Stereoselective synthesis of γ-hydroxynorvaline through combination of organo- and biocatalysis

Article abstract of DOI:10.1039/c4cc06230b

An efficient route for the synthesis of all four diastereomers of PMP-protected α-amino-γ-butyrolacton to access γ-hydroxynorvaline was established. The asymmetric key steps comprise an organocatalytic Mannich reaction and an enzymatic ketone reduction. T

Full text of DOI:10.1039/c4cc06230b

Volume 50 Number 99 25 December 2014 Pages 15637–15824
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Robert C. Simon, Wolfgang Kroutil et al.
Stereoselective synthesis of γ-hydroxynorvaline through combination of
organo- and biocatalysis

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Stereoselective synthesis of c-hydroxynorvaline
through combination of organo- and biocatalysis†
Cite this: Chem. Commun., 2014,
50, 15669
Robert C. Simon,*a Eduardo Busto,a Joerg H. Schrittwieser,a Johann H. Sattler,b
Jorg Pietruszka,c Kurt Fabera and Wolfgang Kroutil*a
¨
Received 8th August 2014,
Accepted 17th September 2014
DOI: 10.1039/c4cc06230b
www.rsc.org/chemcomm
An efficient route for the synthesis of all four diastereomers of PMP-
protected a-amino-c-butyrolacton to access c-hydroxynorvaline
was established. The asymmetric key steps comprise an organo-
catalytic Mannich reaction and an enzymatic ketone reduction.
Three reaction steps could be integrated in a one-pot process,
using 2-PrOH both as solvent and as reducing agent. The sequential
construction of stereogenic centres gave access to each of the four
stereoisomers in high yield and with excellent stereocontrol.
Fig. 1 Selected bioactive compounds containing a-amino-g-butyro-lactones.
from natural sources7 and shows anti-diabetic properties.8 The wide-
spread occurrence of the g-butyrolactone unit has led to the develop-
Progressive legislative regulations with respect to sustainability, safety ment of a number of stereoselective syntheses mainly based on
and quality improvements have changed the focus of contemporary chemo-catalysis,9 while only few biocatalytic strategies have been
chemical research to designing more economic processes. In this developed.10 Most of the procedures either require multiple steps,
context, (asymmetric) catalysis has become the method of choice to are auxiliary-assisted, or based on ex-chiral pool precursors.11 Hence,
replace stoichiometric amounts of reagents and auxiliaries, thereby access to only one or two out of four stereoisomers is provided.
facilitating an efficient and energy saving process.1 While each of the However, as the biological activity of bioactive compounds is related
catalysis sub-fields (metal-, organo- and biocatalysis) has reached an often to one specific unknown stereoisomer,12 a convenient access to
impressive level of sophistication, efficiency can be further increased all four diastereomers, e.g. for SAR-studies, is desired.
by combining methods from these different sub-fields. This allows
In this communication, a novel combination of organo- and
exploiting and combining specific features of each type of catalyst in a biocatalysis is reported, which provides access to optically pure
highly cooperative fashion. For example, numerous reports have been a-amino-g-butyrolactones either in a step-wise fashion or in a
published based on the combination of transition metals and cascade reaction.
organocatalysts;2 furthermore, biocatalytic transformations have been
In our study we focused initially on the bioreduction of
coupled with diverse metals.3 However, the combination of bio- and racemic amino-ketoester rac-1, which is readily available by a
organocatalysts is still scarce.4,5 proline catalysed Mannich reaction.13,14 A set of homo- and
a-Amino-g-butyrolactones are common structural elements heterologously expressed alcohol dehydrogenases (ADHs)15 were
in a series of natural products and pharmaceutical drugs that assayed on analytical scale (5.6 mg, 20 mM) employing 2-propanol
display diverse biological activities (cf. Fig. 1).6 Moreover, the for NAD(P)+ regeneration. Asymmetric reduction of the carbonyl
corresponding amino acid (g-hydroxynorvaline) has been isolated group of amino-ketoester 1 afforded the corresponding diastereo-
mers of alcohol 2, which cyclised spontaneously to the desired
2-amino-lactones 3 (Scheme 1).
