Asymmetric Biocatalytic Synthesis of Fluorinated Pyridines through Transesterification or Transamination: Computational Insights into the Reactivity of Transaminases

Article abstract of DOI:10.1002/adsc.201600835

The synthesis of a family of pyridines bearing a fluorinated substituent on the aromatic ring has been carried out through two independent and highly stereoselective chemoenzymatic strategies. Short chemical synthetic routes toward fluorinated racemic amines and prochiral ketones have been developed, which served as substrates to explore the suitability of lipases and transaminases in asymmetric biotransformations. The lipase-catalyzed kinetic resolution via acylation of racemic amines proceeded smoothly giving conversions close to 50% and excellent enantioselectivities. Alternatively, the biotransamination of the corresponding prochiral ketones was investigated giving access to both optically pure amine enantiomers using transaminases with complementary selectivity. High to quantitative conversion values were achieved, which allowed the isolation of the amines in moderate to high yields (40–88%). A deeper understanding of the latter process was enabled by performing theoretical calculations on thermodynamic and mechanistic aspects. Calculations showed that the biotransamination reactions are highly favoured by the presence of fluorine atoms and the pyridine ring. (Figure presented.).

Full text of DOI:10.1002/adsc.201600835

FULL PAPERS
DOI: 10.1002/adsc.201600835
Asymmetric Biocatalytic Synthesis of Fluorinated Pyridines
through Transesterification or Transamination: Computational
Insights into the Reactivity of Transaminases
Marꢀa Lꢁpez-Iglesias,a,b Daniel Gonzꢂlez-Martꢀnez,a Marꢀa Rodrꢀguez-Mata,a
Vicente Gotor,a Eduardo Busto,c,* Wolfgang Kroutil,b,*
and Vicente Gotor-Fernꢂndeza,*
a
Departamento de Quꢀmica Orgꢂnica e Inorgꢂnica, Instituto Universitario de Biotecnologꢀa de Asturias, Universidad de
Oviedo, 33006 Oviedo, Spain
Fax : (+34)-985-103-446; phone: (+34)-985-103-454; e-mail: vicgotfer@uniovi.es
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, NAWI Graz, BioTechMed Graz,
b
Heinrichstrabe 28, 8010 Graz, Austria
Fax : (+43)-31-6380-9840; phone: (+43)-31-6380-5350; e-mail: wolfgang.kroutil@uni-graz.at
Departamento de Quꢀmica Orgꢂnica I, Facultad de Quꢀmica, Universidad Complutense de Madrid, 28040 Madrid, Spain
c
Fax : (+34)-913-944-103; phone: (+34)-913-944-231; e-mail: bbusto@ucm.es
Received: July 28, 2016; Revised: October 21, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201600835.
Abstract: The synthesis of a family of pyridines bear- amine enantiomers using transaminases with comple-
ing a fluorinated substituent on the aromatic ring has mentary selectivity. High to quantitative conversion
been carried out through two independent and values were achieved, which allowed the isolation of
highly stereoselective chemoenzymatic strategies. the amines in moderate to high yields (40–88%). A
Short chemical synthetic routes toward fluorinated deeper understanding of the latter process was ena-
racemic amines and prochiral ketones have been de- bled by performing theoretical calculations on ther-
veloped, which served as substrates to explore the modynamic and mechanistic aspects. Calculations
suitability of lipases and transaminases in asymmetric showed that the biotransamination reactions are
biotransformations. The lipase-catalyzed kinetic reso- highly favoured by the presence of fluorine atoms
lution via acylation of racemic amines proceeded and the pyridine ring.
smoothly giving conversions close to 50% and excel-
lent enantioselectivities. Alternatively, the biotransa- Keywords: amines; asymmetric synthesis; biocataly-
mination of the corresponding prochiral ketones was sis; computational chemistry; organofluorinated
investigated giving access to both optically pure compounds; pyridines
Introduction
licity, solubility and biological properties,[7] improving
metabolic stability and making possible the discovery
The synthesis of organofluorine compounds has re- of new drug candidates.[8] In this context, the develop-
ceived great attention in recent years due to their ment of heterocyclic systems bearing fluorinated func-
uses in different scientific areas.[1] Fluorinated organic tionalities opens up the access to new valuable mole-
molecules possess a wide range of applications as cules.[9]
agrochemicals,[2] pharmaceuticals,[3] and asymmetric
Biocatalytic methodologies have been described for
catalysts.[4] In a rough estimation, around 20% of the production of biologically active organofluorine
pharmaceuticals and 40% of agrochemicals contain at molecules through selective halogenation using halo-
least one fluorine atom in their structure.[5] The incor- genases.[10] Alternatively, the asymmetrization of pro-
poration of fluorine in organic molecules may lead to chiral or racemic starting materials allows the access
important changes in their stability, bioavailability to enantiopure fluorinated compounds by using other
and binding affinity to receptors.[6] The presence of classes of enzymes such as alcohol dehydrogenases,[11]
fluorine can significantly affect their physicochemical hydrolases[12] or transaminases.[13]
properties such as acidity, hydrogen-bonding, lipophi-
Adv. Synth. Catal. 0000, 000, 0 – 0
1
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
Chiral amines are interesting and versatile com- (Scheme 1, bottom). These methodologies for racemic
pounds with great appeal in both academic and indus- amine syntheses are useful for further application in
trial fields.[14] This structural motif is nowadays consid- enzymatic reactions. On the one hand, the three race-
ered as an interesting scaffold in the preparation of mates will serve as both substrates for lipase-cata-
bioactive molecules.[15] In this context, the develop- lyzed transformations and final products for analytical
ment of stereoselective biocatalytic methods toward purposes in biotransamination experiments. On the
optically active amines has attracted increasing atten- other hand, the intermediate ketones will be the sub-
tion in recent years.[16] The present work combines strates in transaminase-catalyzed reactions.
the relevance that chiral amines offer and the poten-
Having completed the synthesis of the ketones 3a–
tial of compounds bearing fluorine atoms, as both are c and amines 4a–c, two stereoselective enzymatic
challenging items for the scientific community. Hence, strategies were studied: the classical kinetic resolution
a series of pyridylethanamine enantiomers containing through lipase-catalyzed enantioselective acylation of
a fluorine atom or a trifluoromethyl group at the 5- the racemic amines 4a–c and, alternatively, the amina-
or 6-position of the heteroaromatic ring have been tion of prochiral ketones 3a–c using transaminases.
stereoselectively synthesized by two independent en-
zymatic approaches: (a) lipase-catalyzed kinetic reso-
lution via transesterification or (b) highly efficient Asymmetric Synthesis through Lipase-Catalyzed
asymmetric amination employing transaminases.
Resolutions
The potential of lipases in the acylation of racemic
amines has been broadly studied as an efficient strat-
egy for the synthesis of enantiopure amines in organic
Results and Discussion
The synthetic pathway has been chosen in accordance solvents under mild reaction conditions.[16c,17] On this
with the availability of the commercial substrates as basis, the classical kinetic resolution of the fluorinated
well as the viability of the chemical process. Racemic 1-(pyridin-3-yl)ethanamines 4a–c was attempted. The
amines 4a and 4b bearing fluorine atoms at the 5- or initial reaction conditions were rationally established
6-position of the pyridine ring were synthesized in according to the following remarks. First of all, Candi-
moderate yields via a two-step hydroxyalkylation of da antarctica lipase B (CAL-B, Novozyme 435) was
the corresponding 3-bromopyridines 1a and 1b, re- selected as suitable biocatalyst due to the good results
spectively (Scheme 1, top). A subsequent chemical ox- reported in the literature on structurally similar sub-
idation of the resulting racemic alcohols 2a and 2b strates.[18] Ethyl methoxyacetate was chosen as acylat-
using the Dess–Martin reagent in dichloromethane al- ing agent due to the excellent selectivity and activity
lowed the isolation of the ketones 3a and 3b (85–95% values displayed in the resolution of racemic
isolated yield). Then, these ketones were submitted to amines.[19] THF was preferred as solvent owing to sol-
a classical reductive amination employing sodium cya- ubility issues, using a 100 mM substrate concentration
noborohydride and ammonium acetate in methanol. for the study (Table 1).
In the particular case of the trifluoromethylated de-
Enzymatic reactions took place with excellent
rivative 4c, the alternative pathway compromises only enantioselectivity for all the three tested substrates,
two steps: first, addition of methylmagnesium iodide and conversions reached almost the maximum 50%
to the cyano group of the commercially available 6- value in short reaction times (2–7 h, Table 1, en-
(trifluoromethyl)nicotinonitrile (5) and, secondly, the tries 1–3). Thus, this methodology afforded the enan-
reductive amination of the so-obtained ketone 3c tiopure methoxyacetamides (R)-6a–c and the remain-
Scheme 1. Synthesis of ketones 3a–c and racemic amines 4a–c for the study of their enzymatic amination or kinetic resolu-
tion, respectively.
