Amino Aldehydes Revisited

Article abstract of DOI:10.1002/ejoc.201701213

The enzymatic oxidation of amino alcohols was studied to address the long-standing problem of product stability. Amino aldehydes, highly sought and unstable compounds, can be generated under mild conditions if they are immediately protected. Utilizing a r

Full text of DOI:10.1002/ejoc.201701213

A Journal of
Accepted Article
Title: Amino aldehydes revisited
Authors: Luuk Mestrom, Paula Bracco, and Ulf Hanefeld
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10.1002/ejoc.201701213
European Journal of Organic Chemistry
FULL PAPER
Amino aldehydes revisited
Luuk Mestrom*, Paula Bracco and Ulf Hanefeld*
Abstract: The enzymatic oxidation of amino alcohols was
studied to address the long-standing problem of product stability.
Amino aldehydes, the much sought after and unstable
compounds, can be generated under mild conditions when they
2-
O
a
b
HO
PtCl6H2
NH3
PtCl6
OH
2
NH2
are immediately protected. Utilizing
a range of alcohol
glycinal hydrate chloroplatinate
glycinal
dehydrogenases (ADHs) and semicarbazide as scavenger, the
enantioselective synthesis of protected amino aldehydes is
possible. Glycerol dehydrogenase from Gluconobacter oxydans
(GoGDH) displayed excellent enantioselectivity but limited
substrate scope, while horse liver ADH catalyzed a broad range
of conversions with low enantioselectivity.
OH
1/2 O2
O
O
- 2H2O
N
HN
N
N
NH
OH
N
- H2O
NH2 NH2
pyrazine
glycinal
piperazine-
2,5-diol
2,5-dihydro
pyrazine
Introduction
HO
H
N
c
O
H
N
H
- H2O
N
N
The synthesis of unprotected chiral α-amino aldehydes is a
formidable challenge in organic chemistry, in particular due to
their chemical and optical instability. Not surprisingly, the history
of unprotected α-amino aldehydes can be traced back for over a
century.[1] In a predecessor of this journal, H. E. Fischer reported
the isolation of the simplest α-amino aldehyde glycinal hydrate
as a chloroplatinate salt in 1893, as is shown in Scheme 1a.[2] If
not kept under acidic conditions glycinal dimerizes into 2,5-
dihydropyrazine followed by air oxidation into pyrazine (Scheme
1b).[3] Besides the instability of α-amino aldehydes,
intermolecular assembly toward thermodynamically favored
multimers has also been reported for other amino aldehydes.[4]
For instance, γ-amino butyral cyclizes to δ-pyrroline which
exclusively forms trimers in solution (Scheme 1c).[5] For these
reasons, little attention has been given to the production of
unprotected amino aldehydes since they are regarded as
‘impractical as substrates because they are self-reactive’.[6]
Bench-stable amphoteric α-amino aldehydes mask their
unstable functional groups via the formation of an intramolecular
equilibrium, i.e. hemiacetals,[7] as observed with 2-deoxy-2-
amino sugars.[8] Intermolecular masking with an excess of a
nucleophile also enhances the stability of amino aldehydes,
yielding again acetals, cyanohydrins or amine derivatives.[9]
Furthermore, in acidic media optically active α-amino aldehydes
form the ammonium ion and hydrate via the addition of the weak
nucleophile water; no racemization of the α-amino aldehyde
hydrate has been observed.[3b]
N
N
H
NH2
amino
butyraldehyde
pyrroline
dimer
pyrroline
trimer
pyrroline
Scheme 1. First synthesis of α-amino aldehyde glycinal via the formation of
ammonium ion and gem-diol in acidic aqueous media (a) and exemplary
degradation pathways of unprotected amino aldehydes. The intramolecular
condensation followed by oxidative aromatization into pyrazine (b), and the
assembly of larger multimers of cyclic γ-amino aldehyde (c) are depicted.
Alcohol dehydrogenases (ADHs) are nicotinamide
cofactor-dependent oxidoreductases which catalyse the
reversible chemo-, regio-, and stereospecific oxidation of
alcohols to aldehydes under mild reaction conditions.
Subsequent addition of water to the aldehyde results in the
hydrate, frequently again accepted by the enzyme as substrate.
Consequently the corresponding acid is often isolated as the
main product.[10] Moreover, the rate of oxidation is proportional to
the degree of hydration, with α-amino aldehydes typically
showing high hydration constants in water.[11]
The use of HLADH (crude horse-liver extracts) with high
enantioselectivity was reported for the oxidation of β-amino
alcohols for the production of (S)-serine from prochiral serinol
with co-immobilized aldehyde dehydrogenase[12], as were the
enantioselective conversion of alaninol[13], histidinol[14], as well as
phenylglycinol.[3c] To prevent the 4-electron oxidation of β-amino
alcohols to α-amino acids the abovementioned ability of
nucleophiles to stabilize the amino aldehydes was utilized. The
strong nucleophile semicarbazide was employed in aqueous
media for the enzymatic oxidation of enantiopure
(S)-phenylalaninol
to
(S)-phenylalaninal,
producing
(S)-phenylalaninal semicarbazone.[15]
Luuk Mestrom, Dr. Paula Bracco and Prof. Ulf Hanefeld.
Biokatalyse, Afdeling Biotechnologie.
Technische Universiteit Delft
Van der Maasweg 9, 2629HZ Delft (The Netherlands)
E-mail: L.Mestrom@tudelft.nl, U.Hanefeld@tudelft.nl
The mild aqueous conditions employed for ADHs and the
use of semicarbazide as an α-amino aldehyde scavenger
attracted our attention.[11,15] This should enable the first
asymmetric oxidation of rac-β-amino alcohols to chiral α-amino
aldehydes, as is shown in scheme 2. In this study, we report a
HPLC-based screening for the asymmetric oxidation of
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10.1002/ejoc.201701213
European Journal of Organic Chemistry
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rac-phenylalaninol 1 in the presence of semicarbazide. First, 1
was asymmetrically oxidized to, i.e. (R)-phenylalaninal 2, using a
commercially available ADH screening kit (17 different ADHs;
Evocatal) and NAD(P)H oxidase as regeneration system.
