Flow-based enzymatic synthesis of melatonin and other high value tryptamine derivatives: A five-minute intensified process

Article abstract of DOI:10.1039/c9gc01374a

To increase the uptake of biocatalytic processes by industry, it is essential to demonstrate the reliability of enzyme-based methodologies directly applied to the production of high value products. Here, a unique, efficient, and sustainable enzymatic platform for the multi-gram synthesis of melatonin, projected to generate around 1.5 billion U.S. dollars worldwide by 2021, and its analogues was developed. The system exploits the covalent immobilization of MsAcT (transferase from Mycobacterium smegmatis) onto agarose beads increasing the robustness and longevity of the immobilized biocatalyst. The fully-automated process deriving from the integration between biocatalysis and flow chemistry is designed to maximize the overall yields (58-92%) and reduce reaction times (5 min), overcoming the limitation often associated with bioprocesses and bridging the gap between lab scale and industrial production.

Full text of DOI:10.1039/c9gc01374a

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DOI: 10.1039/C9GC01374A
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Flow-based enzymatic synthesis of melatonin and other high
value tryptamine derivatives: a five-minute intensified process
Received 00th January 20xx,
Accepted 00th January 20xx
Martina Letizia Contente,a Stefano Farris,b Lucia Tamborini,*c Francesco Molinari,b and Francesca
Paradisi*a, d
DOI: 10.1039/x0xx00000x
To increase the uptake of biocatalytic processes by industry, it is first source of commercially available melatonin was the animal
essential to demonstrate the reliability of enzyme-based pineal glands.7 However, a major drawback encountered during the
methodologies directly applied to the production of high value extraction process was contamination from animal tissue. Recently,
products. Here, a unique, efficient, and sustainable enzymatic
a microbial cell factory approach for the preparation of
platform for the multi-gram synthesis of melatonin, projected to recombinant melatonin in Saccharomyces cerevisiae was reported,8
generate around 1.5 billion U.S. dollars worldwide by 2021, and its but the engineered yeast strain produced melatonin only in very
analogues was developed. The system exploits the covalent low concentration (15 mg/L), following four enzymatic steps in a 76
immobilization of MsAcT (transferase from Mycobacterium h fermentation process. Consequently, melatonin and its analogues
smegmatis) onto agarose beads increasing the robustness and are almost exclusively chemically synthesized. Industrial procedures
longevity of the immobilized biocatalyst. The fully-automated involve a final step where tryptamines are acetylated with acetic
process deriving from the integration between biocatalysis and acid anhydride or acetyl chloride in anhydrous environment,9,10 at
flow chemistry is designed to maximize the overall yields (58-92%) low temperature11 and using bases to carry out the reaction.9-11
and reduce reaction times (5 min), overcoming the limitation Although these reagents are inexpensive, their environmental
often associated with bioprocesses and bridging the gap between toxicity and hygroscopicity reduce their appeal in greener protocols;
lab scale and industrial production.
moreover, due to their high and non-specific reactivity, low
selectivity is often observed in the acetylation of amines bearing
other reacting functional groups.12 Alternatively, amide bonds can
be formed by using heterogeneous and homogeneous metal
catalysts, which are often expensive and need further purification
steps.13 A green chemistry perspective on catalytic amide bond
formation was recently assessed, pointing out that biocatalysis is
among the techniques better suited to achieve sustainable
processes for amide formation.14 Within this framework, the further
development of biocatalysis in biodegradable, unconventional
reaction media such as deep eutectic solvents (DESs) has been
reported.15,16 Some hydrolases, for example, have proven to be
effective in catalyzing amidation of esters in DESs.17 Following more
traditional methodologies, amide synthesis was achieved in DESs
with Pd/C without further purification steps.18 An acetyltransferase
from Mycobacterium smegmatis (MsAcT) was proposed as
biocatalyst for the synthesis of amides starting from primary amines
and short-chain esters in water.19,20 MsAcT was initially discovered
and fully characterized for its ability to transfer acetyl group to
alcohols.21,22 A very interesting feature of MsAcT is its remarkable
activity even at high substrate concentrations.23 De facto,
biocatalysis can be considered a reliable tool for organic and
industrial chemists only if selectivity and mild operational
conditions (typical of biotransformations) meet green and
intensified processes, characterized by high product concentration
and favourable atom economy.
