Turning on and off a pyridoxal 5'-phosphate mimic using light

Article abstract of DOI:10.1002/anie.201201447

Light is used to toggle a photoresponsive mimic of the cofactor pyridoxal phosphate between two isomers, where only one of them can catalyze the racemization of an amino acid. The system is based on creating and breaking electronic communication between the two catalyst components, i.e., an aldehyde and a pyridinium cation. Copyright

Full text of DOI:10.1002/anie.201201447

Angewandte
Chemie
DOI: 10.1002/anie.201201447
Photoresponsive Molecules
Turning “On” and “Off” a Pyridoxal 5’-Phosphate Mimic Using
Light**
Danielle Wilson and Neil R. Branda*
The use of light to reversibly change the structure of
biologically relevant molecules and turn “on” and “off”
important biochemical functions “on-command” offers the
biomedical end-user a non-invasive, rapid, reversible, spatial
and temporal tool for research and therapy. Applying this
control strategy to macromolecules such as oligonucleotides
and proteins is challenging because their large size and
complexity makes it difficult to target a particular area on the
macromolecules for modification, although several impres-
sive examples have been reported.[1–14] The alternative is the
use of photoresponsive small molecules that play an intimate
role in biochemical processes, as either cofactors or inhibitors.
Not only would these molecular systems be easier to photo-
activate and deactivate than their enzyme partners, they
would also provide a more “universal” method to regulate
biological function because the same cofactor can be involved
in more than one operation.
processes without the presence of an enzyme.[22,23] It is also
the role-model target of the studies described in this report.
The structural features responsible for the action of PLP
are the aldehyde and pyridinium functional groups, which are
electronically connected to each other through bonds
(Scheme 1). This intimate connectivity allows any molecule
Scheme 1. Reaction of an amino acid with PLP showing the aldimine
produced (R=OPO32ꢀ; R’=side chain). The quinonoid structure
formed after removal of the amino acid’s a-hydrogen is also shown.
We have recently described how two different colors of
light can be used to convert a small molecule between two
isomeric forms differing by an order of magnitude in their
ability to act as an inhibitor for human carbonic anhydrase.[15]
While regulating inhibitors is appealing,[16–18] applying the
same strategy to enzyme cofactors would provide control over
a more diverse set of biochemical systems. Cofactors have all
the earmarks of a suitable photoresponsive target. They tend
to be small in size, structurally simple and easy to modify and
study, while still allowing for ultimate control over the
enzyme activity. Here, we take a first logical step by using
a well-known enzyme cofactor as the inspiration in our design
of a proof-of-concept demonstration. We show how our
biomimetic system acts as a photoswitchable catalyst for
a biochemical reaction.[19]
The biologically active form of vitamin B6, pyridoxal 5’-
phosphate (PLP), is a versatile enzyme cofactor responsible
for amino acid metabolism in all organisms from bacteria to
humans.[20] Its participation in a diverse range of enzymatic
reactions including transamination, racemization, decarbox-
ylation, and numerous elimination and replacement processes
makes it unrivalled.[21] It is a particularly inspiring cofactor for
our proof-of-concept design because it can catalyze many
attached to the aldehyde to “sense” the electron withdrawing
nature of the positively charged heterocycle. An example of
this is the aldimine generated when an amino acid condenses
with PLP (Scheme 1) and it is this Schiff base that is
responsible for the enormous range of reactions the cofactor
catalyzes.[24] The pyridinium group in the aldimine makes the
amino acidꢀs a-hydrogen more acidic by stabilizing the
negative charge in the conjugate base through contribution
from a quinonoid structure (Scheme 1).[25,26] The fate of this
intermediate is varied and is responsible for the numerous
biochemical outcomes when PLP is involved.
We have developed a photoresponsive PLP cofactor
mimic whose catalytic activity can be reversibly switched
“on” and “off” when desired by irradiating it with UV and
visible light, respectively. We achieved this goal by taking
advantage of the photoswitchable dithienylethene (DTE)
architecture (Scheme 2), which undergo ring-closing and ring-
opening reactions when exposed to UV and visible light,
respectively.[27–29] The ring-open form of our system (1o) lacks
extensive through-bond communication between its two
“arms” (the ones bearing the pyridinium and aldehyde
functional groups). They are effectively electronically insu-
lated from each other and the cofactor mimic is “inactive”.
Ring-closing with UV light generates a linearly conjugated p-
backbone (shown in bold in structure 1c in the scheme) that
connects the two functional groups and the participation of
a quinonoid intermediate can be expected in the aldimine
produced when isomer 1c reacts with amino acids (shown on
the right of Scheme 2). Only the ring-closed isomer is “active”
and mimics PLP.