Adv. Synth. Catal. 0000, 000, 0 – 0
2
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
Table 1. CAL-B-catalyzed kinetic resolution of racemic
amines 4a–c through enantioselective acylation.[a]
Starting with ketone 3a, good conversion values
were observed in the formation of the amine (S)-4a
with the TAs from Vibrio fluvialis and Chromobacte-
rium violaceum (Table 2, entries 1 and 2), while the
TA from Arthrobacter citreus only led to a 36% con-
version (Table 2, entry 3). For the (R)-selective en-
zymes an 85% conversion was obtained with the TA
from Arthrobacter sp. (Table 2, entry 4), and 69%
with the system b and the mutant ArRmut11-TA
(Table 2, entry 5). In all cases, the amine 4a was iso-
Entry 4a-c
t [h] eeS [%][b] eeP [%][b] c [%][c] E[d]
1
2
3
a (5-F)
b (6-F)
c (6-CF3) 3
7
2
90
96
94
>99
>99
>99
47
49
49
>200
>200
>200
[a]
Reaction conditions: racemic amine 4a–c (100 mM) in
THF, ethyl methoxyacetate (5 equiv.) and a ratio of sub-
strate:CAL-B of 1:1 (w/w) at 308C and 250 rpm.
[b]
Determined by HPLC on a chiral column by analyzing lated in enantiopure form.
the methoxyacetamides or the corresponding acetamides
obtained after derivatization of the enantioenriched
amines (see Supporting Information).
When ketone 3b bearing a fluorine atom substitut-
ing the 6-position of the aromatic ring was subjected
to enzymatic transamination, a significant decrease in
reactivity was noticed (Table 2, entries 6–10). The TA
from Chromobacterium violaceum is linked to the
best results within the (S)-selective enzymes (Table 2,
entry 7), achieving low to moderate conversion values
[c]
[d]
c=eeS/(eeS +eeP).
E=ln[(1Àc)(1ÀeeP)]/ln[(1Àc)(1+eeP)].[20]
ing amines (S)-4a–c in up to 96% ee. Control experi- in all the cases (Table 2, entries 6–8), whereas for the
ments were performed revealing that CAL-B was un- (R)-selective TAs (Table 2, entries 9 and 10) the Ar-
doubtedly responsible for the catalysis. Amide forma- throbacter sp. allowed the formation of the enantio-
tion was not detected in the absence of enzyme, pure amine (R)-4b with a notable 75% conversion
which was expected based on the excellent selectivi- (Table 2, entry 9). Remarkably, the more hindered 6-
ties observed in all cases.
trifluoromethylated pyridine 3c showed an enhanced
reactivity displaying a similar trend as that observed
for substrate 3a. Thus, excellent conversions were ach-
ieved with the (R)-Arthrobacter leading to the amine
(R)-4c in enantiopure form (94%, Table 2, entry 14),
Asymmetric Synthesis Through Biotransamination
In the pursuit of enzymatic strategies that led to theo- and the Chromobacterium violaceum TA allowed the
retically 100% yield in processes toward optically formation of the counterpart (S)-4c in a good extent
active amines, transaminases (TAs) were chosen as al- (80%, Table 2, entry 12).
ternative biocatalysts.[16b,21] This option allowed us to
These promising results led us to make some ad-
take advantage of the synthetic pathway previously justments in the reaction conditions in the belief that
described, as the ketones 3a–c, which are now sub- an improvement in conversions could be achieved by
strates for transamination experiments, were indeed increasing the substrate solubility, a parameter identi-
prepared as intermediates in the synthesis of the fied as key factor of the process. However, the pres-
amines 4a–c studied in lipase-catalyzed acylations. ence of water-miscible organic cosolvents did not
Thus, a number of representative transaminases over- cause significant improvements in the amine forma-
expressed in E. coli were considered, working at tion (Supporting Information, Table S1), and even
a 50 mM substrate concentration (Table 2). These lower conversions were observed when alanine was
transaminases are the (S)-selective ones from Vibrio used as amine donor (Supporting Information,
fluvialis,[22] Chromobacterium violaceum[23] and Ar- Table S2). A similar behaviour was found when the
throbacter citreus,[24] and the (R)-selective ones from amount of enzyme was doubled, even when the pres-
Arthrobacter sp.[25] and its evolved variant called ence of too many immiscible solids in the reaction
ArRmut11[26] used as semipurified form by a heat medium was avoided by employing cell-free extract
treatment (see the Experimental Section).
enzymes obtained after breaking cell walls by sonica-
According to the biocatalyst tolerance, alanine tion cycles of the enzymatic crude (Supporting Infor-
(system a) or an excess of 2-propylamine (1M, system mation, Table S3).
b) was selected as amino source, including in the first
Having obtained some good results but not fully
case a pyruvate removal system formed by NAD+/am- satisfactory ones, we decided to alter the enzyme per-
monium formate/alanine dehydrogenase (AlaDH)/ formance by introducing an initial enzyme rehydra-
formate dehydrogenase (FDH) working in parallel tion step. Therefore, the TA and the buffer were
with the main reaction. Despite the important differ- shaken at 308C and 120 rpm for 30 min prior to their
ences detected in reactivity depending on both the use in the biotransamination experiments (Table 3).
transaminase and the substrate, it is worthwhile to We were pleased to see a significant improvement of
note the excellent enantioselectivity obtained in all conversion values, some of the substrates displaying
cases for the production of amines 4a–c (>99% ee).
even 99% conversions such as for 3a and 3c using
Adv. Synth. Catal. 0000, 000, 0 – 0
3
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
Table 2. Asymmetric biotransamination of ketones 3a–c using different amino sources.[a]
Entry
3a–c
TA
System
c [%][b]
eeP [%][c]
1
2
3
4
5
a (5-F)
a (5-F)
a (5-F)
a (5-F)
a (5-F)
Vibrio fluvialis
(a)
(a)
(a)
(a)
(b)
89
80
36
85
69
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
Chromobacterium violaceum
Arthrobacter citreus
Arthrobacter sp.
Semipurified ArRmut11
6
7
8
9
b (6-F)
b (6-F)
b (6-F)
b (6-F)
b (6-F)
Vibrio fluvialis
(a)
(a)
(a)
(a)
(b)
26
45
<1
75
6
>99 (S)
>99 (S)
n.m.
>99 (R)
n.m.
Chromobacterium violaceum
Arthrobacter citreus
Arthrobacter sp.
10
Semipurified ArRmut11
11
12
13
14
15
c (6-CF3)
c (6-CF3)
c (6-CF3)
c (6-CF3)
c (6-CF3)
Vibrio fluvialis
(a)
(a)
(a)
(a)
(b)
69
80
36
94
85
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
Chromobacterium violaceum
Arthrobacter citreus
Arthrobacter sp.
Semipurified ArRmut11
[a]
Reaction conditions in system a: phosphate buffer (100 mM, pH 7), ketone 3a–c (50 mM), lyophilized cells E. coli/TA
(20 mg), PLP (1 mM), NAD+ (1 mM), l- or d-alanine (250 mM), AlaDH (10 mL, 11 U), FDH (2.6 mg, 11 U), ammonium
formate (150 mM), 24 h at 308C and 120 rpm. Reaction conditions in system b: phosphate buffer (100 mM, pH 7), ketone
3a–c (50 mM), semipurified ArRmut11-TA (500 mL), PLP (0.5 mM), 2-propylamine (1M), 24 h at 458C and 120 rpm.
Conversion values were determined by GC analysis after isolation of products by extraction from basified media (see Ex-
perimental Section).
[b]
[c]
Enantiomeric excess values were measured by GC on a chiral column after the in situ derivatization with acetic anhy-
dride of the resulting amines (see the Supporting Information). n.m.: not measured.
both (S)- and (R)-selective transaminases (Table 3, Similarly, the Arthrobacter sp. TA provided the amine
entries 2 and 3 for 3a, 6 and 7 for 3c). This rehydra- (R)-4c in enantiopure form and very high isolated
1
tion step enhanced the reactivity for the fluorine-sub- yield (88%, entry 7). While the H NMR analysis of
stituted ketone 3b as well (Table 3, entries 4 and 5), the crude reaction mixtures revealed high purity for
keeping intact the exquisite enantioselectivity in all amines (S)-4a, (R)-4a and (R)-4c, the biotransamina-
cases.
tion reaction of ketone 3b with Chromobacterium vio-
With the best results in hand, preparative scale bio- laceum TA did not proceed to completion, so
transformations were performed employing these op- a column chromatography purification was necessary
timized conditions. When TAs from Chromobacteri- to separate the unreacted ketone 3b from the enantio-
um violaceum and Arthrobacter sp. were employed, pure amine (S)-4b. In the latter case only a 40% yield
enantiopure amine (S)-4a as well as the antipode (R)- was obtained.
4a were successfully isolated in good yields (71% and
Next, absolute configurations were assigned
79%, respectively, entries 2 and 3) just by performing through circular dichroism (CD) spectroscopy analy-
a basic extraction after completion of the reaction. ses. A structurally related compound to the family of
Adv. Synth. Catal. 0000, 000, 0 – 0
4
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
Table 3. Asymmetric biotransamination of ketones 3a–c using alanine dehydrogenase system and performing a rehydration
step of the TA before its use in the enzymatic reaction.[a]
Entry
3a–c
TA
c [%][b]
eeP [%][c]
1
2
3
a (5-F)
a (5-F)
a (5-F)
Vibrio fluvialis
Chromobacterium violaceum
Arthrobacter sp.
