Synthesis and characterisation of α-carboxynitrobenzyl photocaged L-aspartates for applications in time-resolved structural biology

Article abstract of DOI:10.1039/c9ra00968j

We report a new synthetic route to a series of α-carboxynitrobenzyl photocaged l-aspartates for application in time-resolved structural biology. The resulting compounds were characterised in terms of UV/Vis absorption properties, aqueous solubility and stability, and photocleavage rates (τ = μs to ms) and quantum yields (φ = 0.05 to 0.14).

Full text of DOI:10.1039/c9ra00968j

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Synthesis and characterisation of a-
carboxynitrobenzyl photocaged L-aspartates for
Cite this: RSC Adv., 2019, 9, 8695
applications in time-resolved structural biology†
a
b
c
d
John J. Zaitsev-Doyle,
Anke Puchert,
Yannik Pfeifer,
Hao Yan,
e
c
df
Briony A. Yorke,
Julia Rehbein,
Henrike M. Muller-Werkmeister,
Nils Huse,
Charlotte Uetrecht,
*
and Marta Sans
¨
g
ab
a
a
*
Arwen R. Pearson
Received 5th February 2019
Accepted 6th March 2019
We report a new synthetic route to a series of a-carboxynitrobenzyl photocaged L-aspartates for
application in time-resolved structural biology. The resulting compounds were characterised in terms of
UV/Vis absorption properties, aqueous solubility and stability, and photocleavage rates (s ¼ ms to ms) and
quantum yields (4 ¼ 0.05 to 0.14).
DOI: 10.1039/c9ra00968j
rsc.li/rsc-advances
In a time-resolved experiment the reactive species have to be fraction of the sample. For state reversible systems stroboscopic
produced instantaneously compared to the timescale of the illumination can result in high populations of particular inter-
reaction being studied. For time-resolved structural biology, mediates.4 However, very few biochemical reactions are easily
time-resolutions spanning many decades in time are required reversible, with the exception of photosensors such as
in order to allow structural biologists to link the ultrafast (fs to rhodopsin8 and photoactive yellow protein,9 or the classical CO
ps) chemical steps with the slower (ns to ms) motions of the release and rebinding to heme groups.7
protein. These slower dynamics are of particular interest as they
are thought to play a key role in modulating the selectivity sensitive moieties, photocages12 (photoremovable protecting
control of enzymatic catalysis.1,2
groups) have received increasing attention in recent years as
As only a small subset of proteins naturally contain photo-
For fast (sub ms) reactions most experiments are some a tool to study structure–function–dynamics relationships in
variation of Porter's pump–probe3–6 approach where the reac- a wide range of biomolecules both in vitro and in vivo,13,14 such
tion is initiated with a short laser pulse that either directly as kinase activation15 and photoinduced gene expression in
triggers the reaction of interest by photolysis or isomer- living cells.16 For time-resolved structural biology experiments
isation,7–9 produces a T-jump,10 or releases a reactive moiety both photocaged ligands that diffuse into or near the active site,
from a photocaged compound.6,11 The two limiting factors of and unnatural amino acids that can be site-specically incor-
such experiments are the time required for reaction initiation, porated into the protein during translation can be used.6,17 An
which can range from a few fs (isomerisation/direct photolysis) ideal photocage for these experiments should have a large
to ms for photocaged compounds, and the degree of reaction absorption above approx. 320 nm, so that the enzyme is not
initiation or quantum yield, which can oen be only a small excited directly, a high quantum yield with very rapid release of
the substrate, be chemically stable in aqueous solution in the
dark, and have good aqueous solubility.
ortho-Nitrobenzyl (oNB) protecting groups are arguably the
most used and best understood class of photocages.12 However,
aThe Hamburg Center for Ultrafast Imaging & Institute for Nanostructure and Solid
State Physics, Luruper Chaussee 149, 22761 Hamburg, Germany. E-mail: arwen.
