Asymmetric synthesis of a high added value chiral amine using immobilized ω-transaminases

Article abstract of DOI:10.3762/bjoc.15.6

Chiral N-heterocyclic molecules and in particular compounds with an amino functional group such as 3-aminopiperidine are valuable intermediates for the production of a large number of bioactive compounds with pharmacological properties. In this paper, the synthesis of both enantiomers of 3-amino-1-Boc-piperidine by amination of the prochiral precursor 1-Boc-3-piperidone using immobilized ω-transaminases (TAs-IMB), isopropylamine as amine donor and pyridoxal-5’-phosphate (PLP) as cofactor is described. Compared to other methods, the present approach affords the target compound in just one step with high yield and high enantiomeric excess starting from a commercial substrate. The reaction was carried out by using different commercially available immobilized enzymes, evaluating the catalytic activity and the enantioselectivity under different experimental conditions. Re-use of the most efficient enzyme was performed both in batch and in a semi-continuous system. The selected biocatalyst showed good stability under the reaction conditions providing consistent results in terms of conversion and enantiomeric excess after several cycles. The reported results may be of practical interest in view of the development of this sustainable approach to an industrial scale.

Full text of DOI:10.3762/bjoc.15.6

Asymmetric synthesis of a high added value chiral amine
using immobilized ω-transaminases
Antonella Petri*1, Valeria Colonna1 and Oreste Piccolo*2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 60–66.
1Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via
doi:10.3762/bjoc.15.6
Giuseppe Moruzzi 13, 56124 Pisa, Italy and 2Studio di Consulenza
Scientifica (SCSOP), Via Bornò 5, 23896 Sirtori (LC), Italy
Received: 23 August 2018
Accepted: 12 December 2018
Published: 07 January 2019
Email:
Antonella Petri* - antonella.petri@unipi.it; Oreste Piccolo* -
contact@scsop.it
Associate Editor: S. Bräse
*
Corresponding author
© 2019 Petri et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
asymmetric catalysis; biotransformations; chiral amine; immobilized
enzymes; ω-transaminases
Abstract
Chiral N-heterocyclic molecules and in particular compounds with an amino functional group such as 3-aminopiperidine are valu-
able intermediates for the production of a large number of bioactive compounds with pharmacological properties. In this paper, the
synthesis of both enantiomers of 3-amino-1-Boc-piperidine by amination of the prochiral precursor 1-Boc-3-piperidone using
immobilized ω-transaminases (TAs-IMB), isopropylamine as amine donor and pyridoxal-5’-phosphate (PLP) as cofactor is de-
scribed. Compared to other methods, the present approach affords the target compound in just one step with high yield and high
enantiomeric excess starting from a commercial substrate. The reaction was carried out by using different commercially available
immobilized enzymes, evaluating the catalytic activity and the enantioselectivity under different experimental conditions. Re-use of
the most efficient enzyme was performed both in batch and in a semi-continuous system. The selected biocatalyst showed good
stability under the reaction conditions providing consistent results in terms of conversion and enantiomeric excess after several
cycles. The reported results may be of practical interest in view of the development of this sustainable approach to an industrial
scale.
Introduction
Enantiomerically pure amines are important precursors to bio- tion. In this context there is an increasing interest in reactions
logically active compounds with different industrial applica- including ω-transaminases (TAs) which have been identified as
tions, including pharmaceuticals, fragrances and agricultural a greener and more sustainable method for chiral amine produc-
products [1]. It is therefore important to develop methods for tion [2-5]. TAs, also known as aminotransferases, are enzymes
their preparation which can be suitable for a large scale produc- capable of transferring an amino group from an amine donor to
60

Beilstein J. Org. Chem. 2019, 15, 60–66.
an acceptor containing a carbonyl functionality in the presence numerous synthetic pathways were tested for the preparation of
of pyridoxal-5'-phosphate (PLP) as a cofactor and the enzymes 3-aminopiperidine and its N-protected precursors in an opti-
are easily regenerated in situ without the need for another en- cally active form by using resolution of racemic mixtures, prep-
zyme. In principle, enantiomerically pure chiral amines can be aration from chiral precursors or asymmetric synthesis from
prepared following two approaches: through kinetic resolution prochiral compounds [20-22].
