Galactose Oxidase Variants for the Oxidation of Amino Alcohols in Enzyme Cascade Synthesis

Article abstract of DOI:10.1002/cctc.201500218

The use of selected engineered galactose oxidase (GOase) variants for the oxidation of amino alcohols to aldehydes under mild conditions in aqueous systems is reported. GOase variant F₂ catalyses the regioselective oxidation of N-carbobenzyloxy (Cbz)-protected 3-amino-1,2-propanediol to the corresponding α-hydroxyaldehyde which was then used in an aldolase reaction. Another variant, M_3-5, was found to exhibit activity towards free and N-Cbz-protected aliphatic and aromatic amino alcohols allowing the synthesis of lactams such as 3,4-dihydronaphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-pot tandem reactions with xanthine dehydrogenase (XDH) or aldehyde oxidase (PaoABC).

Full text of DOI:10.1002/cctc.201500218

DOI: 10.1002/cctc.201500218
Communications
Galactose Oxidase Variants for the Oxidation of Amino
Alcohols in Enzyme Cascade Synthesis
Susanne Herter,[a] Shane M. McKenna,[b] Andrew R. Frazer,[a] Silke Leimkühler,[c]
Andrew J. Carnell,*[b] and Nicholas J. Turner*[a]
The use of selected engineered galactose oxidase (GOase) var-
iants for the oxidation of amino alcohols to aldehydes under
mild conditions in aqueous systems is reported. GOase variant
F2 catalyses the regioselective oxidation of N-carbobenzyloxy
(Cbz)-protected 3-amino-1,2-propanediol to the corresponding
a-hydroxyaldehyde which was then used in an aldolase reac-
tion. Another variant, M3–5, was found to exhibit activity to-
wards free and N-Cbz-protected aliphatic and aromatic amino
alcohols allowing the synthesis of lactams such as 3,4-dihydro-
naphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-
pot tandem reactions with xanthine dehydrogenase (XDH) or
aldehyde oxidase (PaoABC).
Scheme 1. Galactose oxidase (GOase) catalysed oxidation of N-protected
(PG) or free amino alcohols to amino aldehydes in combination with aldo-
lase or xanthine oxidoreductase (XOR) for the synthesis of the amino sugars
and lactams.
enzyme reactions for oxidative cyclisation of unprotected
amino alcohols to give lactams (Scheme 1).
In a move towards designing more sustainable chemistry for
the future, the selective oxidation of alcohols to their more ac-
tivated carbonyl products is a fundamental and central reac-
tion in organic synthesis.[1] Many stoichiometric reagents and
catalysts have been developed, although usually with a view
to single step reactions rather than compatibility with other re-
agents/catalysts for multistep cascade sequences.[2] Additional
challenges are regiocontrol in the oxidation of polyhydroxylat-
ed compounds and also control of the level of oxidation.[3]
Thus, selective chemical oxidation of a diol to an a-hydroxyal-
dehyde may be difficult to achieve in high yield under condi-
tions that would be compatible with subsequent reactions. In
addition, the chemoselective oxidation of amino alcohols and
diols usually requires prior N-carbamoyl/amide protection to
prevent the more reactive amine undergoing oxidation.[4] In
this paper we report enzymatic cascade oxidations using
evolved variants of galactose oxidase (GOase). We show that
the aldehyde products obtained can be directly combined
with both aldolases, for CÀC-bond formation, and in tandem
The aldolase mediated synthesis of amino sugars (iminocycli-
tols) generally N-Cbz-protected amine or azide containing ac-
ceptor aldehydes for coupling with dihydroxyacetone phos-
phate (DHAP).[4c,d,5] However, N-protected acceptor aldehydes
are not commercially available and hence are typically synthes-
ised by chemical oxidation of the N-Cbz-protected aminol in-
volving either (i) trichloroisocyanuric acid/TEMPO/CH2Cl2,[6a]
(ii) IBX/organic solvent/reflux[6b] or (iii) a Dess–Martin periodi-
nane reaction.[5a] The current trend in favour of greener reac-
tions has highlighted biocatalytic methods as a favourable al-
ternative for the synthesis of pharmaceutical building blocks
particularly where multiple protection steps or the use of haz-
ardous reagents are involved. Mifsud et al reported a biocata-
lytic approach towards the oxidation of a N-Cbz-protected
amino alcohol (N-Z-ethanolamine, 5a) using a laccase/TEMPO
mediator system.[7] The product of this reaction has been used
with DHA-dependent fructose-6-phosphate aldolase to pro-
duce an aldol product which was then cyclised by hydrogena-
tion yielding a five-membered iminocyclitol. Notwithstanding
the advantages of laccase-mediator systems (LMS), drawbacks
have to be taken into account in terms of catalytic efficiency,
lack of full mediator regeneration and a possible over-oxida-
tion of aldehydes to carboxylic acids.[8] Moreover, LMS catalyses
the oxidation of alcohols with poor regioselectivity so would
not be an ideal choice for the oxidation of vicinal diols to for
example, an a-hydroxyaldehyde product.
