Carboxymethylproline synthase catalysed syntheses of functionalised N-heterocycles

Article abstract of DOI:10.1039/b924519g

The utility of wild-type and variant carboxymethylproline synthases for biocatalysis was demonstrated by preparing functionalised 5-, 6- and 7-membered N-heterocycles from amino acid aldehydes and (alkylated) malonyl-coenzyme A derivatives; the N-heterocycles produced were converted to the corresponding bicyclic β-lactams by a carbapenem synthetase. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b924519g

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Carboxymethylproline synthase catalysed syntheses of functionalised
N-heterocyclesw
´
Refaat B. Hamed, Jasmin Mecinovic, Christian Ducho,z
Timothy D. W. Claridge and Christopher J. Schofield*
Received (in Cambridge, UK) 23rd November 2009, Accepted 22nd December 2009
First published as an Advance Article on the web 12th January 2010
DOI: 10.1039/b924519g
The utility of wild-type and variant carboxymethylproline synthases
for biocatalysis was demonstrated by preparing functionalised 5-,
6- and 7-membered N-heterocycles from amino acid aldehydes and
(alkylated) malonyl-coenzyme A derivatives; the N-heterocycles
produced were converted to the corresponding bicyclic b-lactams
by a carbapenem synthetase.
report the utilisation of CarB/CarB variants/ThnE for
the chemoenzymatic formation of functionalised 5-, 6- and
7-membered N-heterocycles, which were converted to their
bicyclic b-lactam derivatives by b-lactam synthetase catalysis.
Our results reveal the synthetic versatility of carboxymethyl-
proline synthases and reveal that crotonases are suitable
catalysts for the production of functionalised heterocycles.
Initially, we synthesised and tested analogues of L-GHP 4
with different chain lengths as substrates for CarB and ThnE.
When the higher homologue of 4, L-aminoadipate semi-
aldehyde 9 (Table 1), was synthesised (Scheme S1, ESIw) and
incubated with 3 in the presence of CarB or ThnE, (2S,6S)-6-
(carboxymethyl)-pipecolic acid 10 was isolated (entry 3,
Table 1). The trans-stereochemistry of 10 was assigned by
NMR analyses (Fig. 2, S3 and S5, ESIw). This result contrasts
with that for the lower homologue, L-aspartate semialdehyde
11, which is not a substrate of CarB,11 nor, we found, of ThnE.
We then tested the ability of CarB and ThnE to accept the
7-membered homologue of 4, L-aminopimelate semialdehyde
12,12 as a substrate. The formation of a product with m/z 202
[M + H]+ corresponding to 7-(carboxymethyl)-azepane-2-
carboxylic acid (CMAC) was observed with CarB but not with
ThnE. However, the yield was insufficient for stereochemical
assignment of the product by NMR. Based on analysis of the
CarB crystal structure8 active site variants of CarB were then
prepared, in which residues predicted to be involved in the
binding pocket for 4/12 were substituted with less bulky
residues aiming to provide sufficient space to accommodate
the longer methylene chain of 12 (Fig. 1). Screening of these
variants by analytical LC-MS for the formation of CMAC
from 12 and 3 revealed that the CarB His229Ala variant was
able to form the target compound in a yield B3 times higher
than that obtained with wild-type CarB. The isolated product
of the scaled-up assay of CarBH229A was assigned by NMR
as (2S,7S)-7-(carboxymethyl)-azepane-2-carboxylic acid 13
(Fig. 2, S15 and S16 (ESIw), entry 5, Table 1). The homologue
of 4, L-aminosuberate semialdehyde 14, was synthesised
(Scheme S2, ESIw) and tested as a substrate for CarB/ThnE/
CarB variants in the presence of 3. The incubations led to no
detectable product with the anticipated m/z 216 [M + H]+ by
LC-MS assays.
The essential steps in the biosynthesis of the simplest carbapenem
antibiotic, (5R)-carbapenem-3-carboxylate 1, are catalysed by
three enzymes (CarB, CarA and CarC)1,2 in Pectobacterium
carotovorum. More enzyme catalysed steps are involved in the
biosynthesis of the C-2 and C-6 functionalised carbapenem
thienamycin 2 in Streptomyces cattleya.3 In the biosynthesis of
both 1 and 2, an early step is proposed to be catalysed by a
member of the crotonase superfamily carboxymethylproline
synthase:4 CarB in P. carotovorum5–8 and ThnE in S. cattleya.9
CarB and ThnE convert malonyl-CoA 3 and L-glutamate
semialdehyde 4–5-hydroxyproline 5–pyrroline-5-carboxylate
6 (collectively L-GHP) into (2S,5S)-carboxymethylproline
(t-CMP) 7 (Scheme 1). It is proposed that CarB catalysis
proceeds via decarboxylation of 3 to give an enzyme bound
enolate which reacts with 6 in a C–C bond forming reaction,
followed by thioester hydrolysis of the acyl intermediate to
give 7 and CoASH.5,6
Saturated N-heterocycles are one of the most important
classes of cyclic compounds from a pharmaceutical/biomedical
perspective, with many such ring systems in clinical/preclinical
use. The development of biocatalytic routes for their
production, particularly those suitable for the production of
chiral/functionalised rings, is of interest. The findings5–7,9 that
methylmalonyl- (8), ethylmalonyl- and dimethylmalonyl-CoA
are accepted by CarB upon incubation with L-GHP to give
C6-alkylated-t-CMP stimulated us to investigate other potential
substrate analogues for CarB and ThnE, with a view to
exploring their potential as biocatalysts for the production of
N-heterocycles with ring sizes greater than 5. Excepting for their
use in the desymmetrisation of b-diketones,10 there are few
reported applications of crotonases in biocatalysis. Here we
University of Oxford, Department of Chemistry,
Chemistry Research Laboratory, Mansfield Road, Oxford,
UK OX1 3TA. E-mail: christopher.schofield@chem.ox.ac.uk;
Fax: +44 (0)1865275674; Tel: +44 (0)1865275625
w Electronic supplementary information (ESI) available: Methods and
spectroscopic analyses of products. See DOI: 10.1039/b924519g
z Current address: Georg-August-University Gottingen, Department
¨
of Chemistry, Institute of Organic and Biomolecular Chemistry,
Gottingen, Germany.
¨
In aqueous solutions, amino acid aldehydes exist as an
equilibrium mixture of the aldehyde/hydrated aldehyde, cyclic
hemiaminal and cyclic imine forms (Scheme 1). The latter
form is proposed to be the substrate for CarB.5,6 Although
other factors are likely involved, it is notable that for 11 and
14, which were not substrates, no evidence for the imine form
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Scheme 1 The proposed roles of CarB and ThnE in the biosynthesis of (5R)-carbapenem-3-carboxylic acid 1 and thienamycin 2.
Table 1 Chemoenzymatic synthesis of functionalised 5-, 6- and 7-membered N-heterocycles catalysed by CarB/CarBH229A/ThnE
Substrate
Product
Yieldb (%)
A(n)
B(R)
dra
R1
R2
m
Entry
Enzyme
1
2
4(2)
4(2)
3(H)
8(CH3)
CarB
CarB
7
—
1 : 1
H
CH3
H
H
CH3
H
H
H
CH3
H
H
CH3
H
H
1
1
44
32
17a
17b
10
3
4
9(3)
9(3)
3(H)
8(CH3)
CarB
CarB
—
2 : 1
2
2
11
5
15a
15b
13
5
6
12(4)
12(4)
3(H)
8(CH3)
CarBH229A
CarBH229A
—
—
H
CH3
3
3
6
—
16c
a
b
dr: diastereomeric ratio as determined by 1H NMR spectroscopy under standard incubation conditions. The % overall yield (isolated) was
determined over the following steps: deprotection of amino acid aldehydes, incubation with enzyme, LC-MS purification; products were quantified
by 1H NMR using [2H]4-trimethylsilylpropionate as an external standard. The stereochemistry at C-7/C-8 of 16 was not defined.
c
Fig. 1 A view derived from CarB crystal structure8 with the acetyl-
CoA enolate and the imine form of 12 modelled into the active site. The
position of the acetyl-CoA enolate was fixed by the oxy-anion hole
forming residues (Gly62 and Met108). His229 is one of the closest
Fig. 2 1H NMR spectra for the products of CarB catalysis (7 and 10),
residues to the imine form of 12 (B3.7 A from t-nitrogen of His229 to
CarBH229A catalysis (13), and CarA catalysis (20).
b-carbon of 12).
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synthetase (CarA) from P. carotovorum. CarA catalysed the
conversion of compounds 7, 17a/17b, 10, 15a, and 13 to their
respective bicyclic b-lactam derivatives 18, 19a, 19b, 20, 21,
and 22 as demonstrated by LC-MS analyses (Fig. 3). In case of
10, 15a and 13, the CarA reaction was scaled-up and the
structures of the isolated products were confirmed by NMR
analyses (20: Fig. 2, S19 and S20, 21: Fig. S21 and 22: Fig. S22,
ESIw). In the case of the CarA catalysed reaction of the
mixture of 15a and 15b, we observed selective conversion of
15a to give 21.
In conclusion, we have demonstrated the utility of carboxy-
methylproline synthases as biocatalysts for preparing functionalised
N-heterocycles in a diastereoselective fashion, and that the
products can be converted into the respective bicyclic
b-lactams of potential application in the semisynthesis of
stable b-lactam antibiotics. More generally, the results illustrate
the potential of the crotonase superfamily enzymes for
biocatalysis.
Fig. 3 LC-MS data (negative ion mode) for the bicyclic b-lactams
produced by CarA catalysis.
We thank the BBSRC, the Ministry of Higher Education,
Egypt (R. B. H.), and the Deutsche Akademie der Naturforscher
Leopoldina, Germany (BMBF-LPD 9901/8-137 to C.D.), for
funding, Dr Edward T. Batchelar for helpful discussions, and Dr
Ian Clifton for assistance with Fig. 1.
formation was detected by NMR analyses (pD 7.7). In
contrast, for 4, 9 and 12, which are substrates, the imine form
was detected. The fraction of the imine form (pD 7.7) varied as
follows: 4 (imine to other forms, 49 : 1), 9 (imine to other
forms, B1 : 1) and 12 (imine to other forms, B1 : 9) as
analysed by integrating the imine C–H versus the Ca–H
resonances.
Notes and references
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We then investigated the ability of the carboxymethyl-
proline synthases to catalyse the formation of C-7/C-8
alkylated 6- and 7-membered N-heterocycles, respectively.
Incubation of 9 with CarB and C-2 epimeric methylmalonyl-
CoA 8 led to the observation of the two C-7 methyl-substituted
epimers (15a and 15b) in a B2 : 1 ratio (entry 4, Table 1).
Assignment of the stereochemistry at C-7 was hindered by
rotation about the C-6–C-7 bond; b-lactam ring formation
enabled assignment of the stereochemistry at C-7 of 15a as R
(Fig. S21, ESIw). Incubation of 9 with ThnE and 8 led to no
detectable observation of a product with the anticipated
m/z 202 [M + H]+ by LC-MS. Incubation of 12 with
CarBH229A and 8 led to the observation of a single chromato-
graphic peak with m/z 216 [M + H]+ corresponding to
8-methyl-CMAC 16 (entry 6, Table 1); no detectable product
was observed with wild-type CarB. The yield of 16 was too low
for stereochemical assignment at C-7/C-8 by NMR.
b-Lactams are used therapeutically not only as antibiotics,
but also as protease inhibitors (e.g. inhibition of HIV protease),13
to reduce cholesterol levels14 and as neuroprotective agents.15
Moreover, although many b-lactams including penicillin and
cephalosporins are produced by semisynthesis, others including
carbacephems (which are known to have higher chemical and
serum stability than cephalosporins16) are produced by
expensive total synthesis. The carboxymethyl-functionalised
N-heterocycles, generated in this study, were therefore tested
as substrates for the b-lactam forming enzyme carbapenam
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 1413–1415 | 1415