starting from racemic amines or by asymmetric synthesis
starting from suitable substrates, e.g., the corresponding carbon- In contrast, only few examples have been published on the syn-
yl compounds. In a kinetic resolution, a maximum yield of 50% thesis of this molecule through biotransformations [23,24]. In
of the product can be obtained. Moreover, high quantities of the these procedures TAs are used in commercial free form or iso-
co-product might complicate the product separation and the lated from bacterial sources, both in kinetic resolution starting
recovery of the chiral amine. Thus, the asymmetric synthesis is from the corresponding racemic amine and in asymmetric syn-
generally preferable because a theoretically 100% yield of the thesis starting from the corresponding ketone. The first method,
product is possible. However, this process is reversible and although it proceeds with high enantioselectivity, is a kinetic
therefore the conversion of the substrate is determined by how resolution and as a consequence is limited to 50% maximum
much the equilibrium is shifted towards the formation of the yield of the desired product. The second process employs non-
desired product. For this purpose two different strategies are commercial enzymes which might be a limitation in the
used: (i) removal of the co-product deriving from the amine perspective of a potential large-scale industrial application.
donor (i.e., under reduced pressure or by degradation/transfor- More recently, the synthesis of the (S)-enantiomer of minor ap-
mation) and (ii) use of an excess of the amine donor. The latter plication interest has been described [25]. However, the en-
method might have, however, some drawbacks: the recovery zyme used in this study is not commercially available and no
and purification of the target amine can be difficult depending enantioselectivity of the reaction has been reported.
on the nature of the amine donor used in excess and/or this
excess can cause inhibition of the enzyme. The use of iso- Herein, we describe the use of several immobilized ω-transami-
propylamine (IPA) as amino donor allowed optimizing the use nases in the asymmetric transamination of the selected sub-
of TAs in organic synthesis thanks to its ability to shift the strate 1-Boc-3-piperidone. Selected reaction parameters were
transamination reactions towards complete conversion together studied to optimize the protocol for further scaling-up purposes.
with easy removal of the low boiling point byproduct acetone
[
2-6].
Results and Discussion
In order to obtain enantiomerically pure (R)- and (S)-3-amino-1-
Since (R)- and (S)-selective TAs are available on the market, Boc-piperidine (2), the transamination reaction was studied by
both enantiomers of the amine product are accessible. This using commercially available immobilized TAs (TAs-IMB) and
point is particularly relevant since in most cases the pharmaco- isopropylamine as the amine donor (Scheme 1).
logical activity of a chiral drug is closely related to its absolute
configuration. Recent studies have favored the application of
TAs at the industrial level, so it is not surprising that they are
frequently found among the enzymes designed for the large-
scale synthesis of chiral amines [2,4]. Moreover, immobiliza-
tion of TAs has been developed in order to increase stability,
enzyme recyclability, easy work-up and purification of the
Scheme 1: Transamination reaction of 1-Boc-3-piperidone (1).
product [2-4,7-13]. Recently, we have reported a successful use
of several immobilized TAs with IPA as amine donor [14].
These enzymes are covalently immobilized on an epoxyacry-
Optically active 3-aminopiperidines and their N and/or N'-pro- late resin and offer several advantages over the native ones,
tected analogues, are important intermediates for the synthesis especially in view of a large scale application. In addition to an
of a large number of biologically active compounds used for the enhanced stability at higher temperatures or in organic solvents,
treatment of obesity, type-I and II diabetes mellitus or as immobilization on the support allows an easy recovery of the
psychotropic drugs against depression and schizophrenia enzyme and isolation of the product after the reaction. More-
[
15-19]. In particular (R)-3-amino-1-Boc-piperidine is a useful over, the activity of immobilized TAs used at appropriate con-
precursor of compounds mainly used for the synthesis of dipep- centrations can be comparable to that of the free enzymes [26].
tidyl peptidase IV (DPP-IV) inhibitors, such as alogliptin, The conditions for the application of these enzymes according
linagliptin and other antidiabetic agents [15,17]. Over the years, to the supplier protocol are: 50 mg of substrate (45 mM),
61

Beilstein J. Org. Chem. 2019, 15, 60–66.
