Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal

Article abstract of DOI:10.1016/j.procbio.2016.05.022

The ability of chloroperoxidase (CPO) to catalyze amino alcohol oxidations was investigated. The oxidations of compounds with different configurations with respect to the amine position towards hydroxyl – using H₂O₂ and tert-butyl hydroperoxide (t-BuOOH) – were analyzed in terms of the initial reaction rate, substrate conversion, and CPO operational stability. It was observed that the further the amino group from the hydroxyl, the lower the initial reaction rate. The effect of the amino-protecting group and other substituents (i.e., methyl and hydroxyl) was also examined, revealing an increase in steric hindrance due to the effect of bulky substituents. The observed reaction rates were higher with t-BuOOH, whereas CPO was more stable with H₂O₂. Moreover, CPO stability had to be determined case by case as the enzyme activity was modulated by the substrate. The oxidation of N-Cbz-3-aminopropanol (Cbz, carboxybenzyl) to N-Cbz-3-aminopropanal was investigated. Main operational conditions such as the reaction medium, initial amino alcohol concentration, and peroxide nature were studied. The reaction kinetics was determined, and no substrate inhibition was observed. By-products from a chemical reaction between the formed amino aldehyde and the peroxide were identified, and a novel reaction mechanism was proposed. Finally, the biotransformation was achieved by reducing side reactions and identifying the key factors to be addressed to further optimize the product yield.

Full text of DOI:10.1016/j.procbio.2016.05.022

Accepted Manuscript
Title: Chloroperoxidase-catalyzed amino alcohol oxidation:
substrate specificity and novel strategy for the synthesis of
N-Cbz-3-aminopropanal
Author: Gerard Masdeu M ´ı riam P e´ rez-Trujillo Josep
´
L o´ pez-Sant ´ı n Gregorio Alvaro
PII:
S1359-5113(16)30145-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2016.05.022
PRBI 10696
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
17-3-2016
19-5-2016
20-5-2016
Please cite this article as: Masdeu Gerard, P e´ rez-Trujillo M ´ı riam, L o´ pez-Sant ´ı n
´
Josep, Alvaro Gregorio.Chloroperoxidase-catalyzed amino alcohol oxidation: substrate
specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal.Process
Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.05.022
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Chloroperoxidase-catalyzed amino alcohol oxidation: substrate
specificity and novel strategy for the synthesis of N-Cbz-3-
aminopropanal
a
b
a,
a
Gerard Masdeu, Míriam Pérez-Trujillo, Josep López-Santín, * and Gregorio Álvaro
a
Bioprocess Engineering and Applied Biocatalysis Group. Departament d’Enginyeria
Química, Biològica i Ambiental. Universitat Autònoma de Barcelona, 08193 Bellaterra,
Catalonia, Spain
b
Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, 08193
Bellaterra, Catalonia, Spain
*
Corresponding author:
Josep López-Santín
Bioprocess Engineering and Applied Biocatalysis Group. Departament d’Enginyeria
Química, Biològica i Ambiental. Universitat Autònoma de Barcelona, 08193 Bellaterra,
Catalonia, Spain
Tel.: +34 93 581 1018
Fax: +34 93 581 2013
E-mail: josep.lopez@uab.cat
1

Highlights




Influence of amino alcohols structure on chloroperoxidase specificity was assessed
Effect of peroxide and substrate on the enzyme stability was evaluated
A chemical reaction between the amino aldehyde and peroxides was identified
High N-Cbz-3-aminopropanol conversion was obtained by enzymatic oxidation
Abstract
The ability of chloroperoxidase (CPO) to catalyze amino alcohol oxidations was
investigated. The oxidations of compounds with different configurations with respect to
2 2
the amine position towards hydroxyl – using H O and tert-butyl hydroperoxide (t-
BuOOH) – were analyzed in terms of the initial reaction rate, substrate conversion, and
CPO operational stability. It was observed that the further the amino group from the
hydroxyl, the lower the initial reaction rate. The effect of the amino-protecting group
and other substituents (i.e., methyl and hydroxyl) was also examined, revealing an
increase in steric hindrance due to the effect of bulky substituents. The observed
2 2
reaction rates were higher with t-BuOOH, whereas CPO was more stable with H O .
Moreover, CPO stability had to be determined case by case as the enzyme activity was
modulated by the substrate. The oxidation of N-Cbz-3-aminopropanol (Cbz,
carboxybenzyl) to N-Cbz-3-aminopropanal was investigated. Main operational
conditions such as the reaction medium, initial amino alcohol concentration, and
peroxide nature were studied. The reaction kinetics was determined, and no substrate
inhibition was observed. By-products from a chemical reaction between the formed
amino aldehyde and the peroxide were identified, and a novel reaction mechanism was
proposed. Finally, the biotransformation was achieved by reducing side reactions and
identifying the key factors to be addressed to further optimize the product yield.
2

