The sustainable synthesis of levetiracetam by an enzymatic dynamic kinetic resolution and an: Ex-cell anodic oxidation

Article abstract of DOI:10.1039/d0gc03358h

Levetiracetam is an active pharmaceutical ingredient widely used to treat epilepsy. We describe a new synthesis of levetiracetam by a dynamic kinetic resolution and a ruthenium-catalysed ex-cell anodic oxidation. For the enzymatic resolution, we tailored a high throughput screening method to identify Comamonas testosteroni nitrile hydratase variants with high (S)-selectivity and activity. Racemic nitrile was applied in a fed-batch reaction and was hydrated to (S)-(pyrrolidine-1-yl)butaneamide. For the subsequent oxidation to levetiracetam, we developed a ligand-free ruthenium-catalysed method at a low catalyst loading. The oxidant was electrochemically generated in 86% yield. This route provides a significantly more sustainable access to levetiracetam than existing routes. This journal is

Full text of DOI:10.1039/d0gc03358h

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The sustainable synthesis of levetiracetam by an
enzymatic dynamic kinetic resolution and an
ex-cell anodic oxidation†
Cite this: DOI: 10.1039/d0gc03358h
Sebastian Arndt,‡a Birgit Grill,‡b Helmut Schwab,b,c Georg Steinkellner,b,d
b
Urška Pogorevčnik,b Dominik Weis,a Alexander M. Nauth,a Karl Gruber,
Till Opatz, a Kai Donsbach,e Siegfried R. Waldvogel *a and Margit Winkler
*
b,c
Levetiracetam is an active pharmaceutical ingredient widely used to treat epilepsy. We describe a new
synthesis of levetiracetam by a dynamic kinetic resolution and a ruthenium-catalysed ex-cell anodic oxi-
dation. For the enzymatic resolution, we tailored a high throughput screening method to identify
Comamonas testosteroni nitrile hydratase variants with high (S)-selectivity and activity. Racemic nitrile was
applied in a fed-batch reaction and was hydrated to (S)-(pyrrolidine-1-yl)butaneamide. For the sub-
sequent oxidation to levetiracetam, we developed a ligand-free ruthenium-catalysed method at a low
catalyst loading. The oxidant was electrochemically generated in 86% yield. This route provides a signifi-
cantly more sustainable access to levetiracetam than existing routes.
Received 5th October 2020,
Accepted 3rd December 2020
DOI: 10.1039/d0gc03358h
rsc.li/greenchem
(EF) (calculations in ESI chapter 2†).8 Technical approaches
towards (S)-1 started with the synthesis of 2-aminobutanamide
Introduction
Levetiracetam (S)-1 is an active pharmaceutical ingredient through a Strecker reaction, which is then followed by a chiral
(API) used for treatment and prevention of hypoxic and resolution,9 and by alkylation/acylation of the amino group
ischemic type aggressions of the central nervous system.1 The (Scheme 1, I).10 Although the Strecker reaction was highly
formulated form is known as Keppra®. Levetiracetam is atom-efficient, the resolution and the alkylation were wasteful
applied as a medication for epilepsy6 with a global sale of
770 M€ in 2018.2 Numerous strategies for the synthesis of (S)-1
have been explored as summarised by Kotkar and Sudalai,
including asymmetric hydrogenations, kinetic resolutions,
deracemisation with chiral auxiliaries and proline-catalysed
asymmetric
α-aminooxylation
of
n-butyraldehyde.3
Intramolecular cyclisation and elimination,4 and an asym-
metric Strecker reaction were also reported.5 Recently, (S)-1
was approached by Co(I)-catalysed single electron reduction of
the respective enamide.6 The most efficient approaches to leve-
tiracetam are analysed in view of their sustainability, which
may be expressed by the atom economy (AE)7 and the E-factor
aDepartment of Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-
14, 55128 Mainz, Germany. E-mail: waldvogel@uni-mainz.de
bacib – Austrian Centre of Industrial Biotechnology, Krenngasse 37, A-8010 Graz,
Austria
cInstitute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14,
8010 Graz, Austria. E-mail: margitwinkler@acib.at
dInnophore GmbH, Am Eisernen Tor 3, 8010 Graz, Austria
ePharmaZell GmbH, Hochstrass-Süd 7, 83064 Raubling, Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0gc03358h
Scheme 1 Technical and auxiliary-based approaches to levetiracetam
‡Contributed equally.
