A Benzophenone-Based Photocaging Strategy for the N7 Position of Guanosine

Article abstract of DOI:10.1002/anie.201914573

Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring-nitrogen methylation, such as m⁷G and m¹A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α-hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho-nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site-specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m⁷G and m¹A with spatiotemporal control.

Full text of DOI:10.1002/anie.201914573

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A benzophenone-based photocaging strategy for the N7 position
of guanosine
Authors: Lea Anhäuser, Nils Klöcker, Fabian Muttach, Florian Mäsing,
Petr Spacek, Armido Studer, and Andrea Rentmeister
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914573
Angew. Chem. 10.1002/ange.201914573
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A benzophenone-based photocaging strategy for the N7 position
of guanosine
Lea Anhäuser,[a] Nils Klöcker,[a] Fabian Muttach,[a] Florian Mäsing,[b] Petr Špaček,[a] Armido Studer[b]
and Andrea Rentmeister*[a]
Abstract: The selective modification of nucleobases with photolabile
caging groups provides one of the most versatile strategies to study
and control the processes and interactions of nucleic acids. Numerous
exocyclic and ring positions of nucleobases have been targeted, but
all are formally substituting a hydrogen atom with a photo-caging
group. Nature, however, also explicitly uses ring nitrogen methylation,
such as m7G and m1A, to change the electronic structure and
properties of RNA and control biomolecular interactions essential for
translation and turnover. We report that aryl ketones such as
benzophenone and α-hydroxyalkyl ketone are photolabile caging
groups if installed at the N7 position of guanosine or the N1 position
of adenosine. Common photo-caging groups derived from the ortho-
nitrobenzyl moiety were not suitable for these positions. Both
chemical and enzymatic methods for site-specific modification of N7G
in nucleosides, dinucleotides and RNA were developed, opening the
door to studying the molecular interactions of m7G and m1A with
spatio-temporal control.
introduced into DNA or RNA by polymerases, the 5 position of
pyrimidines is particularly favorable.[7] Recently, also the post-
enzymatic photocaging of DNA was achieved using
methyltransferase (MTase)-catalyzed transfer of PC groups to the
N6 position of adenosines.[8] In all of these cases, the PC group
replaces a hydrogen atom and does not change the charge
distribution of the nucleoside.
However, unlike the chemical modifications with PC groups
developed to date, nature does not only substitute hydrogen
atoms with modifications, but also explicitly uses methylation to
change the electronic structure of the molecule. In naturally
occurring m7G, m1A and m3C, the modifications confer a positive
charge to the nucleobase. Out of these, m7G deserves particular
attention because it is (i) one of the most conserved modified
nucleosides, (ii) installed by numerous MTases in different
organisms and (iii) found in rRNA, tRNA, snRNA as well as part
of the 5′ cap in mRNA.[9] In the latter, m7G is crucial for
coordinating mRNA translation and turnover by several
interactions, such as binding to the translation initiation factor
eIF4E or decapping scavenger enzymes DcpS.[10] Due to its
importance in biology, we chose the N7 position of guanosine for
modification with functional groups with the goal to remove them
upon irradiation with light and recover the free guanosine. In the
5′ cap of mRNAs, the G becomes remethylated in cells.[11] To
generate photocaged guanosines in different contexts, i.e. from
single nucleosides to long mRNAs, we devised both a chemical
and an enzymatic preparative route (Scheme 1). PC groups for
nucleosides that do not substitute a hydrogen atom but instead
introduce a positive charge have not been reported to date to the
best of our knowledge.
