Asymmetric Synthesis of 1-Phenylethylamine from Styrene via Combined Wacker Oxidation and Enzymatic Reductive Amination

Article abstract of DOI:10.1021/acs.joc.8b01247

An enantioselective chemoenzymatic two-step one-pot transformation of styrene to 1-phenylethylamine has been developed based on combining an initial Pd/Cu-catalyzed Wacker oxidation of styrene with a subsequent reductive amination of the in situ formed acetophenone. As a nitrogen source only ammonia is needed. The incompatible catalysts were separated by means of a polydimethylsiloxane membrane, thus leading to quantitative conversion and an excellent enantiomeric excess of the corresponding amine. The overall one-pot process formally corresponds to an asymmetric hydroamination of styrene with ammonia.

Full text of DOI:10.1021/acs.joc.8b01247

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Note
Asymmetric Synthesis of 1-Phenylethylamine from Styrene via
Combined Wacker-Oxidation and Enzymatic Reductive Amination
Florian Uthoff, and Harald Gröger
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01247 • Publication Date (Web): 19 Jul 2018
Downloaded from http://pubs.acs.org on July 19, 2018
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The Journal of Organic Chemistry
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Asymmetric Synthesis of 1-Phenylethylamine from Styrene via Com-
bined Wacker-Oxidation and Enzymatic Reductive Amination
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Florian Uthoff, Harald Gröger*
Chair of Organic Chemistry I, Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
KEYWORDS: Amine dehydrogenase, chemoenzymatic synthesis, enzyme catalysis, one-pot synthesis, Wacker-oxidation.
ABSTRACT: An enantioselective chemoenzymatic two-step one-pot transformation of styrene to 1-phenylethylamine has been de-
veloped based on combining an initial Pd/Cu-catalyzed Wacker-oxidation of styrene with a subsequent reductive amination of the in
situ-formed acetophenone. As a nitrogen source only ammonia is needed. The incompatible catalysts were separated by means of a
polydimethylsiloxane membrane, thus leading to quantitative conversion and excellent enantiomeric excess of the corresponding
amine. The overall one-pot process formally corresponds to an asymmetric hydroamination of styrene with ammonia.
The synthesis of enantiomerically pure amines via novel retro-
synthetic approaches is of high interest since, e.g., such amines
are widely used in the fine chemical, agrochemical and pharma-
idation as the initial step, the formed ketones are in situ con-
verted enantioselectively into the corresponding amine via re-
ductive amination in the presence of an amine dehydrogenase.
This reductive amination step requires ammonia as a nitrogen
source and the reduced form of a cofactor, NADH, which gives
1
ceutical industry. Recently we developed an enantioselective
chemoenzymatic two-step one-pot approach to 1-phenylethyl-
amines starting from styrenes, thus formally corresponding to
an asymmetric hydroamination reaction of alkenes with ammo-
+
its oxidized form, NAD , in this reaction. As such cofactors are
very expensive and high-molecular weight molecules, their use
as reducing agent in stoichiometric amount is not economical.
Thus, in order to use the cofactor in catalytic amount the cofac-
tor NADH is recycled in situ by means of, e.g., a glucose dehy-
drogenase, which catalyzes oxidation of D-glucose to D-gluco-
nolactone and after spontaneous ring-opening to D-gluconic
acid. As D-gluconic acid is neutralized with a base, the whole
reaction is shifted towards the product side in an irreversible
fashion, thus enabling a quantitative formation of the desired
amine product.