[*] D. Wilson, Prof. N. R. Branda
Department of Chemistry and 4D LABS, Simon Fraser University
8888 University Drive, Burnaby, BC V5A1S6 (Canada)
E-mail: nbranda@sfu.ca
[**] This research was supported by the Natural Sciences and Engi-
neering Research Council (NSERC) of Canada, the Canada Research
Chairs Program, and Simon Fraser University.
Our photoresponsive catalyst (1o) was prepared as out-
lined in Scheme 3. (Synthetic procedures and characterization
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201447.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
typical for dithienylethene
photoswitches, there is an
immediate reduction in the
intensity of the high-
energy bands and the
appearance of a new set
of bands in the visible
region of the spectrum
(centered at 635 nm) that
correspond to the ring-
Scheme 2. UV and visible light toggles the dithienylethene structure between its “inactive” ring-open (1o) and
“active” ring-closed (1c) forms and dictates if the pyridinium and aldehyde are insulated or connected to each
other. The structure on the right is the quinonoid that would be generated after deprotonating the a-hydrogen
of the aldimine generated from reaction of compound 1c and an amino acid.
closed
isomer
(1c).
1H NMR
spectroscopy
confirmed the conversion
(1o!1c) and revealed that the photostationary state contains
97% of the ring-closed isomer, the remainder consisting of
the ring-open isomer (1o). Visible light of wavelengths
greater than 490 nm converted the solution back to pale
yellow and regenerated the ring-open isomer (1o).
The ability of our photoresponsive PLP mimic to act as
a controllable catalyst was demonstrated using hydrogen–
1
deuterium exchange experiments and H NMR spectroscopy
on solutions of optically pure l-alanine (Figure 2). This is an
accepted method to monitor the catalytic racemization of
amino acids since racemization is expected to occur through
the planar, achiral sp2 hybridized quinonoid intermediate.
The rates of hydrogen–deuterium exchange and racemization
in a series of amino acid derivatives have been shown to be
nearly equal.[30]
Our experiments were performed by dissolving l-alanine
(1 molar equivalent) and a catalytic amount of either 1o or 1c
(0.2 molar equivalents) in a 1:9 (v/v) mixture of D2O and
[D4]acetic acid at 408C (ꢁ 28C) in a sealed NMR tube. The
solution of the ring-closed isomer was prepared by irradiating
1o with 365 nm light until no changes were observed in the
1H NMR spectrum (typically 120 min at this high concen-
Scheme 3. Synthesis of the photoresponsive catalyst.
1
tration). The partial H NMR spectrum in Figure 2b and c
shows the set of peaks corresponding to the a-hydrogen of l-
alanine. It is clear from this figure that the intensity of the
peaks is greatly reduced when the ring-closed isomer (1c) is
present but the intensity remains the same in the case of the
ring-open isomer (1o). Only the former isomer can induce
deprotonation of the amino acid. This exchange process
presumably occurs through the aldimine intermediate,
although under these conditions this intermediate was not
of all compounds are provided in the Supporting Informa-
tion.) Compound (1o) undergoes light stimulated ring-closing
as is expected for dithienylethenes. This reaction is illustrated
by the changes in the UV/Vis absorption spectra when an
aqueous acetic acid solution of the compound is irradiated
with 365 nm light (Figure 1) and is accompanied by a change
in color of the solution from pale yellow to deep blue. As is
1
observed in the H NMR spectrum. Another indication that
only the ring-closed isomer catalyzes the exchange process is
the conversion of the peak corresponding to the CH3 groups
of l-alanine from a doublet to a singlet as hydrogen–
deuterium exchange proceeds Figure 2d. This spectral
change was not observed when the ring-open isomer (1o)
was used.
After the hydrogen–deuterium exchange catalyzed by the
ring-closed isomer (1c) was nearly complete (95% as
1
confirmed by H NMR spectroscopy), alanine was isolated
1
from the reaction mixture. In the partial H NMR spectrum
Figure 1. a) Changes in the UV/Vis absorption spectrum when
a CH3CO2H/H2O (9:1 v/v) solution (5ꢀ10ꢀ5 m) of 1o is irradiated with
365 nm light. b) The growth of the band at 635 nm corresponding to
the ring-closed isomer (1c), which is present in 97% in the photosta-
tionary state according to 1H NMR spectroscopy.
(D2O) of this sample (Figure 2e bottom), the singlet at
1.21 ppm corresponds to the CH3 group of the amino acid
after hydrogen–deuterium exchange. The smaller peak
appearing at 1.22 ppm corresponds to the residual non-
2
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
after a total of 140 h at 408C). On the other hand, within this
same time period the ring-closed isomer (1c) catalyzes the
exchange to nearly 30%, and when allowed to react for an
additional 90 h at 558C we achieve nearly complete (95%)
hydrogen–deuterium exchange. With no catalyst present
there is essentially 0% exchange showing that the racemiza-
tion of the amino acid substrate on its own is negligible.
