Separation of pyrrolidine allylation products by diastereoselective enzymatic ester hydrolysis

Article abstract of DOI:10.1016/j.tetlet.2004.12.062

A multi-parallel enzyme screen has been used to identify potential catalysts for the selective hydrolysis of diastereomeric esters and then subsequently applied in their separation upon scaleup.

Full text of DOI:10.1016/j.tetlet.2004.12.062

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 945–947
Separation of pyrrolidine allylation products by
diastereoselective enzymatic ester hydrolysis
Varinder K. Aggarwal,a,* Christopher J. Astle,a Hans Iding,b
Beat Wirzb and Mark Rogers-Evansb
aSchool of Chemistry, Bristol University, Cantockꢀs Close, Bristol BS8 1TS, UK
bF. Hoffmann-La Roche Ltd, Basel, CH-4070 Switzerland
Received 25 October 2004; accepted 10 December 2004
Available online 28 December 2004
Abstract—A multi-parallel enzyme screen has been used to identify potential catalysts for the selective hydrolysis of diastereomeric
esters and then subsequently applied in their separation upon scaleup.
Ó 2004 Elsevier Ltd. All rights reserved.
XR′3
Lewis acid
Although enzymes have been used extensively for enan-
tioselective synthesis, they have also occasionally been
used in diastereoselective synthesis.1 In the latter sce-
nario, one diastereomer is transformed by the enzyme
leaving the other diastereomer untouched, thus allowing
facile separation of the two compounds. We recently
encountered a very difficult separation of diastereomers
and considered the use of enzymes to solve the problem.
In this paper we detail a multi-parallel enzyme screen to
identify potential catalysts for the selective hydrolysis of
diastereomeric esters and its application in their separa-
tion on an increased scale.
OR
OR
O
R′
N
N
PG
PG
O
O
1
2
Scheme 1. The allylation reaction.
The far from perfect diastereocontrol in the allylation
reaction requires separation of the diastereomers. Unfor-
tunately, the cis/trans diastereomers produced in the
allylation reaction are not separable by column chroma-
tography, although they can be separated chromato-
graphically after removal of the nitrogen protecting
group, which then requires an additional reprotection
step. We considered a less cumbersome alternative strat-
egy: the use of enzymes. This strategy could result in the
selective hydrolysis of one ester diastereomer to the
carboxylic acid, leaving the other ester diastereomer
intact and therefore allowing an easy separation. Indeed,
separation of a number of enantiomeric and diastereo-
meric mixtures of amino acid derivatives bearing an ester
group (often a methyl ester) has been achieved by the use
of a selective enzymatic ester hydrolysis.7 Performing an
activity screen with a large range of commercially avail-
able enzymes in combination with multi-parallel screen-
ing equipment to investigate the potential enzymatic
separation of the cis/trans diastereomers could reveal a
pragmatic and reproducible method of separation.
Treatment of an aminal such as 1 with a Lewis acid cre-
ates an iminium intermediate, which can then be trapped
by a nucleophile. The reaction of iminium intermediates
with various nucleophiles has been widely reported as
the key step in the synthesis of numerous nitrogen con-
taining compounds.2 A particularly useful case is the
addition of allyltrimethylsilane (or allyltributylstann-
ane) to various N-acyliminium ions bearing an ester side
chain.3 This results in the predominant formation of the
cis diastereomer 24 (80:20 5) (Scheme 1) and the product
has been widely used in alkaloid synthesis. Addition of
other reagents, such as organo-cuprates, result in high
levels of trans diastereoselectivity.3d,6
In order to investigate the possibility of such a method
of separation, preparation of the allylated substrate 8
was required. Treatment of L-pyroglutamic acid 3 with
*
Corresponding author. Tel.: +44 117 954 6315; fax: +44 117 929
8611; e-mail: v.aggarwal@bristol.ac.uk
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.12.062

946
V. K. Aggarwal et al. / Tetrahedron Letters 46 (2005) 945–947
OH
i.
OMe
OMe
ii.
O
O
O
N
N
N
H
H
BOC
O
O
O
4
3
5
iii.
OMe
v.
OMe
iv.
OMe
MeO
HO
N
N
N
BOC
BOC
BOC
O
O
O
8
7
6
Scheme 2. Preparation of allyl-ester 8. Reagents and conditions: (i) SOCl2 (2 equiv), MeOH, rt, 2 h, 70%; (ii) BOC2O (1.2 equiv), DMAP (0.1 equiv),
MeCN, 3 h, 70%; (iii) LiEt3BH (1.2 equiv), THF, À78 °C, 2 h, 86%; (iv) p-TSA (0.1 equiv), MeOH, 20 h, 75%; (v) BF3ÆEt2O (1.03 equiv),
allyltrimethylsilane (4.5 equiv), À78 to 0 °C, Et2O, 17 h, 53%, 70:30 cis/trans (93% isolated yield after BOC-reprotection).
thionyl chloride in methanol gave the methyl ester 4 in
70% yield (Scheme 2). The BOC group was readily intro-
duced to give 5 (70% yield) and subsequent reduction to
the hemi-aminal 6 with Super hydride (lithium triethyl-
borohydride) followed by conversion to the methoxy
aminal in acidic methanol gave the allylation precursor
7 in good yield. The allylation transformation was then
performed, giving 8 in moderate yield (53%) and as a
70:30 mixture of cis:trans isomers. It was found that
the moderate yield was due to N-deprotection occurring
under the reaction conditions, and the yield could be im-
proved by treating the crude material with (BOC)2O.
In order to perform a selectivity screen, it was necessary
to isolate the individual cis/trans ester diastereomers.
Figure 1.
This was achieved by removal of the protecting group,
followed by separation by column chromatography
and reprotection.
for the other diastereomer then this indicates a useful
enzyme for the selective hydrolysis of one diastereomer
in the presence of the other. There were several enzymes
which showed high activity but low selectivity, but grati-
fyingly two selective enzymes were found. An esterase
from Candida lipolytica (Fluka), selectively hydrolysed
the trans diastereomer but not the cis, and a lipase from
Rhizomucor miehei (Novozymes), selectively hydrolysed
the cis diastereomer but not the trans.
With these pure diastereomers in hand, an activity assay
was performed in parallel. One hundred and twenty
commercially available hydrolases (proteases, esterases
and lipases) were combined with 1 mg of ester in the
presence of a red coloured, buffered pH indicator solu-
tion in 96-well plates. In the wells containing an active
enzyme, that is, when ester hydrolysis occurred giving
the corresponding acid, the concurrent pH change re-
sulted in a colour change of red to yellow, indicating a
positive hit (wells E3 and F3, Fig. 1. The control wells
G3 and H3 contain the same enzyme but not the ester
as occasionally the enzyme alone can trigger a colour
change). If the same enzyme produced a negative hit
The separation of a mixture of cis/trans diastereomers
was then investigated on synthetic scale (2.92 g). Treat-
ing the 7:3 cis/trans mixture of diastereomers of 8,
formed in the allylation reaction, with Candida lipoly-
tica esterase, resulted in a highly selective hydrolysis
OMe
OH
i.
+
+
N
N
BOC
BOC
O
O
cis 8
trans 9
OMe
N
BOC
O
8
OH
OMe
ii.
N
N
BOC
BOC
O
O
cis 9
trans 8
Scheme 3. Diastereoselective enzymatic ester hydrolysis. Reagents and conditions: (i) NaCl(aq) (0.1 M), sodium phosphate buffer (pH 7, 0.04 M),
Candida lipolytica esterase (Fluka: 46056), rt, 70 h, cis 8, 86% of theoretical; (ii) NaCl(aq) (0.1 M), sodium phosphate buffer (pH 7, 0.04 M),
Rhizomucor miehei lipase (Novozymes), 43 h, trans 8, 52% of theoretical.

V. K. Aggarwal et al. / Tetrahedron Letters 46 (2005) 945–947
947
of the trans diastereomer allowing the trans carboxylic
References and notes
acid 9 to be washed out in the aqueous phase leaving
highly pure cis 8 in excellent yield (86% of theoretical)
(Scheme 3). Treating the same mixture of diastereomers
with R. miehei lipase resulted in a less selective ester
hydrolysis, with 52% of the trans ester being recovered,
after the cis diastereomer had been completely
hydrolysed.
1. Ohno, A. Rev. Heteroatom Chem. 1999, 20, 29.
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In conclusion, the use of a selective enzymatic ester
hydrolysis complements the allylation reaction perfectly.
The ester moiety, initially responsible for providing
moderately high diastereoselectivities during the allyl-
ation reaction, subsequently provides a handle with
which to remove the unwanted trans diastereomer,
allowing rapid access to enantiomerically and diastereo-
merically pure cis intermediates.
Acknowledgements
4. The origin of the syn selectivity is believed to arise from
activation of the allyl silane by the ester moiety thus
delivering the reagent syn to the ester group: Oba, M.;
Koguchi, S.; Nishiyama, K.; Kaneno, D.; Tomoda, S.
Angew. Chem., Int. Ed. 2004, 43, 2412.
5. The level of syn selectivity varies considerably in the
literature from 80:20 to 95:5 even for the same substrate
(see Refs. 3 and 6)!
6. (a) Wistrand, L.-G.; Skrinjar, M. Tetrahedron 1991, 47,
573; (b) Collado, I.; Ezquerra, J.; Pedregal, C. J. Org.
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Evans, M. Tetrahedron 2004, 60, 647.
We thank Patrick Stocker for aiding with enzymatic
preparative experiments and Hoffman-La Roche for
financial support.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet-
let.2004.12.062. Experimental procedures and full char-
acterisation for all compounds. This material is
available free of charge via the internet.