Expanding dynamic kinetic protocols: Transaminase-catalyzed synthesis of α-substituted β-amino ester derivatives

Article abstract of DOI:10.1039/c3cc46760k

Several α-alkylated β-amino esters have been obtained via DKR processes employing a kit of transaminases and isopropylamine as an amino donor in aqueous medium under mild conditions. Thus, while acyclic α-alkyl-β-keto esters afforded excellent conversions and enantioselectivities, although usually low diastereoselectivities, using more constrained cyclic β-keto esters high to excellent inductions were obtained.

Full text of DOI:10.1039/c3cc46760k

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Expanding dynamic kinetic protocols: transaminase-
catalyzed synthesis of a-substituted b-amino ester
derivatives†
Cite this: Chem. Commun., 2013,
4
9, 10688
Received 4th September 2013,
Accepted 25th September 2013
An ´ı bal Cuetos, Iv a´ n Lavandera* and Vicente Gotor*
DOI: 10.1039/c3cc46760k
www.rsc.org/chemcomm
Several a-alkylated b-amino esters have been obtained via DKR
processes employing a kit of transaminases and isopropylamine as
an amino donor in aqueous medium under mild conditions. Thus,
while acyclic a-alkyl-b-keto esters afforded excellent conversions
and enantioselectivities, although usually low diastereoselectiv-
ities, using more constrained cyclic b-keto esters high to excellent
inductions were obtained.
Scheme 1 General overview on the synthesis of enantioenriched a-alkylated
b-amino esters through o-TA-catalyzed dynamic amination.
The development of enzymatic strategies that enable access to 100%
theoretical yield of stereoisomerically enriched compounds starting
from easily available racemic or prochiral derivatives has recently
attracted much attention due to the simpler isolation and purifica- a-substituted b-amino esters (Scheme 1). These compounds
1
tion techniques required. In this sense, recent advances achieved in have a great relevance since they are present in biologically
7
dynamic systems to get access to enantio- or diastereomerically pure active peptides, and are also valuable for the preparation of
8
9
10
compounds have been described. Thus, performing a kinetic resolu- peptidomimetics, substituted b-lactams and b-amino acids. The
11
tion on a substrate which can simultaneously undergo racemization selective synthesis of these unprotected synthons is challenging,
has led to efficient dynamic kinetic resolutions (DKRs). While and no general strategy comprising the direct asymmetric reductive
2
this methodology has extensively been employed with hydrolases amination of the a-substituted b-keto ester precursors has been
3
12
(
usually in combination with metal-based racemization) and described to date.
oxidoreductases (often combined with racemization under basic
Thus, the synthesis of racemic b-keto esters 1b–k and the
corresponding mixture of syn and anti diastereoisomers of
4
conditions), for other enzymes it has been scarcely exploited.
5
For instance, with o-transaminases (o-TAs, EC 2.6.1.x), some of the a-substituted b-amino esters 2b–k and 3b–k was planned
the most promising biocatalysts applied to the synthesis of chiral (Scheme 2), to optimize the analytical methods to measure the
amines, it is remarkable that only one example described by Kroutil enzymatic conversions and the enantio- and diastereoselectivities.
6
and co-workers can be recognized as a dynamic kinetic resolution.
Compounds 1b–k were achieved through treatment of the b-keto
In this contribution, the authors proposed the DKR of an a-chiral ester precursors with the corresponding alkyl halide in basic
4
a,d
aldehyde through spontaneous racemization combined with the use medium.
of an o-TA to afford, after ring closure, 4-phenylpyrrolidin-2-one by Brandt and co-workers, ultrasonication of these derivatives
with 92% isolated yield and 68% ee. in the presence of benzylamine at 30 1C smoothly afforded the
Due to our previous experience with the synthesis of several (Z)-N-benzylated enamine esters 4b–k in excellent yields. Then,
In a subsequent step, adapting a protocol described
1
3
4a,d
a-alkylated b-keto esters, and since these substrates can racemize the corresponding N-protected b-amino esters syn-5b–k and anti-
at neutral pH, it was decided to study the DKR of these compounds 6b–k were obtained as diastereoisomeric mixtures by reduction
through o-TA-catalyzed amination to obtain the corresponding of the CQC double bond with NaBH(OAc) at 0 1C in high yields. As
3
11d,14
previously described, the syn isomers were preferentially formed.