a Department of Chemistry, University of Graz, NAWI Graz, Heinrichstraße 28,
A number of ADHs transformed rac-1 into the corresponding
A-8010-Graz, Austria. E-mail: wolfgang.kroutil@uni-graz.at,
a-amino-lactones 3 with high conversion (selected results in
Table 1; more details in the ESI†). For example, the (5S)-configured
diastereomers (3S,5S)-3 and (3R,5S)-3 were accessible by the
enzymes ADH-A15a (entry 1) and evo-1.1.03015b (entry 2), whereas
the (5R)-diastereomers (3S,5R)-3 and (3R,5R)-3 were formed utilising
evo-1.1.20015b (entry 5). The enzymes ADH-T15c (entry 3) and
ADH-LS15d (entry 4) gave excellent results, too, with conversions
robert.simon@uni-graz.at
b Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz,
Austria
c
¨
¨
¨
Institut fur Bioorganische Chemie, Heinrich-Heine-Universitat Dusseldorf im
¨
¨
Forschungszentrum Julich, Stetternicher Forst, Geb. 15.8, D-52426-Julich, Germany
† Electronic supplementary information (ESI) available: Detailed description of
each experiment, full characterisation of all compounds and copy of HPLC traces
and NMR data. See DOI: 10.1039/c4cc06230b
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Scheme 1 Alcohol dehydrogenase catalysed reduction of racemic ketoester
rac-1 on analytical scale (20 mM, 5.6 mg). PMP = para-methoxyphenyl.
Table 1 Selected results of the ADH catalysed reduction of ketoester
rac-1 yielding lactone 3 (analytical scale, double determination)a
Fig. 2 Time course of the biocatalytic reduction of racemic and enantio-
pure Mannich-base 1 with the enzyme ADH-LB (double determination).
Entry Enzyme
Conv.b [%] d.r. [syn/anti]b eesyn 3c [%] eeanti 3c [%]
1
2
3
4
5
6
7
ADH-A
evo-1.1.030 99
ADH-T
ADH-LS
evo-1.1.200 99
ADH-LB 49
evo-1.1.270 49
99
52/48
51/49
12/88
71/29
52/48
99/1
499 (3R,5S) 499 (3S,5S)
499 (3R,5S) 499 (3S,5S)
499 (3R,5S) 499 (3S,5S)
499 (3S,5R) 499 (3R,5R)
499 (3S,5R) 499 (3R,5R)
499 (3S,5R) —
83
86
99/1
499 (3S,5R) —
a
Reaction conditions: ketoester rac-1 (5.6 mg, 20 mM), KPi buffer (100 mM,
pH 6.8), NAD(P)+ (1.0 mM), 10 vol% 2-PrOH, ADH (for units see ESI), 30 1C,
700 rpm on a thermoshaker (horizontal position), 24 h. Determined
via GC on an achiral stationary phase. Determined via HPLC on a
b
c
chiral stationary phase.
of 83% and 86%, respectively. In all cases, the enantiomeric
excess (ee) of all diastereomers of lactones 3 was 499%.
Among the enzymes tested, ADH-LB15e and evo-1.1.27015b gave
unexpected results as in both cases mainly one diastereomer (3S,5R)-3
was detected (entries 6 and 7). Since the conversions remained below
50%, we assumed a selective kinetic resolution with respect to the
Fig. 3 Docking study: productive binding mode of Mannich-base (S)-1
(purple) and the cofactor NADP+ (orange) in the active site of ADH-LB.
existing stereogenic centre in b-position to the carbonyl group. In ADH-A and evo-1.1.200. Investigating 2-PrOH as a cosolvent revealed
order to gain a better understanding of such a rare case of distant that a substrate concentration of 30 mM (7.95 mg mLÀ1, 10 vol%
stereorecognition by ADHs,16 further investigations were performed: 2-PrOH) led to completion of the reaction in the case of the
racemic and enantiopure Mannich-bases rac-/(R)-/(S)-1 were incubated (S)-selective ADH-A within 24 h (for details, see ESI†). The (R)-selective
with ADH-LB and the reaction progress was monitored over enzyme evo-1.1.200 transformed even 50 mM (13.3 mg mLÀ1, 30 vol%
time (Fig. 2). Thereby it was found that rac-1 gave the (3S,5R)-3 2-PrOH) to completion within 24 h. The optimum pH was determined
diastereomer as sole product with 40% conversion after eight hours, to be 6.3 in order to keep the partial racemisation of the optically pure
while (R)-1 was not transformed. The pure enantiomer (S)-1 reacted Mannich adduct 1 at a minimum (see ESI†). In the subsequent
fastest, reaching 70% conversion within the same time.
semi-preparative scale transformations [56 mg (S)-1] 499% conver-
The experimental observations were additionally supported sion was achieved; however, ring closure was incomplete, as mainly
by in silico docking studies: only substrate (S)-1 was found to fit in the optically pure amino-alcohol 2 rather than the lactone 3 was
the active site enabling a productive binding mode; the substrate isolated (ca. 50–70%; see ESI†).17 Assuming that the corresponding