Second, in the presence of semicarbazide the produced α-amino
ADH
Conv. (%)
ee 1 (%)b
E-value 1c
rac-1a
010
020
n.d.
n.d.
-
-
-
-
aldehyde
2
was in-situ converted to (R)-phenylalaninal
030
63 ± 2d
9 ± 6 d
n.d.
68 (S)
4
2
-
semicarbazone 3, preventing a second 2-electron oxidation of 2
to (R)-phenylalanine 4. The study was then extended to the
conversion of a broader range of amino alcohols with potential
synthetic applications, such as β-, γ-, δ- and ε- amino alcohols.
040
3 (S)
130
-
140
n.d.
-
99 (R)
-
-
OH
O
O
190 (GoGDH)
200
53 ± 2
n.d.
80
-
H2N
ADH
H2N
ADH
NAD(P)+
H2N
H
OH
NAD(P)+
NAD(P)H
NAD(P)H
NADH
oxidase
NADH
oxidase
210E
(HLADH-E )
47 ± 4e
22 (S)e
2e
1
2
4
O2
O2
H2O
H2O
-H2O
260
270
380
420
430
440
441
442
29 ± 6
n.d.
-
-
-
H
N
NH2
H2N
-
-
O
n.d.
-
H
H2N
N
NH2
N
7± 1
8 (R)
-
10
-
O
3
n.d.
42 ± 1f
48 ± 1d
9± 3
19 (R)f
35 (R)
3 (R)
2f
3
2
Scheme 2. Asymmetric enzymatic oxidation of rac-phenylalaninol 1 with
ADHs to the corresponding α-amino aldehyde 2 and the non-enzymatic
trapping to α-amino semicarbazone 3 in the presence of semicarbazide.
Undesirably, the second enzymatic oxidation leads to the formation of the
α-amino acid 4.
n.d. = not detected
a calculated after 24 h of reaction
b %ee 1 = 100(R – S)(R – S)-1
c E-value 1 = (ln[(1-conv)(1-ee1)]) (ln[(1-conv)(1+ee)])-1
dcalculated after 48 h of reaction
Results and Discussion
First,
a
wide range of commercially available alcohol
e calculated after 2 h of reaction
dehydrogenases (EvocatalTM; Table S1&S2) were evaluated for
the enantioselective oxidation of rac-phenylalaninol 1 in the
presence of semicarbazide by chiral HPLC analysis. From the
17 ADHs that were screened, nine catalysed the conversion of 1
within 48 hours (Table 1 and Fig. S1-S10). In detail, commercial
enzymes 030 and 040 showed poor selectivity towards the
(S)-enantiomer, with moderate and poor conversion respectively.
Additionally, ADH isoenzyme E from Equus caballus (HLADH-E)
showed quantitative conversion, however with poor
enantioselectivity towards the (S)-enantiomer (Fig. S7). The
enantiopreference of HLADH-E for (S)-β-amino alcohols has
been reported earlier for other substrates.[3c, 13] To our delight,
glycerol dehydrogenase from Gluconobacter oxydans (GoGDH)
showed moderate conversion but excellent enantioselectivity for
the (R)-enantiomer. In line with previous observations for
rac-glyceraldehyde, GoGDH shows (R)-selectivity.[16]
f calculated after 6 h of reaction
In order to rigorously establish the enantiomeric excess of the
α-amino aldehyde and α-amino semicarbazone the reference
standards were synthesized according to Scheme 3. Dess-
Martin periodinane (DMP) oxidation of Cbz-(S)-phenylalaninol 5
resulted in 6 with an isolated yield of 74%. To our advantage,
two sequential column purifications with silica gel partially
racemized Cbz-(S)-phenylalaninal
determination of the enantiomers’ retention times as well as their
quantitative calibration for further chiral HPLC analysis (Fig.
6
allowing both the
S33). Deprotection of
6 under acidic conditions showed
quantitative conversion to the stable trifluoroacetate salt of
(S)-phenylalaninal hydrate 7. The deprotection was performed in
deuterated solvents allowing in-situ NMR analysis. Upon
removal of the solvent the α-amino aldehyde 7 degrades into
multiple unidentified side products. However, under acidic
aqueous conditions 7 shows great stability with no degradation
for more than 30 days. Subsequently, the addition of
Table 1. Chiral HPLC screening for the asymmetric oxidation of
rac-phenylalaninol 1 with EvocatalTM ADH screening kit. Conversion and
enantiomeric excess was calculated based on substrate consumption.
Conversion is expressed as average ± standard deviation (n = 3). Reaction
semicarbazide
solution
produces
(S)-phenylalaninal
conditions:
1 mL reaction volume containing 70 mM glycine, 105 mM
semicarbazone 8 quantitatively.
semicarbazide and the internal standard para-toluene sulfonic acid, adjusting
the pH to 9 with aq. NaOH. 1.50 mM rac-phenylalaninol, 2 mM NAD(P)+, 5.0
mg mL-1 NADH oxidase (4.0 U mg-1), and 1.5 mg mL-1 ADH were added and
the mixture incubated at 30 °C at 800 rpm.
