Transaminases: Scheme 1 General Reaction Scheme For - Transaminase-Catalyzed Asymmetric Reductive Amination

383
2.4.3 ø-Transaminases
R. C. Simon, E. Busto, E.-M. Fischereder, C. S. Fuchs, D. Pressnitz, N. Richter, and
W. Kroutil
General Introduction
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ø-Transaminases[1–12] (EC 2.6.1.X) catalyze the reversible transformation of a ketone to an
amine via reductive amination. The nitrogen and the electrons are transferred to the ke-
tone from pyridoxamine 5¢-phosphate (PMP), thereby releasing pyridoxal 5¢-phosphate
(PLP) (Scheme 1).
Scheme 1 General Reaction Scheme for ø-Transaminase-Catalyzed

Asymmetric Reductive Amination
O NH2
ω-transaminase
R1 R2 R1 R2
NH2 H O
OH HO
2−O
3PO OPO32−
N N
PMP PLP
The catalytically required cofactor PLP is ideally recycled to PMP by the same ø-transami-
nase at the expense of an amine donor, which is usually required in stoichiometric
amounts (Scheme 2). Common amine donors employed include alanine or diverse alkyl-
and (arylalkyl)amines.[7,13,14] When using alanine as the amine donor, the thermodynamic
equilibrium is in general on the side of the starting materials (ketone and alanine);[15] con-
sequently, the coproduct, pyruvate, needs to be removed in order to shift the equilibrium
to the product side. This is mainly achieved with a consecutive enzymatic step.[7] More-
over, since ø-transaminases are mostly enantiospecific, enantiopure l- or d-alanine
must be employed. In the case of isopropylamine as amine donor, removal of the coprod-
uct acetone via evaporation is feasible[16] as well as the application of huge amounts there-
of.[17,18] 1-Phenylethylamine is well suited for thermodynamic reasons,[13] but it is the most
expensive choice, especially as only one enantiomer is transformed by stereoselective
ø-transaminases. Thus, either optically pure 1-phenylethylamine is applied or more
workup efforts are required to separate and recover all products.
for references see p 418

384 Biocatalysis 2.4 Transamination and Reductive Amination
Scheme 2 Asymmetric Synthesis of Chiral Amines Cata-

lyzed by ø-Transaminases Employing Alternative Amine
Donors[7,13–18]
O NH2
ω-transaminase
R1 R2 R1 ∗ R2
PMP PLP
O NH2
R3 R3
R3 = Me, CO2H, Ph, etc.
Because ø-transaminase-catalyzed amination reactions are reversible, the enzymes can

also be applied for deamination; starting with a racemic amine, one enantiomer will be
oxidized in a kinetic resolution to the corresponding ketone. In the case of high enantio-
selectivity, the other amine enantiomer remains ideally in optically pure form (Scheme
3). Pyruvate is in general preferred as the amine acceptor for thermodynamic reasons.
Scheme 3 Kinetic Resolution of Racemic Amines Employing ø-Transaminases
NH2 NH2 ω-transaminase NH2 O

+ +
R1 R2 R1 R2 R1 R2 R1 R2
PLP PMP
NH2 O
CO2H CO2H
Regarding the overall efficiency, the reductive amination is generally favored, because
the product can be obtained in up to 100% yield in contrast to only 50% via kinetic resolu-
tion. For the sake of clarity, the cofactor PLP/PMP cycle is not displayed in the schemes
below.
2.4.3.1 Amination of Ketones
2.4.3.1.1 Amination of Linear Monoketones
2.4.3.1.1.1 Amination Employing Alanine as Amine Donor
2.4.3.1.1.1.1 Amination with Lactate Dehydrogenase
One option to shift the unfavorable thermodynamic equilibrium toward product forma-
tion when using alanine as amine donor is to reduce the coproduct pyruvate to lactate via
a lactate dehydrogenase (LDH). For this particular transformation, additional reducing
equivalents in the form of NADH are required; therefore, a further subsystem containing
a third enzyme such as either glucose dehydrogenase (GDH) or formate dehydrogenase
(FDH) is needed. For practical reasons (accessibility of the enzyme, commercial availabil-
ity, etc.), glucose dehydrogenase is most commonly employed, although pH control is re-
quired on a preparative scale due to the formation of gluconic acid.[7,12,17,19–21] The LDH sys-
tem has also been applied in the presence of organic cosolvents such as dimethyl sulfox-
ide (Scheme 4).[19,22–24]
2.4.3 ø-Transaminases 385
Scheme 4 Enzymatic Reductive Amination in Combination with Lactate Dehydrogenase

and NADH Recycling[22,23]
ω-transaminase
O PLP, buffer
NH2
R1 ∗ R1
NH2 O OH
OH OH LDH OH
O O O
NADH NAD+
GDH
gluconolactone glucose
R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Config Ref

[23]
Ph PF 92 (79) >99 (>99) S
[22]
Ph ArR 45 (49) >99 (>99) R
[23]
(CH2)2Ph PF 99 (97) 94 (97) S
[22]
(CH2)2Ph HN 87 (94) >99 (>99) R
[23]
CH2OPh PF >99 (>99) >99 (>99) S
[22]
CH2OPh AT >99 (>99) >99 (>99) R
[22]
CH2OPh HN >99 (>99) >99 (>99) R
[23]
Et PF 30 (23) 99 (96) S
[22]
Et AT 36 (29) >99 (>99) R
[23]
(CH2)5Me PF 95 (73) 99 (98) S
[22]
(CH2)5Me AT 96 (97) >99 (>99) R
[23]
CH2CO2Et VF >99 (>99) >99 (>99) S
[22]
CH2CO2Et AT >99 (>99) >99 (>99) R
[22]
CH2CO2Et ArR >99 (>99) >99 (>99) R
[22]
CH2CO2Et HN >99 (>99) >99 (>99) R
a
Origin of enzymes: PF = Pseudomonas fluorescens; ArR = Arthrobacter sp.;
VF = Vibrio fluvialis; HN = Hyphomonas neptunium; AT = Aspergillus terreus.
b
Conversion determined by achiral GC-FID measurement.
c
Values in parentheses are values obtained for reactions in the presence of
DMSO (15 vol%) as cosolvent.
d
Enantiomeric excess determined by GC on a chiral stationary phase after
derivatization to the corresponding acetamide.
2.4.3.1.1.1.1.1 Preparation of Optically Pure (R)- and (S)-1-Phenylethylamine
Coproduct removal not only shifts the unfavorable thermodynamic equilibrium to the
product side but also prevents possible inhibition of the ø-transaminases by, for example,
pyruvate. Inhibition of the ø-transaminase by pyruvate, as well as by other ketones, may
become an important issue at increased substrate concentration and is therefore of par-
ticular interest.[25] For instance, 1-phenylethylamine (2) is a strong inhibitor for ø-trans-
aminases, although it is still accessible by the transformation of acetophenone (1) in
high substrate concentrations of 50 g/L in a system whereby the formed product is re-
moved from the reaction medium using an acidic ion-exchange resin (Amberlite XAD
1180).[26] After biotransformation, the product can be easily recovered by washing the

resin under basic conditions. Employing this concept, (R)- and (S)-1-phenylethylamine (2)
can be produced in enantiomerically pure form with >90% isolated yield using stereocom-
plementary, commercially available ø-transaminases (Scheme 5).
Scheme 5 Amination of Acetophenone at 50 g/L Substrate Concentration Employing

the Lactate Dehydrogenase System[26]
ω-transaminase
O PLP, buffer
NH2
Ph Ph ∗
1 2 >90%; >99% ee
NH2 O OH
OH OH LDH OH
O O O
NADH NAD+
GDH
(R)- or (S)-1-Phenylethylamine (2); Typical Procedure:[26]

The asymmetric reductive amination was conducted on a 50-mL scale in potassium phos-
phate buffer (100 mM) containing NAD+ (1.0 g/L), PLP (0.5 g/L), glucose (90.0 g/L), d-alanine
(for R-enantiomer) or l-alanine (for S-enantiomer, 90.0 g/L), acetophenone (1; 50 g/L), GDH
(1.0 g/L), LDH (1.0 g/L), ø-transaminase ATA-117 (for R-enantiomer, 5.0 g/L) or ATA-113 (for
S-enantiomer, 5.0 g/L), and ion-exchange resin (Amberlite XAD 1180, 200 g/L). Reactions
were performed at 30 8C and pH 7.5 in a Multimax reactor system with overhead mechan-
ical stirring at 400 rpm until complete conversion, whereby the pH value was controlled
through the automated addition of 2 M aq NaOH. The ion-exchange resin was isolated by
filtration and washed with buffer (pH 11) to recover the optically pure product; yield:
>90%; >99% ee.
2.4.3.1.1.1.1.2 Preparation of an (S)-Rivastigmine Precursor
Rivastigmine {3-[(1S)-1-(dimethylamino)ethyl]phenyl ethyl(methyl)carbamate} is a strong

cholinesterase inhibitor and one of the most potent agents for the treatment of Alzheim-
ers or Parkinsons disease at early stages.[27] In a short chemoenzymatic route, the ø-trans-
aminase originating from Paracoccus denitrificans[28] transforms the ideal ketone precursor
3 into the optically pure amine (S)-4 (76% isolated yield) in combination with the LDH sys-
tem (Scheme 6).[29] Dimethylation of the amine (S)-4 by standard transformations affords
rivastigmine in a short and highly efficient way.[29,30]
Scheme 6 ø-Transaminase-Catalyzed Synthesis of a Key Precursor for Rivastigmine[29]
ω-transaminase
PLP, buffer (pH 7)
Me O 30 oC, 24 h
Me NH2
N O N O
Et Et
O O
3 (S)-4 76%; >99% ee
NH2 O OH
OH OH LDH OH
O O O
NADH NAD+
GDH
3-[(S)-1-Aminoethyl]phenyl Ethyl(methyl)carbamate [(S)-4]; Typical Procedure:[29]

Lyophilized cells of E. coli containing overexpressed ø-transaminase from Paracoccus deni-
trificans (200 mg) were rehydrated in sodium phosphate buffer (100 mM, pH 7; 10 mL) con-
taining PLP (1.0 mM). LDH mix (300 mg, containing LDH, GDH, glucose, and NAD+), l-ala-
nine (220 mg, 2.5 mmol), and ketone 3 (99 mg, 0.45 mmol) were added and the suspension
was shaken at 30 8C and 120 rpm for 24 h. The pH was adjusted to 2 and the mixture was
extracted with EtOAc (2 10 mL) to remove the remaining starting material 3. Then, the
pH was set to >10 with sat. aq K2CO3 and the aqueous phase was extracted with EtOAc (3
10 mL). The combined organic phases were dried (Na2SO4) and the solvent was evaporated
to give the product as a colorless oil; yield: 75 mg (76%); [Æ]D20 –11.2 (c 1.0, MeOH); >99% ee.
2.4.3.1.1.1.1.3 Chemoenzymatic Preparation of a Precursor for the Dual

Orexin Receptor Antagonist MK-6096
MK-6096 (8) is an orexin receptor agonist and hence a potential drug for the treatment of
insomnia. A process for MK-6096 has been developed including an ø-transaminase-cata-
lyzed asymmetric reductive amination as the key step. Notably, the chemoenzymatic syn-
thesis for this pharmaceutical has been established on a kilogram scale (Scheme 7).[31]
In this approach, the chirality on the 6-methylpiperidin-2-one core (6R)-7 is intro-
duced via enzymatic transamination using a commercially available ø-transaminase
(ATA-117). The utilization of d-alanine as the amine source allows the transformation of
the oxo diester 5 into the corresponding amino diester (R)-6, which cyclizes spontaneous-
ly to afford the optically pure lactam (6R)-7 (>99% ee). The thermodynamic equilibrium is
shifted toward product formation for two reasons: on the one hand the amino diester (R)-6
spontaneously cyclizes to the desired piperidin-2-one (6R)-7, avoiding concomitant prod-
uct inhibition. On the other hand, the formed pyruvate is removed employing the LDH
system to circumvent inhibition of the enzyme by pyruvate. This process illustrates the
high applicability of the LDH system in large-scale processes.