2
00 mg of TA enzyme, 1 mM PLP, and 1 mM isopropylamine It should be noted that the TAs-IMB enzymes showed excellent
in triethanolamine buffer (100 mM, pH 7.5) in a total volume of enantioselectivities as demonstrated by the enantiomeric excess
.75 mL. If the substrate is insoluble in the buffer, a DMSO values which were all above 98%. Therefore, these results
5
solution (15% v/v) can be used. These conditions were taken as represent an improvement in the preparation of enantiomerical-
a starting point to evaluate the reactivity and stereoselectivity of ly pure 2 compared with literature data [23]. Höhne et al. ob-
the enzymes in the transamination reaction of substrate 1 and tained only the (R)-enantiomer in 42% yield and 97% ee as a
the results are reported in Table 1.
result of kinetic resolution of 1 using ω-transaminase and pyru-
vate as an amine acceptor. The enantiomeric excess in entries 9
and 10 of Table 1 was not determined due to the presence of
several byproducts.
Table 1: Screening of TAs-IMB for transamination reaction of 1.
entrya
enzyme
time (h)
yield (%)b
ee (%)c
Transamination reactions using these optimized reaction condi-
tions with commercially available TAs-IMB have been per-
formed with other achiral [14] and pro-chiral [27] substrates,
such as for example 4-methoxyphenylacetone. It was found that
quantitative conversions were in some cases obtained only
with (S)-selective TAs, confirming that it is difficult to predict
the reactivity of the enzymes by changing the nature of the sub-
strate.
1
2
3
4
5
6
7
8
9
ATA-025-IMB
ATA-013-IMB
ATA-415-IMB
ATA-303-IMB
ATA-P1-G05-IMB
ATA-260-IMB
ATA-256-IMB
ATA-254-IMB
ATA-301-IMB
ATA-234-IMB
21
52
99
65
81
90
99
80
80
94
90
97
>99 (R)
>99 (R)
>99 (R)
>99 (R)
98 (S)
70
360
313
336
336
336
360
360
>99 (S)
>99 (S)
>99 (S)
n.d.d
10
n.d.d
After these initial investigations at room temperature, four
promising enzymes were selected (i.e., three (R)-selective and
one (S)-selective) which allowed to obtain high conversions and
enantiomeric excesses in the transamination reaction of 1.
aReaction conditions: 50 mg of substrate (9 g/L, 45 mM, DMSO
solution), 200 mg of enzyme, 550 rpm, 25 °C. bDetermined by HPLC
analysis. cDetermined by HPLC analysis after derivatization. dn.d.: not
determined due to the presence of several byproducts.
As can be seen in Table 1, ATA-025-IMB was the most effi- In order to enhance reaction rates, an optimization study
cient among the (R)-selective enzymes since it led to high was undertaken within the stability range of the enzyme.
conversion and enantiomeric excess in the shortest reaction time According to the supplier, the used TAs-IMB showed a higher
(
Table 1, entry 1). Accordingly, ATA-P1-G05-IMB was the stability in the range between 30 °C and 55 °C when the model
most efficient in order to obtain the (S)-enantiomer (Table 1, compound acetophenone was used as substrate. We were
entry 5).
interested in exploring the behavior of these enzymes
using 1 as starting material. Transamination reactions
In contrast, the (S)-selective enzymes ATA-260-IMB, ATA- catalyzed by ATA-025-IMB, ATA-415-IMB, ATA-013-IMB,
56-IMB and ATA-254 allowed for the formation of the prod- and ATA-P1-G05-IMB were repeated under the same
2
uct with high enantioselectivity (>99%) but longer reaction experimental conditions described above with the exception
times were required to achieve high yields of up to 94% of a higher reaction temperature. The results are shown in
(
Table 1, entries 6, 7, and 8).
Table 2.
Table 2: Investigation of the temperature effect on the transamination reaction of 1.
entrya
enzyme
temperature (°C)
time (h)
yield (%)b
ee (%)c
1
2
3
4
5
6
7
8
ATA-025-IMB
ATA-025-IMB
ATA-415-IMB
ATA-415-IMB
ATA-013-IMB
ATA-013-IMB
ATA-P1-G05-IMB
ATA-P1-G05-IMB
35
50
35
50
35
50
35
50
5
99
99
99
99
93
99
92
99
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
98 (S)
3
26
24
51
28
75
50
98 (S)
aReaction conditions: 50 mg of substrate (9 g/L, 45 mM, DMSO solution), 200 mg of enzyme, 550 rpm. bDetermined by HPLC analysis. cDetermined
by HPLC analysis after derivatization.