2
nia (Scheme 1, Part A). In detail, this process is based on com-
3
bining a Pd/Cu-catalyzed Wacker-oxidation of styrenes and a
subsequent enzymatic transamination of the ketones formed as
intermediates. Due to deactivation of the transaminase (TA) by
the Cu-component, the catalyst components have been embed-
ded in different compartments within one reactor by means of
PDMS-membranes according to a concept, which was devel-
4
oped by the Bowden group for combining non-compatible
chemical reactions and introduced by our group to the field of
5
chemoenzymatic synthesis. Although high conversion and en-
antioselectivities were obtained in this one-pot amine synthe-
sis, limitations of this process are the high enzyme loading for
Scheme 1. Concepts for asymmetric chemoenzymatic one-
pot approaches towards amines starting from alkenes
2
the transaminase as well as the use of the two organic reagents
L-alanine and D-glucose in stoichiometric amounts in combina-
tion with the need for two further enzymes (lactate dehydrogen-
ase (LDH), glucose dehydrogenase (GDH)) in order to shift the
equilibrium towards the product side. Thus, simplification of
the one-pot synthesis by minimizing the number of required en-
zymes as well as by replacement of L-alanine by ammonia as a
more preferred nitrogen donor were considered as major tasks
for process improvement. Addressing these challenges, in the
following we report our achievements on realizing such a more
simplified process concept being capable to convert styrene
with ammonia into 1-phenylethyl-1-amineꢀ(R)-3 in a more effi-
cient and practical one-pot fashion while keeping conversion as
well as enantioselectivity in an excellent range. Toward this
end, we replaced the transaminase by an amine dehydrogenase
6
(
AmDH) as a biocatalyst, which enables us to use ammonia
instead of L-alanine and to avoid the use of a lactate dehydro-
genase as a further enzyme component. The overall concept is
shown in Scheme 1, Part B. Accordingly, after the Wacker-ox-
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The initial step of our work consisted in identifying a suitable
Page 2 of 6
pass the membrane whereas water-soluble components are re-
tained by this membrane, the metal salts as well as the enzymes
remain in their aqueous compartment. The concept for this che-
moenzymatic one-pot process with compartmentalization of the
catalysts by means of a PDMS-thimble is shown in Scheme 3.
As mentioned above, in the field of chemoenzymatic synthesis
this concept has been applied successfully by our group for the
combined use of metal-type Wacker-catalysts with an alcohol
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amine dehydrogenase for being coupled with the Wacker-cata-
lysts, thus enabling the envisaged chemoenzymatic one-pot
transformation of styrenes to 1-arylethyl-1-amines. Among the
various representatives of the enzyme class of amine dehydro-
7
genases we choosed a double mutant of a leucine dehydrogen-
ase from Exigobacterium sibiricum (EsLeuDH-DM) developed
by Xu et al. as this enzyme turned out to be able for catalyzing
7
a
2,5
the reductive amination of acetophenones. In addition, in re-
cent own work we utilized this enzyme successfully in process
dehydrogenase and transaminase. In addition, the Greaney
and Micklefield groups applied this concept for combining a
8
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development of the reductive amination of acetophenoneꢀ2.
halogenase with a Suzuki-cross coupling reaction.
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The initial reaction conditions for our current study with this
amine dehydrogenase were based on the previously described
reaction conditions. A high ammonia concentration combined
with an enzymatic cofactor recycling via oxidation of glucose
push the equilibrium to the product side. The cofactor is thus
needed only in catalytic amounts for quantitative conversion.
Scheme 3. Concept of Combining Wacker-Oxidation and
Enzymatic Reductive Amination via Catalyst Compartmen-
talization
7
In the next step, the compatibility of the amine dehydrogenase
with the metal components of the chemocatalytic system for the
Wacker-oxidation were studied since such a compatibility rep-
resents the prerequisite for combining these two reactions in a
“classic” one-pot synthesis. Previous works with an alcohol de-
hydrogenase and a transaminase showed that palladium salts
have minor negative effects on enzymes, whereas copper causes
2
,5
a dramatic loss of enzyme activity. In order to be able to de-
termine even a slight biocatalyst deactivation, the reaction time
was reduced in a way that a conversion of less than 100% was
obtained. In detail, 79% conversion were obtained after 38 h in
combination with 99% ee (Scheme 2). The presence of palla-
dium (II) chloride leads to almost equal conversion (77%) and
no decreased selectivity. However, a complete loss activity was
observed when copper chloride (CuCl) was added (Scheme 2),
thus being in accordance with previous studies on reduced en-
zyme activities in the presence of copper salts when using other
With regard to preparative applications of this compartmentation
technique, diffusion through the membrane can represent a critical
2
,5
step. Previous work showed that co-solvents enable an increased
diffusion rate. However, the presence of a co-solvent might affect
catalyst or substrate stability. Here, DMSO and MeOH were cho-
2
sen as promising candidates known from our previous work to
2
,5
enzyme classes. The results shown in Scheme 2 illustrate that
the amine dehydrogenase is not compatible with the copper
component of the catalytic system for the Wacker oxidation.
