One-pot combination of enzyme and Pd nanoparticle catalysis for the synthesis of enantiomerically pure 1,2-amino alcohols

Article abstract of DOI:10.1039/c3gc41666f

One-pot combinations of sequential catalytic reactions can offer practical and ecological advantages over classical multi-step synthesis schemes. In this context, the integration of enzymatic and chemo-catalytic transformations holds particular potential for efficient and selective reaction sequences that would not be possible using either method alone. Here, we report the one-pot combination of alcohol dehydrogenase-catalysed asymmetric reduction of 2-azido ketones and Pd nanoparticle-catalysed hydrogenation of the resulting azido alcohols, which gives access to both enantiomers of aromatic 1,2-amino alcohols in high yields and excellent optical purity (ee >99%). Furthermore, we demonstrate the incorporation of an upstream azidolysis and a downstream acylation step into the one-pot system, thus establishing a highly integrated synthesis of the antiviral natural product (S)-tembamide in 73% yield (ee >99%) over 4 steps. Avoiding the purification and isolation of intermediates in this synthetic sequence leads to an unprecedentedly low ecological footprint, as quantified by the E-factor and solvent demand.

Full text of DOI:10.1039/c3gc41666f

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Volume 15 | Number 12 | December 2013 | Pages 3279–3490
ISSN 1463-9262
PAPER
Joerg H. Schrittwieser, Frank Hollmann et al.
One-pot combination of enzyme and Pd nanoparticle catalysis for the synthesis
of enantiomerically pure 1,2-amino alcohols

Green Chemistry
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One-pot combination of enzyme and Pd nanoparticle
catalysis for the synthesis of enantiomerically pure
1,2-amino alcohols†
Cite this: Green Chem., 2013, 15, 3318
Joerg H. Schrittwieser,*a Francesca Coccia,b Selin Kara,a Barbara Grischek,c
Wolfgang Kroutil,c Nicola d’Alessandrod and Frank Hollmann*a
One-pot combinations of sequential catalytic reactions can offer practical and ecological advantages over
classical multi-step synthesis schemes. In this context, the integration of enzymatic and chemo-catalytic
transformations holds particular potential for efficient and selective reaction sequences that would not
be possible using either method alone. Here, we report the one-pot combination of alcohol dehydrogen-
ase-catalysed asymmetric reduction of 2-azido ketones and Pd nanoparticle-catalysed hydrogenation of
the resulting azido alcohols, which gives access to both enantiomers of aromatic 1,2-amino alcohols in
high yields and excellent optical purity (ee >99%). Furthermore, we demonstrate the incorporation of an
upstream azidolysis and a downstream acylation step into the one-pot system, thus establishing a highly
integrated synthesis of the antiviral natural product (S)-tembamide in 73% yield (ee >99%) over 4 steps.
Avoiding the purification and isolation of intermediates in this synthetic sequence leads to an unprece-
dentedly low ecological footprint, as quantified by the E-factor and solvent demand.
Received 15th August 2013,
Accepted 11th September 2013
DOI: 10.1039/c3gc41666f
www.rsc.org/greenchem
different types of catalysts is less explored. This is particularly
true for combinations of chemical catalysts and enzymes,
Introduction
The integration of several chemical transformations into one- which are often complicated by divergent reaction conditions
pot processes (often referred to as ‘domino’, ‘tandem’, or and detrimental interactions of the bio- and chemo-catalysts.4
‘cascade’ systems) offers advantages with respect to oper- Nevertheless, several excellent recent studies aimed at bringing
ational simplicity, operating time and costs, safety, and the chemo- and biocatalysis closer together clearly demonstrate
consumption of energy and materials.1 From an ecological per- the potential of chemo-enzymatic one-pot systems.5 In this
spective, the one-pot combination of catalytic methods2 is par- context, true cascades – in which the biocatalytic and chemical
ticularly appealing, and recent years have brought about reactions proceed concurrently – definitely represent the most
remarkable developments in the use of heterogeneous, homo- elegant examples, but they are also most prone to the above-
geneous, organo- and biocatalysts in cascade systems.3 mentioned difficulties.6 Sequential chemo-enzymatic one-pot
However, these approaches typically remain within one indi- reactions are more easily realised and offer essentially the
vidual ‘subfield’ of catalysis, while the one-pot combination of same environmental advantages (e.g. reduction of solvent use
due to elimination of work-up and purification steps).4a,c,7
The 2-amino-1-aryl alcohol moiety is a common structural
motif in biologically active compounds,8 as it forms the basic
scaffold of adrenergics (e.g. the hormones adrenaline and nor-
adrenaline, β-adrenergic blockers, and anti-asthma drugs),
amphenicol antibiotics, and several bioactive natural products
(Fig. 1). In addition, 1,2-amino alcohols have found broad use
as chiral ligands and auxiliaries in asymmetric synthesis.9 For
both pharmaceutical and synthetic applications, a high optical
purity of chiral amino alcohols is desired, causing the need for
highly selective asymmetric syntheses of these compounds.
Chemical methods – such as asymmetric hydrogenation10 or
transfer hydrogenation11 of 2-amino ketones or their synthetic
equivalents – have been intensively studied in recent years, yet
aDepartment of Biotechnology, Delft University of Technology, Julianalaan 136, 2628
BL Delft, The Netherlands. E-mail: j.schrittwieser@tudelft.nl, f.hollmann@tudelft.nl;
Fax: (+31) 152 781415; Tel: (+31) 152 781957
bSchool of Advanced Studies in Science, University “G. d’Annunzio” of
Chieti-Pescara, Via dei Vestini 31, 66013 Chieti, Italy
cDepartment of Chemistry, Organic & Bioorganic Chemistry, University of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
dDepartment of Engineering and Geology (INGEO), University “G. d’Annunzio” of
Chieti-Pescara, Viale Pindaro 42, 65127 Pescara, Italy
†Electronic supplementary information (ESI) available: Complete results of ADH
screening, additional optimisation studies, characterisation data of Pd-NPs,
additional details on the environmental assessment of tembamide syntheses,
additional experimental procedures, full characterisation data of compounds
isolated from the chemo-enzymatic transformations, EATOS and Excel files used
in the environmental impact assessment. See DOI: 10.1039/c3gc41666f
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corresponding commercially available bromo ketone 1e. The
ecological benefits of this one-pot concept are demonstrated
by a basic environmental impact assessment.
Results and discussion
Azido alcohol hydrogenation catalysed by metal nanoparticles
In our one-pot approach, we envisioned combining the asym-
metric reduction of 2-azido ketones catalysed by alcohol dehy-
drogenases (ADHs) with azide hydrogenation catalysed by
recently reported17 lignin-stabilised metal nanoparticles (NPs).
Since the latter reaction had not been investigated before,
we began our studies by screening six different metal nanopar-
ticle preparations (Pd or Pt, stabilised by three different lignin
varieties) in the hydrogenation of 2-azido-1-phenylethanol 3a.
Phosphate buffer containing 5% (v/v) of 2-propanol as organic
co-solvent was chosen as a reaction medium to ensure compat-
ibility with the conditions for the enzyme-catalysed step. As
shown in Table 1, Pd-LC and Pd-LK nanoparticles (for expla-
nation of nanoparticle types, see Table 1 footnotes) gave the
best results, leading to full conversion within 4 h (entries
1 and 3). Minor amounts of unidentified side products were
formed in all reactions, but subsequent experiments showed
Fig. 1 Examples of biologically active 1,2-amino alcohols (2-amino-1-aryl
alcohol motif highlighted in bold).
they often fail to afford enantiomerically pure products. As a
result, classical resolution is still a widely used technique in
the preparation of chiral 1,2-amino alcohols, either for upgrad-
ing the ee of an optically enriched product or for resolving the
racemate.10a
Biocatalysis, on the other hand, offers several options for
the preparation of optically pure 1,2-amino alcohols in
general, such as kinetic resolution or dynamic kinetic resolu-
tion of the racemate using lipases,12 oxidative kinetic resolu-
tion of N-protected amino ketones by Baeyer–Villiger
monooxygenases,13 the combination of aldolase-catalysed C–C
bond formation and enzymatic decarboxylation,14 or the com-
bination of lyase-catalysed C–C bond formation with reductive
amination catalysed by ω-transaminases.15 However, biocataly-
tic asymmetric methods for the preparation of 2-amino-1-aryl
alcohols in particular are comparably scarce.12e,14,15a,16a
Herein, we describe a chemo-enzymatic approach for the
asymmetric synthesis of this important substance class: a
sequential one-pot combination of stereoselective azido ketone
reduction by alcohol dehydrogenases and subsequent azide
hydrogenation by palladium nanoparticles (Scheme 1), which
provides access to both enantiomers of 2-amino-1-aryl alcohols
in high yields and with excellent optical purities. The inte-
gration of an upstream azidolysis step into the one-pot process
and in situ benzoylation of the crude amino alcohol have also
been achieved, enabling the one-pot asymmetric synthesis of
the natural product tembamide (Fig. 1) in four steps from the
Table 1 Hydrogenation of 3a to 4a catalysed by different types of lignin-stabi-
lised metal nanoparticlesa
Entry
NP typeb
Conversionc [%]
Selectivityc [%]
1
2
3
4
5
6
7
Pd–LC
Pd–LA
Pd–LK
Pt–LC
Pt–LA
Pt–LK
Pd–LKd
>99
95
>99
52
97
97
86
89
85
89
85
87
>99
>99
a Conditions: 100 mM 3a, 0.5 mM NPs, 10 bar H2, 30 °C, 4 h. For
details, see the Experimental section. b Lignin varieties: LC
=
sulfonated lignin with Ca2+ counterions, LA = sulfonated lignin with
NH4+ counterions, LK = low-sulfonate Kraft lignin. c Determined by
GC–FID analysis. d Reaction under basic conditions (pH 9).
Scheme 1 Chemo-enzymatic approach towards optically pure 1,2-amino alcohols via azidolysis, alcohol dehydrogenase (ADH) catalysed asymmetric reduction,
and Pd nanoparticle (Pd-NP) catalysed azide hydrogenation.
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that by carrying out the reduction under basic conditions 100 mM concentration of 2a (Suppl. Fig. 1 and 2, ESI†), and a
(pH 9) the chemoselectivity can be raised further, such that good performance over 24 h of reaction time (Suppl. Fig. 3 and
2-amino-1-phenyl-ethanol 4a was the only product detectable 4, ESI†). The substrate scope of ADH-A and KRED-NADH-110
by GC–FID and NMR analysis (Table 1, entry 7). The hydrogen- was investigated using the representative 2-azido-1-aryletha-
ation of 3b–f under identical conditions proceeded with the nones 2a–f as substrates. Both biocatalysts converted all six
same high level of selectivity, affording the corresponding ketones tested with excellent stereoselectivity and with similar
1,2-amino alcohols 4b–f in essentially pure form.18
relative rates; only the reduction of 2f by KRED-NADH-110 was
surprisingly slow in comparison to the other 2-azido ketones
(Fig. 2). Test transformations with 100 mM substrate concen-
tration and optimised enzyme loadings (ADH-A: 1.5–2.5 mg
mL−1, KRED-NADH-110: 0.2–1.0 mg mL−1; for details see ESI†)
proceeded to complete conversion within 20 h in all cases.