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10.1002/ejoc.201701213
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FULL PAPER
52% conversion (R)-phenylalanine 4 is produced with 94% ee
within 6 hours. The enantiomeric excess obtained was the same
as in presence of semicarbazide, indicating little product
racemization occurred by the action of the nucleophilic agent
semicarbazide in-situ. Furthermore, HPLC analysis showed no
free (R)-phenylalaninal in solution (detection limit < 5 µM). Thus
the addition of a nucleophile such as semicarbazide can
effectively be used to change the outcome of the reaction. Either
a chiral α-amino aldehyde is trapped or the oxidation proceeds
towards the acid.
Pd/C, H2
O
H
HO
DMP
1.05 eq. TFA
DCM, r.t.
NHCbz
NHCbz
6
5
74% isolated yield
D2O: D8-dioxane
(1:1)
O
O
H
N
N
D
H2N
ND2
DO
OD
N
NH2
H
ND
3 TFA
TFA
ND3
D2O: D8-dioxane
(3:1)
8
7
>99% conversion
35% ee
>99% conversion
35% ee
Scheme 3. Chemical synthesis of the reference compounds (S)-(+)-
phenylalaninal hydrate 7 and (S)-(-)-phenylalaninal semicarbazone 8 from N-
Cbz-phenylalaninol 5.
With the reference standards in hand, the enantioselective
oxidation of rac-phenylalaninol 1 to (R)-aldehyde 2 catalyzed by
GoGDH was investigated in more detail (Fig. 1). After 6 hours of
reaction time, 44% conversion with 94% ee for (R)-1 was
observed, corresponding to E = 72. Importantly, racemization via
enolization of the (R)-α-amino semicarbazone 3 and (R)-alanine
4 did not occur over 24 hours of reaction in aqueous media. The
free (R)-aldehyde 2 was efficiently scavenged from solution
using a large molar excess of semicarbazide (> 100). Indeed,
81% of the (R)-2 was efficiently scavenged from the aqueous
solution with semicarbazide yielding (R)-3, while only 19%
overoxidation to (R)-alanine 4 occurred.
Figure 2. Asymmetric oxidation of rac-phenylalaninol catalysed by GoGDH in
absence of semicarbazide (< 0.2 mM product magnified in the inset). Reaction
conditions: A reaction solution of 1.0 mL containing 100 mM glycine (adjusted
to pH=9), 2.5 mg mL-1 NADH oxidase (15 U mL-1), 2.0 mM NADP+, 1.5 mg
GoGDH mL-1, 1.8 mM rac-phenylalaninol with para-toluene sulfonic acid
monitored for 24 h, at 800 rpm and 30 °C. No (S)- and (R)-phenylalaninal were
observed.
Utilizing the most active (HLADH-E) and the most selective
(GoGDH) enzyme of our screening (Table 1) we continued to
explore their scope for amino alcohols, including β-, γ-, δ- and
ε-amino alcohols. The oxidation of enantiopure or achiral amino
alcohols was monitored spectrophotometrically. In contrast to
GoGDH, the assay showed that the crude cell-free extract (CFE)
of commercial HLADH-E (Evocatal) generated
a
high
background activity. Therefore, recombinantly expressed and
freshly purified HLADH-E (Histag-labeled) was tested instead.
As shown in Figure 3, pure HLADH-E displayed a high
specific activity towards a wide variety of amino alcohols (8 to
18). In agreement with the previous HPLC-based screening on
rac-1 (Table 1), HLADH-E confirmed once more its poor
(S)-selectivity. Interestingly, the same performance was
observed for phenylglycinol 8. Surprisingly, our result contradicts
earlier research[3c], where an E-value of 32 for the opposite
enantiomer (R)-8 is reported. The observed discrepancies could
be due to the use of crude horse liver extract in the earlier work.
The presence of other oxidoreductases in the complex protein
mixtures can significantly influence the reaction outcome. As a
general result from the substrate screening, it can be said that
unprotected β-amino alcohols were better accepted although
with poor (S)-selectivity, as compared to their protected
counterpart, N-Cbz-β-amino alcohols (10 and 11). However the
Figure 1. Asymmetric oxidation of rac-phenylalaninol to (R)-phenylalaninal
and (R)-phenylalaninal semicarbazone catalysed by GoGDH (< 0.2 mM
product magnified in the inset). Reaction conditions: A reaction solution of 1.0
mL containing 100 mM glycine (adjusted to pH=9), 2.5 mg mL-1 NADH oxidase
(15 U mL-1), 2.0 mM NADP+, 1.5 mg GoGDH mL-1, 1.8 mM rac-phenylalaninol,
100 mM semicarbazide HCl with para-toluene sulfonic acid as internal
standard for 24 h, at 800 rpm and 30 °C.
When the reaction was performed without the α-amino
aldehyde scavenging agent semicarbazide, the initial rate did
not significantly change (Fig. 2). This suggests that even the
large excess of semicarbazide did not influence the enzymes. At
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10.1002/ejoc.201701213
European Journal of Organic Chemistry
FULL PAPER
enantiopreference was retained. The glycerol analogues, such Conclusions
as 1-amino-2,3-propanediol 13 and 2-amino-1,3-propanediol 14,
proved to be converted by this enzyme with a moderate activity.
On the other hand, linear amino alcohols, such us β–15, γ-16,
Since H. E. Fischer reported the isolation of glycinal little
attention has been given in the asymmetric synthesis of α-amino
aldehydes under mild aqueous conditions. In this study, we have
shown that glycerol dehydrogenase from Gluconobacter
δ-17 to ε-18 derivatives were readily oxidized.
A slight
preference towards γ- followed by ε- and last the δ- and β- amino
alcohol was observed, in line with the polymerization instability
of these kind of substrates (Scheme 1c).
