A protection strategy substantially enhances rate and enantioselectivity in ω-transaminase-catalyzed kinetic resolutions

Article abstract of DOI:10.1002/adsc.200800030

The kinetic resolution of 3-aminopyrrolidine (3AP) and 3-aminopiperidine (3APi) with ω-transaminases was facilitated by the application of a protecting group concept. 1-N-Cbz-protected 3-aminopyrrolidine could be resolved with >99% ee at 50% conversion, the resolution of 1-N-Boc-3-aminopiperidine yielded 96% ee at 55% conversion. The reaction rate was up to 50-fold higher by using protected substrates. Most importantly, enantioselectivity increased remarkably after carbamate protection compared to the unprotected substrates (86 vs. 99% ee). Surprisingly, benzyl protection of 3AP had no influence on enantioselectivity. A possible explanation for this observation could be the different flexibility of the benzyl- or carbamate-protected 3AP as confirmed by NMR spectroscopy.

Full text of DOI:10.1002/adsc.200800030

COMMUNICATIONS
DOI: 10.1002/adsc.200800030
A Protection Strategy Substantially Enhances Rate and
Enantioselectivity in w-Transaminase-Catalyzed Kinetic
Resolutions
Matthias Hçhne,a Karen Robins,b and Uwe T. Bornscheuera,*
a
Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University,
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
Fax : (+49) 3834–86–80066; e-mail: uwe.bornscheuer@uni-greifswald.de
Lonza AG, Valais Works, 3930 Visp, Switzerland
b
Received: January 16, 2008; Revised: February 10, 2008; Published online: March 20, 2008
Abstract: The kinetic resolution of 3-aminopyrroli-
dine (3AP) and 3-aminopiperidine (3APi) with w-
transaminases was facilitated by the application of a
protecting group concept. 1-N-Cbz-protected 3-ami-
nopyrrolidine could be resolved with >99% ee at
50% conversion, the resolution of 1-N-Boc-3-ami-
nopiperidine yielded 96% ee at 55% conversion.
The reaction rate was up to 50-fold higher by using
protected substrates. Most importantly, enantiose-
lectivity increased remarkably after carbamate pro-
tection compared to the unprotected substrates (86
vs. 99% ee). Surprisingly, benzyl protection of 3AP
had no influence on enantioselectivity. A possible
explanation for this observation could be the differ-
ent flexibility of the benzyl- or carbamate-protected
3AP as confirmed by NMR spectroscopy.
mellitus types I and II[4] or as psychotropic drugs
against depression and schizophrenia.[5] Various syn-
theses using optically active 3AP and 3APi or their
protected analogues have been reported in literature.
There are three different synthetic routes: substitu-
tion, cyclization and classical resolution of the race-
mate with resolving agents. Most of them have one
ore more drawbacks: substitution reactions via azide
displacement[1,6] require 1-N-protected mesylated or
tosylated alcohol, which is either expensive or must
be prepared via multistep syntheses. Furthermore, the
substitution proceeds via an inversion of the configu-
ration, so that an unwanted non-selective SN1-reaction
has to be avoided. In these methods, enantiomeric
purity of the products has not been characterized via
chromatographic techniques. The 1-N-,3-N-double
protected 3AP/3APi can be obtained by cyclization
starting from optically pure aspartate or glutamate.[7]
Other routes from amino acids involve a reduction of
the cyclized dione-compound.[8] All cyclizations, how-
ever, are elaborate multistep syntheses with either
low to moderate yield or moderate enantiomeric puri-
ties. From (S)-2,4-diaminobutyric acid, (S)-3AP can
Keywords: amines; enantioselectivity; enzyme cat-
alysis; protecting groups; transaminase
Optically pure 3-aminopyrrolidine (3AP) and 3-ami- be obtained in only two steps, but the yield is low.[9]
nopiperidine (3APi) (Scheme 1) have become attrac- The crystallization processes[10] suffer from the fact
tive synthons for the synthesis of a broad range of that the maximum yield is below 50% and that the re-
biologically active pharmaceuticals such as Tosufloxa- cycling and recovery of the resolving agent and the
cin, Clinafloxacin[1] and cephalosporin derivatives.[2] antipode are laborious.
Other compounds containing a 3AP- or 3APi-residue
w-Transaminases are known to convert amines with
are interesting for the treatment of obesity[3], diabetes usually high to excellent enantioselectivity in either a
kinetic resolution of a racemic amine or in an asym-
[11]
metric synthesis starting from the prochiralketone.