90
99 (71)
99 (79)
>99 (S)
>99 (S)
>99 (R)
4
5
b (6-F)
b (6-F)
Chromobacterium violaceum
Arthrobacter sp.
88 (40)[d]
92
>99 (S)
>99 (R)
6
7
c (6-CF3)
c (6-CF3)
Chromobacterium violaceum
Arthrobacter sp.
>99
>99 (88)
>99 (S)
>99 (R)
[a]
Reaction conditions: lyophilized cells E. coli/TA (20 mg) rehydrated in phosphate buffer (100 mM, pH 7) for 30 min at
308C and 120 rpm, ketone 3a–c (50 mM), PLP (1 mM), NAD+ (1 mM), l- or d-alanine (250 mM), AlaDH (10 mL, 11 U),
FDH (2.6 mg, 11 U), ammonium formate (150 mM), 24 h at 308C and 120 rpm.
Conversion values were determined by GC analysis after isolation of products by extraction in basified media (see Exper-
imental Section). Isolated yields in parentheses.
Enantiomeric excess values were measured by GC on a chiral column after an in situ derivatization with acetic anhydride
of the resulting amines (see Supporting Information).
A further purification step by column chromatography was performed in order to separate the product from the unreact-
ed ketone.
[b]
[c]
[d]
pyridines studied here, 1-(pyridin-3-yl)ethan-1-amine mum at 265.6 nm for (S)-4d and a positive maximum
(4d), whose optical rotation values were already re- for its counterpart (R)-4d at 264.2 nm (Figure 1, left).
ported in literature[27] was chosen as reference. Firstly, Qualitative comparison of the CD spectra measured
optically active (S)- and (R)-4d were isolated by car- for both enantiomers of reference pyridine 4d with
rying out a 50 mg scale-up of the enzymatic transami- the corresponding spectra of the unknown pyridines
nation employing the Chromobacterium violaceum 4a–c (Figure 1, right) allowed us to confirm their ab-
and Arthrobacter sp. TAs, respectively. Then, CD ex- solute configurations, which are in agreement either
periments were performed observing a negative mini- with the expected selectivity of the transaminases or
Figure 1. CD spectra recorded for reference compounds (S)- and (R)-4d (left), and enantiopure amines (R)-4a, (S)-4b and
(R)-4c (right) obtained through selected biotransamination reactions. Optical rotation values for amines 4a–c in >99% ee:
[a]2D0: +36.2 (c 0.5, CHCl3) for (R)-4a; [a]2D0: À28.2 (c 0.5, CHCl3) for (S)-4b; [a]D20: +20.0 (c 0.5, CHCl3) for (R)-4c.
Adv. Synth. Catal. 0000, 000, 0 – 0
5
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
with Kazlauskasꢄ rule[28] for the optically active
amines obtained through lipase-catalyzed reactions.
Furthermore, the thermodynamic study was extend-
ed for the structurally analogues acetophenones 3e–g
At this point all the amines 4a–c have been isolated (Scheme 2, bottom), the amine formation being in all
with excellent enantiomeric excess, but still no quanti- cases less favourable in comparison with the corre-
tative transformations have been developed for all sponding pyridine pairs 3a–c. The influence of the ni-
the substrates, something that is significant enough to trogen atom was experimentally confirmed in the bio-
cause a decrease in the isolated yield of the 6-F-sub- transamination of 3g employing both 2-isopropyla-
stituted amine 4b. For that reason, we decided to in- mine and alanine as amine donors, which led to mod-
vestigate more deeply the biotransamination of 3a– erate conversions that are far from the values ob-
c based on computational calculations. The thermo- tained for the corresponding pyridine 3c (Supporting
chemical study highlights substantial differences in Information, Tables S4 and S5, respectively). We have
the thermodynamic stabilization of the fluorinated recently reported a similar tendency of stabilization
pyridines, which are reflected in the calculated DG of by the pyridine ring in comparison with benzene de-
the amination reaction for the different ketone/amine rivatives, in this case due to the formation of an intra-
pairs at the M06-2X/6-311+ +G(3df,2p) level molecular hydrogen bond.[29]
(Scheme 2, top). The values revealed that the global
In our interest in having a deep knowledge of the
equilibrium using isopropylamine as amine donor is process, key intermediates of the known reaction
shifted toward the amine formation for 4a and 4c de- mechanism in the Chromobacterium violaceum TA
rivatives, while for pyridine 4b this was found to be active site were also analyzed through different com-
unfavourable (see additional information in the Sup- putational approaches (Suppporting Information, Fig-
porting Information, Table S8). Interestingly, a relative ures S2 and S3).[30] The ketimine and aldimine inter-
energy difference of 7.5 kJmolÀ1 was found between mediates were selected for the study of each sub-
5-F and 6-F substituted pyridines (3a and 3b, respec- strate, which is covalently bonded to the PLP cofactor
tively) in the transamination reaction.
(Figure 2, left).
Scheme 2. Global Gibbs free reaction energy in the transamination of ketones 3a–c and 3e–g with 2-propylamine calculated
at the M06-2X/6-311+ +G(3df,2p) level.
Figure 2. Key intermediates in the transamination mechanism (left); and the crystallographic position of the PLP in complex
with Lys288 (light blue) and the docked conformations of the aldimines and ketimines in the active site of Chromobacterium
violaceum TA with substitutions: 5-F (A–dark blue; K–purple), 6-F (A–yellow; K–orange) and 6-CF3 (A–red; K–green).
Adv. Synth. Catal. 0000, 000, 0 – 0
6
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
Initially, docking experiments were performed pyridoxal ring located at the crystallographic position
using the AutoDock software (see the Supporting In- (Figure 2, right).
formation, Figure S4, for details)[31] finding in the
The ONIOM(M06-2X/6-31G(d,p):Amber) method
most stable conformations of the intermediates the was employed to optimize the active site geometries
Figure 3. Non-covalent interaction analyses of the optimized geometry within the Chromobacterium violaceum TA active
site of the aldimines with substitutions (A) 5-F; (C) 6-F; (E) 6-CF3; and the ketimines with substitutions (B) 5-F; (D) 6-F;
(F) 6-CF3. Non-covalent attractive interactions appear in green (weak) or blue (strong) and the repulsive ones in red. Sub-
strates are shown in ball-and-stick representation while the cofactor and the enzyme residues are shown as sticks.
Adv. Synth. Catal. 0000, 000, 0 – 0
7
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
of each docked intermediate (Supporting Informa-
Despite the differences between the dispositions of
tion, Figures S5 and S6), after which a non-covalent the three studied substrates, all of them showed fa-
interaction analysis was carried out with NCIPLOT[32] vourable interactions that enabled the transamination
in order to highlight the main interactions between reaction to occur smoothly within the active site of
the pyridines and the closest residues of the TA the TA from Chromobacterium violaceum. On the
active site (Supporting Information, Figure S7).
other hand, in contrast with our previous investigation
The previous model of the active site was employed with a set of pyridylalkylamines,[29] in the present
for a new DFT calculation at the M06-2X/6-31G(d,p) work the hydrogen bonds were established to be in-
level of theory in order to obtain the wavefunction of termolecular between the intermediate species and
the optimized geometry and perform a frequency key active site residues.
computation (Supporting Information, Figure S8 and
Table S6). The non-covalent interactions around the
substrate were computed with NCIPLOT using the Conclusions
SCF densities (Supporting Information, Figure S9 and
Table S7). Figure 3 shows an enlargement of the zone Enzymes have been used in the asymmetric key step
where the most representative non-covalent interac- for the synthesis of valuable fluorinated amines. The
tions were found for each substrate, depicting the development of a straightforward synthetic pathway
weak attractive interactions in green, while the stron- led to suitable substrates for lipase- and transami-
gest ones are shown in blue. The difference of energy nase-catalyzed transformations, which has made possi-
between the two intermediates within the active site ble the preparation of a family of optically active pyr-
was found to be favourable for the ketimine in 3a idylethanamines bearing a fluorine atom at the 5- or
(À11.2 kJ/mol) and 3c (À6.0 kJ/mol), which is in 6-position, or a trifluoromethyl group at the 6-posi-
agreement with the results reported by Cassimjee tion.
et al. for acetophenone transamination.[30] On the con-
On the one hand, classical kinetic resolutions
trary, the aldimine of 3b was more stable than the cor- through Candida antarctica lipase B-catalyzed acyla-
responding ketimine, with an energy difference of tion reached conversions close to the ideal 50%. Both
18.9 kJ/mol.
the methoxyacetamides and the remaining amines
The nitrogen of the 5-F substituted pyridine forms were obtained with excellent enantioselectivity under
a strong hydrogen bond with the indole N–H of tryp- mild reaction conditions in tetrahydrofuran as solvent.