Keywords: chloroperoxidase (CPO); amino alcohol oxidation; substrate specificity for
chloroperoxidase; N-Cbz-3-aminopropanal synthesis; aldehyde–peroxide reaction; D-
fagomine precursor.
Chemical compounds studied in this article: N-Cbz-2-aminoethanol (PubChem CID:
2
80458), N-Cbz-3-aminopropanol (PubChem CID: 562256), N-Cbz-3-aminopropanal
(PubChem CID: 10398106), N-Cbz-3-aminopropanoic acid (PubChem CID: 736104),
3
1
-amino-1-propanol (PubChem CID: 9086), 4-amino-2-butanol (PubChem CID:
70254), N-Cbz-2-hydroxy-3-aminoethanol (PubChem CID: 3362144), N-Fmoc-4-
aminobutanol (PubChem CID: 57365309), N-Cbz-5-aminopentanol (PubChem CID:
072414), N-Cbz-6-aminohexanol (PubChem CID: 24890214).
4
3

1
. Introduction
Chloroperoxidase (CPO, EC 1.11.1.10), isolated from the fungus Caldariomyces
fumago, is one of the most versatile heme-containing enzymes as it catalyzes a wide
1
–3
variety of reactions, apart from its natural halogenation.
The halide-independent
reactions catalyzed by this heme protein include oxidative dehydrogenation, H
2
O
2
4–9
dismutation, and oxygen transfer reactions (e.g. alcohol oxidation). Although CPO
catalyzes alcohol oxidation without requiring cofactors, it uses peroxides, such as
hydrogen peroxide (H
2 2
O ) or tert-butyl hydroperoxide (t-BuOOH), as electron
acceptors. Although peroxides are required for this reaction, they rapidly deactivate
1
0–12
CPO.
Thus, the peroxide nature and its rate of addition to the reactor are key
operation parameters due to their effect on CPO stability.
In this work, we performed CPO-catalyzed oxidation of amino alcohols to yield amino
aldehydes. Amino aldehydes are used as substrates of aldolases for aldol addition of
dihydroxyacetone (DHA) or dihydroxyacetone phosphate (DHAP) to yield aldol
13–17
adducts.
These compounds are precursors of the well-known iminocyclitols, which
bind strongly to glycosidases and glycotransferases. Accordingly, they have been
1
8–21
extensively studied for their antiviral, anticancer, and antidiabetic effects.
However, only few studies have been published on CPO oxidation of amino alcohols,
2
2,23
which were restricted to α-amino alcohols.
The ability of the enzyme to oxidize
amino alcohols in other positions – particularly focusing on β-amino alcohols – is worth
investigating so as to discover new valuable intermediates and products.
One reaction of particular interest is the CPO-catalyzed oxidation of N-Cbz-3-
aminopropanol (Cbz, carboxybenzyl) in order to yield N-Cbz-3-aminopropanal, an
intermediate for the aldol addition of DHA catalyzed by D-fructose-6-phosphate
aldolase (FSA, EC 4.1.2.). The Cbz protection of the amino group is necessary to avoid
4

Schiff base formation between the amine and aldehyde groups of the formed amino
aldehyde. The above coupled reaction produces the Cbz-aldol adduct precursor of D-
fagomine, an iminocyclitol with significant therapeutic potential. This nutraceutical
product reduces the health risks associated with excessive intake of fast-digestible
2
4–27
carbohydrates, or an excess of potentially pathogenic bacteria.
In recent studies, some authors have chemically synthesized protected amino
2
8–30
aldehydes.
Nevertheless, oxidative biocatalysis may have certain advantages over
the chemical process: higher selectivity and hence more control and predictability of the
obtained product structure, as well as fewer chemical reagents used. In a recent study,
N-Cbz-3-aminopropanol oxidation was reported to be catalyzed by horse liver alcohol
3
1
dehydrogenase; however, in this case, cofactor NADH regeneration was required.
In the present study, the substrate specificity in amino alcohol oxidation catalyzed by
CPO is analyzed. For this purpose, several amino alcohol compounds are intended to be
oxidized by targeting their molecular substituents. Different configurations according to
the hydroxyl position towards amine (α to ε) are considered in terms of the substrate
conversion rate and enzyme stability. The effect of peroxide nature is also examined. In
addition, a novel enzymatic procedure for the synthesis of N-Cbz-3-aminopropanal is
2 2
presented, using CPO as the biocatalyst and H O or t-BuOOH as the peroxide. The
main operational conditions (reaction medium, substrate concentration, and peroxide
nature) are determined.
2
. Materials and methods
2
.1. Materials
CPO from C. fumago (chloride–hydrogen peroxide oxidoreductase, EC 1.11.1.10) was
supplied by Chirazyme Labs (Greenville, NC, USA) as a solution of partially purified
−1
−1
enzyme (9.54 mg protein ml ) with a specific activity of 1400 U mg protein.
5