Light is a versatile regulatory element because it is fully
orthogonal to most cellular components, noninvasive, controllable
in timing and localization to tissues, cells and even subcellular
compartments.[1] To gain optical control over nucleic acids and the
respective
biomolecular
interactions,
irreversible
photo(de)activation (“uncaging”) has been applied to nucleosides,
nucleotides and oligonucleotides.[1-2] Various photolabile
protecting groups have been placed either in the oligomer
backbone or on the nucleobases and their removal by light of a
defined wavelength ≥365 nm was proven to be compatible with
living cells and organisms.[3] The ortho-nitrobenzyl (ONB) group
is the most widely used photocaging (PC) group, but numerous
alternative scaffolds, derived from coumarins, quinolines,
dibenzofuran or the piperonyl group have been explored.[1-2]
Typically, these PC groups are installed at exocyclic
heteroatoms, such as the O4 in thymidine or the N4 of cytidine.[4]
In addition, the N3 position of thymidine was successfully used in
optochemical biology.[3b] In purines, the exocyclic O6 of guanosine
and N6 of adenosine are preferred sites for installation of
photocaging groups.[5] In the case of guanosine, other positions
like the N1, C8 or the exocyclic N2 position have been explored
albeit to a much lower extent.[6] For chemo-enzymatic approaches,
where photo-caged NTPs are synthesized and enzymatically
First, we explored the chemical photocaging of guanosine 1
using the well-known ortho-nitrobenzyl (ONB) group as well as
para-nitrobenzyl (PNB) as negative control. The N7 of guanosine
is the most potent nucleophile in DNA and RNA.[12] However,
although N7G methylation was used in Maxam Gilbert
sequencing,[13] the chemical modification of N7 has not been
exploited to manipulate and control interactions by orthogonal
[a]
Dr. L. Anhäuser, N. Klöcker, Dr. F. Muttach, Dr. Petr Špaček, Prof.
Dr. A. Rentmeister
Institut für Biochemie
Westfälische Wilhelms-Universität Münster
Wilhelm-Klemm-Str. 2, 48149 Münster (Germany)
E-mail: a.rentmeister@uni-muenster.de
Dr. Florian Mäsing, Prof. Dr. Armido Studer
Organisch-Chemisches Institut
[b]
Scheme 1. Concept for chemical and enzymatic photocaging of the N7 of
guanosine using classical ortho-nitrobenzyl (ONB) group or aryl ketones such
as benzophenone (BP) to generate the respective nucleoside, 5′ cap or RNA.
N7-BP-modified guanosine is uncaged by subsequent irradiation with light (λmax
= 365 nm).
Westfälische Wilhelms-Universität Münster
Corrensstrasse 40, 48149 Münster (Germany
Supporting information for this article is given via a link at the end of
the document.
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triggers such as light. Building on the good nucleophilicity of the
N7 position of guanosine, we reacted guanosine with the
respective PC-bromides 2a-b and obtained the expected N7-
ortho-nitrobenzyl- (N7-ONB) and N7-para-nitrobenzyl-(N7-PNB)
guanosines 3a-b (Figure 1A, Figure S3-S5) in good yields (3a:
53% and 3b: 56%).[14] Subsequent irradiation of 3a, however, did
not yield the desired photocleavage to release guanosine 1 but
instead a different main product according to HPLC (Figure 1B,
Figure S6). Mass analysis revealed a mass of m/z = 347.1475
indicating a mass loss of m/z = 71.98 from 3a, which would
correspond to loss of CO and CO2 (Figure 1C). After isolating the
product, we could identify the structure of 4 containing a guanidine
moiety based on NMR and UHPLC-MS/MS analyses (Figure S7,
S50-55). Importantly, the controls 3b as well as unmodified 1
were stable when irradiated under the same conditions
(Figure S8-S9). These data indicate that the N7 position of
guanosine can be readily derivatized with the well-known ONB
group, however the photocleavage leads to an unusual product
instead of guanosine, limiting its application in photocaging
3c with light (λmax = 365 nm) in buffer containing EDTA and
glycerol, we found free guanosine 1 and 4-methylbenzophenone
5 as cleavage products, indicating that BP can be used as
photocaging group for the N7 position (Figure S11, Figure S10A-
C). To learn more about the cleavage mechanism, we tested the
components of the reaction buffer in more detail. We found that
the cleavage reaction did not occur in plain water but when EDTA,
glycerol or especially cell lysate were present, suggesting that
hydrogen abstraction occurs from these buffer components or in
the case of cell lysate other components (Figure 2B, Figure S11).