Scheme 7 A Biocatalytic Key Step in the Chemoenzymatic Synthesis of the Orexin Receptor
Antagonist MK-6096[31]
ATA-117, PLP
buffer (pH 7.4)
O CO2Me NH2 CO2Me
30 oC, 42 h
HN
CO2Me CO2Me CO2Me
2 2
O
5 (R)-6 (6R)-7
NH2 O OH
LDH
OH OH OH
O O O
NADH NAD+
GDH
N O
HN
CO2Me O N
N N F
O
(6R)-7 8 MK-6096
Methyl (6R)-6-Methyl-2-oxopiperidine-3-carboxylate [(6R)-7]; Typical Procedure:[31]

In a 100-L Bchi jacketed reactor with overhead stirring Na2HPO4 • H2O (852 g, 5.4 mol),
d-alanine (7.2 kg, 81 mol), glucose monohydrate (6.48 kg, 32.4 mol), NAD+ (22.5 g,
0.034 mol), and PLP (45 g, 0.18 mol) were added to H2O (45 L) at 30 8C. The pH was adjusted
to 7.4 with NaOH and ATA-117 (450 g), LDH (9 g, Biocatalytics cat.# LDH-101), and GDH
(45 g, Biocatalytics cat.# GDH-103) were rinsed into the vessel with H2O (2.5 L). Then, oxo
diester 5 (4.5 kg, 22.3 mol) and H2O (2.5 L) were added. The mixture was stirred for 42 h
and the pH was controlled using 5 M aq NaOH. NaCl (19.4 kg) was added and the pH was
adjusted to 3.5 with 5 M aq HCl (6.0 L). For extraction, MeCN (20 L) was added and the mix-
ture was stirred for 10 min and allowed to settle for 1 h without agitation. The MeCN
phase was removed and the aqueous layer was then re-extracted with MeCN. The organic
phases were combined, filtered through Solka Floc, and concentrated to remove H2O and
MeCN. The remaining oil containing high levels of heterogeneous NaCl was dissolved in
EtOAc (50 L), filtered, and concentrated under reduced pressure; yield (including NaCl):
5.5 kg (74%); >99% ee.
2.4.3.1.1.1.2 Amination with Alanine Dehydrogenase
2.4.3.1.1.1.2.1 (S)-Amination
An alternative method to shift the equilibrium involves the recycling of l-alanine from
pyruvate using three different types of enzymes:[17,32] an ø-transaminase, an alanine dehy-
drogenase (AlaDH), and either glucose dehydrogenase (GDH) or formate dehydrogenase
(FDH). As the AlaDH recycles pyruvate to l-alanine by consuming NADH and ammonia,
additional recycling of NADH becomes necessary. The advantage of this system is that
the amine donor alanine is recycled during the reaction and not consumed as in the reac-
tions described in Section 2.4.3.1.1.1.1.3. However, to date, only l-alanine dehydrogenases
are known, implying that only l-alanine can be recycled. Examples of amines (S)-9 prepar-
ed using this approach are given in Scheme 8.[17,23,32,33]
Scheme 8 (S)-Amination in Combination with the L-Alanine Dehydrogenase Recycling

System[17,23,32,33]
O ω-transaminase NH2
PLP
R1 R1
(S)-9
NH2 O
CO2H CO2H
L-AlaDH
H2O NH4+
NADH NAD+
FDH or
formate CO2
GDH
or or
glucose gluconolactone
R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Ref

[32]
Pr ATA-113 92 93
[33]
BM >99 >99
[33]
ArS 15 >99
[33]
CV >99 >99
[32]
Et ATA-113 >99 >99
[23]
VF 25 (15) >99 (93)
[23]
PF 15 (9) 99 (97)
[32]
(CH2)5Me ATA-113 >89 >99
[23]
VF 80 (19) 93 (92)
[23]
PF 78 (72) >99 (98)
[33]
BM 64 >99
[23]
(CH2)2Ph VF 97 (45) 84 (86)
[23]
PD 81 (23) 92 (92)
[23]
PF 98 (92) 84 (86)
[33]
CV 86 50
[17]
Ph ATA-113 95 >99
[23]
VF 65 (23) 99 (>99)
[23]
PD 33 (15) >99 (>99)
[23]
PF 67 (56) >99 (>99)
[32]
4-MeOC6H4CH2 ATA-113 >99 >99
[33]
BM 54 >99
[33]
AD 36 >99
[33]
CV 97 >99
[32]
CH2OPh ATA-113 50 86


[23]
VF >99 (>99) 97 (96)
[23]
PD 99 (>99) 98 (98)
[23]
PF >99 (>99) >99 (>99)
[32]
CH2CO2Et ATA-113 >99 94
[23]
VF >99 (>99) 98 (>99)
[23]
PD >99 (73) >99 (>99)
[23]
PF >99 (>99) 94 (88)
[33]
CH2OMe BM 94 >99
[33]
ArS 64 >99
[33]
CV 52 94
a
Origin of enzymes: VF = Vibrio fluvialis; PD = Paracoccus denitrificans;
PF = Pseudomonas fluorescens; BM = Bacillus megaterium; AD = Alcali-
genes denitrificans; CV = Chromobacterium violaceum; ArS = Arthro-
bacter citreus; ATA-113 = commercial enzyme available from Codexis.
b
c
Values in parentheses are values obtained for reactions in the pres-
ence of DMSO (15 vol%) as cosolvent.
d
Enantiomeric excess determined by GC on a chiral stationary phase
after derivatization to the corresponding acetamide.
(S)-1-(4-Methoxyphenyl)propan-2-amine (9, R1 = 4-MeOC6H4CH2); Typical Procedure:[33]

Lyophilized cells of E. coli containing overexpressed ø-transaminase from Chromobacteri-
um violaceum (400 mg) were rehydrated in phosphate buffer (100 mM, pH 7; 40 mL) con-
taining PLP (1.0 mM), ammonium formate (750 mg, 300 mM), NAD+ (1.0 mM), l-alanine
(1.0 g, 280 mM), l-AlaDH (200 L, 80 U), FDH (400 L, 88 U), and 1-(4-methoxyphenyl)pro-
pan-2-one (500 mg, 76 mM). The reaction was terminated after 24 h by addition of 1 M aq
HCl (5 mL) and the mixture was subsequently extracted with EtOAc (40 mL). The remain-
ing acidic aqueous phase was adjusted to pH 12 with 10 M aq NaOH. The resulting soln
was extracted with CH2Cl2 (2 15 mL). The combined organic phases were dried (Na2SO4)
and the solvent was removed under reduced pressure to afford the optically pure product
as a colorless oil; yield: 488 mg (97%); [Æ]D20 –34 (c 1.2, CHCl3); >99% ee.
2.4.3.1.1.1.2.2 (R)-Amination
In general, ø-transaminases giving Æ-chiral primary amines in the R configuration (e.g.,

10) require d-alanine as amine donor. Consequently, the l-alanine dehydrogenase em-
ployed in the previous chapter is not suitable for recycling pyruvate to d-alanine. Howev-
er, l-alanine dehydrogenase still removes pyruvate to give l-alanine, therefore avoiding
enzyme inhibition (Scheme 9).[22] Because no d-alanine dehydrogenase is yet known, the
system is less efficient compared with applications leading to (S)-amines.
Scheme 9 (R)-Amination in Combination with L-Alanine Dehydrogenase[17,22]
PLP
R1 R1
(R)-10
NH4+ H2O
NH2 O NH2
L-AlaDH
CO2H CO2H CO2H
NADH NAD+
FDH or
formate CO2
GDH
or or
glucose gluconolactone

[22]
Pr ArR 87 (89) >99 (>99)
[22]
AT 98 (89) >99 (>99)
[22]
HN 26 (25) >99 (>99)
[22]
Et ArR 18 (43) >99 (>99)
[22]
AT 46 (69) >99 (>99)
[22]
HN 14 (10) >99 (>99)
[22]
(CH2)5Me ArR 75 (57) >99 (>99)
[22]
AT 92 (86) >99 (>99)
[22]
HN 65 (76) >99 (>99)
[22]
(CH2)2Ph ArR 74 (90) >99 (>99)
[22]
AT 96 (84) >99 (>99)
[22]
HN 89(77) >99 (>99)
[17]
Ph ATA-117 95 >99
[22]
ArR 53 (44) >99 (>99)
[22]
AT 54 (42) >99 (>99)
[22]
MeOC6H4CH2 ArR >99 (>99) >99 (>99)
[22]
AT >99 (95) >99 (>99)
[22]
HN 45 (51) >99 (>99)
[22]
CH2OPh ArR 99 (>99) >99 (>99)
[22]
AT >99 (>99) >99 (>99)
[22]
HN >99 (>99) >99 (>99)
[22]
CH2CO2Et ArR >99 (>99) >99 (>99)
[22]
AT >99 (>99) >99 (>99)
[22]
HN >99 (>99) >99 (>99)


[22]
CH2OMe ArR 98 (>99) >99 (>99)
[22]
AT >99 (>99) >99 (>99)
[22]
HN >99 (>99) >99 (>99)
a
Origin of enzymes: ArR = Arthrobacter sp.; AT = Aspergillus terreus;
HN = Hyphomonas neptunium; ATA-117 = commercial enzyme origi-
nating from Arthrobacter sp.
b
c
Values in parentheses are values obtained for reactions in the pres-
ence of DMSO (15 vol%) as cosolvent.
d
Enantiomeric excess determined by GC on a chiral stationary phase
after derivatization to the corresponding acetamide.
(R)-4-Phenylbutan-2-amine [10, R1 = (CH2)2Ph)]; Typical Procedure:[22]

Lyophilized cells of E. coli containing overexpressed ø-transaminase from Aspergillus ter-
reus (20 mg) were rehydrated in phosphate buffer (100 mM, pH 7, containing 1.0 mM PLP
and 1.0 mM NAD+; 1 mL) for 30 min at 30 8C. Ammonium formate (150 mM), d-alanine
(250 mM), l-AlaDH (12 U), FDH (11 U), and 4-phenylbutan-2-one (50 mM) were added. The
mixture was shaken at 30 8C and 120 rpm. The reaction was terminated after 24 h by addi-
tion of 10 M aq NaOH (200 L) and the mixture was extracted with EtOAc (2 500 L). The
combined organic phases were dried (Na2SO4) and the solvent was removed under re-
duced pressure affording the optically pure product.
2.4.3.1.1.2 Amination Employing Isopropylamine as Amine Donor
Although, as described in the previous sections, the removal of pyruvate allows the unfa-
vorable equilibrium to be shifted toward product formation, the requirement of addition-
al enzymes besides the ø-transaminase increases the complexity of the amination in gen-
eral. Consequently, isopropylamine seems to be a suitable alternative to alanine because
it is achiral, volatile, and inexpensive. The equilibrium is shifted either by using a large
excess of isopropylamine[17,18] or by removing the oxidized coproduct (acetone) via a gas
sweep,[16] under reduced pressure,[34] or using a multienzyme network.[35] Although isopro-
pylamine has multiple advantages over alanine, this amine source is, however, accepted
only by a limited number of ø-transaminases. This limitation can be circumvented by em-
ploying rational protein design to develop novel catalysts with the ability to accept this
amine source.[16] The multigram-scale synthesis of sitagliptin [12, R1 = 2-oxo-2-{3-(trifluo-
romethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl}ethyl; R2 = 2,4,5-F3C6H2CH2], a
therapeutically useful drug for the treatment of diabetes mellitus type 2, is based on the
amination of prositagliptin ketone [11, R1 = 2-oxo-2-{3-(trifluoromethyl)-5,6-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl}ethyl; R2 = 2,4,5-F3C6H2CH2] using isopropylamine as
amine source (Scheme 10).[16] By using directed evolution techniques, a solvent and tem-
perature tolerant catalyst (ArRmut11) has been developed which is ideal for the amina-
tion of the bulky ketone precursor. The amination proceeds smoothly at high substrate
concentration (200 g/L), satisfying the catalyst requirement for drug production in terms
of substrate/catalyst ratio (» 2600 mol/mol) and turnover frequency (163 h–1),[36] giving the
enantiopure amine in excellent overall yield. Moreover, the evolved biocatalyst (ArR-
mut11) has also been successfully employed for the preparation of the fluorinated
amine 12 (R1 = Ph; R2 = CF3), which is not accessible by traditional chemical reductive am-
ination approaches. Perfect stereoselectivity is also observed for the preparation of
1-phenylethylamine (12, R1 = Ph; R2 = Me), however, a depletion of the enantiomeric ex-
cess is detected for substrates bearing long flexible chains such as phenoxy- and methoxy-
acetone derivatives 11 (R1 = CH2OPh; R2 = Me and R1 = CH2OMe; R2 = Me, respectively).[22] A
dynamic protocol has been established for the amination of a panel of racemic Æ-alkylated
-oxo esters {e.g., 11 [R1 = Me; R2 = (S)-CH(Et)CO2Et]} using a set of commercially available
ø-transaminases.[37] As a general trend, excellent conversions and enantiomeric excesses
are achieved for acyclic derivatives, though with moderate diastereoselectivity except in a
few cases.[5]
Scheme 10 Amination of Linear Ketones Using Isopropylamine[16,22,37]
ω-transaminase
O NH2 PLP NH2 O
+ +
R1 R2 R1 R2
11 12
R1 R2 ø-Transaminasea Conditions Conversion ee Ref
(%) (%)
N 2,4,5-F3C6H2CH2 ArRmut11 50% DMSO, 90–95 >99 [16]