(selected key-intermediates). X = Cl, Br.
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Table 1 Calculated atom efficiencies and E-factors (for details see ESI,
chapter 2†)
Synthetic route via
AE [%]
EF [kg kg−1
]
Overall yield [%]
Strecker synthesis
α-Halogenation
Evans’ auxiliary
Sulfinimine auxiliary
Ugi reaction
Ru catalysis
TM hydrogenation
Our approach
12
5
5
0.8
38
3
39
44
52
95
21
22
37
18
5
25
18
44
465
572
1544
5110
151
21
Comparability might be limited by scale- and optimisation effects, and
by the choice of the starting material. TM = transition metal, AE =
atom efficiency, EF = E-factor.
and low-yielding. Thus, this route afforded 21% overall yield
(Table 1). Another technical approach used 2-halobutanoic
acid, which was accessed by α-halogenation. Subsequent
nucleophilic substitution, a chiral resolution, and finally an
amidation yielded the product (Scheme 1, II).11 The larger step
count, harsher reaction conditions and more extensive use of
chemicals rendered this route less efficient. Overall, 22% of
(S)-1 was obtained at an AE of 5%.
Academic strategies relied upon the use of chiral auxiliaries,
such as oxazolidinones (Scheme 1, III) or sulfinimines.12
Those routes gave indeed higher yields, but the removal of the
auxiliary and the large number of synthetic steps again
resulted in a low overall efficiency. For this reason, those
methods are economically prohibitive.
A particularly short method employed the Ugi three-com-
ponent reaction that, given by the low number of synthetic
steps, seemed very efficient at first sight (Scheme 1, IV).13
Revising the resolution,14 however, a very poor overall yield of
Scheme 2 (a–d) Transition metal-catalysed or electrochemical
approaches to levetiracetam (selected intermediates). Ox. = oxidation,
sel. = selective, ee = enantiomeric excess.
5% was obtained accompanied by an uneconomic use of a TEMPO-related mediator (Scheme 2a, step II).22 A patent
chemicals. Moreover, toxic 2,2,2-trifluoroethanol was used as elaborates on an enzymatic approach to levetiracetam and
the solvent. The syntheses of the auxiliaries themselves were involved an (S)-selective nitrile hydratase (NHase) in a classical
not taken into account, which would have impaired the enzymatic resolution of (rac)-6, leading to a maximum theore-
metrics further.15 Catalytic consecutive double-bond migration tical yield of 50% after a low yielding two-step chemical syn-
and oxidative cleavage of (S)-2 under ruthenium catalysis was thesis of the oxo-nitrile precursor (Scheme 2c).23
reported (Scheme 2a), wherein levetiracetam was obtained in
NHases exhibit a broad substrate scope for catalytic enzy-
25% over six steps.16 The extensive use of chemicals resulted matic transformations of nitriles to the corresponding
in by far the lowest sustainability of this route within our com- amides.24 The perfectly atom economic reaction proceeds in
parison. Furthermore, an unacceptable co-solvent according to water – which is both the reagent and solvent, under ambient
pharmaceutical guidelines was used (CCl4).17 Bandichhor sep- conditions. Nitrile hydratases are applied on industrial scale
arately reported the oxidation of (S)-3 and obtained levetirace- e.g. for the production of the bulk chemical acrylamide.25
tam in 42% after amidation of (S)-4.18 Rhodium or cobalt-cata- Likewise, electrolyses are highly atom-efficient, inherently safe,
lysed approaches were reported by Shevlin and Chirik for the and environmentally benign as they do not require primary
enantioselective hydrogenation of dehydro-levetiracetam oxidants. Electricity is inexpensive, readily available, and sus-
(5).6,19–21 Reviewing this pathway,20,21 levetiracetam was tainable if produced from renewable energy sources.26
obtained in 18% yield, with 39% AE, and with an EF of 151, Combining these two technologies offers new avenues for sus-
which still is poor for a technical scale (Scheme 2b). It should tainable production processes.
