One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine

Article abstract of DOI:10.1039/c4gc02325k

We describe novel chemoenzymatic routes to (S)-benzylisoquinoline and (S)-tetrahydroprotoberberine alkaloids using the enzymes transaminase (TAm) and norcoclaurine synthase (NCS) in a one-pot, one-substrate 'triangular' cascade. Employment of up to two C-

Full text of DOI:10.1039/c4gc02325k

Green Chemistry
COMMUNICATION
One-pot triangular chemoenzymatic cascades for
the syntheses of chiral alkaloids from dopamine†
Cite this: Green Chem., 2015, 17, 852
B. R. Lichman,a E. D. Lamming,b T. Pesnot,b J. M. Smith,a H. C. Hailes*b and
Received 25th November 2014,
Accepted 12th December 2014
J. M. Ward*a
DOI: 10.1039/c4gc02325k
www.rsc.org/greenchem
We describe novel chemoenzymatic routes to (S)-benzylisoquino-
Norlaudanosoline (tetrahydropapaveroline) 1a is a BIA
line and (S)-tetrahydroprotoberberine alkaloids using the enzymes found in mammals: it is the precursor to ‘endogenous’ mor-
transaminase (TAm) and norcoclaurine synthase (NCS) in a one- phine,5 and also has a number of neuronal effects including
pot, one-substrate ‘triangular’ cascade. Employment of up to two roles in Parkinson’s disease13 and drug addiction.14 Syntheti-
C–C bond forming steps allows for the rapid generation of mole- cally, 1a is a versatile BIA precursor, and consequently it has
cular complexity under mild conditions.
been employed in synthetic biology/metabolic engineering
routes to various BIAs.15 BIA 1a can be accessed by a Pictet–
Spengler condensation between dopamine 2a and 3,4-dihydoxy-
phenylacetaldehyde 3a.16
Tetrahydroprotoberberine alkaloids (THPBs, berbines) are a
subgroup of the BIAs found in both plants and animals, and
include compounds such as canadine17 and spinosine.18a The
THPB moiety can be accessed from 1a via a Pictet–Spengler
reaction with formaldehyde.18 The two compounds directly
formed by this reaction (4 and 5) have been shown to have
potential in cancer chemotherapeutics.19 The regioselectivity
of this chemical Pictet–Spengler reaction (major product:
10,11-dihydroxy 4) contrasts with the regioselectivity of the
plant berberine-bridge enzyme (BBE), which typically forms
9,10-dihydroxy-THPBs from a N-methylated BIA precursor.20
Thus chemical approaches to THPBs provide a complementary
method to the reported BBE approach.
Introduction
In order to minimise waste products and reduce the usage of
finite resources, chemistry must find ‘green’ alternatives to tra-
ditional synthetic methods.1 The employment of enzymes as
catalysts offers significant potential in this regard as enzymes
often demonstrate exquisite chemo- and enantio-selectivity,
and operate in mild conditions.2 Furthermore, using enzymes
in one-pot cascades can enable the formation of highly
complex compounds from cheap starting materials, frequently
without the requirement of intermediate isolation or func-
tional group protection strategies.3
Benzylisoquinoline alkaloids (BIAs) are a large, diverse
family of natural products found in both plants4 and animals.5
Many BIAs have pharmacological activities—these include
analgesic,6 antimicrobial7 and antitumor8 effects—and conse-
quently are common synthetic targets.9 A key step in the syn-
thesis of many of these compounds is the formation of the
tetrahydroisoquinoline (THIQ) moiety via a Pictet–Spengler
condensation.10 Recently, a mild one-pot biomimetic approach
to the formation of THIQs was developed, using phosphate
buffer as a catalyst.11 There has also been recent progress
made in the enzymatic synthesis of diverse chiral THIQs using
the plant enzyme norcoclaurine synthase (NCS).12
Triangular cascade design
In this work, we present one-pot two-enzyme one-substrate
syntheses of (S)-1a and (S)-1b using the enzymes transaminase
(TAm) and NCS. The reaction involves in situ generation and
utilisation of reactive aldehyde species. We also demonstrate a
one-pot, two-enzyme, three-step chemoenzymatic synthesis of
the THPB (S)-4. This synthesis uses the same cascade route as
for (S)-1a, but involves the sequential addition of formal-
dehyde to trigger
(Scheme 1).
a second Pictet–Spengler cyclisation
Previous attempts to achieve this type of cascade in vitro
and in vivo have utilised a monoamine oxidase (MAO) as the
catalyst in the first step, converting 2a to 3a. Several MAO
approaches have focused on forming rac-1a through a spon-
taneous Pictet–Spengler reaction (typically phosphate cata-
lysis).16 A number of in vivo metabolic cascades forming
aDepartment of Biochemical Engineering, University College London, Bernard Katz
Building, Gordon Street, London, WC1H 0AH, UK. E-mail: j.ward@ucl.ac.uk
bDepartment of Chemistry, University College London, Christopher-Ingold Building,
20 Gordon Street, London, WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4gc02325k
852 | Green Chem., 2015, 17, 852–855
This journal is © The Royal Society of Chemistry 2015