Monochlorodimedone (1,1-dimethyl-4-chloro-3,5-cyclohexadione) and 1-fluoro-2,4-
dinitrobenzene (DNFB) were obtained from Fluka (Milwaukee, WI, USA). N-Cbz-2-
aminoethanol (benzyl N-(2-hydroxyethyl)carbamate) (1, see Table 1), N-Cbz-3-
aminopropanol (benzyl N-(3-hydroxypropyl)carbamate) (2.a), 3-amino-1-propanol
(
2.b), N-Fmoc-4-aminobutanol (3), N-Cbz-5-aminopentanol (4), N-Cbz-6-aminohexanol
5), N-Cbz-3-aminopropanal (3-[(Benzyloxycarbonyl)amino] propionaldehyde), N-Cbz-
(
3
-aminopropanoic acid (Z-β-Ala-OH), tert-butyl hydroperoxide (t-BuOOH, 70 wt. % in
O), hydrogen peroxide (H , 30 wt. % in H O), and trifluoroacetic acid (TFA) were
H
2
2
O
2
2
purchased from Sigma Aldrich (St. Louis, MO, USA). 4-Amino-2-butanol (2.c) was
procured from Acros Organics (Geel, Belgium). N-Cbz-2-hydroxy-3-aminoethanol
(
2.d) was supplied by Dr. Clapés from the Biotransformation and Bioactive Molecules
Research Group (CSIC, Barcelona, Spain). High-performance liquid chromatography
HPLC)-grade acetonitrile was obtained from Carlo Erba (Milan, Italy). Milli-Q-grade
(
water was used for analytical HPLC. All deuterated solvents for nuclear magnetic
resonance (NMR) analysis were purchased from Cortecnet (Voisins-le-Bretonneux,
France). All other reagents (analytical grade) were commercially available products.
2
.2. CPO stability
The CPO stability was determined by measuring its activity along time. For simplicity,
the chlorination activity test was performed. The CPO half-life time (t1/2) was
experimentally estimated from activity versus time data.
Enzymatic activity was determined by the decrease in absorbance at 278 nm due to the
−
1
−1 32
conversion of monochlorodimedone (MCD, ε278 =12.2 mM
cm )
to
1
dichlorodimedone (DCD), according to the method of Hager and Morris. The
enzymatic assay contained 100 mM potassium phosphate buffer of pH 2.75, 0.16 mM
MCD, 20 mM KCl, 2 mM hydrogen peroxide, and 50 µL of the sample with CPO, in a
6

total volume of 1 mL. The absorbance was measured at 25 °C, using a Cary 50 Bio UV–
Visible Spectrophotometer (Varian, Palo Alto, CA, USA). One activity unit of CPO is
defined as the amount of enzyme required for the conversion of 1 μmol of MCD per
minute at pH 2.75 and 25 °C. The standard deviation of the CPO activity test was
calculated from measurements performed by duplicate.
2
.3. Amino alcohol oxidation: monitoring the concentration of substrates and products
The concentrations of amino alcohols and N-Cbz-3-aminopropanol-oxidized products
N-Cbz-3-aminopropanal and N-Cbz-3-aminopropanoic acid) were measured by HPLC
(
analysis in a HPLC Dionex UltiMate 3000 with UltiMate 3000 Variable Wavelength
Detector. For chromatographic separation, a reversed-phase column (XBridge BEH
C18, 130Å, 5 µm, 4.6 × 250 mm) from Waters (Milford, MA, USA) was used. The
reaction samples were dissolved in acetonitrile (MeCN), which deactivates the enzyme
and arrests the reaction. All separations were performed by injecting 30 μL of the
−1
sample at a flow rate of 1 mL min at 30 °C. For all substrates, the solvent system
consisted of solvent A (0.1% (v/v) TFA in H O) and solvent B (0.095% (v/v) TFA in
MeCN/H O 4:1 (v/v)). Samples were eluted using a specific multistep gradient for each
substrate molecule: (1) gradient from 20% to 36% in 24 min, λ = 200 nm; (2.a, N-Cbz-
-aminopropanal, N-Cbz-3-aminopropanoic acid) gradient from 5% to 31% B in 0.35
2
2
3
min and isocratic elution at 30% B over 14.65 min, λ = 254 nm; (2.d) gradient from 5%
to 28% B in 0.35 min and isocratic elution at 30% B over 9.65 min, λ = 254 nm; (3)
gradient from 20% to 50% B in 1.5 min and isocratic elution at 30% B over 18.5 min, λ
=
200 nm; (4) gradient from 20% to 60% B in 1.5 min and isocratic elution at 30% B
over 7.5 min, λ = 200 nm; and (5) gradient from 20% to 65% B in 1.5 min and isocratic
elution at 30% B over 8.5 min, λ = 200 nm.
7