Dpto. de Qu ´ı mica Org a´ nica e Inorg a´ nica, Universidad de Oviedo. c/Juli ´a n Claver ´ı a 8,
3006, Oviedo, Spain. E-mail: lavanderaivan@uniovi.es, vgs@uniovi.es;
Fax: +34 985 103448; Tel: +34 985 103452
Electronic supplementary information (ESI) available: Experimental proce-
In the last step, deprotection of these compounds under hydrogena-
tion conditions afforded the racemic a-substituted b-amino esters
syn-2b–k and anti-3b–k in high yields. A series of alkyl derivatives
3
†
2
1
13
were synthesized changing the a-alkyl chain (R = Me, Et, Bn)
dures, enzymatic results, analytics and copies of H- and C-NMR spectra of
the novel compounds. See DOI: 10.1039/c3cc46760k
3
i
and the ester alkyl moiety (R = Me, Et, Pr). Furthermore, cyclic
1
0688 Chem. Commun., 2013, 49, 10688--10690
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Then, different reaction conditions were explored to optimize
the transamination process using a-ethylated keto ester 1c as a
substrate. After enzymatic screening, a few TAs were chosen as
suitable catalysts since they afforded a diastereomeric mixture of
2c and 3c with high conversions, excellent ee and moderate to
high de. Some parameters such as pH, temperature, biocatalyst
loading and addition of basic resins were studied to improve the
DKR conditions (see Tables S4 and S5 in ESI†), but no positive
effect was observed. Therefore, 30 1C and pH 7.5 were selected as
the best conditions to perform these transformations.
As a further step, the TA-catalyzed reactions using a-alkylated
b-keto esters 1b–i were attempted to have an overview of the effect
that could present: (a) the alkyl group at the a-position, and (b) the
substitution in the ester moiety. The results are shown in Table 1
and Tables S6–S13 in ESI.† In all cases it was found that 12 TAs
showed (S)-stereopreference while other 12 were (R)-selective. It
was remarkable that with just few exceptions (ATA-007, ATA-009,
Scheme 2 Synthesis of racemic aliphatic a-substituted b-amino ester derivatives and ATA-117), these biocatalysts afforded high conversions for
through an enamine formation-reduction protocol.
these substrates and were highly specific for the amination of the
carbonyl group, showing excellent selectivities at position 3, just
obtaining in most cases 2 out of 4 diastereoisomers.
compounds 1j and 1k were also tried as substrates to study the
effect of constrained systems in these dynamic processes.
Regarding the (S)-selective family of transaminases, generally the
syn-(2R,3S) isomers were provided in slight excess (up to 60/40) and
To perform these biocatalyzed transformations, a series of very high conversions (around 90%). Among them, ATA-224, ATA-234,
s
2
4 commercially available transaminases, the Codex Transaminase TA-P1-F12, and TA-P1-G05 were the most active. (S)-Selective
Screening Kit, was tried as most of them are able to work under enzymes TA-P1-A06, TA-P1-G06 and ATA-103 afforded preferentially
very convenient conditions using isopropylamine in molar anti-(2S,3S)-3b, 3c, 3f, and 3i with moderate to high diastereomeric
1
5
excess as an amino donor. In a first set of experiments, ratios. (R)-Selective biocatalysts also showed a slight stereo-
unsubstituted ethyl acetoacetate 1a was studied as a substrate preference for the formation of the enantiopure syn-(2S,3R) amino
for these TAs (Tables S2 and S3 in ESI†) using alanine or esters (usually up to 60/40) with excellent conversions (>90%),
isopropylamine as an amino donor. Better conversions were especially ATA-015, ATA-016, ATA-024, ATA-025, and ATA-033.
achieved in the second case utilizing an excess (1 M) of the amine Although showing lower conversions, TA-P2-A07 was the most
in 100 mM phosphate buffer, pH 7.5, and in the presence of PLP selective enzyme for most of the keto esters. In contrast, ATA-301
ꢀ
1
(
(
1 mM) and DMSO (2.5% v v ) for solubility reasons. Various presented an opposite diastereopreference for anti-(2R,3R), in
S)- and (R)-selective enzymes were detected showing very high some cases higher than 80/20. For some TAs, e.g. TA-P1-A06 and
to excellent conversions and stereoselectivities. Especially the ATA-301, it remained clear that the presence of a benzyl moiety
last ones are interesting since not many (R)-selective TAs are at the a-position could largely influence the diastereoselectivity
1
6
described in the literature.
of the process (Fig. S1 and S2 in ESI†).