pearson@cfel.de; marta.sans.valls@cfel.de
simple oNBs are not well suited for time-resolved structural
biology, for example due to their absorbance maximum at
260 nm and slow photocleavage, which occurs on the milli-
second timescale.18 Modied oNB cages, for example with
a carboxyl substitution at the benzylic site to improve aqueous
solubility and reduce photocleavage times to the microsecond
timescale, have therefore been explored by a number of groups
and have great potential for time-resolved structural
biology.19–21
bDepartment of Physics and Centre for Hybrid Nanostructures, University of Hamburg,
Luruper Chaussee 149, 22761 Hamburg, Germany
cUniversity of Potsdam, Institute of Chemistry, Physical Chemistry, Karl-Liebknecht-
Str. 24-25, Golm, 14476 Potsdam, Germany
dHeinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistrasse
52, 20251 Hamburg, Germany
eSchool of Chemistry, University of Leeds, Leeds LS2 9JT, UK
fEuropean XFEL GmbH, Holzkoppel 4, 22869 Schenefeld, Germany
g
¨
¨
¨
Fachbereich fur Chemie und Pharmazie, Universitat Regensburg, Universitatsstrasse
31, 93053 Regensburg, Germany
Here we chose 1-(aCNB)-L-aspartate (1a, Fig. 1) as a starting
point.20 As inclusion of a methylenedioxy ring in oNB cages
† Electronic supplementary information (ESI) available: NMR stability studies,
laser ash photolysis data, quantum yield determination, and compound
synthesis and characterisation. See DOI: 10.1039/c9ra00968j
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Conveniently, steps (i)–(iv) can be telescoped to afford 6 with
only a single purication step and the nal compounds were
obtained in good to very good yields (48–74% over 7 steps).
For time-resolved applications, photocages must be stable
under dark conditions in either solid state, or in organic or
aqueous solution, depending on the application. To test
aqueous stability, hydrolysis was followed by 1H NMR spec-
troscopy, with a water suppression pulse sequence. The meth-
ylenedioxy substituted photocage (1b) was much more stable
than the parent a-carboxynitrobenzyl (1a) (Table S1 in the ESI†)
and the hydrolysis was found to be retarded with decreasing pH.
The decreased stability at high pH is due to a neighbouring
group participation mechanism: the a-carboxylate attacks the
ester carbonyl intramolecularly, forming a mixed anhydride
which is readily cleaved. When protonated at lower pH, the a-
carboxylate can no longer attack in this way.25
Fig.
1 a-Carboxynitrobenzyl photocaged L-aspartate compounds
synthesised and investigated in this work: a-carboxynitrobenzyl
(aCNB, 1a), a-carboxynitropiperonyl (aCNP, 1b) and p-bromo-a-car-
boxynitrobenzyl (BraCNB, 1c) L-aspartate.
Photocaged compound 1b displayed the expected additional
absorption band at lmax ¼ 365 nm, with a relatively high
extinction coefficient (~4000 Mꢀ1 cmꢀ1 – approx. 10-fold that of
1a, 1c or other oNB photocages without either methylenedioxy
or dimethoxy substitution patterns at that wavelength). In
addition, the photoreaction (Scheme 2) was investigated by
laser ash photolysis spectroscopy. Excitation at 355 nm (in
aerated phosphate buffer at pH 7) of compound 1b resulted in
a transient absorption at 460 nm which decayed with a time
constant s ¼ 199 ꢁ 6 ns (Fig. S8 in the ESI†). We believe this to
be the initially formed aci-nitro species,§ which is deprotonated
on this timescale (Il'ichev and co-workers reported a similar
time constant for the deprotonation of the aci-nitro formed
aer excitation of o-nitrobenzyl methanol).18 The aci-nitro/
nitronate species are understood to be key intermediates in
oNB photoreactions.12,18 Formation and deprotonation of the
initial aci-nitro is followed by decay of the nitronate species,
observed at 430 nm, which ts to an exponential decay function
with a time constant of 0.87 ms (see Fig. 2). In the case of
compounds 1a and 1c, however, the nitronate decay kinetics
were much faster and followed a bi-exponential decay (see Table
1). The two components may be due to the formation of both
isomers about the C]C double bond. The decay of the nitro-
nate is oen assumed to be the rate determining step, and
Jayaraman showed this to be the case with g-CNB-L-glutamate
(in aq. phosphate buffer at pH 7) using both laser ash
photolysis and time-resolved IR spectroscopy.26 However, Il'i-
chev and co-workers demonstrated, for o-nitrobenzyl methanol,
that the rate-determining step is pH-dependent (the nitronate
decay is acid-catalysed and the benzisoxazolol ring-opening is
base-catalysed – at pH # 4, the ring-opening is the slower step)
and a hemiacetal intermediate can persist when the leaving
group is poor.18 Thus, the measured time constants give only an