N
N pH 8.5, 45 8C, 15 h
N
F3C
[16]
Ph CF3 ArRmut6 30% DMSO, 99 >99
pH 8.5, 60 8C, 24 h
[22]
Ph Me ArRmut11 20% DMSO, pH 11, 85 >99
45 8C, 24 h
[22]
CH2OPh Me ArRmut11 20% DMSO, pH 11, >99 80
45 8C, 24 h
[22]
CH2OMe Me ArRmut11 20% DMSO, pH 11, >99 >99
45 8C, 24 h
Et
Me TA-P1-A06 2.5% DMSO, 87 >99b [37]
CO2Et pH 7.5, 30 8C, 24 h

a
Origin of enzymes: ArRmut11 = Arthrobacter sp. round 11 mutant; ArRmut6 = Arthrobacter sp. round 6 mutant;
TA-P1-A06 = Codex transaminase screening kit.
b
Diastereomeric ratio (anti/syn) 84:16.
(R)-3-Amino-1-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-
(2,4,5-trifluorophenyl)butan-1-one [12, R1 = 2-Oxo-2-{3-(trifluoromethyl)-5,6-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl}ethyl; R2 = 2,4,5-F3C6H2CH2; Sitagliptin];
Typical Procedure:[16]
The reaction was run in a vessel fitted with a mechanical stirrer, temperature probe, pH
probe, and base addition line. The pH was kept between 8.4 and 8.6 by addition of 4 M aq
iPrNH2. H2O was added to the vessel (1.92 L), followed by Et3N (109 mL, 0.82 mol,
0.33 equiv), and 4 M aq iPrNH2 (1.64 L, 6.56 mol). The pH was adjusted to 8.5 using 12 M
aq HCl (424 mL). The reactor was then charged with PLP (6.7 g, 0.027 mol) followed by
ø-transaminase ArRmut11 (40 g). The vessel was placed on the reactor block with the tem-
perature probe, base addition line, and pH probe, and the mechanical stirrer was set to
400 rpm. DMSO (2.22 L) was added and the reactor was heated to 45 8C. After the temper-
ature had stabilized, the pH control loop was turned on and adjusted to pH 8.5. At this
point, stirring was increased to 600 rpm, but tip speed was kept below 2 m/s to avoid vor-
texing. Then, the ketone substrate 11 [R1 = 2-oxo-2-{3-(trifluoromethyl)-5,6-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl}ethyl; R2 = 2,4,5-F3C6H2CH2] as hemihydrate (1.0 kg,
2.46 mol) was dissolved in DMSO (1.11 L) and this soln was added to the reactor over
2–3 h. The reactor was stirred at 45 8C for another 13 h with acetone removal being accom-

plished with 300 Torr vacuum and 2 fps N2 sweep. After 15 h (total reaction time), the re-
action was at 90–95% conversion as judged by HPLC analysis. Then, the pH loop control
was turned off and 12 M aq HCl was added until pH 2–3. The mixture was then aged at
45 8C and 1000 rpm for 2 h. The batch was cooled to rt and iPrOH (3 L) was added, followed
by iPrOAc (3 L). The pH of the aqueous phase was then adjusted to 11 with 19 M aq NaOH.
The mixture was shaken at 20–45 8C (heat may be used to break the emulsion) and then
allowed to settle and separate. The organic phase was set aside and the aqueous layer was
extracted with an iPrOH/iPrOAc mixture (1:4; 3 L). The combined organic extracts were
washed with brine (pH 11; 3 L) and the soln was assayed for yield: 872–902 g (87–90%).
2.4.3.1.1.2.1 Amination Followed by Spontaneous Cyclization
The amination of ketones followed by spontaneous ring-closure reaction of the amine
moiety with an ester, or by another nucleophilic substitution, provides straightforward
access to complex heterocyclic compounds without requiring the isolation or purification
of the unstable intermediates. Moreover, isopropylamine (as the amine donor) can be pro-
vided at low concentration because the irreversible cyclization step drives the reaction to
completion. Using this approach, the amination of ethyl 5-oxohexanoate (Table 1, entries
1 and 2) has been reported at a concentration of 50 g/L.[26] The spontaneous cyclization of
the so-obtained amino ester affords 6-methylpiperidin-2-one, a valuable building block
for the preparation of natural products. Furthermore, the amination of a chlorinated ke-
tone (entry 3) using an engineered enzyme as catalyst enables the efficient synthesis of a
pharmaceutically relevant pyrrolidine.[16] Finally, the multigram chemoenzymatic syn-
thesis of a precursor of Suvorexant, a potent orexin receptor antagonist bearing a diaze-
pane moiety for the treatment of primary insomnia, is achieved by this method (entry 4).
The amination of the ketone precursor directly affords the diazepane ring in good yield
through a spontaneous ring-closure reaction.[38]
Table 1 Amination with Isopropylamine as Amine Donor Followed by Spontaneous Ring-

Closure Cyclization[16,26,38]
ω-transaminase
O PLP NH2 spontaneous R1
∗
R1 R2 R1 R2 N
H
NH2 O
Entry Starting Material ø-Transaminasea Product Yield (%) ee (%) Ref
O
[26]
1 ATA-117 >90 >99
CO2Et O N
H
O
[26]
2 ATA-114 >90 >99
CO2Et O N
H
O HN
Cl [16]
3 ArRmut6 57 >99
F F
MsO HN
O
N N
[38]
4 ArRmut11 62 >99
N O N O
Cl Cl
a
Origin of enzymes: ArRmut11 = Arthrobacter sp. round 11 mutant; ArRmut6 = Arthrobacter sp.
round 6 mutant; ATA-114 = Codex transaminase screening kit; ATA-117 = Codex transaminase
screening kit.
5-Chloro-2-[(5R)-5-methyl-1,4-diazepan-1-yl]benzoxazole Hydrochloride (Table 1, Entry 4);

A 1-L, three-necked flask was charged with iPrNH2 • HCl (25.8 g, 270 mmol) and 0.1 M aq
triethanolamine (525 mL). PLP • H2O (750 mg, 2.82 mmol) and ø-transaminase ArRmut11
(3.0 g) were added to this soln and the suspension was stirred until all components were
dissolved. The soln was heated to 40 8C and the pH was adjusted to 9.5 with 4 M aq iPrNH2.
A soln of the methanesulfonate starting material (15.0 g, 41.6 mmol) in DMSO (225 mL)
was added via syringe over 6 h, and the resulting mixture was stirred for an additional
5 h. The soln was poured into a separatory funnel (3 L) and extracted with iPrOAc/iPrOH
(1:1; 1.5 L). The aqueous layer was extracted again with iPrOAc/iPrOH (4:1; 750 mL). The
organic phases were combined, washed with brine (750 mL), and concentrated with
iPrOH flushing to establish a 45-mL soln in iPrOH, which was then treated with a 4.6 M
soln of HCl in iPrOH (9.94 mL, 45.7 mmol) via dropwise addition. The resulting soln was
stirred vigorously while iPrOAc (52 mL) was added slowly over 5 h, creating a slurry of the
HCl salt of the amine. The slurry was then slowly cooled to 0 8C and stirred overnight. At

this time the slurry was filtered and dried with a stream of N2 over the filter pad, provid-
ing the product as a colorless crystalline solid; yield: 7.80 g (62%); >99% ee; [Æ]D25 –4.67 (c 1,
MeOH).
2.4.3.1.1.2.2 Amination of In Situ Formed Ketones
The excellent compatibility between different biocatalysts opens the possibility of com-
bining enzymatic reactions to develop efficient one-pot cascade reactions. This concept
becomes particularly interesting when the reaction intermediates are unstable or diffi-
cult to obtain through alternative synthetic procedures.[1] For instance, the coupling of
two biocatalytic steps has been successfully used for the preparative-scale (15 mmol) pro-
duction of (R)- and (S)-homoalanine (15), which are valuable building blocks for the prep-
aration of antibiotics. [39] The coupled system avoids the handling of the expensive and un-
stable oxo acid 14 which is produced from readily available l-threonine (13) using a threo-
nine deaminase as catalyst. Moreover, the reductive amination of the oxo acid affords the
l- or d-isomers, depending on the ø-transaminase used [ArRmut11 (Arthrobacter sp. round
11 mutant) or OATA (ø-transaminase from Ochrobactum anthropi)], in good isolated yield
(78%) and enantiopure form (Scheme 11).
Scheme 11 Cascade Coupling of a Threonine Deaminase with Enantiocomplementary

ø-Transaminases To Yield Homoalanine[39]
NH2
threonine deaminase
CO2H
− NH3
OH
13
OATA
PLP, iPrNH2 NH2
buffer (pH 7), 37 oC
78%; >99% ee CO2H

O (S)-15
CO2H ArRmut11
PLP, iPrNH2 NH2
buffer (pH 7), 37 oC
14 CO2H
78%; >99% ee
(R)-15
(R)- or (S)-2-Aminobutanoic Acid (15); General Procedure:[39]

l-Threonine (13; 1.79 g, 15.0 mmol, 300 mM) was dissolved in phosphate buffer (50 mM,
pH 7; 50 mL) at 37 8C containing iPrNH2 (1.84 mL, 22.5 mmol, 450 mM), PLP (6.2 mg,
0.025 mmol, 0.5 mM), threonine deaminase (250 U, 5 U/mL), and ø-transaminase (OATA
or ArRmut11, 500 U, 10 U/mL). The mixture was stirred magnetically until the conversion
exceeded 99%. After that time, the pH of the mixture was adjusted to 1.0 by adding 5 M aq
HCl for protein precipitation. The mixture was filtered through a glass-fritted filter funnel
to remove the protein precipitate. The filtrate was loaded onto a glass column packed
with Dowex 50WX8 cation-exchange resin (40 g), followed by washing with 0.1 M aq HCl
(120 mL), and H2O (120 mL), and subsequent elution with 10% aq NH3 (145 mL) to afford
the product as a colorless solid; yield: 78%; >99% ee.
2.4.3.1.1.3 Amination Employing 1-Phenylethylamine as Amine Donor
Even though isopropylamine is an excellent amine donor, it is not accepted by every