explore the compatibility of the amine dehydrogenase with
these organic co-solvents. The results are shown in Figure 1.
Figure 1. Impact of different Organic Co-Solvents (in [%])
on the Stability of the Amine Dehydrogenase
Scheme 2. Studies on the Compatibility of the Amine Dehy-
drogenase with Palladium and Copper Ions of the Catalyst
for Wacker-Oxidation
conversion
enantiomeric excess
100
80
60
40
20
In order to overcome this hurdle of incompatibility, the com-
partmentalization concept applied successfully for other che-
moenzymatic syntheses has been tested. This compartmentali-
zation is based on the use of a polydimethylsiloxane (PDMS)-
membrane as so-called “thimbles”, which separates the reactor
in two compartments. Since only hydrophobic components can
0
%
%
%
%
%
%
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%
0 %
0 %
0 %
ter 10
ter 20
ter 30 ter 40
wa wa
MeOH 4
wa
wa
MeOH 1 DMSO 10
MeOH 2 DMSO 20
MeOH 3
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The Journal of Organic Chemistry
The amount of co-solvent was increased successively to 40% of
Wacker-oxidation of styrene with a subsequent reductive ami-
nation of the in situ-formed acetophenone. The latter step pro-
ceeds in a high enantioselective fashion in the presence of an
amine dehydrogenase by consuming glucose as a co-substrate
for in situ-cofactor recycling. As a nitrogen source only ammo-
nia is needed. As the enzyme turned out not to be compatible
with the catalytic system of the Wacker-oxidation, compart-
mentalization of the chemo- and biocatalysts by means of pol-
ydimethylsiloxane-based thimbles was carried out, leading to
the desired amine with quantitative conversion and excellent
enantiomeric excess. The overall one-pot process formally cor-
responds to an asymmetric hydroamination of styrene with am-
monia. The tandem reaction has been demonstrated with sty-
rene as a model substrate in this work but other amines can be
expected to be accessible by this concept as well since a broad
substrate scope has been already described for the individual
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the reaction volume. For each buffer/co-solvent mixture, a re-
action was carried out with the same volume of water as refer-
ence to eliminate the effect of lower total ammonia concentra-
tion. Figure 1 shows that the selectivity of the amine dehydro-
genase remains completely unaffected in the presence of meth-
anol. At a volume fraction of 30ꢀ% of MeOH, the relative con-
version begins to decrease and at 40ꢀ% no conversion was de-
tected. For DMSO, even the addition of 10ꢀ% of the reaction
volume leads to a strong decrease in conversion. The product
was only detected in traces and thus could not be used for a de-
termination of selectivity. An increase in the DMSO concentra-
tion leads to a further decrease in the range of the detection limit
of the product. Thus, DMSO is not suitable as a co-solvent for
this amine dehydrogenase system. For further experiments, the
biotransformations have been conducted in an aqueous medium
containing 20% MeOH since this is the maximum concentration
of a co-solvent not showing a negative impact on the biotrans-
formation within this enzymatic cascade.
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two reactions, namely Wacker oxidation and amine dehydro-
6
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genase-catalyzed reductive amination. Thus, this type of pro-
cess has the potential to be used as a platform technology for
the synthesis of a broad range of amines when starting from dif-
ferent substituted styrenes within a one-pot process.