However, asymmetric synthesis is hampered by an un-
favorable equilibrium. Only one report for the kinetic
resolution of racemic 3AP with an w-transaminase
from Vibrio fluvialis was published, but the reaction
was very slow and the enantioselectivity was not ex-
amined.[12] Indeed, initialexperiments carried out by
Scheme 1. Principle of kinetic resolution of unprotected and
1-N-protected 3-aminopyrrolidines and -piperidines with w-
transaminase.
us using two w-transaminases from Vibrio fluvialis
(Vfl-TA)[13] and Alcaligenes denitrificans (Ade-TA)[14]
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Matthias Hçhne et al.
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Table 1. Results of kinetic resolutions of unprotected and
protected substrates.
Entry
Ade-TA
Vfl-TA
Conversion [%] ee [%][a] Conversion [%] ee [%][a]
1a
1b
1c
1d
1e
1f
52
50
50
51
54
55
89
81
98
99
55
49
50
50
54
55
86
77
90
>99
76
72
97[b]
96[b]
[a]
The (R)-enantiomer is the main component.
Assumed to have (R)-configuration.
[b]
Figure 1. Influence of the protecting group on the reaction
rate in the kinetic resolutions of 1a–f using w-transaminases
Ade-TA or Vfl-TA.
confirmed that the selectivity of both enzymes in the
kinetic resolution of 1a was not satisfactory. An enan- derivatized with o-phthaldialdehyde (OPA) prior to
tiomeric excess of 97% ee could only be achieved CE analysis, but luckily, derivatization proceeded fast
after >60% conversion (data not shown) which re- under mild conditions in aqueous media.[19]
duces the yield of the desired isomer to <40%.
Next, the relative reaction rates were determined
In order to enhance the enantioselectivity and using 5 mM (R,S)-1a–1f and pyruvate as amino ac-
hence increase the yield of the desired enantiomer, ceptor employing the two w-transaminase as model
we considered the use of protection groups to facili- biocatalyst (Figure 1). We were pleased to find that
tate a better distinction between the enantiomers. the introduction of a protecting group (benzyl, Boc or
This strategy has scarcely been investigated, but could Cbz) had a substantialimpact on the reaction rates,
be successfully applied to biotransformations with a which were almost 50-fold increased (1d vs. 1a using
lipase[15] and monooxygenases.[16] In the desymmetri- Vfl-TA). It also turned out that the five-membered
zation of 2,4,6-trifunctionalized tetrahydropyrans with ring pyrrolidines are preferred over the six-membered
Pseudomonas cepacia lipase, the unprotected 2,6-- piperidines by both enzymes.
A
Table 1 shows the effect of the protection on enan-
with low selectivity to the monoacetate. In contrast, tiomeric purity of the amines at ~50% conversion.
excellent enantiomeric purities were obtained upon The unprotected 3AP 1a and 3APi 1e were converted
acetalprotection of the keto function, which coudl
with only moderate to good selectivity. Interestingly,
easily be removed from the product in a subsequent benzyl-, Boc- and Cbz-protection of 3AP resulted in
step. Later, this approach was particularly applied in remarkably different enantiomeric purities at 50%
the field of biohydroxylation with monooxygenases conversion independent of the w-transaminase used.
and named docking/protecting (d/p) group concept.[16] Benzyl( 1b) protection of 3AP led to only moderate
The introduction of the d/p group enabled the enan- enantiomeric excess of 77 or 81% ee for Vfl-TA or
tioselective biohydroxylation of non-activated carbon Ade-TA, respectively. On introducing the carbamate
atoms in cyclic or aliphatic compounds like ketones, group (1c), a significantly enhanced enantioselectivity
aldehydes, acids and alcohols. More recently, this con- was found and the best opticalpurity ( >99% ee) was
cept was also applied to the preparation of hydroxy- achieved with the Cbz-group (1d). In case of the Boc-
lated N-heterocycles[17] and to the bioconversion of protected 3APi 1f no rate enhancement was ob-
O-protected b-hydroxynitriles to yield b-hydroxyalka- served, but the protection led to a remarkable in-
noic acids and their amide derivatives, respectively.[18] crease in enantiomeric excess from 72 to 97% ee at
Protection generally resulted in a dramatic increase in similar conversion.
rate, regio- and stereoselectivity in most of the reac-
A possible explanation of the presence and the
tions. Thus, we subjected several N-protected deriva- nature of the protecting group on the enantioselectivi-
tives of 3AP and 3APi (Scheme 1) to w-transaminase- ty can be retrieved from the substrate structure. In
catalyzed kinetic resolutions.
