Substrate-product analogue inhibitors of isoleucine 2-epimerase from: Lactobacillus buchneri by rational design

Article abstract of DOI:10.1039/c9ob01823a

A rational approach that may be applied to a broad class of enzyme-catalyzed reactions to design enzyme inhibitors affords a powerful strategy, facilitating the development of drugs and/or molecular probes of enzyme mechanisms. A strategy for the developm

Full text of DOI:10.1039/c9ob01823a

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DOI: 10.1039/C9OB01823A
ARTICLE
Substrate-Product Analogue Inhibitors of Isoleucine 2-Epimerase
from Lactobacillus buchneri by Rational Design
Noa T. Sorbara,a Joshua W.M. MacMillan,b Gregory D. McCluskey,a and Stephen L. Bearne*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
A rational approach that may be applied to a broad class of enzyme-catalyzed reactions to design enzyme inhibitors affords
a powerful strategy, facilitating the development of drugs and/or molecular probes of enzyme mechanisms. A strategy for
the development of substrate-product analogues (SPAs) as inhibitors of racemases and epimerases is elaborated using
isoleucine 2-epimerase from Lactobacillus buchneri (LbIleE) as a model enzyme. LbIleE catalyzes the PLP-dependent,
reversible, racemization or epimerization of nonpolar amino acids at the C-2 position. The enzyme plays an important role
in the biosynthesis of branched-chain D-amino acids and is a potential target for the development of antimicrobial agents.
3-Ethyl-3-methyl-L-norvaline (Ki = 2.9 ± 0.2 mM) and 3-ethyl-3-methyl-D-norvaline (Ki = 1.5 ± 0.2 mM) were designed as SPAs
based on the movement of the sec-butyl side chain of the substrate L-Ile during catalysis, and were competitive inhibitors
with binding affinities exceeding that of L-Ile by 1.3- and 2.5-fold, respectively. Surprisingly, these compounds were not
substrates, but the corresponding compounds lacking the 3-methyl group were substrates. Unlike serine, glutamate, and
proline racemases, which exhibit stringent steric requirements at their active sites, the active site of LbIleE was amenable to
binding bulky SPAs. Moreover, LbIleE bound the SPA 2,2-di-n-butylglycine (Ki = 11.0 ± 0.2 mM) as a competitive inhibitor,
indicating that the hydrophobic binding pocket at the active site was sufficiently plastic to tolerate gem-dialkyl substitution
at the -carbon of an amino acid. Overall, these results reveal that amino acid racemases/epimerases are amenable to
inhibition by SPAs provided that they possess a capacious active site.
DOI: 10.1039/x0xx00000x
the R- and S-pockets, as demonstrated for mandelate
racemase8,9 and -methylacyl-coenzyme A (CoA) racemase
(AMACR).1,2 Such SPAs combine the structural attributes of the
two ligands and enhanced binding affinity is anticipated due to
the additional binding determinants, provided that the added
steric bulk is tolerated. Unfortunately, application of this
inhibitor design strategy to glutamate,10 serine,11 and proline11
racemases yielded inhibitors that exhibited either poor or only
modest binding affinities relative to their substrates due to the
compact nature of their active sites.
Recently, kinetic and structural studies on a pyridoxal 5-
phosphate (PLP)-dependent isoleucine 2-epimerase from
Lactobacillus buchneri (E.C. 5.1.1.21, LbIleE) were reported.12-14
This enzyme participates in the production of branched-chain D-
amino acids (D-BCAAs) in lactic acid bacteria by catalyzing the
reversible racemization or epimerization of nonpolar L-amino
acids at the C-2 position via a 2-base mechanism.15 While LbIleE
preferentially catalyzes the epimerization of L-Ile and D-allo-Ile
with Lys 280 and Asp 222 acting as the Brønsted base catalysts
that abstract the C2-proton from L-Ile and D-allo-Ile, respectively
(Scheme 1),14 it also catalyzes the racemization or epimerization
of norvaline, norleucine, Val, 2-aminobutanoic acid, Leu, Phe,
Met, and L-allo-Ile, but with varying levels of reduced specific
activity.12 The broad substrate specificity of LbIleE, along with
X-ray crystallographic studies,14 suggested that the binding
pocket for the nonpolar side chain was quite roomy, and hence
the enzyme might be amenable to inhibition by SPAs. Indeed,
structures of LbIleE with the bound reaction-intermediate
Introduction
Inhibitor design, through a single, rational approach applied to
broad class of enzymes sharing similar active site
architectures, furnishes powerful strategy for the
a
a
development of lead compounds for the development of drugs
and/or molecular probes of enzyme mechanisms. Recently, we
described a new strategy for designing inhibitors of racemases
and epimerases with capacious and plastic active sites.1,2 Since
enzymes catalyzing racemization and epimerization reactions
bind their enantiomeric or epimeric substrates in a mirror-
image orientation,3,4 the inversion of stereochemistry may be
accompanied by a movement of two groups attached to the
stereocenter through the active site between the so-called R-
and S-pockets during catalysis, particularly in the absence of
specific polar interactions.5-7
Hence, substrate-product
analogues (SPAs) that incorporate structural features of both
the substrate and product of enzyme-catalyzed racemization or
epimerization reactions may serve as inhibitors. Previously, we
reported that gem-disubstituted SPA inhibitors may be
designed that present binding determinants simultaneously to
a. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax,
NS, B3H 4R2, Canada; E-mail: sbearne@dal.ca
b. Department of Chemistry, Dalhousie University, Halifax, NS, B3H 4R2, Canada
†Electronic Supplementary Information (ESI) available: 1H NMR, 13C NMR, and mass
spectra, HPLC chromatograms, enzyme purification SDS-PAGE, and kinetic plots. See
DOI: 10.1039/x0xx00000x
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Organic & Biomolecular Chemistry
R
Asp
222
Lys
280
Asp
222
Lys
280
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DOI: 10.1039/C9OB01823A
LbIleE
LbIleE
NH2
NH3
NC
NC
N
H
Ph
O
OH
O
O–
H
H
+
+
+
3
NH3
NH3
O
O
O–
O–
H
H
+
+
+
N
H
PLP
PLP
N
O
O
L-isoleucine
D-allo-isoleucine
(2R,3S)
R
R
R
H
O–
O–
(2S,3S)
i
ii
+
HCl·H2N
Ph
a: 84%
b: 91%
a: 100%
b: 78%
H2N
Scheme 1 Reaction catalyzed by LbIleE
N
H
Ph
N
Ph
O
H
H
2
O
1
4
a, R = H; b, R = CH3
analogues (i.e., reduced imines) PLP-L-Ile (PDB 5WYA)14 and PLP-
D-allo-Ile (PDB 5WYF)14 revealed that the nonpolar side chain is
bound in a large hydrophobic cavity and moves through this
cavity during catalysis (Fig. 1A), while the imino-PLP moiety and
the carboxylate group of the substrate remain bound at their
respective binding sites. Recognizing that the entire sec-butyl
side chain of the substrate does not move entirely to a new
location within the hydrophobic cavity, but is only slightly
displaced, we designed SPAs that would effectively fill the
hydrophobic cavity (Fig. 1B).