Asymmetric azido ketone reduction catalysed by ADHs
Next, we turned our attention to the ADH-catalysed asymmetric
reduction of aromatic 2-azido ketones. Although there is litera-
ture precedence for this transformation,19 a broad survey of
suitable enzymes has not yet been reported. Therefore, we
carried out an extensive screening of 79 commercial ADHs of
Chemo-enzymatic one-pot transformations
unspecified origin and four bacterial ADHs (ADH-A from Rho- After suitable ADHs had been identified, we proceeded with
dococcus ruber DSM 44541, TbADH from Thermoanaerobium combining the enzymatic azido ketone reduction and Pd-NP-
brockii, LkADH from Lactobacillus kefir, LbADH from Lactobacil- catalysed azide hydrogenation in a one-pot sequence. The bio-
lus brevis)20 in the reduction of 2a to 3a. The screening was catalytic reduction was run to completion (20 h) before adjust-
performed at 50 mM concentration of 2a and in the presence ing the pH of the reaction mixture to 9 and adding a Pd-NP
of 5% (v/v) 2-propanol, the latter serving both as co-solvent stock solution. First experiments showed that the activity of
and co-substrate. Table 2 shows selected results of the ADH the nanoparticles was not impaired by the presence of the
screening (for complete results, see ESI†). As a general trend, enzymes, and complete reduction of the intermediate azido
we identified more anti-Prelog-selective21,22 ADHs (18) with alcohols 3a–f was achieved in 4 h. However, in addition to the
activity on 2a than Prelog-selective ones (9), and on average, expected products 4, the corresponding 2,2-dimethyloxazoli-
the former showed about 5–10 times higher activity than the dines 5 were also formed in minor amounts (4–10%), appar-
latter. Only eleven enzymes afforded optically pure 3a, whereby ently by the reaction of 4 with acetone generated as by-product
four gave the (R)-enantiomer and seven the (S)-enantiomer.23 in the ADH-catalysed reduction (Scheme 2).24 When the bio-
Based on the observed activities and stereoselectivities, we con- reduction was carried out under reduced pressure, so as to
sidered the commercial ADHs listed in Table 2 (entries 5–8) as remove the acetone from the solution, the one-pot sequence
well as ADH-A (entry 1), the most promising biocatalysts for afforded essentially pure 1,2-amino alcohols 4a–f. In semi-
the reaction under study.
preparative-scale experiments (5 mL volume, 0.5 mmol sub-
A further selection was made according to the enzymes’ tol- strate converted), the desired products could be isolated in
erance for higher substrate concentrations and their oper- good yield and excellent enantiomeric excess (Table 3). Only
ational stability. We identified ADH-A and KRED-NADH-110 as the isolated yields of 4f fall behind compared to the other
the most robust of the investigated Prelog- and anti-Prelog- amino alcohols, which we attribute to the high aqueous solu-
selective enzymes, respectively, showing reasonable activity at bility and the resulting difficult extraction of 4f.
Table 2 Activity and stereoselectivity of selected ADHs in the reduction of 2a to 3aa
Entry
Enzyme
Source/supplier
Cofactorb
Conv.c [%]
Act.d [U mg−1
]
eee [%]
1
2
3
4
5
6
7
8
ADH-A
TbADH
LkADH
LbADH
KRED-NADH-110
KRED-P3-B03
evo-1.1.030
evo-1.1.200
R. ruber
T. brockii
L. kefir
L. brevis
Codexis
Codexis
evocatal
evocatal
NADH
12
<1
2
2
58
5
0.5
<0.1
0.1
0.1
2.4
0.2
0.3
3.1
>99 (R)
nd f
98 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
>99 (S)
NADPH
NADPH
NADPH
NADH
NADPH
NADH
7
73
NADH
a Conditions: 50 mM 2a, 0.1 mM NAD+, 0.1 mg mL−1 ADH preparation, 5% (v/v) 2-PrOH, 30 °C, 2 h. For details, see the Experimental section.
b Preferred cofactor according to literature or supplier’s information. c Determined by GC–FID analysis. d Apparent activity [µmol product/
(min mg enzyme preparation)] calculated from conversion. e Enantiomeric excess of 3a determined by chiral-phase GC–FID analysis. f nd =
not determined.
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Table 3 Chemo-enzymatic two-step one-pot transformation of azido ketones
2a–f into amino alcohols 4a–fa
GC yield Isol. yield
ee (4)d
[%]
Entry Subst. ADH
(4)b [%]
(4)c [%]
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
2a
2b
2c
2d
2e
2f
ADH-A
ADH-A
ADH-A
ADH-A
ADH-A
ADH-A
KRED-NADH-110
KRED-NADH-110
KRED-NADH-110
KRED-NADH-110
KRED-NADH-110
97
98
99
99
98
>99
98
98
98
98
99
87 (60 mg) >99 (R)
89 (76 mg) >99 (R)
86 (67 mg) >99 (R)
85 (64 mg) >99 (R)
85 (71 mg) >99 (R)
55 (35 mg) >99 (S)e
86 (59 mg) >99 (S)
78 (67 mg) >99 (S)
88 (68 mg) >99 (S)
86 (65 mg) >99 (S)
87 (73 mg) >99 (S)
38 (24 mg) >99 (R)e
9
10
11
12
KRED-NADH-110 >99
a Conditions: 100 mM 2, 0.1 mM NAD+, 0.2–2.5 mg mL−1 ADH
preparation, 0.5 mM Pd-NPs, 5% (v/v) 2-PrOH, 30 °C, 24 h. For details,
see the Experimental section. b Relative amount of 4 in the crude
product determined by GC–FID analysis. Rest to 100% is the
corresponding 2,2-dimethyloxazolidine 5. c Isolated yield of pure 4
after column chromatography; semi-preparative scale (5 mL; 0.5 mmol
substrate). d Enantiomeric excess of 4 as determined by chiral GC–FID
analysis
after
conversion
into
the
corresponding
2,2-
dimethyloxazolidine 5. e Switch in substituent priorities according to
Cahn–Ingold–Prelog rules.
Fig. 2 Activity of ADH-A and KRED-NADH-110 in the reduction of azido
ketones 2a–f. Apparent activity [µmol product/(min mg enzyme preparation)]
determined from conversion after 2 h; conditions: 50 mM 2, 0.1 mM NAD+,
0.1 mg mL−1 ADH preparation, 5% (v/v) 2-PrOH, 30 °C. For details, see the
Experimental section.
ketones 2 from the corresponding halo ketones 1 into the one-
pot system. As a first test, 1a was reacted at 60 °C with 1.2
equiv. of NaN3 in buffer containing 5% (v/v) 2-propanol and
varied amounts of potassium iodide as a nucleophilic substi-
tution catalyst. With 5 and 10 mol% of iodide, the reaction
proceeded almost equally fast, and 90% conversion was
achieved within 4 h (Suppl. Fig. 5, ESI†). Transferring the same
conditions (10 mol% KI) to the autoclave setup used for the
one-pot sequence, the azidolysis reaction of 1a–e proceeded to
completion within 5 h, while the furan derivative 1f only
required 2 h for full conversion. In addition, we found that the
ADH-catalysed reduction could be performed on the crude
reaction mixture of the azidolysis step, as a minor decrease in
enzyme activity was easily compensated by slightly raising the
enzyme loading. Hence, the sequential combination of all
three reactions (azidolysis, ADH reduction, and hydrogenation)
proved feasible. All steps of the one-pot process could be run
to complete conversion, and no accumulation of any side pro-
ducts was observed. Consequently, the 1,2-amino alcohols 4a–f
were obtained with the same level of chemical and enantio-
Scheme 2 Rationalisation of the formation of 2,2-dimethyloxazolidines 5 in
the ADH/Pd-NP one-pot sequence.
To further prove the preparative value and the scalability of meric purity as in the two-step process (see Table 4).
the one-pot two-step reaction system, we performed the conver-
An exemplary gram-scale conversion (75 mL, 7.5 mmol sub-
sion of azidoketone 2b into 1,2-amino alcohol (R)-4b on gram strate) of 1c into (R)-4c using ADH-A (2.0 mg mL−1) as a bio-
scale (75 mL, 7.5 mmol substrate). ADH-A (1.5 mg mL−1) was catalyst provided the 1,2-amino alcohol in 84% isolated yield
used as a biocatalyst, and the target compound was isolated in (0.97 g) and optically pure form (ee >99%).
84% yield (1.08 g) and >99% ee.
Finally, we wanted to apply our newly developed chemo-
Encouraged by the positive results of the ADH/Pd-NP com- enzymatic one-pot reaction system to the asymmetric synthesis
bination, we sought to integrate the in situ formation of azido of a biologically active molecule. As a target compound, we
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compound. We chose Sheldon’s E-factor26 (mass of waste pro-
duced per mass of desired product) as a simple metric. We
also decided to exclude solvents from the E-factor analysis and
calculate the solvent demand as a second, independent indi-
cator, since solvent waste and non-volatile waste (particularly
salts) require very different processing. Both metrics can only
provide a rough estimation of environmental impact, as they
do not take into account the chemical composition (and hence
the toxicity) of the waste, the energy demand of the involved
processes, or the waste generated in the preparation of starting
materials and catalysts. On the other hand, such a basic analy-
sis is easily performed, and can thus serve as a quick eco-
assessment of several synthetic options.
Table 5 provides an overview of the environmental perform-
ance of the six synthetic sequences under investigation. The
chemo-enzymatic four-step one-pot system presented herein
achieves the second-highest yield and has clear environmental
advantages over the previously published procedures. Only the
synthesis developed by Brown et al. reaches comparable values
for yield, E-factor and solvent demand; however, it requires
highly toxic hydrogen cyanide as a reagent in the asymmetric
key step.
Table 4 Chemo-enzymatic three-step one-pot transformation of halo ketones
1a–f into amino alcohols 4a–fa
GC yield Isol. yield
ee (4)d
[%]
Entry Subst. ADH
(4)b [%]
(4)c [%]
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1a
1b
1c
1d
1e
1f
ADH-A
ADH-A
ADH-A
ADH-A
ADH-A
ADH-A
KRED-NADH-110
KRED-NADH-110 >99
KRED-NADH-110 >99
KRED-NADH-110 >99
KRED-NADH-110 >99
KRED-NADH-110 >99
>99
>99
>99
>99
>99
>99
99
83 (57 mg) >99 (R)
75 (63 mg) >99 (R)
89 (69 mg) >99 (R)
81 (61 mg) >99 (R)
75 (63 mg) >99 (R)
39 (25 mg) >99 (S)e
76 (52 mg) >99 (S)
80 (69 mg) >99 (S)
79 (61 mg) >99 (S)
82 (62 mg) >99 (S)
86 (72 mg) >99 (S)
36 (23 mg) >99 (R)e
9
10
11
12
a Conditions: 100 mM 1, 120 mM NaN3, 10 mM KI, 0.1 mM NAD+,
0.3–4.0 mg mL−1 ADH preparation, 0.5 mM Pd-NPs, 5% (v/v) 2-PrOH,
60–30 °C, 26–29 h. For details, see the Experimental section. b Relative
amount of 4 in the crude product determined by GC–FID analysis.
Rest to 100% is the corresponding 2,2-dimethyloxazolidine 5. c Isolated
yield of pure 4 after column chromatography; semi-preparative scale
(5 mL; 0.5 mmol substrate). d Enantiomeric excess of 4 as determined
by chiral GC–FID analysis after conversion into the corresponding 2,2-
dimethyloxazolidine 5. e Switch in substituent priorities according to
Cahn–Ingold–Prelog rules.