3
3
2
.b and 2.c were analyzed via a well-established derivatization method with DNFB. In
both cases, 50 μL of the sample was mixed with 80 μL of DNFB (37.6 mM in acetone)
and 20 μL of 1 M NaHCO . The mixture was incubated for 1.5 h at 40 °C. The reaction
3
was terminated by the addition of 40 μL of 1 M HCl and 200 μL of MeCN. Samples
were eluted with a gradient from 31.3% to 48.8% in 25 min, λ = 360 nm.
A quantitative analysis of all compounds was performed by prior calibration with
standards of known concentration. The concentration values were converted into molar
amount (µmol); that is, the volume of the peroxide added in pulses via a microburette
and the volume of the reaction medium withdrawn for each analysis (<10% of the initial
volume) were considered. The standard deviation of molar quantities was calculated
from HPLC measurements performed by duplicate.
2
.4. CPO-catalyzed oxidation of selected amino alcohols
2
2
The reaction conditions were adapted from a previous study of our research group.
The reaction medium was prepared by dissolving the amino alcohol in 100 mM sodium
acetate buffer (pH 5.0) and ethyl acetate (90:10 v/v) in a final volume of 5 mL. The
substrate concentration range was 5 mM for α/β-amino alcohols or 0.5 mM for γ/δ/ε-
amino alcohols. This difference in the order of magnitude was due to solubility
restrictions. All reactions were performed at 25 °C and 1000 rpm of orbital stirring
−1
(
MultiTherm™, Benchmark Scientific, Edison, NJ, USA), and 450 U mL of CPO was
used. The reaction was initiated by a pulse of 0.8 mM peroxide and carried out by
−1
continuous addition of 3 mM h
peroxide using a single-syringe automatic
microburette (Crison Instruments, Barcelona, Spain). A linear fitting of substrate
concentration versus time, at a substrate conversion <10%, was used to estimate initial
reaction rates.
8

Two reaction controls were performed for each substrate oxidation to prevent false-
positive results: (a) the substrate was incubated in a reaction buffer, without enzyme or
peroxide. This control checked the stability of the substrate itself under reaction
2 2
conditions. (b) The substrate was incubated in a reaction buffer with peroxide (H O /t-
−1
BuOOH) addition at 3 mM h and in the absence of enzyme. This second control
accounted for possible chemical reactions between the substrate and peroxide, in the
3
4–38
absence of enzyme.
2
.5. CPO-catalyzed oxidation of N-Cbz-3-aminopropanol
The reaction medium contained N-Cbz-3-aminopropanol dissolved in 100 mM sodium
acetate buffer (pH 5.0) in a final volume of 5 mL. The initial substrate concentration
range was 5–38 mM. For reactions performed with an organic solvent – ethyl or butyl
acetate – in monophasic or biphasic systems, the initial N-Cbz-β-aminopropanol
concentration was set at maximum solubility in each medium: 38 mM in an aqueous
buffer, 53 mM in an acetate buffer saturated with ethyl acetate, 69 mM in an acetate
buffer saturated with butyl acetate, and 85 mM in a biphasic medium of aqueous buffer
−1
and ethyl acetate (90:10 v/v). In addition, 450 U ml of CPO was used. The reaction
performance (peroxide addition, temperature, and agitation) was the same as previously
mentioned.
Oxidation reactions at high enzyme load and high peroxide–substrate ratio were carried
out at an initial substrate concentration of 15 mM. N-Cbz-3-aminopropanol was
dissolved in 100 mM sodium acetate buffer (pH 5.0) in a final volume of 0.3 mL,
−1
containing 350 mM t-BuOOH. For this purpose, 15,000 U mL of CPO was used. The
reaction was performed in duplicate at 25 °C and 1000 rpm of orbital stirring.
2
.6. Amino aldehyde–peroxide chemical reaction
9

N-Cbz-3-aminopropanol (β-OH), N-Cbz-3-aminopropanal (β-CHO), and N-Cbz-3-
aminopropanoic acid (β-COOH) were incubated at maximum concentration (38, 17, and
1
1 mM, respectively) in 5 mL of 100 mM sodium acetate buffer (pH 5.0). The reaction
was allowed to take place under orbital agitation in a MultiTherm™ device overnight
−1
for 19 h. Peroxide was continuously added to the reactor at 3 mM h .
2
.7. Identification of side-reaction products (compounds 6–8)
Products 6–8 were identified either by HPLC–mass spectrometry (MS)–MS or by MS.
NMR analyses of the three molecules confirmed the proposed structure. Further
3
9
information is presented in the related data article.
3
. Results and discussion
.1. Substrate specificity in CPO-catalyzed amino alcohol oxidations
3
The amino alcohol oxidative capacity of CPO was investigated by the oxidation of
compounds 1–5 to amino aldehydes (see Table 1). These compounds are amino alcohols
with similar hydrocarbon chains selected due to (i) the distinct amine position toward
hydroxyl, (ii) presence or absence of an amino-protecting group, and (iii) other specific
carbon substitutions. Each substrate oxidation was performed by continuous addition of
oxidants such as hydrogen peroxide or t-BuOOH, as explained in the section “Materials
and Methods.”
As seen in Table 2, CPO could accept nearly all compounds 1–5 as substrates to
catalyze their oxidation, with 2.d and 3 being the only non-converted molecules. For all
0
substrates, the initial reaction rates (r ) and conversion at the reaction end point (time at
which all substrate was converted or no remaining CPO activity was detected) are
described in relation to compound structural issues. The operational stability of CPO
toward peroxide nature was also compared.
1
0