a
Table 1 Selected results from the transamination of racemic acyclic a-alkyl-b-keto esters 1b–i using isopropylamine as an amino donor (t = 24 h)
(
S)-Selective
(R)-Selective
b
c
c
b
c
c
Entry
1b–i
1b
Enzyme
c (%)
Ratio 2/3
ee 2 (%)
ee 3 (%)
Enzyme
c (%)
Ratio 2/3
ee 2 (%)
ee 3 (%)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
TA-P1-A06
ATA-113
TA-P1-A06
ATA-231
TA-P1-A06
—
TA-P1-G05
TA-P1-A06
ATA-224
—
TA-P1-G05
—
ATA-103
ATA-103
TA-P1-F12
88
89
87
88
98
—
88
93
93
—
94
—
88
94
98
34/66
57/43
16/84
54/46
58/42
—
56/44
29/71
55/45
—
57/43
—
54/46
23/77
58/42
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
—
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
—
>99 (2R,3S)
—
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
>99 (2S,3S)
>99 (2S,3S)
>99 (2S,3S)
>99 (2S,3S)
>99 (2S,3S)
—
>99 (2S,3S)
>99 (2S,3S)
>99 (2S,3S)
—
>99 (2S,3S)
—
>99 (2S,3S)
>99 (2S,3S)
>99 (2S,3S)
ATA-301
TA-P2-A07
ATA-301
TA-P2-A07
ATA-024
ATA-301
TA-P2-A07
ATA-301
TA-P2-A07
ATA-301
ATA-025
ATA-301
TA-P2-A07
—
25
61
21
47
>99
39
79
41
50
47
95
35
53
—
17/83
58/42
20/80
62/38
56/44
36/64
55/45
32/68
58/42
32/68
54/46
38/62
54/46
—
n.d.
>99 (2R,3R)
>99 (2R,3R)
98 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
>99 (2R,3R)
—
>99 (2S,3R)
n.d.
1c
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
>99 (2S,3R)
—
1d
1e
1f
1
1
1
1
1
1
1g
1h
1i
ATA-301
89
59/41
>99 (2S,3R)
>99 (2R,3R)
a
b
c
For other enzymatic results and experimental conditions, see ESI. Measured by GC. Measured by chiral GC.
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 10688--10690 10689

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2
6
7, 3769; (c) Y.-W. Kim, J.-W. Park and M.-J. Kim, ChemCatChem,
Scheme 3 Synthesis of (1S,2S)-3k through a DKR process with a transaminase
2011, 3, 271.
using isopropylamine as an amino donor.
3 Recent examples: (a) M. Egi, K. Sugiyama, M. Saneto, R. Hanada,
K. Kato and S. Akai, Angew. Chem., Int. Ed., 2013, 52, 3654;
(
b) C. Kim, J. Lee, J. Cho, Y. Oh, Y. K. Choi, E. Choi, J. Park and
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Next, it was envisaged that the employment of more con-
strained derivatives could improve the diastereoselectivity of
the process while maintaining the excellent ee value. Therefore,
two cyclic compounds (1j and 1k) were tried as possible amino
acceptors for these TAs (Tables S14 and S15 in ESI†).
4
Examples employing alcohol dehydrogenases: (a) A. Cuetos, A. Rioz-
Mart ´ı nez, F. R. Bisogno, B. Grischek, I. Lavandera, G. de Gonzalo,
W. Kroutil and V. Gotor, Adv. Synth. Catal., 2012, 354, 1749;
(
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5
0, 8387; (e) C. Rodr ´ı guez, G. de Gonzalo, A. Rioz-Mart ´ı nez, D. E. Torres
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For 1j generally lower conversions (o40%) were observed, but
several (S)-selective biocatalysts could afford preferentially the anti-
0
(
2R,1 S) isomer, while (R)-TAs did not show good selectivities. Better
results were obtained for keto ester 1k, finding that ATA-113 and
TA-P1-G05 were able to produce anti-(1S,2S)-3k with very high
conversions, ee (>99%) and de (94–96%). Thus, a 50 mg-scale
reaction was achieved using TA-P1-G05 obtaining by simple
acid–base extraction the chiral amino ester (1S,2S)-3k with a
5 Recent revisions: (a) M. H ¨o hne and U. T. Bornscheuer, in Enzyme
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6
D. Koszelewski, D. Clay, K. Faber and W. Kroutil, J. Mol. Catal. B:
Enzym., 2009, 60, 191.
1
7
good yield and excellent ee and de (Scheme 3).