Differentiation of the E-factor into the contributions of the
reaction itself (excess of reagents, coupled products, by-
products, catalysts) and the down-stream processing reveals the
main advantage of the one-pot concept: the elimination of the
isolation and purification steps leads to significant reductions
in waste generation, which for all syntheses except the one
chose (S)-tembamide, a naturally occurring benzamide deriva- reported by Yadav et al. (which uses large quantities of carrot
tive, for which antiviral (HIV) activity has been reported.25 The root as a catalyst), is mainly linked to work-up and purification
synthesis required benzoylation of amino alcohol (S)-4e as the rather than loss of material in the reaction itself (Fig. 3). Never-
final step, which was achieved by simply adding a solution of theless, our synthesis also features the lowest reaction-linked
benzoyl chloride (1.2 eq.) in MTBE to the alkaline, aqueous E-factor contribution (3.2) of the six procedures analysed.
reaction mixture obtained after the Pd-NP-catalysed hydrogen-
Differentiation by the type of waste (see Suppl. Fig. 10, ESI†)
ation step (Scheme 3). A gram-scale reaction (50 mL, 5.0 mmol shows that silica gel contributes substantially to the E-factor of
substrate, 0.5 mg mL−1 KRED-NADH-110 as biocatalyst) the syntheses that make use of it. Those sequences that do not
yielded 0.98 g (73% from 1e) of (+)-(S)-tembamide, thus provid- require chromatographic purifications (Baeza et al., Brown
ing access to this natural product in a four-step one-pot et al., and the present work), mainly produce inorganic salts
operation.
(NaBr, NaCl, NaOH, Na2SO4, MgSO4, and others) as waste,
which are arguably less problematic.
Environmental impact assessment
Finally, a more detailed analysis of the different types of sol-
vents used (see Suppl. Table 7, ESI†) shows that our chemo-
enzymatic sequence generally employs more environmentally
acceptable solvents31 (mostly water, ethyl acetate, and ethanol)
than the other processes, especially because it avoids the use of
chlorinated solvents and does not require eluents for chromato-
graphy, which often contain large amounts of hexane.
Because of its highly ‘integrative’ nature we considered the
tembamide synthesis a suitable test case for assessing the eco-
logical benefits of the multi-step one-pot concept. Therefore,
we performed a basic environmental impact analysis, in which
we compared our four-step one-pot preparation of tembamide
to previously reported asymmetric syntheses of this
Scheme 3 Asymmetric synthesis of (S)-tembamide in a chemo-enzymatic four-step one-pot sequence.
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Table 5 Environmental impact comparison of catalytic asymmetric syntheses of tembamide
Solventd
Article
Stepsa
Asymmetric key step
Yieldb [%]
E-factorc
[mL g−1
]
Ref.
Present work
Lee et al. 2007
Baeza et al. 2005
4 (1)
5 (5)
3 (2)
Asymmetric ketone reduction (ADH)
73
62
65
11.1
57.8
23.3
309
1600
1031
—
27
28
Asymmetric ketone reduction (Rh catalyst)
Asymmetric cyano-O-phosphorylation
(Lewis acid/Brønsted base catalyst)
Kamal et al. 2004
Yadav et al. 2001
Brown et al. 1993, 1994
5 (4)
3 (2)e
3 (3)
Enantioselective transesterification (lipase)
Asymmetric ketone reduction (carrot root)
Asymmetric hydrocyanation (peptide catalyst)
42
85
72
114.9
97.5
14.6
1801
826
483
29
16b
30
a Total number of chemical transformations. The number of steps carried out individually (with product isolation) is given in parentheses.
b Overall yield. c Overall E-factor (excluding solvents). d Overall solvent demand. e Please note that this synthesis starts from 2-azido ketone 2e,
which is not commercially available, and therefore also needs to be synthesised.
terms of yield, but especially regarding its ecological impact,
as quantified by E-factor and solvent demand. Hence, our
study highlights the advantages of chemo-enzymatic one-pot
processes in the multi-step synthesis of chiral compounds,
and since it uses catalysts that are either commercially avail-
able or easily prepared, we believe that it will also be of practi-
cal value to synthetic chemists.
Experimental
General materials and methods
Unless otherwise noted, reagents and organic solvents were
obtained from chemical suppliers in reagent grade quality and
Fig. 3 Contributions of reaction steps, product isolation steps, and product
used without further purification. Petroleum ether (boiling
purification steps to the overall E-factor (excluding solvents) of catalytic asym-
range 40–60 °C) and ethyl acetate used for extraction and
metric syntheses of tembamide.
column chromatography were purchased in technical grade
quality and were distilled prior to use. Pro analysi (p.a., >99%
purity) grade solvents were used for handling the 1,2-amino
alcohols 4a–f to avoid any undesired oxazolidine formation
Conclusions
In summary, we have developed chemo-enzymatic one-pot
reaction sequences that provide access to enantiomerically
pure 1,2-amino alcohols either in two steps from the corres-
ponding 2-azido ketones or in three steps from 2-halo ketones.
The biocatalytic reduction of 2-azido ketones using alcohol
dehydrogenases (ADHs) is the asymmetric key step in these
processes, and by selecting suitable ADHs, both enantiomers
of the target compounds can be obtained in excellent enantio-
meric excess (ee >99%). The 2-amino-1-arylethanol derivatives
4a–f thus prepared are important building blocks in pharma-
ceutical research, for instance in the synthesis of anti-inflam-
matory, antiviral, or antitumour agents.32
Furthermore, the one-pot concept has been applied to the
asymmetric synthesis of the antiviral natural product (S)-tem-
bamide, obtained in 73% yield over four steps and >99% ee
from commercially available bromo ketone 1e. This synthesis
reaches high catalyst turnover (TON = 200 for Pd, 1000 for
NAD+, several 10 000 for the ADH),33 uses only a small excess
of reagents (1.2 eq. of NaN3 and BzCl), and affords a chemi-
cally pure product after a final recrystallisation as the sole puri-
fication step. Due to these features, our method compares
favourably with previous syntheses of tembamide not only in
due to contaminating acetone. Sulfonated lignin with either
calcium or ammonium counterions was obtained from Burgo
Group S.p.A., Tolmezzo, Italy. Low-sulfonate Kraft lignin was
obtained from Sigma-Aldrich. The halo ketones 1a–e as well as
both enantiomers of amino alcohol 4a were obtained commer-
cially; all other substrates and reference compounds were syn-
thesised as described in the ESI.†
The proprietary enzymes used in this study are part of the
Codexis Codex KRED screening kit, the Almac selectAZyme
CRED screening kit, and the evocatal ADH screening kit. The
ADHs from Lactobacillus kefir and Thermoanaerobium brockii
were obtained from Sigma-Aldrich. ADH-A from Rhodococcus
ruber DSM 44541 and LbADH from Lactobacillus brevis were
heterologously expressed in E. coli as described in the ESI.†
Hydrogenation reactions, as well as analytical-scale (2 mL)
and semi-preparative scale (5 mL) chemo-enzymatic one-pot
transformations were carried out in magnetically stirred stain-
less steel autoclaves (16 mL total volume) that are part of a
HEL PolyBlock8 parallel reactor system, and reactor tempera-
ture as well as stirring speed were controlled using the associ-
ated HEL WinISO software (v. 2.3.85.1). Gram-scale chemo-
enzymatic one-pot transformations were carried out in a
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mechanically stirred Parr 4560 series stainless steel autoclave approx. 650 mM). Potassium phosphate buffer (425 µL;
(452HC2 bomb cylinder, 160 mL total volume), and the reactor 100 mM, pH 7.0, 1 mM MgSO4) and a stock solution of ADH
temperature was controlled using a Parr 4841 heater/controller. (1 mg mL−1; final conc. 0.1 mg mL−1) and NAD(P)+ (0.7 mg
Non-enzymatic reactions were generally stirred at 500 rpm, mL−1, 1 mM; final conc. 100 µM) in potassium phosphate
while biotransformations were stirred at 300 rpm (to minimise buffer (50 µL) were added, and the mixture was shaken at
mechanical stress).
30 °C and 1000 rpm on a thermoshaker for 2 h. The reaction
Thin layer chromatography was carried out on silica gel mixture was extracted with EtOAc (800 µL), the extract was
60 F254 plates (Merck) and compounds were visualized either dried over MgSO4 and conversion as well as product ee were
by UV or by dipping into cerium ammonium molybdate stain determined by GC–FID analysis.
[50 g L−1 (NH4)6Mo7O24·4H2O, 2 g L−1 Ce(SO4)2·4H2O in 10%
Investigation of the substrate scope of ADH-A and KRED-
(v/v) sulfuric acid] or basic permanganate stain (50 g L−1 NADH-110. In a microcentrifuge tube (2 mL), azido ketone
Na2CO3, 10 g L−1 KMnO4, 0.85 g L−1 NaOH in demineralised 2a–f (25 µmol; final conc. 50 mM) was dissolved in 2-propanol
water). Melting points were determined in open capillary tubes (25 µL; final conc. 5% v/v, approx. 650 mM). Potassium phos-
1
on a Büchi B-540 apparatus and are uncorrected. H-NMR and phate buffer (425 µL; 100 mM, pH 7.0, 1 mM MgSO4) and a
13C-NMR spectra were recorded in CDCl3 solution on a Bruker stock solution of ADH (1 mg mL−1; final conc. 0.1 mg mL−1
)
Avance 400 instrument at 400 and 100 MHz, respectively. and NAD(P)+ (0.7 mg mL−1, 1 mM; final conc. 100 µM) in pot-
Chemical shifts are given in parts per million (ppm) relative to assium phosphate buffer (50 µL) were added, and the mixture
the residual CHCl3 peak (1H: δ = 7.26 ppm, 13C: δ = 77.2 ppm) was shaken at 30 °C and 1000 rpm on a thermoshaker for 2 h.
and coupling constants (J) are reported in Hertz (Hz). Specific The reaction mixture was extracted with EtOAc (800 µL), and
optical rotation values [α]2D0 were determined on a Perkin-Elmer the extract was dried over MgSO4. Conversion was determined
Model 343 Polarimeter at 20 °C and a wavelength of 589 nm by GC–FID analysis, while product ee was determined either by
(sodium D-line) using a cuvette of 1 dm path length.
GC–FID analysis (3a, 3e, 3f) or by HPLC analysis (3b–d).
Test transformations to ensure complete conversion under
the conditions of the one-pot sequence were carried out in
Azido alcohol hydrogenation catalysed by metal nanoparticles
Screening of metal nanoparticles for activity and selectivity small-scale autoclave reactors (16 mL): azido ketone 2a–f
in the hydrogenation of azido alcohol 3a. In a small-scale (200 µmol; final conc. 100 mM) was dissolved/dispersed in
autoclave reactor (16 mL), 2-azido-1-phenylethanol (3a; 32 mg, 2-propanol (100 µL; final conc. 5% v/v, approx. 650 mM). Potass-
200 µmol; final conc. 100 mM) was dissolved in 2-propanol ium phosphate buffer (1.7 mL; 100 mM, pH 7.0, 1 mM MgSO4)
(100 µL; final conc. 5% v/v). Potassium phosphate buffer (1.72 and a stock solution of ADH (2–25 mg mL−1; final conc.
or 1.82 mL, depending on nanoparticle type; 100 mM, pH 7.0, 0.2–2.5 mg mL−1, see Table 6) and NAD(P)+ (0.7 mg mL−1
,
1 mM MgSO4) and a stock solution of metal nanoparticles in 1 mM; final conc. 100 µM) in potassium phosphate buffer
water (Pd: 180 µL of a 5.6 mM stock, Pt: 83 µL of a 12 mM (200 µL) were added, and the mixture was stirred at 30 °C and
stock; final conc. 0.5 mM) were added, and the mixture was 300 rpm for 20 h. The reaction mixture was transferred to micro-
stirred at 30 °C and 500 rpm under hydrogen atmosphere centrifuge tubes, the product was extracted into EtOAc (2 ×
(10 bar) for 4 h. The reaction mixture was extracted with EtOAc 1 mL), and the extract was dried over MgSO4. Conversion was
(800 µL), the extract was dried over MgSO4 and conversion was determined by GC–FID analysis, while product ee was deter-
determined by GC–FID analysis.
mined by GC–FID analysis (3a, 3f) or HPLC analysis (3b–e).
Investigation of the tolerance of Pd nanoparticles towards
the presence of ADHs and NAD(P)+. Reactions were set up as
described above, but contained 0.1–1.5 mg mL−1 of different
ADHs and 100 µM NAD+ or NADP+.