Over the past decade, it has become evident that D-amino
acids play important regulatory roles in bacteria.16,17 Indeed, D-
BCAAs such as D-Ile, D-Leu, and D-Val are involved in regulating
remodeling of the cell wall in Vibrio cholera,18 and D-Leu plays a
role in biofilm disassembly in Bacillus subtilis.19 Consequently,
enzymes catalyzing the biosynthesis of D-BCAAs are attractive
targets for the development of antimicrobial agents.16,20
R
R
iv, v
a: iv. 90%, v. 74%
b: iv. 76%, v. 72%
iii
a: 66%
b: 87%
H2N
HO
NH2
NH2
O
O
5
6
Scheme 2 Synthesis of the (2S)-substrate-product analogues (6a,b). Key: (i) NaCN,
MeOH:H2O (1:1); (ii) conc. H2SO4; (iii) H2 (60 psi), Pd/C, MeOH, rt, 2 days; (iv) conc. HCl,
reflux; and (v) SPE, lyophilization.
R
NC
NC
N
H
Ph
8
+
R
R
R
i
ii
+
HCl·H2N
Ph
a: 97%
b: 88%
H2N
a: 81%
b: 68%
N
H
Ph
N
Ph
O
H
H
7
O
1
9
a, R = H; b, R = CH3
R
R
iv, v
iii
a: 82%
a: iv. 79%, v. 100%
b: iv. 56%, v. 70%
H2N
HO
NH2
NH2
b: 100%
O
O
10
11
Scheme 3 Synthesis of the (2R)-substrate-product analogues (11a,b). Key: (i) NaCN,
MeOH:H2O (1:1); (ii) conc. H2SO4; (iii) H2 (60 psi), Pd/C, MeOH, rt, 2 days; (iv) conc. HCl,
reflux; and (v) SPE, lyophilization.
Herein, we report the inhibition of an amino acid racemase
responsible for the production of D-BCAAs by the first rationally
designed SPAs that exceed the binding affinities of the
corresponding substrates.
Results and discussion
Syntheses
Based on the superposition of the X-ray crystal structures of
LbIleE with the bound reaction-intermediate analogues PLP-L-
Ile (PDB 5WYA) and PLP-D-allo-Ile (PDB 5WYF) (Fig. 1A),14
compounds 6a, 6b, 11a, and 11b (Fig. 1B) were designed to
present alkyl groups simultaneously to the (S)- and (R)-
hydrophobic pockets that are occupied by the alkyl side chain
of L-Ile or D-allo-Ile, respectively. The synthesis of the SPAs 6a,b
and 11a,b are outlined in Schemes 2 and 3, respectively.
Starting from either 2-ethylbutyraldehyde (1a) or 2-ethyl-2-
methylbutyraldehyde (1b), a chiral Strecker reaction was
conducted using (S)-(–)--methylbenzylamine to selectively
yield crystals of the nitrile diastereomers 2a and 2b,
respectively, as described by Resnick and Galante.21
Alternatively, use of (R)-(+)--methylbenzylamine selectively
yielded crystals of the nitrile diastereomers 7a and 7b,
respectively. Diastereomers 3a,b and 8a,b were removed with
the solvent upon filtration. Subsequently, the nitriles 2a,b and
7a,b were converted to their corresponding amides 4a,b and
Fig. 1 Design of substrate-product analogues (SPAs). (A) Stereoview showing the
superposition of the reaction-intermediate analogues PLP-L-Ile (2S,3S; purple) and
PLP-D-allo-Ile (2R,3S; olive) (stick representation) bound at the active site of LbIleE
(PDB 5WYA and 2WYF, respectively).14 The carboxylate group and the imino-PLP
moiety remain fixed, while the nonpolar side chain is free to move within its
hydrophobic binding pocket during catalysis. The 2S- and 2R-specific Brønsted
acid-base catalysts Lys 280 and Asp 222, respectively, are also shown in stick
representation. (B) The structures of the SPAs, based on the observed change in
location of the nonpolar side chain accompanying catalysis, are shown. Note the
similarity between the superposed structures in panel A and the structures of 6a,b
and 11a,b. (C) The gem-disubstituted SPA, designed based on movement of the
entire nonpolar side chain, is shown. (D) Examples of gem-disubstituted SPAs,
based on movement of the entire nonpolar side chain, are shown.
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Table 1 Alkyl-based substrates of LbIleEa
9a,b, respectively, by treatment with concentrated H2SO4 for 24
– 48 h. Generally, longer reaction times were required for the
compounds with the bulkier amino acid side chain (i.e., b
series). Removal of the chiral auxiliary was effected by catalytic
hydrogenation over 5% Pd/C for 24 – 48 h to yield the amino
acid amides 5a,b and 10a,b. Finally, the amino acid amides
were refluxed in concentrated HCl, followed by solid phase
extraction (SPE) and lyophilization to furnish the pure amino
acids 6a,b and 11a,b. The yields for each of the steps ranged
between 56% to ~100% and were similar to those reported by
Resnick and Galante21 for both the a and b series of compounds.
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–1
DOI: 10.1039/C9OB01823A
substrate
Km, mM
kcat, s
kcat/Km,
mM–1s–1
67 ± 4
L-isoleucine
D-allo-isoleucine
3.8 ± 0.2
(5.00 ± 0.08)b
11 ± 1
(13.2 ± 0.6)b
7.0 ± 0.8
4.3 ± 0.6
4.9 ± 0.7
7 ± 1
253 ± 14
(126 ± 4)b
447 ± 42
(235 ± 7)b
215 ± 31
137 ± 14
148 ± 12
202 ± 16
(25 ± 1)b
40 ± 2
(18 ± 1)b
31 ± 3
32 ± 3
31 ± 6
29 ± 3
3-ethyl-L-norvaline (6a)
3-ethyl-D-norvaline (11a)
L-2-cyclohexylglycine
D-2-cyclohexylglycine
a
Values of the kinetic constants are the means, and errors are the SD from 3
experiments. b Kinetic parameters reported by Mutaguchi et al.12 The reported
kcat and kcat/Km values were per tetramer, but have been adjusted here to reflect
the turnover number per active site.
1
All synthesized compounds were characterized by H and 13C
NMR spectroscopy and mass spectrometry (MS). Details are
presented in ESI,† Figs. S1 – S41.
The enantiomeric excess (e.e.) was estimated for 6a,b and
11a,b using HPLC to separate the isoindole derivatives prepared
by reacting the compounds with o-phthaldialdehyde in the
presence of either N-acetyl-L-cysteine (6a and 11a) or N-
isobutyryl-L-cysteine (6b and 11b) as shown in ESI,† Figs. S42
and S43. Overall, e.e. values of 96.3%, 100%, 99.1%, and 99.5%
were obtained for 6a, 6b, 11a, and 11b, respectively, with the
pairs 6a and 11a, and 6b and 11b, giving equal and opposite
circular dichroism spectra (ESI,† Fig. S44).
Representative Michaelis-Menten plots for all substrates
examined are shown in ESI,† Fig. S46.
Unlike L-Ile and D-allo-Ile, which are diastereomers, 6a and
11a, as well as L- and D-2-cyclohexylglycine, are enantiomers.
Consequently, the overall equilibrium constant (Keq) for
interconversion of the enantiomeric pairs should be unity.
Indeed, from the Haldane relationship (Keq = [D]/[L] =
(kcat/Km)LD/(kcat/Km)DL),22 the values of Keq for the
interconversion of 6a and 11a, and L- and D-2-cyclohexylglycine,
are 1.0 ± 0.1 and 1.1 ± 0.2, respectively.
Substrate studies
Following an approach similar to that described by Awad et al.,13
we inserted a codon-optimized synthetic open reading frame
encoding LbIleE into a pET-28a(+) plasmid. The resulting
pET28a(+)-wtLbIleE plasmid encodes an LbIleE fusion protein
bearing an N-terminal His6-tag, which allowed for purification to
≥97% using immobilized metal ion affinity chromatography
(ESI,† Fig. S45).
