Norcoclaurine Synthase-Mediated Stereoselective Synthesis of 1,1'-Disubstituted, Spiro- And Bis-Tetrahydroisoquinoline Alkaloids

Disubstituted, Spiro- and Bis-Tetrahydroisoquinoline Alkaloids

Research Article

Norcoclaurine Synthase-Mediated Stereoselective Synthesis of 1,1’-

∥

Jianxiong Zhao, Daniel Men

dez-San

chez, Rebecca Roddan, John M. Ward, and Helen C. Hailes*

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sı Supporting Information

ABSTRACT: The Pictet−Spenglerase norcoclaurine synthase (NCS) catalyzes the formation of

S)-norcoclaurine, an important intermediate in the biosynthetic pathway of benzylisoquinoline

(

alkaloids. NCS has been used as a biocatalyst with meta-hydroxy phenethylamines and aldehydes

for the preparation of single-isomer tetrahydroisoquinoline alkaloids (THIAs). Recently, it was

also reported that some ketones can be accepted as substrates, including 4-substituted

cyclohexanones and phenyl acetones. Here, we report the use of wild-type NCS and selected

variants with aliphatic, cyclic, α-substituted cyclic, heterocyclic, and bicyclic ketones to access

challenging non-natural THIAs. Remarkably, fused bicyclic ketones as well as diketones could also be accepted by some of the NCS

variants, and in silico modeling was used to provide insights into the rationale for this.

KEYWORDS: biocatalysis, norcoclaurine synthase, Pictet−Spengler, tetrahydroisoquinoline, in silico modeling

INTRODUCTION

present as part of a mixture of components, and low extraction

■

yields are obtained by the end of the process. To overcome

these limitations, diﬀerent synthetic strategies have been

developed. To date, there are many reported approaches to

access THIAs, although the Bischler−Napieralski cyclization/

13,14

Alkaloids are found primarily in plants and are particularly

prevalent in certain families of ﬂowering plants. Tetrahydroi-

soquinoline alkaloids (THIAs) are a structurally diverse class

of compounds, with a history of human use dating back

reduction sequence or Pictet−Spengler reactions (PSRs)

thousands of years. In addition to their prominent role in

have been most frequently utilized. For asymmetric synthetic

strategies, transfer hydrogenation with transition metals and

chiral ligands have been successfully employed with a range of

traditional medicine, THIAs have a wide variety of

pharmacological applications, for example, as antitussives,

antimicrobials, and antispasmodics.

studies have also uncovered new potential applications in

treating cancer, malaria, and many other diseases.

−3

Recently, preliminary

15,16

substrates for the synthesis of a variety of THIAs.

Although

−9

many of these methods are very useful, protection of phenolic

groups is typically required; reaction eﬃcacy varies, as does the

stereoselectivities achieved; and the approach cannot be used

to generate 1,1′-disubstituted THIAs. In order to access these,

Among

all THIAs, a particularly interesting group are the 1,1′-spiro-

tetrahydroisoquinolines, such as the natural products ocho-

tensine 1 and the related N-oxide 2, as well as the

antiplasmodial compound 3 (Figure 1 ). Indeed, when 3 was

Bro̷nsted acid or Lewis acid catalysts have been utilized

(

Scheme 1a); however, harsh reaction conditions are normally

17−20

required.

More recently, the use of aqueous phosphate

media (Scheme 1b) has enabled access to a range of 1,1′-

disubstituted THIAs under mild conditions. Here, the main

limitation is that racemic products are formed, unless single-

isomer chiral substrates are used, which could also lead to the

formation of diastereoselective products.