We could also reversibly turn “on” and “off” our catalyst
by using UV and visible light (Figure 2g). When a solution of
l-alanine is treated with the ring-open isomer (1o) under the
same conditions as described previously, as expected there is
little observable hydrogen–deuterium exchange. After expo-
sure of the sample to 365 nm light, which generates the ring-
closed isomer (1c), we observed immediate exchange. Con-
verting the catalyst back to its ring-open form with visible
light reduces the exchange to its originally low rate. The rate
can be increased again by using UV light.
The relative rates of deuterium exchange reflect the
differences in electronic properties of the ring-open and ring-
closed isomers of our PLP mimic and demonstrate the success
of our concept. The higher rate of exchange when 1c is
present supports the design strategy wherein the pyridinium is
able to communicate through the DTE backbone with the
aldehyde only in this isomeric state. This provides evidence
for a dramatic increase in activity when in the ring-closed
form and potentially offers new means to control enzymatic
processes by modifying appropriate cofactors with photo-
responsive components. Although rates of racemization are
low, the system demonstrated in our studies is the first
example of photoswitchable PLP mimic that can act as
a catalyst.
Figure 2. a) The racemization of l-alanine can be indirectly assessed
by monitoring the exchange of the a-hydrogen by a deuterium in the
presence of 1o or 1c. The ring-closed isomer was prepared by
1
irradiating (1o) with 365 nm light for 120 min. b–d) Partial H NMR
spectra of CD3CO2D/D2O (9:1 v/v) solutions of l-alanine (0.1m)
containing 0.2 equiv of b) 1o and c,d) 1c at 408C (ꢁ28C). In each
case, the top spectrum corresponds to the solution at the beginning of
the experiment and the bottom spectrum is after 20 days. e) The
alanine isolated after reaction with 1c (for an additional 90 h at 558C)
with (top) and without (bottom) 0.1 molar equivalents of sodium [(R)-
1,2-diaminopropane-N,N,N’,N’-tetraacetato]samarate(III). Both sam-
ples were basified to pH 10–11 with NaOD (40% w/w in D2O) to
ensure binding to the chiral shift reagent. f) Percent hydrogen–
Received: February 21, 2012
Revised: March 3, 2012
Published online: && &&, &&&&
~
deuterium exchange of the l-alanine solutions without catalyst ( ), in
*
*
the presence of 0.2 equiv of catalyst, 1o ( ) or 1c ( ) over the first
140 h. g) Changes in the percent hydrogen–deuterium exchange of
CD3CO2D/D2O (9:1 v/v) solution of l-alanine (0.2m) containing
0.2 equiv of 1o in response to alternating cycles of irradiation with UV
and visible light. The timescale referred to on the x-axis is the total
reaction time at 408C and does not include 1H NMR acquisition time
and irradiation times. The 1H NMR measurements and sample irradi-
ations were conducted at room temperature where the rate of hydro-
gen–deuterium exchange is insignificant.
Keywords: biomimetics · enzymatic catalysis · photochemistry ·
.
photochromism
[1] A. A. Beharry, G. A. Woolley, Chem. Soc. Rev. 2011, 40, 4422 –
4437.
[2] D. L. Fortin, T. W. Dunn, A. Fedorchak, D. Allen, R. Montpetit,
M. R. Banghart, D. Trauner, J. P. Adelman, R. H. Kramer, J.
Neurophysiol. 2011, 106, 488 – 496.
[3] T. Fehrentz, M. Schçnberger, D. Trauner, Angew. Chem. 2011,
123, 12362 – 12390; Angew. Chem. Int. Ed. 2011, 50, 12156 –
12182.
[4] F. Bonardi, G. London, N. Nouwen, B. L. Feringa, A. J. M.
Driessen, Angew. Chem. 2010, 122, 7392 – 7396; Angew. Chem.
Int. Ed. 2010, 49, 7234 – 7238.
[5] Y. Hachikubo, S. Iwai, T. Q. P. Uyeda, Biotechnol. Bioeng. 2010,
106, 1 – 8.
deuterated alanine, which is actually an overlapping doublet.
The addition of an NMR chiral shift reagent ([(R)-1,2-
diaminopropane-N,N,N’,N’-tetraacetato] samarate(III)) to
a solution of the isolated amino acid in D2O produces two
well-resolved diastereomeric complexes with unique chemical
1
shifts in the H NMR spectrum as shown in (Figure 2e top).
Each singlet can be assigned to the CH3 group of d-alanine
and l-alanine, and their approximately equal intensity con-
firms that racemization has occurred.