Previously, Mutaguchi et al.12 characterized the activity of
LbIleE with L-Ile and D-allo-Ile as substrates. Our His6-tagged
variant of LbIleE exhibited similar affinities for these substrates,
but the kcat values were ~2-fold greater than those reported by
Mutaguchi et al.12 (Table 1). Consequently, the efficiency
(kcat/Km) of our LbIleE was also increased by ~2-fold. Mutaguchi
et al.12 also demonstrated that LbIleE exhibits a broad substrate
specificity, catalyzing the racemization of a variety of amino
acids bearing linear and branched alkyl side chains such as
norvaline, norleucine, Val, 2-aminobutanoic acid, Leu, and allo-
Ile, although with ~20-60% reduced specific activity relative to
L-Ile. To explore the ability of the hydrophobic pocket to
accommodate larger alkyl groups, we explored the ability of the
LbIleE to act on the enantiomers of 2-cyclohexylglycine. We
found that LbIleE was able to catalyze the racemization of L- and
Inhibition by substrate-product analogues 6b and 11b
Compounds 6b and 11b were designed to mimic the structure
suggested from the superposition of the alkyl side chains of the
PLP-L-Ile and PLP-D-allo-Ile complexes observed in the X-ray
crystal structures (Fig. 1A & 1B).14 Surprisingly, the addition of
a single methyl group to the alkyl side chain of the substrates 6a
and 11a resulted in 6b and 11b acting as competitive inhibitors
of LbIleE with Ki values of 2.9 mM and 1.5 mM, respectively
(Table 2 and ESI,† Figs. S47 and S48). SPAs are expected to
exhibit enhanced binding affinity due to their additional binding
determinants, provided that the added steric bulk does not
compromise binding. Apparently, the additional methyl group
in 6b and 11b affords additional van der Waals interactions to
promote binding so that these compounds bind slightly better
than their corresponding substrates L-Ile and D-allo-Ile,
respectively. Many amino acid racemases, including LbIleE,
exhibit rather weak binding of their substrates, often with Km
values in the millimolar range. Hence, it is not surprising that
the binding affinities of the SPAs are also in the low millimolar
range.
Table 2 Alkyl-based substrate-product analogue inhibitors of LbIleEa
D-2-cyclohexylglycine
with
catalytic
efficiencies
of
approximately 30 mM–1s–1 (Table 1), which was not surprising
since LbIleE also exhibits a low racemase activity with Phe.12
Moreover, this observation suggests that LbIleE should be able
to accommodate the additional steric bulk of compounds 6a,b
and 11a,b within its hydrophobic binding pocket. Indeed, both
compounds 6a and 11a were substrates for LbIleE, exhibiting
catalytic efficiencies similar to those of L- and D-2-
cyclohexylglycine and D-allo-Ile (Table 1).
inhibitor
IC50, mM
–
Ki, mM
2.9 ± 0.2
1.5 ± 0.2
144 ± 8b
19 ± 2b
3-ethyl-3-methyl-L-norvaline (6b)
3-ethyl-3-methyl-D-norvaline (11b)
2,2-diethylglycine (12)
2,2-di-n-propylglycine (13)
2,2-di-n-butylglycine (14)
–
334 ± 18
43 ± 5
18 ± 2
11.0 ± 0.2
a Values are the means, and errors are the SD from 3 experiments. b Calculated
from the observed IC50 value using eqn. 4 with Km = 3.8 mM and [L-Ile] = 5.0 mM.
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Examination of 6b and 11b as potential substrates
inhibit LbIleE activity (see Fig. 1D). Representative IC50
determination curves are shown in ESI,† Fig. S50. 2,2-
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DOI: 10.1039/C9OB01823A
Since 6a and 11a were both substrates for LbIleE, we tested 6b
and 11b as potential substrates. Even when the concentration
of the enzyme was increased from 2 µg/mL to 20 µg/mL (i.e.,
10-fold above its regular assay concentration), no turnover was
observed using the HPLC-based assay. This observation
suggested that the introduction of the additional methyl group
in 6b and 11b (cf. 6a and 11a) either resulted in a binding
orientation wherein the -proton of 6b and 11b was no longer
correctly positioned for abstraction by the enantiospecific
Brønsted base, or abstraction of the -proton could occur but
congestion of the hydrophobic pocket at the active site arising
from the additional steric bulk of the side chain obviated the
ability of the compound to undergo a Walden inversion. To
distinguish between these two possibilities, we conducted a
hydrogen-deuterium (H-D) exchange experiment. Using 1H
NMR spectroscopy to follow H-D exchange of the -proton in
deuterated assay buffer in the presence of 362 µg/mL enzyme,
we observed almost complete replacement of the -proton in
6a (8.5 (± 0.1)% H remaining) and 11a (6.6 (± 0.2)% H remaining)
over 1 h as evidenced by the marked reduction in the area under
the doublet at 3.76 ppm, corresponding to the -proton (ESI,†
Fig. S49). On the other hand, under identical conditions, the
integral of the singlet at 3.57 ppm, corresponding to the -
proton in 6b and 11b, remained at 100 (±6)% and 98 (±5)%,
respectively, indicating that no H-D exchange with the -proton
was detected for the 3-methyl-containing compounds over the
same time period, during which the enzyme was fully active.
Thus, 6b and 11b are not substrates for LbIleE (or they are
extremely unreactive) since the enzyme is unable to catalyze
their deprotonation. This remarkable observation suggests
that, unlike the substrates 6a and 11a, the orientation of 6b and
11b at the active site is altered by the additional methyl group
such that neither Asp 222 nor Lys 280 (Scheme 1) can interact
with the -proton and/or that formation of a Schiff base with
bound PLP does not occur. Similarly, Mutaguchi et al.12
observed that tert-Leu was not a substrate. These observations
suggest that the presence of a quaternary -carbon is not
compatible with catalysis. An alternative possibility is that
deprotonation occurs, but internal return occurs so rapidly that
H-D exchange with the conjugate acid of the Brønsted base
cannot occur. However, this seems unlikely since Lys 280 is a
polyprotic base and, upon abstraction of the -proton, would
exist as Lys-ND2H+, thereby having a 2/3 probability of
incorporating a deuterium upon internal return.23
Diethylglycine (12) proved to be an extremely weak inhibitor of
LbIleE, while 2,2-di-n-propylglycine (13) was a modest inhibitor
(Table 2). The fact that LbIleE bound 2,2-di-n-propylglycine
suggested that the very weak binding of 2,2-diethylglycine was
not due to geminal substitution of the ethyl groups on the -
carbon, but arose from the lack of van der Waals interactions
with the larger hydrophobic binding pocket. 2,2-Di-n-
butylglycine (14) was shown to be a competitive inhibitor (see
ESI,† Fig. S51) and, interestingly, bound to LbIleE with an affinity
similar to that observed for the substrate D-allo-Ile (cf. Km
value). These observations suggest that there is sufficient
plasticity and/or room at the active site of LbIleE to
accommodate amino acid analogues with gem-dialkyl
substitution on the -carbon. This behavior differs markedly
from that observed previously for the PLP-dependent serine
racemase from Saccharomyces pombe, which was not able to
bind the gem-disubstituted SPA -(hydroxymethyl)serine.11
Consequently, better binding affinity might be obtained with
bulkier aliphatic side chains, however, the low solubility of such
inhibitors becomes an impediment.