ø-transaminase and a huge excess is generally required to shift the equilibrium toward
product formation. Of all the alternative amine donors tested to date, 1-phenylethylamine
has shown excellent properties from a thermodynamic point of view compared with the
analogous process using isopropylamine.[13,14] Exploiting this energetic advantage, the re-
ductive amination of a panel of aliphatic ketones has been performed successfully requir-
ing only small excesses of the amine donor. By suitable selection of the ø-transaminase
(OATA for the S- or ArRmut11 for the R-enantiomer), the asymmetric synthesis of the (R)-
or (S)-amines 16 can be successfully achieved (Scheme 12). Furthermore, (R)-3-fluoroala-
nine (16, R1 = CH2F; R2 = CO2H) can be readily synthesized through amination of 3-fluoro-
pyruvate using Vibrio fluvialis ø-transaminase as catalyst.[40] The reaction must be per-
formed in a two-phase system to avoid inhibition of the enzyme by acetophenone. This
approach is also suitable for the multigram preparation of valuable drug precursors
such as (S)-1-(5-fluoropyrimidin-2-yl)ethanamine (16, R1 = 5-fluoropyrimidin-2-yl; R2 =
Me), a building block for the preparation of the kinase inhibitor AZD1480.[41] Inhibition
effects observed at high substrate concentration (0.35 M) are avoided by adding toluene
as a second phase.
Scheme 12 Amination of Linear Ketones Using 1-Phenylethylamine as Amine Donor[13,40,41]
ω-transaminase
O NH2 PLP NH2 O
+ +
R1 R2 Ph ∗ R1 ∗ R2 Ph
16
R1 R2 Config of ø-Transaminasea Conditions Conversion ee Config Ref

1-Phenylethylamine (%) (%) of 16
[13]
Et Me rac OATA 15% DMSO, 91 >99 S
pH 8.6, 37 8C
[13]
Et Me rac ArRmut11 15% DMSO, 93 >99 R
pH 8.6, 37 8C
[13]
iPr Me rac OATA 15% DMSO, 92 >99 S
pH 8.6, 37 8C
[13]
iPr Me rac ArRmut11 15% DMSO, 82 >99 R
pH 8.6, 37 8C
[13]
CH2OMe Me rac OATA 15% DMSO, 94 >99 S
pH 8.6, 37 8C
[13]
CH2OMe Me rac ArRmut11 15% DMSO, 96 >99 R
pH 8.6, 37 8C
[40]
CH2F CO2H S VF 50% isooc- 96 >99 R
tane, pH 7,
37 8C
N [41]
Me S VF 25% toluene, 99 >99 S
N
pH 7.5, 29 8C
F
a
Origin of enzymes: ArRmut11 = Arthrobacter sp. round 11 mutant; OATA = Ochrobactum anthropi; VF = Vibrio
fluvialis.

(S)-1-(5-Fluoropyrimidin-2-yl)ethylamine Hydrochloride [16 • HCl, R1 = 5-Fluoropyrimidin-

2-yl; R2 = Me]; Typical Procedure:[41]
(S)-1-Phenylethylamine (68.0 mL, 0.53 mol) was added to a soln of KH2PO4 (13.21 g,
0.097 mol) in H2O (1020 mL). The pH was adjusted to 7.5 by addition of AcOH (26.0 mL,
0.45 mol). PLP • H2O (0.643 g, 2.4 mmol) was added, followed by 2-acetyl-5-fluoropyrimi-
dine (68.0 g, 0.49 mol), a buffered soln of ø-transaminase VF (182 mL, 35.3 kU), and tolu-
ene (340 mL). The mixture was adjusted to pH 7.5 with K2CO3 (6.71 g, 0.053 mol) and then
kept at 29 8C for 18 h with stirring. Harborlite (12.0 g) was added, and the mixture was
stirred for a further 15 min before being filtered. The filter bed was washed with a mix-
ture of H2O (136 mL) and toluene (136 mL). The organic layer was discarded. K2CO3
(134.14 g, 0.98 mol) was added to the aqueous phase followed by a soln of Boc2O (116.5g,
0.53 mol) in 2-methyltetrahydrofuran (544 mL). The biphasic mixture was stirred at 40 8C
for 2 h. Harborlite (12.0 g) was added, and stirring was continued for an additional 15 min.
The mixture was filtered and the filter bed was washed with a mixture of H2O (136 mL)
and 2-methyltetrahydrofuran (136 mL). The aqueous layer was extracted with 2-methyltet-
rahydrofuran (272 mL). The organic layers were combined and distilled at atmospheric
pressure until the internal temperature reached 98 8C (760 mL distillate collected). The re-
sidual soln was treated with a 5.55 M soln of HCl in iPrOH (238 mL, 1.32 mol). The mixture
was heated to 40 8C for 18 h, whereby the amine hydrochloride precipitated. Heptane
(204 mL) was added over 15 min and the resulting suspension was stirred at 20 8C for
16 h. The mixture was filtered and the isolated amine hydrochloride was washed with
2-methyltetrahydrofuran (102 mL) and dried under reduced pressure; yield: 68.2 g (77.1%).
2.4.3.1.1.4 Amination in Organic Solvents
Transformations employing ø-transaminases are generally performed in aqueous media;

however, the mostly lipophilic substrates are only moderately soluble in water. Addition-
ally, because the commonly employed pH for enzymatic transformations is <10, the prod-
uct amine is generally protonated, necessitating a more laborious work up which requires
at least basification of the reaction mixture and extraction with organic solvents. Conse-
quently, protocols employing ø-transaminases exclusively in organic solvents are de-
sired. Using a freeze-dried, cell-free ø-transaminase preparation, various ketones are suc-
cessfully aminated employing isopropylamine as amine donor in tert-butyl methyl ether
at a water activity (aw) of 0.6 (Scheme 13).[42] Interestingly, every ø-transaminase tested ac-
cepts isopropylamine in tert-butyl methyl ether, although some of these ø-transaminases
do not accept this donor when applied in buffer. Notably, thermodynamically favored
amines 17 are prepared by using just 3 equivalents of isopropylamine, leading to conver-
sions of up to >99%. In the case of thermodynamically less favored amines, isopropyl-
amine is required in excess to increase the conversion. It must be stated, that the prepa-
ration of the freeze-dried, cell-free ø-transaminase preparation needs special attention;
thus, a homogeneous solution of the catalyst and the time of freeze-drying significantly
influences the activity of the preparation.
Scheme 13 Enzymatic Amination in tert-Butyl Methyl Ether[42]
ω-transaminase
O PLP, t-BuOMe
NH2
R1 R2 R1 ∗ R2
17 >99% ee
NH2 O
R1 R2 iPrNH2 Time ø-Transaminasea Conversion ee Config Ref

(Equiv) (h) (%) (%)
[42]
CH2OMe Me 3 2 ArR >99 >99 R
[42]
CH2OMe Me 3 4 ArS >99 >99 S
[42]
CH2CO2Et Me 3 24 ArR >99 >99 R
[42]
CH2CO2Et Me 3 12 ArS 88 >99 S
[42]
Ph CH2OH 3 24 ArR 98 >99 S
[42]
Ph CH2OH 3 72 CV 7 >99 R
[42]
CH2OPh Me 3 9 ArR >99 >99 R
[42]
CH2OPh Me 3 24 CV >99 >99 S
[42]
Ph Me 3 72 ArR 43 >99 R
[42]
Ph Me 3 72 ArS 39 >99 S
[42]
(CH2)2Ph Me 3 72 ArR 48 >99 R
[42]
(CH2)2Ph Me 15 24 ArS 75 >99 S
[42]
(CH2)5Me Me 3 72 ArR 41 >99 R
[42]
(CH2)5Me Me 3 72 ArS 44 >99 S
a
Origin of enzymes: ArR = R-selective ø-transaminase from Arthrobacter sp. (1.60 U/mg of
crude preparation); CV = S-selective ø-transaminase from Chromobacterium violaceum (2.04
U/mg of crude preparation); ArS = HisTag Arthrobacter citreus (0.74 U/mg of crude prepara-
tion).
(S)-1-Methoxypropan-2-amine Hydrochloride (17 • HCl, R1 = CH2OMe; R2 = Me);

The cell-free, freeze-dried S-selective ø-transaminase ArS (452 mg) was incubated in a
50-mL glass flask with water-saturated t-BuOMe (22.6 mL) at 25 8C and 900 rpm for
30 min. An additional amount of H2O was added (0.361 mL to reach finally aW 0.6) and
the sample was shaken for another 30 min to equilibrate the system, before 1-methoxy-
propan-2-one (100 mg) and iPrNH2 (0.289 mL, 3 equiv) were added. The mixture was shak-
en at 900 rpm at 25 8C. After 4 h, quantitative conversion (>99%) was detected by GC. The
enzyme preparation was removed by filtration and the organic phase was dried (Na2SO4).
The ethereal soln was distilled to remove the remaining iPrNH2 and the acetone coprod-
uct. The total volume was reduced to 5–10 mL by distillation. The organic mixture was
cooled to 4 8C and a 2 M ethereal soln of HCl (2 mL) was added. The soln became cloudy
due to formation of the hydrochloride salt. The remaining solvent was evaporated yield-
ing a pale yellow oil, which was washed with pentane (2 ); yield: 84%. The product
formed a pale yellow solid upon standing overnight at 4 8C.

2.4.3.1.2 Amination of Diketones
2.4.3.1.2.1 Monoamination of 1,5-Diketones
Regioselective transformations are highly desired in organic synthesis because they allow
elaborate protecting group strategies and additional purification steps to be avoided. For
example, various substituted 1,5-diketones 18 with R1 being either a linear or branched
alkyl, aryl, or cyclic residue are regioselectively converted into the enantiopure 2,3,4,5-
tetrahydropyridines (˜1-piperideines) 19 by employing enantiocomplementary ø-trans-
aminases (Scheme 14). The enzymatic reductive amination proceeds almost exclusively
at the sterically less demanding (ø-1) carbonyl residue to afford an amino ketone, which
cyclizes spontaneously to the corresponding six membered imine 19. Notably, even bare-
ly soluble substrates can be transformed by the addition of organic solvents such as di-
methylformamide or dimethyl sulfoxide.[43] Using alanine as amine donor, the equilibri-
um is shifted to the product side either by recycling the formed pyruvate to alanine via
AlaDH or reduction to lactate via LDH (see Sections 2.4.3.1.1.1.2.1 and 2.4.3.1.1.1.1). The
cyclic imines are obtained with excellent conversion and optical purity, and high regiose-
lectivity.[43–45] As the products are rather volatile and prone to racemization, it is recom-
mended that further transformations be performed immediately.
Scheme 14 Regioselective Asymmetric Reductive Monoamination of 1,5-Diketones Cata-

lyzed by ø-Transaminases[43–45]
O O
R1
18
OH
ω-transaminase
NH2
buffer, PLP
NAD+, FDH
O 30 oC, 24 h
OH
O
NH2 O O NH2
∗ R1 ∗ R1
− H2O − H2O
∗ ∗
N R1 N R1
19
R1 ø-Transaminasea Conversion Regioselectivity ee (%) of Config of Ref

(%) 19 19
[44]
Pr CV >99 >99:1 >99 S
[44]
Pr AT >99 >99:1 >99 R
b [43]
(CH2)8Me ArS >99 >99:1 >99 S
c [43]
(CH2)8Me ArR >99 >99:1 >99 R
[44]
cyclopropyl VF >99 >99:1 >99 S
[44]
cyclopropyl HN >99 >99:1 >99 R
[44]
iPr ArS 97 >99:1 >99 S
[44]
iPr AT 93 >99:1 >99 R
[44]
iBu BM >99 >99:1 >99 S
[44]
iBu HN >99 >99:1 >99 R
[44]
Ph PF >99 >99:1 98 S
[44]
Ph ArR >99 >99:1 >99 R
a
Origin of enzymes: CV = Chromobacterium violaceum; AT = Aspergillus terreus; VF = Vibrio fluvia-
lis; HN = Hyphomonas neptunium; ArS = Arthrobacter citreus; BM = Bacillus megaterium; PF =
Pseudomonas fluorescens; ArR = Arthrobacter sp.
b
Reaction performed in the presence of 20 vol% DMF.
c
Reaction performed in the presence of 20 vol% DMF and 5 vol% heptane.
Functionalized chiral piperidines can be found in a vast number of bioactive compounds

and natural products.[46] Moreover, the scaffold is present in numerous synthetic proto-
cols and ranks among the most common skeletal fragments found in nature. Numerous
(asymmetric) synthetic strategies to access this scaffold have been published based on chi-
ral pool precursors, catalytic transformations, and auxiliary controlled methods.[47–49] The
2,3,4,5-tetrahydropyridines (˜1-piperideines) derived from the ø-transaminase-catalyzed
monoamination are useful precursors for the synthesis of a series of diastereo- and enan-
tiomerically pure 2,6-disubstituted piperidines; palladium-catalyzed hydrogenation pro-
vides the optically pure cis-diastereomers, whereas the anti-isomers are accessible
through a Lewis acid mediated change in conformation during the reduction step.[50,51]
Consequently, the natural alkaloids dihydropinidine (21A)[44,45] and epi-dihydropinidine
(21B),[45] derived from 2,3,4,5-tetrahydropyridine (S)-20, and other related natural prod-
ucts[43] are constructed within only three steps starting from commercial sources with
full stereocontrol and with high selectivity (Scheme 15).
Scheme 15 Synthesis of Dihydropinidine and epi-Dihydropinidine[44,45]
1. Pd/C, H2, rt, 4 h

