A Photoregulated Racemase Mimic

Article abstract of DOI:10.1002/anie.202012124

The racemase enzymes convert L-amino acids to their D-isomer. The reaction proceeds through a stepwise deprotonation–reprotonation mechanism that is assisted by a pyridoxal phosphate (PLP) coenzyme. This work reports a PLP–photoswitch–imidazole triad where the racemization reaction can be controlled by light by tweaking the distance between the basic residue and the reaction centre.

Full text of DOI:10.1002/anie.202012124

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Accepted Article
Title: A photoregulated racemase mimic
Authors: Monochura Saha, Munshi Sahid Hossain, and Subhajit
Bandyopadhyay
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A photoregulated racemase mimic
Monochura Saha, Munshi Sahid Hossain, Subhajit Bandyopadhyay*
Abstract: The racemase enzymes convert L-amino acids to their D-
isomer. The reaction proceeds through a stepwise deprotonation-
reprotonation mechanism that is assisted by a pyridoxalphosphate
(PLP) coenzyme. This work reports a PLP-photo switch-imidazole
triad where the racemization reaction can be controlled by light by
tweaking the distance between the basic residue and the reaction
centre.
The active form of vitamin B6, pyridoxal-5-phosphate (PLP), is
one of the most versatile coenzymes used by nature in a plethora
of biosynthetic transformations.1 The diversity of reactions
2-3
promoted by the coenzyme includes transamination,
decarboxylation, racemization of amino acids,4-5 catabolism of
neurotransmitters, and -elimination of amino acids such as
serine. The structural features of the PLP consist of a central
pyridinium moiety that acts as an electron sink functionalized with
a formyl unit, a phenolic OH, a phosphorylated primary alcohol,
and a methyl substitution (Figure 2A). In terms of the reactivity,
both the pyridinium unit and the formyl units are crucial, whereas
the role of the –OH/O− and the –OPO3 are primarily for the
2-
Figure 1. (A) Active site structure of a racemase having a PLP coenzyme
43
stabilization of the intermediates. The PLP-aldehyde group hooks
up as a Schiff’s base with an active site lysine residue of the
enzymes.6 The -amino group of the amino acid substrates
displace the lysine and form an aldimine Schiff’s base with the
amino acid.7-9
(box) (PDB ID 4ysn)
inset: proximity of two bases Lys and Tyr of the
enzyme to the PLP. (B) Our artificial photoregulated PLP racemase.
The aldimine intermediate is crucial and is common for all PLP-
mediated reactions.10-11 Peptidoglycans on the bacterial cell
surface contain the unusual D-alanine amino acid residues.
Biosynthetically, the D-alanine isomers are formed upon
racemization of the naturally abundant L-alanine using the
bacterial cellular machinery using alanine racemase, a PLP-
dependent enzyme. In terms of the mechanism (Figure 2), the
formation of the alanine-PLP Schiff’s base causes a lowering of
pKa of the chiral α-proton, which is subsequently abstracted by a
basic amino acid residue at the racemase active site.12
Reprotonation from the opposite face and subsequent hydrolysis
generates the D-isomer.13
Figure 2. A putative mechanism of L-alanine racemization.
In his Nobel lecture, Feringa highlighted on the fundamental
challenges of controlling molecular functions using dynamic
molecular systems.14 Photochromic systems are excellent
examples of dynamic molecular systems.15
Upon photoisomerization, they generate light-induced molecular
motions which can be exploited to accomplish targeted
functions.16-21 Photoregulation of biological functions is an
emerging area of immense importance.22 Use of bistable
photochromic systems for such external regulation offers the
additional advantage of reversibility with the non-invasive light as
stimulus which additionally offers its precise temporal and spatial
control of the activity. Either a photochromic unit attached to a
protein can be used to regulate its activity23-26, or the control can
be achieved by using photochromic inhibitors.27 The mechanism
of racemization involves the PLP covalently attaching itself to the
L-alanine as a Schiff’s base (Figure S16) forming the L-Ala-
aldimine intermediate (Figure 6B). The CH of the aldimine is
then deprotonated by a basic residue present at the active site
(Figure 1A) leading to the destruction of the chirality of the entity
and thereby forming a highly delocalized prochiral carbanionic
quinonoid intermediate.28 Reprotonation of this achiral quinoidal
carbanion from the other face of the Cyields the racemic amino
acid.32 A "two-base" mechanism involves a basic residue (Tyr)
Monochura Saha, Munshi Sahid Hossain, Subhajit
Bandyopadhyay
Department of Chemical Sciences, Indian Institute of Science
Education and Research (IISER) Kolkata, Mohanpur, Nadia, West
Bengal 741246, India.
E-mail: sb1@iiserkol.ac.in
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Figure 4. Photoisomerization studies of triad 1 under 366 nm UV light and
466 nm blue light respectively. (A) Schematic re-presentation of
photoisomerization; (B) Trans-1 to cis-1 conversion; (C) cis-1 to trans-1
conversion.