GoGDH again showed excellent (R)-selective conversion
of rac-1 with an E-value of 87. However, despite the structural
oxydans
catalyzes
the
asymmetric
oxidation
of
(R)-phenylalaninol 1 to (R)-phenylalaninal 2 with modest activity
and excellent enantioselectivity. On the other hand, horse liver
ADH displays high activity in the oxidation of amino alcohols,
making amino aldehydes accessible. However, only modest
enantioselectivity was observed for this enzyme. Overall careful
reaction engineering, enzyme engineering, and judicious choice
of additives is essential for success. Even a century after the
first reports on amino aldehydes they still pose many questions.
homology of phenylglycinol
8 and phenylalaninol 1, 8 is
essentially not converted. Also, the insignificantly lower
conversion of serine 9 harboring a negative charge indicates a
low substrate tolerance of GoGDH. Nevertheless, the
hydrophobic N-Cbz-2-aminopropanol 10 was slowly converted
with (R)-selectivity, as was achiral N-Cbz-2-aminoethanol 11.
Possibly, the α-amino group is not an essential hydrogen bond
donor/acceptor and selectivity is dominated by hydrophobic
interactions within GoGDH. In line with this view, the hydrophilic
glycerol analogues 13 and 14 are sluggishly converted. The
apparent link between hydrophobicity and reaction rate becomes
evident with the elongation of the aliphatic carbon chain when
testing β-15, γ-16, δ-17 to ε-18 amino alcohols. GoGDH has
been reported to have a broad substrate specificity for a wide
range of polyols, aromatic-, and aliphatic alcohols.[16] This is not
extended towards amino alcohols. The unselective HLADH-E
displays a similarly wide substrate scope to amino alcohols as it
does to other substrates.[17]
Experimental Section
Chemicals 1-amino-2,3-propanediol (99%, Sigma-Aldrich), 3-nonanone
(99.5%, Sigma-Aldrich), acetone (>99.5%, Sigma-Aldrich), acetophenone
(>98%, Sigma-Aldrich), benzaldehyde (99.5%, Sigma-Aldrich), benzoyl
chloroformate (>98%, Sigma-Aldrich), benzoylformate (97%, Sigma-Aldrich),
benzyl alcohol (99%, Sigma-Aldrich), benzylacetone (99%, Acros), bovine
albumin serum (BioRad), butanolamine (Acros, 99%), Cbz-(S)-phenylalaninol
(97%, 99% ee, Sigma-Aldrich), Cbz-(R)-phenylalaninol (98%, 99% ee, Sigma-
Aldrich), cyclohexanone (99.8%, Acros), Dess-Martin periodinane (97%,
Sigma-Aldrich), D8-dioxane (Cortecnet, 99.9%), D2O (Cortecnet, 99.9%),
diacetyl (97%, Sigma-Aldrich), dichloromethane (99.8%, VWR), dipotassium
phosphate (Merck, 99%), dithiothreitol (>99.5%, Sigma-Aldrich), (R)-
phenylalaninol (98%,Sigma-Aldrich), (R)-phenylglycinol (Sigma-Aldrich), D-
serine (99%, Sigma-Aldrich), ethanol (Merck Millipore, absolute),
ethanolamine (99%, Acros), ethyl acetate (99.7%, Sigma-Aldrich), glycine
(>99%, Sigma-Aldrich), ethyl acetate (98%, Sigma-Aldrich), levulinic acid (98%,
Acros), (S)-phenylalaninol (98%, Sigma-Aldrich), (S)-phenylglycinol (98%,
Sigma-Aldrich), (S)-serine (99%, Sigma-Aldrich), magnesium chloride
hexahydrate (>99%, J.T. Baker), methanol (BioSolve), methyl acetoacetate
(>99%, Sigma-Aldrich), methyl benzoyl formate (98%, Sigma-Aldrich), methyl
tert-butyl
ether
(99.5%,Sigma-Aldrich),
MOPS
(3-(N-
morpholino)propanesulfonic acid) (Bio-Rad Laboratories), NAD+ (98%, Roth),
NADP+ hydrate (>98%, Sigma-Aldrich), octanal (99%, Sigma-Aldrich), pentane
(98%, VWR), pentanolamine (Acros, 99%), phenylpyruvic acid (Sigma-Aldrich,
99%), potassium phosphate (99%, Merck), propanolamine (Acros, 99%), p-
toluene sulfonic acid monohydrate (98.5%, Sigma-Aldrich), semicarbazide
hydrochloride (>99%, Sigma-Aldrich), serinol (95%, Sigma-Aldrich), sodium
thiosulfate
(VWR, 99.9%),
trans-2-hexenal
(99%,
Sigma-Aldrich),
triethanolamine (99.5%, Sigma-Aldrich), triethylamine (>99%, Sigma-Aldrich).
The solvents methyl tert-butyl methyl ether, diethyl ether, pentane, and ethyl
acetate were distilled before use.
Enzymes & Kits The Alcohol Dehydrogenase Screening Kit from EvocatalTM
containing 17 different preparations of 100 mg lyophilized alcohol
dehydrogenase, specifically named evozyme cat. no. 1.1.010, 1.1.020,
1.1.030, 1.1.040, 1.1.130, 1.1.140, 1.1.190, 1.1.200, 1.1.210, 1.1.260, 1.1.270,
1.1.280, 1.1.380, 1.1.420, 1.1.430, 1.1.440, 1.1.441 and glucose
dehydrogenase 1.1.442, were used without further purification. The His6-tag
purified HLADH-E was kindly produced by Dr. Sandy Schmidt at the TU Delft
with a plasmid provided by Prof. Rother (Forschungszentrum Jülich, GmbH,
Germany), NADH oxidase PRO-NOX(001) was purchased from Novozymes
and assayed before use. The bicinchoninic acid (BCA) protein quantitation kit
was purchased from Thermo Scientific (Carlsbad, USA) and performed as
manufacturer’s protocol.
Analytical instruments The multi-mode spectrophotometer plate reader
(Synergy 2TM
, BioTek) was used for spectrophotometric measurements.