Chemo-enzymatic one-pot transformations
One-pot, two-step transformation of azido ketones 2a–f into
Hydrogenation of azido alcohols 3a–f catalysed by Pd nano- amino alcohols 4a–f (analytical scale, 2 mL). A small-scale
particles. Reactions were set up as described above, using autoclave reactor (16 mL) was charged with the azido ketone 2
200 µmol (final conc. 100 mM) of azido alcohols 3a–f as sub-
strates. After 4 h, the reaction mixture was transferred to micro-
Table 6 ADH concentrations used for the two-step one-pot transformation of
centrifuge tubes, the product was extracted into EtOAc (2 ×
1 mL), and the extract was dried over MgSO4. Evaporation of the
solvent under reduced pressure afforded the crude amino alco-
hols 4a–f. The conversion as well as the purity of the crude pro-
ducts were determined by GC–FID and NMR analysis.
azido ketones 2a–f into amino alcohols 4a–f
c(ADH-A)a
c(KRED-NADH-110)a
Substrate
[mg mL−1
]
[mg mL−1
]
2a
2b
2c
2d
2e
2f
1.5
1.5
1.5
1.5
2.5
1.5
0.2
0.2
0.2
0.2
0.4
1.0
Biotransformations
Screening of ADHs for activity and stereoselectivity in the
reduction of azido ketone 2a. In a microcentrifuge tube
(2 mL), 2-azidoacetophenone (2a; 4 mg, 25 µmol; final conc.
a Final concentration of crude ADH preparation in the reaction
50 mM) was dissolved in 2-propanol (25 µL; final conc. 5% v/v, mixture.
3324 | Green Chem., 2013, 15, 3318–3331
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(200 µmol; final conc. 100 mM), 2-propanol (100 µL; final CDCl3): δ [ppm] = 2.25 (3H, br s, OH, NH2), 2.80 (1H, dd, J =
conc. 5% v/v, approx. 650 mM), potassium phosphate buffer 12.8 Hz, 7.7 Hz, CH2), 2.93 (1H, dd, J = 12.9 Hz, 3.9 Hz, CH2),
(1.7 mL; 100 mM, pH 7.0, 1 mM MgSO4) and a stock solution 4.62 (1H, dd, J = 7.8 Hz, 4.0 Hz, CH-OH), 7.26–7.37 (5H, m, Ar).
of ADH (ADH-A: 15–25 mg mL−1, final conc. 1.5–2.5 mg mL−1
Codexis KRED-NADH-110: 2.0–10 mg mL−1
;
13C-NMR (100 MHz, CDCl3): δ [ppm] = 49.3, 74.4, 125.9, 127.5,
,
final conc. 128.4, 142.6. GC–MS (EI, 70 eV): m/z = 137 (M+, <1), 118 (3),
0.2–1.0 mg mL−1; see Table 6) and NAD+ (0.7 mg mL−1, 1 mM; 107 (31), 91 (11), 79 (100), 77 (79), 65 (6), 51 (37). The NMR
final conc. 100 µM) in potassium phosphate buffer (200 µL), data are in accordance with literature values.36
and the mixture was stirred at 30 °C and 300 rpm. The vent
(S)-2-Amino-1-phenylethanol [(S)-4a]. 59 mg (86%) off-white
valve of the autoclave reactor was connected to a vacuum solid. mp: 61–62 °C (lit.34 57–59 °C). TLC (silica, MTBE–
pump via a pressure control valve, and the reactor was evacu- MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = +58.5 (CHCl3, c =
ated to 200 mbar. After a reaction time of 18 h, the tempera- 1.53); lit.35 +60.6 (CHCl3, c = 0.5). NMR and MS data were in
ture was raised to 45 °C (to increase acetone evaporation) and accordance with those of the opposite enantiomer.
stirring was continued for 2 h. A sample (50 µL) was taken,
(R)-2-Amino-1-(4-chlorophenyl)ethanol
[(R)-4b]. 76
mg
extracted with EtOAc (800 µL), the extract was dried over (89%) off-white solid. mp: 94–95 °C (lit.37 92–94 °C). TLC
MgSO4 and conversion was determined by GC analysis. The (silica, MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = −64.5
reaction mixture was then made alkaline (pH 9) by the (CHCl3, c = 1.08); lit.38 (S) +67.4 (CHCl3, c = 0.35). 1H-NMR
addition of 4 M aq. NaOH solution (25 µL), a stock solution of (400 MHz, CDCl3): δ [ppm] = 2.30 (3H, br s, OH, NH2), 2.73
Pd nanoparticles (180 µL of a 5.6 mM stock; final conc. (1H, dd, J = 12.8 Hz, 7.8 Hz, CH2), 2.92 (1H, d, J = 11.3 Hz,
0.5 mM) was added, and the mixture was stirred for another CH2), 4.57 (1H, dd, J = 8.0 Hz, 3.9 Hz, CH-OH), 7.26 (2H, d, J =
4 h at 30 °C and 500 rpm under hydrogen atmosphere 8.3 Hz, Ar-o), 7.31 (2H, d, J = 8.1 Hz, Ar-m). 13C-NMR
(10 bar). The reaction mixture was extracted with EtOAc (100 MHz, CDCl3): δ [ppm] = 49.2, 73.5, 127.2, 128.5, 133.1,
(800 µL), the extract was dried over MgSO4 and conversion as 141.1. GC–MS (EI, 70 eV): m/z = 171 (M+, 1), 143 (5), 141 (16),
well as product ee were determined by GC analysis.
115 (4), 113 (13), 77 (100), 51 (28), 50 (14). The NMR data are
One-pot, two-step transformation of azido ketones 2a–f into in accordance with literature values.36
amino alcohols 4a–f (semi-preparative scale, 5 mL). A small-
(S)-2-Amino-1-(4-chlorophenyl)ethanol [(S)-4b]. 67 mg (78%)
scale autoclave reactor (16 mL) was charged with the azido- off-white solid. mp: 94–95 °C (lit.37 92–94 °C). TLC (silica,
ketone 2 (500 µmol; final conc. 100 mM), 2-propanol (250 µL; MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = +64.7
final conc. 5% v/v), potassium phosphate buffer (4.25 mL; (CHCl3, c = 0.71); lit.38 +67.4 (CHCl3, c = 0.35). NMR and MS
100 mM, pH 7.0, 1 mM MgSO4) and a stock solution of ADH data were in accordance with those of the opposite
(ADH-A: 15–25 mg mL−1, final conc. 1.5–2.5 mg mL−1; Codexis enantiomer.
KRED-NADH-110: 2.0–10 mg mL−1, final conc. 0.2–1.0 mg
(R)-2-Amino-1-(4-fluorophenyl)ethanol [(R)-4c]. 67 mg (86%)
mL−1; see Table 6) and NAD+ (0.7 mg mL−1, 1 mM; final conc. off-white solid. mp: 83–84 °C (lit.38 63–65 °C). TLC (silica,
100 µM) in potassium phosphate buffer (500 µL), and the MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = −66.8
mixture was stirred at 30 °C and 300 rpm. The vent valve of the (CHCl3, c = 1.08); lit.38 (S) +40.8 (EtOH, c = 1.68). 1H-NMR
autoclave reactor was connected to a vacuum pump via a (400 MHz, CDCl3): δ [ppm] = 2.24 (3H, br s, OH, NH2), 2.75
pressure control valve, and the reactor was evacuated to (1H, dd, J = 12.8 Hz, 7.9 Hz, CH2), 2.94 (1H, dd, J = 12.6 Hz, 3.9
200 mbar. After a reaction time of 18 h, the temperature was Hz, CH2), 4.59 (1H, dd, J = 8.0 Hz, 3.9 Hz, CH-OH), 7.03 (2H, t,
raised to 45 °C (to increase acetone evaporation) and stirring J = 8.5 Hz, Ar-m), 7.31 (2H, dd, J = 8.3 Hz, 5.4 Hz, Ar-o).
was continued for 2 h. The reaction mixture was then made 13C-NMR (100 MHz, CDCl3): δ [ppm] = 49.3, 73.6, 115.2 (d, JCF
alkaline (pH 9) by the addition of 4 M aq. NaOH solution = 21.3 Hz), 127.5 (d, JCF = 8.0 Hz), 138.3 (d, JCF = 3.1 Hz), 162.2
(65 µL), a stock solution of Pd nanoparticles (450 µL of a (d, JCF = 245 Hz). GC–MS (EI, 70 eV): m/z = 155 (M+, 1), 125
5.6 mM stock; final conc. 0.5 mM) was added, and the mixture (39), 123 (16), 109 (14), 97 (100), 95 (41), 77 (56), 75 (22), 70 (8),
was stirred for an additional 4 h at 30 °C and 500 rpm under 57 (11), 51 (25), 50 (14). The NMR data are in accordance with
hydrogen atmosphere (10 bar). The reactor was depressurised, literature values.38
4 M aq. NaOH solution (500 µL) was added, and the reaction
(S)-2-Amino-1-(4-fluorophenyl)ethanol [(S)-4c]. 68 mg (88%)
mixture was saturated with NaCl. The product was extracted off-white solid. mp: 83–84 °C (lit.38 63–65 °C). TLC (silica,
into EtOAc (4 × 5 mL), the combined extracts were dried over MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = +66.7
MgSO4 and evaporated under reduced pressure to give the (CHCl3, c = 0.90); lit.38 +40.8 (EtOH, c = 1.68). NMR and MS
crude amino alcohols as orange oils or solids. Column chrom- data were in accordance with those of the opposite
atography (∼0.6 g of silica gel 60 in a Pasteur pipette; MTBE → enantiomer.
MTBE–MeOH–NH4OH = 90/9/1) afforded the pure amino
(R)-2-Amino-1-(4-tolyl)ethanol [(R)-4d]. 64 mg (85%) off-
alcohols 4a–f.
white solid. mp: 59–60 °C (lit.37 67–69 °C). TLC (silica, MTBE–
(R)-2-Amino-1-phenylethanol [(R)-4a]. 60 mg (87%) off-white MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = −65.5 (CHCl3, c =
solid. mp: 63–64 °C (lit.34 57–59 °C). TLC (silica, MTBE– 1.00); lit.39 −32 (CHCl3, c = 5.0). H-NMR (400 MHz, CDCl3): δ
1
MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = −58.8 (CHCl3, c = [ppm] = 2.19 (3H, br s, OH, NH2), 2.35 (3H, s CH3), 2.79 (1H,
1.31); lit.35 (S) +60.6 (CHCl3, c = 0.5). 1H-NMR (400 MHz, dd, J = 12.8 Hz, 7.8 Hz, CH2), 2.93 (1H, d, J = 11.1 Hz, CH2),
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4.58 (1H, dd, J = 7.8 Hz, 4.0 Hz, CH-OH), 7.16 (2H, d, J = (5 mL), and the mixture was stirred at 30 °C and 300 rpm. The
7.7 Hz, Ar-m), 7.23 (2H, d, J = 7.6 Hz, Ar-o). 13C-NMR vent valve of the autoclave reactor was connected to a vacuum
(100 MHz, CDCl3): δ [ppm] = 21.1, 49.3, 74.3, 125.8, 129.1, pump via a pressure control valve, and the reactor was evacu-
137.1, 139.7. GC–MS (EI, 70 eV): m/z = 151 (M+, 3), 122 (15), ated to 200 mbar. After a reaction time of 18 h, the temp-
121 (100), 119 (12), 93 (93), 91 (96), 77 (75), 65 (28), 51 (18). erature was raised to 45 °C and stirring continued for 2 h. The
The NMR data are in accordance with literature values.10d
reaction mixture was cooled to room temperature, and com-
(S)-2-Amino-1-(4-tolyl)ethanol [(S)-4d; CAS 149403-05-4]. plete consumption of the 2b was verified by TLC analysis. The
65 mg (86%) off-white solid. mp: 59–60 °C (lit.37 67–69 °C). TLC reaction mixture was then made alkaline (pH 9) by the
(silica, MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = +65 addition of 4 M aq. NaOH solution (1 mL), a stock solution of
(CHCl3, c = 1.00); lit. (R)39 −32 (CHCl3, c = 5.0). NMR and MS Pd nanoparticles (6.7 mL of a 5.6 mM stock; final conc.
data were in accordance with those of the opposite enantiomer.
