r. with CvTAm CCL for isomers (1S,3R):(1R,3R)‐7, whereas the CnTYR‐CvTAm‐(potassium phosphate) KPi chemoenzymatic cascade gave 7 in a 65:35 d .r. (1S,3R):(1R,3R). The preference for the (1S)‐ stereochemistry was consistent with the preferred selectivity of WT‐NCS, while the decrease in selectivity at C‐1 in CCL and KPi can be attributed to the background PS reaction [27].

TYR‐TAm‐NCS cascades to give 7. Reaction conditions: 6 (10 mM), TYR lysates (10%, v/v), PLP (1 mM), DMSO (10% v/v), TAm lysates (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) in water or KPi buffer (50 mM, pH 7.5), 37°C, 18 h. aYields were calculated against product standards by HPLC analysis; diastereomeric ratios were determined by HPLC analysis and 1H NMR spectroscopy.

We have also previously reported that substituted arylethylamines can lead to stereoselectivities through favoured conformations due to pseudo‐equatorial orientations in the transition state [17], which is consistent with the increased 1R‐product in the KPi‐mediated reactions. Overall, this approach demonstrates the broad feasibility of the cascades with functionalized amines and highlights the potential to readily access THIQs in high stereoselectivities.

Conclusion

3

In conclusion, a TAm‐NCS cascade towards a variety of THIQs was developed. Variation of the substrate concentrations and TAms were explored to achieve higher cross‐PS product yields. The TAm‐NCS cascade described demonstrated applications with a variety of nonfunctionalized amines and functionalized amines, and the cascade was extended to incorporate a novel arylethylamine precursor. Overall, the cascades provide an efficient route to prepare (1S)‐6,7‐dihydroxy‐THIQ alkaloids starting from low cost and readily available amines, through the in situ sustainable preparation of aldehydes.

Experimental Section [33]

4

General Experimental Information

4.1

All chemicals and solvents used were purchased from Sigma–Aldrich and Alfa Aesar. Nuclear magnetic resonance (NMR) spectroscopy: ^1^H and ^13^C NMR spectra were recorded using Bruker Advance 500, 600, and 700 MHz spectrometers at 298 K. The chemical shifts (in ppm) were determined relative to tetramethylsilane set at 0 ppm and referenced to residual, protonated NMR solvents. Coupling constants in ^1^H NMR spectra are defined as J and measured in Hertz (Hz) and are described as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), etc. 2D NMR spectra: ^1^H‐^1^H COSY, ^1^H‐^13^C HSQC, ^1^H‐^13^C HMBC, and ^1^H‐^1^H NOESY were used for compound identification when required. Infrared spectra: Infrared spectra were recorded on a Bruker Alpha Platinum‐ATR machine.

Liquid chromatography‐mass spectrometry (LC‐MS): LC‐MS was performed on an Agilent 1100 Series System with a Finnigan LTQ mass spectrometer. An ACE 5 C18 reverse phase column (50 mm × 2.1 mm, 5 μm) was adopted with a mobile phase of eluent A (H_2_O with 0.1% (v/v) formic acid) and eluent B (acetonitrile) over 5 min with a flow rate of 0.2 mL/min, following a gradient of A/B = 95%/5%–5%/95% at 0–4 min, A/B = 5%/95%–95%/5% at 4–4.5 min, A/B = 95%/5% at 4.5–5 min. The sample injection volume was 10 μL. Chemical compounds were measured in a positive ion mode, and the operating conditions of the ESI interface were set to a capillary temperature of 300°C, capillary voltage of 9 V, spray voltage of 4 kV, sheath gas of 40, auxiliary gas of 10, sweep gas 0 arbitrary units. High‐resolution mass spectrometry (HRMS): HRMS experiments were performed by the UCL Chemistry Mass spectrometry service, using EI or CI on a MAT900 or a Waters LCT Premier Q‐TOF for ESI. HPLC methods are described in the Supporting Information.