The chemical structures of compounds 1, 2.a, 4, and 5 differ only in the n value of CH
groups and therefore in the amine position toward the hydroxyl. According to the initial
reaction rate analysis, the highest r was observed for substrate 1 and the lowest for 5
using either H or t-BuOOH as the oxidant. Thus, these results revealed that the
further the amino group from the hydroxyl in the substrate molecule, the lower the r
2
0
2
O
2
0
.
Conversely, the presence of amino-protecting groups altered the reaction course in
terms of the initial rate and maximum substrate conversion. Thus, the Cbz group in
substrate 2.a reduced r
amino protection, r increased 1.4-fold with H
BuOOH. Moreover, for compound
0
compared with the non-protected substrate 2.b; due to non-
as the oxidant and 3.1-fold with t-
which was protected by
0
2 2
O
3,
fluorenylmethyloxycarbonyl (Fmoc) instead of Cbz, no oxidation was observed,
presumably because the protecting group was bulky.
1
The extra methyl substituent in substrate 2.c R compared with 2.b had a different
impact on the initial rate depending on the oxidant used. The lower initial rate with t-
BuOOH can be attributed to the steric hindrance produced by the methyl group. Finally,
no conversion was observed for substrate 2.d with any peroxide, probably due to steric
2
hindrance or changes in electron density caused by the presence of the CH OH group.
Oxidant selection affected the operational stability of CPO and consequently the
reaction time course, as reflected by enzyme half-life values obtained in each case. In
2 2
many substrate reactions, t1/2 of CPO was higher with H O than with t-BuOOH, thus
achieving longer reaction periods; in other cases, t1/2 was practically the same for both
peroxides. For Cbz-protected substrates, conversion of similar order of magnitude was
achieved for both oxidants, due to lower reaction rates and higher enzyme stability with
2 2
H O as the peroxide.
1
1

Substrate species were also found to have a modulatory effect on enzyme stability.
Under the same reaction conditions, the specific substrate determined the CPO
deactivation degree. As an example, t1/2 of CPO was 7.8-fold higher for compound 2.b
than 4 with H
enzyme is well established.
.2. N-Cbz-3-aminopropanol oxidation catalyzed by CPO
2 2
O as the oxidant. This stabilizing effect of the substrate toward the
4
0,41
3
As mentioned previously, our target reaction in the present work is the enzymatic
oxidation of N-Cbz-3-aminopropanol (β-OH, also compound 2.a) to yield N-Cbz-3-
aminopropanal (β-CHO) (see Scheme 1). The possible CPO-catalyzed oxidation of the
amino aldehyde can yield N-Cbz-3-aminopropanoic acid (N-Cbz-β-alanine, β-COOH),
an undesirable compound if the target is the aldehyde product or an eventual coupling
with the FSA-catalyzed aldol addition. Several assays were performed to determine the
main operational conditions for β-OH oxidation. According to above CPO-catalyzed
oxidation of amino alcohols, t-BuOOH was preliminarily chosen as the peroxide for the
0
main reaction as higher r values were obtained with this oxidant.
The solubility of β-OH in an aqueous buffer is moderate (maximum 38 mM in 100 mM
sodium acetate buffer of pH 5.0). To overcome this limitation, three other media with
selected eco-friendly solvents were used: (i) acetate buffer saturated with ethyl acetate,
(ii) acetate buffer saturated with butyl acetate, and (iii) biphasic medium of aqueous
buffer and ethyl acetate (90:10 v/v). All four media were tested for the CPO operational
stability and substrate oxidation. The reactions were performed at an initial β-OH
concentration corresponding to maximum solubility in each medium.
The enzyme half-life time increased from 3.4 h in an aqueous buffer to 5.1 h in a
biphasic medium, as presented in Fig. 1.A. The saturated media stabilities were similar
to that measured for the acetate buffer. However, the oxidation curves of the reactions
1
2