[a] Dr. S. Herter, Dr. A. R. Frazer, Prof. N. J. Turner
School of Chemistry, Manchester Institute of Biotechnology
University of Manchester
131 Princess Street, M1 7DN Manchester (United Kingdom)
E-mail: nicholas.turner@manchester.ac.uk
[b] S. M. McKenna, Dr. A. J. Carnell
Department of Chemistry, Robert Robinson Laboratories
University of Liverpool
Crown Street, L69 7ZD Liverpool (United Kingdom)
E-mail: a.j.carnell@liverpool.ac.uk
We have previously developed variants of GOase, such as
[9]
M3–5, which is able to oxidise both primary and secondary al-
[10]
cohols, as well as GOase F2 which has an increased activity
[c] Prof. S. Leimkühler
towards the non-natural substrate glucose. To determine
whether these galactose oxidase variants possessed activity to-
wards amino alcohols and diols, we firstly examined the wild-
type (wt) enzyme as well as variants M3–5 and F2 towards a set
Institute of Biochemistry and Biology, University of Potsdam
Maulbeerallee 2,d-14476 Potsdam (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500218.
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Table 1. Specific activities of wild-type (wt) GOase and variants M3–5 and
F2 towards free and N-Cbz-protected amino alcohols. Cbz=carbobenzy-
loxy.
Table 2. Kinetic constants determined for GOase variant F2 towards (S)-
and (R)-1, rac-1, rac-2 and of variants F2 and M3–5 towards 5a.[a]
Compound
Variant
Km
[mM]
Vmax
[U mgÀ1
kcat
[sÀ1
kcat/Km
[sÀ1 mMÀ1
]
]
]
(S)-1
(R)-1
rac-1
rac-2
5a
F2
F2
F2
F2
F2
M3–5
7.66
8.13
6.42
7.41
167.81
67.25
0.027
0.019
0.019
0.149
1.87
0.031
0.022
0.022
0.171
2.13
0.004
0.003
0.003
0.023
0.013
0.046
5a
2.71
3.09
[a] Conditions: ABTS/horseradish peroxidase-coupled assay (200 mL) in
NaPi (50 mm, pH 7.4) with substrates (S)-1, (R)-1, rac-1 and rac-2 dissolved
in NaPi and substrate 5a dissolved in DMSO (co-solvent concentration
thoroughly adjusted to 6%, v/v), l=420 nm, 308C (Figure S8–S13 in the
Supporting Information).
[a]
Specific activity [mU mgÀ1
GOase M3–5
]
Compound
GOase wt
GOase F2
Galactose
(S)-1
(R)-1
rac-2
4a
4b
4c
4d
5a
310
0
0
0
0
0
0
0
0
6
7
35
49
14
13
31
19
330
30
41
142
22
20
46
60
127
35
78
higher velocity (Vmax), turnover (kcat) and catalytic efficiency
(kcat/Km) for rac-2, highlighting that N-Cbz protection facilitates
a significant improvement in catalytic turnover. In contrast to
diol substrates 1 and rac-2, both variants revealed a notably
lower affinity (Km) towards N-Z-ethanolamine 5a. However,
M3À5 had a 2.5-fold higher affinity (Km) together with a 3.5-fold
elevated catalytic efficiency (kcat/Km) in comparison to F2.