Staining SDS-PAGE gels were performed using SimplyBlue SafeStain (Novex).
The pH electrode (Metrohm) was calibrated before use with standards of pH =
4.00 and pH 7.00 with an R2 > 0.98. A HPLC system (Shimadzu) connected to
a LC-20AD pump, SIL-20A HT auto-sampler, SPD-10A UV-VIS detector, and
a CTO-20AC temperature control unit, Crownpak® Cr (+) column (150 x 4 mm)
with HClO4 (pH = 1.00, 0.6 mL min-1, 15 °C, 210 nm) as a mobile phase was
used for tr = 13.2 min for (S)-phenylalaninal, tr = 14.1 min for (R)-phenylalaninal,
tr = 21.3 min for (R)-phenylalanine, tr = 23.6 min for (R)-phenylalaninol, tr = 25.8
min for (S)- phenylalaninol, tr = 33.0 min for (S)-phenylalanine, tr = 34.6 min for
Figure 3. Spectrophotometric activity assay of purified HLADH-E and GoGDH
(Evocatal 190) catalyzing the oxidation of a wide range of amino alcohols.
NADPH formation was monitored at λ = 340 nm and 20°C for 1 min. Assay
conditions: 0.25 mM NADP+, GoGDH (2.0 mg mL-1) or HLADH-E (0.17 mg
mL-1), glycine buffer (50 mM, pH = 9.0), 5 mM substrate or 2.5 mM for the N-
Cbz-β-amino alcohols. Benzyl alcohol was used as a positive control.
(R)-phenylalaninal semicarbazone, tr
semicarbazone. Para-toluene sulfonic acid tr = 7.1 min was used as an
internal standard for rac-phenylalaninol and rac-phenylalanine. HPLC
system (Shimadzu) connected to a LC-20AD pump, SIL-20A HT auto-sampler,
= 41.1 min for (S)-phenylalaninal
A
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10.1002/ejoc.201701213
European Journal of Organic Chemistry
FULL PAPER
Chiralcel® OD-H column (250 x 4.6 mm) with 10:90 isopropanol/heptane as
mobile phase was monitored at 254 for the chiral separation of Cbz-(R)-2-
amino propanol and Cbz-(S)-2-amino propanol. NMR spectra were recorded
using an Agilent 400 MHz (1H, 9.4 Tesla) spectrometer operating at 399.67
MHz for 1H at 298K and were subsequently interpreted using MNOVA. Water
SPD-10A UV-VIS detector, and
a
CTO-20AC temperature control unit,
128.55, 128.27, 128.12, 127.18 (Carom), 67.10 (CHCO), 61.06 (CH2COONH),
35.38 (CH2CH) ppm.
In correspondence to others[19] 1H NMR: CDCl3 δ = 9.57 (s, 1 H), 7.37–7.09
:
(m, 10 H), 5.40 (d, J = 6.6 Hz, 1 H), 5.07 (s, 2 H), 4.46 (q, J = 6.7 Hz, 1 H),
3.12 (dd, J = 13.8, 6.8 Hz, 1H), 3.05 (dd, J = 13.8, 6.8 Hz, 1 H) ppm. 13
C
NMR: δ = 199.0, 156.0, 136.2, 135.5, 129.3, 128.8, 128.6, 128.3, 128.2, 127.2,
suppression was performed using
a
PRESAT pulse sequence in sealed
67.1, 61.1, 35.3 ppm.
Wilmad® screw-cap NMR tubes (Sigma Aldrich). Coupling constants were
expressed in Hertz (Hz).
Synthesis of (S)-2-amino-3-phenylpropanal hydrate trifluoroacetate. A
synthetic protocol from literature was adopted to produce the trifluoroacetate
BCA assay Protein content was determined with the BCA protein quantitation
kit (Thermo Scientific, Carlsbad, USA). Standard curves were prepared with
BSA in the range of 0.01 – 2 mg mL−1 in (poly)styrene 96 wells plate. Samples
were measured in triplicates and monitored at 562 nm utilizing a microtiter
plate spectrophotometer (Synergy 2, BioTek)
salt of (S)-2-amino-3-phenylpropanal hydrate in-situ[3b]
. To a glass vial
equipped with a magnetic stirrer 30.0 mg Cbz-(S)-2-amino-3-phenylpropanal
(111 µmol, 1 eq.) was dissolved in 1 mL of 1:1 mixture of D8-dioxane:D2O. To
this solution 11 µL TFA (101.8 µmol, 13.9 mg, 1.1 eq) and 5.0 mg Pd/C were
added under H2 atmosphere (1 atm). The reaction was monitored with silica
TLC (EtOAc:MeOH 95:5) to completion after 18h, filtered, and analysed with
NMR and HPLC.
SDS-PAGE Protein samples were denatured using XT sample buffer (BioRad)
supplied with XT reducing agent (BioRad) at 95 °C for 15 minutes. Gel
electrophoresis was performed with Criterion XT 4-12% Bis-Tris precast gels
(Bio-Rad) using MOPS buffer (Bio-Rad). The gels were run at 150 V for 40 to
60 minutes and stained with SimplyBlue SafeStain (Novex). The Precision
Plus ProteinTM Unstained Standard (Bio-Rad) was used to determine the
molecular weight of the protein.
