Amination of ω-Functionalized Aliphatic Primary Alcohols by a Biocatalytic Oxidation-Transamination Cascade

Article abstract of DOI:10.1002/cctc.201500589

Amination of non-activated aliphatic fatty alcohols to the corresponding primary amines was achieved through a five-enzyme cascade reaction by coupling a long-chain alcohol oxidase from Aspergillus fumigatus (LCAO-Af) with a ω-transaminase from Chromobacterium violaceum (ω-TA-Cv). The alcohol was oxidized at the expense of molecular oxygen to yield the corresponding aldehyde, which was subsequently aminated by the PLP-dependent ω-TA to yield the final primary amine product. The overall cascade was optimized with respect to pH, O₂ pressure, substrate concentration, decomposition of H₂O₂ (derived from alcohol oxidation), NADH regeneration, and biocatalyst ratio. The substrate scope of this concept was investigated under optimized conditions by using terminally functionalized C₄-C₁₁ fatty primary alcohols bearing halogen, alkyne, amino, hydroxy, thiol, and nitrile groups.

Full text of DOI:10.1002/cctc.201500589

DOI: 10.1002/cctc.201500589
Communications
Amination of w-Functionalized Aliphatic Primary Alcohols
by a Biocatalytic Oxidation–Transamination Cascade
[b]
[b]
[a, b]
[b]
Mathias Pickl, Michael Fuchs, Silvia M. Glueck,
and Kurt Faber*
th
Dedicated to Wolf-Dieter “Woody” Fessner on the occasion of his 60 birthday.
Amination of non-activated aliphatic fatty alcohols to the cor-
responding primary amines was achieved through a five-
enzyme cascade reaction by coupling a long-chain alcohol ox-
idase from Aspergillus fumigatus (LCAO_Af) with a w-transami-
nase from Chromobacterium violaceum (w-TA_Cv). The alcohol
was oxidized at the expense of molecular oxygen to yield the
corresponding aldehyde, which was subsequently aminated by
the PLP-dependent w-TA to yield the final primary amine prod-
uct. The overall cascade was optimized with respect to pH, O2
pressure, substrate concentration, decomposition of H O (de-
transaminases with other enzymes, such as acetohydroxyacid
synthase, transketolase, various hydrolases, and alcohol dehy-
drogenases enabled the synthesis of (chiral) amine deriva-
[
7]
tives.
The direct transformation of alcohols to amines is only feasi-
[
8]
ble by metal catalysts, no enzyme is known for this reaction.
However, biocatalytic two-step oxidation–reductive amination
sequences are known. Oxidation of an alcohol by an alcohol
dehydrogenase yields the corresponding aldehyde/ketone,
which can be reductively aminated by an w-transaminase. The
elegance of this redox-neutral process is the internal cofactor
recycling, in which NADH generated during alcohol oxidation
is employed in the reductive amination step. This concept has
been successfully applied to a broad range of linear and cyclic
aliphatic primary and secondary alcohols, aryl-alkanols, benzylic
2
2
rived from alcohol oxidation), NADH regeneration, and biocata-
lyst ratio. The substrate scope of this concept was investigated
under optimized conditions by using terminally functionalized
C4–C11 fatty primary alcohols bearing halogen, alkyne, amino,
hydroxy, thiol, and nitrile groups.
[
7]
alcohols, and a,w-diols for the synthesis of (di)amines.