Herein we show our first results on the application of a set
of commercially available o-TAs to provide several a-alkylated
b-amino esters using a dynamic protocol. These highly inter-
esting targets are difficult to synthesize by other methodologies
and in most cases the synthetic routes involve several steps
or the employment of harsh conditions. Thus, a series of acyclic
7 E. Juaristi, in Enantioselective Synthesis of b-Amino Acids, ed.
E. Juaristi and V. A. Soloshonok, Wiley & Sons, 2nd edn, 2005, p. 1.
8
(a) D. Seebach and J. Gardiner, Acc. Chem. Res., 2008, 41, 1366;
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b-Amino Acids, ed. E. Juaristi and V. A. Soloshonok, Wiley & Sons,
nd edn, 2005, p. 527.
P. A. Magriotis, Angew. Chem., Int. Ed., 2001, 40, 4377.
(
2
9
10 G. Cardillo and C. Tomasini, Chem. Soc. Rev., 1996, 25, 117.
a-alkyl-b-keto esters were synthesized and used as substrates 11 (a) T. Kano, Y. Yamaguchi and K. Maruoka, Chem.–Eur. J., 2009, 15, 6678;
(
2
b) M. Periasamy, S. Suresh and S. S. Ganesan, Tetrahedron: Asymmetry,
006, 17, 1323; (c) A. J. McNeil, G. E. S. Toombes, S. M. Gruner,
for these enzymes, finding that although excellent conversions
and ee were achieved for many of them, diastereoselectivities
remained modest with few exceptions. As an additional extension,
two constrained cyclic derivatives were employed to study their
effect in the transamination reactions. Gratifyingly, racemic ethyl
E. Lobkovsky, D. B. Collum, S. V. Chandramouli, B. J. Vanasse and T. A.
Ayers, J. Am. Chem. Soc., 2004, 126, 16559; (d) C. Cimarelli and
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S. Walters, J. Chem. Soc., Perkin Trans. 1, 1994, 1141.
2 However, o-TAs have proven to be excellent catalysts applied to the
synthesis of enantiopure a-unsubstituted b-amino acid derivatives
through amination of the corresponding carbonylic precursors, see:
J. Rudat, B. R. Brucher and C. Syldatk, AMB Express, 2012, 2, 11.
3 C. A. Brandt, A. C. M. P. da Silva, C. G. Pancote, C. L. Brito and
M. A. B. da Silveira, Synthesis, 2004, 1557.
4 C. Bartoli, C. Cimarelli, E. Marcantoni, G. Palmieri and M. Petrini,
J. Org. Chem., 1994, 59, 5328.
5 K. E. Cassimjee, C. Branneby, V. Abedi, A. Wells and P. Berglund,
Chem. Commun., 2010, 46, 5569.
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H. Jochens, K. Robins and U. T. Bornscheuer, Nat. Chem. Biol., 2010,
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P. N. Devine, G. W. Huisman and G. J. Hughes, Science, 2010, 329, 305.
7 2-Aminocyclopentanecarboxylate derivatives are useful monomers
to synthesize constrained peptides, see: S. H. Choi, I. A. Guzei,
L. C. Spencer and S. H. Gellman, J. Am. Chem. Soc., 2009, 131, 2917.
2
-oxocyclopentanecarboxylate showed excellent ee and de under
our dynamic conditions, so the corresponding amino ester anti-
1S,2S)-3k could be synthesized at a higher scale. With these
(
preliminary results in hand, the application of these biocatalysts
over new constrained substrates will be preferred, and due to the
development of highly efficient tools in the Molecular Biology
1
16b,18
field,
the design of novel transaminases that could provide
1
1
1
1
specifically each diastereoisomer in enantiomerically pure form
seems to be highly feasible in the near future.
A.C. thanks the Principado de Asturias for his predoctoral
fellowship Severo Ochoa. I. L. thanks the Spanish MICINN for
personal funding (Ram o´ n y Cajal Program). Financial support
from MICINN (Project MICINN-12-CTQ2011-24237) is gratefully
acknowledged.
Notes and references
1
1
(a) E. Garc ´ı a-Urdiales, I. Lavandera and V. Gotor, in Enzyme Catalysis
in Organic Synthesis, ed. K. Drauz, H. Gr o¨ ger and O. May, Wiley-VCH,
3
rd edn, 2012, p. 43; (b) E. Garc ´ı a-Urdiales, I. Alfonso and V. Gotor, 18 D. Koszelewski, M. G o¨ ritzer, D. Clay, B. Seisser and W. Kroutil,
Chem. Rev., 2011, 111, PR110; (c) N. J. Turner, Curr. Opin. Chem. ChemCatChem, 2010, 2, 73.
1
0690 Chem. Commun., 2013, 49, 10688--10690
This journal is c The Royal Society of Chemistry 2013