0.5 mM), was added, and the mixture was stirred for an
(R)-2-Amino-1-(4-methoxyphenyl)ethanol [(R)-4e]. 71 mg additional 16 h (overnight) at 30 °C and 500 rpm under hydro-
(85%) off-white solid. mp: 102–103 °C. TLC (silica, MTBE– gen atmosphere (10 bar). Complete consumption of 3b was
MeOH–NH4OH = 90/9/1): Rf = 0.09. [α]2D0 = −61.4 (CHCl3, c = verified by TLC analysis, 4 M aq. NaOH solution (10 mL) was
0.93); lit.38 −39.9 (CHCl3, c = 1.03). 1H-NMR (400 MHz, CDCl3): added, and the reaction mixture was saturated with NaCl. The
δ [ppm] = 1.94 (3H, br s, OH, NH2), 2.79 (1H, dd, J = 12.8 Hz, product was extracted into EtOAc (5 × 40 mL; phase separation
7.8 Hz, CH2), 2.97 (1H, dd, J = 12.5 Hz, 4.0 Hz, CH2), 3.81 (3H, accelerated by centrifugation), and the combined extracts were
s, OCH3), 4.59 (1H, dd, J = 7.9 Hz, 4.1 Hz, CH-OH), 6.90 (2H, d, dried over MgSO4 and evaporated under reduced pressure to
J = 8.7 Hz, Ar-m), 7.29 (2H, d, J = 8.8 Hz, Ar-o). 13C-NMR give 1.30 g of an orange solid. Column chromatography (silica
(100 MHz, CDCl3): δ [ppm] = 49.3, 55.3, 74.1, 113.8, 127.1, gel 60, MTBE → MTBE–MeOH–NH4OH = 90/9/1) afforded 1.08 g
134.6, 159.1. GC–MS (EI, 70 eV): m/z = 167 (M+, 3), 138 (10), (84%) of (R)-4b as an off-white solid. mp: 94–95 °C (lit.37
137 (100), 109 (42), 94 (43), 77 (40), 66 (14), 65 (10), 51 (9). The 92–94 °C). TLC (silica, MTBE–MeOH–NH4OH = 90/9/1): Rf =
NMR data are in accordance with literature values.10d
0.11. [α]2D0 = −64.4 (CHCl3, c = 0.94); lit.38 (S) +67.4 (CHCl3, c =
(S)-2-Amino-1-(4-methoxyphenyl)ethanol [(S)-4e; CAS 46084- 0.35). NMR and MS data were in accordance with those obtained
19-9]. 73 mg (87%) off-white solid. mp: 102–103 °C. TLC for the product of the semi-preparative scale experiment.
(silica, MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.09. [α]2D0 = +61.6
One-pot, three-step transformation of halo ketones 1a–f into
(CHCl3, c = 1.01); lit. (R)38 −39.9 (CHCl3, c = 1.03). NMR and amino alcohols 4a–f (analytical scale, 2 mL). A small-scale
MS data were in accordance with those of the opposite autoclave reactor (16 mL) was charged with the halo ketone 1
enantiomer.
(200 µmol; final conc. 100 mM), 2-propanol (100 µL; final
(S)-2-Amino-1-(2-furyl)ethanol [(S)-4f]. 35 mg (55%) yellow- conc. 5% v/v, approx. 650 mM) and potassium phosphate
ish solid. mp: 80–81 °C. TLC (silica, MTBE–MeOH–NH4OH = buffer (1.9 mL; 100 mM, pH 7.0, 1 mM MgSO4) containing
90/9/1): Rf = 0.14. [α]2D0 = −33.5 (CHCl3, c = 0.93); lit.40 −38.6 NaN3 (7.8 mg mL−1, 120 mM) and KI (1.7 mg mL−1, 10 mM),
1
(CHCl3, c = 1.80). H-NMR (400 MHz, CDCl3): δ [ppm] = 2.53 and the mixture was stirred at 60 °C and 500 rpm for 2–5 h. A
(3H, br s, OH, NH2), 2.98 (2H, d, J = 5.8 Hz, CH2), 4.61 (1H, t, sample (50 µL) was taken, extracted with EtOAc (800 µL), the
J = 5.8 Hz, CH-OH), 6.24 (1H, d, J = 3.3 Hz, Ar-3), 6.32 (1H, dd, extract was dried over MgSO4 and conversion was determined
J = 3.2 Hz, 1.8 Hz, Ar-4) 7.35 (1H, d, J = 1.8 Hz, Ar-5). 13C-NMR by GC analysis. Additional 2-propanol (100 µL; to supplement
(100 MHz, CDCl3): δ [ppm] = 46.0, 68.2, 106.3, 110.2, 142.0, the material lost through evaporation) and a stock solution of
155.5. GC–MS (EI, 70 eV): m/z = 127 (M+, 12), 98 (82), 97 (86), ADH (ADH-A: 20–30 mg mL−1, final conc. 2.0–3.0 mg mL−1
;
81 (8), 69 (33), 53 (17), 51 (15), 42 (24), 41 (100). The NMR data Codexis KRED-NADH-110: 3.0–15 mg mL−1
are in accordance with literature values.41 0.3–1.5 mg mL−1; see Table 7) and NAD+ (0.7 mg mL−1, 1 mM;
, final conc.
(R)-2-Amino-1-(2-furyl)ethanol [(R)-4f]. 24 mg (38%) yellow- final conc. 100 µM) in potassium phosphate buffer (200 µL)
ish solid. mp: 81–82 °C. TLC (silica, MTBE–MeOH–NH4OH = were added, and the mixture was stirred at 30 °C and 300 rpm.
90/9/1): Rf = 0.14. [α]D20 = +33.6 (CHCl3, c = 0.85); lit. (S)40 −38.6
(CHCl3, c = 1.80). NMR and MS data were in accordance with
those of the opposite enantiomer.
Table 7 ADH concentrations used for the three-step one-pot transformation of
The ADH concentrations used for the one-pot, two-step
transformation of azido ketones 2a–f into amino alcohols 4a–f
are summarised in Table 6.
halo ketones 1a–f into amino alcohols 4a–f
c(ADH-A)a
c(KRED-NADH-110)a
Substrate
[mg mL−1
]
[mg mL−1
]
One-pot, two-step transformation of azido ketone 2b into
amino alcohol (R)-4b (gram scale, 75 mL). An autoclave
reactor (160 mL) was charged with 2-azido-4′-chloroacetophe-
none (2b; 1.47 g, 7.5 mmol; final conc. 100 mM), 2-propanol
(3.75 mL; final conc. 5% v/v), potassium phosphate buffer
(66.25 mL; 100 mM, pH 7.0, 1 mM MgSO4) and a stock solu-
tion of ADH-A (113 mg; final conc. 1.5 mg mL−1) and NAD+
2a
2b
2c
2d
2e
2f
2.0
2.0
2.0
2.0
3.0
2.0
0.3
0.3
0.3
0.3
0.5
1.5
a Final concentration of crude ADH preparation in the reaction
(6 mg; final conc. 100 µM) in potassium phosphate buffer mixture.
3326 | Green Chem., 2013, 15, 3318–3331
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The vent valve of the autoclave reactor was connected to a
(S)-2-Amino-1-phenylethanol [(S)-4a]. 52 mg (76%) off-white
vacuum pump via a pressure control valve, and the reactor was solid. mp: 61–62 °C (lit.34 57–59 °C). TLC (silica, MTBE–
evacuated to 200 mbar. After a reaction time of 18 h, the temp- MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = +59.0 (CHCl3, c =
erature was raised to 45 °C (to increase acetone evaporation) 1.02); lit.35 +60.6 (CHCl3, c = 0.5). NMR and MS data were in
and stirring was continued for 2 h. A sample (50 µL) was accordance with those obtained for the product of the two-step
taken, extracted with EtOAc (800 µL), the extract was dried over one-pot sequence.
MgSO4 and conversion was determined by GC analysis. The
(R)-2-Amino-1-(4-chlorophenyl)ethanol
[(R)-4b]. 63
mg
reaction mixture was then made alkaline (pH 9) by the (75%) off-white solid. mp: 93–95 °C (lit.37 92–94 °C). TLC
addition of 4 M aq. NaOH solution (25 µL), a stock solution of (silica, MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = −64.2
Pd nanoparticles (180 µL of a 5.6 mM stock; final conc. (CHCl3, c = 1.08); lit. (S)38 +67.4 (CHCl3, c = 0.35). NMR and
0.5 mM) was added, and the mixture was stirred for another MS data were in accordance with those obtained for the
4 h at 30 °C and 500 rpm under hydrogen atmosphere product of the two-step one-pot sequence.
(10 bar). The reaction mixture was extracted with EtOAc
(S)-2-Amino-1-(4-chlorophenyl)ethanol [(S)-4b]. 69 mg (80%)
(800 µL), the extract was dried over MgSO4 and conversion as off-white solid. mp: 94–95 °C (lit.37 92–94 °C). TLC (silica,
well as product ee were determined by GC analysis.
MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = +64.4
One-pot, three-step transformation of halo ketones 1a–f into (CHCl3, c = 1.10); lit.38 +67.4 (CHCl3, c = 0.35). NMR and MS
amino alcohols 4a–f (semi-preparative scale, 5 mL). A small- data were in accordance with those obtained for the product of
scale autoclave reactor (16 mL) was charged with the halo the two-step one-pot sequence.
ketone 1 (500 µmol; final conc. 100 mM), 2-propanol (250 µL;
(R)-2-Amino-1-(4-fluorophenyl)ethanol [(R)-4c]. 69 mg (89%)
final conc. 5% v/v, approx. 650 mM) and potassium phosphate off-white solid. mp: 82–83 °C (lit.38 63–65 °C). TLC (silica,
buffer (4.9 mL; 100 mM, pH 7.0, 1 mM MgSO4) containing MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = −66.9
NaN3 (7.8 mg mL−1, 120 mM) and KI (1.7 mg mL−1, 10 mM), (CHCl3, c = 1.04); lit. (S)38 +40.8 (EtOH, c = 1.68). NMR and MS
and the mixture was stirred at 60 °C and 500 rpm for 2–5 h. data were in accordance with those obtained for the product of
After cooling to room temperature, any larger agglomerates of the two-step one-pot sequence.
solid 2 were broken into smaller pieces using a stirring rod,
(S)-2-Amino-1-(4-fluorophenyl)ethanol [(S)-4c]. 61 mg (79%)
additional 2-propanol (250 µL; to supplement the material lost off-white solid. mp: 83–84 °C (lit.38 63–65 °C). TLC (silica,
through evaporation) and a stock solution of ADH (ADH-A: MTBE–MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = +66.7
20–35 mg mL−1, final conc. 2.0–3.5 mg mL−1; Codexis KRED- (CHCl3, c = 1.04); lit.38 +40.8 (EtOH, c = 1.68). NMR and MS
NADH-110: 3.0–15 mg mL−1, final conc. 0.3–1.5 mg mL−1; see data were in accordance with those obtained for the product of
Table 7) and NAD+ (0.7 mg mL−1, 1 mM; final conc. 100 µM) in the two-step one-pot sequence.
potassium phosphate buffer (500 µL) were added, and the
(R)-2-Amino-1-(4-tolyl)ethanol [(R)-4d]. 64 mg (85%) off-
mixture was stirred at 30 °C and 300 rpm. The vent valve of the white solid. mp: 56–58 °C (lit.37 67–69 °C). TLC (silica, MTBE–
autoclave reactor was connected to a vacuum pump via a MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = −64.5 (CHCl3, c =
pressure control valve, and the reactor was evacuated to 1.02); lit.39 −32 (CHCl3, c = 5.0). NMR and MS data were in
200 mbar. After a reaction time of 18 h, the temperature was accordance with those obtained for the product of the two-step
raised to 45 °C (to increase acetone evaporation) and stirring one-pot sequence.
was continued for 2 h. The reaction mixture was then made
(S)-2-Amino-1-(4-tolyl)ethanol [(S)-4d]. 62 mg (82%) off-
alkaline (pH 9) by the addition of 4 M aq. NaOH solution white solid. mp: 57–58 °C (lit.37 67–69 °C). TLC (silica, MTBE–
(65 µL), a stock solution of Pd nanoparticles (450 µL of a MeOH–NH4OH = 90/9/1): Rf = 0.11. [α]2D0 = +64.8 (CHCl3, c =
5.6 mM stock; final conc. 0.5 mM) was added, and the mixture 1.00); lit. (R)39 −32 (CHCl3, c = 5.0). NMR and MS data were in
was stirred for an additional 4 h at 30 °C and 500 rpm under accordance with those obtained for the product of the two-step
hydrogen atmosphere (10 bar). The reactor was depressurised, one-pot sequence.