With this optimized biotransformation towards the desired
compartmentalization concept in hand, next a one-pot transfor-
mation by using a Wacker-oxidation and a biocatalytic reduc-
tive amination was performed under these buffer-MeOH condi-
tions (Scheme 4). The advantageous solvent mixture consisting
of MeOH and water was also used for the Wacker-oxidation,
and both reactions were compartmentalized by means of a
PDMS membrane. In Figureꢀ2 the result of the one-pot process
is shown. A quantitative conversion leads to the desired amine
EXPERIMENTAL SECTION
General information. Chemicals for this work were purchased
from Sigma-Aldrich, Alfa Aesar, Roth, Merck, VWR, Acros Organ-
ics, Amano Enzyme Inc. and Oriental Yeast Co., Fisher Scientific
and used without further purification. All HPLC chromatograms
were recorded on
a LC-Net II / ADC Machine and
(
R)-3 in good yield and excellent enantiomeric excess of 99%
pumps (PU-2080) of the company JASCO. Chiral columns (Chi-
ralpak AD-H) for analytical separation of the enantiomers via
HPLC are commercially available from Daicel Chemical Indus-
tries.
A typical procedure of the Wacker oxidation is described in the
following. Styreneꢀ1 (115 µL, 1.00 mmol, 1.00 eq.) is added to a
ee. The yield of 76ꢀ% is limited by the Wacker oxidation which
gives a range of by-products.
Scheme 4. Optimized One-Pot Process of a Formal Asym-
metric Hydroamination by Combining Wacker-Oxidation
and Reductive Amination with an Amine Dehydrogenase
mixture of PdCl
2
(8.7 mg, 0.05 mmol, 0.05 eq) and CuCl (99.9 mg,
O (100 µL). The
1
.00 mmol, 1.00 eq.) in MeOH (700µL) and H
2
reaction is carried out under an atmosphere of oxygen (provided via
a balloon) at room temperature. After 24 h the reaction is termi-
nated by adding H
ride, and the organic phase is dried with Na
2
O. The mixture is extracted by methylene chlo-
SO . Then, the solvent
2
4
is evaporated in vacuo, and the resulting crude product is analyzed
by NMR (with tert-BuOH as an internal standard). The conversion
related to the formation of the product is 86 %. For a detailed prod-
1
0
uct ratio and its explanation see Supporting Information and ref. .
Thus, an efficient transformation of styreneꢀ1 to (R)-1-phenyle-
thylamineꢀ3 has been developed in spite of the fact that the cop-
per ions of the Wacker-oxidation are not compatible with the
amine dehydrogenase utilized in the second reaction step. The
quantitative conversion and excellent ee-value show that by
means of a PDMS-based compartmentalization such a one-pot
transformation of styreneꢀ1 to 1-phenyl-ethyl-1-amineꢀꢀ(R)-3
can be realized, leading to the desired chiral amine with high
enantioselectivity when starting from a non-functionalized al-
kene. It should be added that in previous work re-usability of
the PDMS-thimble with the interior phase bearing the Wacker
oxidation catalyst system therein was demonstrated with ten re-
action cycles in total and without catalyst leaching and loss of
1
ar
H-NMR (500 MHz, CDCl
3
): 훿 (ppm) = 6.80-7.30 (m, 5 H, H ),
2
.61 (s, 3 H). All signals are in agreement with the data reported in
2
,11
literature.
Biocatalyst preparation. The construction of the biocatalyst was
described before and is used in this work the same way. Also the
8
commercial resources are exactly the same.
8
a) Transformation of plasmid into E.ꢀcoli (according to ref. ). After
digestion 10ꢀµL plasmid DNA was added to chemical competent
cells (50ꢀµL) and incubated for 30 min on ice. The cells were heated
at 42ꢀ°C for 90 sec and incubated again for five minutes on ice.
Afterwards 1 mL of LB media was added. The mixture was heated
for three hours at 37ꢀ°C and 800 rpm. Subsequently, the cells were
cultured on LB agar plates with suitable antibiotic and incubated
overnight at 37ꢀ°C.