Melatonin (N-acetyl-5-methoxytryptamine) is a natural mammalian
hormone that plays a crucial role in regulating the human body’s
circadian sleep-wake cycle.1 Clinically, it exhibits positive effects as
a sleep aid2,3 and a powerful antioxidant as an over-the-counter
dietary supplement.4 Recent studies on tryptamine derivative redox
activity show their involvement to re-establish the equilibrium
between pro- and antioxidants. Additionally, the fact that
melatonin readily crosses the blood-brain-barrier suggests that it
could be an adjuvant in treating oxidative stress, a key factor of
neurodegenerative disorders.5 Thanks to its antiestrogenic
properties as well as its ability to improve the efficacy and reduce
the side effects of conventional antioestrogens, melatonin can be
safely associated with breast cancer therapies currently in use.6 The
a. School of Chemistry, University of Nottingham, University Park, Nottingham, NG7
2RD, UK
b. Department of Food, Environmental and Nutritional Sciences (DeFENS), University
of Milan, via Mangiagalli 25, 20133, Milan, Italy
c. Department of Pharmaceutical Sciences (DISFARM), University of Milan, via
Mangiagalli 25, 20133, Milan, Italy
E-mail: lucia.tamborini@unimi.it
d. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
3012 Bern, Switzerland
E-mail: francesca.paradisi@dcb.unibe.ch
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Integration of biocatalytic methods and continuous flow reactors Additionally, spatial distribution of fluorophore-labelled enzyme
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(either micro- or meso-reactors) is designed to intensify the overall was investigated (see Supporting informatiDoOnI):.1M0.1s0A3c9T/Cis9GloCc0a1l3iz7e4Ad
process and to overcome low productivity, which is one of the more across the porous surface of the agarose beads favoring an intimate
common limitation of biocatalysis.24 In this work, we have contact with the substrates (Figure 2). The spatial enzyme
developed a unique, rapid, and robust enzymatic method to organization remained stable after two-week storage
prepare melatonin and analogous amides using immobilized MsAcT demonstrating that the enzyme is covalently bound to the matrix
in a flow reactor to set-up highly productive biotransformations on and unable to migrate.
a multi-gram scale.
Firstly, a highly tailored immobilization of the octameric MsAcT, key
to achieve a robust and durable catalyst, was developed. To date,
MsAct has been immobilized onto carbon nanotubes to fulfil its
perhydrolase activity,25 and siliceous monolithic microreactors have
been used for the acetylation of neopentylglycol.26 Although
continuous-flow microchannel reactors can be efficient, the
synthesis and functionalization of silica monoliths is anything but
eco-friendly (e.g., requirement of HNO3, high temperature, inert
atmosphere, long reaction times). In the present study, strategies
forming covalent bonds between the matrix and the enzyme (see
Supporting Information) were screened to achieve a durable
Figure 2. Confocal microscopy of glyoxyl agarose particles immobilizing MsAcT (1
mg/gmatrix) 20x magnification.
catalyst to be used specifically at high flow rate for scalable
Batch reactions to test the catalytic performance of agarose
immobilized MsAcT were carried out. Three amines (2-
phenylethylamine, p-ethylaniline, 3,4-dimethoxybenzylamine),
productivity.27,28 Hydrophilic carriers (i.e., agarose, cellulose, 3-
aminopropylic silica, epoxy resins) with different pore diameter (40-
60/10-20 nm) and spacer size were chosen. Hydrophobic supports
already studied using the free MsAcT, were selected.20 The reaction
have been avoided as they can create non-specific interactions
was carried out in phosphate buffer (0.1 M pH 8.0) using non-
between the enzyme and non-functionalized hydrophobic regions
activated acyl donors (ethyl formate, ethyl acetate, ethyl
of the carrier.25 Different concentration of MsAcT were tested (1, 2,
propionate) and activated esters (vinyl acetate, vinyl propionate).