4 M aq. NaOH solution (500 µL) was added, and the reaction
(R)-2-Amino-1-(4-methoxyphenyl)ethanol [(R)-4e]. 71 mg
mixture was saturated with NaCl. The product was extracted (85%) off-white solid. mp: 102–103 °C. TLC (silica, MTBE–
into EtOAc (4 × 5 mL), the combined extracts were dried over MeOH–NH4OH = 90/9/1): Rf = 0.09. [α]2D0 = −61.4 (CHCl3, c =
MgSO4 and evaporated under reduced pressure to give the 0.93); lit.38 −39.9 (CHCl3, c = 1.03). NMR and MS data were in
crude amino alcohols as orange oils or solids. Column chrom- accordance with those obtained for the product of the two-step
atography (∼0.6 g of silica gel 60 in a Pasteur pipette; MTBE → one-pot sequence.
MTBE–MeOH–NH4OH = 90/9/1) afforded the pure amino alco-
(S)-2-Amino-1-(4-methoxyphenyl)ethanol [(S)-4e]. 72 mg
hols 4a–f.
(86%) off-white solid. mp: 103–104 °C. TLC (silica, MTBE–
(R)-2-Amino-1-phenylethanol [(R)-4a]. 57 mg (83%) off-white MeOH–NH4OH = 90/9/1): Rf = 0.09. [α]2D0 = +61.4 (CHCl3, c =
solid. mp: 62–63 °C (lit.34 57–59 °C). TLC (silica, MTBE– 1.24); lit. (R)38 −39.9 (CHCl3, c = 1.03). NMR and MS data were
MeOH–NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = −58.8 (CHCl3, c = in accordance with those obtained for the product of the two-
1.31); lit. (S)35 +60.6 (CHCl3, c = 0.5). NMR and MS data were in step one-pot sequence.
accordance with those obtained for the product of the two-step
one-pot sequence.
(S)-2-Amino-1-(2-furyl)ethanol [(S)-4f]. 25 mg (39%) yellow-
ish solid. mp: 79–81 °C. TLC (silica, MTBE–MeOH–NH4OH =
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90/9/1): Rf = 0.14. [α]D20 = −33.9 (CHCl3, c = 0.77); lit.40 −38.6
One-pot, four-step transformation of halo ketone 1e into (S)-
(CHCl3, c = 1.80). NMR and MS data were in accordance with tembamide [CAS 15779-24-5] (gram scale, 50 mL). An auto-
those obtained for the product of the two-step one-pot clave reactor (160 mL) was charged with the 2-bromo-4′-meth-
sequence.
oxyacetophenone (1e; 1.15 g, 7.5 mmol; final conc. 100 mM),
(R)-2-Amino-1-(2-furyl)ethanol [(R)-4f]. 23 mg (36%) yellow- 2-propanol (2.5 mL; final conc. 5% v/v, approx. 650 mM) and
ish solid. mp: 80–81 °C. TLC (silica, MTBE–MeOH–NH4OH = potassium phosphate buffer (47.5 mL; 100 mM, pH 7.0, 1 mM
90/9/1): Rf = 0.14. [α]2D0 = +33.8 (CHCl3, c = 0.75); lit. (S)40 −38.6 MgSO4) containing NaN3 (0.39 g, 6 mmol; final conc. 120 mM)
(CHCl3, c = 1.80). NMR and MS data were in accordance with and KI (0.08 g, 0.5 mmol; final conc. 10 mM), and the mixture
those obtained for the product of the two-step one-pot was stirred at 60 °C and 500 rpm for 5 h. The reaction mixture
sequence.
was cooled to room temperature, and complete consumption
The ADH concentrations used for the one-pot, three-step of 1e was verified by TLC analysis. Any larger agglomerates of
transformation of halo ketones 1a–f into amino alcohols 4a–f solid 2e were broken into smaller pieces using a stirring rod,
are summarised in Table 7.
additional 2-propanol (2.5 mL; to supplement the material lost
One-pot, three-step transformation of halo ketone 1c into through evaporation) and a stock solution of Codexis KRED-
amino alcohols (R)-4c (gram scale, 75 mL). An autoclave NADH-110 (25 mg; final conc. 0.5 mg mL−1) and NAD+
reactor (160 mL) was charged with the 2-bromo-4′-fluoro- (3.3 mg; final conc. 100 µM) in potassium phosphate buffer
acetophenone (1c; 1.63 g, 7.5 mmol; final conc. 100 mM), (5 mL) were added, and the mixture was stirred at 30 °C and
2-propanol (3.75 mL; final conc. 5% v/v, approx. 650 mM) and 300 rpm. The vent valve of the autoclave reactor was connected
potassium phosphate buffer (71.25 mL; 100 mM, pH 7.0, to a vacuum pump via a pressure control valve, and the reactor
1 mM MgSO4) containing NaN3 (0.59 g, 9 mmol; final conc. was evacuated to 200 mbar. After a reaction time of 18 h, the
120 mM) and KI (0.13 g, 0.75 mmol; final conc. 10 mM), and temperature was raised to 45 °C (to increase acetone evapor-
the mixture was stirred at 60 °C and 500 rpm for 5 h. The ation) and stirring was continued for 2 h. The reaction mixture
reaction mixture was cooled to room temperature, and the was cooled to room temperature, and complete consumption
complete consumption of 1c was verified by TLC analysis. of 2e was verified by TLC analysis. The reaction mixture was
Any larger agglomerates of solid 2c were broken into smaller then made alkaline (pH 9) by the addition of 4 M aq. NaOH
pieces using a stirring rod, additional 2-propanol (3.75 mL; to solution (1 mL), a stock solution of Pd nanoparticles (6.7 mL
supplement the material lost through evaporation) and a of a 5.6 mM stock; final conc. 0.5 mM) was added, and the
stock solution of ADH-A (150 mg; final conc. 2.0 mg mL−1
)
mixture was stirred for an additional 4 h at 30 °C and 500 rpm
and NAD+ (5.3 mg; final conc. 100 µM) in potassium phos- under hydrogen atmosphere (10 bar). Complete consumption
phate buffer (5 mL) were added, and the mixture was stirred of 3e was verified by TLC analysis, 4 M aq. NaOH solution
at 30 °C and 300 rpm. The vent valve of the autoclave reactor (1.5 mL) and a solution of benzoyl chloride (0.84 g, 6.0 mmol)
was connected to a vacuum pump via a pressure control in MTBE (15 mL) were added, and stirring was continued at
valve, and the reactor was evacuated to 200 mbar. After a reac- room temperature for 2 h. Complete consumption of 4e was
tion time of 18 h, the temperature was raised to 45 °C (to verified by TLC analysis, EtOAc (20 mL) was added, and the
increase acetone evaporation) and stirring was continued for phases were separated (accelerated by centrifugation). The
2 h. The reaction mixture was cooled to room temperature, aqueous phase was extracted with EtOAc (3 × 30 mL; phase
and complete consumption of 2c was verified by TLC analy- separation accelerated by centrifugation), the combined
sis. The reaction mixture was then made alkaline (pH 9) by extracts were washed with 1 M aq. NaOH solution (40 mL) and
the addition of 4 M aq. NaOH solution (1 mL), a stock solu- brine (10 mL), and dried over MgSO4. The solvent was evapor-
tion of Pd nanoparticles (6.7 mL of a 5.6 mM stock; final ated under reduced pressure to give 1.40 g of an off-white
conc. 0.5 mM) was added, and the mixture was stirred for an solid. Recrystallisation (EtOH–water = 8/2) afforded 0.98 g
additional 4 h at 30 °C and 500 rpm under hydrogen atmos- (73%) of (S)-tembamide as a colourless, crystalline solid. mp:
phere (10 bar). Complete consumption of 3c was verified by 148–149 °C (lit.27 145–147 °C). TLC (silica, MTBE–MeOH–
TLC analysis, 4 M aq. NaOH solution (10 mL) was added, and NH4OH = 90/9/1): Rf = 0.10. [α]2D0 = +54.9 (CHCl3, c = 0.52); lit.29
the reaction mixture was saturated with NaCl. The product +56.9 (CHCl3, c = 0.54). 1H-NMR (400 MHz, DMSO-d6): δ [ppm]
was extracted into EtOAc (5 × 40 mL; phase separation accel- = 3.28–3.35 (1H, m, CH2), 3.43–3.49 (1H, m, CH2), 3.73 (3H, s,
erated by centrifugation), and the combined extracts were OCH3), 4.74 (1H, dt, J = 7.7 Hz, 4.7 Hz, CH-OH), 5.41 (1H, d, J =
dried over MgSO4 and evaporated under reduced pressure to 4.4 Hz, OH), 6.90 (2H, d, J = 8.6 Hz, Ar-m), 7.29 (2H, d, J = 8.6
give 1.06 g of an orange solid. Column chromatography (silica Hz, Ar-o), 7.45 (2H, t, J = 7.3 Hz, Ar-m′), 7.51 (1H, t, J = 7.2 Hz,
gel 60, MTBE → MTBE–MeOH–NH4OH = 90/9/1) afforded Ar-p′), 7.84 (2H, dd, J = 7.0 Hz, 1.6 Hz, Ar-o′), 8.46 (1H, t, J = 5.7
0.97 g (84%) of (R)-4c as an off-white solid. mp: 83–84 °C Hz, NH). 13C-NMR (100 MHz, DMSO-d6): δ [ppm] = 47.7, 55.0,
(lit.38 63–65 °C). TLC (silica, MTBE–MeOH–NH4OH = 90/9/1): 70.7, 113.4, 127.1, 127.2, 128.2, 131.0, 134.6, 135.8, 158.3,
Rf = 0.10. [α]2D0 = −66.5 (CHCl3, c = 0.92); lit. (S)38 +40.8 166.4. GC–MS (EI, 70 eV): m/z = 271 (M+, <1), 150 (39), 137 (34),
(EtOH, c = 1.68). NMR and MS data were in accordance with 135 (76), 134 (100), 109 (20), 105 (57), 94 (19), 77 (57), 66 (7),
those obtained for the product of the semi-preparative scale 51 (15). The NMR data are in accordance with the literature
experiment.
values.42
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Environmental impact assessment
63, 149–156; (c) E. Burda, W. Hummel and H. Gröger,
Angew. Chem., Int. Ed., 2008, 47, 9551–9554.