1H-NMR (400 MHz, D2O:D8-dioxane (1:1)): 7.60–7.42 (m, 5H, CHarom), 5.28 (d,
J = 3.3 Hz, 1H, CHC(OD)2), 3.63 (m, 1 H, CHND3), 3.31 (dd, J = 14.2 Hz, J =
6.0 Hz, 1H, CHHCH), 3.10-3.08 (dd, J = 14.2 Hz, J = 8.3 Hz, 1H, CHHCH).
13CNMR (D2O:D8-dioxane (1:1): δ = 134.36, 128.32, 128.20, 127.99, 126.33
(Carom), 86.43 (C(OD)2), 56.26 (CHND3), 33.01 (CH2CH). HPLC: 35% ee for
(S)-2-amino-3-phenylpropanal: tr = 12.8 min for (S)-2-amino-3-phenylpropanal,
tr = 13.8 min for (R)-2-amino-3-phenylpropanal. HPLC conditions: Crownpak®
Cr (+) column (4 x 150 mm); flow-rate 0.6 mL min-1; UV 210 nm; aq. HClO4
(pH = 1), 15°C.
Calibration HPLC. Rac-phenylalaninol and rac-phenylalanine were calibrated
using in HClO4 (pH = 1.00) with the internal standard pTSA (60.5 µg mL-1).
Calibration curves with external standards were prepared of (S)-phenylalaninal
(35% ee) between a range of 0.030 and 2.50 mM in aq. HClO4 (pH = 1.00)
and (S)-phenylalaninal semicarbazone (35% ee) between a range of 2.58 mM
Synthesis of (S)-2-amino-3-phenylpropanal semicarbazone. (S)-2-amino-
3-phenylpropanal hydrate trifluoroacetate (4.5 mg, 17.1 μmol, 1 eq.) in a glass
vial equipped with a magnetic stirrer was dissolved in 0.15 mL of 1:1 mixture
of D8-dioxane:D2O. Then 0.15 mL semicarbazide HCl in D2O (0.37 M, 89 μmol,
10.0 mg, ~5 eq., pH = 2) was added and stirred at room-temperature. The
reaction mixture was analysed with NMR and HPLC.
and 0.01 mM in HClO4 (pH
= 1.00) containing 100 mM semicarbazide
hydrochloride to prevent any hydrolysis to the α-amino aldehyde.
Spectrophotometric activity assays The activity of all proteins utilized in the
Alcohol Dehydrogenase screening kit from EvocatalTM were assayed
spectrophotometrically before the HPLC screening. Spectrophotometric
assays were performed in polyacrylate 1 cm cuvettes by monitoring the
consumption of 0.25 mM NAD(P)H at 340 nm at 20°C. NAD(P)H oxidase
activity of PRO-NOX(001) was monitored in glycine buffer (50 mM, pH 9.0)
before HPLC screening. Reactions were started by addition of the enzyme
1H-NMR (400 MHz, D2O:D8-dioxane (3:1)): 7.28–7.15 (m, 6H, HC=N, CHarom),
4.25 – 4.21 (m, 1H, CHCH2), 3.07–2.96 (s, 2H, CHCH2). 13C-NMR (100 MHz,
D2O:D8-dioxane (3:1)):
δ = 157.47 (NDCOND2), 138.69 (CH=N), 132.92,
128.38, 127.87, 126.54 (Carom), 51.23 (CHND3), 35.54 (CH2CH). HPLC:
Enantiomeric excess 35% for (S)-2-amino-3-phenylpropanal: tr = 12.8 min for
(S)-2-amino-3-phenylpropanal, tr = 13.8 min for (R)-2-amino-3-phenylpropanal.
Enantiomeric excess 32% for (S)-2-amino-3-phenylpropanal semicarbazone: tr
= 34.2 min for (R)-2-amino-3-phenylpropanal semicarbazone, tr = 42.4 min for
(S)-2-amino-3-phenylpropanal semicarbazone. HPLC conditions: Crownpak®
Cr (+) column (4 x 150 mm); flow-rate 0.6 mL min-1; UV 210 nm; aq. HClO4
(pH = 1), 15°C.
solution and measured over
out at least in triplicate.
1 min. All measurements were carried
Chiral HPLC screening assay (Table 1): A stock solution containing glycine
buffer, semicarbazone, and the internal standard para-toluene sulfonic acid
was prepared freshly and corrected to a final pH of 9.0 with a NaOH solution.
Separate stock solutions of rac-phenylalaninol, NADP+, NAD(P)H oxidase
(100 mg mL-1, 4.0 U mg-1) were prepared before use. The protein content of
the alcohol dehydrogenases from the Evocatal ADH screening kit was
determined with the BCA assay and normalized to 15 mg mL-1 (MiliQ). The 1
mL reaction solutions containing 70 mM glycine, 105 mM semicarbazide, 1.50
mM rac-phenylalaninol, 2 mM NAD(P)+, 5 mg mL-1 NADH oxidase (4.0 U mg-1),
and 1.5 mg mL-1 ADH were incubated at 30 °C at 800 rpm. Samples were
collected by quenching 50 µl of the reaction solution with 500 µl HClO4 (pH =
1) in a 1.5 mL Eppendorf tube, followed by centrifugation for 1 minute at 13000
rpm. The supernatant was transferred into a polypropylene HPLC microvial
(Grace) and analysed by HPLC. HPLC: tr = 7.1 min for para-toluene sulfonic
acid, tr = 13.2 min for (S)-phenylalaninal, tr = 14.1 min for (R)-phenylalaninal, tr
Synthesis of N-carbobenzyloxy-2-amino alcohols for the activity assay.
A round-bottom flask containing ethanolamine, (S)- or (R)-2-amino propanol
(>99% ee, 20 mmol) in DCM (25 mL) under dry N2 was cooled to 0 °C in an
ice bath. To this solution triethylamine was added drop-wise, followed by the
drop-wise addition of benzyl chloroformate (26 mmol) under dry N2 over 30
minutes. The mixture was stirred at 0°C for 1 h and monitored using TLC (1:1
EtOAc:pentane) on silica gel F254. The mixture was quenched with water (25
mL) and extracted four times with 25 mL DCM. The combined organic layer
was washed with brine and dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure. The yellow oil was purified by column
chromatography (silica gel, EtOAc: pentane, 1:1). The desired product was
obtained as a white solid. The compound was analysed with chiral HPLC and
NMR.