Concluding remarks
The rational design of SPA inhibitors of racemases and
epimerases generally assumes that two groups on the carbon
undergoing stereoinversion remain fixed in orientation through
their interactions with binding determinants at the active site,
while the hydrogen and a motile group formally switch
positions. Consequently, this motile group travels between the
R- and S-binding pockets that recognize the motile group of the
corresponding enantiomers. Gem-disubstituted SPAs may be
designed that simultaneously present the motile group to both
of these binding pockets.1,2,8-11
The additional binding
interactions should result in an inhibitor binding affinity that
exceeds the binding affinity of the substrate, provided that the
active site can tolerate the additional steric bulk. In the present
study, the 2,2-dialkylglycines conform to this design strategy
with 2,2-di-n-butylglycine binding with an affinity similar to that
of the substrates. However, superposition of the structures of
the LbIleE complexes14 with the bound PLP-L-Ile and PLP-D-allo-
Ile analogues clearly indicated that stereoinversion is
accompanied by only partial motion of the sec-butyl side chain
of L-Ile or D-allo-Ile during catalysis (Fig. 1A). Therefore, we
modified the SPA inhibitor design strategy and prepared
analogues that mimicked the partial displacement of the sec-
butyl group (Fig. 1B). Indeed, LbIleE bound compounds 6b and
11b with affinities that slightly exceeded those exhibited for the
corresponding substrates, thereby validating this new variation
of the SPA inhibitor design strategy. The inhibitors described in
the present work may serve as tools for studying the
metabolism of D-BCAAs or their regulatory roles in bacteria.16,20
Furthermore, they may furnish a route to lead compounds for
the development of antimicrobial agents16,20 or agents to
control bacterial biofilm formation.19
Inhibition by gem-dialkyl glycines
Typically, the design of SPAs involves the geminal substitution
on the stereogenic center undergoing inversion.1,2,8-11 The
corresponding analogue for LbIleE is shown in Fig. 1C. We
initially avoided this type of analogue because of our previous
observations that amino acid racemases seem to have sterically
restrictive active sites that may not tolerate gem-dialkyl
substitutions at the -carbon of the amino acid substrate. To
explore the feasibility of such SPAs as inhibitors of LbIleE, we
examined the ability of several gem-disubstituted glycines to
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in a 2-L Erlenmeyer flask. The culture was incubated at 37 °C
Experimental
View Article Online
with continuous shaking at 250 rpm unDtiOl It:h10e.1O03.9D/.C9wOaBs01~802.36A.
IPTG was added (final concentration 1.0 mM) and the culture
was incubated overnight at 20 °C. The cells were then harvested
by centrifugation (3000 × g, 10 min, 4 °C) and the cell pellet was
re-suspended in ice-cold binding buffer [Tris-HCl buffer (20 mM,
pH 7.9) containing NaCl (500 mM) and imidazole (5 mM)]. The
cells were lysed using sonication with 5 × 10 s bursts with 1 min
cooling intervals, at a constant power setting of 5.5 using a
Branson Sonifier 250. The soluble cell extract was clarified by
ultracentrifugation (110 000 × g, 30 min, 4 °C) and the
supernatant was loaded onto a column containing His·Bind
resin (2.5 mL) (Novagen/Millipore (Canada) Ltd., ON). The
column was then eluted with binding buffer (25 mL) followed by
wash buffer [Tris-HCl buffer (20 mM, pH 7.9) containing NaCl
(500 mM) and imidazole (70 mM)] (15 mL). The enzyme was
eluted with strip buffer [Tris-HCl buffer (20 mM, pH 7.9)
containing NaCl (500 mM) and EDTA (100 mM)] (6 mL). Upon
elution, the enzyme was dialyzed (MWCO 10 kDa) against
citrate buffer (200 mM, pH 5.5) containing PLP (0.10 mM)] and
DTT (1.0 mM) and then stored at –20 °C. The purity of the
enzyme (≥ 97%) was assessed using SDS-PAGE (8% acrylamide)
General information
2-Ethylbutyraldehyde, D-allo-isoleucine, and L-isoleucine were
purchased from TCI America (Portland, OR). L-2-Cyclohexyl-
glycine and D-2-cyclohexylglycine were purchased from
Oakwood Products, Inc. (Estill, SC). 2-Amino-2-ethylbutanoic
acid (2,2-diethylglycine) and 2-propyl-D,L-norvaline (2,2-di-n-
propylglycine) were purchased from Acrotein ChemBio Inc.
(Hoover, AL). 5-Aminononane-5-carboxylic acid hydrochloride
(2,2-di-n-butylglycine) was purchased from Toronto Research
Chemicals (Toronto, ON). All other chemicals were purchased
from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada).
Dichloromethane (DCM) was dried and distilled over calcium
hydride prior to use. Concentration under reduced pressure
refers to the removal of solvent using a rotary evaporator. All
NMR spectra were obtained using a Bruker AVANCE 300 or 500
MHz spectrometer. Chemical shifts (δ in ppm) for proton (1H)
spectra are reported relative to the residual solvent signal for
CDCl3 (δ 7.26), DMSO-d6 (δ 2.50), or HOD (δ 4.79).24 Chemical
shifts (δ in ppm) for carbon (13C) spectra are reported relative
to the residual solvent signal for CDCl3 (δ 77.16) or DMSO-d6 (δ
39.52).24 High resolution (HR) electrospray ionization (ESI) mass
spectra (MS) were collected using a Bruker microTOF Focus
orthogonal ESI-TOF mass spectrometer instrument operating in
either negative or positive ion mode as indicated in the
experimental procedures below. Abbreviations used for
reporting NMR spectral data are: bm, broad multiplet; bs, broad
singlet; bt, broad triplet; d, doublet; dd, doublet of doublets; m,
multiplet; q, quartet; s, singlet; and t, triplet. For high-
performance liquid chromatography (HPLC) analyses, a Waters
510 pump and 680 controller were used for solvent delivery.
Injections were made using a Rheodyne 7725i sample injector
fitted with a 20-L injection loop. Analytes were detected using
a Waters 474 scanning fluorescence detector.
with staining by Coomassie blue R-250.25
Protein
concentrations were determined from the intrinsic enzyme
absorbance at 280 nm using an extinction coefficient of ε = 49
280 M–1cm–1 (assuming all Cys reduced), which was estimated
using
the
ProtParam
program
from
ExPasy
(web.expasy.org/protparam/).26
Alternatively, protein
concentrations were determined using the Bio-Rad Protein
Assay (Bio-Rad Laboratories, Mississauga, ON) with bovine
serum albumin (BSA) standards. The (His)6-tag was not
removed from the enzyme.
Enzyme assays
The epimerization of L-Ile and D-allo-Ile was followed using a
reversed-phase (RP) HPLC-based assay similar to that described
by Mutaguchi et al.12 Assay solutions (total volume of 850 µL)
contained L-Ile or D-allo-Ile (2.0 – 50.0 mM) and LbIleE (2.0
µg/mL) in citrate (assay) buffer (200 mM, pH 5.5) containing PLP
(0.1 mM). The reaction was incubated for 4 min at 37 °C and
terminated by removing a 500-µL aliquot and mixing it with 50%
trichloroacetic acid (125 µL). Following centrifugation (15 min,
10 000  g, 4 °C) of this solution, the supernatant (500 µL) was
added to NaOH (1 M, 300 µL). The neutralized samples (20 µL)
were added to 140 µL of borate-NaOH buffer (0.1 M, pH 10.4)
followed by 40 µL of the freshly prepared derivatization solution
(20 mg N-acetyl-L-cysteine and 16 mg o-phthaldialdehyde in 2
mL methanol) and the reaction allowed to proceed for 1 min.