A possible cleavage mechanism is shown in Figure 2C.
Upon irradiation with λmax = 365 nm, benzophenone generates a
triplet ketone, which abstracts a hydrogen atom from a C–H bond
of EDTA or glycerol.[16] The ketyl radical thus formed does not
engage in a C–C cross link, but the positive charge at the N7
position favors fragmentation to recover guanosine 1 along with
the radical cation A. Single electron reduction of A by the EDTA
or glycerol derived radical and tautomerization eventually leads to
5.
Figure 1. Chemical modification of the N7 position of guanosine derivatives with
the common ONB group and subsequent irradiation. (A) Concept. (B) HPLC
analysis before and after irradiation of 3a at 365 nm. (C) Mass analysis of 4
(calculated mass of [C15H19N6O4]+ = 347.1462 [M]+, found: 347.1475).
approaches.
We therefore turned away from the classical PC groups and
contemplated alternative light-sensitive groups as potential
position-specific PC groups. Photoactivatable aryl ketone
derivatives such as benzophenone (BP) are widely used
biochemical probes.[15] BP can be manipulated in ambient light
and activated at λ ~ 360 nm. It is chemically stable and reacts
preferentially with unreactive C-H bonds, which is widely used to
study protein-protein but also protein-RNA interactions.[16] The
substituents on BP can affect the photochemistry significantly and
electron-withdrawing groups increase the efficiency of H-
abstraction.[15] We therefore reasoned that the positive charge at
the N7 position might lead the radical to a different reaction
pathway and therefore considered aryl ketone derivatives, such
as BP or a 2-hydroxy-2-methyl-1-phenylpropan-1-one group
(HAK), as a potential PC groups for the N7 position of guanosine.
We chemically synthesized N7-BP-guanosine 3c (Figure S3-S4)
and tested its uncaging properties (Figure 2A). After irradiation of
Figure 2. Chemical preparation of N7-BP-guanosine and subsequent
irradiation. (A) Concept. (B) HPLC analysis before and after irradiation of 3c at
365 nm in aqueous solution with different additives. (C) Postulated mechanism
for the photocleavage.
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To test whether this strategy can be applied to the other purine
nucleosides, we synthesized N1 benzophenone-modified
adenosine (7) (Figure S4).[17] The corresponding methylated
nucleoside m1A has recently been identified in mRNA and its
functional role is still investigated.[18] This compound also has a
positive charge, suggesting that photouncaging of aryl ketone
derivatives might be possible in an analogous way. Indeed, we
observed that irradiation of 7 with light of 365 nm led to complete
formation of adenosine 6 and release of 5 similar to N7-
photocaged guanosine (Figure S12-S13).
remained stable when irradiated under these conditions, in line
with our results from the guanosine (Figure S16,S24).
The benzophenone group, however, was again suitable for
photouncaging at the N7G of the 5′ cap. Specifically, 10d was
almost completely reacted (79% decrease) when irradiated in
buffer, and GpppA 8 was formed as main product (Figure 3E,
Figure S18A-B). As in the case of the nucleoside, addition of
buffer was required to yield 8 and release 4-methylbenzophenone
5 (Figure S25-S26). Importantly, the uncaged product 8 could be
re-modified enzymatically using the BP-group, confirming that the
To examine whether our strategy can be extended to more
complex molecules, we chose the dinucleotide GpppA 8, which is
the hallmark structure of the 5′ cap found in eukaryotic mRNAs.