In order to overcome these issues, the use of biocatalysis has

emerged as a powerful tool to generate high-value single

isomer products. For instance, imine reductases (IREDs) have

been used to produce enantiopure THIAs from the

Figure 1. Examples 1,1′-spiro tetrahydroisoquinoline alkaloids 1−3.

used in in vitro assays against P. falciparum, compared to a

range of other THIAs, the antimalarial activity signiﬁcantly

increased, and it was suggested that this was due to the rigidity

Received: October 29, 2020

Revised: December 2, 2020

in this spiro analog. The structurally related Erythrina class of

alkaloids, also spiro-THIAs, has furthermore been used for a

plethora of, for example, ethnomedicine applications.

Traditionally, many alkaloids such as morphine have been

extracted from plant materials. However, typically, they are

XXXX American Chemical Society

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Research Article

Scheme 1. Nonenzymatic and Enzymatic Routes to 1-

Monosubstituted and 1,1′-Disubstituted PSRs

RESULTS AND DISCUSSION

■

In a previous work, it was reported that NCS enzymes could

accept phenyl acetones. Initial studies here focussed on NCS

reactivities toward dopamine 4 with phenylketone 5a and

aliphatic methyl ketones 5b-e to give THIAs 6a-6e to

understand the structure−activity trends, and also methods

were developed to help determine the stereoselectivities of the

reactions. While 5a readily formed 6a as previously described

(

87% isolated yield), for methyl ketones 5b-e, they were

screened against WT-TfNCS and variants A79I, A79F, L76A,

L76V, F80L, M79F, and Y108F as cell lysates. From this, the

highest performing variant was identiﬁed (Supporting

Information Figure S1.1 ), reaction conditions were optimized,

including the addition of the antioxidant sodium ascorbate to

avoid side reactions due to the oxidation of catechols, and

THIAs were generated (Scheme 2). Reactions were monitored

Scheme 2. Use of TfNCSs with Methyl Ketones 5a-5e to

Synthesize 6a-6e

Parts (a) and (b) previous routes to 1,1′-disubstituted THIAs, (c)

use of imine reductases to enantiopure THIAs, and (d) this work

using norcoclaurine synthase (NCS) to 1,1′-disubstituted THIAs.

corresponding dihydroisoquinoline (Scheme 1c). Pictet−

Spenglerases are lyases (EC 4) that have been used as

biocatalysts for the stereoselective synthesis of a range of

4−26

alkaloids.

The main advantage of this group of enzymes is

that no cofactor such as NADPH is required, which reduces

the overall cost of processes compared to other biocatalysts

that require, for example, glucose dehydrogenase redox

cofactor recycling systems. Synthesis of functionalized starting

materials for enzymes such as IRED is also required.

The Pictet−Spenglerase NCS catalyzes the PSR between

dopamine and 4-hydroxylphenylacetaldehyde (4-HPAA) to

form the key compound (S)-norcoclaurine, which is then

biosynthetically converted into more than 2500 other alkaloids

such as morphine. Despite the acceptance of a range of

Reaction conditions: Small-scale reactions were performed using 4

(10−15 mM), ascorbate (10−15 mM), methyl ketone (10−150

mM), and dimethyl sulfoxide (DMSO, 10%) at 37 °C in HEPES

buﬀer (100 mM pH 7.5) using the NCS and time indicated. NCS:

25−32

aldehydes by NCS,

Previously, some α-ketoacid acceptance was described, and

very few ketones have been tolerated.

puriﬁed NCS (1.6 mg/mL), NCS lysate (1.7 mg/mL NCS).

recently, it was reported that several unactivated ketones have

Reaction time: 1 day; 7 days; or more. Reaction conversions by:

been accepted in high yields. For this, wild-type (WT)

Thalictrum ﬂavum NCS (Tf NCS) was used, together with the

selected single-point active-site mutants, with some variants

leading to higher product yields with cyclic ketones (e.g.,

A79F), while others were more productive toward the methyl

ketones (e.g., A79I), and computational substrate docking

experiments highlighted putative orientations of the imine

dopamine depletion by HPLC analysis, HPLC analysis against

product standards. Determination of ees: Marfey’s reagent and H

NMR spectroscopy, chiral HPLC. Isolated yields are given in

parenthesis. For further details and larger scale reactions for

characterization purposes, see the Supporting Information.