be pointed out that N-heterocyclic carbene or chiral phosphine
In conclusion, technical routes to levetiracetam are more
ligands are expensive. Their molar price may be higher than sustainable than ‘academic’ routes, but still suffer from low
that of the noble transition metal itself. An alternative electro- overall yields. Yet half of the material is lost in a resolution if a
chemical oxidation of (S)-3 was developed by Stahl et al. using recycling of the wrong enantiomer is impossible. Chiral auxili-
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ary-based routes indeed circumvent the resolution, but these tor before.32 Cyanide indeed inhibited CtNHase (ESI Fig. S4†)
methods are accompanied by a large step-count. Unfavourably, whereas pyrrolidine and propanal did not (ESI Fig. S5†). In
the synthesis and the removal of the auxiliary are extremely successive optimisation of reaction conditions including
uneconomic. Catalytic routes ought to feature higher atom increased substrate concentrations, we aimed for minimal
efficiencies and a better waste management in theory, but the cyanide concentrations by pH control and concomitantly
elaborate syntheses of starting materials, the utilisation of adapted buffer capacities in analytical scale reactions. In doing
chiral auxiliaries, and the employment of expensive ligands so, product levels could be increased to more than 40% at
eliminate those advantages. Furthermore, they suffer from 50 mM substrate load, and 15% at 200 mM substrate concen-
high overall costs that impede the application on a technical tration while product ee ranged from 70–90% (ESI Fig. S6†).
scale.
Time resolved monitoring of 7 hydration showed that the reac-
The aim of this work was to find both a sustainable and tion was very fast, but ceased within a short period of time
economical route for the synthesis of levetiracetam. From a (ESI, Fig. S7†). The reason was not fast deactivation of
retro-synthetic point of view, we envisioned a Strecker reaction CtNHase,30,31 but the formation of a side product: propanal
with the benefit of low-priced and readily available starting and cyanide form 2-hydroxybutanenitrile – a competitive sub-
materials. As opposed to previous routes, the pyrrolidine strate for CtNHase which gives 2-hydroxybutanamide upon
moiety shall be directly installed saving one synthetic step. hydration, as confirmed by HPLC-MS (ESI Fig. S8†). Formation
The Strecker reaction is telescoped by an enzymatic dynamic of (S)-8, enantioselectivity and the substrate selectivity needed
kinetic resolution of 2-(pyrrolidine-1-yl)butanenitrile (rac)-7 to to be increased to create a potent biocatalyst suited for indus-
the corresponding amide (S)-8. Finally, we envisioned an trial application. We therefore focused on CtNHase on the
electrochemical stereo-conservative and regiospecific oxidation molecular level, docked (S)-7 into the active site of a model
of (S)-8 in position α to the amine (Scheme 2d).
(ESI Fig. S11†) and identified amino acid residues within 4 Å
around the docked compound as potential targets to shape the
active site for improved (S)-7 binding. Putative Cobalt-binding
residues, strictly conserved residues in the protein family and
Arg52 in the β-subunit (a residue likely involved in proton
Results and discussion
A highly (S)-selective NHase27 should convert (S)-7 to (S)-8 and transfer33) were excluded.
we hypothesised that the undesired (R)-enantiomer would dis-
We created site saturation libraries for ten positions: Q93,
integrate in aqueous solution and subsequently form (rac)-7 W120, P126, K131 and R169 of the α-subunit and M34, F37,
again – the prerequisite for a dynamic kinetic resolution (DKR) L48, F51 and Y68 of the β-subunit. To screen for improved var-
to theoretically yield 100% of (S)-8 (Scheme 1d). A DKR similar iants,
a colorimetric assay was established. Specifically,
to that described for (R)-mandelamide28 or 2-aminophenylace- amides can be transacylated by amidases in the presence of
tamide29 is not feasible with the racemic oxo-nitrile (rac)-6, hydroxylamine to give hydroxamic acids, which can then be
since conditions for its racemisation are incompatible with the visualised as iron-complex (Scheme 3).34 Using an (S)-selective
enzymatic nitrile hydration step.