and 5 mg/gmatrix) for each carrier; the highest recovered activity
The substrate concentration was fixed at 0.25 M (> 30 g/L), while
(73%), was obtained at low enzyme concentration using activated
10% v/v (~1 M) of acyl donor was used, observing maximum
glyoxyl agarose as support (see Supporting Information). This can
conversions between 75 and 100% in times ranging between 30 min
be explained taking into consideration that MsAcT is a large
and 2 h (see Supporting information). It is of note that the
molecule (72 x 72 x 60 Å) which needs tight association of dimer
immobilized system afforded much higher activity and substrate
pairs to work. The supports showing the best retained activity
tolerance than the free enzyme: in fact, the enzyme loading (0.2
(>30%) were examined before and after enzyme immobilization by
mg/mL) required for this level of reactivity was five times lower
scanning electron microscopy (SEM) to detect any surface change
than for free MsAcT, even if a 5-fold higher substrate concentration
(see Supporting information). In Figure 1, the characterization of
was used.
the agarose matrix is reported. Particles supported with
1
To demonstrate the robustness and longevity of the immobilized
biocatalyst, stability of the immobilized enzyme towards different
esters (acting as acyl donors) and temperature was investigated.
Agarose-immobilized MsAcT showed a remarkable increase of the
tolerance to the assayed esters (more than 50% of retained activity
after 24 h) and slightly higher thermal stability when incubated up
to 70 °C (see Supporting information).
mgMsAcT/gmatrix did not present any aggregation phenomena, thus
maximizing the contact area during the biotransformation. While
epoxy resins seem to have the same behavior, SEM analysis of
cellulose demonstrates that the fibers create aggregates with less
surface available for the interaction with the substrates (see
Supporting information).
Despite amide formation being dramatically favored over hydrolysis
due to the characteristic hydrophobicity of the active site, product
cleavage was observed over prolonged times; accordingly,
continuous product removal can guarantee high yield of the desired
amide. With this in mind and taking into consideration that flow
processing has also the potential to accelerate biotransformations
(due to enhanced mass transfer and better control of the reaction
parameters), a continuous flow reactor using immobilized MsAcT
was set-up and optimized for the preparation of N-(2-
Figure 1. SEM images of agarose matrix. Line a: activated glyoxyl agarose before
phenylethyl)acetamide using EtOAc as acetylating agent.
A
MsAcT immobilization 60x; 500x; 1000x magnification respectively. Line b: activated
segmented liquid-liquid phase composed of buffer/EtOAC 90:10
was flowed into the packed-bed column containing immobilized
MsAcT. Keeping constant the residence time (5 min), concentration
of the amine in the feeding flow was varied. Complete conversion
glyoxyl agarose after MsAcT immobilization (1 mg/gmatrix
) 60x; 500x; 1000x
magnification respectively.
2 | J. Name., 2012, 00, 1-3
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COMMUNICATION
was observed using an amine feed between 0.25-0.50 M (30-60 Using EtOAc as acetyl donor (10% v/v) and 0.5 M amine solutions,
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g/L). When 2-phenylethylamine concentration was increased up to melatonin analogues were prepared with vDerOyI:g1o0o.1d03i9so/Cla9tGeCd0y1i3e7l4dAs
1.0 M (120 g/L), the biphasic flow was slightly modified to ensure a (70%, 62% and 60% respectively) with 5-minute residence time
constant excess of acyl donor (85:15 aqueous phase/EtOAc), (further increasing the reaction time did not give any significantly
allowing for N-acetylation with 90% molar conversion.
better result). Activated vinylOAc was also employed for the N-
acetylation of 0.5 M tryptamines: the desired amides were rapidly
produced (5 min of residence time) with excellent conversion (95%,
96% and 89%, respectively). Downstream liquid extraction afforded
HN
NH2
Buffer
(EtOAc)
O
Amine
Solution A
imm-MsAcT
N
N
H
H
solvent
removal
Tryptamine
N-Acetyl tryptamine
1 mg/g
25 °C
Amide
HN
NH2
O
O
O
Organic phase
MsAcT, EtOAc/VinylOAc
buffer pH 8.0
EtOAc
Solution B
N
H
N
H
5-Methoxytryptamine
Melatonin
NH2
HN
O
HO
HO
Scheme 1. Flow synthesis of amides. Solution A: 0.5 M or 1.0 M amine solution in
phosphate buffer (0.1 M, pH 8.0) containing 5-10% DMSO. Solution B: EtOAc or
vinylOAc. T= 25 °C, P= atmospheric pressure.