An aliquot (10 µL) was then transferred to 990 µL of sodium
acetate buffer (100 mM, pH 5.9) and analyzed using RP-HPLC
(20-L injection volume) on a Kinetex C18 column (5 m; 100 Å;
250  4.6 mm; Phenomenex, Torrance, CA) equipped with a
guard column. The isoindole derivatives were eluted under
isocratic conditions at a flow rate of 0.45 mL/min with sodium
acetate buffer (50 mM, pH 5.9):methanol (49:51) and detected
using fluorescence detection (ex, 337 nm; em, 454 nm). The
LbIleE production
The open reading frame (ORF) encoding isoleucine 2-epimerase
from Lactobacillus buchneri JCM 1115 (Genbank KC413940.1)
(LbIleE) was synthesized and codon-optimized by Bio Basic
Canada Inc. (Markham, ON, Canada) and inserted into a pET-
28a(+) plasmid (Novagen/Millipore (Canada) Ltd., ON) between
the SacI and XhoI sites of the multiple cloning region to generate
the pET-28a(+)-wtLbIleE plasmid. This construct encodes wild-
type
LbIleE
with
an
N-terminal
(His)6-tag
(MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELM1–
LbIleE), where M1 denotes the initial Met of wild-type LbIleE.
The stop codon of the ORF was utilized so that the C-terminal
(His)6-tag was not present. LbIleE was overexpressed in and
purified from Escherichia coli BL21(DE3) cells transformed with
the plasmid following a procedure modified from that described
by Awad et al.13 Briefly, two disposable sterile culture tubes (10
mL) containing lysogeny broth (LB, 5 mL) and kanamycin (15
g/mL) were inoculated with a glycerol stock solution (10 L
each) and incubated overnight at 37 °C with continuous shaking
at 250 rpm. The culture (10 mL) from both tubes was then used
to inoculate LB broth (1.0 L) containing kanamycin (15 g/mL)
This journal is © The Royal Society of Chemistry 20xx
Org. Biomol. Chem., 2019, 00, 1-3 | 5
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ARTICLE
Organic & Biomolecular Chemistry
chromatograms were generated and integrated using
PeakSimple software v. 4.32 (Mandel Scientific, Guelph, ON,
Canada). Initial velocities were calculated using standard curves
with the product of the reaction being monitored, i.e., L-Ile (0.5
– 6.0 mM), D-allo-Ile (0.5 – 6.0 mM), L-2-cyclohexylglycine (0.4 –
1.4 mM), D-2-cyclohexylglycine (0.4 – 1.4 mM), 6a (0.2 – 2.4
mM), and 11a (0.2 – 2.2 mM) derivatized in the same manner
as described above.
View Article Online
DOI: 10.1039/C9OB01823A
IC50
[S]
퐾i =
(4)
1 +
(
[퐾m]
)
Hydrogen-deuterium exchange
Water was removed from the assay buffer or solutions of assay
buffer containing either 6a, 11a, 6b, or 11a (10.0 mM in 10 mL)
by lyophilization. The dry powder was then dissolved in D2O
(7.5 mL) and the solvent was removed by lyophilization. This
procedure was then repeated. The resulting powder was then
dissolved in D2O, the solution was adjusted to pD 5.5 using DCl
(pH 5.1 on the pH meter),29 and the solution was brought to a
volume of 10 mL in a volumetric flask by addition of D2O.
Solutions (650 µL) of deuterated assay buffer alone, or of 6a,
11a, 6b, or 11a (10.0 mM) in deuterated assay buffer were
placed in an NMR tube. LbIleE (100 µL of a 2714 µg/mL stock
solution in assay buffer containing H2O) was added and the tube
was incubated at 37 °C for 1 h in a water bath. The samples
were then analyzed using 1H NMR spectroscopy. The extent of
H-D exchange was estimated by comparing the integration of
the doublets at 3.76 ppm arising from the -proton of 6a and
11a with the integration of the overlapping triplets of the CH3
groups centered at 0.90 ppm. Similarly, the integration of the
singlet at 3.57 ppm arising from the -proton of 6b and 11b
were compared with the integration of the singlet
corresponding to the CCH3 at 0.9 ppm and the overlapping
triplets arising from the two CH2CH3 groups centered at 0.80
ppm.
Kinetic studies
The Michaelis-Menten equation (eqn. 1) was fit to the initial
velocity data using nonlinear regression analysis and the
program KaleidaGraph v. 4.02 from Synergy Software (Reading,
PA), where vi is the initial velocity, Vmax is the apparent maximal
velocity at saturating concentrations of substrate, Km is the
apparent Michaelis constant, and [S] is the substrate
concentration.
푉max[S]
(1)
푣i =
퐾m + [S]
The values of kcat were calculated by dividing the observed Vmax
values by the enzyme concentration (protomer MW = 53 487
Da). The concentration ranges of L-Ile (20.0 – 50.0 mM), D-allo-
Ile (20.0 – 50.0 mM), L-2-cyclohexylglycine (2.0 – 10.0 mM), D-2-
cyclohexylglycine (2.0 – 10.0 mM), 3-ethyl-L-norvaline (6a) (2.0
– 20.0 mM), and 3-ethyl-D-norvaline (11a) (2.0 – 50.0 mM) were
as indicated. Inhibition experiments with 3-ethyl-3-methyl-L-
norvaline (6b) (0, 5.0, and 10.0 mM) and 3-ethyl-3-methyl-D-
norvaline (11b) (0, 5.0, and 10.0 mM) were conducted using
concentrations of L-Ile between 5.0 and 50.0 mM and a
concentration of LbIleE of 2.0 µg/mL. Inhibition experiments
with 2,2-di-n-butylglycine (14) (0, 5.4, and 10.8 mM) were
conducted using concentrations of L-Ile between 2.5 and 20.0
mM and a concentration of LbIleE of 2.0 µg/mL. The apparent
Km/Vmax values were obtained from direct fits of eqn. 1 to initial
velocity data (i.e., Michaelis-Menten plots) using nonlinear
regression analysis and the KaleidaGraph software.
Competitive inhibition constants (Ki) were determined by re-
plotting these apparent Km/Vmax values against inhibitor
concentration in accord with eqn. 2.
Syntheses
Compound 1a30 and 2a – 6a21 were synthesized as described in
the literature. In general, compounds 2 – 6 and 7 – 11 were
prepared following the protocols adapted from Resnick and
Galante.21
3-Ethyl-L-norvaline (6a).21 Compound 5a (2.85 g, 20 mmol),
prepared as described by Resnick and Galante,21 was mixed with
concentrated HCl (50 mL) and the mixture was refluxed for 24
h. Upon cooling to room temperature, white crystals formed.
After 30 min, the white crystals (3.23 g, 90%) were collected by
suction filtration and air-dried. The hydrochloride salt was then
converted to the free amino acid by dissolving the crystals (1.52
g, 8.4 mmol) in water (4 mL) with gentle heating followed by
passage through a reversed-phase, solid-phase extraction
column (60 mL/10 g, C18-E, 55 µm, 70 Å, Phenomenex,
Torrance, CA). Fractions (3 mL) containing the amino acid
(yellow spots on a silica TLC sheet developed with a potassium
dichromate dip) were combined and the aqueous solvent was
removed by lyophilization to yield a white powder: yield 0.90 g
푉max[S]
푣i =
[I]
(2)
퐾m 1 +
+ [S]
(
)
퐾i
The IC50 values for the inhibition of the LbIleE by the gem-
dialkylglycines 12, 13, and 14, relative to L-Ile (5.0 mM), were
determined by fitting the relative velocities (vi/vo) obtained at
various concentrations of 12 (50.0 – 400.0 mM), 13 (5.0 – 40.0
mM), and 14 (2.0 – 13.0 mM) to eqn. 3, where the IC50 value is
the concentration of inhibitor that yields vi/vo = 0.5.27
1
(74%); m.p. 200 °C; H NMR (500 MHz, DMSO-d6) δ 8.43 (br s,
NH2, 2H), 3.81 (d, J = 3.5 Hz, CHNH2, 1H), 1.69-1.78 (m,
CHCHNH2, 1H), 1.19-1.30 (m), 1.30-1.46 (m) and 1.46-1.57 (m)
(2  CH2, 4H), 0.90 (t, J = 7.4 Hz, CH3) and 0.88 (t, J = 7.4 Hz CH3)
(6H); 13C NMR (126 MHz, DMSO-d6) δ 170.54 (COOH), 53.78
(CHCOOH), 42.29 (CHCHNH2), 21.80 (CH2), 21.59 (CH2), 11.60
(CH3), 11.44 (CH3) ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for
푣i
IC50
(3)
=
푣o IC50 + [I]
The IC50 values were used to estimate the values for the
inhibition constants using the Cheng-Prusoff equation and
assuming competitive inhibition (eqn. 4).28
6 | Org. Biomol. Chem., 2019, 00, 1-3
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Organic & Biomolecular Chemistry
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ARTICLE
C7H16NO2, 146.1181, found 146.1171; [M+Na]+ calcd for removed in vacuo to yield a white solid: yield 0.94Vgie(w9A6r%tic)le;Omnl.ipne.