2. HCl/Et2O
Cl−
N Pr
H H
21A•HCl 94%; dr (syn/anti) 99:1
N Pr
1. Et3Al, LiAlH4, −78 oC, 3 h

(S)-20
2. HCl/Et2O
Cl−
N Pr
H H
21B•HCl 91%; dr (syn/anti) 18:82

(S)-2-Methyl-6-propyl-2,3,4,5-tetrahydropyridine (19, R1 = Pr); Typical Procedure:[44]

Lyophilized cells of E. coli containing overexpressed ø-transaminase from C. violaceum
(225 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM; 10 mL) con-
taining PLP (1.0 mM), NAD+ (1.0 mM), ammonium formate (150 mM), FDH (11 U), AlaDH
(12 U), and d/l-alanine (500 mM) at 22 8C for 30 min. Nonane-2,6-dione (78 mg, 0.5 mmol)
was added and the mixture was shaken at 30 8C for 26 h. Sat. aq Na2CO3 (1.00 mL) was
added and the mixture was extracted with Et2O (4 5 mL). The soln containing the enan-
tiopure product was ready for further transformations, but could be characterized by
careful concentration on a rotary evaporator (650 mbar, 35 8C).
(2S,6R)-2-Methyl-6-propylpiperidine Hydrochloride [21A • HCl; (–)-Dihydropinidine
Hydrochloride]; Typical Procedure:[44]
The crude soln from the biotransformation containing the corresponding cyclic imine
(S)-2-methyl-6-propyl-2,3,4,5-tetrahydropyridine [(S)-20] was treated with Pd/C (10% wt).
The mixture was stirred vigorously and a stream of H2 was bubbled through the soln for
4 h. After full conversion, which was detected via GC or GC/MS, the soln was filtered
through a small plug of Celite and cooled to 0 8C. A soln of ethereal HCl (~5 equiv) was
added. The solvent was removed and the precipitate was collected to give the product as
a colorless solid; yield: 82 mg (94%); dr (syn/anti) 99:1; mp 243–245 8C; [Æ]D20 –12.2 (c 0.5,
EtOH).
(2S,6S)-2-Methyl-6-propylpiperidine Hydrochloride [21B • HCl; (–)-epi-Dihydropinidine];

CAUTION: Solid lithium aluminum hydride reacts vigorously with a variety of substances, and
can ignite on rubbing or vigorous grinding.
CAUTION: Short-chain trialkylaluminums are pyrophoric. Appropriate safety precautions and

procedures should be adopted when handling and disposing of these reagents.
The crude soln from the biotransformation containing the corresponding cyclic imine
(S)-2-methyl-6-propyl-2,3,4,5-tetrahydropyridine [(S)-20] was concentrated with a rotary
evaporator (35 8C, 650 mbar). The residue was diluted with anhyd THF (10 mL) and cooled
to –78 8C and a 1 M soln of Et3Al in hexane (5.0 mL) was added dropwise. The mixture was
stirred for an additional 10 min and 2 M LiAlH4 in THF (1.25 mL, 2.5 mmol) was added.
After the mixture had been stirred for 3 h at –78 8C, a saturated soln of potassium sodium
tartrate (Rochelles salt; 30 mL) was added and the mixture was allowed to warm to rt. The
aqueous phase was extracted with t-BuOMe (4 10 mL) and the combined organic layers
were dried (MgSO4). Precipitation with ethereal HCl afforded the product; yield: 81 mg
(91%); dr (syn/anti) 18:82. Recrystallization (CHCl3) gave the diastereomerically pure prod-
uct as a colorless solid; mp 165–167 8C; dr (syn/anti) 1:99; [Æ]D30 –3.9 (c 0.89, EtOH).
2.4.3.1.3 Amination of Cyclic Monoketones
Cyclic structures are often a key feature of bioactive compounds and natural products, in
particular cyclic chiral amines are interesting targets for the development of new phar-
maceuticals.
2.4.3.1.3.1 Amination of Unsubstituted and 3-Substituted Monocyclic Ketones
Apart from the case of cyclohexanone (22, R1 = R2 = R3 = R4 = R5 = H), which leads to achiral
cyclohexylamine (23, R1 = R2 = R3 = R4 = R5 = H),[32,52] chiral 3-substituted cyclohexylamine
derivatives 23 are prepared from ketones 22 via an enzymatic cascade utilizing ø-trans-
aminases in the key step (Scheme 16).[53] By choosing an R- or S-selective ø-transaminase,
either enantiomer can be obtained. The amination is performed using either alanine
(LDH/GDH system, see Section 2.4.3.1.1.1.1) or isopropylamine (see Section 2.4.3.1.1.2) as
amine donor.
Scheme 16 Enzymatic Asymmetric Reductive Amination of Monocyclic Ketones[32,52,53]
O NH2
ω-transaminase
PLP
R1 R1
R5 R5
R2 R2
R3 R4 R3 R4
NH2 O
22 23
R6 ∗ R6
R1 R2 R3 R4 R5 R6 ø-Transaminasea Conversion (%) de (%) Ref

b [52]
H H H H H CO2H CV – –
[32]
H H H H H CO2H ATA-113 >99 –
[53]
H H H H CH2CO2Me CO2H VF >99 >99 (1S,3S)
[53]
H H H H CH2CO2Me Me ArRmut11 96 >99 (1S,3R)
[53]
Me Me H H CH2CO2Me CO2H VF 25 >99 (1S,5R)
[53]
Me Me H H CH2CO2Me Me ArRmut11 69 >99 (1S,5R)
[53]
H H Me Me CH2CO2Me CO2H VF >99 99 (1S,5S)
[53]
H H Me Me CH2CO2Me Me ArRmut11 >99 >99 (1S,5R)
a
Origin of enzymes: CV = Chromobacterium violaceum; VF = Vibrio fluvialis; ArRmut11 = R-selec-
tive variant from Arthrobacter sp.; ATA-113 = commercial enzyme available from Codexis.
b
Only initial rates were published.
Methyl [(1S,5R)-5-Amino-3,3-dimethylcyclohexyl]acetate Hydrochloride (23 • HCl, R1 = R2 =

Me; R3 = R4 = H; R5 = CH2CO2Me); Typical Procedure:[53]
Lyophilized E. coli BL21 (DE3) cells containing the recombinant R-selective ø-transaminase
variant from Arthrobacter sp. (ArRmut11; 20 mg/mL) were rehydrated in phosphate buffer
(100 mM, pH 8.0, 1.0 mM PLP) containing iPrNH2 (1 M). After 15 min of rehydration at 30 8C
and 120 rpm, methyl [(S)-3,3-dimethyl-5-oxocyclohexyl]acetate (22, R1 = R2 = Me; R3 = R4 =
H; R5 = CH2CO2Me; 34 mg, 0.2 mmol) was added. The mixture (total volume 1 mL) was
shaken at 500 rpm and 45 8C for 16 h in an Eppendorf orbital shaker. The reaction was
stopped by adding sat. aq NaHCO3 (100 L), and the mixture was extracted with EtOAc
(3 500 L). The organic layers of four analytical scale reactions were combined and
dried (Na2SO4). EtOAc was removed under reduced pressure, and the crude product was
dissolved in Et2O and a 1 M soln of HCl in Et2O was added to precipitate the product as
the hydrochloride. The solvent was removed under reduced pressure and, after washing
with Et2O, the product was obtained as colorless crystals; yield: 39 mg (94%).
2.4.3.1.3.2 Amination of 2-Substituted Monocyclic Ketones via Dynamic

Kinetic Resolution
Dynamic kinetic resolution involves a kinetic resolution coupled to a racemization and

has been successfully established for the reductive amination of ethyl 2-oxocyclopentane-
carboxylate. Various R- and S-selective ø-transaminases have been tested in the dynamic
kinetic resolution, but most enzymes give the amine product 24 either in moderate yield
or do not show good selectivity. However, ø-transaminases ATA-113 and TA-P1-G05 afford

the anti amino ester (1S,2S)-24 with high conversions, enantiomeric excess (>99%), and
diastereomeric excess (94–96%). Scheme 17 outlines the results of a preparative-scale con-
version using TA-P1-G05 as catalyst.[37]
Scheme 17 Chemoenzymatic Synthesis of Ethyl 2-Aminocy-

clopentanecarboxylate via ø-Transaminase-Catalyzed Dynam-
ic Kinetic Resolution[37]
O NH2
ω-transaminase
CO2Et PLP CO2Et
rac (1S,2S)-24 58%;
>99% ee; 98% de
NH2 O
Ethyl (1S,2S)-2-Aminocyclopentanecarboxylate [(1S,2S)-24]; Typical Procedure:[37]

In a 15-mL Falcon tube, ø-transaminase P1-G05 (12.5 U) and ethyl 2-oxocyclopentanecar-
boxylate (50 mg, 0.32 mmol, 25 mM) were dissolved in phosphate buffer (100 mM, pH 7.5;
5 mL) containing PLP (1.0 mM) and iPrNH2 (1.0 M), and EtOH (125 L, 2.5 vol%). The reac-
tion was shaken at 30 8C and 250 rpm for 24 h and the mixture was subsequently acidified
with 1 M aq HCl (5 mL) and extracted with EtOAc (2 10 mL). The aqueous phase was basi-
fied with sat. aq K2CO3 until pH 10–11 and then extracted with EtOAc (3 10 mL). The or-
ganic layers were dried (Na2SO4) and the solvent was evaporated under reduced pressure
to give the product with excellent purity; yield: 58%; >99% ee; 98% de.
2.4.3.1.3.3 Amination of Bicyclic Ketones
In addition to monocyclic ketones, early studies reported that various ø-transaminases

show activity toward 1-amino-1,2,3,4-tetrahydronaphthalene (1-aminotetralin) and 1-ami-
noindane in deamination assays.[54] Subsequently, an evolved thermostable ø-transami-
nase isolated from Arthrobacter citreus was employed to afford a substituted (S)-aminotet-
ralin stereoselectively.[55] Moreover, various tetrahydronaphthalenones and dihydroben-
zopyranone derivatives are transformed by several ø-transaminases to give the corre-
sponding enantiopure amines (Table 2).[24] The amination of the ketones is performed
using either alanine or isopropylamine as amine donor. The coproduct pyruvate is re-
moved from the system by either a lactate dehydrogenase (LDH) or is recycled by alanine
dehydrogenase (AlaDH). For NADH cofactor recycling, a glucose dehydrogenase (GDH) or a
formate dehydrogenase (FDH) is employed. Furthermore, a new ø-transaminase from
Burkholderia vietnamiensis has been identified as being able to transform (S)-1-amino-
indane and (S)-1-amino-1,2,3,4-tetrahydronaphthalene into the corresponding ketones.[56]
Table 2 Enzymatic Amination of Tetrahydronaphthalenones and Dihydrobenzopyranones[19,22,24,54–57]
PLP, pH 7.0, 30 oC
R1 R1
NH2 O
R2 R2
R2 = CO2H, Me
ø-Transaminasea
Entry Starting Material Product Conversion ee Ref
(%) (%)
O NH2
[19,24,55,57]
1 ArS >99 >99
O NH2
[19,24,55,57]
2 ArR 14 76
O NH2
[24]
3 ArS 90 >99
MeO MeO
O NH2
[24]
4 ArRmut11 >99 >99
MeO MeO
MeO O MeO NH2