62

Beilstein J. Org. Chem. 2019, 15, 60–66.
In all cases the increase in temperature resulted in a decrease in
reaction time compared to the reaction at room temperature
(
Table 1). It should be noted that in all reactions the product
was obtained with high enantioselectivity (98% or higher).
Among the (R)-selective enzymes the best was ATA-025-IMB
as it gave in 3 hours at 50 °C 99% yield with >99% ee. ATA-
4
15-IMB and ATA-013-IMB were also sensitive to the temper-
ature effect; however, complete conversion of 1 was reached
with these enzymes at 50 °C in 24 and 28 hours, respectively.
The decrease in the reaction time observed with ATA-P1-G05-
IMB in the reactions carried out at 35 °C and at 50 °C with
respect to those at room temperature is particularly significant.
Nevertheless, the time necessary for complete conversion was
higher than that observed for (R)-selective enzymes at the same
temperature. From the obtained results of the investigation of
the temperature effect in the transamination reaction of 1 it was
concluded that TAs-IMB exhibited higher activities at higher
temperatures without loss of efficiency and selectivity.
Figure 1: Reuse of ATA-025-IMB in five consecutive cycles in the
transamination reaction of 1 in batch system (reaction time: 30 minutes
in cycles 1, and 2 and 1 hour in cycles 3–5; temperature: 50 °C).
The good results obtained in the batch recycling reactions
prompted us to investigate the possibility of a recycling process
Having in hand the good results obtained both in terms of activ- under flow conditions. For this purpose a microreactor was built
ity and enantioselectivity, in particular with ATA-025-IMB at consisting of a PEEK column which was filled with the en-
5
0 °C, optimization of the reaction regarding the recycling of zyme. The reaction mixture was circulated within the reactor by
the enzyme was studied. Indeed, one of the advantages of using using a suitable pump. Both the PEEK column and the reaction
immobilized enzymes is the easy recovery of the enzyme at the mixture were thermostated at a temperature of 50 °C. The
end of the reaction by simple filtration and its reuse in succes- progress of the reaction was monitored with the same methods
sive reactions.
used for the previously described reactions, by withdrawing
aliquots of the reaction mixture. After 4 hours, when a com-
As previously indicated, in the transamination reaction it is plete conversion was achieved, the reaction was interrupted.
necessary to use DMSO as a co-solvent to solubilize the sub- The reaction mixture was then removed and the column was
strate 1, which is insoluble in an aqueous medium. In batch washed first with a 5% (v/v) aqueous solution of DMSO
recycling reactions, it was tested if the reused enzyme would be in H2O/triethanolamine buffer at pH 7.5 followed by
partially deactivated after several reaction cycles in the pres- H2O/triethanolamine buffer at pH 7.5 for the quantitative isola-
ence of the organic solvent. Therefore, the amount of enzyme tion of the reaction product. The column was then used in five
was increased in order to reduce the reaction time and as a consecutive reactions under the same experimental conditions.
consequence to limit the contact time of the enzyme with The results are shown in Figure 2.
DMSO. The enzymatic transamination reaction was carried out
as previously described at 50 °C but using 1 g of enzyme. At the In all cycles, the same values of conversion and enantiomeric
end of the reaction, the enzyme was filtered, washed and reused excess as in the batch reactions were obtained. These results are
in five consecutive reactions under the same experimental of interest for application as they show that the activity of the
conditions (Figure 1).
enzyme was maintained even after several hours of continuous
operation at 50 °C in the presence of DMSO. It is also note-
Under these experimental conditions, complete conversion was worthy that when the reactor was stored at 4 °C and reused after
obtained after 30 minutes of reaction in the first and in the several months under the same experimental conditions for
second cycle. In the following reactions, complete conversion further five runs on the substrate 1, no relevant changes in activ-
was reached after 1 hour. The enantiomeric excess of (R)-2 was ity and selectivity were obtained.