Enzyme Preparation

4.2

Enzyme sequences of WT‐Δ29TfNCS, CvTAm (CV2025), pQR2189‐TAm, and pQR2208‐TAm have been reported previously [22, 28]. WT‐Δ29TfNCS was expressed and purified following a previously reported method and it was used as a solution in HEPES buffer (100 mM, pH 7.5) containing 10% glycerol [26]. Generally, an aliquot of glycerol stocks of E. coli BL21 (DE3) cells, transformed with plasmids containing WT‐Δ29TfNCS genes, was grown in TB medium containing 50 μg/mL of kanamycin at 37°C until OD_600_ > 1. IPTG was then added (final concentration 1 mM) and the induction was carried out at 25°C, 250 rpm for 5 h. Cell pellets were collected by centrifugation and lysed by Bugbuster (Novagen Bugbuster 10X Protein Extraction Reagent). Sonication was also used for cell lysis. The supernatant was filtered through a 0.22 μm cellulose acetate syringe filter (Millex‐GP) and passed through a pre‐equilibrated Ni‐NTA column (pre‐equilibration buffer: 100 mM HEPES, 100 mM NaCl, 20 mM imidazole, pH 7.5). Then the column was eluted with pre‐equilibration buffer, wash buffer (100 mM HEPES, 100 mM NaCl, 40 mM imidazole, pH 7.5), and finally protein elution buffer (100 mM HEPES, 100 mM NaCl, 500 mM imidazole, pH 7.5). Purified NCS was desalted using the desalting column (GE Healthcare Sephadex G‐25 M) and exchanged into storage buffer (100 mM HEPES, pH 7.5). Glycerol was added (final concentration 10% v/v) and purified NCS was stored at −80°C [34].

CvTAm, pQR2189‐TAm, and pQR2208 TAm cell lysates were prepared as follows: an aliquot of glycerol stocks of E. coli cells containing CvTAm, pQR2189, and pQR2208 plasmids were cultured in TB medium (50 μg/mL kanamycin, 10 mL) overnight at 37°C. The starter culture was diluted (1 mL starter in 100 mL TB medium, 50 μg/mL kanamycin) and shaken at 37°C until the OD_600_ exceeded 0.8. IPTG (100 µL, 1 M) was then added (final concentration, 1 mM) and the culture was shaken at 25°C overnight, followed by harvesting the cells by centrifugation and frozen. Cell pellets obtained from 50 mL of the TB growth culture were thawed in HEPES buffer (100 mM, pH 7.5, 5 mL) and sonicated. After centrifugation, the supernatant was directly used as the TAm clarified cell lysate.

Desalting CvTAm lysates: A SephadexTM G‐25 in the PD‐10 desalting column (GE Healthcare Life Sciences, Germany) was washed with water and HEPES buffer (50 mM, pH 7.5) using three column volumes. The concentrated eluent containing enzymes was loaded on the column and washed into 3–5 mL HEPES buffer (50 mM, pH 7.5). The enzyme was stored at −20°C. The concentration of proteins was measured at OD_595_ using a NanoDrop or using a standard Bradford calibration curve against BSA (Bovine Serum Albumin).

THIQ Acid–Base Extraction Method

4.3

This purification method has been described previously [26]. Briefly, the enzyme reaction mixture was quenched with aq. HCl (10%), adjusted to pH 7.5–8 and extracted with ethyl acetate (3×). The combined organic extracts were washed with saturated NaCl solution, dried and evaporated in vacuo. The residue obtained was resuspended in dilute aq. HCl (1 M) and dimethyl carbonate (DMC). The aqueous phase was washed with DMC and the combined DMC extracts evaporated under vacuum at 55°C to give the products.

Representative Products Characterized

4.4

(1S)‐1‐Phenethyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4b) [35]. A mixture of 3‐phenylpropylamine 2b (2.8 μL, 0.020 mmol), sodium pyruvate (0.10 mL of a 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL) and water (0.20 mL) were shaken at 37°C for 3 h. The product 4b was formed in 40% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 8.2 min, method 6) to give 4b as a white solid. Rt = 5.2 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 7.33–7.20 (5H, m, Ph‐H), 6.67 (1H, s, 8‐H), 6.63 (1H, s, 5‐H), 4.42–4.39 (1H, m, 1‐H), 3.57 (1H, dt, J = 12.5, 6.2 Hz, 3‐HH), 3.37 (1H, dt, J = 12.5, 6.2 Hz, 3‐HH), 3.02 (1H, dt, J = 17.1, 6.2 Hz, 4‐HH), 2.93 (1H, dt, J = 17.1, 6.2 Hz, 4‐HH), 2.86–2.76 (2H, m, 2′‐H_2_), 2.31 (1H, ddt, J = 15.7, 10.9, 5.7 Hz, 1′‐HH), 2.24–2.16 (1H, m, 1′‐HH); ^13^C NMR (126 MHz; CD_3_OD) δ 146.8, 145.9, 141.6, 129.7, 129.3, 127.5, 123.8, 123.6, 116.2, 113.8, 56.2, 40.8, 37.1, 32.4, 25.6; m/z [ES+] 270 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 270.1487. [C_17_H_20_NO_2_+H]^+^ requires 270.1488.