Figure 3. Photoregulation of L-Ala to D-Ala transformation with the PLP-
azobenzene-imidazole triad 1.
that deprotonates the chiral proton of the Schiff’s base, followed
by the conjugate acid of a second basic residue (Lys) that delivers
the proton to resulting prochiral carbanionic intermediate from the
opposite face.31
nitrobenzyl imidazole precursors with glucose/NaOH as the
reducing agent (scheme S4).36 The PLP unit was synthesized
from the commercially available pyridoxal hydrochloride upon
treatment with propargyl bromide under the basic conditions at
room temperature. An equilibrium was observed between the
hemi-acetal and the aldehyde forms of the coenzyme. The
precursor 2 was obtained as crystals in the hemiacetal form
(Figure S24, CCDC 1978370). The details of the multistep
synthesis of the intermediates compound 2 and 3, as well as the
other precursors have been provided in the supporting
information.
The PLP is stabilized by a network of hydrophobic, hydrogen
bonding, and other non-covalent interactions. The reaction
mechanism was thoroughly established by evidence from X-ray
crystal structure29-30 and detailed spectroscopic studies.
An innovative way to control a bio-inspired activity with light was
demonstrated by Branda with the design and demonstration of a
de novo photochromic pyridoxal phosphate (PLP) mimic.34-35
Light regulated enzyme mimics of various enzymes such as
glycosidase and carbonic anhydrase mimic has been reported
recently.33,18
In this work, instead of altering the PLP scaffold, a photochromic
PLP-azobenzene-imidazole triad was designed to regulate the
position of the general basic residue responsible for the
racemization using light as the stimuli (Figure 3). In the cis-1
isomer, the imidazole of the histidinyl unit is in close proximity of
the racemizable CH of the L-Ala aldimine Schiff’s base (Figure
S16 and Figure 6B) whereas, in the trans form, the basic unit is
far and cannot reach the C-proton. The cis form can, therefore,
effectively deprotonate the amino acid leading to its racemization,
whereas the trans-form is less effective in terms of the
deprotonation and, consequently for the racemization as well.
Thus, this photochromic PLP-azobenzene-imidazole triad offers a
reversible photomodulated control on the activity of the racemase
enzyme mimic.
The trans triad 1 displayed absorption bands with λmax at 230, 250,
319, and 431 nm. The band at 319 nm (ε = 13,300 M-1cm-1)
corresponds to a π–π* transition, and the band at 431 nm is a
symmetry forbidden n–π* band (ε = 470 M−1cm−1). Irradiation with
366 nm light under cold conditions (273 K) and monitoring by UV-
Vis spectroscopy revealed that trans-1 (30 µM, in CH3CN)
gradually isomerized to the cis-1 upon exposure to UV light
(λ=366 nm, irradiation power of 2.3 mW cm-2) within 45 min
(Figure 4B). Upon photoisomerization of the trans-1, the
absorption maxima at 319 nm gradually decreased, and a new
peak appeared at 431 nm (ε = 730 M-1.cm-1) corresponding to the
cis-1 isomer. The reversal of the cis-1 to the trans-1 form was
achieved upon the exposure of the sample to blue light (λ = 466
nm, irradiation power = 0.5 mW cm−2) (Figure 4C). Under
continuous irradiation, the cis form was quantitatively converted
to the trans photoisomer within 250 seconds with an 85%
composition of the cis-rich photostationary state (PSS), as
encountered with the azobenzene photochromic system.37-38
Reversibility studies also performed for ten cycle and observed
no fatigue resistance (Figure S14). The photoswitching behavior
The PLP based azobenzene triad 1 was synthesized by a
Cu(I)catalyzed azide-alkyne “click” cycloaddition reaction
between the PLP derivative 2 and the imidazole-azide based
azobenzene precursor 3 (Scheme S1). The product was purified
by preparative TLC and was characterized thoroughly by various
spectroscopic techniques and by the mass spectrometry (Figure
S3, S4 and Figure S11). The azobenzene unit was prepared via
the reduction of the corresponding m-nitrobenzyl azide and m-
1
of the triad 1 in methanol-d4 was also studied by the H NMR
spectroscopy (Figure S13).
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Figure 6. (A) Scheme for the racemization; (B) FTIR spectra of intermediate
(triad-1, compound 7) Schiff’s base with L-alanine; (C) Michaelis-Menten
plot of recemization with catalyst-1.
Figure 5. CD spectra for the racemization of L-Alanine with (A) trans-1,
and (B) with cis-1. (C) HPLC traces of Alanine under the racemization
conditions of PLP-conjugated cis-1, the retention time of L-Alanine and D-
Alanine are 3.85 min and 3.45 min respectively in the chiral-HPLC (1:1
isocratic mixture of CH3CN/water with 1% TFA).
at 201 nm having a positive Cotton effect, commonly observed for
the L-alanine in the CD spectra, was found to diminish gradually
with time (Figure 5B).