Alcohol oxidases represent an attractive, but underrepre-
sented, alternative to (thermodynamically disfavoured) nicoti-
namide-dependent alcohol oxidation catalyzed by alcohol de-
hydrogenases. These enzymes are commonly flavin- or Cu-de-
Biocatalytic cascades have emerged as a time-, resource-, and
[1]
cost-saving strategy in bioorganic synthesis. The use of sever-
al enzymes in a one-pot fashion avoids purification/isolation of
[
9]
(
unstable) intermediates and the associated unavoidable loss
pendent and use O as an electron acceptor. Two-electron
2
of material. Numerous examples of multienzymatic processes
of ever increasing complexity for the production of valuable
transfer yields H O as a byproduct, which is destroyed by cata-
2
2
lase or by the horseradish peroxidase (HRP)/2,2’-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid (ABTS) system. This
method is a “green” alternative to traditional protocols, which
require transition metals, dimethylsulfoxide (e.g. Swern, Pfitz-
[
2]
[3]
compounds indicate that the areas of “systems biocatalysis”
[4]
and pathway engineering are beginning to merge. The syn-
thesis of amines dominates current cascade design because
the occurrence of amines is underrepresented in the pool of
renewable carbon sources, in contrast to their frequent need
[
10,11]
[12]
ner–Moffat oxidation),
or nitroxyl radicals (e.g. TEMPO).
Recently, we established a two-step one-pot oxidation–trans-
[5]
I
in chemical synthesis.
amination cascade based on Cu-dependent galactose oxidase
[
13]
For instance, terminal alkylamino functionalization of alkanes
and fatty acid methyl esters was achieved by combining an
alkane monooxygenase (AlkBGT) and a w-transaminase in
(GOase) in combination with a w-transaminase.
Dictated
by the substrate characteristics of GOase from Fusarium
NRRL 2903, only electronically activated benzylic and cinnamic
alcohols were accepted, and this method was not applicable
to nonactivated aliphatic (fatty) alcohols. To broaden the sub-
strate scope of this protocol, a search for a suitable alcohol ox-
idase revealed a putative flavin-dependent long-chain alcohol
oxidase from Aspergillus fumigatus (LCAO_Af) as a promising
[6]
a single designed whole-cell system. The coupling of a w-
[
a] Dr. S. M. Glueck
Austrian Centre of Industrial Biotechnology (ACIB GmbH)
Petersgasse 14, 8010 Graz (Austria)
[
14]
[
b] M. Pickl, Dr. M. Fuchs, Dr. S. M. Glueck, Prof. Dr. K. Faber
Department of Chemistry, Organic&Bioorganic Chemistry
University of Graz
candidate.
The enzyme shows homology and sequence
identity (30–40%) with other flavoprotein alcohol oxidases and
contains the conserved flavin-binding domain (pfam00732) of
the glucose–methanol–choline (GMC) oxidase family. LCAO
shows activity in the H O /HRP/ABTS and supplementary flavin
Heinrichstrasse 28, 8010 Graz (Austria)
Fax: (+43)316-380-9840
E-mail: Kurt.Faber@Uni-Graz.at
2
2
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500589.
adenine dinucleotide (FAD) enhances the activity of the
enzyme (data not shown). Preliminary results reported the oxi-
dation of C –C fatty alcohols yielded the corresponding alde-
ꢀ
2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
6
8
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
hydes. Reductive amination of aliphatic aldehydes through w-
[
15]
transaminases is well known. To shift the unfavorable equi-
ChemCatChem 2015, 7, 3121 – 3124
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Communications
conditions and at elevated
pressure, which revealed an
optimal performance at 2–
4
bar (Table S4).
7
) To evaluate the overall per-
formance of the optimized
cascade reaction compared
to the initial conditions, the
amine formation was fol-
lowed over time. Engineer-
ing of the reaction parame-
Scheme 1. Biocatalytic oxidation–transamination of w-functionalized nonactivated fatty alcohols.
librium towards the amine, the well-established l-alanine
donor system, employing an alanine dehydrogenase (Ala-DH)
combined with an adequate NADH-recycling system, complet-
ters led to a considerable improvement of the overall effi-
ciency with full conversion of 1-hexanol to the correspond-
ing amine within 10 h (Figure S4).