catalyzed in ethyl or butyl acetate (saturated or biphasic) were not as expected with low
β-OH conversion (see Fig. 1.B). Maximum initial rate and final β-OH conversion were
noted for the aqueous buffer. Conversion in acetate buffer was 21%, but <10% in other
cases. This result can be attributed to two possible factors: (a) a protective effect of the
substrate toward the enzyme (maximum initial substrate concentration in each case) or
(b) a likely t-BuOOH partition between the two phases leading to lower peroxide
concentration in the aqueous phase. In turn, this would explain the lower deactivation of
CPO observed as well as the low oxidation rate in the presence of an organic phase.
To establish the appropriate initial β-OH concentration for the biotransformation,
several reactions in the aqueous buffer were carried out at different initial substrate
concentrations: 10, 16, 19, 26, and 36 mM. The half-life time of CPO increased with the
initial substrate concentration in the range studied (see Fig. 2). It was 1.6-fold higher at
3
8 mM than at 11 mM. These results were in concordance with the low t1/2 values
obtained for the oxidation of compounds 1–5 (initial substrate concentration was 0.5–5
mM). Again, the data revealed the protective effect of the substrate on the operational
stability of the enzyme.
However, substrate inhibition in CPO-catalyzed reactions has been reported
4
2,43
previously.
acetate buffer. The initial reaction rates were calculated and adjusted to a Michaelis–
Menten model (see Fig. 3). The estimated K and rmax values were 34.0 mM and 5.4
Nevertheless, it was not significant in the solubility range of β-OH in
M
−1
mM h , respectively.
Two reactions were carried out in 100 mM acetate buffer at the same substrate
−1
concentration (38 mM β-OH) and peroxide addition rate (3 mM h ), each one with a
different oxidant (t-BuOOH or H ). The evolution of enzymatic activity over time
2
O
2
1
3

(see Fig. 4.A), β-OH conversion, and formation of β-CHO and β-COOH (see Fig. 4.B)
were evaluated.
The CPO half-life time increased 2.4-fold when hydrogen peroxide was used instead of
t-BuOOH; however, as expected, the initial β-OH oxidation rate was lower with H
Nevertheless, both oxidants led to similar degrees of final β-OH conversion degree
CPO remained active for a longer time with H ). β-CHO production was faster and
higher with t-BuOOH, but it conducted to high amino acid yield.
.3. Identification of side reactions in CPO-catalyzed oxidation of N-Cbz-3-
2 2
O .
(
2 2
O
3
aminopropanol
On carefully evaluating the mole number of converted substrate and products obtained
with reaction time (see Fig. 4.B), we found a mass imbalance: (a) using t-BuOOH at t =
7
h, 45.5 µmol of oxidized β-OH yielded only 18.7 µmol of products (16.1 µmol β-CHO
and 2.6 µmol β-COOH). (b) Using H at t = 24h, 41.3 µmol of oxidized β-OH
2
O
2
yielded only 13 µmol of products (11.5 µmol β-CHO and 1.5 µmol β-COOH).
Therefore, in addition to the reactions presented in Scheme 1, secondary reactions
conducting to by-products were assumed to take place. This was hypothesized to be a
chemical reaction between any of the three compounds and the peroxide. To confirm
this, each molecule was incubated separately in acetate buffer for 19 h with the addition
−
1
2 2
of 3 mM h H O or t-BuOOH. Both β-OH and β-COOH were not oxidized by any
peroxygen species, but the amino aldehyde did react with the two peroxides: (i) 13.5%
of initial β-CHO reacted with t-BuOOH after 19-h incubation and (ii) 87.1% of β-CHO
2 2
reacted with H O . A white precipitate was observed in the reactor when hydrogen
peroxide was used. This chemical reaction between the amino aldehyde and the
peroxide could explain the mass balance non-closure. This type of reaction has been
44,45
described elsewhere.
1
4

Thus, a novel reaction scheme was proposed (see Scheme 2). It includes one by-product
when the selected oxidant is t-BuOOH and two with H . Then, for t-BuOOH, the
putative product is benzyl (3-(tert-butylperoxy)-3-hydroxypropyl)carbamate
, compound 6). For H reactions, the proposed hydroxy alkyl peroxide –
benzyl (3-hydroperoxy-3-hydroxypropyl)carbamate (C11 , compound 7) – can
further react with another amino aldehyde molecule and form a dialkyl peroxide
dibenzyl (peroxybis(3-hydroxypropane-3,1-diyl))dicarbamate; C22 , compound
). The dialkyl peroxide is only formed with H , as the hydroxy peroxy intermediate
2
O
2
(
C
15
H23NO
5
2 2
O
H15NO
5
(
28 2 8
H N O
8
2 2
O
formed by t-BuOOH is not sufficiently chemically reactive to react with another
aldehyde. To characterize these molecules, all three compounds were obtained in the
absence of salts, which might interfere in the identification analysis. The concentration
of each target compound was maximized for the identification according to the mole
ratio of peroxide/β-CHO. Compounds 6–7 were soluble under reaction conditions,
whereas 8 was highly insoluble and formed a solid precipitate.
Compounds 6–8 were identified by MS and NMR spectroscopy. The exact mass of
compound 6 was obtained by MS: 320.1469 (ESI+, positive electrospray ionization).
1
The chemical structure of molecule 6 was elucidated by H NMR spectroscopy, via one-
dimensional (1D)-selective nuclear Overhauser effect spectroscopy (NOESY)
experiments. Reaction intermediate 7 was detected, and its mass was identified by
HPLC-MS (264.0842). By HPLC–MS/MS, the main ion fragment was identified
(201.0769), which confirmed the presence of the Cbz substituent. The concerted
1
13
analysis of 1D and 2D NMR experiments allowed full H and C NMR characterization
of 7, confirming its molecular structure. Finally, compound 8 was analyzed by MS–
ESI+ (471.1744). The chemical structure of 8 was obtained by 1D, 2D, and diffusion-
3
9
ordered spectroscopy (DOSY) NMR experiments.
1
5

