Identification and application of enantiocomplementary lactamases for Vince lactam derivatives

Article abstract of DOI:10.1002/cctc.201402077

Four enzymes showing hydrolytic activity on derivatives of 2-azabicyclo[2.2.1]hept-5-en-3-one (Vince lactam) were successfully identified through analysis of protein crystal structure and amino acid sequence alignments. Enantiocomplementary activities were observed on Vince lactam and its saturated analog 2-azabicyclo[2.2.1]heptan-3-one with non-heme chloroperoxidase (CPO-T) from Streptomyces aureofaciens, cyclic imide hydrolase (CIH) from Pseudomonas putida, polyamidase (NfpolyA) from Nocardia farcinica, and amidase (AMI) from Rhodococcus globerulus, and perfect kinetic resolution was achieved (E>200). Computational analysis of amide bond resonance stabilization in lactams correlated well with the overall reactivity pattern of the lactams as a function of ring size and strain. The biocatalysts cloned and investigated in this study could be of interest for the synthesis of enantiopure carbocyclic nucleoside analogues.

Full text of DOI:10.1002/cctc.201402077

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DOI: 10.1002/cctc.201402077
Identification and Application of Enantiocomplementary
Lactamases for Vince Lactam Derivatives
[
a, b]
[a]
[a]
[a]
[b]
Zeinab Assaf,
Elisabeth Eger, Zeljko Vitnik, Walter M. F. Fabian, Doris Ribitsch,
[b, c]
[a]
[a]
Georg M. Guebitz,
Kurt Faber, and Mꢀlanie Hall*
Four enzymes showing hydrolytic activity on derivatives of 2-
azabicyclo[2.2.1]hept-5-en-3-one (Vince lactam) were success-
fully identified through analysis of protein crystal structure and
amino acid sequence alignments. Enantiocomplementary activ-
ities were observed on Vince lactam and its saturated analog
Numerous chemical strategies have been developed for the
synthesis of enantiopure carbocyclic nucleosides, such as Mit-
[5]
sunobu and transition-metal-mediated reactions. The imple-
mentation of biocatalysis, in contrast, provides an attractive, ef-
ficient, and greener alternative. Chemoenzymatic processes
have therefore been developed on the basis of the kinetic res-
olution of racemic Vince lactam, which generates two enantio-
pure compounds of synthetic value (Schemes 1 and 2). Several
sources of enantiospecific enzymatic activities have been re-
ported: 1) (+)-g-Lactamase activities have been identified in
2-azabicyclo[2.2.1]heptan-3-one with non-heme chloroperoxi-
dase (CPO-T) from Streptomyces aureofaciens, cyclic imide hy-
drolase (CIH) from Pseudomonas putida, polyamidase (NfpolyA)
from Nocardia farcinica, and amidase (AMI) from Rhodococcus
globerulus, and perfect kinetic resolution was achieved (E>
[6]
200). Computational analysis of amide bond resonance stabili-
whole cells of Pseudomonas cepacia, Pseudomonas solanacea-
[7]
zation in lactams correlated well with the overall reactivity pat-
tern of the lactams as a function of ring size and strain. The
biocatalysts cloned and investigated in this study could be of
interest for the synthesis of enantiopure carbocyclic nucleoside
analogues.