1
DOI: 10.1039/C9OB01823A
C7H15NO2Na, 168.1000, found 168.0999.
87 °C; H NMR (300 MHz, DMSO-d6) δ 7.17 (s, CONH2, 1H), 6.80
(2S)-3-Ethyl-3-methyl-2-{[(1S)-1-phenylethyl]amino}-
(s, CHNH2, 1H), 2.96 (s, CHNH2, 1H), 1.45 – 1.16 (m, CH2, 4H),
pentanenitrile (2b). Aqueous HCl (2.9 M, 13.8 mL) was added 0.83 – 0.67 (m, CH3C and 2  CH3CH2, 9H); 13C NMR (75 MHz,
to a solution of methanol (200 mL) and water (186 mL) followed DMSO-d6) δ 176.53 (CONH2), 59.03 (CHNH2), 38.24 (CCH2),
by (S)-(–)-α-methylbenzylamine (5.1 mL, 40 mmol, 1.0 equiv.). 27.24 (CH2), 26.73 (CH2), 19.61 (CH3C), 7.94 (CH3CH2), 7.70
2-Ethyl-2-methylbutanal (5.66 mL, 40 mmol, 1.0 equiv.) was (CH3CH2) ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for C8H19N2O,
then added followed by NaCN (1.96 g, 40 mmol, 1.0 equiv.). 159.1497, found 159.1489.
After stirring for 48-60 h to allow for adequate asymmetric
3-Ethyl-3-methyl-L-norvaline (6b). Compound 5b (3.70 g,
crystallization to occur, white crystals were collected by suction 23 mmol) was mixed with concentrated HCl (50 mL) and the
1
filtration: yield 8.90 g (91%); m.p. 51 °C; H NMR (300 MHz, mixture was refluxed for 24 h. Upon cooling to room
DMSO-d6) δ 7.32 (m, ArH, 4H), 7.25 (m, ArH, 1H), 3.86 (qd, J = temperature, white crystals formed. After 30 min, the product
6.4, 1.9 Hz, CHPh, 1H), 2.87 (d, J = 12.3 Hz, CHCN, 1H), 2.70 (dd, was collected by suction filtration and air-dried to yield white
J = 12.3, 1.5 Hz, NH, 1H), 1.49 – 1.27 (m, CH3CH and CH2, 6H), crystals: yield 3.46 g (76%). The hydrochloride salt was then
1.31 – 1.12 (m, CH2, 1H), 0.85 (s, CH3C, 3H), 0.59 (m, 2  CH3CH2, converted to the free amino acid by dissolving the crystals
6H); 13C NMR (75 MHz, DMSO-d6) δ 144.54 (CPh), 128.86 (2  (0.7028 g, 3.6 mmol) in water (4 mL) with gentle heating
CPh), 127.80 (CPh, 127.40 (2  CPh), 120.27 (CCN), 56.98 (CHPh), followed passage through
a reversed-phase, solid-phase
55.97 (CHCN), 38.99 (CCH2), 28.05 (CH2), 27.09 (CH2), 24.69 extraction column (60 mL/10 g, C18-E, 55 µm, 70 Å). Fractions
(CH3CH), 20.75 (CH3C), 7.91 (CH3CH2), 7.76 (CH3CH2) ppm. (3 mL) containing the amino acid (yellow spots on a silica TLC
HRMS-ESI+ (m/z): [M+H]+ calcd for C16H25N2, 245.2018, found sheet developed with a potassium dichromate dip) were
245.2003.
3-Ethyl-3-methyl-N2-[(1S)-1-phenylethyl]-L-norvalinamide
combined and the aqueous solvent was removed by
lyophilization to yield a white powder: yield 0.41 g (72%); m.p.
(4b). Concentrated H2SO4 (4 mL per gram of reactant; 655 183 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.36 (br s, NH2, 2H), 3.60
mmol, 18.2 equiv.) was added to the amino nitrile 2b (8.90g, 36 (s, CHNH2, 1H), 1.35-1.48 (2  CH2, 4H), 0.90 (s, CH3C, 3H), 0.793
mmol, 1.0 equiv.) and stirred for 72 h. The reaction mixture was (t, J = 7.4 Hz, CH2CH3) and 0.789 (t, J = 7.5 Hz CH2CH3) (6H); 13
C
then pipetted onto ice (75 mL) followed by addition of NMR (126 MHz, DMSO-d6) δ 169.94 (COOH), 57.99 (CHCOOH),
concentrated NH4OH (aq.) (1 mL aliquots with cooling by 37.72 (CCHCOOH), 27.27 (CH2), 26.84 (CH2), 19.85 (CH3C), 7.51
additional ice) until the pH was 8. The resulting white gummy (CH3), 7.47 (CH3) ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for
product and opaque white solution were poured into a C8H18NO2, 160.1338, found 160.1336.
separatory funnel with EtOAc used to transfer the gummy
(2R)-3-Ethyl-2-{[(1R)-1-phenylethyl]amino}pentanenitrile
product. The solution was then extracted with EtOAc (3 100 (7a). Aqueous HCl (2.9 M, 6.9 mL) was added to a solution of
mL) and the combined organic layers were dried over methanol (100 mL) and water (93 mL) followed by (S)-(–)-α-
anhydrous Na2SO4. In some preparations, a minor amount of methylbenzylamine (2.55 mL, 20 mmol, 1.0 equiv.). 2-Ethyl-
reactant remained, which was removed by column butanal (2.46 mL, 20 mmol, 1.0 equiv.) was then added followed
chromatography on silica using hexanes:EtOAc (8:2). The by NaCN (0.98 g, 20 mmol, 1.0 equiv.). After stirring for 48 h to
reactant (2b) and product (4b) had Rf values of 0.88 and 0.19, allow for adequate asymmetric crystallization to occur, the
respectively. Removal of the solvent in vacuo yielded a white white crystals were collected by suction filtration: yield 4.45 g
1
solid: yield 7.46 g (78%); m.p. 114 °C; 1H NMR (300 MHz, DMSO- (97%); m.p. 58 °C; H NMR (300 MHz, DMSO-d6) δ 7.39 – 7.27
d6) δ 7.36 – 7.24 (m, ArH and CONH, 5H), 7.20 (m, ArH, 1H), 6.99 (m, ArH, 4H), 7.32 – 7.19 (m, ArH,1H), 3.88 (qd, J = 6.5, 2.2 Hz,
(br s, CONH, 1H), 3.47 (q, J = 6.5 Hz, CHPh, 1H), 2.54 (br s, CHPh, 1H), 3.01 (dd, J = 11.6, 5.5 Hz, CHCN 1H), 2.92 (dd, J =
CHCONH2, 1H), 2.01 (d, J = 8.9 Hz, NH, 1H), 1.54 – 1.36 (m, CH2) 11.6, 2.3 Hz, NH, 1H), 1.65 – 1.35 (m, CHCH2 and CH2CH3) and
and 1.31 – 1.07 (m, CH3CH and CH2) (7H), 0.74 (s, CH3C, 3H), 0.57 1.34 – 1.18 (m, CH3CHPh and CH2CH3) (8H), 0.73 (m, 2  CH2CH3,
(t, J = 7.5 Hz, CH3CH2, 3H), 0.47 (t, J = 7.4 Hz, CH3CH2, 3H); 13C 6H); 13C NMR (75 MHz, DMSO-d6) δ 144.68 (CPh), 128.93 (2 
NMR (75 MHz, DMSO-d6) δ 175.48 (CONH2), 145.77 (CPh), CPh), 127.71 (CPh), 127.14 (2  CPh), 120.67 (CCN), 56.71 (CHPh),
127.96 (2  CPh), 127.00 (2  CPh), 126.61 (CPh), 63.75 51.25 (CHCN), 43.83 (CHCHCN), 24.92 (CH3CHPh), 22.22
(CHCONH2), 56.03 (CHPh), 37.84 (CCH2), 27.14 (CH2), 26.93 (CH2), (CH2CH3), 21.93 (CH2CH3), 11.26 (CH2CH3), 10.92 (CH2CH3).