intermediates in the active site. In silico docking experiments

and protein X-ray crystallographic studies have also been

performed to understand better the NCS mechanism and role

to determine conversion yields by high-performance liquid

chromatography (HPLC) analysis (dopamine depletion or

product formation) and/or H NMR spectroscopy using an

32,35,36

of key active site residues.

internal standard as indicated because of the reported

challenges of product isolation which frequently lowers the

isolated yield. Larger scale reactions were also performed for

Here, our aim was to fully explore the ketone substrate

promiscuity of NCSs to enhance the applications of using this

PSR more widely synthetically. As part of this, a more eﬀective

method for determining reaction stereoselectivities was

required. Remarkably, using a wide range of cyclic, acyclic,

fused bicyclic ketones and diketones, THIAs were synthesized

and the reaction conditions were optimized and successfully

applied to several phenethylamines (Scheme 1d). Importantly,

some of the THIAs synthesized also have important

bioactivities.

8,22

product characterization purposes.

It was clear that

regardless of the NCS variant used, increasing the length of

the alkyl chain decreased the THIA yield. For example with 5b,

6b was formed in 70% conversions, while with 5c, the

conversion decreased to 37% for 6c, with the best variants

A79F and A79I, respectively (Scheme 2). Indeed, no reaction

was observed with 3-methylbutan-2-one, longer aliphatic chain

methyl ketones, and 1-cyclic-methylketones, presumably

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because of unfavorable steric interactions (Scheme 2,

Supporting Information Figure S1.2). Interestingly, a 75%

conversion was observed with methoxyacetone 5d to give 6d,

although problems were encountered during its puriﬁcation,

while hydroxylated methyl ketones were not accepted.

Benzylacetone 5e gave 6e in conversions of 61%, perhaps

reﬂecting its structural similarity to the natural substrate 4-

HPAA. Overall, isolated yields were in the range of 4−29%.

Previously, the stereoselectivity of the 1,1′-disubstituted

THIAs produced by NCS was determined by chiral HPLC,

compared to racemic standards. The absolute stereoselectivity

of the major isomer was also assigned based upon the known

Scheme 3. Use of TfNCSs with Cyclic Ketones 5f-5k to

Synthesize 6f-6k and 3

selectivity with aldehydes. However, for 6b-6e chiral

separations could not be achieved using a range of chiral

HPLC columns and methods. Alternative methodologies were

therefore investigated using 6a as the ees, which had previously

been established, including the preparation of Moshers’s

amide which has been reported with THIAs formed using

NCS and aldehydes. However, with the more sterically

congested C-1 center, the H NMR spectrum of the Mosher’s

products could not readily be assigned because of the amide

rotamers formed. An alternative method to determine ees is the

38,39

use of Marfey’s reagent,

which avoids the amide rotamer

problems and is attached via S Ar reaction. Using (rac)-6a,

formed via the recently reported basic potassium phosphate

(

KPi) reaction conditions, and (1S)-6a generated using

Reaction conditions: Small-scale reactions were performed using 4, 2-

TfNCS (95% ee via chiral HPLC analysis ), protocols were

developed using Marfey’s reagent which conﬁrmed an ee of

(

3-hydroxyphenyl)ethylamine or 5-(2-aminoethyl)-2-ﬂuorophenol

10−15 mM), ascorbate (10−15 mM), ketone (10−150 mM), and

3% (Supporting Information Figure S1.3).