amidase in the screening assay provides additional selection
Screening a panel of known and new NHases,30 we identi- for (S)-8 and strictly (S)-selective Rhodococcus erythropolis
fied the thermotolerant cobalt-dependent Comamonas testoster- amidase35 (ReAmd) was chosen for this step. The 10 libraries
oni NHase (CtNHase) as the most promising enzyme candidate were screened in 96 deep-well plates. As an example, the
(ESI Table S1†).31 It showed activity from pH 6.0 to 9.5 (ESI results for position β-L48 are shown in the ESI (Fig. S12†).
Fig. S2†), hydrolysed 7 in the temperature range between 25
Cell suspensions of the most promising variants from this
and 50 °C (ESI Fig. S3†), and showed the highest (S)-selectivity screening were used for biotransformation reactions (ESI
among the candidates with 89% ee. At 10 mM (rac)-7 substrate chapter 5.1 and 5.2†) to simultaneously determine concen-
concentration the product was detected in 1.1 mM concen- tration of 8 and the corresponding ee by chiral HPLC. In this
tration (ESI Table S1†). This small amount of detected product re-screening step, false positives generated by potentially
indicated a strong need for a deeper understanding of the formed 2-hydroxybutaneamide were unambiguously elimi-
limitations of the reaction system.
nated. Whereas variants in the α-subunit showed little effect,
We investigated potential product inhibition and found several mutants from the β-subunit gave increased formation
that up to 25 mM, no inhibition by (rac)-8 was obvious. of 8 and/or ee. Most promising hits were analysed by sequen-
Aminonitrile 7 is in equilibrium with its three components
propanal, pyrrolidine and hydrocyanic acid in aqueous solu-
tion, which is the key requirement for a dynamic kinetic
resolution. The equilibrium is pH dependent, with high pH
values favouring the formation of 7. On the one hand, the
amounts of pyrrolidine, propanal and cyanide in the reaction
solution depend on the pH, on the other hand, they also influ-
ence the pH. Each of these components may inhibit CtNHase,
and especially cyanide has been reported as an NHase inhibi- Scheme 3 Coupled assay to screen for nitrile hydratase activity.
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Table 2 Selected re-screening results
cing (for a selected example see Table S3†). Hydrophobic
amino acids instead of Phe in position 51 increased product
levels up to twofold with ee of up to 92% at 50 mM of (rac)-7.
Random engineering by error prone PCR was used to comp-
lement the rational protein engineering approach.36 To
increase the throughput, the above described assay was estab-
lished on colony level (ESI chapter 8.2†). Colonies are attached
to a membrane and separated from their growth medium.
First, the membranes are treated with substrate solution. In
the second phase they are transferred to a solution of amidase
and hydroxylammonium chloride. Finally, the red complex is
developed by transfer of the membrane attached colonies to
acidic FeSO4 solution. Instead of the full length sequence, four
short stretches in CtNHase lining the active site were defined
to decrease the library size and therefore reduce the number of
Anal. yielda
[%]
ee(S)
[%]
Entry
CtNHase
Library
1
2
3
4
5
6
7
8
Wild-typeb
—
24
26
62
30
50
24
45
57
65
50
9
85
96
90
91
91
92
90
93
90
95
98
94
98
99
βL48R
Random β1
Random β1
Random β1
Random β1
Random β1
Random β1
Random α1c
Random α2c
Random β2c
β1-Focused
β1-Focused
Combined
Combined
βF51I
βF51L
βF51V/ βE70L
βH53L/ βG54V
βN43I/ βG54C
αV110I/ βF51L
αP121T/ βF51L
βF51L/ βH146L/ βF167Y
βL48R/ βF51I/ βG54I
βF51V/ βG54V
βP121T/ βL48Rb
βL48R/ βG54Vb
9
10
11
12
13
14
66
42
31
variants that must be screened. Random libraries were con- a (rac)-7 (100 mM), resting cells (8.5 mg mL−1), Tris-HCl buffer
(500 mM pH 7.0), 25 °C, 700 rpm, 2 h. b Cells cultivated in shake
structed for each stretch (coloured elements in Fig. 1).