Green Chemistry
Communication
removes equal quantities of molecules from both sides of the
TAm equilibrium. This three-sided ‘triangular’ cascade design
results in a system with high atom economy.3
Results and discussion
The first step towards establishing the one-pot cascade system
was the identification of a TAm with good activity towards 2a.
The screening reaction was conducted in phosphate buffer,
which enabled the in situ formation of rac-1a from 2a and 3a
(Table 1). The screen identified two TAms with activity towards
2a at pH 7.5: CV2025 (Chromobacterium violaceum)21 and
PP_3718 (Pseudomonas putida), providing 21% and 15% con-
version of 1a from 50 mM 2a respectively. Trace activities were
found in PP_0596 (P. putida), SaV_2612 (Streptomyces avermiti-
lis) and VF_JS17 (Vibrio fluvialis)22 (Table 1, ESI† Fig. S1). We
selected the best performing TAm, CV2025, for use in further
studies.
In order to form (S)-1a from 2a two aspects of the previous
TAm screening reaction conditions were modified. Changing
the buffer from phosphate to HEPES removed most of the
background chemical reaction11 and adding purified
Δ29TfNCS enabled catalytic formation of the chiral product
(S)-1a from 2a. Optimum reaction conditions were determined
by varying the concentrations of 2a, NCS and TAm present. A
balance between reaction components was crucial to ensure
the rates of the two steps were matched; a build-up of 3a
would cause an increase in undesirable side-reactions includ-
ing the non-enzymatic Pictet–Spengler condensation, which
would reduce the final enantiomeric excess (ee) of the product.
BIA (S)-1a was produced from 2a with very good conversions
and excellent enantioselectivity. In all cases, an increase in
TAm concentration resulted in a greater consumption of 2a,
Scheme 1 Overview of the biocatalytic and non-enzymatic cascades
presented in this work, including the ‘triangular’ cascade.
(S)-reticuline report the use of recombinant NCS as the Pictet–
Spengler catalyst. However, it is not clear that NCS is active in
these systems: any chirality reported seems to be the result of
(S)-selective enzymes downstream of NCS.15a–d
Here, we employ a TAm for the conversion of 2a to 3a (step
1), which then combine to form 1a (step 2). TAms have an
advantage over MAOs in this system: the conversion of 2a to 3a
with TAms can be controlled by stoichiometrically limiting the
quantity of co-substrate (in this instance, pyruvate) available.
Furthermore, we avoid the problems associated with driving
TAm reactions to completion, as the Pictet–Spengler reaction
Table 1 TAm mediated synthesis of rac-1a from 2aa
Entry
TAm
Organism
Uniprot entry name
Gene name
Conv.b (%)
1
2
3
4
5
6
7
8
9
Empty vector
BSU_09260
CV_202521
Dgeo_1416
KPN_00255
PP_0596
n.d.c
n.d.
21
n.d.
n.d.
Trace
14
Trace
n.d.
Trace
Bacillus subtilis
YHXA_BACSU
yhxA
Chromobacterium violaceum
Deinococcus geothermalis
Klebsiella pneumoniae
Pseudomonas putida
Pseudomonas putida
Streptomyces avermitilis
Streptomyces avermitilis
Vibrio fluvialis
Q7NWG4_CHRVO
Q1IYH3_DEIGD
A6T537_KLEP7
Q88Q98_PSEPK
Q88GK3_PSEPK
Q82JZ2_STRAW
Q82ER2_STRAW
F2XBU9_VIBFL
CV_2025
argD/lysJ
gabT
PP_0596
PP_3718
SAV_2612
SaV_4551
JS17
PP_3718
SaV_2612
SaV_4551
VF_JS1722
10
a 50 mM 2a, 25 mM pyruvate, 1 mM PLP, 10% v.v−1 TAm, 37 °C, 4 h. b Concentration of rac-1a, determined by analytical HPLC. c n.d. = not
detected. See ESI Fig. S1 for reaction time course.
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Green Chem., 2015, 17, 852–855 | 853