N
H
N
H
Serotonin
N-Acetyl serotonin
Continuous product removal completely eliminated hydrolysis of
the newly formed amide, improving the overall process yield. The
process was further implemented with the addition of an in-line
extraction. The biphasic system exiting from the flow reactor was
continuously separated using a Zaiput liquid/liquid separator and
the desired amide was recovered in the organic phase, significantly
simplifying and accelerating the work-up procedure, as no further
purification was required. The designed process can be considered
an ultra-efficient, closed-loop strategy,29 as all the materials
involved in the biosynthesis can be reused. The recovered waste
water containing the unreacted amine can be easily re-circulated in
the system, while evaporation of the organic phase allows for the
recovery of ethyl acetate, used as acyl donor. Further optimization
of the reaction conditions (such as addressing the buffer
formulation and shifting towards more volatile mixtures) is ongoing.
The operational stability of the immobilized MsAcT under
continuous flow was also assayed by running the reactor over
prolonged time. Samples (4 mL) were collected and analyzed by
HPLC. The consistency of the results obtained over time was
verified in terms of N-(2-phenylethyl)acetamide produced, showing
excellent stability under the chosen working conditions. A total
volume of more than 400 mL was collected without any loss of
enzymatic activity. This allowed for an extensive utilization of the
pre-packed column with an unprecedented amide production of 56
g x die (using 1.6 mg of enzyme), thus demonstrating the
intensification of the process with excellent atom efficiency and
automation.
Figure 3. Flow production of melatonin analogues
products with high chemical purity (>95%) and 90%, 92% and 85%
isolated yields, respectively. Notably, N-acetylation of tryptamines
catalyzed by MsAcT occurred with complete chemoselectivity, since
no reaction involving the phenolic OH was observed, demonstrating
that our process can be considered a clean, mild and virtually zero-
waste procedure for amide production.
Finally, continuous flow-based preparation of melatonin was carried
out for 24 hours (Table 1), showing that multi-gram synthesis of
melatonin was achievable using less than 2 mg of enzyme in a 3.5
mL reactor: 19 or 30.2 g of melatonin were isolated after
automated work up described above.
Table 1. Continuous preparation (24 h) of melatonin (0.5 M, 95 g/L feed) in flow
reactor. Reactor volume 3.5 mL, residence time 5 min.
M. c.a
Space time yield
(g/d)
Catalyst productivity
(mmol/mg)
Acetyl donor
EtOAc
(%)
62
96
19.0
30.2
67
vinylOAc
104
aDetermined by HPLC
Conclusions
The high substrate concentrations (and relative yields), together
with the remarkable operational stability of the immobilized system
described in this work, help to overcome the perception about the
inefficiency of biocatalysis when compared with conventional
chemical strategies. The method described is remarkably intensified
and can compete with conventional (bio)processes for the
The established methodology was then applied to the synthesis of
the target pharmaceuticals; N-acetyl amines derived from
commercially available tryptamine, 5-methoxy triptamine and
serotonin (Figure 3). Melatonin analogues, as well as playing an
important biological role as agonist of melatonin receptors, are
promising neuroprotectant drugs for the treatment of
neurodegenerative disorders30,31
.
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Conflicts of interest
There are no conflicts to declare.
Notes and references
This project was supported by the European Union’s Horizon 2020
research and innovation programme under the Marie Skłodowska-
Curie grant agreement N. 792804 AROMAs-FLOW (M.L.C.). The
authors wish to thank Resindion S.R.L. for donating the EP403/S,
HFA403/S, EC-HFA/S, EC-EP/S resins and UNITECH NO LIMITS for
the SEM and confocal microscopy experiments.
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