Specifically, the regions were amino acid 70–110 (α1) and
flasks. c Libraries based on mutant βF51L.
120–175 (α2) in the α-, and 30–71 (β1) and 124–170 (β2) in the
β-subunit, respectively. The β1 library was based on wild-type
CtNHase, whereas the other three libraries were generated on lower than that of the parent βF51L (entry 9). The hits of
β1 variant βF51L. At least 11 000 clones per library were library β2 contained mutations in numerous positions,
screened and 900–1700 variants per library were used for re- however, only one combination with improved ee was found
screening in the liquid format as described for the site-satur- (entry 10). Associating most impact on CtNHase stretch β1, we
ation libraries. Hit positions were evaluated on the 500 µL screened a focused library that aimed for combinations of the
scale to determine concentration of 8 and ee by chiral HPLC. three critical positions 48, 51 and 54, respectively (ESI chapter
Library β1 revealed a high number of improved clones, 9.3†). Substitutions in pos. 48 lead to ees up to 99%, however,
whereby position F51 was found in 16 of 26 sequenced clones, product formation were strongly compromised (Table 2, entry
with substitutions to either Ile, Leu or Val (Table 2, entries 11). Around 60% of amide 8 were obtained (based on rac-7
3–5) alone or in combination with other amino acid exchanges addition) with βF51V variants combined with Ile, Arg or Val in
or silent mutations. Also, substitution of βG54 was found 9 pos. 54 (entry 12). Finally, combining the most promising
times (entries 6 and 7). The highest ee was found for variant mutations from all libraries revealed 10 variants with ees
βL48R (96.1%). In contrast, the α1 library consisted of many >98.5%. Most of them combined mutations of L48 and G54
parents and only pos 110 carried an Ile in 7 of 16 clones (entry (entry 14). Mutations in position 51 alone lead to both, an
8). This position significantly increased product levels as com- increase in selectivity and activity, but in combination with
pared to parent βF51L and slightly the product ee. Similarly, other mutations, activity decreased below wild-type level or ees
library α2 revealed a substitution of P121 by Ser, Val or Thr did not reach beyond 94% (data not shown). The best variants
beneficial for the formation of 8, whereas ees were typically and relevant control strains were compared in reactions which
were supplemented with 1 equivalent of propanal (Fig. 2) to
push the equilibrium towards (rac)-7 formation and compensate
for the volatility of propanal. All variants displayed ee >98%. The
double mutant αP121T/ βL48R showed two-fold increased
product formation in comparison to the wild-type. Three double
mutants based on the βL48P exchange reached ee 99.8% or
higher. Clearly, position 48 in the β-subunit was responsible for
the most significant increase in enantioselectivity, potentially
due to improved contact of the amino acid side chain to (S)-7.
On the preparative scale, (rac)-7 was hydrated to (S)-8 in pH
controlled stirred 50 mL volume fed-batch reactions, using
whole cell biocatalysts (ESI chapter 10†). Variant P121T/L48R
showed the highest concentration of the desired product on
analytical scale and hydrated (rac)-7 (1.3 g substrate in total) to
(S)-8 (1.1 g, 95% ee) on a 50 mL scale. The more selective
variant L48R/G54V gave (S)-8 in 98% ee (0.78 g at 1.21 g sub-
Fig. 1 Homology model of CtNHase. The four regions targeted by
strate load). The yields of (S)-8 were defined by the degree of
random mutagenesis are shown in colour. The cobalt ion is displayed as
pink sphere, region α1 in blue, α2 in green, β1 in orange and β2 in red.
cyanohydrin hydration (Scheme 4) and reached 73.3% analyti-
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CCl4/MeCN/H2O, a low to neutral pH, and low temperatures.