>
99% in all cases. These results showed that ATA-025-IMB
can be reused in subsequent runs without loss of selectivity and In order to optimize the reaction, some scale-up tests were per-
therefore looks promising for the development of a large-scale formed using 200 mg of ATA-025-IMB and a substrate concen-
reaction. Furthermore, it should be noted that the selected en- tration of 40 g/L. Compared to the reactions carried out with
zyme allowed obtaining the (R)-enantiomer which is of greater smaller amounts of substrate, the reaction was slower which is
industrial interest than the (S)-enantiomer.
most likely due to low solubility of the starting material under
63

Beilstein J. Org. Chem. 2019, 15, 60–66.
measured on an Anton Paar MCP 300 polarimeter. Thin-layer
chromatography (TLC) analyses were performed on silica gel
6
0 F254 plates using EtOAc/EtOH 1:1 as eluent and visual-
izing compounds by exposure to 0.2% ninhydrin solution and
UV light or I2 vapor. The transamination reactions were per-
formed in an Eppendorf ThermoMixer C which combines tem-
perature control and mixing.
Chromatographic analysis
Analyses of the reactions were performed on a Jasco HPLC
system with a Gemini C18 column (150 × 4.6 mm). Elution was
carried out at 1 mL/min with detection at 220 nm and a column
temperature of 25 °C. The eluent was H2O/CH3CN/DEA
7
0:30:0.1. Authentic standards were analyzed before analysis of
Figure 2: Reuse of ATA-025-IMB IMB in five consecutive cycles in the
transamination reaction of 1 in a flow system (reaction time: 4 hours;
temperature: 50 °C).
the reaction mixtures.
The enantiomeric excesses were determined after derivatization.
these experimental conditions. After 24 hours, complete conver- An aliquot (50 mg) of the crude sample and 100 mg K2CO3
sion of 1 was obtained but in addition to the desired product were added to a 15 mL volumetric flask and dissolved in 1 mL
(
R)-2 some byproducts were also observed, confirming possible of CH3CN and 4 mL H2O. Benzyl chloroformate (100 μL) was
degradation of the substrate in aqueous media [25]. However, slowly added dropwise and the solution was sonicated for
R)-2 was obtained in 70% isolated yield and >99% ee. Finally, 5 minutes. After the addition of 1 mL ethyl acetate the solution
(
the product was converted into the corresponding dihydrochlo- was sonicated for further 5 minutes and extracted with ethyl
ride and compared with an authentic commercial sample in acetate (1 mL). The organic phase was dried over Na2SO4,
order to confirm the enantiomeric excess and absolute configu- filtered and after evaporation of the solvent diluted with the
ration.
mobile phase. Enantiomer separation was achieved on a Jasco
HPLC system and a Lux-Cellulose 3 (250 × 4.6 mm) column
with a flow rate of 0.5 mL/min, detection at 216 nm and column
Conclusion
Several (S)- and (R)-selective immobilized transaminases have temperature of 25 °C. The eluent was hexane/isopropanol 9:1.
been investigated for the synthesis of a high added value chiral The racemic compound was used as reference. The absolute
amine from the corresponding commercial ketone employing configuration was assigned by comparison of elution order of
isopropylamine as amine donor. Due to the excellent enantiose- an authentic standard.
lectivity of the selected enzymes, the target molecule could be
prepared with high yield and excellent enantiomeric excess. The General procedure for the transamination reaction
stability of the enzyme with respect to the temperature and the with immobilized enzymes
organic solvent allowed its reuse both in batch and flow system. In a similar manner as previously described in [14], to 5 mL of
a triethanolamine buffer (100 mM, pH 7.5) containing iso-
Experimental
propylamine (1.1 M), TA-IMB enzyme (200 mg) and PLP
General
(1.4 mM) were added. The mixture was stirred at 35 °C and
The chemicals used in this study were purchased from Sigma- 550 rpm for 5 minutes, and then a preheated solution (35 °C) of
Aldrich, Fluka or Alfa Aesar and used as received unless other- 1-Boc-3-piperidone (1, 0.26 mmol, 45 mM) in DMSO (750 μL,
wise stated. An authentic sample of (R)-3-aminopiperidine 13% v/v) was added. The reaction was stirred at 35 °C and
dihydrochloride was obtained from Cangzhou Senary Chemi- 550 rpm in an open vessel for 24 hours. The reaction was moni-
cals S. & T. CO., LTD (China). Codex® ATA-IMB Screening tored by HPLC analysis and by TLC. At the end of the reaction,
Kit was purchased from Purolite. The enzyme loading was the enzyme was filtered under vacuum and washed with
about 100 mg protein per gram of wet resin. 1H and 13C NMR triethanolamine buffer (100 mM, pH 7.5, 3 × 2 mL). The recov-
spectra were recorded at room temperature at the field indicat- ered enzyme was suspended in buffer and stored at 4 °C. HCl
ed on a Bruker Avance II spectrometer. Multiplicities for (4 M) was added to the reaction mixture to reach pH 2 and the
1
H NMR couplings are shown as s (singlet), d (doublet), aqueous layer was extracted with CH2Cl2 (2 × 5 mL) to remove
m (multiplet). Chemical shifts (in ppm) are referenced to any starting material. After that, the pH of the solution was
residual protonated solvent. The optical rotations were changed to pH 13 by addition of KOH and the aqueous layer
64

Beilstein J. Org. Chem. 2019, 15, 60–66.