Scheme 2, both enantiomers of (un)protected 3AP
For the analysis of conversion and especially optical are shown. To allow conversion of the amines, their
purity, an analytical method using capillary electro- amino group has to be positioned towards the C4’ of
phoresis (CE) was developed. The protected amines the bound PLP-cofactor. The moderate selectivity for
are UV-detectable at 200 nm and CE with highly sul- 3AP and 3APi could be explained by the fact that the
fated a-, b- or g-cyclodextrin as chiral selectors only possibility for the enzyme to discriminate the en-
turned out to be a powerfultechnique for the separa-
antiomers is the recognition of the endocyclic nitro-
tion of the amines. The unprotected amines had to be gen, which is protonated in neutralsoultion. In con-
808
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A Protection Strategy Substantially Enhances Rate and Enantioselectivity
COMMUNICATIONS
On the other hand, the introduction of a carbamate
residue creates a strong limitation of the possible ro-
tations: due to the partialC6-N1 doubel bond charac-
ter, the carbamate group as well as C2 and C5 of the
ring form a plane geometry (marked as gray rectangle
in Scheme 2). Thus, only two stable ring conforma-
tions are possible, whereas C3 and C4 are below or
above the plane, respectively, and vice versa. Indeed,
this assumption could be confirmed by NMR spec-
troscopy: two discrete signals for each of the ring car-
bons were observed in 13C NMR spectra in CDCl3 as
well in D2O (at pH 8, 258C). This indicates that two
relatively stable positions for the carbonyl group
occur, in which the oxygen atom can be syn- or anti-
oriented with respect to the amino group. As expect-
ed for rotamers, elevating the temperature accelerates
their interconversion so that the carbon signalpairs
are merged into one broad signalfor each carbon
atom. An additionalfeature for stereodiscrimination
could be the capability of the enzyme to form an H-
bond with the carbonylgroup of the carbamate func-
tion. As a result of this rigid geometry, the discrimina-
tion of the enantiomers of 1c and 1d is superior com-
pared to the benzyl-protected or unprotected 3AP.
For preparative scale kinetic resolution of 1c, the
influence of pH of the transamination with Ade-TA
was investigated and pH 8 was found as optimum
Scheme 2. Comparison of the structuralfeatures of protect-
ed and unprotected 3AP with respect to stereodiscrimina-
tion by the enzyme.
trast, the presence of a bulky protecting group at this value (Figure 2, top). In a preparative scale kinetic
position allows a better binding of the substrate by
the enzyme, as indicated by the increasing relative ac-
tivities. Furthermore, positioning the amino group of
both enantiomers towards the cofactor would lead to
a different orientation of the protection group, thus
increasing the chance for a selective binding of one
enantiomer. At this point the question remains why
the Boc- and Cbz-protection led to a drastically en-
hanced enantioselectivity, but on the other hand, no
increase of selectivity was observed with the benzyl
group at all. This can be explained by the different
flexibility of the benzyl- and carbamate-protected
substrates. Both the unprotected and benzyl-protected
3AP have no restrictions in terms of conformational
freedom of the five-membered ring, so five envelope
and five half-chair conformations with slightly differ-
ent energies are possible. The bond of the benzyl
group to the endocyclic nitrogen is freely rotable, so
that the phenylring of both enantiomers coudl be
positioned in a similar orientation, thus preventing an
exact stereodiscrimination (Scheme 2).
The amino groups of both enantiomers are orien-
tated in the same direction to show the possibilities
for an enzymatic stereodifferentiation. Two rotamers
of 1c and 1d are designated as cis and trans with re-
spect to the orientation of the carbonyloxygen to the
Figure 2. Top : Influence of pH on the kinetic resolution of
amino group. The atoms in the gray rectangle form a
planar geometry [PLP=cofactor pyridoxal5 ’-phos-
phate].
1c. Enantiomeric excess was determined after 4 h. Bottom:
Progress of the enantiomeric excess in the preparative-scale
kinetic resolution of 1c.
Adv. Synth. Catal. 2008, 350, 807 – 812
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809

Matthias Hçhne et al.