rum (referred to as ENZA 20), and Pseudomonas fluorescens
[1c]
(ENZA 22). Related (+)-enantioselective enzymes have been
[8]
isolated from Sulfolobus solfataricus, Comamonas acidovor-
[9]
[10]
ans, and Bradyrhizobium japonicum and were cloned and
overexpressed in E. coli. 2) Microorganisms exhibiting (À)-g-lac-
[7]
tamase activity include Rhodococcus sp. (ENZA 1) and Aureo-
[1c]
bacterium sp. (ENZA 25). Analysis of the crystal structure of
(À)-g-lactamase isolated from Aureobacterium sp. attributed
the protein to the a/b-hydrolase fold family and supported
The bicyclic g-lactam 2-azabicyclo[2.2.1]hept-5-en-3-one (Vince
lactam, 1a) and derivatives thereof are versatile synthons that
have been exploited in enantiomerically pure form as precur-
sors for the preparation of carbocyclic nucleoside analogues
that possess therapeutic properties ranging from antibiotic ac-
[11]
a mechanism based on a Ser-His-Asp catalytic triad. Addi-
tionally, promiscuous lactamase activity on Vince lactam and
a few derivatives thereof (including 2-azabicyclo[2.2.1]heptan-
3-one, N-tert-butoxycarbonyl-protected and N-acetylated Vince
lactam) was identified in a range of commercially available hy-
[
1]
tivity to antitumor and antiviral activities. The (À)-enantiomer
of Vince lactam, for instance, was employed as a precursor in
the synthesis of purine carbonucleosides, including (À)-carbo-
[12]
drolytic enzymes (esterases, lipases, and proteases). Some of
these biocatalysts, regardless of their enantioselectivity, have
been characterized and protein structures are available. How-
ever, enantiocomplementary enzymes, applicable to the syn-
thesis of a broad set of Vince lactam derivatives that would
allow a more systematic use of biocatalysis in carbocyclic nu-
cleoside synthesis, are lacking. We therefore initiated a broad
search for potential new enzyme candidates, with focus on
Vince lactam derivatives so far not known to be substrates in
enzyme-catalyzed lactam hydrolysis. Non-heme cofactor-free
[
2,3]
vir and abacavir, both active against HIV.
The hydrolyzed
Vince lactam amino acid product (i.e., 1b) has also been used
as a starting material in the synthesis of pharmaceutical tar-
[
4]
gets, such as melogliptin and MK-0812 (Scheme 1).
[a] Dr. Z. Assaf, E. Eger, Dr. Z. Vitnik, Prof. Dr. W. M. F. Fabian, Prof. Dr. K. Faber,
Dr. M. Hall
Department of Chemistry
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: melanie.hall@uni-graz.at
[
13]
chloroperoxidase (CPO-T) from Streptomyces aureofaciens,
[14]
polyamidase (NfpolyA) from Nocardia farcinica,
dase E2 (MAE2) from Bradyrhizobium japonicum,
malonami-
[15]
amidase
AMI) from Rhodococcus globerulus, and cyclic imide hydro-
[b] Dr. Z. Assaf, Dr. D. Ribitsch, Prof. Dr. G. M. Guebitz
[16]
(
ACIB GmbH c/o
[17]
lase (CIH) from Pseudomonas putida were all cloned, overex-
pressed in E. coli, and purified, and their catalytic activity and
enantiospecificity were evaluated. In addition, computational
analysis of the resonance stabilization of the lactam amide
bond was used to rationalize the differences observed in
lactam reactivity.
Petersgasse 14, 8010 Graz (Austria)
[
c] Prof. Dr. G. M. Guebitz
Institute for Environmental Biotechnology
University of Natural Resources and Life Sciences
Konrad Lorenz Strasse 20, 3430 Tulln (Austria)
Supporting information (cloning and enzyme purification methods, com-
putational details, analytical methods, and synthesis of reference materi-
als) for this article is available on the WWW under http://dx.doi.org/
Five proteins were selected for initial lactamase activity
screening based on amino acid sequence or structure similarity
10.1002/cctc.201402077.
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lactamase activity on rac-1a. CIH and NfpolyA were
excellent catalysts, as they both displayed high con-
version levels and enantioselectivity (Table 1, entries 1
and 4). Whereas CIH was highly selective for (À)-1a
leading to perfect kinetic resolution [(1S,4R)-1b in
>
99% ee, E>200], NfpolyA gave access to the oppo-
site enantiomer (1R,4S)-1b in 96%ee (E=115). CPO-T
and AMI both showed moderate (À)- and (+)-enan-
tiopreference, respectively. At elevated conversions
(
66 and 76%, respectively; Table 1, entries 2 and 5),
nonhydrolyzed substrate 1a could be obtained in
enantiomerically pure form (>99%ee). A minimal
level of activity was observed with MAE2 (5% conver-
sion; Table 1, entry 3), which did not allow accurate
determination of the ee value for 1b (at the GC de-
tection limit for chiral measurements). The reaction
did not show higher conversion levels after a reaction
Scheme 1. Vince lactam (1a) and corresponding hydrolyzed product 1b used for the
generation of drugs and clinical candidates.