E-factor calculations were performed using the EATOS (v. 1.1)
software tool,43 while solvent demand was calculated using
Microsoft Excel (determining the solvent use of each step sepa-
rately and carrying forward the solvent demand of all inter-
mediates). All reactions were treated as proceeding to complete
conversion, hence all losses in yield are accounted for as
‘unknown by-products’. In cases where the exact quantities of
reagents or auxiliary materials were not given in the literature,
the following estimations were used: solvents for diluting solu-
tions: 3 times the initial volume, Celite for filtration: 0.1 g mL−1
of filtrate, solvents for extraction: 50 mL g−1 of crude product,
aqueous solutions for washing extracts: 20% of extract volume,
except brine: 10% of extract volume, anhydrous salts for drying
extracts: 0.02 g mL−1 of extract volume, solvents for recrystallisa-
tion: 10 mL g−1 of crude product, silica gel for chromatography:
20 g g−1 of crude product, eluents for chromatography: 500 mL
g−1 of crude product (total eluent volume). The EATOS and
Excel files used for the calculations are available as ESI.†
5 For recent reviews, see: (a) A. C. Marr and S. Liu, Trends Bio-
technol., 2011, 29, 199–204; (b) P. N. R. Vennestrøm,
C. H. Christensen, S. Pedersen, J.-D. Grunwaldt and
J. M. Woodley, ChemCatChem, 2010, 2, 249–258;
(c) H. C. Hailes, P. A. Dalby and J. M. Woodley, J. Chem.
Technol. Biotechnol., 2007, 82, 1063–1066.
6 For recent reviews on the combination of chemo- and bio-
catalysis for dynamic kinetic resolution in organic media,
see: (a) P. Hoyos, V. Pace and A. R. Alcántara, Adv. Synth.
Catal., 2012, 354, 2585–2611; (b) J. H. Lee, K. Han,
M. J. Kim and J. Park, Eur. J. Org. Chem., 2010, 999–1015;
(c) B. Martín-Matute and J. E. Bäckvall, Curr. Opin. Chem.
Biol., 2007, 11, 226–232; (d) H. Pellissier, Tetrahedron, 2008,
64, 1563–1601. For recent examples of the concurrent com-
bination of chemo- and biocatalysis in aqueous or bi-
phasic media, see:
(e) Z. J. Wang, K. N. Clary,
R. G. Bergman, K. N. Raymond and F. D. Toste, Nat. Chem.,
2013, 5, 100–103; (f) V. Köhler, Y. M. Wilson,
M. Dürrenberger, D. Ghislieri, E. Churakova, T. Quinto,
L. Knörr, D. Häussinger, F. Hollmann, N. J. Turner and
T. R. Ward, Nat. Chem., 2013, 5, 93–99; (g) F. G. Mutti,
A. Orthaber, J. H. Schrittwieser, J. G. de Vries, R. Pietschnig
and W. Kroutil, Chem. Commun., 2010, 46, 8046–8048.
Acknowledgements
This study has been financed by the Austrian Science Fund
(FWF project J3244-N17). The authors would like to thank
Florian Mayer for assistance with the NMR measurements,
Maarten Gorseling and Remco van Oosten for general techni-
cal assistance, and Caroline Paul for proof-reading the
manuscript.
7 For selected recent examples of the sequential combination
of chemo- and biocatalysis in aqueous medium, see: ref. 4a
and 4c; (a) A. Cuetos, F. R. Bisogno, I. Lavandera and
V. Gotor, Chem. Commun., 2013, 49, 2625–2627;
(b) J. S. Willemsen, J. C. M. van Hest and F. P. J. T. Rutjes,
Chem. Commun., 2013, 49, 3143–3145; (c) J. M. Foulkes,
K. J. Malone, V. S. Coker, N. J. Turner and J. R. Lloyd, ACS
Catal., 2011, 1, 1589–1594; (d) H. Maid, P. Böhm,
S. M. Huber, W. Bauer, W. Hummel, N. Jux and H. Gröger,
Angew. Chem., Int. Ed., 2011, 50, 2397–2400; (e) M. Kraußer,
T. Winkler, N. Richter, S. Dommer, A. Fingerhut,
W. Hummel and H. Gröger, ChemCatChem, 2011, 3, 293–
296; (f) K. Tenbrink, M. Seßler, J. Schatz and H. Gröger,
Adv. Synth. Catal., 2011, 353, 2363–2367; (g) G. Rulli,
N. Duangdee, K. Baer, W. Hummel, A. Berkessel and
H. Gröger, Angew. Chem., Int. Ed., 2011, 50, 7944–7947;
(h) P. N. R. Vennestrøm, E. Taarning, C. H. Christensen,
S. Pedersen, J.-D. Grunwaldt and J. M. Woodley, Chem-
CatChem, 2010, 2, 943–945; (i) E. Burda, W. Bauer,
W. Hummel and H. Gröger, ChemCatChem, 2010, 2, 67–72;
( j) V. Gauchot, W. Kroutil and A. R. Schmitzer, Chem.–Eur.
J., 2010, 16, 6748–6751; (k) W. Szymanski, C. P. Postema,
C. Tarabiono, F. Berthiol, L. Campbell-Verduyn, S. De
Wildeman, J. G. De Vries, B. L. Feringa and D. B. Janssen,
Adv. Synth. Catal., 2010, 352, 2111–2115; (l) K. Baer,
M. Kraußer, E. Burda, W. Hummel, A. Berkessel and
H. Gröger, Angew. Chem., Int. Ed., 2009, 48, 9355–9358;
(m) A. Prastaro, P. Ceci, E. Chiancone, A. Boffi, R. Cirilli,
M. Colone, G. Fabrizi, A. Stringaro and S. Cacchi, Green
Chem., 2009, 11, 1929–1932; (n) R. M. Haak, F. Berthiol,
T. Jerphagnon, A. J. A. Gayet, C. Tarabiono, C. P. Postema,
Notes and references
1 A. Bruggink, R. Schoevaart and T. Kieboom, Org. Process
Res. Dev., 2003, 7, 622–640.
2 (a) L. M. Ambrosini and T. H. Lambert, ChemCatChem,
2010,
2,
1373–1380;
(b)
A.
M.
Walji
and
D. W. C. MacMillan, Synlett, 2007, 1477–1489;
(c) J.-C. Wasilke, S. J. Obrey, R. T. Baker and G. C. Bazan,
Chem. Rev., 2005, 105, 1001–1020; (d) J. M. Lee, Y. Na,
H. Han and S. Chang, Chem. Soc. Rev., 2004, 33, 302–312.
3 For recent reviews, see: (a) H. Pellissier, Tetrahedron, 2013,
69, 7171–7210; (b) I. Oroz-Guinea and E. García-Junceda,
Curr. Opin. Chem. Biol., 2013, 17, 236–249; (c) J. Park
and S. Hong, Chem. Soc. Rev., 2012, 41, 6931–6943;
(d) H. Pellissier, Adv. Synth. Catal., 2012, 354, 237–294;
(e) R. C. Wende and P. R. Schreiner, Green Chem., 2012, 14,
1821–1849; (f) Ł. Albrecht, H. Jiang and K. A. Jørgensen,
Angew. Chem., Int. Ed., 2011, 50, 8492–8509; (g) E. Ricca,
B. Brucher and J. H. Schrittwieser, Adv. Synth. Catal., 2011,
353, 2239–2262; (h) C. Grondal, M. Jeanty and D. Enders,
Nat. Chem., 2010, 2, 167–178; (i) M. J. Climent, A. Corma
and S. Iborra, ChemSusChem, 2009, 2, 500–506.
4 (a) I. Schnapperelle, W. Hummel and H. Gröger, Chem.–
Eur. J., 2012, 18, 1073–1076; (b) M. Poizat, I. W. C.
E. Arends and F. Hollmann, J. Mol. Catal. B: Enzym., 2010,
This journal is © The Royal Society of Chemistry 2013
Green Chem., 2013, 15, 3318–3331 | 3329

View Article Online
Paper
V. Ritleng, M. Pfeffer, D. B. Janssen, A. J. Minnaard,
Green Chemistry
2011, 155, 320–329; (c) C. U. Ingram, M. Bommer,
M. E. B. Smith, P. A. Dalby, J. M. Ward, H. C. Hailes and
G. J. Lye, Biotechnol. Bioeng., 2007, 96, 559–569.
B. L. Feringa and J. G. de Vries, J. Am. Chem. Soc., 2008,
130, 13508–13509.
8 S. C. Bergmeier, Tetrahedron, 2000, 56, 2561–2576.
9 For reviews, see: (a) J. L. Vicario, D. Badía, L. Carrillo,
E. Reyes and J. Etxebarria, Curr. Org. Chem., 2005, 9, 219–
235; (b) K. Everaere, A. Mortreux and J. F. Carpentier, Adv.
Synth. Catal., 2003, 345, 67–77; (c) D. J. Ager, I. Prakash and
D. R. Schaad, Chem. Rev., 1996, 96, 835–876.
16 (a) G. Sello, F. Orsini, S. Bernasconi and P. D. Gennaro,
Tetrahedron: Asymmetry, 2006, 17, 372–376; (b) J. S. Yadav,
P. T. Reddy, S. Nanda and A. B. Rao, Tetrahedron: Asymme-
try, 2001, 12, 3381–3385; (c) P. A. Procopiou, G. E. Morton,
M. Todd and G. Webb, Tetrahedron: Asymmetry, 2001, 12,
2005–2008.
10 For a review, see: (a) F. D. Klingler, Acc. Chem. Res., 2007, 17 (a) F. Coccia, L. Tonucci, D. Bosco, M. Bressan and
40, 1367–1376. For recent examples, see: (b) R. Kuwano,
N. Kameyama and R. Ikeda, J. Am. Chem. Soc., 2011, 133,
7312–7315; (c) J. F. McGarrity and A. Zanotti-Gerosa, Tetra-
hedron: Asymmetry, 2010, 21, 2479–2486; (d) G. Shang,
N. d’Alessandro, Green Chem., 2012, 14, 1073–1078;
(b) F. Coccia, L. Tonucci, N. d’Alessandro,
P. d’Ambrosio and M. Bressan, Inorg. Chim. Acta, 2013,
399, 12–18.
D. Liu, S. E. Allen, Q. Yang and X. Zhang, Chem.–Eur. J., 18 Notably, this high chemoselectivity is in stark contrast to
2007, 13, 7780–7784; (e) A. Hu and W. Lin, Org. Lett., 2005,
7, 455–458; (f) A. Lei, S. Wu, M. He and X. Zhang, J. Am.
Chem. Soc., 2004, 126, 1626–1627; (g) T. Morimoto,
the hydrogenation of 3b over Pd/C in methanol, where
reductive dehalogenation was observed and 4a·HCl was
formed as the sole product in quantitative yield.
K. Yoshikawa, M. Murata, N. Yamamoto and K. Achiwa, 19 For studies using isolated enzymes, see: ref. 7a;
Chem. Pharm. Bull., 2004, 52, 1445–1450; (h) T. Ohkuma,
D. Ishii, H. Takeno and R. Noyori, J. Am. Chem. Soc., 2000,
122, 6510–6511.