COMMUNICATIONS
Table 2. Separation conditions for the analysis of enantio-
meric purity of amines by capillary electrophoresis.
resolution (Figure 2, bottom), 0.85 mmol 1c was re-
solved and at a conversion of ꢀ50%, 1c could be iso-
lated having an enantiomeric purity of 98.7% ee. The
isolated yield for the amine 1c and the ketone 2c
were 39% and 44%, respectively. A preparative kinet-
ic resolution of 1f with Ade-TA yielded 42% of 1f
with 97% ee. Deprotection can easily be performed
using standard methods.[20]
In summary, we have shown that, by means of pro-
tection, 1c and 1f can be efficiently resolved using w-
transaminases on a preparative scale. This is the first
time that the d/p concept was applied to transami-
nase-catalyzed synthesis. The reaction rate could be
dramatically enhanced by using protected 3AP. Al-
though this was not observed for 3APi, the enantiose-
lectivity was still remarkably enhanced for both 3AP
and 3APi after introducing the carbamate protecting
group. Although kinetic resolutions are limited to
50% yield, this enzymatic approach is an easy way for
the synthesis of enantiopure 3-aminopyrrolidines.
Compound Cyclodextrin Capillary Length
[cm]
Migration
Time
[min][a,b]
to detector total( R)
(S)
1a
HSgCD
HSbCD
HSgCD
HSaCD
HSgCD
HSgCD
10
50
10
20
10
10
30
60
30
30
30
30
4.4
6.3
7.7
10.6
5.9
8.4
4.7
6.2
6.4
11.1
5.1
8.9
1b[c]
1c
1d
1e
1f[d]
[a]
The migration order was determined using commercially
available optically pure reference compounds.
Voltage applied: 15 kV except for 1d (10 kV).
Before rinsing the capillary with the cyclodextrine con-
taining background electrolyte, the capillary was rinsed
for 15-20 min with 0.1M NaOH.
The migration order has not been determined. It is as-
sumed that in the kinetic resolution with (S)-selective-
transaminases the (R)-enantiomer is enriched, which is
the faster migrating enantiomer.
[b]
[c]
[d]
Experimental Section
pH 3.0), containing the analyte. For the analysis of 1a and
1e, the compounds were derivatized following a published
protocol[19] with some modification. An aliquot (100 mL) of
the reaction mixture was given to sodium borate buffer
(500 mL, 100 mM, pH 10) and OPA reagent (300 mL) was
added (80 mg OPA, 20 mL 2-mercaptoethanol, 10 mL aceto-
nitrile). The sample was incubated for 30 min at 378C and
the derivatives were subsequently extracted by the addition
of NaOH (70 mL 15M) and chloroform (1 mL). The organic
phase was then mixed with triethylammonium phosphate
buffer (100 mL, 10 mM, pH 3), and washed two times with
chloroform (500 mL). To 100 mL of the amine in the trieth-
Chemicals and Enzymes
All chemicals were from Sigma–Aldrich, Germany, except
for (R,S)-1-N-Boc-3-aminopiperidine hydrochloride which
was from Fluorochem (Germany) and 1-N-benzyl-3-amino-
pyrrolidine which was from ABCR, Germany. The w-trans-
ACHTREUNGaminases from V. fluvialis and A. denitrificans were obtained
from Julich Chemical Solutions (now Codexis Inc.).
Analysis of Optical Purity
AHCTREyUNG lammonium phosphate phase, 1,3,6,8-pyrenetetrasulfonate
The enantiomeric excesses of 1c and 1f were analysed by
gas chromatography. After extraction of the amine with di-
chloromethane, derivatization to the trifluoroacetamide was
performed by adding a 20-fold excess of trifluoroacetic an-
hydride. After purging with nitrogen to remove excess anhy-
dride and residualtriful oroacetic acid, the derivatized com-
pound was dissolved in dichloromethane (50 mL) and ana-
lyzed using a Shimadzu GC14A equipped with a heptakis-
(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-cyclodextrin
column (25 m”0.25 mm). The retention times for 1c were
13.5 min for the (S)-enantiomer and 19 min for the (R)-en-
antiomer at an oven temperature of 1808C (absolute config-
urations were assigned using commercially available optical-
ly pure standards). Compound 1f was separated at 1608C,
the retention times were 6.4 and 7.0 min.
Alternatively, the optical purity could also be determined
by capillary electrophoresis (see below). All CE measure-
ments were done with a PACE-MDQ system equipped with
a fused silica capillary (50 mm inner diameter). For the de-
termination of the enantiomeric excess of 1b–d and 1f, the
reaction was stopped by adding an aliquot of the reaction
mixture (100 mL) to NaOH solution (100 mL, 0.1M), fol-
lowed by an extraction with dichloromethane (200 mL). Sub-
sequently, 100 mL of the organic layer were extracted with
triethylammonium phosphate buffer (200 mL, 10 mM,
(2 mL, 10 mM) as migration standard was added. Enantio-
mer separation was achieved by the addition of 5% highly
sulfated-a-, -b- or -g-cyclodextrin (HSa/b/gCD), Beckman-
Coulter) to the running buffer as chiral selector. An aper-
ture with a 200 mm slit was used and the capillary tempera-
ture was set to 158C. The compounds were detected at
200 nm. All important parameters and migration times are
shown in Table 2. The separation protocol was programmed
as recommended by the manufacturer.