searches by using enzymes active on Vince lactam or amides
[
18]
as templates (searches conducted with BLAST
and PDBe-
[
19]
Fold ). These proteins were chosen on the basis of the follow-
[13]
ing considerations: CPO-T from Streptomyces aureofaciens,
despite its moderate level of amino acid sequence identity
68%), shares striking structural similarities to (À)-g-lactamase
from Aureobacterium sp. [0.53 root mean square deviation
RMSD) and 0.92 Q-score (quality function of Ca alignment) in
structure alignment search using PDBeFold]. NfpolyA from No-
(
[
11]
(
[
14]
cardia farcinica, besides its activity on nylon, displays aryl
acylamidase activity. It belongs to the amidase signature family
of proteins and shares high sequence identity (95%) with w-
[
20]
octalactam hydrolase from Rhodococcus sp. Oct1, which is
active on large monocyclic lactams. MAE2 from Bradyrhizobium
[
15]
japonicum is an enzyme specific for the hydrolysis of the
amide bond in malonamate. MAE2 was selected through struc-
ture similarity search by using the 6-aminohexanoate cyclic
dimer hydrolase from Flavobacterium sp. KI72 as a template
Scheme 2. Kinetic resolution of Vince lactam (1a) catalyzed by lactamases
and reactive Vince lactam derivatives 2a–4a identified in this study.
(
2.1 RMSD and 0.52 Q-score, with 24% overall sequence identi-
time of 7 days, and similar results were obtained with (+)-1a
and (À)-1a employed separately (data not shown). These re-
sults were further confirmed by applying the five enzymes to
the hydrolysis of enantiopure (À)-1a (full conversion by CIH
and CPO-T, 53% conversion by AMI, 4 and 2% conversion by
MAE2 and NfpolyA, respectively; Table S1, Supporting Informa-
tion). Next, the hydrolytic activity of the five enzymes was in-
vestigated on several Vince lactam derivatives (Scheme 2) pre-
pared by hydrogenation (see rac-2a), N-methylation (see rac-
3a), and epoxidation (see rac-4a) of rac-1a. The reduction of
the C=C bond in rac-1a to produce rac-2a was found to have
a strong influence on all enzymatic activities (Table 1, en-
tries 6–9). CPO-T now showed perfect kinetic resolution of rac-
2a with conserved enantiopreference for (À)-2a (E>200;
Table 1, entry 6), whereas AMI displayed excellent opposite
(+)-stereoselectivity with somewhat reduced activity (E>200;
Table 1, entry 8). Compound rac-2a was poorly converted by
(+)-selective NfpolyA (6% conversion; Table 1, entry 9) and sur-
prisingly unreactive with CIH (conversion <1%; Table 1,
entry 7). These data were confirmed by the absence of activity
ty). AMI from Rhodococcus globerulus is encoded by a gene
that coexists with alkylaldoxime dehydratase and nitrile hydra-
tase genes in a gene cluster responsible for alkylaldoxime me-
[
16]
tabolism in Rhodococcus globerulus. It is 97% identical at the
amino acid sequence level to an amidase from Rhodococcus
[
21]
sp. N-771, which is structurally related to MAE2 (1.51 RSMD
[17]
and 0.48 Q-score). Finally, CIH from Pseudomonas putida was
chosen on the basis of its hydrolytic activity on cyclic imides,
dihydrouracil, and hydantoin, all of which are compounds that
are structurally related to lactams. All five proteins were suc-
cessfully cloned, overexpressed with a His tag, and employed
in lactamase activity screening (Scheme 2) in purified form (see
the Supporting Information for protein purification proce-
dures). The substrate scope of NfpolyA, AMI, MAE2, CIH, and
CPO-T in lactam hydrolysis reactions was investigated by set-
ting up a screening system with 2–5 mm pure enzyme in aque-
ous buffer (50 mm phosphate buffer, pH 7.5, 308C).
First, the reaction was performed with racemic Vince lactam
(
rac-1a) as a model substrate. Overall, all five enzymes showed
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was highly active on rac-4a (47% conversion after a reaction
time of 2 h) but E values never exceeded 7 (Table S2). Addition-
Table 1. Hydrolysis of racemic Vince lactam (1a) and derivatives 2a--4a
by using purified proteins.