[16]
I
ed the overall cascade (Scheme 1). In contrast to Cu -depen-
dent GOase, where alcohol oxidation selectively stops at the al-
dehyde stage, undesired over oxidation of aldehydes to car-
boxylic acids (a common phenomenon for flavin-dependent al-
To evaluate the substrate tolerance (see Table 1) of the bio-
catalytic oxidation–transamination cascade reaction, primary al-
cohols with a chain length ranging from C –C and derivatives
4
11
[
17]
cohol oxidases) by LCAO_Af had to be taken into account.
bearing a halogen, alkyne, amino, hydroxy, thiol, and nitrile
group were subjected to the enzymatic amination cascade.
In the first screening, various unfunctionalized linear aliphat-
ic alcohols with a chain length ranging from four to eleven
carbon atoms were tested, and the performance was measured
as overall conversion to the corresponding amine 1b–8b
(Table 1, entries 1–8). Although short 1-butanol (1a) was con-
The overall performance of the cascade was tuned with re-
spect to the following parameters by using 1-hexanol as sub-
strate (detailed data are given in the Supporting Information):
1
2
) For the removal of H O , disproportionation catalyzed by
2 2
catalase from Micrococcus lysodeiktikus or two-electron
transfer mediated by HRP employing ABTS as an electron
sink worked equally well. The latter (chromophoric) ap-
proach has advantages in screening conditions, whereas
the former is more suitable for preparative-scale transfor-
mations (Supporting Information, Table S1).
Table 1. Substrate scope of the biocatalytic oxidation–transamination
cascade.
) For the reductive amination step, various nicotinamide co-
factor recycling systems based on glucose/glucose dehy-
drogenase (GDH), formate/formate dehydrogenase (FDH),
and phosphite/phosphite dehydrogenase (PtDH) were com-
pared: The phosphite/PtDH system was the least efficient,
whereas the formate/FDH system led to significant over ox-
idation of the aldehyde intermediate to yield the undesired
carboxylic acid. The best results were obtained with glu-
cose/GDH by using standard conditions, but this result was
not further optimized (Table S2).
Entry
Substrate
n
Product
[%]
Byproduct
[%]
[a]
R
1
2
3
4
5
6
7
8
9
10
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11 a
12a
13a
14a
15a
16a
3
4
5
6
7
8
9
10
5
5
7
5
5
5
5
5
H
H
H
H
H
H
H
H
1b
2b
3b
4b
5b
6b
7b
8b
20
75
>99
>99
>99
82
46
16
74
98
1c
2c
3c
4c
5c
6c
7c
8c
<1
<1
<1
<1
<1
5
[b]
[
[
[
c]
c]
c]
3
4
5
6
) A significant advantage of separate overexpression of the
oxidase and the w-transaminase is the possibility to adjust
the ratio of both enzymes. The best results were obtained
when LCAO Af and w-TA Cv (employed as whole lyophi-
lized cells) were used in a ratio of 2:1 (Table S3).
20
26
Br
Cl
Cl
9b
9c
<1
<1
<1
<1
<1
n.c.
n.c.
n.c.
10b
11 b
12b
13b
14b
15b
16b
10c
11 c
12c
13c
14c
15c
16c
1
1
96
42
>99
1
1
1
2
3
4
CꢁN
) The pH profile was investigated within a range of pH 5–12
CꢁCH
(
Figure S2), which revealed that the efficiency of the cas-
NH
2
n.c.
n.c.
n.c.
cade continuously increased from pH 6 (conversion <2%)
to a maximum at pH 10, followed by a sharp drop at
pH 12.
15
OH
SH
16
Reaction conditions: Sodium phosphate buffer (100 mm, pH 10), sub-
+
strate (10 mm), l-alanine (100 mm), glucose (80 mm), NAD (2 mm), NH
67 mm), PLP (2 mm), FAD (1 mm), LCAO_Af (40 mg whole lyophilized
E. coli cells), w-TA_Cv (20 mg whole lyophilized E. coli cells), GDH (2 U),
Ala-DH (0.013 U), catalase (1700 U), 20 h at RT, 170 rpm and 2 bar O
4
Cl
) The efficiency of the cascade was very sensitive towards
elevated product concentrations, deduced from the fact
that increasing amounts of substrate (10–75 mm) led to
a steady decrease in conversion and gave comparable
levels of product (ꢀ18–20 mm, Figure S3).