tophan 60 (Figure 3A and B), which is also present on On the other hand, the use of transaminases acting
the 6-F substituted pyridine but displaying a slightly with complementary stereoselectivity in biotransami-
weaker bond strength (Figure 3C and D). Specifically, nation experiments allowed the synthesis of both
the hydrogen bond distances and angles are 2.16 ꢅ amine enantiomers with excellent conversions and
and 159.38 for the 5-F-aldimine (Figure 3A), the complete selectivity in all cases. Therefore, the de-
values being 2.32 ꢅ and 156.58 in the case of the 6-F- sired enantiopure amines were isolated in moderate
aldimine (Figure 3C). On the other hand, the distan- to good yields. Transamination reactions have been
ces are shorter in both the ketimine structures with investigated in depth in order to get a better under-
values of 2.01 ꢅ (159.38) and 2.10 ꢅ (154.48) for the standing of the biocatalytic process through computa-
5-F and 6-F derivatives, respectively (Figure 3B and tional studies of the global equilibrium shift and the
D). Besides, the 6-F-aldimine shows an additional N– main interactions in the enzymatic active site. Thus,
H···F bond with the arginine 416 (2.11 ꢅ, 169.38), calculations confirmed that the presence of both the
which could be responsible for the higher stabilization fluorine and the pyridyl nitrogen of the studied struc-
of the aldimine over the ketimine mentioned above.
tures favoured the stabilization of the intermediates
Interestingly, the pyridine hydrogen bond is not ob- in the active site of the Chromobacterium violaceum
served for the hindered nitrogen atom of the 6-CF3 al- TA through different non-covalent interactions and
dimine derivative (Figure 3E) and it is weak in the therefore promoting the transamination reaction.
corresponding ketimine derivative (2.54 ꢅ, Fig-
ure 3F), while a new strong interaction between two
fluorine atoms and the Arg416 is responsible for the
stabilization of these intermediates. Thus, three N–
H···F bonds were found in the aldimine between the
two fluorine atoms mentioned before and two hydro-
gens of the residue (2.18 ꢅ, 146.48; 2.08 ꢅ, 143.18;
2.02 ꢅ, 136.08; Figure 3E). For the corresponding ke-
timine (Figure 3F), non-hydrogen bond strong elec-
trostatic interactions were established between the
guanidinium cation and the trifluoromethyl group.
Experimental Section
Enzyme Activity
Candida antarctica lipase type B (CAL-B, Novozym 435,
73000 PLU/g) was kindly donated by Novozymes. TAs from
Chromobacterium violaceum (2.1 U/mg), Vibrio fluvialis
(1.3 U/mg), Arthrobacter citreus (0.9 U/mg) and Arthrobact-
er species (2.2 U/mg) were overexpressed in E. coli and
used as lyophilized cells, while the evolved TA ArRmut11
Adv. Synth. Catal. 0000, 000, 0 – 0
8
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
(16 U/mg) was overexpressed in E. coli and purified by
a heat protocol. See the Supporting Information for further
details.
tered and the solvent removed by distillation under reduced
pressure to afford the ketones 3a and 3b as yellow solids;
yield: 85–95%.
1-(5-Fluoropyridin-3-yl)ethan-1-one (3a): Yellow solid;
yield: 85%; mp 48–508C; 1H NMR (300.13 MHz, CDCl3):
General Procedure for the Synthesis of the Alcohols
2a and 2b.
3
4
d=2.64 (s, 3H, H-2), 7.90 (ddd, JF,H =8.7 Hz, JH,H =2.8 Hz,
4
4JH,H =1.7 Hz, 1H, H-4’), 8.63 (d, JH,H =2.7 Hz, 1H, H-6’),
8.96 (s, 1H, H-2’); 13C NMR (75.5 MHz, CDCl3): d=27.1
A
1.6M solution of n-BuLi in n-hexane (1.76 mL,
2
(CH3, C-2), 121.8 (d, JF,C =18.7 Hz, CH, C-4’), 133.7 (C-3’),
2.81 mmol) was added dropwise to a solution of the corre-
sponding bromopyridine 1a or 1b (450.5 mg, 2.56 mmol) in
anhydrous Et2O (13 mL) at À788C under an argon atmos-
phere. The mixture was stirred at that temperature for
142.4 (d, 2JF,C =23.6 Hz, CH, C-6’), 145.7 (d, 4JF,C =4.0 Hz,
CH, C-2’), 159.6 (d, 1JF,C =259.4 Hz, C-5’), 195.4 (C, C-1);
HR-MS (ESI+): m/z=140.0510, calcd. for (C7H7FNO)+
(M+H)+: 140.0506.
30 min. Then,
a
solution of acetaldehyde (360 mL,
1-(6-Fluoropyridin-3-yl)ethan-1-one (3b): yellow solid;
6.39 mmol) in anhydrous Et2O (1 mL) was slowly added to
the resulting yellow suspension maintaining the inert atmos-
phere. The resulting solution was stirred at À788C for addi-
tional 1.5 h. After this time, H2O (10 mL) was added, lead-
ing to an orange suspension that was allowed to reach room
temperature. Then, an aqueous 1M HCl solution was added
until pH 8 and the product was extracted with Et2O (8ꢆ
10 mL). The organic layers were combined, dried over
Na2SO4, filtered and the solvent removed by distillation
under reduced pressure. The reaction crude was purified by
column chromatography on silica gel (80% EtOAc/hexane)
to afford the alcohols 2a and 2b as yellow oils; yield: 48–
68%.
yield: 95%; mp 47–498C; 1H NMR (300.13 MHz, CDCl3):
3
3
d=2.62 (s, 3H, H-2), 7.02 (dd, JH,H =8.5 Hz, JF,H =2.6 Hz,
1H, H-5’), 8.36 (ddd, 3JH,H =8.5 Hz, 4JF,H =7.7 Hz, JH,H
=
4
2.6 Hz, 1H, H-4’), 8.80 (d, 4JH,H =2.4 Hz, 1H, H-2’);
13C NMR (75.5 MHz, CDCl3): d=26.8 (CH3, C-2), 110.1 (d,
2JF,C =37.3 Hz, CH, C-5’), 131.1 (d, 4JF,C =4.4 Hz, C, C-3’),
141.3 (d, 3JF,C =9.4 Hz, CH, C-4’), 149.5 (d, 3JF,C =16.4 Hz,
1
CH, C-2’), 165.7 (d, JF,C =246.4 Hz, C-6’), 195.0 (C-1); HR-
MS (ESI+): m/z=140.0512, calcd. for (C7H7FNO)+ (M+
H)+: 140.0506.
Procedure for the Synthesis of 1-[(6-Trifluoromethyl)-
pyridine-3-yl]ethan-1-one (3c)
1-(5-Fluoropyridin-3-yl)ethan-1-ol (2a): Light yellow oil-
1
solid; yield: 48%; H NMR (300.13 MHz, CDCl3): d=1.40
3
3
(d, JH,H =6.5 Hz, 3H, H-2), 4.86 (q, JH,H =6.5 Hz, 1H, H-1),
A 3M solution of methylmagnesium iodide in Et2O (2.4 mL,
7.34 mmol) was added dropwise to a solution of the carboni-
trile 5 (317 mg, 1.84 mmol) in anhydrous Et2O (6.11 mL) at
08C and under a nitrogen atmosphere. An extra milliliter of
anhydrous Et2O was added to drag out the organomagnesi-
um reagent remaining in the addition funnel, and the mix-
ture was stirred at room temperature for 4 h. A colour
change was observed from the starting yellow solution to an
intense dark red one during this time. The reaction was
poured into a saturated aqueous NH4Cl solution (6 mL) at
08C and concentrated HCl was added until strongly acidic
(pH 1). The resulting mixture was stirred overnight at room
temperature and then basified until pH 9 by addition of an
aqueous NH3 solution. The reaction was extracted with
Et2O (3ꢆ15 mL), the combined organic layers were dried
over Na2SO4, filtered and the solvent removed by distillation
under reduced pressure. The crude product was purified by
column chromatography on silica gel (20% EtOAc/hexane)
to afford the ketone 3c as a pale yellow solid; yield: 153 mg
(44%); mp 57–598C; 1H NMR (300.13 MHz, CDCl3): d=
5.05 (brs, 1H, OH), 7.41 (dt, 3JF,H =9.3 Hz, 4JH,H =2.2 Hz,
4
1H, H-4’), 8.12 (d, JH,H =2.6 Hz, 1H, H-6’), 8.18 (s, 1H, H-
2’); 13C NMR (75.5 MHz, CDCl3): d=25.3 (CH3, C-2), 66.9
(CH, C-1), 120.4 (d, 2JF,C =18.3 Hz, CH, C-4’), 136.4 (d,
2JF,C =24.0 Hz, CH, C-6’), 142.8 (d, JF,C =3.6 Hz, CH, C-2’),
4
3
1
144.0 (d, JF,C =2.5 Hz, C, C-3’), 159.8 (d, JF,C =258.1 Hz, C,
C-5’); HR-MS (ESI+): m/z=142.0674, calcd. for
(C7H9FNO)+ (M+H)+: 142.0663.