Since site-specific chemical modification is not possible in this
case, we devised an enzymatic approach, exploiting the site-
specificity of the cap N7-methyltransferase Ecm1 together with its
cosubstrate promiscuity.[19] To this end, we synthesized analogs
of the natural cosubstrate S-adenosyl-L-methionine (AdoMet),
suitable for transfer of the classical ONB-based and novel BP-
free and functional GpppA
photocleavage of the BP group (Figure S18C).
8
had been formed after
To test whether other aryl ketone derivatives could also be
suitable for photocaging, we used the α-hydroxyalkyl ketone
(HAK) substituent, which is known to normally react according to
Norrish type I to give the acylradical and ketylradical.[20] We
enzymatically produced N7-HAK-GpppA 10e from GpppA 8 using
Ecm1 and AdoHAK 9e (Figure S14-S15,S27). Irradiation of 10e
with light of 312 nm in the reaction mixture led to recovery of
almost 60% of 5 (Figure S28). UHPLC-MS analysis verified the
photocleavage to GpppA 8 and small amounts of side-products,
namely the N7-benzoic acid-modified cap analogue 11e,a and the
based photocaging groups, i.e. AdoONB 9a, AdoPNB 9b and
19c]
AdoANB 9c as well as AdoBP 9d (Figure S14).[8,
These
AdoMet analogues are well accepted by the highly promiscous
Ecm1 resulting in efficient formation of the desired products N7-
PC-GpppA 10a-d (Figure 3A-B, Figure S15), according to HPLC
and UHPLC-MS analysis (Figure 3C, Figures S16-S19). In line
with the results obtained for the nucleoside, irradiation of N7-
ONB-GpppA 10a did not yield the uncaged 8 but instead a product
11a with a mass loss of m/z = 71.98 (Figures 1C-D, Figure S20).
Similarly, irradiation of N7-ANB-GpppA 10c revealed a new
product 11c with a mass loss of m/z = 71.98 (Figure S17,S19E).
More detailed analyses of 10a and 11a by enzymatic digestion
into single nucleosides using Snake Venom phosphodiesterase
revealed that the mass loss occurred exclusively at the N7-ONB-
guanosine moiety of the dinucleotide (Figure S21-S23). The
controls, PNB-caged GpppA 10b as well as uncaged GpppA 8,
corresponding
N7-benzaldehyde-modified
one
11e,b
(Figure S29-S230). These data show that the HAK group is also
a photocaging group for the N7 position of guanosine nucleotides,
suggesting that our approach can be extended to other aryl
ketones.
Furthermore, we enzymatically modified plasmid DNA at the
N6 position of deoxyadenosine using MTase TaqI and AdoBP 9d,
which worked efficiently (Figure S31). As expected, no uncaging
and thus no plasmid degradation were observed after irradiation
with light of 365 nm in buffer (Figure S31B). This showed that the
positive charge at the N7 position is required for the
photocleavage of aryl ketone derivatives such as BP.
Figure 3: Enzymatic modification of N7 position of 5′ cap structure GpppA. (A) Concept. (B) Panel of PC groups tested and summary of irradiation results. (B) HPLC
analysis of enzymatic reaction of 8 to 10a and subsequent irradiation at 365 nm. (D) Mass analysis of 11a (calculated mass of [C25H33N11O16P3]+ = 836.1314, found:
836.1315). (E) HPLC analysis of enzymatic reaction of 8 to 10d before and after irradiation at 365 nm in buffer. (F) Mass analysis after irradiation, 8: Calculated
mass of [C20H28N10O17P3]+ = 773.0841 [M+H]+, found: 773.0855. (* = impurities of 9a)
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To study whether our strategy is applicable to photocaging
of oligonucleotides, we used a panel of short 5–21 nucleotide long
RNAs with internal guanosine sites (Figure S33A). These RNA
oligonucleotides were incubated with BP-bromide 2c, resulting in
N7G but not N1A modification according to UHPLC-MS analysis
after digestion and dephosphorylation to single nucleosides. The
modified RNAs were then irradiated with light of 365 nm in buffer
containing EDTA and single nucleosides were analyzed by
UHPLC-MS. The data showed that BP was removed from N7-BP-
guanosine after irradiation, suggesting that photocaging and
uncaging is possible also in the context of RNA oligonucleotides
(Figures S10D-E,S32-S39). Neither the reaction with 2c nor the
irradiation at λmax = 356 nm led to RNA degradation as shown in
denaturing PAGE analysis (Figure S38). Furthermore, the N7-
modified caps were successfully used for in vitro transcription to
produce long reporter mRNAs (>1000 nt) and these also
remained intact after irradiation under the same conditions
(Figure 4B).
that the common ONB group is not suitable for N7G. We
developed both a chemical and an enzymatic strategy to
photocage and release N7G, presenting the first photocaged
nucleoside that strictly affecting the Hoogsteen recognition site of
G to the best of our knowledge. Hoogsteen interactions are
important in RNA biology, e.g. in riboswitches, ribozymes or
formation of G quadruplexes. Furthermore, due to the biological
importance of m7G in the 5′ cap and the enzymatic remethylation
process in nature, our approach significantly expands the
chemical biology toolbox and opens the door to control the
functions of the 5′ cap with spatio-temporal control in the biological
context. The photocaging of N7G and N1A can be further
improved by testing additional aryl ketone derivatives that are
excited at longer wavelengths and exploiting the two-photon
excitation properties of BP in the future.[22]
Acknowledgements
Finally, we tested whether photocaging the N7 position of
guanosine can be used to block a biological function (Figure 4A).
The 5′ cap is involved in several interactions, most notably eIF4E
for translation and decapping enzymes, such as DcpS, for RNA
turnover.[10] These interactions require N7 methylation and
unmodified caps have been show to become remethylated in the
cytoplasm.[11] We measured the Kd values of recombinantly
produced eIF4E and the inactive variant DcpS (H277N) for the
native and modified cap and found that these proteins are not
binding to N7-BP-modified-GpppA, whereas m7GpppA is bound,
showing a Kd value in the sub-micromolar range—in line with
reports in the literature (Figures S40-S41, Table S1).[10d, 21] After
irradiation and enzymatic remethylation mimicking the cellular
remethylation, the binding to both proteins was fully restored
(Figure 4C-D). These data show that benzophenone can be used
to block and release biologically relevant functions.
The authors thank Dr. W. Dörner, A.-M. Dörner, S. Hüwel and S.
Wulff for experimental assistance and Dr. M. Teders for helpful
discussion (all WWU). We thank the Glorius group, Dr. F. Höhn
and the technical workshop for providing and building custom-
made LED boxes (all WWU Münster).This work was supported by
the Deutsche Forschungsgemeinschaft [RE2796/6-1], the ERC
and the Fonds der Chemischen Industrie (doctoral fellowship to
L.A. and Dozentenpreis to A.R.). We thank Prof. C. Lima (Sloan
Kettering Institute) for a plasmid coding for DcpS (H277N).
Keywords: N7G • photocaging • RNA modification •
benzophenone • RNA
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10.1002/anie.201914573
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
A
strategy was developed to
Lea Anhäuser,[a] Nils Klöcker,[a] Fabian
Muttach,[a] Florian Mäsing,[b] Petr
Špaček,[a] Armido Studer[b] and Andrea
Rentmeister*[a]
chemically or enzymatically photocage
the N7 position of nucleosides,
dinucleotides and RNA, which could be
removed upon irradiation with λmax
=
Page No. – Page No.
365 nm. The well-known ortho-
nitrobenzyl photocaging groups were
A benzophenone-based photocaging
strategy for the N7 position of
guanosine
unsuitable,
however
aryl-ketone
derivatives as the benzophenone
group were identified as potential
photocaging group for purine imine
positions.
This article is protected by copyright. All rights reserved.