extracted with CH2Cl2 (4 × 5 mL). The combined organic (c 0.31, EtOH) for the (R)-enantiomer, lit. [28]: [α]D20 = +26.0
extracts were dried over Na2SO4, filtered and then evaporated (c 0.308, EtOH) for the (S)-enantiomer.
under reduced pressure to yield 3-amino-1-Boc-piperidine (2).
Each of the crude reaction mixtures were analyzed by NMR and Synthesis of (R)-3-aminopiperidine dihydrochloride
HPLC.
In a vial, 100 mg (0.5 mmol) of (R)-1-Boc-3-aminopiperidine
(R)-2, ee >99%) were dissolved in 100 μL EtOH. To the result-
ing solution, 400 μL of a solution of acetyl chloride (2.8 mmol)
(
Reuse of immobilized TA in batch system
The transamination reaction of 1-Boc-3-piperidone (1) was con- in EtOH (1:1) was gradually added at 0 °C. The reaction mix-
ducted using 1 g of ATA-025-IMB under the experimental ture was then stirred at room temperature. A white solid settled
conditions described above. Upon complete conversion, the en- down and indicated the completion of the reaction. The super-
zyme was filtered, washed with triethanolamine buffer natant was decanted and the solid was washed with cold ethanol
(
100 mM, pH 7.5) and reused under the same reaction condi- (2 × 100 μL). The solid was dried under vacuum to yield
tions for five subsequent reactions. The reaction mixtures were (R)-3-aminopiperidine dihydrochloride. 1H NMR (400 MHz,
extracted and analyzed as described above.
D2O) δ 3.73 (m, 2H), 3.47–3.44 (m, 1H), 3.06–2.89 (m, 2H),
.19–2.17 (m, 1H), 2.03–2.00 (m, 1H), 1.76–1.73 (m, 1H),
2
General procedure for the transamination reaction
with immobilized TA in flow system
1.61–1.64 (m, 1H); 13C NMR (101 MHz, D2O) δ 45.61, 44.94,
43.41, 26.26, 20.15; [α]D20 = −0.74 (c 0.5, CH3OH), lit. [22]:
ATA-025-IMB (0.8 g) was packed into a PEEK column which [α]D20 = −0.80 (c 0.5, CH3OH) for the (R)-enantiomer.
was then connected to a suitable pump. Triethanolamine buffer
(
(
100 mM, pH 7.5) containing isopropylamine (1.1 M) and PLP Acknowledgements
1.4 mM) was circulated through the column at 35 °C, at a flow This study was financially supported by research funding from
rate of 4 mL/min prior to use. Then a preheated solution of the University of Pisa, Italy (Fondi di Ateneo; Progetti di
1
1
-Boc-3-piperidone (1, 0.26 mmol, 45 mM) in DMSO (750 μL, Ricerca di Ateneo-PRA) and by the Studio di Consulenza
3% v/v) was added. The reaction mixture contained in an open Scientifica (SCSOP), Sirtori (LC), Italy. Giulia Masia is grate-
vessel was pumped through the column at 50 °C. The reaction fully acknowledged for her contribution to the preparation of
was monitored by HPLC analysis and TLC. Upon complete (R)-3-aminopiperidine dihydrochloride.
conversion, the column was washed with triethanolamine buffer
(
100 mM, pH 7.5) to quantitatively recover the product. The ORCID® iDs
column was stored at 4 °C. The eluted solution and washings
were extracted as described above. The column was reused in
Oreste Piccolo - https://orcid.org/0000-0001-8948-2218
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7
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