154
132
132
5b
5c
0
0
[a] Conditions: ABTS/horseradish peroxidase-coupled assay (200 mL) in
NaPi (50 mm, pH 7.4) with substrates applied in 5 mm concentration (5%
v/v DMSO), l=420 nm, 308C.
Encouraged by the kinetic studies, variant F2 was applied to
the oxidation of diol rac-2 to give a-hydroxyaldehyde rac-3
and variant M3–5 was used for the oxidation of alcohol 5a to
give 6a on analytical scale. High (78%, [S]=20 mm) to quanti-
tative conversions (100%, [S]=7.5 mm) of rac-2 were obtained
depending on initial substrate concentrations (Table S1, Fig-
ure S18 in the Supporting Information). Despite improved ki-
netic constants of GOase M3–5 towards 5a, compared with var-
iant F2 (cf. Table 2), this amino alcohol was transformed with
lower conversions (75–43%, [S]=7.5-20 mm) (Table S2, Fig-
ure S18 in the Supporting Information). In order to exemplify
the potential application of GOase variant F2 as a biocatalyst
for the synthesis of a-hydroxyaldehyde rac-3 from N-Cbz-pro-
tected amino diol rac-2, analytical scale reactions ([S]=7.5 mm,
48 h) were supplemented with 1 equiv. of DHAP and rabbit
muscle aldolase (Scheme 2). After 18 h, LC/MS analysis revealed
a new peak possessing a m/z of 392.0 [MÀH+] which corre-
sponds to the phosphorylated aldol product 3(S), 4(R), 5(S,R)-7
as a mixture of diastereoisomers (m/ztheoret. 393.28) (Figure S19
in the Supporting Information). An adjustment of the pH value
to pH 4.8 and addition of acid phosphatase resulted in com-
plete consumption of the previously observed aldol product
peak. Subsequent hydrogenation according to Concia et al.[5a]
produced a peak which mass (m/z 208.3 [M+H]+, m/z 230.2
[M+Na]+) corresponded to an authentic standard of the enan-
tiopure amino sugar N-(2-hydroxyethyl)-1-deoxynojirimycin
(C5=S) (m/ztheoret. 207.11; Figure S20,21 in the Supporting Infor-
mation) present as one of the diastereoisomers of 9.
of amino alcohols and diols with or without N-Cbz-protection
(Table 1). Wild-type GOase showed no activity towards any of
the various amino alcohols. However, GOase enzyme variants
M3–5 and F2 showed good to moderate activity against all of
the compounds investigated. Both variants revealed activity
against enantiomeric diols (S)- and (R)-1. GOase F2 showed
higher activity against both enantiomers than M3–5; however
the latter enzyme was more enantioselective. Also, aliphatic
amino alcohols 4a–d showed slightly higher activity with var-
iant F2 and this was more pronounced with the longer chain
substrate 4d. In general, initial investigations clearly indicated
N-Cbz-protection to improve oxidation of aliphatic alcohol
moieties (rac-2, 5a–c) as compared to non-protected deriva-
tives, with variant F2 having almost 3-times the specific activity
towards diol rac-2 compared to M3–5. However, GOase variant
M3À5 exhibited highest specific activities towards N-Cbz-pro-
tected alcohols 5a–c. Also of note for this variant was a 26-
fold increase in activity relative to the activity towards the nat-
ural substrate galactose.
To confirm these preliminary findings, kinetic constants for
the GOase variant F2 towards (S)- and (R)-1, rac-1 as well as
rac-2 were measured. In addition, kinetic studies for substrate
5a with both F2 and M3–5 were carried out (Table 2). Interest-
ingly, GOase F2 displayed similar affinities (Km) and catalytic effi-
ciencies (kcat/Km) for diols (S)- and (R)-1 as well as rac-1, con-
firming that F2 does not differentiate between these enantio-
mers. N-Cbz-protection present in substrate rac-2 did not lead
to a remarkable change of affinity (Km) compared to non-pro-
tected substrates 1. However, we identified an almost 8-fold
In the present work, the synthesis of other amino sugars
was not pursued although we believe that either of the GOase
enzymes would be suitable for the generation of aldolase ac-
ceptor aldehydes from amino alcohols 1–5 based on enzyme
activity profiles determined (cf. Table 1).
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We have previously demon-
strated that 3,4-dihydroisoquino-
line (DHIQ) 18 is an excellent
substrate for E. coli XDH when
used in a cascade with mono-
amine oxidase (MAO-N) which
catalyses the oxidation of tetra-
hydroisoquinolines.[9c] Hence, we
tested (2-(2-aminoethyl)phenyl)-
methanol 16 as a substrate for
the GOase M3–5-XDH cascade as
a route to 3,4-dihydroisoquino-
lin-1(2H)-one 19 (Scheme 4). We
found that the oxidation of 16
by GOase M3–5 was remarkably
Scheme 2. Galactose oxidase F2 catalysed oxidation of N-Cbz-3-amino-1,2-propanediol rac-2 to aldolase acceptor
aldehyde rac-3 and subsequent steps for the synthesis of the amino sugar 9 (C5=S and R). Cbz=carbobenzyloxy.
Oxidative
cyclisations
of
amino alcohols to give benzo-
fused or simple aliphatic lactams
have previously been reported
using Cp*Rh[11] or a Ru com-
plexes respectively.[12] These re-
Scheme 4. GOase M3–5-XDH catalysed synthesis of 3,4-dihydroisoquinolin-1(2H)-one 19.
actions were carried out in or-
ganic solvents and required
harsh conditions of temperature and pressure. Having estab-
lished the oxidation of amino alcohols by engineered variants
of GOase we considered reaction sequences in which the ini-
tially formed amino aldehyde product could undergo cyclisa-
tion to generate an intermediate imine followed by subse-
quent oxidation to form a lactam. For the second step we tar-
geted molybdenum-dependent enzymes known as xanthine
oxidoreductases (XORs), including xanthine dehydrogenase/ox-
idase (XDH/XO) and aldehyde oxidase (PaoABC). From drug
metabolism studies it is known that XORs have the ability to
catalyse the oxidation of aldehydes to acids and cyclic imines
to lactams.[13]
pH sensitive within the range pH 7.0–8.5, with a pH of 7.0 re-
quired to achieve full conversion to DHIQ 18 (Table S4 in the
Supporting Information). Formation of 18 is presumed to
occur by cyclization of the initially formed amino aldehyde 17.
GOase M3–5 and E. coli XDH were then combined in a one-
pot reaction at pH 7.0 for the direct synthesis of the lactam 19
with an overall conversion of 69%. HPLC analysis showed that
DHIQ 18 was only present in trace amounts under the cascade
conditions and was consumed immediately by XDH upon for-
mation (Figure S23 in the Supporting Information). NMR analy-
sis of DHIQ 18 in deuterated potassium phosphate buffer at
pH 6.0–8.0 indicated no amino aldehyde or DHIQ hemi-aminol
present under the conditions employed (Figure S7 in the Sup-
porting Information). Only DHIQ signals were observed which
suggests that DHIQ is the true substrate for XDH rather than
the amino aldehyde. Under the cascade conditions,
Initially we investigated the GOase-XDH cascade for oxida-
tion of 2-(2-aminophenyl)ethan-1-ol 11 to give indan-2-one 15,
a core motif in a number of anticancer drugs (Scheme 3). The
GOase M3–5 oxidised amino alcohol 16 more slowly
than with GOase M3–5 alone in which over the same
time period DHIQ 18 was obtained in quantitative
conversion (Table S4 and S5 in the Supporting Infor-
mation). This difference is possibly due to the inhibi-
tion of the GOase M3–5 by the lactam product 19.
We then turned our attention to a series of aliphat-
ic amino alcohols 4b–d for cascade oxidative cyclisa-
tion reactions (Table 3). Both GOase M3–5 and F2 and
Scheme 3. Attempted GOase M3–5-XDH cascade to form indan-2-one 15.
three different xanthine oxidoreductase enzymes
were tested: bovine xanthine oxidase (XO), E. coli
xanthine dehydrogenase (XDH) and E. coli periplas-
GOase M3–5 catalysed step gave good to excellent conversions
(84–100%) to amino aldehyde 12 at [S]=1–5 mm with lower
conversions at [S]>7.5 mm (55% conversion; Table S3). How-
ever, the initially formed 3H-indole 13 readily tautomerised to
1H-indole 14 under the reaction conditions employed.
mic aldehyde oxidase (PaoABC).[14] Conversions were deter-
mined by HPLC (Figure S3 and S4 in the Supporting Informa-
tion). Neither the bovine XO nor the XDH enzyme gave any
lactam products at pH 7.0–8.5 (Table S6). However the combi-
nation of GOase M3–5 and PaoABC was more promising. Sub-
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GOase amino-aldehyde products, to afford valerolactam and 2-
pyrrolidone in up to 86% conversion.
Table 3. GOase-PaoABC cascade for the oxidative cyclisation of linear ali-
phatic amino alcohols 4b-d to give 2-pyrrolidone 20b and valerolactam
20c.[a]
Acknowledgements
The research leading to these results received support from the
Innovative Medicines Initiative Joint Undertaking under the grant
agreement no. 115360 (Chemical manufacturing methods for the
21st century pharmaceutical industries, CHEM21), resources of
which are composed of financial contribution from the European
Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA
companies’ in-kind contributions (to S.H), the Science and Indus-
try Endowment (SIEF, to A.R.F.), the Engineering and Physical Sci-
ences Research Council (EPSRC, to S.M.M. and A.J.C.) and The
Royal Society Wolfson Merit Award (to N.J.T.).
Entry
Substrate
GOase
pH
Conversion
20b–d [%][b]
1
2
3
4
5
6
7
8
4b
4b
4b
4b
4b
4c
4c
4c
4c
4c
4d
4d
M3–5
M3–5
M3–5
M3–5
F2
M3–5
M3–5
M3–5
M3–5
F2
7.0
7.5
8.0
8.5
8.5
7.0
7.5
8.0
8.5
8.5
8.5
8.5
10
41
56
85
22
trace
3
20
26
8
9
Keywords: aldehyde oxidase
· amino alcohols · cascade
10
11
12
reactions · enzyme catalysis · lactams
M3–5
F2
0
0
[a] Reaction conditions: 7 mm 4b–d, 103 mL GOase M3–5 or F2
(3.7 mgmLÀ1), 33 mL catalase (3.3 mgmLÀ1), 5 mL E. coli PaoABC
(13.3 mgmLÀ1), KPi buffer (300 mL final volume). [b] Yields calculated by
calibration curves of 2-pyrrolidone 20b and valerolactam 20c (Fig-
ure S3,S4 in the Supporting Information).
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(entry 6) but at pH 8.5 the conversion increased to 26%
(entry 9). With the shorter chain homologue 4b, the same pH
effect was evident (entries 1–4), however in this case the con-
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The longer chain substrate 4d (n=5) gave no conversion to
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compared with valerolactam 20c presumably reflects the more
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oxidation by GOase at a 2.5 fold higher rate compared to 4b
(cf. Table 1). For the cascade reaction GOase M3–5 is operating
at above its pH optimum whereas PaoABC is known to work
over a broad pH range. An alternative explanation is that the
increase in pH facilitates cyclisation of the assumed amino-al-
dehyde intermediate giving a higher concentration of the
imine for the PaoABC enzyme. We also tried the GOase F2
mutant with PaoABC which gave lower conversions for 4b and
c (entries 5 and 10) and no conversion for 4d (entry 12). Again
the results with 4b,c are contrary to what would have been ex-
pected from the specific activities of the GOase step (cf.
Table 1).
In summary the present work has identified two variants of
GOase that are useful biocatalysts for the oxidation of amino
alcohols and amino diols to produce aldehydes for subsequent
enzymatic transformation. GOase can be used with N-Cbz pro-
tected or free amino groups, affording flexibility in synthesis.
Furthermore, we have shown that a previously unexploited al-
dehyde oxidase, PaoABC from E. coli, can be used in a tandem
cascade to oxidise cyclic imines, formed by cyclisation of the
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Received: March 3, 2015
Published online on June 26, 2015
ChemCatChem 2015, 7, 2313 – 2317
www.chemcatchem.org
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