[6] J. H. Harvey, D. Trauner, ChemBioChem 2008, 9, 191 – 193.
[7] X. Liang, K. Fujioka, Y. Tsuda, R. Wakuda, H. Asanuma,
Nucleic Acids Symp. Ser. 2008, 52, 19 – 20.
[8] P. Gorostiza, M. Volgraf, R. Numano, S. Szobota, D. Trauner,
E. Y. Isacoff, Proc. Natl. Acad. Sci. USA 2007, 104, 10865 –
10870.
The rate of deuterium exchange was monitored in situ by
recording the decrease in integration under the curve of the
peaks in the spectrum corresponding to the a-hydrogen of the
amino acid (4.13 ppm) as it is converted to a-deuterium. A
plot of percentage of hydrogen–deuterium exchange over
time (Figure 2 f) emphasizes the minimal racemization of l-
alanine when 1o is present (less than 3% exchange observed
[9] M. Bose, D. Groff, J. Xie, E. Brustad, P. G. Schultz, J. Am. Chem.
Soc. 2006, 128, 388 – 389.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
[10] I. Hamachi, T. Hiraoka, Y. Yamada, S. Shinkai, Chem. Lett. 1998,
537 – 538.
[11] I. Willner, S. Rubin, Angew. Chem. 1996, 108, 419 – 439; Angew.
Chem. Int. Ed. Engl. 1996, 35, 367 – 385.
[12] I. Willner, S. Rubin, A. Riklin, J. Am. Chem. Soc. 1991, 113,
3321 – 3325.
[19] For an excellent review on the use of light to control catalysis,
see: R. S. Stoll, S. Hecht, Angew. Chem. 2010, 122, 5176 – 5200;
Angew. Chem. Int. Ed. 2010, 49, 5054 – 5075.
[20] A. C. Eliot, J. F. Kirsch, Annu. Rev. Biochem. 2004, 73, 383 – 415.
[21] D. E. Metzler, M. Ikawa, E. E. Snell, J. Am. Chem. Soc. 1954, 76,
648 – 652.
[13] H. Kaufman, S. M. Vratsanos, B. F. Erlanger, Science 1968, 162,
1487 – 1489.
[22] H. Hayashi, H. Wada, T. Yoshimura, N. Esaki, K. Soda, Annu.
Rev. Biochem. 1990, 59, 87 – 110.
[14] U. Krauss, T. Drepper, K.-E. Jaeger, Chem. Eur. J. 2011, 17,
2552 – 2560.
[23] T. F. Buckley, H. Rapoport, J. Am. Chem. Soc. 1982, 104, 4446 –
4450.
[15] D. Vomasta, C. Hçgner, N. R. Branda, B. Kçnig, Angew. Chem.
2008, 120, 7756 – 7759; Angew. Chem. Int. Ed. 2008, 47, 7644 –
7647.
[16] D. Pearson, A. D. Abell, Chem. Eur. J. 2010, 16, 6983 – 6992.
[17] A. D. Abell, M. A. Jones, A. T. Neffe, S. G. Aitken, T. P. Cain,
R. J. Payne, S. B. McNabb, J. M. Coxon, B. G. Stuart, D. Pearson,
H. Y. Y. Lee, J. D. Morton, J. Med. Chem. 2007, 50, 2916 – 2920.
[18] P. R. Westmark, J. P. Kelly, B. D. Smith, J. Am. Chem. Soc. 1993,
115, 3416 – 3419.
[24] H. Hayashi, J. Biochem. 1995, 118, 463 – 473.
[25] M. D. Toney, Biochim. Biophys. 2011, 1814, 1407 – 1418.
[26] M. D. Toney, Arch. Biochem. Biophys. 2005, 433, 279 – 287.
[27] H. Tian, S. Yang, Chem. Soc. Rev. 2004, 33, 85 – 97.
[28] M. Irie, Molecular Switches, Wiley-VCH, Weinheim, 2001.
[29] M. Irie, Chem. Rev. 2000, 100, 1685 – 1716.
[30] H. Matsuo, Y. Kawazoe, M. Sato, M. Ohnishi, T. Tatsuno, Chem.
Pharm. Bull. 1967, 15, 391 – 398.
4
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Photoresponsive Molecules
D. Wilson, N. R. Branda*
&&&& — &&&&
Turning “On” and “Off” a Pyridoxal 5’-
Phosphate Mimic Using Light
Light is used to toggle a photoresponsive
mimic of the cofactor pyridoxal phos-
phate between two isomers, where only
one of them can catalyze the racemiza-
tion of an amino acid. The system is
based on creating and breaking electronic
communication between the two catalyst
components, i.e., an aldehyde and a pyr-
idinium cation.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!