(
2
;
n.c.=no conversion, [a] conversion refers to the formed amine as deter-
mined by GC-MS analysis after derivatization; [b] used in optimization
studies; [c] owing to peak tailing, the accuracy for the determination of
the corresponding carboxylic acid was Æ5%.
) Since the solubility of the oxidant O in aqueous systems is
2
limited, experiments were conducted under atmospheric
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Communications
verted only moderately (conv.=20%), significantly enhanced
amine formation was observed by increasing the chain length,
leading to quantitative conversions for C –C alcohols (3a–5a,
entries 3–5). This effect nicely correlates with the fact that
a “long-chain” alcohol oxidase was used in the oxidation step.
Beyond this range, conversions gradually decreased again and
reached only 16% for 1-undecanol (8a). The decrease in amine
formation for long-chain alcohols was accompanied by signifi-
cant over oxidation to the corresponding carboxylic acid (6c–
(100 mm), ammonium chloride (67 mm), and d-glucose (80 mm) in
sodium phosphate buffer (500 mL, 100 mm, pH 10.0) was added.
Alanine dehydrogenase (Ala-DH) from B. subtilis (0.013 U), glucose
dehydrogenase (GDH from DSM, 2 U), and catalase (1700 U) from
M. lysodeikticus were added. Finally, the substrate (10 mm) was
added and the reaction mixture was placed into an oxygen pres-
6
8
[19]
sure chamber. The apparatus was primed with oxygen (technical
grade) for about 1 min and pressurized to 2 bar. The reaction mix-
ture was shaken at RT and 170 rpm for 24 h. The conversion was
determined by GC-MS analysis after derivatization of the corre-
sponding primary amine with ethyl(succinimidooxy)formate (see
the Supporting Information).
8
c), which indicates that long-chain aldehydes are less well ac-
cepted by the w-TA, in addition to possible solubility issues.
Various functional groups were introduced at the C-terminal
end of 1-alkanols, encompassing halogen, amino, alkyne, thiol,
and nitrile functionalities (entries 9–16). For the w-halo-1-alco-
hols, good-to-excellent conversions were obtained [74% for 9a
Acknowledgements
Funding by the Austrian Science Fund (FWF) within the DK Mo-
lecular Enzymology (project W9) and an Erwin-Schrçdinger fel-
lowship (J3466), and the Austrian BMWFW, BMVIT, SFG, Standor-
tagentur Tirol, Government of Lower Austria and ZIT through the
Austrian FFG-COMET-Funding Program is gratefully acknowl-
edged.
(
entry 9) and nearly full conversion for the C and C chloro an-
6 8
alogues 10a and 11a (entries 10, 11). The introduction of a ter-
minal nitrile group possessing similar properties as a halogen
group (i.e. a pseudohalogen) led to a significant decrease in
conversion (12a, conv. 42%, entry 12), whereas a terminal eth-
ynyl group of similar size resulted in full conversion (13a,
conv.>99%, entry 13).
The limitations of the cascade are set by the incompatibility
of amino, hydroxy (diol), and thiol substituents (14a–16a, en-
tries 14–16). Steric factors can be neglected, therefore, the
nonacceptance of substrates bearing polar end groups is most
likely because of their heavy hydration in aqueous medium.
The same is true for secondary aliphatic and activated benzylic
or allylic alcohols, which were not converted independent of
their E/Z configuration (Figure S5).
Keywords: alcohol oxidase
cascade · transaminase
· amination · biocatalysis ·
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3124ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim