Enantioselective biocatalytic oxidative desymmetrization of substituted pyrrolidines

Article abstract of DOI:10.1002/anie.200906655

"Chemical Equation Presented" Center stage: Additions of nitrogen-centered radicals to cyanamide compounds provided the first radical synthesis of aromatic polycyclic guanidine derivatives (see scheme). Modular assembly of the substrates allows for a rapid increase of the molecular complexity of scaffolds, which have potential applications for medicinal chemistry.

Full text of DOI:10.1002/anie.200906655

Communications
DOI: 10.1002/anie.200906655
Chiral Imines
Enantioselective Biocatalytic Oxidative Desymmetrization of
Substituted Pyrrolidines**
Valentin Kꢀhler, Kevin R. Bailey, Anass Znabet, James Raftery, Madeleine Helliwell, and
Nicholas J. Turner*
3
À
Direct activation of sp C H bonds a to a nitrogen atom
represents an attractive strategy for functionalization of
amines, especially those found in 5- and 6-membered ring
biocatalysts, which use molecular oxygen as oxidant, have
been subjected to several rounds of directed evolution such
that they now possess broad substrate specificity and high
enantioselectivity.[4a,b] However, to date we have not exam-
ined their ability to catalyse the desymmetrization of sym-
metrical amines. The MAO-N D5 variant was found to exhibit
good activity towards a range of substituted pyrrolidines 1–10
as determined by a colorimetric assay (Figure 1). In general,
increasing the substrate bulk and lipophilicity seemed to
correlate with higher rates of oxidation.
heterocycles.[1] In particular, C H activation by oxidation,
À
followed by nucleophilic addition, generates products of an
overall oxidative Strecker or Mannich process. Recent reports
have described the use of various metal-based oxidants to
achieve this transformation although there are few examples
of catalytic and/or enantioselective processes.[2] The enantio-
selective oxidation of N-protected pyrrolidines has been
reported using manganese–salen catalysts and iodosobenzene
as the stoichiometric oxidant.[3] Herein we report the
enantioselective enzyme-catalyzed desymmetrization of a
range of unprotected pyrrolidines to the corresponding D1-
pyrrolines (Scheme 1), which serve as useful building blocks
for the synthesis of l-proline analogues of high enantiomeric
purity.
Scheme 1. Oxidative desymmetrization of pyrrolidines.
Figure 1. Relative rates of oxidation with MAO-N D5/air for amines 1–
11. Rates were determined by absorbance measurements in a horse-
radish peroxidase coupled colorimetric assay with cell-free extracts.
Assays were performed at 10 mm substrate concentration, 308C and
pH 8.0. Rates are relative to (S)-a-methylbenzylamine (11; rate=100).
We have previously reported the use of monoamine
oxidases (MAO-N from Aspergillus niger) for the deracem-
ization of primary, secondary, and tertiary amines.[4] These
[*] Dr. V. Kꢀhler, Dr. K. R. Bailey, A. Znabet, Prof. Dr. N. J. Turner
School of Chemistry, University of Manchester
Manchester Interdisciplinary Biocentre
131 Princess Street, Manchester, M1 7DN (UK)
Fax: (+44)161-275-1311
E-mail: nicholas.turner@manchester.ac.uk
To determine the enantioselectivity of the MAO-N
catalysed oxidations, the required racemic imine standards
were prepared by elimination of HCl from the corresponding
N-chloroamines in methanolic KOH.[5] Interestingly, after
distillation of the concentrated methanolic solution of imine
12, spontaneous crystallization occurred in the distillate. The
racemic crystals consisted of homochiral trimers as shown by
X-ray crystallography.[6]
Pyrrolidine 4 was selected for initial studies, and biotrans-
formations were carried out with E. coli cells expressing
MAO-N in aqueous phosphate buffer (100 mm) and moni-
tored by GC-FID. Reactions at 20 mm substrate concentra-
Dr. J. Raftery, Dr. M. Helliwell
School of Chemistry, University of Manchester
Oxford Road, Manchester, M13 9PL (UK)
[**] We thank the BBSRC for financial support and Dr. Reuben Carr
(Ingenza) for helpful advice. V.K. is grateful for a postdoctoral
fellowship from the Swiss National Science Foundation and
Novartis Foundation. A.Z. thanks the Netherlands Organisation of
Scientific Research for a Mosaic-Fellowship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906655.
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Chemie
tion ran to high conversion (> 98%, based on 4) within 7 h
(1 mmol scale). The enantiomeric excess (ee) of the imine 12,
isolated by simple extraction (77% yield), was determined as
94% after derivatization to the trifluoroacetamide of the
corresponding a-amino nitriles (see below for assignment of
the absolute configuration).[7] Recrystallization of the trimer
of 12 from EtOAc improved the ee to at least 98%.
Optimization of this process yielded 51% of trimeric imine
12 with an ee of 98% (5 mmol scale). Similarly imines 13 and
14 were obtained from pyrrolidines 8 and 7, respectively, in
greater than 99% ee (Figure 2).
diastereoselective formation of racemic bicyclic amino acids
or derivatives starting from 3,4-substituted meso-pyrrolidines
has been demonstrated previously[5a] and has also been
described in a recent patent for the synthesis of the proline
analogue in boceprevir (16).[9b]
The diastereomeric ratio (d.r.) values obtained in the
addition of cyanide to the imines turned out to be strongly
dependent on the reaction conditions. In the buffered
aqueous medium of the biotransformation, addition of HCN
occurs initially with high diastereoselectivity (ꢀ 96:4) but the
kinetic product epimerizes rapidly at 378C (d.r. at equilibri-
um = 66:34). On the other hand, treatment of a solution of the
racemic imine 12 in CH2Cl2 with TMSCN/MeOH at room
temperature gave a d.r. of 97:3.
A similar d.r. was observed when the concentrated TBME
(methyl tert-butyl ether) extracts of the biotransformation
reaction were treated with TMSCN/MeOH in CH2Cl2 (d.r. =
96:4). Hydrolysis of the amino nitrile in aqueous HCl led,
after ion exchange chromatography, to the free amino acid 17
in 94% ee and 51% overall yield. Recrystallization of 17 from
EtOH/TBME gave the amino acid 17 in a d.r. of 150:1 and an
ee of 98% (Scheme 2).
Figure 2. Left: Imines isolated by extraction from biotransformation
mixtures. Biotransformation conditions: substrate: 20 mm (4, 8) or
10 mm (7), wet cells containing MAO-N D5: 100 gLÀ1, 100 mm KPO4
buffer, 378C, 250 rpm, pH 8.0 at start. Reactions were carried out on a
1 mmol scale (7, 8) or on a 2 mmol scale (4). The ee values were
determined after derivatization with: 1) TMSCN/MeOH (1.3 equiv)
and 2) TFAA (5 equiv) by chiral GC. [a] As determined in the concen-
trated extract, see Supporting Information. Right: Crystal structure of
trimeric 12 (single enantiomer). TMSCN=cyanotrimethylsilane;
TFAA=trifluoroacetic anhydride.
Scheme 2. Stereoselective synthesis of amino acid 17. Diastereomeric
ratios were determined by 1H NMR spectroscopy, the ee of 17 by chiral
HPLC.
To demonstrate the application of these D1-pyrrolines of
high enantiomeric purity we decided to investigate the
addition of HCN, which allows access to 3,4-substituted
proline analogues as exemplified by those found in hepatitis C
virus protease inhibitors telaprevir (15; Vertex Pharmaceut-
icals)[8] and boceprevir (16; Schering Plough; Figure 3).[9] The
Biotransformations of amines 3, 5, and 6 led to the
formation of the corresponding imines in high ee (ꢀ 98%)
with some side products, presumably due to decomposition of
the D1-pyrrolines. The addition of 2 equivalents HCN at the
start of the biotransformation allowed the isolation of the
corresponding a-amino nitriles as diastereomeric mixtures,
which were converted to trifluoroacetamides 18–20 for
analysis. Similarly the N-methylated amino acid 21 can be
obtained starting from N-methylated amine 9 in 98% ee
(Figure 4). The diastereoselectivity of HCN addition deter-
mined for the preparation of standards rac-18–20 in CH2Cl2
with TMSCN/MeOH was consistently higher (Figure 5).
To establish the absolute configuration of the imines
derived from the MAO-N reactions, a sample of d,l-17 was
treated with d-amino acid oxidase and analysed by chiral
HPLC. Oxidation of the peak corresponding to d-17 assigned
the product derived from the biotransformation as l-17
(Scheme 2). In summary, a method for the preparation of D1-
pyrrolines with very high enantiomeric excesses by enzymatic
oxidation of 3,4-substituted meso-pyrrolidines has been
developed and applied to the synthesis of amino nitriles and
nonproteinogenic amino acids.
Figure 3. Hepatitis C virus protease inhibitors telaprevir (15) and
boceprevir (16).
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Communications
chromatography over silica (EtOAc/petrol ether (b.p. 40–608C) =
1:5). Yield: 70% (based on 5, mixture of diastereomers).
Received: November 25, 2009
Published online: February 9, 2010
Keywords: asymmetric synthesis · biocatalysis · imines ·
.
oxidation
Figure 4. Biotransformation conditions: substrate: 20 mm (5, 6, 9) or
10 mm (3), 2 equiv KCN, wet cells containing MAO-N D5: 100 gLÀ1
,
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Figure 5. Diastereoselectivity of HCN addition for racemic samples
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Experimental Section
Representative procedure for the direct formation of a-amino nitriles
from pyrrolidines: cis-8-azabicyclo[4.3.0]non-3-ene (5; 124 mg,
1.01 mmol) and KCN (130 mg, 2.00 mmol) were dissolved in KPO4
buffer (5.0 mL, 1m, pH 8.0) and demineralized water, and the
pH value of the resulting solution was adjusted to pH 8.0 by addition
of aqueous HCl. This solution was combined with the cell pellet from
E. coli cultures (5.0 g) expressing MAO-N D5. The mixture was
homogenized by shaking, transferred to a 500 mL screw cap bottle
and the total volume adjusted to 50 mL by addition of demineralized
water. The mixture was agitated in a shaking incubator at 378C/
250 rpm. Workup was performed after 2 h: the mixture was centri-
fuged at 3220 g and 48C for 60 min, and the supernatant was
subsequently separated and extracted with tert-butyl methyl ether
(3 ꢀ 50 mL). The combined organic extracts were dried with Na2SO4
and concentrated by means of a rotary evaporator. The crude a-
amino nitrile was treated with TFAA (5 equiv) in dry CH2Cl2 at room
temperature for 1 h. Removal of volatiles on the rotary evaporator
left the crude trifluoroacetamide 20, which was purified by column
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J. M. Eckert, G. S. Wong, H. C. Lee, N. C. Erickson, J. A. Vance,
P. C. Nirchio, J. Weber, J.-S. D. Tsai, Z. Nanfei (Schering
Corporation), WO2007075790, 2007.
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