Biomimetic synthesis of 2-substituted N-heterocycle alkaloids by one-pot hydrolysis, transamination and decarboxylative Mannich reaction

Article abstract of DOI:10.1039/C8CC06759G

Heterocycles based on piperidine and pyrrolidine are key moieties in natural products and pharmaceutically active molecules. A novel multi-enzymatic approach based on the combination of a lipase with an α,ω-diamine transaminase is reported, opening up the synthesis, isolation and characterisation of a broad range of 2-substituted N-heterocycle alkaloids.

Full text of DOI:10.1039/C8CC06759G

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. L. Galman, I.
Slabu, F. Parmeggiani and N. J. Turner, Chem. Commun., 2018, DOI: 10.1039/C8CC06759G.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 5
View Article Online
DOI: 10.1039/C8CC06759G
Journal Name
COMMUNICATION
Biomimetic synthesis of 2-substituted N-heterocycle alkaloids
by one-pot hydrolysis, transamination and decarboxylative
Mannich reaction
Received 00th January 20xx,
Accepted 00th January 20xx
James L. Galman,a Iustina Slabu,a Fabio Parmeggiani,a Nicholas J. Turner*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Heterocycles based on piperidine and pyrrolidine are key moieties
in natural products and pharmaceutically active molecules. A
novel multi-enzymatic approach based on the combination of a
lipase with an α,ω-diamine transaminase is reported, opening up
the synthesis, isolation and characterisation of a broad range of 2-
substituted N-heterocycle alkaloids.
Me
N
H
N
O
O
hygrine
norsedaminone
Me
N
R
N
Ph Ph
H
N
OH
Ph
OH
Nitrogen-containing heterocyclic compounds (alkaloids)
represent a diverse range of natural products found as
secondary metabolites in various kingdoms of life, notably in
approximately 20% of all plant species.1 Alkaloids often
possess potent biological activities which make them highly
attractive targets for the development of pharmaceutical
drugs such as antimalarial (e.g., quinine), anticancer (e.g.,
homoharringtonine) and antibacterial (e.g., chelerythrine)
agents. Other alkaloids have shown to act as stimulants (e.g.,
caffeine, nicotine), narcotics (e.g., cocaine, morphine) and as
poisons (e.g., coniine). As a result, many have found usage in
traditional medicine, as well as in modern drug development
as essential building blocks and pharmacophores for medicinal
chemistry. In a recent survey of U.S. FDA approved drugs, 59%
contained at least one nitrogen heterocycle.2
O
n
n = 1, 2
R = H, Me
pipradrol
atropine
O
OH
Ph
H
H
N
N
OH
OH
OMe
O
methylphenidate
rimiterol
Scheme 1. Representative natural products and APIs based on
2-substituted pyrrolidine/piperidine alkaloids derived from the
cyclic imines of lysine or ornithine.
psychostimulants, adrenergic and cycloplegic drugs)2,4 make
these compounds attractive targets for total synthesis
(Scheme 1).
Pyrrolidine and piperidine type alkaloids, in particular, are
abundant in Nature, and are derived from lysine and the non-
proteinogenic amino acid ornithine, respectively. The
biosynthetic pathway involves initial decarboxylation of these
amino acid precursors followed by oxidative deamination to
form reactive cyclic intermediates (Scheme 1). Subsequent
Mannich-type nucleophilic addition provides the 2-substituted
pyrrolidine/piperidine carbon frameworks which are the basis
of a diverse range of alkaloids containing 5-membered rings,
such as tropanes and pyrrolizidines, and 6-membered rings,
such as quinolizidine and lycopodium alkaloids.3 The frequent
occurrence of these structural ring motifs in a large number of
pharmaceutical products (such as antidepressants,
These compounds and their analogues have been the subject
of many synthetic studies, most often based on Mannich-type
reactions, both with and without the presence of an
organocatalyst (Scheme 2). One reported method involves a
one-pot three-component Mannich reaction and cyclisation,
mediated by an excess of L-proline, although no
stereoselectivity was observed.5 Similarly, direct oxidative
coupling of tertiary amines with non-activated ketones has
been described with catalytic L-proline in combination with
vanadium acetate; however, this reaction suffers from a
limited substrate scope and afforded only modest isolated
yields.6 Another common synthetic approach uses the Henry-
Nef protocol involving the generation of nitroalkenes followed
by acid hydrolysis; this method requires several synthetic steps
and multiple chromatographic separations leading to a poor
average overall yield (~40%) of the alkaloid, in N-protected
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
View Article Online
DOI: 10.1039/C8CC06759G
COMMUNICATION
Journal Name
utilise linear diamines but differ in their acceptor
substrates.15,16 The different substrate specificities indicate
distinct evolution histories with possible common degradation
pathways between these microorganisms.17 In this study, we
focused on YgjG, which exhibits a strict preference for linear
diamines, with α-ketoglutarate (or pyruvate) as acceptor. The
ygjG gene from E.coli K-12 was overexpressed in E. coli BL21
(DE3) and, in order to provide an easy to use biocatalyst for
preparative chemistry, the cell-free lysate was employed
instead of the purified enzyme. Preliminary experiments were
performed using diamine
2 in the presence of pyruvate as the
amino acceptor to generate the reactive imine intermediate
4,
followed by a Mannich-type addition with benzoylacetic acid
6a to access the alkaloid norsedaminone 8a. A one-pot tandem
enzyme cascade was envisaged, avoiding the sequential
addition of reagents because of the tendency for the reactive
imine intermediates to polymerize under the reaction
conditions.15 However, attempts at performing this cascade
using YgjG and 6a (Table 1, entry 1) were unsuccessful,
affording unsatisfactory amounts of the alkaloid product, due
to the spontaneous loss of the carboxyl moiety via
decarboxylation.18,19
Scheme 2. Strategies for the synthesis of 2-substituted
pyrrolidine/piperidine alkaloids.
form.7 Finally, a recent strategy involved a decarbonylative
Mannich reaction that requires the conversion of protected
prolines to the corresponding acid chlorides, which undergo a
decarbonylative C-C bond formation reaction with methyl
ketones, providing a direct access to various N-protected
pyrrolidine alkaloids.8
To overcome this problem, we decided to generate labile 6a in
situ from its commercially available ethyl ester 5a, via the
addition of a lipase. Previous reports have described the
transaminase mediated asymmetric amination of both ester 5a
and acid 6a, which would lead to undesirable side products in
our cascade.19,20 However, YgjG showed no activity towards 5a
An alternative approach for the synthesis of substituted
piperidines and pyrrolidines relies on the use of engineered
biocatalysts. The expanding toolbox of enzymes available for
the synthesis of chiral amines has allowed the development of
new synthetic routes for asymmetric amine synthesis based on
ω-transaminases,9 reductive aminases,10 ammonia lyases,11 as
well as deracemisation and dynamic kinetic resolution
processes (e.g., with monoamine oxidases12 or hydrolases13).
Biocatalytic strategies for the synthesis of these classes of
alkaloids rely on mimicking the fundamental lysine and
ornithine catabolism pathway. The first step involves the
Table 1. Optimisation of the lipase-transaminase cascade.a
conversion of 1,4-diaminobutane (putrescine,
diaminopentane (cadaverine, ) to cyclic imines 1-pyrroline
and 1-piperideine , respectively. These reactive intermediates
1) or 1,5-
2
3
4
Entry
Substrates
(ratio)
Lipase
Product distribution [%]b
are highly susceptible to nucleophilic attack, and the Mannich
addition of 3-ketoacids followed by decarboxylation would
7a-8a
5
9a
92
29
29
15
30
29
12
8
6a
3
1
2
2
2
2
2
2
2
2
2
1
+
+
+
+
+
+
+
+
+
+
6a (1 : 1)
5a (1 : 1)
5a (1 : 1)
5a (1 : 1)
5a (1 : 1)
5a (1 : 1)
5a (1 : 1)
5a (1.5 : 1)
5a (1 : 1.5)
5a (1.5 : 1)
none
PPL
PSL
provide
a variety of 2-substituted pyrrolidine/piperidine
2
29
15
59
11
22
79
89
90
98
42
55
26
59
49
9
3
2
1
3c
4
analogues. Recently, diastereomerically pure 2,6-disubstituted
piperidines have been synthesised via an ω-transaminase
mediated aza-Michael reaction, in a one-pot reaction starting
from complex diketone precursors.14 Herein, we describe an
alternative approach towards the production of
pyrrolidine/piperidine alkaloids that mimics the natural
biosynthetic pathway, by assembling an enzyme cascade
consisting of a transaminase and a lipase, employing biogenic
diamines and readily available 3-ketoesters as substrates.
In previous studies we investigated class III α,ω-diamine
transaminase (α,ω-DTA) genes spuC (part of the polyamine
uptake and utilization pathway in Pseudomonas putida) and
ygjG (the complementary gene in Escherichia coli), which both
CALB
F-AP15
TLL
5c
6
7
8
9
10
CALBd
CALBd
CALBd
CALBd
8
1
a: Expt. cond.: 5.0-7.5 mM substrates, 7.5 mM pyruvate, 1 mM PLP,
0.5% v/v DMSO, 200 µL/mL lysate, 2 mg/mL lipase, 50 mM Tris-HCl
buffer, pH 9.0, 30°C, 250 rpm, 18 h. b: Measured by reverse-phase
HPLC. c: <5% of 5a was left unreacted. d:
5 mg/mL lipase
concentration.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 3 of 5
View Article Online
DOI: 10.1039/C8CC06759G
Journal Name
COMMUNICATION
and 6a, likely due to the small size and greater hydrophobicity Table 2. One-pot biocatalytic cascade synthesis of
of the active site entrance compared to other class III piperidine/pyrrolidine alkaloids 7-8a-i from diamines 1-2 and
transaminases.21 Several commercially available lipases known aromatic ketoacids 5a-i a
.
to have broad substrate tolerance (see ESI for the full list) were
tested with an equimolar mixture of amine and ketoester
substrates (Table 1, entries 2-6). The product distribution of
the lipase reactions gave modest conversions to the piperidine
alkaloid 8a whilst still yielding undesirable acetophenone side
product 9a resulting from the partial decarboxylation of 6a. Of
the commercial enzymes tested, the immobilised CALB
(Novozyme 435) gave the highest product conversion (entry 4,
59%). Encouraged by these preliminary results, we increased
the lipase concentration and a higher percentage of 8a was
attained (entry 7). Further increases in lipase concentration
had a slightly negative influence on the production of 8a (data
not shown), and this observation could result from a higher
rate of hydrolysis. Increasing the concentration of either
5a gave a similar increase of conversion to 8a (entries 8-9).
Thus, given the higher cost of 5a compared to , we decided to
2 or
2
use an excess of diamine in all subsequent experiments. Under
these optimised conditions, we also tested natural diamine
Diamine Ketoester
Product distribution [%]b
substrate
1 to ensure this reaction was not limited to 2, and
7a-i
98
99
99
84
88
88
80
87
99
–
–
–
–
–
–
–
–
–
8a-i
–
–
–
–
–
–
–
–
9a-i
1
1
1
8
9
8
14
5
1
6a-i
1
0
0
9
3
4
5
8
the system furnished pyrrolidine alkaloid 7a, with near perfect
conversion (entry 10, 98%).
1
5a
5b
5c
5d
5e
5f
5g
5h
5i
Building on the successful results obtained with Mannich-type
addition processes under optimised conditions, the reaction
was tested with both diamines (1-2) and a panel of aromatic 3-
ketoesters (5a-i), to assess the general scope of the reaction.
The results (Table 2) revealed that both
1 and 2 could react
with a variety of ketoesters bearing electron-donating
–
0
2
5a
5b
5c
5d
5e
5f
5g
5h
5i
89
89
95
64
54
65
55
73
89
8
5
3
15
44
33
43
27
9
3
6
2
21
2
2
1
0
2
substituents (5b-d) or halogens (5e-i) at various positions on
the aromatic ring, yielding the corresponding 2-substituted
pyrrolidines and piperidines 7-8a-i with moderate to excellent
conversions (54-99%). Most notably, we demonstrated the
facile and efficient synthesis of the natural product
ruspolinone 7c and its piperidine analogue 8c (71 and 69%
isolated yields, respectively).
In order to demonstrate the preparative synthesis of products
accessible via this method, we performed large-scale reactions
with selected aromatic ketoesters from the previous panel (5a-
a: Expt. cond.: 7.5 mM diamine, 5.0 mM ketoester, 7.5 mM pyruvate, 1
mM PLP, 0.5% v/v DMSO, 200 µL/mL lysate, 5 mg/mL CALB, 50 mM
Tris-HCl buffer, pH 9.0, 30°C, 250 rpm, 18 h. b: Measured by reverse-
phase HPLC.
c
) and we also expanded the substrate scope to aliphatic
ketoesters (5j-m). Substrate concentrations were increased (25
mM ketoester, 30 mM diamine) to facilitate isolation and
purification of the products (Scheme 3). Following a simple
work-up procedure involving quenching of the reaction
mixture with aqueous HCl, the aromatic derivatives could be
isolated easily in good overall yields (60-81%), while for the
smaller and more hydrophilic aliphatic derivatives yields were
lower (50-78%) due to the higher solubility of the targeted
compounds.‡ These preparative scale reactions demonstrate
the effectiveness of our one-pot system to provide access to
piperidine/pyrrolidine alkaloid scaffolds for synthetic
applications.
with hygrine and pelletierine reported to racemise via a retro
aza-Michael reaction.22 Attempts to deracemise some of these
products using the well-established monoamine oxidase
(MAO-N)
or
6-hydroxy-D-nicotine
oxidase
(HDNO)
deracemisation protocols proved to be unsuccessful, with only
compounds 8a-c generating low ee values (<10%, data not
shown).
Lastly, we considered N-methylputrescine 10 as an alternative
substrate for the transaminase step. In Nature, 10 is
oxidatively deaminated by methylputrescine oxidase23 to
generate a highly reactive pyrrolinium cation, a central
metabolic precursor belonging to tropane and pyrrolidine
alkaloids biosynthesis. To our delight, α,ω-DTA YgjG readily
All compounds were isolated as racemates as expected, based
on
the
known
propensity
of
2-substituted
piperidine/pyrrolidine alkaloids to racemise readily at basic pH,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
View Article Online
DOI: 10.1039/C8CC06759G
COMMUNICATION
Journal Name
Scheme 3. Preparative scale one-pot biocatalytic synthesis of
Notes and references
selected piperidine/pyrrolidine alkaloids (with isolated yields).
‡
Unexpectedly, pelletierine 8j could not be obtained in
sufficient purity, in spite of multiple attempts to isolate it.
This is likely due to known degradation (see for instance: E.
C. Carlson, L. K. Rathbone, H. Yang, N. D. Collett and R. G.
Carter, J. Org. Chem., 2008, 73, 5155) and high volatility,
leading to significant loss of product.
1
2
3
4
5
6
J. Ziegler and P. J. Facchini, Annu. Rev. Plant Biol., 2008, 59,
735–769.
E. Vitaku, D. T. Smith and J. T. Njardarson, J. Med. Chem.,
2014, 57, 10257.
P. M. Dewick, in Medicinal Natural Products, John Wiley &
Sons Ltd., Chichester, UK, 2013.
R. Vardanyan, Classes of Piperidine-Based Drugs (ch. 10), in:
Piperidine-Based Drug Discovery, Elsevier, 2018.
S. Chacko and R. Ramapanicker, Tetrahedron Lett., 2015, 56
2023.
,
A. Sud, D. Sureshkumar and M. Klussmann, Chem. Commun.,
2009, 7345, 3169.
7
8
C. Bhat and S. G. Tilve, Tetrahedron Lett., 2011, 52, 6566.
Y.-C. Shih, P.-H. Tsai, C.-C. Hsu, C.-W. Chang, Y. Jhong, Y.-C.
Chen and T.-C. Chien, J. Org. Chem., 2015, 80, 6669.
I. Slabu, J. L. Galman, R. C. Lloyd and N. J. Turner, ACS Catal.,
9
2017, 7, 8263.
10 G. A. Aleku, S. P. France, H. Man, J. Mangas-Sanchez, S. L.
Montgomery, M. Sharma, F. Leipold, S. Hussain, G. Grogan
and N. J. Turner, Nat. Chem., 2017, 9, 961.
11 F. Parmeggiani, N. J. Weise, S. T. Ahmed and N. J. Turner,
Chem. Rev., 2018, 118, 73.
12 D. Ghislieri, A. P. Green, M. Pontini, S. C. Willies, I. Rowles, A.
Frank, G. Grogan and N. J. Turner, J. Am. Chem. Soc., 2013,
135, 10863.
13 J. Paetzold and J. E. Bäckvall, J. Am. Chem. Soc., 2005, 127
,
17620.
14 E. O’Reilly, C. Iglesias, D. Ghislieri, J. Hopwood, J. L. Galman,
catalysed the deamination of 10, expanding the substrate
scope to N-methylalkaloids. As a representative example,
hygrine 11 was obtained under the same conditions, in 75%
isolated yield (Scheme 3).
R. C. Lloyd and N. J. Turner, Angew. Chem. Int. Ed., 2014, 53
2447.
,
15 I. Slabu, J. L. Galman, N. J. Weise, R. C. Lloyd and N. J. Turner,
ChemCatChem, 2016, , 1038.
8
In summary, we have designed and tested
a novel
16 J. L. Galman, I. Slabu, N. J. Weise, C. Iglesias, F. Parmeggiani,
R. C. Lloyd and N. J. Turner, Green Chem., 2017, 19, 361.
17 L. a Nahum, S. Goswami and M. H. Serres, Physiol. Genomics,
2009, 38, 250.
18 J. Kim, D. Kyung, H. Yun, B. K. Cho, J. H. Seo, M. Cha and B. G.
Kim, Appl. Environ. Microbiol., 2007, 73, 1772.
19 S. Mathew, S. Jeong, T. Chung, S. Lee and H. Yun, Biotechnol.
J., 2016, 11, 185.
20 O. Buß, M. Voss, A. Delavault, P. Gorenflo, C. Syldatk, U.
Bornscheuer and J. Rudat, Molecules, 2018, 23, 1211.
21 H. J. Cha, J. Jeong, C. Rojviriya and Y. Kim, PLoS One, 2014, 9,
multienzymatic protocol for the synthesis of 2-substituted and
N-substituted piperidine/pyrrolidine alkaloids, in a one-pot
fashion, under mild conditions and starting from commercially
available substrates. The products isolated and characterised
are natural alkaloids or analogs, many of which have been
shown to possess a wide range of biological activities (e.g.,
anticoagulant,24 antimicrobial against pathogenic yeasts,25
anthelmintic against parasitic worms26).
e113212.
22 M. R. Monaco, P. Renzi, D. M. Scarpino Schietroma and M.
Bella, Org. Lett., 2011, 13, 4546.
23 H. D. Boswell, B. Dräger, W. R. McLauchlan, A. Portsteffen, D.
J. Robins, R. J. Robins and N. J. Walton, Phytochemistry,
1999, 52, 871.
24 J. M. Grisar, G. P. Claxton, K. T. Stewart, R. D. MacKenzie and
T. Kariya, J. Med. Chem., 1976, 19, 1195.
25 A. Al-Shamma, S. D. Drake, L. E. Guagliardi, L. A. Mitscher and
J. K. Swayze, Phytochemistry, 1982, 21, 485.
Acknowledgements
N.J.T. acknowledges the ERC for the award of an Advanced
Grant. J.L.G. acknowledges the support of the BIOINTENSE
project, financed through the European Union 7th Framework
Programme (grant agreement no. 312148). I.S. acknowledges a
CASE award from BBSRC and Dr. Reddy’s (grant code
BB/K013076/1).
26 L. H. Yan, F. Dagorn, E. Gravel, B. Séon-Méniel and E.
Poupon, Tetrahedron, 2012, 68, 6276.
Conflicts of interest
The authors declare no conflict of interest.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C8CC06759G
Table of contents entry
A novel multi-enzymatic approach enabled the facile synthesis of a broad range of biologically active
2-substituted piperidine and pyrrolidine alkaloids.