Analysis of Conversion
Conversions were determined by the measurement of the
residual amine by capillary electrophoresis. The reaction
was stopped by adding an aliquot of the reaction mixture
(40 mL) to HClsoul tion (160 mL, 15 mM) containing a-meth-
ylbenzylamine (1 mM) as internal standard for the analysis
of 1c and 1f. For 1b, the amine 1d was used as internalstan-
dard and vice versa. The capillary was dynamically coated
with CElixir (MicroSolvTech) according to the manufactur-
erꢁs instructions to avoid adsorption of proteins to the capil-
lary wall. The separation was done on a Beckman PACE-
MDQ system equipped with a fused silica capillary (60 cm
length, 10 cm to the detector, 50 mm inner diameter) and a
810
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A Protection Strategy Substantially Enhances Rate and Enantioselectivity
COMMUNICATIONS
PDA detector. A pressure of 0.5 psi was applied for 5 s for
the injection. The background electrolyte contained triethyl-
ACHTREUNGammonium phosphate (50 mM, pH 3.0). A voltage of 30 kV
was started by the addition of Ade-TA solution (2 mL) and
stirred with a magnetic stirrer bar. After 19 h, the enantio-
meric excess reached 97% ee at a conversion of 55%. The
proteins of the reaction mixture were removed after addi-
tion of ice-cold acetone (100 mL) and incubation for 4 h at
48C. After evaporation of acetone, the amine was extracted
as described above. The yield of 1f was 42%. 1H- and
13CNMR spectra confirmed the structure of 1f.
was applied for four minutes for separation, the compounds
1c and 1f were detected at 190 nm and 1a, 1b and 1d at
200 nm. For the measurement of the concentration of 1a
and 1e, an indirect detection method was developed. The
compounds had to be extracted from a sample (100 mL) by
the addition of NaOH (50 mL 15M) and chloroform
(300 mL). The chloroform phase (200 mL) was subsequently
extracted with phosphoric acid (5 mM). This solution was in-
jected into the capillary (5 s, 1 psi). The background electro-
lyte contained m-xylenediamine phosphate (10 mM, pH 2.5).
The capillary had a total length of 40 cm, and an effective
length of 30 cm. A voltage of 15 kV was applied for 6 min
for separation.
1f: 1H NMR (300 MHz, CDCl3): d=1.42 (s, 9H, CH3),
1.60–1.68 (m, 2H, CH3CH3CH3), 1.83–1.92 (m, 2H,
CHCH2CH3), 2.53 (s, 2H, NH2), 2.69–2.82 (m, 1H,
CHNH2), 3.76–3.90 (m, 4H); 13C NMR (CDCl3): d=23.6
(CH2CH2CH2), 28.3 (CH3), 43.7 (NCH2CH2), 47.5 (CHNH2),
52.2 (NCH2CH), 79.3 [CCATHRE(UNG CH3)3], 154.8 (NCOO).
Biocatalysis
References
All kinetic resolutions were done at 378C, 600 rpm, in
sodium phosphate buffer (50 mM, pH 8), containing pyri-
doxal5 ’-phosphate (0.1 mM), racemic amine (5 mM) and
pyruvic acid (10 mM) unless otherwise indicated.
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added and the pH adjusted to 8. After heating to 378C, the
reaction was started by the addition of Ade-TA solution
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HCl(5M), and the ketone 2c was extracted five times with
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1
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2.59 (t, 2H, J=7,8 Hz, COCH2), 3.75 (s, 2H, COCH2NCO2),
3.78 (t, 2H, J=7,8 Hz, CH2CH2NCO2); 13C NMR (DMSO-
d6): d=28.4 (CH3), 37.1 (CH2), 42.4 (CH2), 52.6 (CH2), 80.4
[C
ACHTREUNG(CH3)3], 154.4 (NCOO), 211.0 (CH2COCH2).
1
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34.8 (CHNH2CH2CH2), 43.9+44.3 (CH2CH2NCO2), 50.5+
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Preparative-Scale Resolution of 1f
This was performed similar as described for 1a: 1-N-Boc-3-
aminopiperidine hydrochloride (0.25 mmol) and pyruvic
acid (0.25 mmol) were dissolved in phosphate buffer
(25 mL, 50 mM, pH 8), containing PLP (10 mM). After pH
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