1-(6-Fluoropyridin-3-yl)ethan-1-ol (2b): Light yellow oil-
1
solid; yield: 68%; H NMR (300.13 MHz, CDCl3): d=1.45
(d, 3JH,H =6.5 Hz, 3H, H-2), 3.46 (brs, 1H, OH), 4.90 (q,
3
3JH,H =6.5 Hz, 1H, H-1), 6.86 (dd, 3JH,H =8.4 Hz, JF,H
=
2.7 Hz, 1H, H-5’), 7.80 (dt, 3JH,H =8.1 Hz, 4JF,H =8.1 Hz,
4JH,H =2.5 Hz, 1H, H-4’), 8.06 (s, 1H, H-2’); 13C NMR
(75.5 MHz, CDCl3): d=25.3 (CH3, C-2), 67.2 (CH, C-1),
109.5 (d, 2JF,C =36.9 Hz, CH, C-5’), 139.0 (d, 3JF,C =8.0 Hz,
4
3
CH, C-4’), 139.1 (d, JF.C =4.4 Hz, C, C-3’), 144.7 (d, JF,C
=
1
14.2 Hz, CH, C-2’), 162.9 (d, JF,C =238.9 Hz, C, C-6’); HR-
MS (ESI+): m/z=142.0660, calcd. for (C7H9FNO)+ (M+
H)+: 142.0663.
3
2.69 (s, 3H, H-2), 7.81 (d, JH,H =8.0 Hz, 1H, H-5’), 8.40 (dd,
4
3JH,H =8.1 Hz, 4JH,H =1.6 Hz, 1H, H-4’), 9.17 (d, JH,H
=
1.3 Hz, 1H, H-2’); 13C NMR (75.5 MHz, CDCl3): d=27.1
(CH3, C-2), 120.7 (d, 4JF,C =2.5 Hz, CH, C-5’), 121.2 (q,
1JF,C =274.6 Hz, CF3), 134.0 (C-3’), 137.3 (CH, C-4’), 150.0
General Procedure for the Synthesis of the Ketones
3a and 3b
2
(CH, C-’), 151.3 (q, JF,C =35.2 Hz, C-6’), 195.6 (C-1); HR-
Dess–Martin reagent (2.65 g, 6.25 mmol) was added to a so-
lution of the corresponding alcohol 2a or 2b (0.589 g,
4.17 mmol) in CH2Cl2 (42 mL). The mixture was stirred at
room temperature for 2 h. After this time, the reaction was
quenched by adding an aqueous NaHCO3/Na2S2O3 solution
(20 mL, 1:1 v/v of aqueous saturated solutions) and stirred
for additional 5 min until complete disappearance of the
solid. The mixture was extracted with CH2Cl2 (3ꢆ20 mL),
the organic layers were combined, dried over Na2SO4, fil-
MS (ESI+): m/z=190.0479, calcd. for (C8H7F3NO)+ (M+
H)+: 190.0474.
General Procedure for the Synthesis of the Racemic
Amines 4a–c
Ammonium acetate (335 mg, 4.34 mmol) and sodium cyano-
borohydride (55 mg, 0.87 mmol) were added to a solution of
Adv. Synth. Catal. 0000, 000, 0 – 0
9
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
the corresponding ketone 3a–c (0.43 mmol) in anhydrous
MeOH (1.4 mL). The resulting mixture was stirred during
14 h at room temperature. After this time, the solvent was
removed by distillation under reduced pressure and the re-
action crude was purified by column chromatography on
silica gel (5% MeOH/CH2Cl2), yielding the corresponding
racemic amines 4a–c as yellow oils; yield: 40–55%.
General Procedure for the Asymmetric
Transamination of Ketones 3a–c Employing
Lyophilized Cells and Alanine Dehydrogenase as
Regeneration System
The lyophilized cells of E. coli containing overexpressed
transaminases (20 mg) were suspended in a 100 mM phos-
phate buffer pH 7 (440 mL) and the mixture was stirred at
308C and 120 rpm for 30 min. To the resulting suspension,
the ketones 3a–c (0.05 mmol, 50 mM), ammonium formate
(100 mL of 1.5M solution in a 100 mM phosphate buffer
pH 7; final concentration 150 mM), alanine (250 mL of 1M
solution in phosphate buffer 100 mM pH 7; final concentra-
tion 250 mM), NAD+ (100 mL of 10 mM solution in
1-(5-Fluoropyridin-3-yl)ethan-1-amine (4a): yellow oil;
yield: 47%; 1H NMR (300.13 MHz, CDCl3): d=1.38 (d,
3
3JH,H =6.6 Hz, 3H, H-2), 1.82 (bs, 2H, NH2), 4.21 (q, JH,H
=
3
4
6.6 Hz, 1H, H-1), 7.45 (dt, JF,H =9.6 Hz, JH,H =2.0 Hz, 1H,
4
H-4’), 8.32 (d, 4JH,H =2.8 Hz, 1H, H-5’), 8.38 (t, JH,H
=
1.8 Hz, 1H, H-2’); 13C NMR (75.5 MHz, CDCl3): d=25.8
2
(CH3, C-2), 48.6 (CH, C-1), 120.3 (d, JF,C =18.1 Hz, CH, C-
4
4’), 136.7 (d, 2JF,C =23.4 Hz, CH, C-6’), 143.9 (d, JF,C
=
a
100 mM phosphate buffer pH 7; final concentration
1
1 mM), PLP (100 mL of 10 mM solution in a 100 mM phos-
phate buffer pH 7; final concentration 1 mM), formate dehy-
drogenase (FDH, 2.6 mg, 11 U) and alanine dehydrogenase
(AlaDH, 10 mL, 11 U) were successively added. d- or l- ala-
nine was used as amine donor depending on the (R) or (S)-
transaminase selectivity, respectively.
The resulting mixture was shaken at 308C and 120 rpm
for 24 h. After this time, the reaction was quenched by
adding an aqueous 4M NaOH solution (400 mL) and ex-
tracted with EtOAc (3ꢆ500 mL). The organic phases were
combined and dried over Na2SO4. The reaction crude was
analyzed through GC to determine conversion values, re-
quiring an in situ derivatization for the measurement of the
enantiomeric excess.
3.7 Hz, CH, C-2’), 144.8 (C-3’), 159.9 (d, JF,C =256.5 Hz, C-
5’); HR-MS (ESI+): m/z=141.0823, calcd. for (C7H10FN2)+
(M+H)+: 141.0823.
1-(6-Fluoropyridin-3-yl)ethan-1-amine (4b): Yellow oil;
yield: 55%; 1H NMR (300.13 MHz, CDCl3): d=1.39 (d,
3JH,H =6.6 Hz, 3H, H-1), 1.94 (brs, 2H, NH2), 4.38 (1H, H-
3
3
1), 6.88 (dd, JH,H =8.5 Hz, JF,H =2.5 Hz, 1H, H-5’), 7.83 (dt,
3JH,H =8.1 Hz, 4JF,H =8.1 Hz, 4JH,H =2.5 Hz, 1H, H-4’), 8.16
4
(d, JH,H =2.0 Hz, 1H, H-2’); 13C NMR (75.5 MHz, CDCl3):
2
d=21.2 (CH3, C-2), 48.1 (CH, C-1), 110.0 (d, JF,C =37.5 Hz,
CH, C-5’), 140.0 (C-3’), 140.1 (d, 3JF,C =8.1 Hz, CH, C-4’),
3
1
146.6 (d, JF,C =14.8 Hz, CH, C-2’), 163.5 (d, JF,C =240.2 Hz,
C-6’); HR-MS (ESI+): m/z=141.0835, calcd. for
(C7H10FN2)+ (M+H)+: 141.0823.
1-[(6-Trifluoromethyl)pyridine-3-yl]ethan-1-amine
Yellow oil; yield: 40%; H NMR (300.13 MHz, CDCl3): d=
(4c):
1
General Procedure for the Asymmetric
Transamination of Ketones 3a–c Catalyzed by
Semipurified ArRmut11-TA
3
3
1.41 (d, JH,H =6.6 Hz, 3H, H-2), 4.27 (q, JH,H =6.6 Hz, 1H,
H-1), 7.63 (d, 3JH,H =8.1 Hz, 1H, H-5’), 7.90 (dd, JH,H
=
3
4
4
8.1 Hz, JH,H =2.0 Hz, 1H, H-4’), 8.68 (d, JH,H =2.0 Hz, 1H,
H-2’); 13C NMR (75.5 MHz, CDCl3): d=22.2 (CH3, C-2),
Ketones 3a–c (0.05 mmol, 50 mM) were suspended in
a 100 mM phosphate buffer pH 7 (500 mL) containing PLP
(1 mM; final concentration 0.5 mM) and 2-propylamine
(2M; final concentration 1M). Then, semipurified
ArRmut11-TA (500 mL) was added and the mixture was
shaken at 458C and 120 rpm for 24 h. The reaction was
quenched by adding aqueous an aqueous 4M NaOH solu-
tion (400 mL), extracted with ethyl EtOAc (3ꢆ500 mL). The
organic phases were combined and dried over Na2SO4. Re-
action crude was analyzed through GC to determine conver-
sion values, requiring an in situ derivatization for the mea-
surement of the enantiomeric excesses.
1
48.8 (CH, C-1), 120.8 (CH, C-5’), 121.2 (q, JF,C =274.1 Hz,
2
CF3), 136.4 (CH, C-4’), 138.8 (C-3’), 148.2 (q, JF,C =35.1 Hz,
C-6’), 148.9 (CH, C-2’); HR-MS (ESI+): m/z=191.0787,
calcd. for (C8H10F3N2)+ (M+H)+: 191.0791.