An experimental verification of the proposed reaction mechanism was required to
ensure its consistency. The test was performed using hydrogen peroxide, because the
secondary reactions in this case are more significant. β-OH (380 µmol) in 10 mL of
−1
acetate buffer was oxidized by CPO with the addition of 3 mM h H
2.1 µmol of β-OH underwent conversion. Only 63.6 µmol of aldehyde and acid
products were obtained; 18.5 µmol was missing. The 18.5 µmol represented compounds
and 8. Under the reaction conditions, the concentration of by-product 7 was negligible
HPLC analysis quantification). Applying the proposed mechanism scheme and
2 2
O . After 24 h,
8
7
(
molecular weight of by-product 8, 4.15 mg was missing (equivalent to 9.25 µmol of by-
product 8). The reaction medium was carefully filtered, the filter and the empty reactor
vessel were dried at 35 °C overnight, and the weight increase was considered to be the
weight of compound 8 (4.0 ± 0.6 mg). Hence, the proposed mechanism was confirmed.
3
.4. Intensification of the CPO-catalyzed oxidation of N-Cbz-3-aminopropanol
The abovementioned results of β-OH oxidation indicated a necessary compromise
between CPO stability, rates of substrate oxidation and key product formation, and the
presence of side reactions. High substrate conversion was achieved by increasing the
enzyme load and the peroxide/substrate ratio. Although hydrogen peroxide is an eco-
friendly oxidant with a weaker enzyme-deactivating effect, t-BuOOH was the chosen
peroxide because it leads to higher initial oxidation rate and a lower degree of side
reactions.
The reaction was performed under conditions indicated in the section “Materials and
Methods.” The results are shown in Fig. 5. In this case, 87% β-OH conversion was
achieved after 60 min of reaction. In terms of the target product formation, yields of
1
6% β-CHO and 47% β-COOH were obtained. As can be seen in Fig. 5, the product
selectivity was in favor of the acid product. It could be driven toward the amino
1
6

aldehyde by coupling the CPO-catalyzed reaction with a consecutive one, thus
preventing the formation of compound 6.
4
. Conclusions
The amino alcohol oxidative capacity of CPO regarding substrate structure was studied.
It is worth noting that CPO accepts α to ε configurations as substrates for the reaction.
The effect of this configuration on enzyme catalysis was investigated. It was found that
the further the amino group from the hydroxyl in the substrate molecule, the lower the
initial reaction rate. The influence of substituents on the amino alcohol molecule was
also discussed, especially with respect to the amino-protecting group. The presence of
this substituent was significant in the reaction progression, which prevented the
chemical reaction between the amino alcohol and the peroxide. However, the reaction
rates and final conversions were lower when the amino group was protected.
The deactivation of CPO by peroxides was also examined. Higher stability was
observed with hydrogen peroxide than t-BuOOH in many reactions. Nevertheless, the
substrate species influenced enzyme operational stability: the substrate was found to
have a modulatory effect. Therefore, the exact deactivation of CPO should be
determined case by case, depending on the substrate.
Furthermore, N-Cbz-3-aminopropanal was successfully synthesized by a novel strategy
using N-Cbz-3-aminopropanol as the starting material and CPO as the biocatalyst. Main
operational conditions, such as reaction medium and initial substrate concentration,
were studied. The Michaelis–Menten kinetics parameters were estimated. A secondary
chemical reaction between the formed amino aldehyde and peroxides was identified,
and a new reaction scheme was proposed and validated.
Finally, the extent of the target reaction was determined: high amino alcohol conversion
was achieved, but with an amino aldehyde selectivity of only 18 %. Hence, further
1
7

research must focus on driving the oxidation in the desired synthetic direction, avoiding
the mentioned undesirable reaction. One-pot coupling of the former oxidation and the
FSA-catalyzed aldol addition of DHA to yield the D-fagomine precursor is currently
being investigated.
Acknowledgments
We thank Dr. Alba Eustaquio and Dr. Maria Jesús Ibarz at Servei d’Anàlisi Química of
the Universitat Autònoma de Barcelona for their assistance on mass spectrometry
analyses. This work was supported by Spanish MINECO (project number CTQ2014-
5
3114R) and cofinanced by European Regional Development Fund (ERDF).
The Department of Chemical, Biological and Environmental Engineering of UAB
constitutes the Biochemical Engineering Unit of the Reference Network in
Biotechnology (XRB) and the research group 2014 SGR 452, Generalitat de Catalunya.
Financial support from Spanish MINECO for the PhD scholarship of Gerard Masdeu is
acknowledged.
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2
3