The sequence of the CD spectra after 2h displayed a spectrum
close to the baseline with a decline of the characteristic L-alanine
peak indicating the racemization (Figure S18). To validate
whether racemization has indeed taken place, the aqueous
solution of the reaction with cis-1 was subjected to HPLC analysis
with a reverse phase chiral column. Under the HPLC conditions
(Figure S20), the peak for the L-alanine standard solution had a
retention time of 3.85 min whereas a standard D-alanine had a
retention time of 3.45 min (Figure 5C).
The aliquot taken out of the reaction after 1.5 h of the reaction in
the presence of the cis-1 PLP-conjugate displayed two peaks –
one corresponding to the L-alanine and the other corresponding
to the D-alanine with an enantiomeric ratio of 70:30 (±2) (L>D).
The aliquot collected after 3 h of the reaction displayed an
enantiomeric ratio of 52/48 (±2) (L:D) of the two isomers indicating
the racemization. The rate of racemization with the cis-1 PLP-
conjugate was 1.5 x 103 times faster compared to the trans-1
(Table S1, Figure S19). To quantify the intramolecular catalytic
efficiency of the imidazole unit present in the active catalyst,
compared to the intermolecular reaction with imidazole, the
effective molarity (kintra/kinter) was estimated and found to be 1.91
In the presence of 366 nm ultraviolet light, the signals at  7.83,
7.34 and 7.20 corresponding to the aromatic protons of the
azobenzene unit gradually diminished and growth of three new
peaks in the upfield region at 7.08, 7.02, 6.60 for the same sets
of protons were observed.39-41
The free PLP and its Schiff’s base are in equilibrium with the
multiple tautomeric forms, which are not easily distinguishable in
the absorption spectra.42 Nevertheless, various attempts were
made to capture the formation of the Schiff base of the triad 1 with
L-alanine. Finally, the initial intermediate (compound 7) formed in-
situ was detected by FTIR spectroscopy and mass spectrometry
(Figure 6B, Figure S16-S17).
The activity of our PLP based enzyme mimic was monitored with
the catalyst in both the forms (trans-1 and cis-1) using circular
dichroism (CD) and also by HPLC analysis in the dark as well as
under 366 nm constant irradiation (Table S1, Figure S19B). Thus,
the solution of L-alanine (0.15 mM) in the presence of trans-1 (30
L; buffered with PBS at pH 7.0) was incubated at 298 K and the
aliquots were monitored by CD spectroscopy at various time
intervals and no noticeable spectral changes at 201 nm attributed
to the L-alanine was observed even after 3 h (Figure 5A).
Interestingly, in case of cis-1, spectral changes were observed
owing to the racemization. As a control, similar experiments were
carried out with imidazole and without any catalyst in the buffer
solution. The outcome of the experiments was found to be close
to the trans-1 (Figure S18C and S18D). To substantiate the
racemization, the aqueous solution of the reaction with cis-1 was
subjected to HPLC analysis with a reverse phase chiral column.
Under the HPLC conditions (Figure S20), the peak for the L-
alanine standard solution had a retention time of 3.85 min
whereas a standard D-alanine had a retention time of 3.45 min
(Figure 5C) the PLP-containing triad 1 (both trans/cis). However,
when the same experiment was carried out using cis-1, the peak
Table 1. Kinetic parameters of racemization with the catalyst (cis-1) in PBS
buffer, pH 7.0
kcat
kuncat
kcat/kuncat
Km
kcat/Km
(min-1)
(min-1)
(mM)
(min-1mM-1)
0.3915
0.00002 1.8×104
0.13
3.25
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x 103 M. The kinetic parameters of the racemization process were
also determined using the cis-catalyst with different substrate
concentrations. The kinetics data exhibited a Michaelis-Menten
behaviour (Figure 6C). Albeit the natural enzymes are far superior
compared to this mimic (Table S2), the catalyst cis-1 displays an
enzyme-like activity as is evident from the data in Table 1.
Although establishment of the details of the mechanism including
the rate-determining step require further studies, a key step for
the racemase activity is the base-induced deprotonation from the
amino acid-PLP aldimine. For our active-site mimic, in the cis
isomer the base reaches an optimum orientation and the distance
for an intramolecular deprotonation from the aldimine, whereas,
in the trans-isomer, the base and the aldimine units are far apart.
This makes the only cis-isomer an efficient active-form and the
activity could thus be turned on and off by light (Figure 6A).
Various thermodynamic parameters, including ΔEact, ΔH#, ΔS#
and t1/2 were calculated from the variable temperature kinetics
data. (Figure S22A and S22B, Table S3).
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Acknowledgements
This work was financially supported by DST-SERB, Govt. of India
(Grant No. EMR/2017/003720). MS is supported by an INSPIRE
doctoral fellowship by DST-India and MSH is funded by CSIR
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
An azobenzene based artificial
PLP racemase showed an
Monochura Saha, Munshi Sahid
Hossain, Subhajit Bandyopadhyay*
excellent catalytic efficiency with L-
alanine in the photoisomerized cis
form owing to close proximity of
the catalytic units.
Page No. – Page No.
A photoregulated racemase mimic
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