[24]
5 VF >99 >99
MeO O MeO NH2

[24]
6 ArRmut11 >99 >99
O NH2
[22,24,56]
7 VF 22 98
O NH2
[22,24,56]
8 ArRmut11 26 >99
O NH2
[22,24,56]
9 BV 51 >99
O NH2
[24]
10 VF 56 >99
O O

Table 2 (cont.)
Entry Starting Material ø-Transaminasea Product Conversion ee Ref
(%) (%)
O NH2
[24]
11 ArRmut11 54 >99
O O
O NH2
[24]
12 ArS >99 >99
O O
O NH2
[24]
13 AN 22 >99
O O
a
Origin of enzymes: ArS and ArR = S- and R-selective enzymes from Arthrobacter; ArRmut11 = variant of (R)-Ar-
throbacter sp.; VF = Vibrio fluvialis; AN = Aspergillus niger; BV = Burkholderia vietnamiensis.
(R)-2,3-Dihydro-4H-1-benzopyran-3-amine Hydrochloride (Table 2, Entry 12);

Lyophilized cells of E. coli containing overexpressed ø-transaminase ArS (1.0 g) were dis-
rupted in lysis buffer [16 mL of lysis buffer per gram of lyophilized cells, phosphate buffer
pH 7.0, 100 mM, 1.0 mM EDTA, 1.0 mM PLP, lysozyme from chicken egg white (1.06 mg/
mL >40 000 U/mg), and 1.0 mM phenylmethanesulfonyl fluoride]. The preparation was
shaken on an orbital shaker at 150 rpm and 22 8C for 3 h. The suspension was centrifuged
(18 000 rpm, 15 min, 4 8C) and the supernatant was lyophilized yielding a powder of the
crude extract. In a round-bottomed flask, an aliquot of the lyophilized extract of ArS
(270 mg, 1.26 U/mg crude preparation) was dissolved in phosphate buffer (pH 7, 100 mM,
PLP 1 mM; 13.5 mL). l-Alanine (301 mg, 3.375 mmol), LDH (90 U/mL), GDH (30 U/mL), and
NADH (1 mM) were added. Finally, 2,3-dihydro-4H-1-benzopyran-3-one (100 mg,
0.675 mmol) was dissolved in DMSO (135 L) and added to the aqueous soln. The flask
was shaken on an orbital shaker at 150 rpm at 0 8C for 24 h. The mixture was basified
with 10 M aq NaOH (2.7 mL) and extracted with EtOAc (3 5 mL). The combined organic
phases were dried (Na2SO4) and reduced to a small volume (2 mL). After the mixture had
been cooled to 4 8C, an ethereal soln of 2 M HCl (1.0 mL) was added to form the correspond-
ing hydrochloride. The organic solvent was then evaporated and the residual oil was dried
under reduced pressure. The crude product was recrystallized (anhyd Et2O, 2 mL), yielding
the optically pure product as a yellow solid; yield: 98.2 mg (78%); [Æ]D20 +46.4 (c 1.00,
MeOH); >99% ee.
2.4.3.1.3.4 Amination of Multicyclic (Nonsteroidal) Ketones
In addition to mono- and bicyclic ketones, ø-transaminases have also been applied for the
amination of multicyclic ketones. For example, a very efficient route for the synthesis of
(R)-ramatroban, a pharmaceutical used in the treatment of allergic rhinitis and asthma,
has been established employing an ø-transaminase for the asymmetric key step.[58] The
developed route gives the desired R-enantiomer with excellent isolated yield of up to
95% and an enantiomeric excess of >97% (Table 3, entries 1–3), replacing a chemoenzy-
matic four-step synthesis using lipases or oxidoreductases.[59] A further very interesting
example is the chemoenzymatic multistep synthesis of the CRTH2 antagonist MK-7246.
Here, a chemical route (18 steps, ~10% overall yield) was optimized by reducing the num-
ber of steps to eight, mainly due to the direct installation of the required chiral amine
functionality from the prochiral ketone precursor (entry 4).[60] This enabled a more scala-
ble and cost-efficient manufacturing route to MK-7246.
Table 3 Asymmetric Reductive Amination of Multicyclic Ketones Using ø-Transaminases[58,60]
ω-transaminase
O PLP
NH2
R1 R2 R1 ∗ R2
NH2 O
3 3
R R
Entry Starting Material R3 ø-Transaminasea Product Yield (%) ee (%) Ref
O NH2
[58]
1 Ph ArRmut11 96 >97
N N
H H
O NH2
2 CO2H ArS 90b 66 [58]
N N
H H
O NH2
[58]
3 CO2H CV 62 >97
N N
H H
O H2N
N N
[60]
4 Me CDX-017 76 >99
EtO2C EtO2C
a
Origin of enzymes: ArRmut11 = variant of R-selective ø-transaminase from Arthrobacter sp.; ArS = Arthrobacter
citreus; CV = Chromobacterium violaceum; CDX-017 = R-selective ø-transaminase variant available from Codexis.
b
Only conversion was reported.
Ethyl [(R)-7-Amino-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]acetate Hydrochloride

(Table 3, Entry 4); Typical Procedure:[60]
For the preparation of the triethanolamine/iPrNH2 buffer, triethanolamine (0.32 mL,
2.4 mmol) was dissolved in H2O (24 mL). The soln was cooled (5 8C) and iPrNH2 (3.11 mL,
76 mmol) was added over 20 min. MsOH (2 mL) was added to adjust the pH to 9.0 and the
soln was then charged at rt to a 50-mL Multimax vessel equipped with an overhead stirrer,
thermocouple, pH probe, and N2 inlet. PLP (40 mg, 0.16 mmol) was added, and the mixture
was gently agitated to give a clear yellow soln at pH 8.0–8.3. CDX-017 enzyme (115 mg) was
then added and dissolved with gentle agitation. DMSO (7.5 mL) was added over 5 min to
the agitated soln (400 rpm) at rt and the mixture was heated to 45 8C. The pH was adjusted
to 8.5 using 4 M aq iPrNH2. A soln of ethyl (7-oxo-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-
yl)acetate (2.67 g, 9.44 mmol) in DMSO (8 mL + 0.5 mL rinse) was added over 4 h via a sy-
ringe pump, and the reaction was kept at 45 8C for 48 h under constant N2 sweep and pH

control (pH 8.3–8.5) by adding 4 M aq iPrNH2. The mixture was cooled to rt and adjusted to
pH 7.0 using MsOH (0.45 mL), and then cooled to 12 8C, acidified to pH 2.0 with another
portion of MsOH (0.2 mL), and kept for 3 h at 12 8C and 1000 rpm. The mixture was fil-
tered, rinsing with a chilled mixture of H2O/DMSO/MsOH (60:40:1; 3 10 mL). The filtrate
was washed with cyclopentyl methyl ether (40 mL) and the organic layer was discarded.
Cyclopentyl methyl ether (70 mL) was added to the aqueous layer and the mixture was ad-
justed to pH 10.5 using 5 M aq NaOH (5.5 mL). The layers were separated and the aqueous
layer was extracted with cyclopentyl methyl ether (40 mL). The combined organic layers
were washed with sat. aq NaCl (20 mL), concentrated, and flushed with cyclopentyl meth-
yl ether (24 mL). The soln was then cooled to 0 8C and seeded with the product salt (24 mg),
and a 5.5 M soln of HCl in iPrOH (1.76 mL) was added over 2 h until a nominal pH of 6.0 was
reached. The slurry was further aged at 0 8C for 1 h, filtered, washed with cyclopentyl
methyl ether (2 5 mL), and dried at rt under reduced pressure to give the product as a
cream powder; yield: 2.27 g (76%); >99% ee.
2.4.3.1.3.5 Amination of Steroids and Related Compounds
Steroids and steroidal compounds are further interesting classes of target molecules. Ste-
roids have, based on their natural function as secondary metabolites controlling a variety
of biological processes, enormous potential as pharmaceuticals. Indeed, among the 200
top-selling drugs in 2010, 13% were steroids and derivatives thereof.[61]
For instance, the ø-transaminases ATA-113 and ATA-117 and the ø-transaminase
from Vibrio fluvialis have been successfully applied for the preparation of amine steroid
derivatives giving both enantiomers in moderate conversions (37–49%, Table 4, entries
4–6).[62] In further examples, the ø-transaminase ArRmut11 has been employed for the
preparation of various 17-Æ-amino steroids, compounds recently highlighted as potent in-
hibitors of steroid sulfatase, a target in the treatment of breast cancer.[63–66] In this ap-
proach, steroids can be prepared on a preparative scale with high isolated yields (83–
89%) and excellent selectivity (entries 1–3).[67]
Table 4 Asymmetric Reductive Amination of Steroids and Related Compounds with ø-Transaminases[62,67]
ω-transaminase
O PLP
NH2
R1 R2 R1 R2
NH2 O
R3 R3
Entry Starting Material R3 ø-Trans- Product Yield Config Ref

aminasea (%)
O NH2
H H
1 Me ArRmut11 83 17-Æ-Rb [67]
H H H H
HO HO
O NH2
H H
2 Me ArRmut11 85 17-Æ-Rb [67]
H H H H
HO HO
Table 4 (cont.)
Entry Starting Material R3 ø-Trans- Product Yield Config Ref
aminasea (%)
O NH2
H H
3 Me ArRmut11 89 17-Æ-Rb [67]
H H H H
HO HO
H H
Cbz Cbz
N N
H H
H H
O O
4 CO2H VF 49c R [62]

H H
H H
H H
H H
O H2N
Cbz Cbz
N N
H H
H H
O O
5 CO2H ATA-113 37c S [62]

H H
H H
H H
H H
O H2N
Cbz Cbz
N N
H H
H H
O O
6 CO2H ATA-117 5c S [62]

H H
H H
H H
H H
O H2N
a
Origin of enzymes: VF = Vibrio fluvialis; ArRMut11 = R-selective ø-transaminase variant from Arthrobacter sp.;
ATA-113 and ATA-117 are commercial enzymes available from Codexis.
b
dr 1:99.
c
Only conversions determined by HPLC were reported.
17Æ-Amino-5Æ-androstan-3-ol (Table 4, Entry 3); Typical Procedure:[67]

iPrNH2 (1.72 mL, 20.1 mmol) and PLP (5 mg, 0.02 mmol) were dissolved in distilled H2O
(11.2 mL) and the pH was adjusted to 10.0 with aq HCl. trans-Androsterone (50 mg,

0.17 mmol) in DMF (7 mL) was added. The reaction was started by addition of freeze-dried
E. coli cells containing overexpressed ArRmut11 (500 mg) and shaken at 45 8C. After 3 d,
the reaction was stopped by addition of 2 M aq HCl (6 mL) and the mixture was extracted
with EtOAc (2 10 mL). The remaining aqueous phase was treated with 10 M aq NaOH
(1 mL) and extracted again with EtOAc (4 10 mL). The combined organic layers were
dried (Na2SO4), filtered, and concentrated under reduced pressure. The product, a brown-
ish solid, was purified by flash column chromatography (CH2Cl2/MeOH/Et3N 90:9:1). The
product was obtained as a colorless, amorphous solid; yield: 43.7 mg (89%); [Æ]D25 –12.5 (c
0.8, MeOH).
2.4.3.2 Amination of Aldehydes
The amination of aldehydes has been investigated mainly in the course of the character-
ization of novel transaminases during the determination of their substrate scope and ki-
netic studies.[28,52,68–70] In general, as to be expected, aldehydes are more active amine ac-
ceptors than ketones or oxo acids.[68,71] However, the ø-transaminase-catalyzed amination
of aldehydes on a preparative scale has been less investigated compared to the amination
of ketones.
In one, preparative, example a ª-aminobutyric acid (GABA) derivative was accessed,
which is a key precursor for a series of potent drugs for the treatment of various diseases
concerning the central nervous system (e.g., epilepsy, Huntingtons and Parkinsons dis-
ease).[72] In the first report of dynamic kinetic resolution mediated by ø-transaminases, a
chemoenzymatic concept was employed for the synthesis of the GABA precursors (R)- and
(S)-4-phenylpyrrolidin-2-one (27) (Scheme 18).[73] In this approach, the chiral ª-amino ester
26 is formed by transaminase-catalyzed amination of racemic aldehyde rac-25. The ami-
nation product 26 cyclizes spontaneously to the corresponding lactam 27, which can be
transformed into a GABA derivative by subsequent ring opening. Due to the spontaneous
racemization of substrate 25, the reaction proceeds via dynamic kinetic resolution and
hence complete conversion can be obtained.
Scheme 18 Amination of an Æ-Chiral Aldehyde in a Dynamic Kinetic Resolution[73]
ω-transaminase
PLP, buffer (pH 7) H
H O H 2N O
O 15% DMSO O N
30 oC, 24 h
∗
Ph OEt Ph ∗ OEt
Ph
rac-25
26 27
NH2 O OH
LDH
OH OH OH
NADH
O O recycling O
ø-Transaminasea Cosolvent (vol %) Conversionb (%) Yield (%) eec (%) Config Ref
[73]
VF DMSO (10) 98 90 6 S
[73]
ATA-114 DMSO (10) 99 80 45 S
[73]
ATA-117 none 98 93 61 R
[73]
ATA-117 DMSO (10) >99 95 65 R
a
Origin of enzymes: VF = Vibrio fluvialis; ATA-114 and ATA-117 are commercial enzymes.
b
Determined by GC.
c
Determined by HPLC on a chiral stationary phase.
(R)-4-Phenylpyrrolidin-2-one [(R)-27]; Typical Procedure:[73]