25.47 (CH3CH), 19.75 (CH3C), 7.67 (CH3CH2), 7.56 (CH3CH2) ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for C15H23N2, 231.1861, found
HRMS-ESI+ (m/z): [M+H]+ calcd for C16H27N2O, 263.2133, found 231.1865.
263.2126.
3-Ethyl-N2-[(1R)-1-phenylethyl]-D-norvalinamide
(9a).
3-Ethyl-3-methyl-L-norvalinamide (5b). MeOH (75 mL) was Concentrated H2SO4 (2 mL per gram of reactant; 166 mmol, 8.7
carefully added to 5% Pd on charcoal (1.87 g) under a flow of equiv.) was added to the amino nitrile 7a (4.45 g, 19 mmol, 1.0
argon. Compound 4b (7.46 g, 28 mmol) was added and the equiv.) and stirred for 36 h. The reaction mixture was then
mixture was shaken (Parr Shaker) for 72 h under an atmosphere pipetted onto ice (75 mL) followed by addition of concentrated
of hydrogen (60 psi). The reaction mixture was then filtered NH4OH (aq.) (1 mL aliquots with cooling by additional ice) until
through a pad of Celite and the Celite was washed with MeOH the pH was 8. The resulting white gummy product and opaque
(50 mL). After the solvent was removed in vacuo, the resulting white solution were poured into a separatory funnel with EtOAc
solid was dissolved in DCM (10 mL) and the solvent was again used to transfer the gummy product. The solution was then
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Organic & Biomolecular Chemistry
extracted with EtOAc (3  75 mL) and the combined organic stirring for 48-60 h to allow for adequateVieawsyArmticmle Oentlrinice
layers were dried over anhydrous Na2SO4. Removal of the crystallization to occur, the product waDsOcIo: l1l0e.c10te39d/Cb9yOBsu01c8t2io3nA
solvent in vacuo gave a clear oil: yield 1.64 g (81%); 1H NMR (500 filtration giving white crystals: yield 4.28 g (88%); m.p. 51 °C; 1H
MHz, DMSO-d6) δ 7.36 – 7.26 (m, ArH and CONH, 5H), 7.24 – NMR (300 MHz, CDCl3) δ 7.42 – 7.22 (m, ArH, 5H), 4.06 (q, J =
7.17 (m, ArH, 1H), 7.02 – 6.98 (m, CONH, 1H), 3.55 (q, J = 6.5 Hz, 6.5 Hz, CHPh, 1H), 3.01 (s, CHCN, 1H), 1.25-1.57 (m, CH3CHPh, 2
CHPh, 1H), 2.62 (m, CHCONH2, 1H), 2.06 (br d, J = 5.4 Hz, NH,  CH2, and NH, 8H), 0.94 (s, CH3CH, 3H), 0.69 (m, 2  CH3CH2,
1H), 1.47 (m, CHCH2CH3, 1H), 1.32 – 1.16 (m, 2  CH3CH2 and 6H); 13C NMR (75 MHz, CDCl3) δ 143.64 (CPh) 128.74 (2  CPh),
CH3CHPh, 9H – 2H for overlapping t of EtOAc = 7H), 0.72 (t, J = 127.81 (CPh), 127.41 (2  CPh), 120.33 (CCN), 56.88 (CHPh), 56.21
7.1 Hz, CH2CH3, 3H), 0.61 (t, J = 7.0 Hz, CH2CH3, 3H); 13C NMR (CHCN), 39.22 (CCHCN), 28.21 (CH2), 27.41(CH2), 25.16
(126 MHz, DMSO-d6) δ 176.57 (CONH2), 145.87 (CPh), 128.01 (2 (CH3CHPh), 20.58 (CH3C), 7.88 (CH3CH2), 7.70 (CH3CH2) ppm.
 CPh), 126.84 (2  CPh), 126.59 (CPh), 61.07 (CHCONH2), 56.15 HRMS-ESI+ (m/z): [M+H]+ calcd for C16H25N2, 245.2018, found
(CHPh), 44.24 (CHCH2), 25.24 (CH3CHPh), 21.78 (CH2), 20.92 245.2014.
(CH2), 11.35 (CH2CH3), 10.97 (CH2CH3) ppm. HRMS-ESI+ (m/z):
[M+H]+ calcd for C15H25N2O, 249.1967, found 249.1970.
3-Ethyl-3-methyl-N2-[(1R)-1-phenylethyl]-D-norvalinamide
(9b). Concentrated H2SO4 (4 mL per gram of reactant; 315
3-Ethyl-D-norvalinamide (10a). MeOH (40 mL) was carefully mmol, 18.2 equiv.) was added to the amino nitrile 7b (4.28 g, 18
added to 5% Pd on charcoal (0.53 g) under a flow of argon. mmol, 1.0 equiv.) and stirred for 72 h. The reaction mixture was
Compound 9a (3.51 g, 14 mmol) was added and the mixture was then pipetted onto ice (75 mL) followed by addition of
shaken (Parr Shaker) for 72 h under an atmosphere of hydrogen concentrated NH4OH (aq.) (1 mL aliquots with cooling by
(60 psi). The reaction mixture was then filtered through a pad additional ice) until the pH was 8. The resulting white gummy
of Celite and the Celite was washed with MeOH (50 mL). After product and opaque white solution were poured into a
the solvent was removed in vacuo, the resulting solid was separatory funnel with EtOAc used to transfer the gummy
dissolved in DCM (10 mL) and the solvent was again removed in product. The solution was then extracted with EtOAc (3 100
1
vacuo to give a white solid: yield 1.68 g (82%); m.p. 115 °C; H mL) and the combined organic layers were dried over
NMR (500 MHz, DMSO-d6) δ 7.33 (s, CONH2, 1H), 6.93 (s, CHNH2, anhydrous Na2SO4. Note that in some preparations, a minor
1H), 3.12 (d, J = 4.7 Hz, CHNH2, 1H), 1.41-1.50 (m, CHCH2, 1H), amount reactant remained, which was removed by column
1.25-1.38 (m) and 1.10-1.22 (m) (2  CH2, 4H), 0.86 (t, J = 7.4 Hz, chromatography on silica using hexanes:EtOAc (8:2). The
CH3, 3H), 0.82 (t, J = 7.4 Hz, CH3, 3H); 13C NMR (126 MHz, DMSO- reactant (2b) and product (4b) had Rf values of 0.88 and 0.19,
d6) δ 177.30 (CONH2), 55.79 (CHNH2), 44.24 (CHCH2), 22.20 respectively. Removal of the solvent in vacuo gave a white
(CH2), 20.83 (CH2), 11.65 (CH3), 11.58 (CH3). HRMS-ESI+ (m/z): solid: yield 3.15 g (68%); m.p. 111 °C; 1H NMR (300 MHz, DMSO-
[M+H]+ calcd for C7H17N2O, 145.1341, found 145.1334.