indication of the release rate of photocages investigated in this
work. Nevertheless, carboxylate is a good leaving group and our
experiments were carried out in buffer at neutral pH. Therefore,
affords absorption at longer wavelengths,19 we combined 1a
with an a-carboxy substitution and synthesised a-carboxyni-
tropiperonyl L-aspartate (1-(aCNP)-L-aspartate, 1b). Concur-
rently, we also synthesised a p-bromo derivative (1c), as this has
been previously reported to improve the rate of oNB photo-
cleavage.22 We chose L-aspartate as the leaving group in this
study as a photocaged amino acid exemplar system. a-Carbox-
ylate caged amino acids are potential photocaged enzyme
substrates, for example for amino acid decarboxylases. An
analogous synthetic approach to that reported here can also be
used to deliver g-carboxylate caged amino acids that can serve
as enzyme substrates or be incorporated into a peptide back-
bone during translation or peptide synthesis. In our current
work we are exploring the application of these photocages to
studying the mechanism of the E. coli enzyme L-aspartate alpha-
decarboxylase, which catalyses the irreversible conversion of L-
aspartate to b-alanine and carbon dioxide, the rst dedicated
step in pantothenate and coenzyme A biosynthesis.23
The synthesis of a-carboxynitrobenzyl L-aspartate is sum-
marised in Scheme 1. In comparison with the synthesis of
Grewer and co-workers,‡,20 this improved route avoids the use of
stoichiometric in situ generated HCN. Following a literature
procedure,24 trimethylsilyl cyanide was added to commercially
available o-nitrobenzaldehydes (2) to afford the corresponding
racemic trimethylsilyl (TMS) protected cyanohydrins (3). These
were hydrolysed with concentrated HCl to afford o-nitro-
mandelic acids (4), which were then acetylated to give 5. Next,
the carboxyl groups were protected with tert-butyl tri-
chloroacetimidate to give 6 and treatment with catalytic Cs2CO3
in methanol afforded the alcohols (7).25 These were coupled to
a commercially available amino acid derivative (N-Boc 4-tBu L-
aspartate) using EDC and DMAP to give the esters (8), as a 1 : 1
diastereomeric mixture, which were deprotected in the nal
step with TFA/CH2Cl2 to afford the photocaged L-aspartates (1).
‡ Regarding Grewer's synthesis,20 we found that cyanohydrin formation was not
complete within 1.5 h (overnight is sufficient) and 1 h at reux (100 ꢂC bath
temp.) – rather than 8 h at 80 ꢂC – was enough for hydrolysis of the amide to
afford 4a.
§ Il'ichev and co-workers argue that, though the Z-isomer about the aci-nitro
group would form rst, both E- and Z-forms would rapidly equilibrate in
aqueous solution via solvent caged H3O+ and would not be spectroscopically
distinguishable (except perhaps using ultrafast techniques).18
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Scheme 1 Synthesis of photocaged L-aspartates 1a–1c (structures of final products shown in Fig. 1). (i) TMSCN, MeCN, 18 h, r.t. (ii) HCl, 24 h, 0–
100 ꢂC. (iii) Ac2O, 18 h, r.t. or 4 h, 60 ꢂC. (iv) TBTA, benzene, 2 d, r.t., (6a, 72%, 6b, 85%, 6c, 66%; 4 steps). (v) Cs2CO3, MeOH, 1 h, r.t. (vi) N-Boc
4-tBu L-aspartate, DMAP, EDC$HCl, CH2Cl2, 24 h, 0 ꢂC – r.t., (8a, 81%, 8b, 88%, 8c, 73%; 2 steps). (vii) TFA, CH2Cl2, 24 h, r.t., quant. TMSCN ¼
trimethylsilyl cyanide, TBTA ¼ tert-butyl trichloroacetimidate, DMAP ¼ 4-(dimethylamino)pyridine, EDC ¼ 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, TFA ¼ trifluoroacetic acid.
Scheme 2 Proposed mechanistic pathway (compound 1b is shown as an example) of the nitrobenzyl photocleavage reaction. Photoinduced
HAT leads to the aci-nitro/nitronate intermediates, which subsequently cyclise before releasing the leaving group (LG). Refer to the text for
a detailed discussion.
it is likely that product release coincides with nitronate decay in effective quantum yield of 1b was only 5%, half that of 1a. The
this case.
aCNP cage would therefore be more suited to applications
Contrary to our expectation, the pBr-aCNB (1c) caged where longer wavelength UV light is required, and high
compound did not display faster nitronate decay kinetics downstream time-resolution is not required, or applications
compared to aCNB (1a). Both compounds 1a and 1c absorb involving continuous irradiation. The low quantum yields are
about an order of magnitude less than 1b, however their also non-ideal for time-resolved crystallographic applications
kinetics appear to be a hundred fold faster, with quantum where the resulting electron density is an average of all
yields of about 10% and 14%, respectively. Conversely, the molecules in the X-ray illuminated volume (i.e. both caged and
uncaged), and improving them is therefore a future goal.
