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10.1002/anie.202104476
Angewandte Chemie International Edition
COMMUNICATION
in vitro: although it has been reported in the N-methylation of
THIQs,[31] this is not practicable for wider applications. In previous
work, integration of MTs into a linear cascade with two further
enzymes overcame this issue using a methionine adenosyl
transferase (MAT E.C. 2.5.1.6) and a methylthioadenosine
nucleosidase (MTAN, E.C. 3.2.2.9).[33] MATs generate SAM from
ATP and L-methionine, both of which are less expensive and
importantly more stable than SAM. When SAM is used for the
methylation reaction, (S)-adenosyl homocysteine (SAH) is formed
as a by-product. SAH, which would otherwise inhibit the
methylation, can then be cleaved by the MTAN into (S)-ribosyl
homocysteine (SRH) and adenine (Figure 1a). For the
supply/SAH degradation system, enzymes MAT and MTAN, both
from E. coli (EcMAT and EcMTAN, respectively) were selected.[33]
Secondly, clarified lysates were used instead of pure enzymes to
avoid the requirement for enzyme purification steps. Thirdly, to
further streamline the process, the methylation cascade reaction
was coupled directly to the NCS-catalysed PS reaction in a one-
pot two-step cascade, obviating the need for purification of the
THIQ intermediates. The methylation of representative substrates
(S)-1, (S)-6, 9, 10 and (S)-11 was explored first (Scheme 1: e.e.s
for (S)-1 (>97%) and (S)-6 (>98%) are shown in the SI). Here it
was essential that all the dopamine was consumed in the TfNCS
catalysed reaction before the MT step to avoid the methylation of
unreacted dopamine,[33] which could interfere with the
regiochemical outcome.
(Table 1). As expected, the regiochemistry of the reaction was
strongly dependent on the nature of the side chain, which directed
the methylation preferentially at the 6-OH for substrates (S)-1, (S)-
6, and (S)-11 and surprisingly preferably on the 7-OH for
substrates 9 and 10. Interestingly, when 9 or 10 underwent the
same reaction conditions with MxSafC, the 6-OH was methylated
instead preferentially and derivatives 6-OMe-9 or 6-OMe-10
respectively were obtained in good yield and excellent
regioselectivities (Table 1). This unique performance with a switch
in the regioselectivities for both RnCOMT and MxSafC further
confirms the complementarity of these two enzymes.[33]
With the challenge to demonstrate the potential of this
approach, the methylation with RnCOMT was further coupled with
a multistep in vitro enzyme cascade for the in situ generation of
norcoclaurine (S)-2 (generated in >97% e.e.) using Candidatus
nitrosopumilus salaria BD31Q tyrosinase (CnTYR) and
Enterococcus faecalis DC32 tyrosine decarboxylase (EfTyrDC),
together with a versatile transaminase from Chromobacterium
violaceum[34] (CvTAm) and wild-type TfNCS enzyme.[17] A total of
seven enzymes as clarified cell-lysates were used in the same
pot, achieving the conversion of L-tyrosine into (S)-coclaurine (S)-
6-OMe-2 in an unique one-pot three step cascade (Scheme 2a).
Table 1. Isolated yields and regioisomeric ratios for the preparative scale
cascade reactions described in Schemes 1-3.
Regioisomeric
ratio[b]
(6-OMe:7-
OMe)
(a)
Substrate
Product
MT
Yield[a]
HO
RnCOMT MeO
HO
HO
TfNCS
NH2
NH
NH
RCHO
MAT
MTAN
HO
HO
55%
(96%)
(S)-1
(S)-2
(S)-6-OMe-1
(S)-6-OMe-2
(S)-6-OMe-2
RnCOMT
RnCOMT
95:5
95:5
R
R
dopamine
(S)-6-OMe-1 R = CH2Ph
(S)-6-OMe-6 R = C5H11
(S)-6-OMe-11 R =CH2CH2Ph
(S)-1 R = CH2Ph
(S)-6 R = C5H11
55%
(92%)
(S)-11 R = CH2CH2Ph
RnCOMT
43%
(89%)
(S)-2
(S)-2
MxSafC[c]
MxSafC[c]
60:40
na
(b)
or
R1O
R2O
HO
HO
HO
MxSafC
TfNCS
6%
(9%)[d]
NH
n
NH2
(S)-6,7-(OMe)2-2
NH
n
O
MAT
MTAN
HO
dopamine
34%
(64%)[e]
RnCOMT
n
(S)-3
(S)-3
(S)-6,3'-(OMe)
(S)-6,4'-(OMe)
2
-3
-3
RnCOMT
95:5
100
9 n = 2
10 n = 1
7-OMe-9 n = 2, R1 = H, R2 = Me
7-OMe-10 n = 1, R1 = H, R2 = Me
MxSafC
Cj-6-OMT
then
MxSafC
6-OMe-9 n = 2, R1 = Me, R2 = H
6-OMe-10 n = 1, R1 = Me, R2 = H
27%
(70%)[e]
2
19%
(27%)
Scheme 1. Four-enzyme cascades for the synthesis of: (a) THIQs (S)-6-OMe-
1, (S)-6-OMe-6, and (S)-6-OMe-11, and (b) THIQs 6-OMe-9, 7-OMe-9, 6-OMe-
10, and 7-OMe-10 from 9 and 10 respectively.
(S)-5
(S)-5
(S)-6-OMe-5
(S)-6-OMe-5
RnCOMT
MxSafC
95:5
95:5
20%
(31%)
44%
(90%)
With the architecture of the cascades defined, a biocatalytic
scale-up of the methylated THIQs was attempted in a single pot.
Starting from dopamine (5-20 mM) and an excess of the
corresponding carbonyl compound, the THIQs (S)-1, (S)-6, 9,10
and (S)-11 were generated with high enantiopurity (for single
isomer compounds) and the methylation step initiated by the
addition of RnCOMT clarified lysate (10% v/v) in the presence of
ATP, L-methionine and the SAM supply/SAH degradation system
enzymes EcMAT (10% v/v) and EcMTAN (2.5% v/v). Using
RnCOMT the methylated products (S)-6-OMe-1, (S)-6-OMe-6, 7-
OMe-9, 7-OMe-10, and (S)-6-OMe-11 were isolated with good
yields and high regioselectivities with only 5-10% of the opposite
regioisomer detected by 1H NMR spectroscopy in most cases
(S)-6
(S)-6-OMe-6
7-OMe-9
RnCOMT
RnCOMT
90:10
10:90
9
40%
56%
9
6-OMe-9
MxSafC
90:10
10:90
(94%)[f]
45%[g]
(90%)
10
7-OMe-10
RnCOMT
30%[g]
(89%)
10
6-OMe-10
MxSafC
90:10
85:15
33%
(40%)
(S)-11
(S)-6-OMe-11
RnCOMT
3
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