5
membrane performance. Thus, in analogy also for this one-pot
8
b) Protein expression (according to ref. ) typically in a 500 mL
process recyclability of the Wacker oxidation catalyst (as the
highest priced component of this process) can be expected.
scale. TB medium with kanamycin (50ꢀµg/mL) were inoculated
with 1% overnight culture. The cultures were grown at 37ꢀ°C and
1
80ꢀrpm. When the culture reached an OD of 0.5, cell cultures were
In conclusion, an enantioselective chemoenzymatic two-step
one-pot transformation of styrene to 1-phenylethyl-amine has
been developed based on combining an initial Pd/Cu-catalyzed
induced with 200ꢀµL of IPTG (1M). For expression temperature
was reduced to 20ꢀ°C. Afterwards cells were harvested (4000ꢀg,
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4
ꢀ°C, 30 min). For purification cells were suspended in binding
buffer (25% cell suspension) and digested under ultrasound
3x3ꢀmin, 5x10 cycles). The suspension was centrifuged at
0,000ꢀg for 20 min. The pellet was discarded and the crude extract
as a substrate. First, styrene (56 µL, 0.5 mmol, 1.00 eq.) is added
to a mixture of PdCl (4.5 mg, 0.03 mmol, 0.05 eq) and CuCl
(49.5 mg, 0.5 mmol, 1.00 eq.) in MeOH and H O (350 µL: 50 µL,
1
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2
(
2
2
7:1) which is prepared in a PDMS thimble. The reaction is done
under an atmosphere of oxygen (provided via a balloon) at RT. Af-
ter 24 h the exterior volume is filled with MeOH (3.97ꢀmL) and the
complete reactor is stirred for 19ꢀh at room temperature. Before
adding the enzymatic solution, ammonia (9.7ꢀmL, 19.38ꢀmmol,
2 M, pHꢀ9.5) and glucose (625ꢀµL, 0.05ꢀmmol, 1 M) are dissolved
in an AmDH crude extract (5.16ꢀmL, 0.06 M phosphate buffer,
pH 7.0, 0.23 U/µmolsubstrate). To the solution are given a solution of
was used for biotransformation without further purification.
c) Activity assay (according to ref. ). For the activity assay the ox-
idation of NADH to NAD+ was measured by decrease in absorb-
ance at 340 nm at 30ꢀ°C. The enzyme activity is defined as µmol
min . The extinction coefficient is 6.3*10 L mol cm . The reac-
tion was performed in a 1 mL cuvette consisting of 980 µL buffer
2
8
-1
3ꢀ
-1
-1
M ammonium chloride buffer, pH 9.5 with ketone (5–20 mM),
0ꢀµL NADH (10 M, final concentration 0.1 mM) and 10 µL en-
+
1
cofactor NAD (250ꢀµL, 1ꢀµmol, 50ꢀmM) and glucose dehydrogen-
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zyme crude extract. The activity was measured with a V-630
UV/Vis spectrophotometer from JASCO.