Communication
Green Chemistry
Table 2 One pot synthesis of (S)-BIAsa
Amine
[mM]
CV2025
NCS
Conv.b
[%]
eec
[%]
(v.v−1) [%]
[µg mL−1
]
Scheme 2 One-pot chemoenzymatic synthesis of (S)-4 and (S)-5.
Reaction conditions: (a) 20 mM 2, 10 mM sodium pyruvate, 500 µg mL−1
NCS and 20% v.v−1 CV2025 lysate, 50 mM HEPES pH 7.5, 37 °C, 3 h. (b)
40 mM formaldehyde, 1 M sodium phosphate, pH 6, 30 min, 37 °C.
2a
2a
2a
2a
2a
2a
2a
2a
2a
2a
2a
2a
2b
20
20
20
20
20
20
50
50
50
50
50
50
20
10
20
30
10
20
30
10
20
30
10
20
30
20
100
100
100
500
500
500
100
100
100
500
500
500
500
74 (74)
86 (89)
82 (93)
77 (82)
87 (92)
84 (95)
36 (42)
55 (66)
70 (78)
42 (48)
65 (73)
72 (85)
56 (57)
99
99
99
99
99
98
98
97
99
98
98
96
90
of the system is established by NCS, the subsequent addition
of formaldehyde does not affect the ee: only the (S)-enantiomer
of the major product 4 was observed by chiral HPLC.
The major regioisomer 4 formed by this method has 10,11-
dihydroxy regiochemistry. BBE, a plant enzyme which catalyses
the formation of (S)-THPBs en route to berberine, typically
forms products with 9,10-dihydroxy regioselectivity.20 This
enzyme has recently been used in a novel in vitro deracemisa-
tion cascade to produce (S)-THPBs.20b The synthesis presented
here and the BBE catalysed cascade can be seen as comp-
lementary methods, providing efficient chemoenzymatic
routes to 10,11-dihydroxy-(S)-THPBs and 9,10-dihydroxy-(S)-
THPBs respectively.
Finally, to demonstrate the preparative potential of our cas-
cades, we performed syntheses of (S)-1a and (S)-4 on a
0.5 mmol scale. The concentrations of components in these
cascades were the same as those used in the micro-scale cas-
cades (Scheme 2). The synthesis scaled with no complications:
conversion of 2a to (S)-1a was achieved in 2 hours with 86%
conversion and 62% isolated yield (>95% ee). (S)-4 was formed
from 2a in 2.5 hours with 47% conversion and 42% isolated
yield (>95% ee). The identities of these products were verified
by NMR.
a General reaction conditions: 2 equivalents 2a or 2b, 1 equivalent
pyruvate, purified Δ29TfNCS and CV2025 lysate, 50 mM HEPES pH
7.5, 37 °C, 3 h. b Determined by analytical HPLC. Conversions in
brackets refer to depletion of primary amine. c Determined by chiral
HPLC of the crude product.
generally leading to an increase in product formation
(Table 2). However, at lower concentrations of 2a (20 mM) the
use of 20% v.v−1 CV2025 lysate, rather than 30%, was optimal.
The enantioselectivity was excellent under all conditions. In
terms of conversion, the effect of NCS concentration was more
apparent with higher concentrations of dopamine. The best
conditions identified were those with 20 mM 2a, 500 µg mL−1
NCS and 20% v.v−1 CV2025 lysate, which provided an 87% con-
version of (S)-1a from 2a with an ee of 99%.
To demonstrate the versatility of this system, it was used to
synthesise (S)-1b from 2-(3-hydroxyphenyl)ethylamine 2b. The
para-hydroxyl group of 2a is not required for the Pictet–
Spengler condensation, and thus 2b is a suitable substrate for
NCS.12b Using the optimum conditions determined previously
for 2a, (S)-1b was formed from 2b with a fair conversion of
56% and a high ee of 90%. The lower conversion and ee com-
pared to (S)-1a is possibly reflective of a poorer affinity of
TfNCS towards for 2b compared to the natural substrate 2a.
In order to form THPBs (S)-4 and (S)-5, a one-pot synthesis
of (S)-1a was conducted (as described above) and formal-
dehyde was added to the reaction after 3 hours. This triggered
the second Pictet–Spengler condensation and resulted in the
formation of (S)-4 and (S)-5 in a ratio of approximately 7 : 1
(Scheme 2). This cascade features 2 enzymes, 3 steps and the
formation of 4 bonds, including 2 C–C bonds. The overall reac-
tion occurred with good conversion: the second Pictet–Spen-
gler step alone (from (S)-1a) provided 74% conversion (64%
and 9% for (S)-4 and (S)-5 respectively), which translates as an
overall 64% conversion (56% and 8%) from 2a. As the chirality
Conclusions
In summary, we have developed one-pot syntheses of chiral
alkaloids, using the enzymes TAm and NCS in a ‘triangular’
cascade. BIA (S)-1a was formed from 2a with very good conver-
sion and excellent enantioselectivity in only 2 hours. (S)-1b
was formed in a similar manner with fair conversion and a
high ee. A chemical extension to the cascade enabled the for-
mation of (S)-4 in a one-pot three-step reaction from the low-
cost starting material 2a, again with excellent enantio-
selectivity and an overall reaction time of less than 3 hours.
The cascades presented here exhibit high atom economy,
the in situ generation of reactive intermediates, and the rapid
accumulation of molecular complexity through C–C bond
forming steps. Overall, these syntheses demonstrate the
854 | Green Chem., 2015, 17, 852–855
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Green Chemistry
Communication
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