2-Benzyloxycarbonyl-(S)-2-aminopropan-1-ol, isolated yield 1.13 g, 27%.
1H NMR (400 MHz, CDCl3) δ = 7.32 (m, 5H, Ph), 5.16 (br s, 1H, NH), 5.07 (s,
2H, PhCH2), 3.79 (m, 1H, CH), 3.62 – 3.55 (m, 1H, CHHOH), 3.47 (m, 1H,
CHHOH), 3.02 (br s, 1H, OH), 1.12 (d, J = 6.7 Hz, 3H, CH3). 13C NMR (101
MHz, CDCl3) δ = 156.59 (s, C=O), 136.36 (Carom), 128.50 (CHarom), 128.12
(CHarom), 128.07 (CHarom), 66.78 (CH2), 66.52 (CH2), 48.92 (CH), 17.21 (CH3).
=
tr
21.3 min for (R)-phenylalanine, tr
=
23.6 min for (R)-phenylalaninol,
tr 33.0 min for
=
25.8 min for (S)-phenylalaninol,
=
(S)-phenylalanine, tr = 34.6 min for (R)-phenylalaninal semicarbazone, tr = 41.1
min for (S)-phenylalaninal semicarbazone.
Synthesis reference standards
Synthesis of N-Cbz-(S)-2-amino-3-phenylpropanal.
A synthetic protocol
from literature was adopted[18]. Dess-Martin periodinane (3835.1 mg, 9.05
mmol, 2.1 equiv.) was added to a solution of N-Cbz-L-phenylalaninol (>99%
ee, 1.234 g, 4.25 mmol, 1 equiv.) in water-saturated DCM (37.5 mL). The
resulting suspension was stirred at room temperature (23 °C), monitoring the
progress of the reaction by TLC analysis (EtOAc:MeOH 95:5) on silica gel.
Additional 10 mL portion of water-saturated DCM were added until the reaction
was completed in one hour. The reaction mixture was diluted with diethyl ether
(50 mL), and a solution of sodium thiosulfate (11.55 g, 46.55 mmol) in 80%
saturated sodium bicarbonate solution was added. The mixture was stirred
vigorously until both phases were clear. The layers were separated, and the
aqueous layer was extracted with diethyl ether (3x50 mL). The combined
organic layers were washed with saturated aq. NaHCO3 (50 mL), water (50
mL), and brine (50 mL), dried over Na2SO4, and filtered. Concentration in
vacuo afforded crude N-Cbz-L-phenylalaninal as a white solid (891.2 mg, 3.14
mmol, 74% isolated yield). The solution was purified with column
chromatography using silica gel (EtOAc:MeOH 19:1, Rf 0.71). Trace impurities
were removed with a second column using silica gel (MTBE:pentane 1:1, Rf
0.32),dried in vacuo resulting in 41% isolated yield (498.2 mg, 1.75 mmol).
1H-NMR (400 MHz, CDCl3): δ = 9.63 (s, 1H, CHO), 7.38–7.12 (m, 10H,
CHarom), 5.32 (d, J = 6.6 Hz, 1H, CH2NH), 5.11 (s, 2H, COCH2), 4.51 (q, J =
6.7 Hz, 1H, CH2CH), 3.14 (d, J = 4.0 Hz, 2H, CH2CH). 13C-NMR (100 MHz,
CDCl3): δ = 199.81 (CHO), 155.87 (NHCOO), 136.09, 135.38, 129.28, 128.8,
HPLC: tr
= 10.9 min for 2-benzyloxycarbonyl-(S)-2-aminopropan-1-ol with
>99 % ee. HPLC conditions: Chiralcel® OD-H column; flow-rate 1.0 mL min-1;
UV 210 nm; 10:90 isopropanol/heptane, 35°C.
2-Benzyloxycarbonyl-(R)-2-aminopropan-1-ol, isolated yield 1.08 g, 26%.
1H NMR (400 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H, Ph), 5.08 (s, 2H, PhCH2),
4.99 (br s, 1H, NH), 3.82 (m, 1H, CH), 3.62 (m, 1H, CHHOH), 3.50 (m, 1H,
CHHOH), 2.56 (br s, 1H, OH), 1.15 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (101
MHz, CDCl3) δ 156.55 (C=O), 136.33 (Carom), 128.52, 128.08, 128.04 (CHarom),
66.82 (CH2), 48.96 (CH), 17.23 (CH3). HPLC: tr
= 13.3 min for 2-
benzyloxycarbonyl-(R)-2-aminopropan-1-ol with > 99 % ee. HPLC conditions:
Chiralcel® OD-H column; flow-rate 1.0 mL min-1; UV 210 nm; 10:90
isopropanol/heptane, 35°C.
Reported in literature for 2-benzyloxycarbonyl-(R)-2-aminopropan-1-ol[20] 1H
:
NMR (CDCl3) δ = 7.35 (m, 5H, Ph), 5.10 (s, 2H, PhCH2), 4.92 (br s, 1H, NH),
3.83 (m, 1H, CH), 3.66 (m, 1H, CHHOH), 3.53 (m, 1H, CHHOH), 2.40 (br s,
1H, OH), 1.16 (d, 3H, CH3). 13C NMR (CDCl3) δ = 156.5 (C=O), 136.3 (Carom),
128.0, 128.1, 128.5 (CHarom), 66.8 (CH2), 66.6 (CH2), 48.9 (CH2), 17.2 (CH3).