ase (75ꢀµL, 0.19 U/µmolsubstrate). Then, the enzymatic solution is
filled in the exterior volume. The reaction mixture is stirred 48 h at
30 °C. After the complete reaction time, the mixture is extracted by
methylene chloride (3 times). The combined organic layers are
A typical procedure of the enzymatic reductive amination is de-
scribed in the following. Stock solution concentrations are given
in the brackets. First, ammonia (776ꢀµL, 1.55ꢀmmol, 2 M, pHꢀ9.5)
and glucose (50ꢀµL, 0.05ꢀmmol, 1 M) are dissolved in an AmDH
crude extract (413ꢀµL, 0.06 M phosphate buffer, pH 7.0, 0.23
U/µmolsubstrate). To the solution are given a solution of cofactor
2 4
dried with Na SO and separated from the solvent in vacuo. Con-
version is calculated by NMR spectrum of the crude product is pu-
rified by extraction with hydrochloric acid (1ꢀM). The pH of the
aqueous phase is changed by sodium hydroxide solution to 14 and
is extracted by methylene chloride. The enantiomeric excess is de-
termined after derivatization to the corresponding amide by chiral
+
NAD (20ꢀµL, 1ꢀµmol, 50ꢀmM) and glucose dehydrogenase (6ꢀµL,
0.19 U/µmolsubstrate). Then, acetophenone (2, 4.22ꢀµL, 0.04ꢀmmol)
is added to the solution. The reaction mixture is stirred 48 h at
1
HPLC. H-NMR signals of the resulting product (R)-3 within the
3
3
0 °C. After the complete reaction time, the mixture is extracted by
methylene chloride (3 times). The combined organic layers are
dried with Na SO and separated from the solvent in vacuo. A con-
crude product (500 MHz, CDCl
3
): 훿 (ppm) =1.39 (d, JHH=6.7 Hz,
3
ar
3 H), 4.10 (q, JHH=6.7 Hz, 1 H), 7.10-7.30 (m, 5 H, H ). The yield
(46ꢀmg, 76%) has been calculated based on the amount of obtained
amine. All signals agree with them published before in litera-
2
4
version of 96ꢀ% was determined by NMR and an enantiomeric ex-
1
11,12
cess of 99ꢀ% was measured after derivatization on HPLC. H-NMR
ture.
3
(500 MHz, CDCl
3
): 훿 (ppm) =1.39 (d, JHH=6.7 Hz, 3 H, H-9), 4.10
3
ar
(q, JHH=6.7 Hz, 1 H, H-7), 7.10-7.30 (m, 5 H, H ). All signals are
in agreement with the data reported in literature.
ASSOCIATED CONTENT
For HPLC chromatograms detailed product characterization of the
Wacker oxidation see Supporting Information.
1
2
For ee-analysis the amines were transformed to amides. The deri-
vatization of the amine products through acylation with acetic an-
hydride was conducted according to a protocol reported by Kroutil
AUTHOR INFORMATION
Corresponding Author
13
et al. : DMAP (0.8 eq.) is dissolved in acetic anhydride (20.0 eq.).
The amine (1.0 eq.) is dissolved in EtOAc and given to the DMAP
solution. After stirring the mixture at room temperature the reaction
was quenched with water and extracted with methylene chloride.
The crude product was purified by one acidic extraction at pH 1
and one basic (pH 13) extraction (3 times each of them).
*
E-mail: harald.groeger@uni-bielefeld.de.
Author Contributions
The manuscript was written through contributions of all authors.
As a reference for HPLC analytics a racemic mixture of 1-phenyle-
thylamine (rac-3) was acylated after the procedure described
above. The reaction was done in a 100 mg scale and leads into
Funding Sources
See section “Acknowledgment”.
1
quantitative conversion and a very good yield (87 %). H-NMR
ar
(
500 MHz, CDCl
J
3
): 훿 (ppm) = 7.40-7.20 (m, 5 H, H ), 5.14 (qui,
3
3
ACKNOWLEDGMENT
HH=8.0 Hz; 1 H), 1.99 (s, 1 H), 1.50 (d, JHH=5.0 Hz, 1 H). The
measured signals are in agreement with the data reported in litera-
The authors gratefully acknowledge generous support from the
German Federal Ministry of Education and Research (Bundesmin-
isterium für Bildung und Forschung (BMBF) within the project
“Biotechnologie 2020+, Nächste Generation biotechnologischer
Verfahren” (grant number 031A184A).
10i
ture (ref. ). Retention times (by using CO
1.5ꢀmL/min; 20ꢀ°C; 10ꢀmPa back pressure; AD-Hꢀcolumn;
210ꢀnm): t (R): 15.0 min; t (S): 20.3 min.
2
ꢀ/ꢀ2-propanol 95:5;
R
R
Preparation of a PDMS thimble. The preparation of the polydi-
methylsiloxane (PDMS)-thimbles on a 1 mL-scale contains the fol-
lowing major steps: first, the surface of a glass bottle is passivated
by adding five drops of trichloro-(1H,1H,2H,2H-perfluorooc-
tyl)silane (CAS: 102488-47-1) in a desiccator and an incubation
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