(1S)‐1‐Butyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4c) [36]. A mixture of pentylamine 2c (2.3 μL, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL) and water (0.20 mL) were shaken at 37°C for 3 h. The product 4c was formed in 72% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 7.5 min, method 6) to give 4c as an oil. Rt = 4.7 min (analytical HPLC method 2, Supporting Information); ^1^H NMR (500 MHz; CD_3_OD) δ 6.65 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.34 (1H, dd, J = 8.0, 5.0 Hz, 1‐H), 3.51 (1H, dt, J = 12.1, 5.9 Hz, 3‐HH), 3.35–3.28 (1H, m, 3‐HH), 3.02–2.87 (2H, m, 4‐H_2_), 2.06–2.00 (1H, m, 1′‐HH), 1.90–1.84 (1H, m, 1′‐HH), 1.47–1.42 (4H, m, 2′‐H_2_, 3′‐H_2_), 0.99 (3H, t, J = 6.9 Hz, 4′‐H_3_); ^13^C NMR (126 MHz; CD_3_OD) δ 146.7, 145.9, 124.2, 123.5, 116.1, 113.8, 56.6, 40.9, 34.8, 28.5, 25.6, 23.5, 14.1; m/z [ES+] 222 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 222.1488. [C_13_H_20_NO_2_+H]^+^ requires 222.1488.

(1S)‐1‐Pentyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol hydrochloride (4d.HCl) [34]. A mixture of hexylamine 2d (26.5 μL, 0.200 mmol), sodium pyruvate (16.5 mg, 0.150 mmol), PLP (2.5 mg, 0.010 mmol), dopamine hydrochloride 1.HCl (19.0 mg, 0.100 mmol), sodium ascorbate (20.0 mg, 0.100 mmol), DMSO (0.1 mL), WT‐Δ29TfNCS (4.1 mg/mL stock solution, 1.22 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (2 mL) and water (6.65 mL) were shaken at 37°C for 3 h. The workup followed the general acid–base extraction method to obtain 4d.HCl as a yellow solid (16.9 mg, 62%). It could also be purified by preparative HPLC (Rt = 8.0 min, method 6). Rt = 5.1 min (analytical HPLC method 2, Supporting Information); ^1^H NMR (600 MHz; CD_3_OD) δ 6.65 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.34 (1H, dd, J = 7.2, 4.8 Hz, 1‐H), 3.51–3.47 (1H, m, 3‐HH), 3.28–3.25 (1H, m, 3‐HH), 3.04–2.95 (1H, m, 4HH), 2.92–2.84 (1H, m, 4‐HH), 2.05–1.99 (1H, m, CHCHH), 1.92–1.85 (1H, m, CHCHH), 1.54–1.45 (2H, m, CH 2(CH_2_)2_CH_3), 1.45–1.26 (4H, m, (CH 2)2_CH_3), 0.93 (3H, t, J = 7.2 Hz, CH_3_). ^13^C NMR (126 MHz; CD_3_OD) δ 146.6, 145.9, 124.2, 123.5, 116.1, 113.8, 56.6, 40.9, 35.0, 32.7, 26.1, 25.6, 23.4, 14.2; m/z [ES+] 236 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 236.1644. [C_14_H_22_NO_2_+H]^+^ requires 236.1645.

(1S)‐1‐Hexyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4e) [36]. A mixture of heptylamine 2e (2.9 μL, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4e was formed in 81% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt 8.6 min, method 6) to give 4e as a yellow oil. Rt = 5.5 min (analytical HPLC method 2, Supporting Information); ^1^H NMR (500 MHz; CD_3_OD) δ 6.65 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.35–4.32 (1H, m, 1‐H), 3.50 (1H, dt, J = 12.2, 5.9 Hz, 3‐HH), 3.32–3.30 (1H, m, 3‐HH), 3.00–2.97 (1H, m, 4‐HH), 2.92–2.88 (1H, m, 4‐HH), 2.07–1.98 (1H, m, 1′‐HH), 1.92–1.82 (1H, m, 1′‐HH), 1.52–1.49 (2H, m, 2′‐H_2_), 1.39–1.31 (6H, m, 3′‐H_2_, 4′‐H_2_, 5′‐H_2_), 0.94–0.90 (3H, m, 6′‐H_3_); ^13^C NMR (126 MHz; CD_3_OD) δ 146.7, 145.9, 124.2, 123.5, 116.1, 113.8, 56.7, 40.9, 35.1, 32.7, 30.2, 26.4, 25.6, 23.6, 14.3; m/z [ES+] 250 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 250.1800. [C_15_H_24_NO_2_+H]^+^ requires 250.1801.

(1S)‐1‐Ethyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4f). A mixture of propan‐1‐amine 2f (1.6 μL, 0.020 mmol), sodium pyruvate (0.10 mL of a 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), TAm cell lysate (0.20 mL) and water (0.20 mL) were shaken at 37°C for 3 h. The product 4f was formed in 54% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 6.5 min, method 6) to give 4f a white solid. Rt = 3.5 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 6.66 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.29 (1H, dd, J = 8.2, 4.9 Hz, 1‐H), 3.55–3.46 (1H, m, 3‐HH), 3.35–3.28 (1H, m, 3‐HH), 3.03–2.86 (2H, m, 4‐H_2_), 2.15–2.05 (1H, m, 1′‐HH), 1.96–1.86 (1H, m, 1′‐HH), 1.10 (3H, t, J = 7.5 Hz, CH_2_CH 3); ^13^C NMR (126 MHz; CD_3_OD) δ 146.7, 145.8, 124.0, 123.7, 116.2, 113.8, 57.8, 41.0, 27.8, 25.7, 10.0; m/z [ES+] 194 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 194.1176. [C_11_H_15_NO_2_+H]^+^ requires 194.1176.

(1S)‐1‐Isopropyl‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4g). A mixture of 2‐methylpropan‐1‐amine 2g (1.9 μL, 0.020 mmol), sodium pyruvate (0.10 mL of a 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), TAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4g was formed in 47% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 6.9 min, method 6) to give 4g a white solid. Rt = 4.4 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 6.66 (1H, s, 8‐H), 6.62 (1H, s, 5‐H), 4.30 (1H, d, J = 4.8 Hz, 1‐H), 3.56–3.52 (1H, m, 3‐HH), 3.28–3.23 (1H, m, 3‐HH), 3.02–2.95 (1H, m, 4‐HH), 2.88–2.82 (1H, m, 4‐HH), 2.44 (1H, heptd, J = 7.0, 4.8 Hz, 1′‐H), 1.15 (3H, d, J = 7.0 Hz, CHCH 3(CH_3_)), 0.92 (3H, d, J = 7.0 Hz, CHCH_3_(CH 3)); ^13^C NMR (126 MHz; CD_3_OD) δ 146.6, 145.9, 124.5, 123.2, 116.2, 114.0, 62.0, 41.9, 32.1, 25.7, 19.5, 16.3; m/z [ES+] 208 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 208.1332. [C_12_H_17_NO_2_+H]^+^ requires 208.1332.

(1S)‐1‐(Cyclohexylmethyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol hydrochloride (4h.HCl) [14]. A mixture of 2‐cyclohexylethylamine 2h (31.4 μL, 0.200 mmol), sodium pyruvate (16.5 mg, 0.150 mmol), PLP (2.5 mg, 0.010 mmol), dopamine hydrochloride 1.HCl (19.0 mg, 0.100 mmol), sodium ascorbate (20.0 mg, 0.100 mmol), DMSO (0.1 mL), WT‐Δ29TfNCS (2.3 mg/mL stock solution, 2.17 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (2 mL), and HEPES buffer (100 mM, pH 7.5, 5.7 mL) were reacted at 37°C for 3 h. The workup followed the general acid–base extraction method to give 4h.HCl (24.8 mg, 83%). ^1^H NMR (700 MHz; CD_3_OD) δ 6.61 (1H, s, 8‐H), 6.60 (1H, s, 5‐H), 4.42 (1H, dd, J = 9.1, 5.6 Hz, 1‐H), 3.48 (1H, quintet, J = 7.0 Hz, 3‐HH), 3.35–3.27 (1H, m, overlap with CD_3_OD protic solvent residual peak, 3‐HH), 2.94 (2H, m, 4‐H_2_), 1.95–1.93 (1H, m, Cy‐H), 1.86–1.71 (6H, m, 1′‐H_2_ and Cy‐H_4_), 1.59–1.51 (1H, m, Cy‐H), 1.40–1.32 (2H, m, Cy‐H_2_), 1.30–1.21 (1H, m, Cy‐H), 1.10–1.01 (2H, m, Cy‐H_2_); ^13^C NMR (176 MHz; CD_3_OD) δ 146.5, 145.8, 124.7, 123.5, 116.1, 113.8, 53.6, 43.3, 40.5, 34.8, 34.7, 33.4, 27.3, 27.1, 26.8, 25.5; m/z [HRMS ESI+] found [M+H]^+^ 262.1797, C_16_H_24_NO_2_ requires 262.1802.

(1S)‐1‐(Cyclopentylmethyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4i). A mixture of 2‐cyclopentylethan‐1‐amine 2i (2.6 μL, 0.020 mmol), sodium pyruvate (0.10 mL of a 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4i was formed in 37% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 8.1 min, method 6) to give 4i a white solid. Rt = 5.2 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 6.64 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.33 (1H, dd, J = 8.4, 5.2 Hz, 1‐H), 3.58–3.48 (1H, m, 3‐HH), 3.35–3.30 (1H, m, 3‐HH), 3.00–2.88 (1H, m, 4‐H_2_), 2.07–1.89 (5H, m, 1′‐H_2_, cyc‐H), 1.78–1.69 (2H, m, cyc‐H), 1.68–1.60 (2H, m, cyc‐H), 1.29–1.22 (2H, m, cyc‐H); ^13^C NMR (126 MHz; CD_3_OD) δ 146.6, 145.8, 124.5, 123.5, 116.2, 114.0, 55.7, 41.8, 40.5, 37.3, 33.9, 33.1, 26.0, 25.9, 25.5; m/z [ES+] 248 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 248.1645. [C_15_H_21_NO_2_+H]^+^ requires 248.1645.

(1S)‐1‐(Cyclohex‐1‐en‐1‐ylmethyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4k). A mixture of cyclohexene ethamine 2k (2.8 μL, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4k was formed in 31% (by qNMR, see Supporting Information Section 4). It was also purified by preparative HPLC (Rt = 8.3 min, method 8), to give 4k as a yellow oil. Rt = 5.3 min (analytical HPLC method 2, Supporting Information); ^1^H NMR (500 MHz; CD_3_OD) δ 6.67 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 5.69 (1H, s, 2′’‐H), 4.48–4.46 (1H, m, 1‐H), 3.53–3.50 (1H, m, 3‐HH), 3.28–3.24 (1H, m, 3‐HH), 3.04–2.98 (2H, m, 4‐H_2_), 2.46 (1H, dd, J = 14.5, 9.5 Hz, 1′‐HH), 2.26–2.23 (1H, m, 1′‐HH), 2.10–2.02 (4H, m, 3^“^’‐H_2_, 6′’‐H_2_), 1.82–1.59 (4H, m, 4^“^‐H_2_, 5′‐H_2_); m/z [ES+] 260 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 260.1644. [C_16_H_22_NO_2_+H]^+^ requires 260.1645.

(1S)‐1‐(But‐3‐en‐1‐yl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4l) [37]. A mixture of pent‐4‐enylamine 2l (2.3 μL, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 18 h. The product 4l was formed in 72% (by HPLC against product standards), It was also purified by preparative HPLC (Rt = 7.2 min, method 7) to give 4l as an oil. Rt = 4.5 min (analytical HPLC method 2, Supporting Information); ^1^H NMR (700 MHz; CD_3_OD) δ 6.64 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 5,89 (1H, ddt, J = 17.0, 10.6, 6.6 Hz, 3′‐H), 5.16 (1H, J = 17.0 Hz, 4′‐HH), 5.08 (1H, d, J = 10.6 Hz, 4′‐HH), 4.37–4.35 (1H, m, 1‐H), 3.53–3.31 (2H, m, 4‐H_2_), 3.00–2.89 (2H, m, 3‐H_2_), 2.28–2.24 (2H, m, 1′‐H_2_), 2.14–2.08 (1H, m, 2′‐HH), 2.00–1.95 (1H, m, 2′‐HH); ^13^C NMR (126 MHz; CDCl_3_) δ 146.8, 145.9, 137.7, 123.9, 123.6, 116.6, 116.2, 113.9, 56.0, 40.7, 34.4, 30.4, 25.6; m/z [ES+] 220 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 220.1322. [C_13_H_17_NO_2_+H]^+^ requires 220.1332.

(1S)‐1‐(4‐Hydroxybutyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4m). A mixture of 5‐aminopentan‐1‐ol 2m (2.2 μL, 0.020 mmol), sodium pyruvate (0.10 mL of a 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), TAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4m was formed in 46% yield (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 6.5 min, method 6) to give 4m as a yellow oil. Rt = 3.4 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 6.66 (1H, s, 8‐H), 6.61 (1H, s, 5‐H), 4.36 (1H, dd, J = 8.1, 4.9 Hz, 1‐H), 3.61 (2H, t, J = 6.0 Hz, 4′‐H_2_), 3.54–3.49 (1H, m, 3‐HH), 3.34–3.29 (1H, m, 3‐HH), 3.03–2.88 (2H, m, 4‐H_2_), 2.10–2.03 (1H, m, 1′‐HH), 1.94–1.87 (1H, m, 1′‐HH), 1.72–1.50 (4H, m, CH_2_CH_2_); ^13^C NMR (126 MHz; CD_3_OD) δ 146.7, 145.9, 124.2, 123.6, 116.2, 113.9, 62.3, 56.5, 40.9, 34.8, 33.1, 25.6, 22.9; m/z [ES+] 238 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 238.1438. [C_13_H_19_NO_3_+H]^+^ requires 238.1438.

(1S)‐1‐(6‐Aminohexyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (4n). A mixture of 1,7‐diamineheptane 2n (2.6 mg, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), dopamine hydrochloride 1.HCl (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.20 mL) were shaken at 37°C for 3 h. The product 4n was formed in 39% (according to qNMR, see Supporting Information Section 6). It was also purified by preparative HPLC (Rt = 6.2 min, method 8) to give 4n as a colorless oil. Rt = 3.9 min (analytical HPLC method 2); ^1^H NMR (500 MHz; CD_3_OD) δ 6.64 (1H, s, 8‐H), 6.62 (1H, s, 5‐H), 4.36–4.34 (1H, m, 1‐H), 3.52–3.50 (1H, m, 3‐HH), 3.32–3.30 (1H, m, 3‐HH), 3.00–2.91 (4H, m, 4‐H_2_, 6′‐H_2_), 2.07–1.97 (1H, m, 1′‐HH), 1.95–1.85 (1H, m, 1′‐HH), 1.71–1.61 (2H, m, 5′‐H_2_), 1.52–1.35 (6H, m, 2′‐H_2_, 3′‐H_2_, 4′‐H_2_); ^13^C NMR (126 MHz; CD_3_OD) δ 145.1, 144.9, 122.7, 122.3, 114.6, 112.4, 55.0, 39.4, 39.2, 33.3, 32.1, 28.4, 27.1, 26.5, 25.9, 24.7; m/z [ES+] 265 [M+H]^+^; m/z [HRMS ESI+] found [M+H]^+^ 265.1911, C_15_H_25_N_2_O_2_ requires 265.1912.

(1S)‐1‐(7‐Aminoheptyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol hydrochloride (4o.2TFA). A mixture of 1,8‐diamineoctane 2o (29 mg, 0.20 mmol), sodium pyruvate (16.5 mg, 0.15 mmol), PLP (2.5 mg, 0.01 mmol), dopamine hydrochloride 1.HCl (19 mg, 0.10 mmol), sodium ascorbate (20.0 mg, 0.10 mmol), DMSO (0.1 mL), WT‐Δ29TfNCS (7.2 mg/mL stock solution, 0.7 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (2 mL), and HEPES buffer (100 mM, pH 7.5, 7.2 mL) were stirred at 37°C for 4 h. The mixture was diluted with water, centrifuged, treated with aq.HCl (1 M) and centrifuged again. The clear supernatant was purified by preparative HPLC—mobile phase: A (H_2_O with 0.1% TFA) and B (CH_3_CN with 0.1% TFA), A/B = 95%/5% at 0–3 min, A/B = 95%/5% to A/B = 40%/60% at 3–20 min, A/B = 40%/60% to A/B = 95%/5% at 20–21 min, A/B = 95%/5% at 21–22 min; Flow rate: 8 mL/min; Product 4o.2TFA was obtained after freeze‐drying (18.2 mg, 36%). ^1^H NMR (700 MHz; D_2_O) δ 6.73 (1H, s, 8‐H), 6.72 (1H, s, 5‐H), 4.38 (1H, dd, J = 7.7, 4.9 Hz, 1‐H), 3.51–3.48 (1H, m, 3‐HH), 3.32–3.28 (1H, m, 3‐HH), 2.98–2.87 (4H, m, 4‐H_2_ and 7′‐H_2_), 2.02–1.96 (1H, m, 1′‐HH), 1.88–1.83 (1H, m, 1′‐HH), 1.62–1.58 (2H, m, 6′‐H_2_), 1.43–1.28 (8H, m, 2′‐H_2_, 3′‐H_2_, 4′‐H_2_ and 5′‐H_2_); ^13^C NMR (176 MHz; D_2_O) δ 144.4, 143.6, 124.8, 124.4, 116.4, 114.3, 55.6, 40.0, 40.0, 33.6, 28.8, 28.4, 27.2, 26.0, 24.9, 24.6; m/z [HRMS ESI+] found [M+H]^+^ 279.2059, C_16_H_27_N_2_O_2_ requires 279.2067; preparative HPLC retention time 16.0 min.

(1S, 3R)‐1‐(But‐3‐en‐1‐yl)‐3‐(hydroxymethyl)‐1,2,3,4‐tetrahydroisoquinoline‐6,7‐diol (7). A mixture of pent‐4‐enylamine 2l (2.3 μL, 0.020 mmol), sodium pyruvate (0.10 mL of 150 mM stock solution), PLP (0.10 mL of 10 mM stock solution), D‐tyrosinol 6 (0.10 mL of 100 mM stock solution), sodium ascorbate (0.10 mL of 100 mM stock solution), DMSO (0.10 mL), CnTYR cell lysates (0.10 mL), WT‐Δ29TfNCS (5 mg/mL stock solution, 0.10 mL, final concentration 0.5 mg/mL), CvTAm cell lysate (0.20 mL), and water (0.10 mL) were shaken at 37°C for 18 h. The product 7 was formed in 80% yield (by HPLC against product standards). It was also purified by preparative HPLC (Rt = 7.2 min, method 6) to give 7 as an oil. Rt = 4.5 min (analytical HPLC method 2); [α]D ^26^ 39.3 (c 0.28, CH_3_OH); ν_max_ (neat)/cm^−1^ 3289, 3074, 1635; ^1^H NMR (500 MHz; CD_3_OD) δ 6.72 (1H, s, 5‐H), 6.62 (1H, s, 8‐H), 5.91 (1H, ddt, J = 17.0, 10.2, 5.9 Hz, 3′‐H), 5.16 (1H, dd, J = 17.0, 1.7 Hz, 4′‐HH), 5.08 (1H, dd, J = 10.2, 1.7 Hz, 4′‐HH), 4.43–4.29 (1H, m, 1‐H), 3.91 (1H, dd, J = 11.8, 3.9 Hz, CHCHHOH), 3.77–3.74 (1H, m, 3‐H), 3.64 (1H, dd, J = 11.8, 6.1 Hz, CHCHHOH), 2.95 (1H, dd, J = 17.1, 5.7 Hz, 4‐HH), 2.81 (1H, dd, J = 17.1, 10.3 Hz, 4‐HH), 2.31–2.27 (2H, m, 2′‐H_2_), 2.04–1.98 (2H, m, 1′‐H_2_); ^13^C NMR (126 MHz; CD_3_OD) δ 147.0, 145.6, 137.8, 124.0, 122.6, 116.7, 116.4, 114.7, 62.2, 55.5, 52.0, 34.8, 30.9, 27.7; m/z [ES+] 250 [M+H]^+^; m/z [HRMS ES+] found [M+H]^+^ 250.1428. [C_14_H_20_NO_3_+H]^+^ requires 250.1437.