General Procedure for the Kinetic Resolution of
Amines (Æ)-4a–c through CAL-B-Catalyzed
Acylation
Ethyl methoxyacetate (5 equiv.) was added to a suspension
containing the corresponding racemic amine 4a–c (40 mg)
and the enzyme (CAL-B, 40 mg) in anhydrous THF (0.1M)
under an inert atmosphere. The reaction mixture was
shaken at 308C and 250 rpm for the necessary time to ach-
ieve a good kinetic resolution (2–7 h for around 50% con-
version, see Table 1), following the reaction time course by
HPLC analysis. The enzyme was filtered off, washed with
THF (5ꢆ1 mL) and the solvent evaporated under reduced
pressure. The reaction crude was purified by column chro-
matography on silica gel (eluent gradient 5–50% MeOH/
CH2Cl2), affording the corresponding optically active me-
thoxyacetamides (R)-6a–c (6a: 47% yield; 6b: 31% yield;
6c: 36% yield) and the remaining amines (S)-4a–c (4a: 14%
yield; 4b: 28% yield; 4c: 34% yield). For (R)-6a in >99%
ee: [a]2D0: +68.9 (c 1, CHCl3). For (R)-6b in >99% ee: [a]2D0:
+61.4 (c 0.5, CHCl3). For (R)-6c in >99% ee: [a]2D0: +56.5
(c 1, CHCl3).
General Procedure for the Preparative Reductive
Amination of Ketones 3a–c Employing Alanine
Dehydrogenase as Regeneration System
Scale-up was carried out by linear increasing of the reagent
amounts reported before. Thus, lyophilized cells of E. coli
containing overexpressed transaminases (144 mg) were sus-
pended in a 100 mM phosphate buffer pH 7 (3.8 mL) and
the mixture was stirred at 308C and 120 rpm for 30 min. To
the resulting suspension the ketones 3a–c (0.36 mmol,
50 mM), ammonium formate (720 mL of 1.5M solution in
a
100 mM phosphate buffer pH 7; final concentration
150 mM), alanine (1.8 mL of 1M solution in a 100 mM phos-
phate buffer pH 7; final concentration 250 mM), NAD+
(720 mL of 10 mM solution in a 100 mM phosphate buffer
Adv. Synth. Catal. 0000, 000, 0 – 0
10
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
pH 7; final concentration 1 mM), PLP (720 mL of 10 mM so-
lution in a 100 mM phosphate buffer pH 7; final concentra-
tion 1 mM), FDH (19 mg, 79 U) and alanine dehydrogenase
(AlaDH, 72 mL, 79 U) were successively added. d- or l- ala-
nine was used as amine donor depending on the (R) or (S)-
transaminase selectivity, respectively.
The resulting mixture was shaken at 308C and 120 rpm
for 24 h. The reaction was quenched by adding an aqueous
4M NaOH solution until pH around 10 and extracted with
EtOAc (3ꢆ10 mL). The organic phases were combined,
dried over Na2SO4, filtered and the solvent removed under
reduced pressure. Enantiopure amines (S)-4a, (R)-4a and
(R)-4c were isolated as colourless oils (yield: 71–88%). A
further purification by column chromatography on silica gel
(10% MeOH/CH2Cl2) was performed to isolate enantiopure
amine (S)-4b as colourless oil; yield: 40%. For (R)-4a in
>99% ee: [a]2D0: +36.2 (c 0.5, CHCl3). For (S)-4b in >99%
ee: [a]20: À28.2 (c 0.5, CHCl3). For (R)-4c in >99% ee: [a]2D0:
+20.0 D(c 0.5, CHCl3).
Int. Ed. 2014, 53, 8294–8308; i) M. C. Walker, M. C. Y.
Chang, Chem. Soc. Rev. 2014, 43, 6527–6536; j) A. Ko-
perniku, H. Liu, P. B. Hurley, Eur. J. Org. Chem. 2016,
871–886.
[2] P. Jeschke, Pest. Manag. Sci. 2010, 66, 10–27.
[3] a) S. Purser, P. R. Moore, S. Swallow, V. Gouverneur,
Chem. Soc. Rev. 2008, 37, 320–330; b) S. M. Ametamey,
M. Honer, P. A. Schubiger, Chem. Rev. 2008, 108,
1501–1516; c) K. L. Kirk, Org. Process Res. Dev. 2008,
12, 305–321; d) W. K. Hagmann, J. Med. Chem. 2008,
51, 4359–4369; e) C. Huang, J. McConathy, Curr. Top.
Med. Chem. 2013, 13, 871–891; f) J. Wang, M. Sꢂnchez-
Rosellꢁ, J. L. AceÇa, C. del Pozo, A. E. Sorochinsky, S.
Fustero, V. A. Soloshonok, H. Liu, Chem. Rev. 2014,
114, 2432–2506; g) E. P. Gillis, K. J. Eastman, M. D.
Hill, D. J. Donnelly, N. A. Meanwell, J. Med. Chem.
2015, 58, 8315–8359.
[4] D. Cahard, V. Bizet, Chem. Soc. Rev. 2014, 43, 135–147.
[5] a) K. Mꢇller, C. Faeh, F. Diederich, Science 2007, 317,
1881–1886; b) E. A. Ilardi, E. Vitaku, J. T. Njardarson,
J. Med. Chem. 2014, 57, 2832–2842.
Heat Purification Protocol for ArRmut11
[6] H.-J. Bçhm, D. Banner, S. Bendels, M. Kansy, B. Kuhn,
K. Mꢇller, U. Obst-Sander, M. Stahl, ChemBioChem
2004, 5, 637–643.
[7] a) B. E. Smart, J. Fluorine Chem. 2001, 109, 3–11; b) P.
Kirsch, Modern fluoroorganic chemistry: synthesis, reac-
tivity, applications, 2nd edn., Wiley-VCH, Weinheim,
Germany, 2013.
Lyophilized E. coli cells containing overexpressed enzyme
(2 g) were suspended in a 100 mM phosphate buffer pH 7
(20 mL) and the suspension was sonicated with the follow-
ing protocol: 1 s pulse; 4 s pause; 2.5 min; 40% amplitude.
The mixture was centrifuged for 20 min at 18000 rpm and
48C and the supernatant was treated for 30 min at 508C.
After this time the suspension was centrifuged again for
20 min at 18000 rpm and 48C and the supernatant contain-
ing the active enzyme was stored at À188C. Protein concen-
tration: 26.7 mg/mL according to Bradfordꢄs assay. Activity
(determined for the deamination of a-methylbenzylamine
with pyruvate): 16 U/mL.
[8] J. W. Clader, J. Med. Chem. 2004, 47, 1–9.
[9] a) V. A. Petrov, Fluorinated heterocyclic compounds:
Synthesis chemistry, and applications, Wiley-VCH,
Hoboken, New Jersey, USA, 2009; b) V. M. Muzalev-
skiy, A. V. Shastin, E. S. Balenkova, G. Haufe, V. G.
Nenajdenko, Synthesis 2009, 3905–3929; c) F. Meyer,
Chem. Commun. 2016, 52, 3077–3094.
[10] D. OꢄHagan, H. Deng, Chem. Rev. 2015, 115, 634–649.
[11] See for recent articles: a) A. Hibino, H. Ohtake, Pro-
cess Biochem. 2013, 48, 838–843; b) J. Mangas-Sꢂnchez,
E. Busto, V. Gotor, V. Gotor-Fernꢂndez, Org. Lett.
2013, 15, 3872–3875; c) A. S. Rowan, T. S. Moody,
R. M. Howard, T. J. Underwood, I. R. Miskelly, Y. He,
B. Wang, Tetrahedron: Asymmetry 2013, 24, 1369–1381;
d) W. Borze˛cka, I. Lavandera, V. Gotor, Tetrahedron
Lett. 2013, 54, 5022–5025; e) R.-J. Chen, G.-W. Zheng,
Y. Ni, B.-B. Zeng, J.-H. Xu, Tetrahedron: Asymmetry
2014, 25, 1501–1504; f) S. Kara, D. Spickermann, A.
Weckbecker, C. Leggewie, I. W. C. E. Arends, F. Holl-
mann, ChemCatChem 2014, 6, 973–976; g) A. Dꢀaz-Ro-
drꢀguez, J. Iglesias-Fernꢂndez, C. Rovira, V. Gotor-
Fernꢂndez, ChemCatChem 2014, 6, 977–980; h) R. O.
Lopes, A. S. de Miranda, B. Reichart, T. Glasnov, C. O.
Kappe, R. C. Simon, W. Kroutil, L. S. M. Miranda,
I. C. R. Leal, R. O. M. A. de Souza, J. Mol. Catal. B
2014, 104, 101–107; i) C. Rodrꢀguez, W. Borze˛cka, J. H.
Sattler, W. Kroutil, I. Lavandera, V. Gotor, Org.
Biomol. Chem. 2014, 12, 673–681; j) R. O. Lopes, J. B.
Ribeiro, A. S. de Miranda, G. V. V. da Silva, L. S. M.
Miranda, I. C. R. Leal, R. O. M. A. de Souza, Tetrahe-
dron 204, 70, 3239–3242; k) M. Lꢁpez-Iglesias, E.