d6) δ 7.36 – 7.14 (m, ArH, 5H and CONH, 1H), 6.98 (s, CONH, 1H),
3-Ethyl-D-norvaline (11a). Compound 10a (1.68 g, 12 mmol) 3.47 (q, J = 6.5 Hz, CHPh, 1H), 2.54 (s, CHCONH2, 1H), 2.01 (d,
was mixed with concentrated HCl (50 mL) and the mixture was NH, 1H), 1.54 – 1.36 (m, CH2) and 1.30 – 1.08 (m, CH3CHPh and
refluxed for 24 h. Upon cooling to room temperature, white CH2) (6H), 0.74 (s, CH3C, 3H), 0.57 (t, J = 7.4 Hz, CH3CH2, 3H), 0.47
crystals formed. After 30 min, the crystals were collected by (t, J = 7.5 Hz, CH3CH2, 3H); 13C NMR (75 MHz, DMSO-d6) δ 175.46
suction filtration and air-dried: yield 1.68 g (79%). The (CONH2), 145.76 (CPh), 127.95 (CPh), 126.99 (CPh), 126.60 (CPh),
hydrochloride salt was then converted to the free amino acid by 63.74 (CHCONH2), 56.02 (CHPh), 37.83 (CCHCONH2), 27.13
dissolving the crystals (0.80 g, 4.4 mmol) in water (4 mL) with (CH2), 26.92(CH2), 25.46 (CH3CHPh), 19.74 (CH3C), 7.66
gentle heating followed passage through a reversed-phase, (CH3CH2), 7.55(CH3CH2) ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for
solid-phase extraction column (60 mL/10 g, C18-E, 55 µm, 70 Å). C16H27N2O, 263.2123, found 263.2121.
Fractions (3 mL) containing the amino acid (yellow spots on a
3-Ethyl-3-methyl-D-norvalinamide (10b). MeOH (35 mL)
silica TLC sheet developed with a potassium dichromate dip) was carefully added to 5% Pd on charcoal (0.79 g) under a flow
were combined and the aqueous solvent was removed by of argon. Compound 9b (3.15 g, 12 mmol) was added and the
lyophilization to give a white powder: yield 0.64 g (100%); m.p. mixture was shaken (Parr Shaker) for 72 h under an atmosphere
200 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.42 (br s, NH2, 2H), 3.80 of hydrogen (60 psi). The reaction mixture was then filtered
(d, J = 3.5 Hz, CHNH2, 1H), 1.68-1.78 (m, CHCHNH2, 1H), 1.18- through a pad of Celite and the Celite was washed with MeOH
1.30 (m), 1.30-1.45 (m), and 1.45-1.57 (m) (2  CH2, 4H), 0.90 (t, (50 mL). After the solvent was removed in vacuo, the resulting
J = 7.4 Hz, CH3) and 0.88 (t, J = 7.4 Hz CH3) (6H); 13C NMR (126 solid was dissolved in DCM (10 mL) and the solvent was again
MHz, DMSO-d6) δ 170.56 (COOH), 53.81 (CHCOOH), 42.30 removed in vacuo to give a white solid: yield 1.90 g (100%); m.p.
(CHCHCOOH), 21.79 (CH2), 21.59 (CH2), 11.60 (CH3), 11.45 (CH3) 91 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.17 (s, CONH, 1H), 6.80
ppm. HRMS-ESI+ (m/z): [M+H]+ calcd for C7H16NO2, 146.1181, (s, CHNH, 1H), 2.96 (s, CHNH2, 1H), 1.47 – 1.16 (m, 2  CH2, 4H),
found 146.1172.
0.83 – 0.67 (m, CH3C and 2  CH3CH2, 9H); 13C NMR (75 MHz,
DMSO-d6) δ 177.01 (CONH2), 59.52 (CHNH2), 38.73 (CCH3),
(2R)-3-Ethyl-3-methyl-2-{[(1R)-1-phenylethyl]amino}-
pentanenitrile (7b). Aqueous HCl (2.9 M, 6.9 mL) was added to 27.72 (CH2), 27.22 (CH2), 20.09 (CH3C), 8.43 (CH3CH2), 8.18
a solution of methanol (100 mL) and water (93 mL) followed by (CH3CH2). HRMS-ESI+ (m/z): [M+H]+ calcd for C8H19N2O,
(S)-(–)-α-methylbenzylamine (2.55 mL, 20 mmol, 1.0 equiv.). 2- 159.1497, found 159.1492; [M+Na]+ calcd for C8H18N2ONa,
Ethyl-2-methylbutanal (2.83 mL, 20 mmol, 1.0 equiv.) was then 181.1317, found 181.1312.
added followed by NaCN (0.98 g, 20 mmol, 1.0 equiv.). After
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Organic & Biomolecular Chemistry
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Organic & Biomolecular Chemistry
ARTICLE
3-Ethyl-3-methyl-D-norvaline (11b). Compound 10b (1.91 (RGPIN-2016-05083) (S.L.B.) and NSERC-USRAsVie(wNA.Trt.icSle. Oanlninde
DOI: 10.1039/C9OB01823A
g, 12 mmol) was mixed with concentrated HCl (50 mL) and the J.W.M.M).
mixture was refluxed for 24 h. Upon cooling to room
temperature, white crystals formed. After 30 min, the crystals
Notes and references
were collected by suction filtration and air-dried: yield 1.32 g
(56%). The hydrochloride salt was then converted to the free
amino acid by dissolving the crystals (0.70 g, 3.6 mmol) in water
(4 mL) with gentle heating followed passage through a
reversed-phase, solid-phase extraction column (60 mL/10 g,
C18-E, 55 µm, 70 Å). Fractions (3 mL) containing the amino acid
(yellow spots on a silica TLC sheet developed with a potassium
dichromate dip) were combined and the aqueous solvent was
removed by lyophilization to give a white powder: yield 0.40 g
1
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3
4
5
6
1
(70%); m.p. 183 °C; H NMR (500 MHz, DMSO-d6) δ 8.34 (br s,
NH2, 2H), 3.59 (s, CHNH2, 1H), 1.31-1.47 (m, 2  CH2, 4H), 0.90
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Dr. David Jakeman for the use of his hydrogenation
apparatus and Dr. Jan Rainey for use of his lyophilizer. We also
thank the Natural Sciences and Engineering Research Council
(NSERC) of Canada for support through a Discovery Grant
This journal is © The Royal Society of Chemistry 20xx
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Organic & Biomolecular Chemistry
TOC Figure
View Article Online
DOI: 10.1039/C9OB01823A
TOC Statement
Substrate-product analogues, designed based on partial
movement of the sec-butyl side chain during catalysis, inhibit
isoleucine 2-epimerase.
By Noa T. Sorbara, Joshua W.M. MacMillan, Gregory D.
McCluskey, and Stephen L. Bearne
10 | Org. Biomol. Chem., 2019, 00, 1-3
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