2-Benzyloxycarbonyl-2-aminoethan-1-ol, isolated yield 1.01 g, 26%. 1H
NMR (400 MHz, CDCl3) δ = 7.44 – 7.19 (m, 5H, Ph), 5.40 (br s, 1H, NH), 5.08
(s, 2H, PhCH2), 3.65 (m, 2H, NCH2CH2OH), 3.30 (q, J
= 4.8 Hz, 1H,
NCH2CH2OH), 2.89 (br s, 1H, OH).13C NMR (101 MHz, CDCl3) δ = 157.14
(C=O), 136.34 (Carom), 128.51, 128.14, 128.07 (CHarom), 66.86 (OCH2Ph),
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201701213
European Journal of Organic Chemistry
FULL PAPER
62.00 (NCH2CH2OH), 43.44 (NCH2CH2OH). HPLC: tr
=
11.9 min for 2-
[16]
[17]
N. Richter, M. Neumann, A. Liese, R. Wohlgemuth, T. Eggert, W.
Hummel, ChemBioChem 2009, 10, 1888-1896.
benzyloxycarbonyl-2-aminoethan-1-ol. HPLC conditions: Chiralcel® OD-H
column; flow-rate 1.0 mL min-1; UV 210 nm; 10:90 isopropanol/heptane, 35°C.
a) D. Quaglia, M. Pori, P. Galletti, E. Emer, F. Paradisi, D.
Giacomini, Process Biochem. 2013, 48, 810-818; b) S. Kara, D.
Spickermann, J. H. Schrittwieser, A. Weckbecker, C. Leggewie, I.
W. C. E. Arends, F. Hollmann, ACS Catal. 2013, 3, 2436-2439; c)
D. Quaglia, J. A. Irwin, F. Paradisi, Mol. Biotechnol. 2012, 52, 244-
250.
A. G. Myers, B. Zhong, M. Movassaghi, D. W. Kung, B. A. Lanman,
S. Kwon, Tetrahedron Lett. 2000, 41, 1359-1362.
S. Mirilashvili, N. Chasid-Rubinstein, A. Albeck, Eur. J. Org. Chem.
2010, 2010, 4671-4686.
2-Benzyloxycarbonyl-2-aminoethan-1-ol, reported in literature[21] 1H NMR
:
(300 MHz, CDCl3) δ = 7.32 – 7.25 (m, 5H, Ph), 5.39 (br s, 1H, NH), 5.07 (s, 2H,
PhCH2), 3.65 (2H, m, NCH2CH2OH), 3.3 (2H, J = 5.2 NCH2CH2OH), 2.85 (br s,
1H, OH). 13C NMR (75.4 MHz, CDCl3) δ = 157.41 (C=O), 134.64 (Carom),
128.35, 28.43, 128.79 (CHarom), 67.16 (OCH2Ph), 62.37 (NCH2CH2OH), 43.74
(NCH2CH2OH).
[18]
[19]
[20]
[21]
GoGDH catalyzed oxidation of 1 (Fig. 1 & 2): Quantitative HPLC analysis
was performed with altered reaction conditions. A reaction solution of 1.0 mL
containing 100 mM glycine, 100 mM semicarbazide, 1.80 mM rac-
phenylalaninol, 2 mM NADP+, 1.5 mg mL-1 NADH oxidase PRO-NOX(001)
(4.0 U mg-1), pTSA (60 µg mL-1) and 1.0 mg mL-1 ADH were incubated and
worked up under the conditions previously stated. Chemical background
hydrolysis of (S)- and (R)- phenylalaninal semicarbazone due to the addition of
HClO4 during quenching were calculated and subtracted from the reactions
performed.
S. Fernández, R. Brieva, F. Rebolledo, V. Gotor, J. Chem. Soc.,
Perkin Trans. 1 1992, 2885-2889.
D. J. Miller, M. B.-U. Surfraz, M. Akhtar, D. Gani, R. K. Allemann,
Org. Biomol. Chem. 2004, 2, 671-688.
Spectrophotometric substrate screening assay (Fig. 3). The assay was
performed in polyacrylate 1 cm cuvettes by monitoring the conversion of 0.25
mM NAD(P)+ at 340 nm and 20 °C using an extinction coefficient of 6.22 mM−1
cm−1 by GoGDH (2.0 mg mL-1) or HLADH-E (0.17 mg mL-1) in glycine buffer
(50 mM, pH = 9). Final concentration of substrates used: benzyl alcohol, (R)-
phenylalaninol, (S)-phenylalaninol, (R)-serine, (S)-phenylglycinol, (R)-
phenylglycinol, ethanolamine, propanolamine, butanolamine, pentanolamine,
1-amino-2,3-propanediol, serinol, glycerolwas was always fixed to 5 mM with
the exceptions of Cbz-(R)-2-aminopropan-1-ol and Cbz-(S)-2-aminopropan-1-
ol were 2.5 mM was used. The reactions were started by the addition of the
enzyme solution and measured over 1 min. All measurements were carried
out in triplicate.
Acknowledgements
We thank Dr D. Rother for the kind provision with the plasmid
encoding HLADH-E and Dr. S. Schmidt for the expression and
purification of HLADH-E.
Keywords: amino alcohols • amino aldehydes • alcohol
dehydrogenase • asymmetric oxidation • biocatalysis
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The juxtaposition of an amino and
aldehyde functionality predisposes
chiral amino aldehydes as divergent
biocatalysis
Luuk Mestrom*, Paula Bracco
and Ulf Hanefeld*
building
chemistry. In this study, we
established the asymmetric
blocks
within
organic
enzymatic oxidation of β-amino
alcohols to α-amino aldehydes using
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glycerol
dehydrogenase
from
Amino aldehydes revisited
Gluconobacter oxydans with excellent
enantiopurity.
*mild and selective biocatalytic conversion of amino alcohols to amino aldehyde derivatives
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*one or two words that highlight the emphasis of the paper or the field of the study
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