Especially interesting would be a photocage with fast kinetics,
high quantum yield and a high extinction coefficient at ca.
350 nm.
In summary, in the experiments described here we have
presented a new and improved synthetic route to a-carbox-
ynitrobenzyl photocages. These have been fully charac-
terised, showing good aqueous stability in buffer. The a-
carboxynitropiperonyl group shows
a new absorbance
maximum at 360 nm, which is advantageous for time-
resolved structural studies of biomolecules. Work towards
conrming the nitronate decay as the rate determining step
is ongoing, and we are also working towards an asymmetric
synthesis of 2-nitromandelic acid derivatives (4) to address
the issue of the racemic and diastereomeric compounds
presented here.
Fig. 2 Kinetic trace of transient species (assigned to the nitronate)
formed by excitation of 1b with a 355 nm (third harmonic) pulse from
a Nd:YAG laser, 30 mJ pulse energy, 1 cm ꢃ 1 cm cuvette, 0.4 mM 1b
in 5ꢃ PBS buffer, pH 7. Data were fitted with the exponential decay
function DA ¼ aeꢀt/s. The time constant obtained was s ¼ 0.87 ꢁ 0.04
ms. See ESI† for more information.
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Table 1 Photochemical properties and hydrolysis half-lives of photocaged L-aspartates (1)a
Photocaged compound
s1/ms
s2/ms
4365
3355/Mꢀ1 cmꢀ1
t1/2/h (hydrolysis)b
1a
1b
1c
1.5 ꢁ 0.1
870 ꢁ 40
10.4 ꢁ 0.4
n/a
38 ꢁ 1
0.105 ꢁ 0.005
0.048 ꢁ 0.007
0.14 ꢁ 0.02
430
4060
520
13.9 ꢁ 0.2
12.6 ꢁ 0.1
10.2 ꢁ 0.1
1.48 ꢁ 0.08
a
Time constants were derived by exponential tting of kinetic traces from laser ash photolysis data and represent the two biexponential
components of nitronate decay (suspected to coincide with the release of L-aspartate). Quantum yield determinations were carried out with
a 365 nm UV LED, and were measured using phenylglyoxylic acid actinometry and HPLC. Thermal half-lives were obtained by tting 1H NMR
data recorded over 24 h with a water-suppression pulse sequence. See ESI for more details. b Half-life of the less stable diastereoisomer at pH 7.
F. Diederich, E. F. Pai and R. J. D. Miller, Nat. Methods,
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Conflicts of interest
7 F. Schotte, M. Lim, T. A. Jackson, A. V. Smirnov, J. Soman,
J. S. Olson, G. N. Phillips, M. Wulff and P. A. Annrud,
Science, 2003, 300, 1944–1947.
The authors declare no conicts of interest.
Acknowledgements
8 M. Andersson, E. Malmerberg, S. Westenhoff, G. Katona,
¨
M. Cammarata, A. B. Wohri, L. C. Johansson, F. Ewald,
This work was supported by the Cluster of Excellence ‘The
Hamburg Centre for Ultrafast’ of the Deutsche For-
schungsgemeinscha (DFG) – EXC 1074 – project ID 194651731.
B. A. Y. thanks the Wellcome Trust (Grant code 110296/Z/15/Z)
for support. J. R. thanks the DFG (Emmy-Noether, grant code
251211948) for generous funding. The Heinrich Pette Institute,
Leibniz Institute for Experimental Virology is supported by the
Free and Hanseatic City of Hamburg and the Federal Ministry of
Health. We thank Prof Malte Brasholz for the use of laboratory
equipment and helpful discussions. We thank Dr Diana C. F.
Monteiro for helpful discussions. The authors acknowledge the
Scientic Service of Hamburg University's Chemistry Depart-
ment for compound characterisation and we are grateful to
Claudia Wontorra in particular for NMR (hydrolysis studies). J.
J. Z.-D. wishes to thank Dana Komadina, Dr Iosiphina Sarrou
and Prof Henry N. Chapman for the use of their HPLC equip-
ment. The authors wish to thank Prof Godfrey Beddard for
reading the manuscript and providing helpful comments. J. J.
Z.-D. thanks Owen Tuck for help preparing buffers and stan-
dards, and Bastian Wulff for assistance with the operation of
the Applied Photophysics LKS80 instrument used for laser ash
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