Busto, V. Gotor, V. Gotor-Fernꢂndez, J. Org. Chem.
2015, 80, 3815–3824; l) H. Li, J. Moncecchi, M. D.
Truppo, Org. Process Res. Dev. 2015, 19, 695–700;
Acknowledgements
Financial support of this work by the Spanish Ministerio de
Economꢀa y Competitividad (MINECO) (CTQ-2013-44153-
P project) and the Asturian Regional Government (FC-15-
GRUPIN14-002) is gratefully acknowledged. E. B. thanks
MINECO for a postdoctoral contract. M.L.-I. and D.G.-M.
thank Fundaciꢁn para el Fomento en Asturias de la Investi-
gaciꢁn Cientꢀfica Aplicada y la Tecnologꢀa (FICYT), while
M.R.-M. thanks the Spanish Ministerio de Ciencia e Innova-
ciꢁn (MICINN) for predoctoral fellowships.
References
[1] a) D. OꢄHagan, Chem. Soc. Rev. 2008, 37, 308–319;
b) T. Furuya, C. A. Kuttruff, T. Ritter, Curr. Opin.
Drug. Discovery Dev. 2008, 11, 803–819; c) F.-L. Qing,
F. Zheng, Synlett 2011, 1052–1072; d) C. Hollingworth,
V. Governeur, Chem. Commun. 2012, 48, 2929–2942;
e) S. K. Ritter, Chem. Eng. News 2012, 90, 10–17; f) X.-
F. Wu, H. Neumann, M. Beller, Chem. Eur. J. 2012, 7,
1744–1754; g) T. Liang, C. N. Neumann, T. Ritter,
Angew. Chem. 2013, 125, 8372–8423; Angew. Chem.
Int. Ed. 2013, 52, 8214–8264; h) H. Egami, M. Sodeoka,
Angew. Chem. 2014, 126, 8434–8449; Angew. Chem.
Adv. Synth. Catal. 0000, 000, 0 – 0
11
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
m) G-C. Xu, H.-L. Yu, Y.-P. Shang, J.-H. Xum, RSC
Adv. 2015, 5, 22703–22711; n) D. Mꢈndez-Sꢂnchez, J.
Mangas-Sꢂnchez, E. Busto, V. Gotor, V. Gotor-Fernꢂn-
dez, Adv. Synth. Catal. 2016, 358, 122–131; o) S. S. Ri-
beiro, S. P. de Vasconcellos, P. L. Ramos, J. B. da Cruz,
A. L. M. Porto, Curr. Microw. Chem. 2016, 3, 9–13;
p) F. M. Perna, M. A. Ricci, A. Scilimati, M. C. Mena,
I. Pisano, L. Palmieri, G. Agrimi, P. Vitale, J. Mol.
Catal. B 2016, 124, 29–37; q) K. Wu, L. Chen, H. Fan,
Z. Zhao, H. Wang, D. Wei, Tetrahedron Lett. 2016, 57,
899–904.
Org. Process Res. Dev. 2013, 17, 693–700; h) R. E.
Meadows, K. R. Mulholland, M. Schꢇrmann, M.
Golden, H. Kierkels, E. Meulenbroeks, D. Mink, O.
May, C. Squire, H. Straatman, A. S. Wells, Org. Process
Res. Dev. 2013, 17, 1117–1122; i) L. Frodsham, M.
Golden, S. Hard, M. N. Kenworthy, D. J. Klauber, K.
Leslie, C. Macleod, R. E. Meadows, K. R. Mulholland,
J. Reilly, C. Squire, S. Tomasi, D. Watt, A. S. Wells,
Org. Process Res. Dev. 2013, 17, 1123–1130; j) E.-S.
Park, M. S. Malik, J.-Y. Dong, J.-S. Shin, ChemCatChem
2013, 5, 1734–1738; k) M. Pꢉiviç, L. T. Kanerva, Pro-
cess Biochem. 2013, 48, 1488–1494; l) M. Shon, R.
Shanmugavel, G. Shin, S. Mathew, S.-H. Lee, H. Yun,
Chem. Commun. 2014, 50, 12680–12683; m) C. E. Paul,
M. Rodrꢀguez-Mata, E. Busto, I. Lavandera, V. Gotor-
Fernꢂndez, V. Gotor, S. Garcꢀa-Cerrada, J. Mendiola,
ꢋ. de Frutos, I. Collado, Org. Process Res. Dev. 2014,
18, 788–792; n) N. G. Schmidt, R. C. Simon, W. Kroutil,
Adv. Synth. Catal. 2015, 357, 1815–1821; o) A. K.
Holzer, K. Hiebler, F. G. Mutti, R. C. Simon, L. Lauter-
bach, O. Lenz, W. Kroutil, Org. Lett. 2015, 17, 2431–
2433; p) A. Cuetos, M. Garcꢀa-Ramos, E.-M. Fischered-
er, A. Dꢀaz-Rodrꢀguez, G. Grogan, V. Gotor, W. Krou-
til, I. Lavandera, Angew. Chem. 2016, 128, 3196–3199;
Angew. Chem. Int. Ed. 2016, 55, 3144–3147.
[12] See for recent articles: a) S. S. Ribeiro, C. Raminelli,
A. L. M. Porto, J. Fluorine Chem. 2013, 154, 53–59;
b) P. Chun, X. Zhu, J. Mol. Catal. B 2013, 97, 184–188;
c) X. Wei, X. Jiang, L. Ye, S. Yuan, Z. Chen, M. Wu,
H. Yu, J. Mol. Catal. B 2013, 97, 270–277; d) C. Kim, J.
Lee, J. Cho, Y. Oh, Y. K. Choi, E. Choi, J. Park, M.-J.
Kim, J. Org. Chem. 2013, 78, 2571–2578; e) G. Cheng,
B. Xia, Q. Wu, X. Lin, RSC Adv. 2013, 3, 9820–9828;
f) G.-W. Zheng, X.-Y. Liu, Z.-J. Zhang, P. Tian, G.-Q.
Lin, J.-H. Xu, RSC Adv. 2013, 3, 20446–20449; g) S.
Kim, Y. K. Choi, J. Hong, J. Park, M.-J. Kim, Tetrahe-
dron Lett. 2013, 54, 1185–1188; h) Q. Jin, G. Jia, Y.
Zhang, C. Li, Catal. Sci. Technol. 2014, 4, 464–471;
i) D. A. Chaplin, M. E. Fox, S. H. B. Kroll, Chem.
Commun. 2014, 50, 5858–5860; j) M. Sakulsombat, P.
Vongvilai, O. Ramstrçm, Chem. Eur. J. 2014, 20,
11322–11325; k) J. A. Fernꢂndez-Salas, S. Manzini, S. P.
Nolan, Chem. Eur. J. 2014, 20, 13132–13135; l) K. P. J.
Gustafson, R. Lihammar, O. Verho, K. Engstrçm, J.-E.
Bꢉckvall, J. Org. Chem. 2014, 79, 3747–3751; m) O. V.
Kucher, A. O. Kolodiazhnaya, O. B. Smolii, D. V. Pri-
suazhnyk, K. A. Tolmacheva, O. A. Zaporozhets, Y. S.
Moroz, P. K. Mykhailiuk, A. A. Tolmachev, Eur. J. Org.
Chem. 2014, 7692–7698; n) E. Fernꢂndez-ꢊlvaro, J. Es-
quivias, M. Pꢈrez-Sꢂnchez, P. Domꢀnguez de Marꢀa,
M. J. RemuiÇꢂn-Blanco, J. Mol. Catal. B 2014, 100, 1–6;
o) D. Mꢈndez-Sꢂnchez, N. Rꢀos-Lombardꢀa, S. Garcꢀa-
Granda, J. Montejo-Bernardo, A. Fernꢂndez-Gonzꢂlez,
V. Gotor, V. Gotor-Fernꢂndez, Tetrahedron: Asymme-
try 2014, 25, 381–386; p) G. Xu, X. Dai, S. Fu, J. Wu, L.
Yang, Tetrahedron Lett. 2014, 55, 397–402; q) I. M. Fer-
reira, R. H. V. Nishimura, A. B. A. Souza, G. C. Closos-
ki, S. A. Yoshioka, A. L. M. Porto, Tetrahedron Lett.
2014, 55, 5062–5065; r) A. Singh, J. Falabella, T. L. La-
Porte, A. Goswami, Org. Process Res. Dev. 2015, 19,
819–830; s) G. Xu, S. Lan, S. Fu, J. Wu, L. Yang, Tetra-
hedron Lett. 2015, 56, 2714–2719.
[14] T. C. Nugent, Chiral Amine Synthesis: Methods, Devel-
opments and Applications, Wiley-VCH, Weinheim,
2010.
[15] a) M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M.
Keßeler, R. Stꢇrmer, T. Zelinski, Angew. Chem. 2004,
116, 806–843; Angew. Chem. Int. Ed. 2004, 43, 788–824;
b) J. Duan, P. Li, Catal. Sci. Technol. 2014, 4, 311–320;
c) B. M. Paz, H. Jiang, K. A. Jørgensen, Chem. Eur. J.