Racemic 4-oxo ester 25 (100 mg, 0.49 mmol) was suspended in phosphate buffer (100 mM,
pH 7, 15 vol% DMSO, 1 mM PLP; 17 mL) in a 50-mL screw cap tube. d-Alanine (2.25 mmol),
LDH mix (200 mg), and a crude preparation of commercial ø-transaminase ATA-117
(30 mg) were added. The reaction was conducted at 30 8C and 120 rpm for 24 h. The pH
was adjusted to 14 using 10 M aq NaOH and the product was extracted with CH2Cl2 (5
10 mL). The organic phases were combined and the solvent was evaporated to give the
product as a white solid; yield: 92%; [Æ]D20 +22.0 (c 0.5, MeOH).
2.4.3.3 Amination of Alcohols
One of the most attractive routes to prepare amines is the catalytic amination of alcohols,
because alcohols are often readily available, e.g. from renewable sources, on a large
scale.[74] Moreover, ideally external reducing or oxidizing reagents may be avoided as
both alcohol substrate and product amine are in the same oxidation state.[75] Even though
no enzyme is capable of catalyzing the direct amination, various multienzyme one-pot
cascades have been developed to afford the corresponding amines.
For instance, a redox-self-sufficient multienzyme cascade has been designed based
on the “borrowing hydrogen” approach for the amination of primary alcohols without re-
quiring external redox equivalents.[76] In the first step, an alcohol dehydrogenase (ADH)
catalyzes the oxidation of the primary alcohol to the corresponding aldehyde, giving
one equivalent of the reduced cofactor NADH. Next, the ø-transaminase performs the am-
ination of the intermediate aldehyde at the expense of l-alanine. Finally, alanine dehydro-
genase (AlaDH) connects the oxidation and reductive amination step by consuming NADH
obtained in the oxidation for the regeneration of l-alanine from pyruvate. Primary amines
bearing aliphatic or even bulkier aromatic moieties are obtained with excellent conver-
sion from the corresponding alcohols (Table 5). Even more interesting is the application
of this multienzyme network for the diamination of 1,ø-diols which provides straightfor-
ward access to 1,ø-diamines, valuable building blocks for the preparation of polyamide
derivatives (entries 4 and 5). In this particular case, the synthesis of decane-1,10-diamine
has been performed on a preparative scale. Due to the poor solubility of the diol, the ad-
dition of 10% 1,2-dimethoxyethane is required. The desired diamine is obtained in good
yield without detecting traces of the intermediate aldehyde or over-oxidation products.
Alternatively, the intermediate aldehyde can be produced using an oxidase, but in this
case the efficiency is lower because it is necessary to add external redox equivalents.[77]

Table 5 ø-Transaminase-Catalyzed Redox-Neutral Cascade for the Amination of Primary and Secondary
Alcohols[76,78,79]
R1 R2
NH2
ADH ω-transaminase
OH
NADH
O
NAD+ AlaDH O
OH
OH NH2
NH3 H2O O
R1 R2 R1 R2
Entry Starting Material ADHa ø-Transaminaseb Product Conversion (%) Yield (%) Ref
OH NH2
1 hT CV >99 –c [76]
5 5
2 Ph OH hT CV Ph NH2 87 –c [76]
Ph OH Ph NH2
3 hT CV >99 –c [76]
3 3
HO OH H2N NH2
4 hT CV >99 –c,d [76]
8 8
HO OH H2N NH2
5 hT CV 94 70d [76]
10 10
OH NH2
[78]
6 A BM 54 35
5 5
HO H H2N H
O O
7 LR PD (L417M) 7 –c [79]
O O
H OH H OH
a
Origin of alcohol dehydrogenases: hT = Bacillus stearothermofilus; A = Rhodococcus ruber; LR = Leifsonia aquatica.
b
Origin of ø-transaminases: CV = Chromobacterium violaceum; BM = Bacillus megaterium; PD (L417M) = Paracoccus
denitrificans L417M variant.
c
Not isolated.
d
10% DME was added due to solubility reasons.
The amination of secondary alcohols is an even more interesting transformation because

it can provide straightforward access to valuable Æ-chiral amines. In this particular case,
the complexity of the cascade is higher because the stereochemistry of the starting mate-
rial (alcohol), the final product (amine), the cofactor dependence, as well the stereoprefer-
ence of the ADH and the ø-transaminase must be considered. On the other hand, the ex-
perimental data show that the amination of secondary alcohols does not go to comple-
tion, due to thermodynamic reasons, if a borrowing hydrogen approach is used according
to Table 5. For instance, moderate amounts of (S)-octan-2-amine (Table 5, entry 6) are re-
covered in the bioamination of (S)-octan-2-ol. In this case, the amine is isolated in lower
enantiomeric excess (78%) compared with the previous results obtained in the amination
of octan-2-one (>99% ee).[78] This depletion in enantiomeric excess is attributed to the con-
tinuous back and forward reactions of the system. The diamination of cyclic secondary
alcohols such as isosorbide has also been studied, however, in this case the transforma-
tion gives the (2S,5S)-configured amino alcohol (entry 7) with only 7% conversion.[79]
Decane-1,10-diamine (Table 5, Entry 5); Typical Procedure:[76]

Decane-1,10-diol (174.2 mg, 1.0 mmol) was dissolved in DME (2 mL, 10 vol%) in a round-
bottomed flask (50 mL), followed by the addition of a soln of l-alanine (445 mg, 5 mmol)
in H2O (4 mL) and a soln of NH4Cl (320 mg, 6 mmol) in H2O (1.5 mL). The pH was adjusted to
8.5 by addition of 6 M aq NaOH (80 L). NAD+ (10 mg, 12 mol), and PLP (2 mg, 8 mol),
each dissolved in H2O (500 L), additional H2O (3.12 mL), ADH (4 mL, 5 U), ø-transaminase
CV (4 mL, 4 U), and AlaDH (300 mL, 5 U) were added. After the mixture had been shaken at
20 8C and 120 rpm (orbital shaker, Infors Unitron) for 22 h, the product was separated
from the mixture and purified through a weak acid cation exchanger; yield: 121 mg (70%).
2.4.3.4 Resolution of Æ-Chiral Primary Amines
2.4.3.4.1 Kinetic Resolution via Deamination
Enantiopure monocyclic amines can be accessed by kinetic resolution via deamination of

the racemic amines catalyzed by ø-transaminases. For instance, optically pure pyrrolidin-
3-amines 28 (n = 1) and piperidin-3-amines 28 (n = 2) are successfully prepared using such
an enzyme-catalyzed strategy. The cyclic amines are attractive synthons for the prepara-
tion of a broad range of biologically active pharmaceuticals.[80–84] Whereas alternative
chemical strategies such as substitution, cyclization, and classical resolution of the race-
mate with a resolving agent often include multiple steps or lack in selectivity, a kinetic
resolution catalyzed by a very selective enzyme is a powerful alternative route.
Initial experiments using the ø-transaminase from Vibrio fluvialis in the kinetic reso-
lution of racemic pyrrolidin-3-amine (rac-28, R1 = H; n = 1) displayed only very slow reac-
tion rates and poor selectivities.[71] Therefore, a protection group strategy has been devel-
oped to engineer the substrate and thereby to enhance the enantioselectivity and increase
the yield of the desired enantiomer. An efficient resolution has been established on a pre-
parative scale by using tert-butoxycarbonyl as protecting group (Scheme 19), facilitating a
promising route for the synthesis of enantiopure pyrrolidin-3-amines.[85]

Scheme 19 Kinetic Resolution of Monocyclic Amines[85]
H 2N H2N O
ω-transaminase
PLP
N N + N
R1 R1 R1
n n n
rac-28 (R)-28 29
O O
OH OH
O NH2
R1 n ø-Transaminasea Conversion (%) eeb (%) Ref
[85]
H 1 Ad 52 89
[85]
H 1 VF 55 86
[85]
Bn 1 Ad 50 81
[85]
Bn 1 VF 49 77
[85]
Boc 1 Ad 50 98
[85]
Boc 1 VF 50 90
[85]
Cbz 1 Ad 51 99
[85]
Cbz 1 VF 50 >99
[85]
H 2 Ad 54 72
[85]
H 2 VF 54 76
c [85]
Boc 2 Ad 55 97
c [85]
Boc 2 VF 55 96
a
Origin of enzymes: Ad = Alcaligenes denitrificans; VF =
Vibrio fluvialis.
b
R-Enantiomer was the main product.
c
Assumed to have R configuration.
(R)-1-(tert-Butoxycarbonyl)pyrrolidin-3-amine (28, R1 = Boc; n = 1) and 1-(tert-Butoxycar-

bonyl)pyrrolidin-3-one (29, R1 = Boc; n = 1); Typical Procedure:[85]
Racemic 1-(tert-butoxycarbonyl)pyrrolidin-3-amine (rac-28, R1 = Boc; n = 1; 230 mg,
1.24 mmol) was dissolved in phosphate buffer (50 mM, pH 8.0; 122 mL) and, after the addi-
tion of pyruvic acid (109 mg, 1.2 mmol), the pH was adjusted to 8.0. After the mixture had
been heated to 37 8C, the reaction was started by the addition of a soln of ø-transaminase
Ade (3.7 mL) and stirred for 7 h. The pH of the mixture was adjusted to 3 with 5 M aq HCl,
and the ketone was extracted with CH2Cl2 (5 ). Then, the pH of the aqueous phase was
adjusted to 13 and the remaining amine was extracted with CH2Cl2 (4 ). The solvents of
the different fractions were evaporated providing the amine 28 (R1 = Boc; n = 1); yield:
89 mg (39%), and the ketone (29, R1 = Boc; n = 1); yield: 104 mg (44%).
2.4.3.4.2 Deracemization via Deamination/Transamination
Enzymatic deracemization represents an interesting method for the synthesis of enantio-

pure compounds from racemic amines, theoretically providing the optically pure amine
in 100% yield. For example, a process for the deracemization of mexiletine (30) by ø-trans-
aminases in a one-pot two-step procedure leading to either enantiomer in up to >99% con-
version and >99% enantiomeric excess has been described.[20,21] In the first step, kinetic
resolution of the amine 30 is performed (Scheme 20), followed by a stereoselective trans-
amination of the formed intermediate ketone 31 in the second step. The pyruvate con-
sumed in the first step is recycled in situ by an amino acid oxidase (DAAO). To improve the
deracemization approach of mexiletine described above, immobilized ø-transaminase
has been employed, with the enzyme immobilized in a sol-gel/Celite matrix. In contrast
to free enzymes, the sol-gel immobilized ø-transaminases are easily removable by centri-
fugation or filtration after the first step.[86] The sol-gel entrapment represents an easy and
effective way to prepare high-performance amines. Using this approach (S)-mexiletine
[(S)-30] is obtained in enantiomerically pure form and up to 95% isolated yield.[20]
Scheme 20 Deracemization of Mexiletine by an ø-Transaminase-Cata-

lyzed One-Pot Two-Step Procedure[20,21,86]
NH2
O
rac-30
O2
CO2H
(R)-ω-transaminase
DAAO PLP, pH 7.0, 30 oC
NH2
CO2H
H2O2
O NH2
O O
+
31 (S)-30
+
NH4
NH2
CO2H
(S)-ω-transaminase
NADH
L-AlaDH PLP, pH 7.0, 30 oC
recycling
CO2H
H2O
NH2
O
(S)-30 >99% ee; >99% conversion
Recently, various aromatic amines have been deracemized by ø-transaminases, yielding

the corresponding R-enantiomers with good conversions in a simultaneous one-pot cas-
cade (Scheme 21). In the case where the (S)-ø-transaminase from Polaromonas sp. [(S)-PO]

is used, Æ-ketoglutarate is preferentially used as amine acceptor for the deamination in

the kinetic resolution.[87] Æ-Ketoglutarate is poorly or not accepted by the (R)-ø-transami-
nases responsible for the asymmetric synthesis using d-alanine as amine donor.
Scheme 21 Deracemization of Amines[20,21,86,87]
ω-transaminase
amine acceptor
NH2 NH2 O
PLP, pH 7.0, 30 oC
1 2 1 2 + 1
R R R R R R2
rac
enantiocomplementary ω-transaminase
NH2
amine donor, PLP, pH 7.0, 30 oC
R1 R2
R1 R2 ø-Transaminasea Conversion (%) ee (%) Config Ref