Periodate is often used as terminal oxidant.43 The catalysis was
performed by dissolving the hydrated ruthenium(IV) oxide
(RuO2) and the desired oxidant. The mixture was suspended
until the ruthenium tetroxide (RuO4) was formed as indicated
by the appearance of a pale-yellow colour. Subsequently, (S)-8
was added as a solid and the reaction progress was monitored
by GC. After complete reaction, the yield was determined
either by GC using caffeine as an internal standard or the
product was isolated by flash column chromatography. Best
results were obtained for hydrated RuCl3 or RuO2 pre-catalysts
and sodium metaperiodate. The catalytic reactions were per-
formed in mixtures of aqueous and organic solvent such as di-
methylformamide, acetonitrile, or acetone (Table 3, entries
2–4). Ethers, benzene, alcohols and tetrachloromethane were
not investigated for safety- and regulatory reasons.44 A neutral
pH was critical, and an excess of periodate (≥2.6 eq.) as well as
a low temperature (0 °C, Table 3, entries 5 and 6) were
beneficial. The product (S)-1 was formed in 66% yield and it
was isolated in 49% yield and 99.6% ee. A substantial loss of
material was observed in the gas chromatograms and in the
mass balance after flash column chromatography. To rule out
misleading GC data, the intermediate 9 and the potential over-
oxidation product (S)-2-(2,5-dioxopyrrolidin-1-yl)butanamide
were isolated or synthesised, respectively, but the clear appear-
ance of their GC signals indicated a sufficient GC-stability and
response factor. Accordingly, ring opening- and/or polymeris-
ation pathways of the unstable hemi-aminal 9 intermediate
were hypothesised.45 To prevent those side reactions, a bipha-
sic solvent system with ethyl acetate/water was investigated
similar to the protocols of Hamed et al. and Sashida et al.46 Up
to 76% of the product was obtained even at room temperature
and at a low amount of organic solvent (Table 3, entry 7),
Fig. 2 Enantioselective hydration of (rac)-7 (100 μM) by CtNHase var-
iants. Reactions were performed in triplicates in Tris-HCl buffer (500 μM)
at pH 7.0 in the presence of propanal (100 μM) at 25 °C and 700 rpm
for 2 h.
Scheme 4 CtNHase mediated dynamic kinetic resolution.
cal yield (ESI Fig. S14 and 15†) in case of variant P121T/L48R
and 61.7% in case of variant L48R/G54V, respectively.
Table 3 Ru-Catalysed oxidation (selected results, for more details see
ESI chapter 18†)
For the final step towards levetiracetam, a direct electro-
chemical oxidation was envisaged first. Pyrrolidines have been
electrolysed in Shono-type oxidations,37 whereby pyrrolidones
may be obtained in water as reaction medium.38 Stereo-conser-
vative Shono oxidations have been reported for sulphon-
amides.39 Cyclic voltammetry indicated an irreversible oxidation
of (S)-8 in the desired α-position of the amine. Up to 29% of 1
was determined by GC in optimised batch-electrolyses (detailed
in experimental section in ESI, chapter 17†).40 However, chiral
HPLC revealed a racemisation of the product during electrolysis.
We reasoned that an N-radical intermediate must be formed
during the electrolysis causing the racemisation. This prompted
us to pursue an ex-cell electrochemical oxidation.
The ex-cell approach was investigated utilising ruthenium
catalysts. Oxo ruthenium-catalysed α-oxidation of N-protected
pyrrolidines generally feature mild reaction conditions, short
reaction times and high selectivity.41 More importantly, RuO4
is known to perform two consecutive concerted oxidation steps
that should result in the retention of the absolute configur-
ation.42 Reported favourable conditions are solvent mixtures of
GC yield [%]
Entry
Solvent
[v%]
T [°C]
8
9 a
1
∑
1
2
3
4
5
6
7
8
H2O
DMF
—
33
33
33
50
70
33
33
20
20
20
20
0
0
20
20
5
16
1
1
1
0
—
—
9
18
4
4
2
0
—
—
31
37
57
60
65
66
76
74
45
72
62
65
68
66
Isol.