Figure legends
Figure 1. Oxidation of N-Cbz-3-aminopropanol by CPO and addition of t-BuOOH at
−
1
3
mM h in 5 mL of the four assayed reaction media: 100 mM sodium acetate buffer of
pH 5.0 ( ), acetate buffer saturated with ethyl acetate ( ), acetate buffer saturated with
butyl acetate ( ), and biphasic medium buffer/ethyl acetate 90:10 v/v ( ). Initial
substrate concentrations corresponded to maximum solubility in each medium: 38, 53,
−1
6
8, and 85 mM. 450 U mL of CPO was used. (A) Operational stability of soluble
CPO. (B) N-Cbz-3-aminopropanol consumption.
Figure 2. Operational stability of CPO in relation to initial N-Cbz-3-aminopropanol
concentration: 11 mM ( ), 21 mM ( ), 34 mM ( ), and 38 mM ( ). All reactions were
catalyzed in 5 mL of 100 mM sodium acetate buffer (pH 5.0) with the addition of 3 mM
−
1
−1
h t-BuOOH. 450 U mL of CPO was used.
Figure 3. Lineweaver–Burk plot from the Michaelis–Menten adjustment of CPO-
catalyzed oxidation of β-OH. Reactions were performed at initial substrate
concentrations of 10, 16, 19, 26, and 36 mM β-OH in 5 mL of 100 mM acetate buffer
−1
−1
(
pH 5.0) with the addition of 3 mM h t-BuOOH. 450 U mL of CPO was used.
Figure 4. Oxidation of N-Cbz-3-aminopropanol performed in 5 mL of 100 mM sodium
acetate buffer (pH 5.0) with initial substrate concentration set at 38 mM by addition of 3
−
1
−1
mM h t-BuOOH/H
2
O
2
. 450 U mL of CPO was used. (A) Operational stability of
CPO. t-BuOOH ( ); H
2
O
2
( ). (B) Substrate consumption and oxidized product
2 2
formation. t-BuOOH: β-OH ( ), β-CHO ( ), β-COOH ( ); H O : β-OH ( ), β-CHO ( ),
β-COOH ( )
Figure 5. Extent of the N-Cbz-3-aminopropanol oxidation: reaction carried out in 0.3
mL of 100 mM sodium acetate buffer (pH 5.0) with 15 mM β-OH and 350 mM t-
−
1
BuOOH as initial concentrations. 15,000 U mL of CPO was used. β-OH ( ), β-CHO
2
4

(
), β-COOH ( ), operational stability of CPO ( ). Inset: Measured selectivity for Cbz-
amino aldehyde (β-CHO) and Cbz-amino acid (β-COOH) products. Selectivity for
compound 6 (scheme 2) was calculated by difference.
Scheme legends
Scheme 1. CPO-catalyzed oxidation of N-Cbz-3-aminopropanol to N-Cbz-3-
aminopropanal and N-Cbz-3-aminopropanoic acid.
Scheme 2. Novel proposed reaction scheme for N-Cbz-3-aminopropanol oxidation
2 2
catalyzed by CPO with the addition of peroxide (H O /t-BuOOH)
2
5

*Graphical Abstract

TT aa bb ll ee ( 1s ) Process Biochemistry edited
Table 1. List of amino alcohols used as substrates in CPO-catalyzed oxidation reactions
1
2
#
Amino
alcohol
NH–carbon
R
R
n
configuration
1
α
β
β
β
β
γ
H
H
Cbz
Cbz
H
1
2
2
2
1
3
4
5
2
.a
.b
.c
.d
2
H
2
CH
3
H
2
CH
OH
2
Cbz
Fmoc
Cbz
Cbz
3
4
5
H
H
H
δ
ε

Table 2
Table 2. Amino alcohol oxidations catalyzed by CPO using H O or t-BuOOH as
2
2
-1
oxidant at an addition rate of 3 mM h in 5 mL of monophasic medium with 100 mM
acetate buffer of pH 5.0 (ethyl acetate 10%, v/v).
#
Amino
Oxidant: H
2
O
2
Oxidant: t-BuOOH
1

alcohol
1
r0
Conversion CPO t1/2 [h]
r0
Conversion
CPO t1/2 [h]
-
1
[a]
-1
[a]
(
μmol h )
[%]
(μmol h )
[%]
2.2
1.2
1.6
16.6
0
24.4
9.7
44.0
25.7
0
2.0
1.3
7.0
2.5
5.6
0.9
0.9
6.0
3.3
2.5
7.8
4.6
0
18.8
10.9
83.6
77.9
0
0.9
0.8
3.1
3.0
1.0
1.1
1.1
1.0
2
.a
.b
.c
.d
2
2
2
3
4
5
[
[
[
b]
b]
b]
0
0
0
0
1.1
0.1
16.3
12.3
1.3
0.1
16.8
9.4
[
a]
Substrate conversion was determined at the reaction end point by HPLC analysis of
the corresponding amino alcohols.
[b]
Initial reaction rate (r ) values were normalized according to initial substrate
0
concentrations: 5 mM for compounds 1–2.d; 0.5 mM for compounds 35.