Step 1 Step 2
Pr Me ATA-117 ATA-113 99b >99c S [20]
Pr Me ATA-114 ATA-117 87b >99c R [20]
b c [20]
Et Me ATA-117 ATA-113 >99 >99 S
Et Me ATA-114 ATA-117 >99b >99c R [20]
b c [20]
CH2OMe Me ATA-117 ATA-113 >99 >99 S
CH2OMe Me ATA-114 ATA-117 >99b 96c R [20]
b c [20]
Cy Me ATA-117 ATA-113 62 >99 S
b c [20]
Cy Me ATA-114 ATA-117 88 >99 R
b c [20]
Ph Me ATA-117 ATA-113 60 >99 S
b c [20]
Ph Me ATA-114 ATA-117 84 >99 R
Ph Et ATA-117 ATA-113 82b >99c S [20]
b c [20]
Ph Et ATA-114 ATA-117 72 >99 R
(CH2)2Ph Me ATA-117 ATA-113 >99b 5c S [20]
b c [20]
(CH2)2Ph Me ATA-114 ATA-117 >99 >99 R
b c [86]
(CH2)2Ph Me ATA-117 ATA-114 90 >99 S
b c [20,21,86]
2,6-Me2C6H3OCH2 Me ATA-117 ATA-113 >99 >99 S
b c [20,21,86]
2,6-Me2C6H3OCH2 Me ATA-114 ATA-117 81 >99 R
2,6-Me2C6H3OCH2 Me ATA-117 CV 97b >99c S [21]
b c [21]
2,6-Me2C6H3OCH2 Me ATA-117 BM 77 95 S
2,6-Me2C6H3OCH2 Me ATA-117 VF >99b 96c S [21]
b c [21]
2,6-Me2C6H3OCH2 Me CV ATA-117 >99 >99 R
2,6-Me2C6H3OCH2 Me ATA-113 ATA-117 97b >99c R [21]
d d,e [87]
4-HOC6H4 Me (S)-PO (R)-MV >99 >99 R
d d,e [87]
4-HOC6H4 Me (S)-PO (R)-NF >99 >99 R
4-FC6H4 Me (S)-PO (R)-MV 82d >99d,e R [87]
d d,e [87]
4-FC6H4 Me (S)-PO (R)-NF 71 >99 R
3,4-F2C6H3 Me (S)-PO (R)-MV 98d >99d,e R [87]
d d,e [87]
3,4-F2C6H3 Me (S)-PO (R)-NF 87 >99 R
3,5-F2C6H3 Me (S)-PO (R)-MV 87d >99d,e R [87]
d d,e [87]
3,5-F2C6H3 Me (S)-PO (R)-NF 94 >99 R
R1 R2 ø-Transaminasea Conversion (%) ee (%) Config Ref

Step 1 Step 2
4-BrC6H4 Me (S)-PO (R)-MV 90d >99d,e R [87]
4-BrC6H4 Me (S)-PO (R)-NF 97d >99d,e R [87]
d d,e [87]
4-Tol Me (S)-PO (R)-MV >99 >99 R
4-Tol Me (S)-PO (R)-NF 89d 81d,e R [87]
d d,e [87]
3-Tol Me (S)-PO (R)-MV 98 >99 R
d d,e [87]
3-Tol Me (S)-PO (R)-NF 79 >99 R
d d,e [87]
3-NCC6H4 Me (S)-PO (R)-MV >99 >99 R
d d,e
[87]
3-NCC6H4 Me (S)-PO (R)-NF 77 >99 R
a
Origin of enzymes: CV = Chromobacterium violaceum; BM = Bacillus megaterium; VF = Vibrio flu-
vialis; PO = Polaromonas sp.; MV = Mycobacterium vanbaalenii; NF = Neosartorya fischeri; ATA-
113, ATA-114, and ATA-117 are commercial enzymes.
b
c
ee determined by GC on a chiral stationary phase after derivatization as the corresponding
acetamides or trifluoroacetamides.
d
Conversion and ee were determined by HPLC using a Crownpak CR(+) column. Conversion is
the percentage of the remaining amine concentration to the initial amine concentration.
e
ee determined using a C18 symmetry column after GITC derivatization.
(S)-Mexiletine [(S)-30]; Typical Procedure:[21]

Racemic mexiletine (rac-30; 100 mg, 0.46 mmol) was suspended in phosphate buffer (100
mM, pH 7.0, 1 mM PLP; 20 mL). Sodium pyruvate (1.98 mg, 0.02 mmol), d-amino acid ox-
idase from porcine kidney (30 mg, 45 U), and the ø-transaminase ATA-117 (30 mg) were
added. The reaction was shaken at 30 8C and 300 rpm. After the kinetic resolution (24 h)
the mixture was kept at 75 8C for 30 min and cooled to rt, and amino acid dehydrogenase
from Bacillus subtilis (100 L, 40 U), ammonium formate (88.3 mg, 1.4 mmol), NAD+
(13.3 mg, 0.02 mmol), and formate dehydrogenase (200 L, 40 U) were added and the mix-
ture was shaken for 1 h at 30 8C. l-Alanine (178 mg, 2 mmol) and the second ø-transami-
nase from Chromobacterium violaceum (100 mg, E. coli cells) were added and the mixture
was shaken at 30 8C. After 24 h, the pH of the mixture was adjusted to 1 with 5 M aq HCl,
and the remaining ketone was extracted with CH2Cl2 (5 10 mL). After the extraction, no
ketone was detectable in the residual aqueous phase. The pH was adjusted to 12, and the
product was extracted with CH2Cl2 (4 10 mL). The solvent of the combined extracts was
evaporated; yield: 95.2 mg (95%); >99% ee; [Æ]D20 +3.2 (c 2.0, CHCl3).

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Transaminases: Scheme 1 General Reaction Scheme For - Transaminase-Catalyzed Asymmetric Reductive Amination

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Transaminases: Scheme 1 General Reaction Scheme For - Transaminase-Catalyzed Asymmetric Reductive Amination

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383

Scheme 1 General Reaction Scheme for ø-Transaminase-Catalyzed

for references see p 418

Scheme 2 Asymmetric Synthesis of Chiral Amines Cata-

Because ø-transaminase-catalyzed amination reactions are reversible, the enzymes can

Scheme 3 Kinetic Resolution of Racemic Amines Employing ø-Transaminases

NH2 NH2 ω-transaminase NH2 O

2.4.3.1 Amination of Ketones

2.4.3.1.1 Amination of Linear Monoketones

2.4.3.1.1.1 Amination Employing Alanine as Amine Donor

2.4.3.1.1.1.1 Amination with Lactate Dehydrogenase

Scheme 4 Enzymatic Reductive Amination in Combination with Lactate Dehydrogenase

R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Config Ref

2.4.3.1.1.1.1.1 Preparation of Optically Pure (R)- and (S)-1-Phenylethylamine

for references see p 418

Scheme 5 Amination of Acetophenone at 50 g/L Substrate Concentration Employing

(R)- or (S)-1-Phenylethylamine (2); Typical Procedure:[26]

2.4.3.1.1.1.1.2 Preparation of an (S)-Rivastigmine Precursor

Rivastigmine {3-[(1S)-1-(dimethylamino)ethyl]phenyl ethyl(methyl)carbamate} is a strong

Scheme 6 ø-Transaminase-Catalyzed Synthesis of a Key Precursor for Rivastigmine[29]

3 (S)-4 76%; >99% ee

3-[(S)-1-Aminoethyl]phenyl Ethyl(methyl)carbamate [(S)-4]; Typical Procedure:[29]

2.4.3.1.1.1.1.3 Chemoenzymatic Preparation of a Precursor for the Dual

for references see p 418

Methyl (6R)-6-Methyl-2-oxopiperidine-3-carboxylate [(6R)-7]; Typical Procedure:[31]

2.4.3.1.1.1.2 Amination with Alanine Dehydrogenase

Scheme 8 (S)-Amination in Combination with the L-Alanine Dehydrogenase Recycling

R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Ref

for references see p 418

R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Ref

(S)-1-(4-Methoxyphenyl)propan-2-amine (9, R1 = 4-MeOC6H4CH2); Typical Procedure:[33]

In general, ø-transaminases giving Æ-chiral primary amines in the R configuration (e.g.,

Scheme 9 (R)-Amination in Combination with L-Alanine Dehydrogenase[17,22]

CO2H CO2H CO2H

R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Ref

for references see p 418

R1 ø-Transaminasea Conversionb,c (%) eec,d (%) Ref

(R)-4-Phenylbutan-2-amine [10, R1 = (CH2)2Ph)]; Typical Procedure:[22]

2.4.3.1.1.2 Amination Employing Isopropylamine as Amine Donor

Scheme 10 Amination of Linear Ketones Using Isopropylamine[16,22,37]

N 2,4,5-F3C6H2CH2 ArRmut11 50% DMSO, 90–95 >99 [16]

CO2Et pH 7.5, 30 8C, 24 h

for references see p 418

2.4.3.1.1.2.1 Amination Followed by Spontaneous Cyclization

Table 1 Amination with Isopropylamine as Amine Donor Followed by Spontaneous Ring-

Entry Starting Material ø-Transaminasea Product Yield (%) ee (%) Ref

5-Chloro-2-[(5R)-5-methyl-1,4-diazepan-1-yl]benzoxazole Hydrochloride (Table 1, Entry 4);

for references see p 418

2.4.3.1.1.2.2 Amination of In Situ Formed Ketones

Scheme 11 Cascade Coupling of a Threonine Deaminase with Enantiocomplementary

78%; >99% ee CO2H

(R)- or (S)-2-Aminobutanoic Acid (15); General Procedure:[39]

2.4.3.1.1.3 Amination Employing 1-Phenylethylamine as Amine Donor

Even though isopropylamine is an excellent amine donor, it is not accepted by every

Scheme 12 Amination of Linear Ketones Using 1-Phenylethylamine as Amine Donor[13,40,41]

R1 R2 Config of ø-Transaminasea Conditions Conversion ee Config Ref

for references see p 418

(S)-1-(5-Fluoropyrimidin-2-yl)ethylamine Hydrochloride [16 • HCl, R1 = 5-Fluoropyrimidin-

2.4.3.1.1.4 Amination in Organic Solvents

Transformations employing ø-transaminases are generally performed in aqueous media;

Scheme 13 Enzymatic Amination in tert-Butyl Methyl Ether[42]

R1 R2 iPrNH2 Time ø-Transaminasea Conversion ee Config Ref