Isol.
MeCN
Acetone
MeCN
MeCN
EtOAc
EtOAc
a Relative intensity vs. standard, eq. = equivalents, T = temperature,
DMF = dimethylformamide, isol. = isolated yield. Screenings were per-
formed on a 100 mg scale of (S)-8.
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which is favourable from a technical point of view. We (up to 71% isolated yield).52 The electrochemically obtained
reasoned that the RuO4 is transferred into the organic layer periodate was tested in the Ru-catalysed oxidation to give
where, due to the low water content, the intermediate might reproducible results. Finally, the iodate electrolysis was scaled
be protected from side reactions. The more polar product in up in flow electrolysis.53 The electrolysis conditions were
turn is transferred to the aqueous phase where it might be pro- adjusted to an increased current density of j = 100 mA cm−2
tected from over-oxidation. The reaction was scaled up 10-fold (Table 4, entries 4–7) and an applied charge of Q = 4 F
obtaining a reproducible yield of 74% [Table 3, entry 8, 1.0 g (Table 4, entries 8–10). The product was obtained in 78% yield,
(S)-8]. The RuO2 was efficiently recovered by filtration over alu- which corresponds to 48 g of paraperiodate.
minium oxide and the iodate was recovered quantitatively by
crystallisation with methanol (up to 96% isolated yield).
Next, an electrochemical recycling method was developed
for the re-oxidation of the iodate generated in the ruthenium
Conclusions
catalysis. High-grade periodate is expensive and generates sub- In conclusion, we have developed a sustainable synthetic
stantial amounts of waste. Therefore, the recycling decreases pathway to levetiracetam that outcompetes the known synthesis
the E-factor and the costs of the process. The direct oxidation routes in terms of atom efficiency, E-factor and overall yield. In
of common iodide was recently published by our group.47 the first part, a screening method was designed to enable the
Based on our previous research, the iodate was oxidised in screening of tens of thousands of clones for highly (S)-selective
caustic soda. Water is generally considered as an abundant, NHase variants on colony level and allows us to report the first
environmentally benign and non-hazardous solvent.48
A
high throughput engineering study of a NHase. Time consum-
divided electrolysis cell was used equipped with a Nafion ing chiral HPLC analytics only had to be applied for re-screen-
membrane, a stainless steel cathode, and a boron-doped ing purposes of the best variants from rational and random
diamond (BDD) anode.40 Notably, BDD anodes are innovative protein engineering libraries. The selectivity of CtNHase for (S)-
and high performance metal-free electrodes, which are con- 1 versus 2-hydroxybutanenitrile was improved significantly and
sidered to be sustainable since they are made from methane.49 excellent enantiomeric excess of the desired product was
In anodic electro-organic synthesis they proved to be superior achieved with several CtNHase double mutants. In the second
and technically viable as compared to other electrode part, a ruthenium-catalysed ex-cell oxidation was developed that
materials.50 The conditions were optimised by a statistical provides an economical and sustainable access to levetiracetam.
screening approach,51 whereby a robust process was found. The catalysis was performed without the addition of ligands, by
Optimum conditions were found for an applied charge of Q = using a low catalyst load of 0.5 mol%, and by using water as
3 F, a current density of j = 10 mA cm−2, and a hydroxide/ “green” main solvent. Levetiracetam was obtained in up to 76%
iodate concentration of 1 M/0.21 M (Table 4, entries 1–3). yield with full stereo-retention. In addition, the primary oxidant
Paraperiodate (H2IO63−) was obtained in 86% yield and was was electrochemically generated in up to 86%. The recycling
converted to metaperiodate (IO4−) by acidic recrystallisation process was investigated, which drastically eliminated waste and
which formally substituted the primary oxidant periodate by
environmentally benign water. BDD anodes feature a superior
stability compared to commonly used anode materials for this
kind of oxidation. Thus, toxic metal impurities in the terminal
oxidant are prevented allowing the use for the synthesis of regu-
lated products. Furthermore, the method might provide syn-
thetic access to other valuable racetams, such as piracetam, bri-
varacetam, oxiracetam, or nefiracetam.