[
a]
ally,
we
2.2.1]heptan-3-one (rac-6a) and enantiomer (1R,4S,5R,6S)-6a
Figure 1), a valuable precursor also used in the preparation of
tested
(Æ)-exo-cis-5,6-dihydroxy-2-azabicyclo-
[
b]
[22]
Entry Substrate Enzyme Conv. [%]
ee [%]
1b–4b
E
[
(
1
a–4a
1
2
3
4
5
6
7
8
9
1
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-2a
rac-2a
rac-2a
rac-2a
rac-3a
rac-4a
rac-4a
CIH
50
66
5
45
76
50
<1
21
6
50
(+) >99 (1S,4R) >99 >200
carbocyclic nucleosides and obtained by dihydroxylation of 1a
CPO-T
MAE2
NfpolyA
AMI
CPO-T
CIH
AMI
NfpolyA
AMI
CPO-T
AMI
(+) >99 (1S,4R)
52
15
n.d.
115
8
[1a,24]
by using OsO4.
Both were found to be nonsubstrates for
(+)
(À)
5 n.d.
77 (1R,4S)
(À) >99 (1R,4S)
all enzymes tested. Given the enhanced polarity and hydrophi-
licity of 6a relative to those of other derivatives, which was as-
sumed to impede substrate binding, corresponding acetonide
rac-7a was synthesized and tested (Figure 1). This substrate
was also found to be unreactive, whereby the methylenedioxy
moiety certainly rendered the lactam too bulky. MAE2 showed
no activity on substrates 2a–7a. The catalytic mechanism of
this enzyme in the hydrolysis of its natural substrate malona-
mate has been studied, and it has been revealed that Arg158
forms an ion pair with the carboxylate of malonamate via the
guanido moiety. The Arg158 residue is critical for binding and
96
32
(+) >99 (1R,3S) >99 >200
n.d.
(À)
(À)
n.d.
n.d.
26 (1S,3R) >99 >200
6 (1S,3R) >99 >200
[c]
0
(À) >99 (1R,4S) >99 >200
[
[
d]
d]
[e]
1
1
>99
>99
n.d.
n.d.
rac
rac
<1
<1
12
[
a] Conditions: 10 mm substrate, 100 mg enzyme (2–5 mm) in phosphate
buffer (50 mm, pH 7.5), 24 h, 308C, 120 rpm. The ee values were obtained
by GC on a chiral column after double derivatization with TMSCHN and
Ac O in the presence of 4-(N,N-dimethylamino)pyridine and pyridine; au-
2
2
[15b]
thentic reference materials were used for identification (see the Support-
improves substrate recognition and catalytic rate.
The ab-
ing Information). n.d.=Not determined. [b] Conversion calculated by
sence of the carboxylate moiety in all lactams tested presuma-
bly reduced binding affinity and is likely the reason for the in-
activity of MAE2 in the hydrolysis of substrates 2a–7a. In addi-
tion to bicyclic lactams 1a–7a, all five enzymes were investi-
gated on several monocyclic lactams, hydantoin (24a) and 3-
methyl-1-phenylpyrrolidine-2,5-dione (25a), but none of these
compounds showed detectable activity (Figure 1).
[
eesubstrate/(eesubstrate +eeproduct)]. [c] CPO-T, CIH, MAE2, and NfpolyA not
active on rac-3a. [d] Compound 4b was identified by NMR spectroscopy
after scale-up of the enzymatic reaction (no trace amount of remaining
rac-4a was detected). [e] Maximum E value of 7 was reached in the first
7
h of the reaction (Table S2).
from NfpolyA, CIH, and AMI on (À)-2a, whereas full conversion
was observed with CPO-T (Table S1). N-Methylation of 1a had
a drastic effect on the enzymatic activity of all candidates. It
led to complete loss of activity with NfpolyA, CIH, and CPO-T
on rac-3a. AMI, in contrast, displayed perfect kinetic resolution
and (+)-selectivity (50% conversion and E>200; Table 1,
entry 10). No activity was detected with any of the four en-
zymes on (À)-3a (Table S1). Re-
From the results reported here and from analysis of the liter-
ature, it seems that only a handful of proteins can hydrolyze
saturated 2a with moderate to good activity, whereas racemic
epoxy analogue 4a could be fully hydrolyzed by a few pro-
teins. Vince lactam (1a), on the contrary, is extremely reactive
(Table 1). Overall, monocyclic lactams seem to be more difficult
substrates: No enzyme active on g-butyrolactam (8a) and d-va-
markably, reduction of the C=C
bond combined with methyla-
tion of the amide nitrogen atom
led to unreactive substrate rac-
5
a (Figure 1), even with AMI,
which was active on rac-2a and
rac-3a. Further, we investigated
the activity on (Æ)-exo-3-oxa-6-
2,4
azatricyclo[3.2.1.0 ]octan-7-one
(
rac-4a), a compound that offers
effective entry into the synthesis
of 2-deoxy-, 3-deoxy-, and ara-
cyclopentyl carbocyclic nucleo-
[
23]
sides. The racemic epoxide de-
rivative was not converted by
NfpolyA and CIH. In contrast, full
conversion was obtained with
CPO-T and AMI (Table 1, en-
tries 11 and 12), which indicates
a surprising and complete loss
of stereoselectivity upon epoxi-
dation of substrate rac-1a. A
Figure 1. Structures of unreactive Vince lactam derivatives and monocyclic lactams with CPO-T, CIH, NfpolyA,
time study revealed that CPO-T MAE2, and AMI. Hydantoin (24a) is only reactive with CIH and was used to evaluate its activity.
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lerolactam (9a) has been identified yet, and only one protein
has been shown to hydrolyze w-heptalactam (10a), albeit with
Kinetic resolution of Vince lactam derivatives is a synthetically
useful transformation, as it gives access to two enantiopure
building blocks for the synthesis of various pharmaceuticals, in-
cluding carbocyclic nucleosides. Four highly active lactam-hy-
drolyzing enzymes were identified, all of which display excel-
lent enantioselectivities and activities, and this therefore en-
larges the repertoire of proteins with lactamase activity. Impor-
tantly, new Vince lactam derivatives could be hydrolyzed in an
enantiocomplementary fashion by using NfpolyA, AMI, CPO-T,
and CIH. The sequential synthesis of these derivatives in an
enantiopure form (Vince lactam derivatization followed by en-
zymatic kinetic resolution) appears especially valuable for cases
in which synthesis routes are not highly chemoselective (such
[20]
a low activity level (OctHD from Rhodococcus sp. Oct1). The
extreme variability in hydrolytic activities of all enzymes tested
on bicyclic Vince lactam derivatives, combined with the ab-
sence of activity on simpler monocyclic lactams, could be relat-
ed to the chemical stability of the amide bond. The CÀN bond
of the lactam has intrinsic partial double-bond character, and
this resonance stabilization goes in hand with cis–trans isomer-
[
25]
ism (Scheme 3).
[26]
as the synthesis of secondary amines ) and derivatization of
enantiopure compounds (i.e., after kinetic resolution) would
imply loss of material of higher value. The observed resistance
of lactams to enzymatic hydrolysis could be correlated to their
chemical stability, evaluated by computational analysis of the
CÀN double-bond character of the lactam amide bond.
Scheme 3. CÀN double-bond character and cis–trans isomerism of the
amide bond.
To estimate the relevance of the CÀN double-bond character
in several lactams, natural resonance theory analysis was used
to calculate the weight of the respective resonance structures.
This resulted in more than one resonance structure described
by a C=N bond. The sum of the individual weights of these
structures was therefore considered as a measure of the CÀN
double-bond character (see the Supporting Information for de-
tails and Table S2). With higher partial CÀN double-bond char-
acter, nucleophilic attack at the carbon atom is impeded and
the hydrolysis rates decrease. This correlated well with the rela-
tive enzymatic activities, and thus provides a partial explana-
tion for the general trend observed in biohydrolysis reactions
Acknowledgements
This work has been supported by the Federal Ministry of Science,
Research and Economy (BMWFW), the Federal Ministry of Traffic,
Innovation and Technology (BMVIT), the Styrian Business Promo-
tion Agency SFG, the Standortagentur Tirol and ZIT—Technology
Agency of the City of Vienna through the COMET-Funding Pro-
gram managed by the Austrian Research Promotion Agency FFG.
We thank Sabine Zitzenbacher for technical support. Dr. Hꢀlꢁne
Hall is thanked for her help in editing.
(
Figure 2).
Keywords: enantioselectivity · hydrolases · kinetic resolution ·
natural resonance theory · lactams
The practical applicability of these biotransformations was
demonstrated on a preparative scale (0.5 g). Both (À)-1a and
+)-2a could be recovered in >99%ee by using purified Nfpo-
lyA and lyophilized whole cells of E. coli overexpressing CPO-T,
(
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Published online on July 18, 2014
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