2015, 21, 1846–1853; d) H. Ishikawa, S. Shiomi, Org.
Biomol. Chem. 2016, 14, 409–424.
[16] For recent reviews about the biocatalytic synthesis of
chiral amines: a) D. Ghislieri, N. J. Turner, Top. Catal.
2014, 57, 284–300; b) M. Fuchs, J. E. Farnberger, W.
Kroutil, Eur. J. Org. Chem. 2015, 6965–6982; c) O.
Verho, J.-E. Bꢉckvall, J. Am. Chem. Soc. 2015, 137,
3996–4009; d) G. Grogan, N. J. Turner, Chem. Eur. J.
2016, 22, 1900–1907.
[17] a) U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405–
2415; b) F. van Rantwijk, R. A. Sheldon, Tetrahedron
2004, 60, 501–519; c) E. Busto, V. Gotor-Fernꢂndez, V.
Gotor, Chem. Rev. 2011, 111, 3998–4035; d) D.
Mꢈndez-Sꢂnchez, M. Lꢁpez-Iglesias, V. Gotor-Fernꢂn-
dez, Curr. Org. Chem. 2016, 20, 1186–1203.
[13] a) C. K. Savile, J. M. Janey, E. C. Mundorff, J. C.
Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber,
F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman,
G. J. Hughes, Science 2010, 329, 305–309: b) M. Truppo,
J. M. Janey, B. Grau, K. Morley, S. Pollack, G. Hughes,
I. Davies, Catal. Sci. Technol. 2012, 2, 1556–1559;
c) M. D. Truppo, H. Strotman, G. Hughes, ChemCat-
Chem 2012, 4, 1071–1074; d) J. Hopwood, M. D.
Truppo, N. J. Turner, R. C. Lloyd, Chem. Commun.
2011, 47, 773–775; e) M. S. Malik, E.-S. Park, J.-S. Shin,
Green Chem. 2012, 14, 2137–2140; f) J. S. Reis, R. C.
Simon, W. Kroutil, L. H. Andrade, Tetrahedron: Asym-
metry 2013, 24, 1495–1501; g) R. L. Hanson, R. M.
Johnston, S. L. Goldberg, W. L. Parker, A. Goswami,
[18] a) L. E. Iglesias, V. M. Sꢂnchez, F. Rebolledo, V. Gotor,
Tetrahedron: Asymmetry 1997, 8, 2675–2677; b) O.
Torre, E. Busto, V. Gotor-Fernꢂndez, V. Gotor, Adv.
Synth. Catal. 2007, 349, 1481–1488; c) J. B. Crawford,
R. T. Skerlj, G. J. Bridger, J. Org. Chem. 2007, 72, 669–
671; d) K. Ditrich, Synthesis 2008, 14, 2283–2287;
e) L. K. Thalꢈn, D. Zhao, J.-B. Sortais, J. Paetzold, C.
Hoben, J.-E. Bꢉckvall, Chem. Eur. J. 2009, 15, 3403–
3410; f) K. P. J. Gustafson, R. Lihammar, O. Verho, K.
Engstrçm, J.-E. Bꢉckvall, J. Org. Chem. 2014, 79, 3747–
3751; g) F. Xu, Q. Wu, X. Chen, X. Lin, Q. Wu, Eur. J.
Org. Chem. 2015, 5393–5401.
[19] a) F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, J.
Prakt. Chem. 1997, 339, 381–384; b) M. Cammenberg,
Adv. Synth. Catal. 0000, 000, 0 – 0
12
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
asc.wiley-vch.de
K. Hult, S. Park, ChemBioChem 2006, 7, 1745–1749;
c) M. A. J. Veld, K. Hult, A. R. A. Palmans, E. W.
Meijer, Eur. J. Org. Chem. 2007, 5416–5421; d) M. Ro-
drꢀguez-Mata, E. Garcꢀa-Urdiales, V. Gotor-Fernꢂndez,
V. Gotor, Adv. Synth. Catal. 2010, 352, 395–406; e) M.
Paravidino, U. Hanefeld, Green Chem. 2011, 13, 2651–
2657; f) S. Jung, J. Kim, S. Park, RSC Adv. 2013, 3,
2590–2594.
[25] a) Y. Yamada, A. Iwasaki, N. Kizaki, K. Matsumoto, Y.
Ikenaka, M. Ogura, J. Hasegawa, (Kaneka Corpora-
tion), PCT Int. Appl. WO 9848030A1 19981029, 1998;
b) A. Iwasaki, Y. Yamada, N. Kizaki, Y. Ikenaka, J. Ha-
segawa, Appl. Microbiol. Biotechnol. 2006, 69, 499–505;
c) F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler, W.
Kroutil, Adv. Synth. Catal. 2011, 353, 3227–3233.
[26] C. K. Savile, J. M. Janey, E. M. Mundorff, J. C. Moore,
S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J.
Fleitz, J. Brands, P. N. Devine, G. W. Huisman, G. J.
Georges, Science 2010, 329, 305–309.
[20] a) C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J.
Am. Chem. Soc. 1982, 104, 7294–7299; b) Online tool:
K. Faber, W. Kroutil, G. Thallinger, http://biocatalysis.-
uni-graz.at/enantio/cgi-bin/enantio.pl.
[27] H. E. Smith, L. J. Schaad, R. B. Banks, C. J. Wiant, C. F.
[21] a) D. Koszelewski, K. Tauber, K. Faber, W. Kroutil,
Trends Biotechnol. 2010, 28, 324–332; b) S. Mathew, H.
Yun, ACS Catal. 2012, 2, 993–1001; c) M. S. Malik, E.-
S. Park, J.-S. Shin, Appl. Microbiol. Biotechnol. 2012,
94, 1163–1171; d) R. C. Simon, N. Richter, E. Busto, W.
Kroutil, ACS Catal. 2014, 4, 129–143; e) R. C. Simon,
E. Busto, E.-M. Fischereder, C. S. Fuchs, D. Pressnitz,
N. Richter, W. Kroutil, Biocatalysis in Organic Synthe-
sis, in: Science of Synthesis, (Eds.: K. Faber, W.-D. Fess-
ner, N. J. Turner), Georg Thieme Verlag, Stuttgart,
2015, pp 383–420; f) E. Busto, R. C. Simon, N. Richter,
W. Kroutil, in: Green Biocatalysis, (Ed. R. N. Patel),
John Wiley & Sons, Inc. New Jersey, 2016, pp 17–57.
[22] B.-Y. Hwang, B.-K. Cho, H. Yun, K. Koteshwar, B.-G.
Kim, J. Mol. Catal. B 2005, 37, 47–55.
[23] U. Kaulman, K. Smithies, M. E. B. Smith, H. C. Hailes,
J. M. Ward, Enzyme Microb. Technol. 2007, 41, 628–
637.
[24] S. Pannuri, S. V. Kamat, A. R. M. Garcia, (Cambrex
North Brunswick Inc.), PCT Int. Appl. WO
2006063336A2 20060615, 2006.
Jordan, J. Am. Chem. Soc. 1973, 95, 811–818.
[28] R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport,
L. A. Cuccia, J. Org. Chem. 1991, 56, 2656–2665.
[29] M. Lꢁpez-Iglesias, D. Gonzꢂlez-Martꢀnez, V. Gotor, E.
Busto, W. Kroutil, V. Gotor-Fernꢂndez, ACS Catal.
2016, 6, 4003–4009.
[30] a) M. S. Humble, K. E. Cassimjee, M. Hꢌkansson, Y. R.
Kimbung, B. Walse, V. Abedi, H.-J. Federsel, P. Ber-
glund, D. T. Logan, FEBS J. 2012, 279, 779–792;
b) K. E. Cassimjee, B. Manta, F. Himo, Org. Biomol.
Chem. 2015, 13, 8453–8464.
[31] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput.
Chem. 2009, 16, 2785–2791.
[32] a) E. R. Johnson, S. Keinan, P. Mori-Sꢂnchez, J. Con-
treras-Garcꢀa, A. J. Cohen, W. Yang, J. Am. Chem. Soc.
2010, 132, 6498–6506; b) J. Contreras-Garcꢀa, E. R.
Johnson, S. Keinan, R. Chaudret, J.-P. Piquemal, D. N.
Beratan, W. Yang, J. Chem. Theory Comput. 2011, 7,
625–632; c) R. Soria-Martꢀnez, R. Mendoza-Merono, S.
Garcꢀa-Granda, J. Mol. Struct. 2016, 1105, 322–331.
Adv. Synth. Catal. 0000, 000, 0 – 0
13
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!

FULL PAPERS
14 Asymmetric Biocatalytic Synthesis of Fluorinated Pyridines
through Transesterification or Transamination:
Computational Insights into the Reactivity of Transaminases
Adv. Synth. Catal. 2017, 359, 1 – 14
Marꢀa Lꢁpez-Iglesias, Daniel Gonzꢂlez-Martꢀnez,
Marꢀa Rodrꢀguez-Mata, Vicente Gotor, Eduardo Busto,*
Wolfgang Kroutil,* Vicente Gotor-Fernꢂndez*
Adv. Synth. Catal. 0000, 000, 0 – 0
14
ꢃ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!