(a) F. R. Bisogno, E. Garcia-Urdiales, H. Valdes,
I. Lavandera, W. Kroutil, D. Suarez and V. Gotor,
Chem.–Eur. J., 2010, 16, 11012–11019; (b) F. R. Bisogno,
I. Lavandera, W. Kroutil and V. Gotor, J. Org. Chem., 2009,
74, 1730–1732; (c) H. Ankati, Y. Yang, D. Zhu, E. R. Biehl
and L. Hua, J. Org. Chem., 2008, 73, 6433–6436;
(d) K. Edegger, C. C. Gruber, T. M. Poessl, S. R. Wallner,
I. Lavandera, K. Faber, F. Niehaus, J. Eck, R. Oehrlein,
A. Hafner and W. Kroutil, Chem. Commun., 2006, 2402–
2404. For studies using whole microbial cells, see: ref. 16c;
(e) L. C. Fardelone, J. A. R. Rodrigues and P. J. S. Moran,
J. Mol. Catal. B: Enzym., 2006, 39, 9–12; (f) H. Antunes,
L. d. C. Fardelone, J. A. R. Rodrigues and P. J. S. Moran,
11 For recent examples, see: (a) H. Vázquez-Villa, S. Reber,
M. A. Ariger and E. M. Carreira, Angew. Chem., Int. Ed.,
2011, 50, 8979–8981; (b) O. Soltani, M. A. Ariger,
H. Vázquez-Villa and E. M. Carreira, Org. Lett., 2010, 12,
2893–2895; (c) Z. Xu, S. Zhu, Y. Liu, L. He, Z. Geng and
Y. Zhang, Synthesis, 2010, 811–817; (d) M. Watanabe,
K. Murata and T. Ikariya, J. Org. Chem., 2002, 67, 1712–
1715; (e) A. M. Kawamoto and M. Wills, J. Chem. Soc.,
Perkin Trans. 1, 2001, 1916–1928.
12 (a) A. Prechter, H. Gröger and M. R. Heinrich, Org. Biomol.
Chem., 2012, 10, 3384–3387; (b) F. J. Quijada, F. Rebolledo
and V. Gotor, Tetrahedron, 2012, 68, 7670–7674;
(c) R. Lihammar, R. Millet and J. E. Bäckvall, Adv. Synth.
Catal., 2011, 353, 2321–2327; (d) A. Rouf, P. Gupta,
M. A. Aga, B. Kumar, R. Parshad and S. C. Taneja, Tetra-
hedron: Asymmetry, 2011, 22, 2134–2143; (e) A. Chaubey,
R. Parshad, P. Gupta, S. C. Taneja, G. N. Qazi, C. R. Rajan
and S. Ponrathnam, Bioorg. Med. Chem., 2009, 17, 29–34.
Tetrahedron:
Asymmetry,
2004,
15,
2615–2620;
(g) J. S. Yadav, P. T. Reddy, S. Nanda and A. B. Rao, Tetra-
hedron: Asymmetry, 2001, 12, 63–67; (h) A. E. P. M. Sorillha,
M. Marques, I. Joekes, P. José, S. Moran, J. Augusto and
R. Rodrigues, Bioorg. Med. Chem. Lett., 1992, 2, 191–196.
For studies using carrot root as biocatalyst, see: ref. 16b;
(i) J. S. Yadav, S. Nanda, P. Thirupathi Reddy and
A. Bhaskar Rao, J. Org. Chem., 2002, 67, 3900–3903.
13 (a) J. Rehdorf, M. D. Mihovilovic and U. T. Bornscheuer, 20 (a) B. Kosjek, W. Stampfer, M. Pogorevc, W. Goessler,
Angew. Chem., Int. Ed., 2010, 49, 4506–4508; (b) J. Rehdorf,
M. D. Mihovilovic, M. W. Fraaije and U. T. Bornscheuer,
Chem.–Eur. J., 2010, 16, 9525–9535.
K. Faber and W. Kroutil, Biotechnol. Bioeng., 2004, 86, 55–
62; (b) W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil and
K. Faber, Angew. Chem., Int. Ed., 2002, 41, 1014–1017;
(c) W. Hummel, Trends Biotechnol., 1999, 17, 487–492;
(d) W. Hummel, Adv. Biochem. Eng. Biotechnol., 1997, 58,
145–184; (e) R. J. Lamed and J. G. Zeikus, Biochem. J., 1981,
195, 183–190.
14 (a) K. Baer, N. Dückers, W. Hummel and H. Gröger, Chem-
CatChem, 2010, 2, 939–942; (b) J. Steinreiber,
M. Schürmann, F. Van Assema, M. Wolberg, K. Fesko,
C. Reisinger, D. Mink and H. Griengl, Adv. Synth. Catal.,
2007, 349, 1379–1386; (c) J. Steinreiber, M. Schürmann, 21 Due to the effect of the azide group on the Cahn–Ingold–
M. Wolberg, F. Van Assema, C. Reisinger, K. Fesko,
D. Mink and H. Griengl, Angew. Chem., Int. Ed., 2007, 46,
1624–1626.
Prelog priorities, enzymes with a stereoselectivity according
to Prelog’s rule will produce the (R)-enantiomer of 3a.
However, in the suppliers’ catalogs these enzymes are cate-
gorised as (S)-selective, in accordance with the stereoprefer-
ence observed with ‘standard substrates’ such as
acetophenone or 2-octanone.
15 (a) T. Sehl, H. C. Hailes, J. M. Ward, R. Wardenga, E. von
Lieres, H. Offermann, R. Westphal, M. Pohl and D. Rother,
Angew. Chem., Int. Ed., 2013, 52, 6772–6775;
(b) S. Matosevic, G. J. Lye and F. Baganz, J. Biotechnol., 22 V. Prelog, Pure Appl. Chem., 1964, 9, 119–130.
3330 | Green Chem., 2013, 15, 3318–3331
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23 Interestingly, the ADHs from T. brockii and L. brevis, which
have been reported to reduce 2a, did not give substantial con-
version in our screening, despite proving active in a photo-
metric assay on 2-propanol oxidation (for details, see ESI†).
24 The oxazolidine 5 can also be turned into the main
product by adding additional acetone after extraction of the
crude product from the aqueous phase, and preliminary
experiments have shown that more complex oxazolidines
(which may contain a second chiral centre) can also be
accessed by this method.
25 M.-J. Cheng, K.-H. Lee, I.-L. Tsai and I.-S. Chen, Bioorg.
Med. Chem., 2005, 13, 5915–5920.
26 (a) R. A. Sheldon, Chem. Ind., 1992, 903–906;
(b) R. A. Sheldon, Green Chem., 2007, 9, 1273–1283;
(c) R. A. Sheldon, Chem. Commun., 2008, 3352–3365.
Internat. Pat., WO 2012/156367, 2012; (f) J. A. Linn,
T. Longstaff and K. L. Stevens (Smithkline Beecham Corp.),
Internat. Pat., WO 2008/024634, 2008; (g) A. Slassi,
B. Joseph, F. Ma, I. Egle, J. Clayton, M. Isaac and
K. Swierczek (Astrazeneca AB; NPS Pharmaceuticals, Inc.),
Internat. Pat., WO 2007/078523, 2007; (h) A. Ali,
J. M. Napolitano, Q. Deng, Z. Lu, P. J. Sinclair, G. E. Taylor,
C. F. Thompson, N. Quraishi, C. J. Smith and J. A. Hunt
(Merck & Co., Inc.), Internat. Pat., WO 2006/014357, 2006;
(i) P. J. Hergenrother, V. Nesterenko, K. Putt, B. J. Allen,
R. S. Dothager and B. J. Leslie (Univ. Illinois), Internat. Pat.,
WO 2005/044191, 2005; ( j) M. E. Schnute, M. M. Cudahy,
M. Eggen, D. J. Anderson, T. M. Judge, E. J. Kim and
S. A. Collier (Pharmacia & Upjohn, Co.), Internat. Pat., WO
2004/106345, 2004.
27 D.-M. Lee, J.-C. Lee, N. Jeong and K.-I. Lee, Tetrahedron: 33 Since the ADH preparation is a crude cell-free extract, the
Asymmetry, 2007, 18, 2662–2667.
28 A. Baeza, C. Nájera, J. M. Sansano and J. M. Saá, Chem.–
Eur. J., 2005, 11, 3849–3862.
29 A. Kamal, A. A. Shaik, M. Sandbhor and M. S. Malik, Tetra-
hedron: Asymmetry, 2004, 15, 3939–3944.
exact concentration of the productive enzyme is not known.
An approximation assuming 20% target protein content
and a molecular weight of 30 kDa yields a concentration of
3.33 µM, corresponding to a TON of 30 000 at full conver-
sion (100 mM substrate).
30 (a) R. F. C. Brown, A. C. Donohue, W. R. Jackson and 34 A. Kamal, G. B. R. Khanna, T. Krishnaji and R. Ramu,
T. D. McCarthy, Tetrahedron, 1994, 50, 13739–13752; Bioorg. Med. Chem. Lett., 2005, 15, 613–615.
(b) R. F. C. Brown, W. R. Jackson and T. D. McCarthy, Tetra- 35 A. V. Malkov, A. J. P. S. Liddon, P. Ramírez-López,
hedron: Asymmetry, 1993, 4, 205–206.
31 For ecological perspectives of solvent use, see:
L. Bendová, D. Haigh and P. Kočovský, Angew. Chem., Int.
Ed., 2006, 45, 1432–1435.
(a) K. Alfonsi, J. Colberg, P. J. Dunn, T. Fevig, S. Jennings, 36 M. J. Bosiak and M. M. Pakulski, Synthesis, 2011, 316–
T. A. Johnson, H. P. Kleine, C. Knight, M. A. Nagy, 324.
D. A. Perry and M. Stefaniak, Green Chem., 2008, 10, 31–36; 37 L. Kniežo, P. Kristian and S. Velebný, Collect. Czech. Chem.
(b) R. A. Sheldon, Chem. Soc. Rev., 2012, 41, 1437–1451. Commun., 1978, 43, 1917–1923.
32 For recent articles, see: (a) K. P. Bhabak and C. Arenz, 38 B. T. Cho, S. K. Kang and S. H. Shin, Tetrahedron: Asymme-
Bioorg. Med. Chem., 2012, 20, 6162–6170; (b) A. S. Aboraia, try, 2002, 13, 1209–1217.
S. W. Yee, M. S. Gomaa, N. Shah, A. C. Robotham, 39 W. Becker, H. Freund and E. Pfeil, Angew. Chem., 1965, 77,
B. Makowski, D. Prosser, A. Brancale, G. Jones and 1139–1139.
C. Simons, Bioorg. Med. Chem., 2010, 18, 4939–4946; 40 C. Paolucci and G. Rosini, Tetrahedron: Asymmetry, 2007,
(c) M. D. Wittman, B. Balasubramanian, K. Stoffan, 18, 2923–2946.
U. Velaparthi, P. Liu, S. Krishnanathan, J. Carboni, A. Li, 41 A. S. Demir, C. Tanyeli, H. Aksoy, V. Gulbeyaz and
A. Greer, R. Attar, M. Gottardis, C. Chang, B. Jacobson, A. S. Mahasneh, Synthesis, 1995, 1071–1073.
Y. Sun, S. Hansel, M. Zoeckler and D. M. Vyas, Bioorg. Med. 42 R. Somanathan, H. R. Aguilar, R. Ventura and K. M. Smith,
Chem. Lett., 2007, 17, 974–977; (d) V. Nesterenko, K. S. Putt Synth. Commun., 1983, 13, 273–280.
and P. J. Hergenrother, J. Am. Chem. Soc., 2003, 125, 14672– 43 (a) M. Eissen and J. O. Metzger, Chem.–Eur. J., 2002, 8,
14673. For recent patents, see: (e) K. Eis, F. Puehler,
L. Zorn, A. Scholz, P. Lienau, M. J. Gnoth, U. Boemer,
J. Guenther, J. Fanghaenel and D. Knorr (Bayer GmbH),
3580–3585; (b) EATOS: Environmental Assessment Tool for
Organic Syntheses, http://www.metzger.chemie.uni-oldenburg.
de/eatos/english.htm, Accessed 13.06.2013.
This journal is © The Royal Society of Chemistry 2013
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