Table 4 Electrochemical oxidation of iodate and scale-up (selected
results, for more details see ESI, chapter 19†)
LC-PDA yield [%]
V
[mL]
Q
[F]
j
C(NaOH)
[M]
Author contributions
Entry
[mA cm−2
]
IO3
IO4
∑
−
−
S.A. Conceptualization, Investigation, Formal analysis,
Visualization, Writing – original draft, B.G. Investigation, Formal
1
2
3
4
5
6
7
8
9
10
6
6
6
50
50
50
50
500
1000
1000
3
3
3
3
3
3
3
3
3
4
2
10
10
10
3
1
1
1
1
1
1
1
1
1
20
16
11
20
26
34
63
29
37
18
83
83
86
85
73
63
37
70
72
78
103
99
97
105
99
97
100
99
109
96
analysis, Visualization, Writing
– review & editing; H.S.
Conceptualization, Funding acquisition, Writing – review &
editing; G.S. Investigation, Visualization, Writing – review &
editing, U.P. Investigation, D.W. Investigation, A. M. N.
Investigation, Conceptualization, Writing – review & editing, K.G.
Supervision, Writing – review & editing, T.O. Conceptualization,
Funding acquisition, Supervision, Writing – review & editing,
Project administration, K.D. Conceptualization, Resources,
Validation, Writing – review & editing; S.R.W. Conceptualization,
50
100
300
100
100
100
BBD
=
boron-doped diamond, VA
=
stainless steel, rt
=
room
temperature. Reactions were performed with an initial concentration
of iodate of 0.21 M.
Green Chem.
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Paper
Funding acquisition, Project administration, Supervision, Writing
– review & editing. M.W. Conceptualization, Formal analysis, Data
Curation, Funding acquisition, Project administration,
Supervision, Writing – original draft.
2013, 1–6; (b) K. Chandra Babu, R. Buchi Reddy,
K. Mukkanti, K. Suresh, G. Madhusudhan and S. Nigam,
J. Chem., 2013, 2013, 1–5; (c) F. Boschi, P. Camps,
M. Comes-Franchini, D. Muñoz-Torrero, A. Ricci and
L. Sánchez, Tetrahedron: Asymmetry, 2005, 16, 3739–3745;
(d) K. Chandra Babu, R. Buchi Reddy, K. Mukkanti,
G. Madhusudhan and P. Srinivasulu, J. Chem. Pharm. Res.,
2012, 4, 4988–4994.
Conflicts of interest
There are no conflicts to declare.
13 R. Cioc, L. van Schaepkens Riempst, P. Schuckman,
E. Ruijter and R. Orru, Synthesis, 2017, 1664–
1674.
14 M. Forcato, F. Massaccesi, L. Cotarca, I. Michieletto and
P. Maragni, WO2008/12268, 2008.
Acknowledgements
The skilful technical assistance of Karin Reicher and Gernot
A. Strohmeier is gratefully acknowledged. The COMET center acib:
Next Generation Bioproduction is funded by BMVIT, BMDW, SFG,
Standortagentur Tirol, Government of Lower Austria und Vienna
Business Agency in the framework of COMET – Competence
Centers for Excellent Technologies. The COMET-Funding Program
is managed by the Austrian Research Promotion Agency FFG. UP
received funding from the Erasmus+ Program. SRW was funded
by the Deutsche Forschungsgemeinschaft (DFG, German Research
Foundation Wa1276/27-1 and in frame of FOR 2982 – UNODE
Wa1276/23-1).
15 R. A. Sheldon, J. R. Soc., Interface, 2016, 13, 20160087.
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139, 13944–13949.
17 (a) European Medicines Agency, ICH guideline Q3D (R1)
on elemental impurities, can be found under, https://www.
ich.org/page/quality-guidelines;
(b) D. R. Abernethy,
A. J. Destefano, T. L. Cecil, K. Zaidi and R. L. Williams,
Pharm. Res., 2010, 27, 750–755.
18 R. Mylavarapu, R. V. Anand, G. C. M. Kondaiah,
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