occupies therein a pro-R conformation in which the carbonyl methyl ester of 1 would undergo faster lactonisation than the ethyl
group can be activated by two hydrogen bonds through amino analogue, a transesterification step (HCl–MeOH) was integrated into
acids Tyr152 and Ser142 (Fig. 3). Attempts to dock the opposite the reaction sequence. The optimised conditions finally allowed
enantiomer (R)-1 into the active site failed to provide a reasonable conversion of the pure amino-ester (R)-/(S)-1 (up to 4300 mg),
productive binding mode (see ESI†). Hence, the structure of readily accessible by a proline-catalysed Mannich reaction of
ADH-LB allows a chiral recognition of a remote stereocentre acetone and imine 4,14 into the corresponding lactones 3. The
enabling a kinetic resolution.16 Attempts to couple the diaster- syn- and anti-diastereomers were isolated in excellent yields
eoselective reduction of 1 with a racemisation of 1 in a dynamic (78–82%) and with remarkable stereocontrol (de 498 : 2 and ee
kinetic resolution failed so far due spontaneous degradation of 499% in all cases; see Scheme 2).
1 via elimination of anisidine over time.
As an alternative to the conventional step-by-step synthesis
The next step was the synthesis of all four stereoisomers of (Scheme 2) two sequential approaches were established. In a
amino-lactone 3; for this purpose, the biocatalysed reduction first approach, aldimine 4 was formed in 2-PrOH, which also
of ketoester 1 was optimised using alcohol dehydrogenases served as a solvent for the subsequent Mannich reaction and as
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Scheme 3 Synthesis of a-amino-g-butyrolactones by means of two different
approaches: (a) sequential cascade reaction in which the solvent (2-PrOH) was
integrated in the first three steps (5 steps, 2 pots); (b) conventional sequential
reaction sequence with the preformed aldimine (4 steps, 2 pots).
Scheme 2 Synthesis of all four diastereomers of PMP-protected
a-amino-g-butyrolactone 3 through combination of organo- and bio-
catalysis. Reagents and conditions: (a) D-/L-proline (0.25 eq.), DMSO, 3 h, rt;
(b) (R)-/(S)-1 (30 mM), ADH-A, NAD+, buffer (100 mM, pH 6.3), 2-PrOH
(10 vol%), 30 1C, 24 h; (c) (R)-/(S)-1 (50 mM), evo-1.1.200, NAD+, buffer
(100 mM, pH 6.3), 2-PrOH (30 vol%), 30 1C, 24 h; (d) HClÁEt2O (2.0 eq.),
MeOH, 16 h, rt. PMP = para-methoxyphenyl.
the reducing agent for the enzyme-mediated reduction (after dilution
with buffer solution). All three steps were accomplished in one pot, Scheme 4 PMP deprotection to yield natural g-hydroxynorvaline.
after which the transesterification-lactonisation cascade was
performed in a separate vessel. The whole sequence afforded
the syn-configured lactone (3S,5R)-3 in a remarkable 47% yield
with a d.r. of 86/14 in favour of the syn-diastereomer (Scheme 3a:
including the organo- as well as the biocatalysed transforma-
tions were performed in one pot, integrating 2-propanol first as
solvent and then as reducing agent.
5 steps in total, 2 pots, 90% average yield per step). The dimin-
E. B. received funding from the European Commission by a
ished d.r. in comparison to the previous results can be rationa-
Marie Curie Actions-Intra-European Fellowship (IEF) in the project
lised by the usage of 2-PrOH for the proline-catalysed reaction,
‘‘BIOCASCADE’’ (FP7-PEOPLE-2011-IEF). COST Action CM1303
which under these conditions afforded (S)-1 with 72% ee.
‘‘Systems Biocatalysis’’ is acknowledged. J.H.S. has been supported
by the Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and
ZIT through the Austrian FFG-COMET-Funding Program.
In a second approach, the Mannich reaction was carried out
with the preformed aldimine 4 in acetone as solvent, and amino-
ketoester (R)-1 was obtained at prolonged reaction time (16 h) with
499% conversion and in optically pure form (ee 499%). After
evaporation of the solvent, the crude product was subjected to
reduction by ADH-A in buffer–2-PrOH. The final transesterification-
lactonisation cascade furnished the diastereomerically pure amino
lactone (3R,5S)-3 in 51% yield with an excellent d.r. of 99 : 1
(Scheme 3b: 4 steps, 2 pots, 85% average yield per step).
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15672 | Chem. Commun., 2014, 50, 15669--15672
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