Supporting Information

Additional supporting information can be found online in the Supporting Information section. Supporting information includes initial optimisation data, HPLC methods, NMR spectroscopy data, methods for the determination of selected ees and the synthesis of substrates and rac‐products. The authors have cited additional references within the Supporting Information [38, 39]. Supporting Scheme S1. Use of Marfey's reagent to determine the stereoselectivity in the TAm‐NCS cascade with 2h and 1 (0.1 mmol scale) as previously described [29,30]. Green peak: racemic product, red peak enzymatic reaction, (ee = 89%). Supporting Fig. S1: Reaction conditions (0.01 mmol scale): 1 (10 mM), PLP (1 mM), DMSO (10% v/v), CvTAm lysate (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL), in H_2_O, 37°C, 3 h. Supporting Fig . S2: Reaction conditions (0.01 mmol scale): CvTAm lysate (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL), 2a (20 mM), 1 (10 mM, final concentration), sodium pyruvate 3 (15 mM), PLP (1 mM), DMSO (10% v/v) in H_2_O, 37°C. The reaction mixture was incubated for another 3 h after the addition of dopamine. Supporting Fig. S3: Reaction conditions (0.01 mmol scale): 2a (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL), in H_2_O, 37°C, 3 h. The HPLC chromatogram was measured using HPLC method 1. Retention times: Dopamine 1 ‐ 2.4 min, product 4a ‐4.5 min, product 5 ‐ 4.7 min, lactam formed from the cyclisation of 4a – 5.4 min. Supporting Fig. S4: LC‐MS spectrum of the reaction shown in Figure S3 using enzymes pQR2208‐NCS in the cascade. The m/z peaks corresponding to 4a and 5 are shown. Supporting Fig. S5: Reaction conditions (0.005 mmol scale): 5j (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), DMSO (10% v/v), pQR 2208 TAm lysate (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL), in H_2_O, 37°C, 3 h. The HPLC chromatogram was measured using HPLC method 1. Retention times: Product 5 ‐ 4.7 min, 4j ‐ 4.9 min, lactam formed from the cyclisation of 4j – 5.4 min. Supporting Fig. S6: (A) Reaction conditions (0.01 mmol scale): 3‐phenyl‐1‐propylamine 2b (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h; This reaction mixture was used as the racemic standard and analysed directly by HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): 3‐phenyl‐1‐propylamine 2b (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm CCL (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. (C) Reaction conditions were the same as for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using HPLC method 3. Supporting Fig. S7: (A) Reaction conditions (0.01 mmol scale): amylamine 2c (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amylamine 2c (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using (chiral) HPLC method 3. Supporting Fig. S8: (A) Reaction conditions: Rac‐4d was prepared as previously described [27]. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions are given in the main paper (0.1 mmol scale). (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using HPLC method 3. Supporting Fig. S9: (A) Reaction conditions: amine 2e (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2e (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using HPLC method 3. Supporting Fig. S10: (A) Reaction conditions: amine 2f (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2f (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), desalted CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. The ee for (B) was determined using HPLC method 3. Supporting Fig. S11: (A) Reaction conditions: amine 2g (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2g (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), desalted CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. The ee for (B) was determined using HPLC method 3. Supporting Fig. S12: (A) Reaction conditions: amine 2i (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3 with an Astec CHIROBIOTIC T2 column. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2i (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), desalted CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. The ee for (B) was determined using HPLC method 3 with an Astec CHIROBIOTIC T2 column. Supporting Fig. S13: (A) Reaction conditions: amine 2k (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 5. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2k (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using HPLC method 5. Supporting Fig. S14: (A) Reaction conditions: amine 2l (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemate standard and analysed directly using HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2l (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H2O, 37°C, 3 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using (chiral) HPLC method 3. Supporting Fig. S15: (A) Reaction conditions: amine 2m (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 3. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2i (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), desalted CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. The ee for (B) was determined using HPLC method 3. Supporting Fig. S16: (A) Reaction conditions: amine 2n (20 mM), 1 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), TAm lysate (20% v/v), KPi buffer [27] (50 mM, pH 7.5), 37°C, 18 h. This reaction mixture was used as the racemic standard and analysed directly by HPLC following HPLC method 4. (B) Reaction conditions were the same as that for the NMR yield determination (0.01 mmol scale, SI part 4.1): amine 2o (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), dopamine hydrochloride 1. HCl (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CCL CvTAm (20% v/v), WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 3 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Ees for (B) and (C) were determined using (chiral) HPLC method 4. Supporting Fig. S17: (A) Reaction conditions amine 2l (20 mM), D‐tyrosinol 6 (10 mM), sodium pyruvate 3 (15 mM), sodium ascorbate (10 mM), PLP (1 mM), DMSO (10% v/v), CnTYR lysates (10% v/v), CvTAm lysate (20% v/v), KPi buffer (50 mM, pH 7.5), 37°C, 18 h. (B) Reaction conditions: amine 2l (20 mM), sodium pyruvate 3 (15 mM), PLP (1 mM), D‐tyrosinol 6 (10 mM), sodium ascorbate (10 mM), DMSO (10% v/v), CnTYR CCL (10% v/v), CCL CvTAm cell lysate (20% v/v), and WT‐Δ29TfNCS (0.5 mg/mL) and H_2_O, 37°C, 18 h. (C) Reaction conditions were the same as that for B but using desalted CCL CvTAm (20% v/v). Diastereomeric ratios were determined using HPLC method 2.

Funding

University College London Dean's Prize to Jianxiong Zhao; UCL‐China Scholarship Council Joint Research Scholarship to Jianxiong Zhao; Engineering and Physical Sciences Research Council (EP/W019132/1) to Yu Wang; Biotechnology and Biological Sciences Research Council (BB/Y007972/1) to Yeke Ni; Engineering and Physical Sciences Research Council (EP/P020410/1).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material