Reaction stereoselectivities were then established using this

DMSO (10%) at 37 °C in HEPES buﬀer (100 mM pH 7.5) using the

NCS and time indicated. NCS: NCS lysate (1.7 mg/mL NCS; for 3,

method for the NCS reactions with the most productive

variants toward 5b-5e (Scheme 2) and for this, racemic

k 8.5 mg/mL). Reaction time: 1 day, 3 days, 7 days. Reaction

conversions by: amine depletion by HPLC analysis, HPLC analysis

compounds were also synthesized. With 2-butanone 5b, 6b

was formed in a negligible ee, while 5c with a slightly longer

aliphatic chain gave 6c in a 14% ee. Clearly, where similar sized

groups are present either side of the carbonyl moiety, there is

little stereodiﬀerentiation in the active site. The incorporation

of the methyl ether in 5d improved the ee in 6d to 69%,

reﬂecting the impact of possible hydrogen bonding in the side

chain on the stereochemistry. Surprisingly, benzyl acetone 5e

only gave 6e in 15% ee. Following the stereopreference for the

WT and NCS variants with aldehydes, the major isomers were

assigned as having the (1S)-stereochemistry, other than 6d,

which because of the Cahn−Ingold−Prelog priority rules was

assigned as (1R)-6d.

Previously, it was reported that NCS enzymes could readily

accept several 4-substituted cyclohexanones, but we were

curious as to whether the presence of diﬀerent heteroatoms on

the cyclohexane ring or diﬀerent ring sizes could aﬀect the

reaction. For the cyclic ketones leading to 11−15, as before,

they were screened against WT Tf NCS and available variants

as lysates to identify the highest performing variant for use in

reactions (Supporting Information Figure S1.4 ). In previous

works using the 4-substituted cyclohexanones, A79F was noted

as a productive variant, as was the case here using 4 and 1-N-

Boc-4-piperidone 5f: with A79F, 6f was formed, with a large

Boc group at the 4-position relative to the carbonyl group, in a

conversion yield of 73% after 72 h (48% isolated yield)

against product standards, H NMR spectroscopy against an internal

standard. Determination of ees: ^hMarfey’s reagent and H NMR

spectroscopy. Isolated yields are given in parenthesis. For larger-scale

reactions for characterization purposes, see the Supporting

Information.

priority rules was assigned as (1R)-6g. Despite the good

reactivities observed with 5f and 5g, the corresponding

analogues with N-benzyl groups did not seem to be accepted,

which may have been due to substrate solubility issues

(Supporting Information Figure S1.2). Next, tetrahydro-4H-

pyran-4-one 5h was investigated with 4 and several variants

readily gave 6h, including A79I in 56% conversion yield (32%

isolated yield). Alternative ring sizes using 4 with cyclo-

butanone 5i and cyclopentanone 5j gave 6i and 6j after 24 h in

52 and 70% conversions, respectively, using A79F, and both

were isolated in −50% yield. Interestingly, the ﬁve-membered

ring analogue of 5g, 1-N-Boc-3-pyrrolidinone, was not

accepted, nor was the analogue 1-N-benzyl-3-pyrrolidinone

(Supporting Information Figure S1.2 ). In addition to using

alternative cyclic ketones, selected substituted phenethyl-

amines were investigated. 2-(3-Hydroxyphenyl)ethylamine

and a ﬂuorinated analogue were prepared following reported

procedures and reacted with cyclohexanone 5k and A79F to

give 3 and WT to give 6k, in 54 and 84% conversion yields (51

and 69% isolated yields), respectively. Compound 3 is

particularly interesting, as it has been reported to possess

good antimalarial properties compared to other THIAs

(Scheme 3). When 1-N-Boc-3-piperidone 5g was reacted with

, again with A79F, the conversion to give 6g after 72 h was

3% (51% isolated yield) and an ee of 78% was observed.

screened. Overall, it was clear that a range of cyclic ketones

Following the general stereochemical preference for aldehydes,

the major isomer in 6g due to the Cahn−Ingold−Prelog

were accepted by the NCSs and cell-free lysates could readily

be used for these reactions; however, rings possessing bulkier

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side groups were not tolerated, most likely because of steric

factors, and so this was explored in more detail with α-

substituted cyclohexanones.

observed with aldehydes and NCS. Interestingly, in a previous

work, (3R)-methylcyclohexanone was accepted by A79F-

Tf NCS to give the (1R,3’R)-product, but with the methyl

group at the 3-position, diﬀerent steric restraints will be acting

In a previous work, it was reported that α-substituted cyclic

ketones yielded no products most likely because of steric

within the active site.

reasons. However, as α-methyl-substituted aldehydes can be

A computational docking study also suggested that the imine

intermediate formed between dopamine and (2R)-5l was

preferred in the active site, compared to (2S)-5l, with the

methyl group adopting an equatorial conformation (Support-

ing Information Figure S1.6). Because A79 is located at the

entrance of the active site, the improved reactivity of A79F

toward 5l could be explained by the bulkier amino acid residue

helping to orientate the imine intermediate into a reactive

conformation, promoting the cyclization to give 6l. The

acceptance of 5l by NCS highlights its ability to achieve both

enantioselective and stereoselective PSRs, which is chemically

challenging to achieve by other methods. In addition to 5l, 2-

ethylcyclohexanone was also used and a small amount of the

corresponding THIA was detected by LC−MS but could not

be isolated.

accepted by NCS, and the acceptance of α-substituted

cyclohexanones by NCS could be useful in developing routes

to the erythrina alkaloids, this was explored further. Initial

attempts using 2-chlorocyclohexanone led to substrate

degradation, and no THIA product was generated. However,

with 2-methylcyclohexanone 5l and WT-NCS, THIA for-

mation was noted by LC−mass spectrometry (MS). Reactions

were optimized, particularly using higher equivalents of ketone,

higher enzyme concentrations, longer reaction times, diﬀerent

cosolvents (DMSO gave the highest yields), and enzyme

variants. Variants A79I, A79F, Y108F, and WT that gave rise to

some of the higher conversions after 24 h were used in

reactions for longer periods of time, leading to improved

conversions: a preparative scale reaction, for example, with rac-

l (A79F, 7 d) gave 6l in 15% isolated yield; longer reaction

Next, more challenging bicyclic ketone substrates were

times reported higher conversions of >70%, but they were

evaluated as unsatisfactory for synthetic applications (Support-

ing information Figure S1.5 ). In theory, up to four stereo-

isomers could be generated, but chiral HPLC analysis

suggested that there was predominantly one diastereoisomer

present. To conﬁrm whether one isomer of 5l was accepted, a

two-step one-pot reaction cascade was developed. Compound

explored aﬀording a family of otchosentine 1 natural product

analogues. Again, the use of a range of NCS variants was

explored, and the highest yielding variants used are given in

Scheme 5 (also see Supporting Information Figure S1.7 ).

Remarkably, when using β-tetralone 5m, 6m was formed in

79% conversion yield and 86% ee using A79I. Using other β-

tetralones 5n-5p, good conversions and isolated yields were

achieved, together with similar ees. Indeed, 6-substituted-β-

tetralones with electron-withdrawing chloro- or bromo-groups

gave 6n with WT-NCS and 6o with A79I in 50 and 54%

isolated yields, respectively. The use of 5p with an electron-

donating group at the 7-methoxy-position was also readily

accepted, giving 6p in 55% isolated yield with M97F. The

puriﬁcation of THIAs 6m-6p was readily achieved by acid−

base extraction procedures without the requirement for

chromatographic puriﬁcations, enabling the facile scalability

of such approaches. All NCS variants used, however, failed to

accept β-tetralone-bearing methoxy groups at C-5 or C-8

(

2R)-5l was prepared from 2-methyl-2-cyclohexen-1-one 7

using the ene-reductase NCR from Zymomonas mobilis

expressed in E. coli (and NADPH and G6PDH cofactor

recycling system), and then A79F-TfNCS was added to give 6l

(Scheme 4a). Both products formed using (rac)-5l and (2R)-5l

Scheme 4. Use of 5l and Key NMR Analyses to Establish the

Stereochemistry of 6l

(

Supporting Information Figure S1.2), presumably because

steric reasons when entering the active site. Assignment of the

major enantiomer shown (Scheme 5) was tentatively made

based upon preference for formation of the (S)-isomer with

25,26

aldehydes.

2-Indanone 5q was readily accepted to give 6q

in 76% conversion yield and 59% isolated yield.

To understand better the results with these challenging

bicyclic ketones, some docking experiments were performed.

Here, both the aﬃnity energies and key distances of the ﬁve

NCS mutants used (A79I, A79F, M97V, F80L, and L76V) and

the WT-NCS (Supporting information Figures S1.8 and S1.9)

with imine intermediates toward 6m and 6o were determined.

Previous works have described the ‘dopamine-ﬁrst mechanism’

where the Pictet−Spengler cyclization is triggered by the

deprotonation of the (dopamine-derived) meta-OH by Lys122,

(

a) A79F-TfNCS-catalyzed PSR between 4 and (rac)- 5l or (2R)-5l

generated from 2-methyl-2-cyclohexen-1-one and the ene-reductase

NCR; (b) C-2 Me signals in the H NMR spectra of 6l starting from

(

rac)-5l (green) and (2R)-5l (red); and (c) Key H- H NOESY

correlations in 6l produced from (rac)-5l.

and the crystal structure containing a mimic highlighted this

35,36

gave the same single product by H NMR spectroscopy (see

key interaction with bond distances of ∼3 Å.

Notably, with

the methyl signal region in Scheme 4b). H NMR

spectroscopic analysis revealed key Nuclear Overhauser eﬀects

NOEs) between 2-H and 8’-H and 3’-H and 2-CH , and

because (2R)-5l was readily accepted by A79F-TfNCS and the

equatorial C-2-methyl substituent is more likely, the stereo-

chemistry of 6l was assigned as (1’S,2R), consistent with the

NOE data (Scheme 4c) and the S-selectivity normally

both intermediates modeled (Figure 2), they ﬁtted into the

active site of all the structures, but A79I gave either the

shortest distance (∼3 Å) from meta-OH to the Lys122 residue

combined with a folded conformation (6o) or one of the best

binding aﬃnities and a folded conformation (6m) with meta-

OH to Lys122 approaching ∼4.0 Å when a weak interaction

occurs. Indeed, the modeling with L76V to give 6m gave a less

(

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for characterization purposes, see the Supporting Information .

Research Article

Scheme 5. Use of Tf NCSs with Dopamine and Tetralones

Finally, it was decided to explore the use of diketones 5r-5t

and 1,2-, 1,3-, and 1,4-cyclohexanedione to establish whether

mono-PSR products could be formed with ketone function-

alities for further derivatization. With 5r and 5s , no PSR

products were observed (Supporting Information Figure S1.2).

With cyclohexan-1,4-dione, surprisingly, we observed forma-

tion of both mono- and a symmetrical di-PSR-product, with

the selectivity depending on the NCS variant used (Scheme 6).

m-5p and Indanone 5q to Synthesize Spiro-THIA

Alkaloids

Scheme 6. Application of the Tf NCS-Catalyzed PSR

between Dopamine and Diﬀerent di-Ketones to Synthesize

,1′-Disubstituted and Spiro-THIA Alkaloids

Reaction conditions: Reactions were performed using 4 (10−15 mM),

ketone 5r-5u (10−150 mM), puriﬁed NCS (1.6 mg/mL), sodium

ascorbate (1 eq with respect to 4), and 10% (v/v) DMSO in HEPES

Reaction conditions: Small-scale reactions were performed using 4,

ascorbate (10−15 mM), ketone 5m-5q (10−150 mM), and DMSO

buﬀer (pH 7.5, 100 mM) at 37 °C for 1 day. Conversion yield,

(

10%) at 37 °C in HEPES buﬀer (100 mM pH 7.5) using the NCS

isolated yield.

and time indicated. NCS: NCS lysate (1.7 mg/mL: for 6n-q 8.5 mg/

mL). Reaction time: 1 day, 3 days, 7 days. Reaction conversions by:

dopamine depletion by HPLC analysis, HPLC analysis against

Moderate conversions were recorded in all cases (20−35%).

Notably, WT-TfNCS gave mono-product 6t-mono: 6t-dimer

in a ratio of 8:1, and 6t-mono was isolated in 28% yield.

Enzyme variants with larger residues at positions toward the

entrance to the active site (A79I, A79F) gave similar product

ratios of 9:1. However, replacement of the bulky phenylalanine

residue with leucine in F80L reversed the selectivity for 6t-

mono: 6t-dimer to 1:3, enabling 6t-dimer to be isolated more

easily, and the structure shown, which was the same with all

mutants, was consistent with the spectroscopic data. Both the

formation of di-PSR products and changes in such reaction

selectivities are unprecedented, and control reactions gave no

PSR products. Compound 6t-mono was also observed to form

some of the hydrate by NMR spectroscopy.

product standards. Determination of ees: Marfey’s reagent and H

NMR spectroscopy (compared to racemic standards). Isolated yields

are given in parenthesis. For further details and larger-scale reactions

Figure 2. (A) Predicted conformation of the imine-β-tetralone

intermediate to give 6m in the A79I-TfNCS (modeled) active site

and key residues. (B) Predicted conformation of the imine-β-

bromotetralone intermediate in the A79I-TfNCS active site to give 6o

As an alternative, diketone (1R,4R)-5u was also used. Up to

70% yields of 6u were observed after 20 h with the most

productive variant A79F (Supporting Information Figure

S1.10), and the formation of one isomer was observed. The

product 6u was puriﬁed by preparative HPLC and isolated in

(

subunit A, 5N8Q). Reaction intermediates were docked in the

TfNCS active site (subunit A, 5N8Q) using AUTODOCK VINA

and UCSF Chimera. For further details, see the Supporting

Information .

34% yield. Based on the selectivity observed with aldehydes

and the α-methyl cyclohexanone, the isomer formed was

assigned as (1R,2S,4R)-6u with the new (2S)-spiro-sterocenter

at C-2/C-1′. No disubstituted PSR products were formed with

any of the mutants screened, presumably because of steric

eﬀects with 5u.

In order to probe further the results with these diketones,

some molecular docking experiments with the 6t-dimer were

carried out. Using WT-Tf NCS (subunit A, 5N8Q), 6t-dimer

did not ﬁt into the active site, presumably indicating the

preference for the 6t-mono that was formed. Furthermore,

favorable binding aﬃnity, which was consistent with the lower

conversions observed. The modeling overall revealed that

productive conformations of the imine intermediates can

remarkably ﬁt into the WT-NCS active site and that of

variants, with meta-OH proximal to Lys122 and the bicyclic

system occupying the entrance into the active site, thus

highlighting many interesting potential applications of these

enzymes.

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modeling with F80L (homology model) allowed entrance of

the imine diproduct intermediate into the active site, which

must occur after the ﬁrst reaction to aﬀord 6t-dimer (Figure

Authors

Jianxiong Zhao − Department of Chemistry, University

College London, London WC1H 0AJ, United Kingdom

Daniel Mendez-Sa

nchez − Department of Chemistry,

University College London, London WC1H 0AJ, United

Kingdom

Rebecca Roddan − Department of Chemistry, University

College London, London WC1H 0AJ, United Kingdom;

Institute for Structural and Molecular Biology, Department of

Biological Sciences, Birkbeck, University of London, London

WC1E 8HX, United Kingdom

John M. Ward − Department of Biochemical Engineering,

University College London, London WC1E 6BT, United

Kingdom

Figure 3. (A) Predicted conformation of the imine diproduct

intermediate to give 6t-dimer in the F80L-Tf NCS (modeled) active

site and key residues. (B) Predicted conformation of the imine

diproduct intermediate to give 6t-dimer in the WT-TfNCS active site

https://pubs.acs.org/10.1021/acscatal.0c04704

(

subunit A, 5N8Q). Reaction intermediates were docked in the

Author Contributions

J.Z. and D.M.-S. contributed equally. J. Z. and D. M.-S.

performed chemical syntheses, chemical characterization, and

enzymatic assays and R.R. expressed TfNCS mutants and

performed enzymatic assays. The project was supervised by

J.M.W and H.C.H. All authors have given approval to the ﬁnal

version of the manuscript.

TfNCS active site (subunit A, 5N8Q) using AUTODOCK VINA

and UCSF Chimera. For further details, see the Supporting

Information .

∥

no suitable potential binding modes because long distances

between the mechanistically important meta-OH and key

residue Lys122. This could explain the preference of the WT-

TfNCS for 6t-mono formation. Also, modeled A79I and A79F

with the dimer intermediate showed either poorer binding

aﬃnities or longer meta-OH to Lys122 distances, or both.

In summary, the Pictet−Spenglerase NCS was found to

accept a large range of unnatural substrate ketones to

synthesize 1,1′-substituted and spiro-THIAs. Several selected

NCS variants, in particular A79I/F, F80L, M97F, and Y108F,

were identiﬁed to eﬀectively catalyze the synthesis of more

than 20 diverse THIAs from linear aliphatic, α-substituted,

cyclic, and bicyclic ketones and diketones. The key parameter

for reactivity with these substrates is, besides a folded

productive conformation in the active site and good binding

aﬃnity, a short distance between the mechanistically important

Lys122 and (dopamine or more generally the phenethylamine)

meta-OH to initiate the reaction. If steric or other factors

prevent this key interaction, then it is not possible for the

reaction to proceed. This study also highlights the high degree

of enzyme promiscuity displayed by NCSs and good

stereoselectivities that can be achieved. NCS is proving to be

a very useful, sustainable catalyst to access 1,1′-substituted and

spiro-THIAs which are chemically challenging to synthesize.

Funding

This work was funded by a UCL Dean’s Prize and UCL-China

Scholarship Council Joint Research Scholarship to J. Z.

Funding from the Biotechnology and Biological Sciences

Research Council (BBSRC) to D. M.-S. (BB/N01877X/1) is

gratefully acknowledged. This work was also funded by a

Birkbeck Anniversary PhD scholarship to R.R as part of the

London Interdisciplinary Doctoral Program. We also acknowl-

edge 700 MHz NMR equipment support by EPSRC (EP/

P020410/1).

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank K. Karu (UCL Mass Spectrometry Facility) and A.

E. Aliev (UCL NMR Facility) in the Department of Chemistry

at UCL.

ABBREVIATIONS

■

NCS,norcoclaurine synthase; PSR,Pictet-Spengler reaction;

THIA,tetrahydroisoquinoline alkaloids; Tf ,Thalictrum flavum;

-HPAA,4-hydroxyphenylacetaldehyde; IRED,imine reductase;

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acscatal.0c04704 .

■

NMR,nuclear magnetic resonance; de,diastereomeric excess;

ee,enantiomeric excess; GDH,glucose dehydrogenase;

G6PDH,glucose-6-phosphate dehydrogenase; HEPES,4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC,high

performance liquid chromatography; DMSO,dimethylsulfox-

ide; LC−MS,liquid chromatography-mass spectrometry;

WT,wild-type.

Experimental methods, supporting ﬁgures and tables and

chemical characterization are given (PDF ).

AUTHOR INFORMATION

■

REFERENCES

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