Genetic Code Expansion Facilitates Position-Selective Labeling of RNA for Biophysical Studies

Article abstract of DOI:10.1002/chem.201904623

Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic aci

Full text of DOI:10.1002/chem.201904623

A Journal of
Accepted Article
Title: Genetic code expansion facilitates position-selective labeling of
RNA for biophysical studies
Authors: Andreas Hegelein, Diana Müller, Sylvester Größl, Michael
Goebel, Martin Hengesbach, and Harald Schwalbe
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To be cited as: Chem. Eur. J. 10.1002/chem.201904623
Link to VoR: http://dx.doi.org/10.1002/chem.201904623
Supported by

10.1002/chem.201904623
Chemistry - A European Journal
FULL PAPER
Genetic code expansion facilitates position-selective labeling of
RNA for biophysical studies
Andreas Hegelein+[a], Diana Müller+[a], Sylvester Größl[b], Michael Göbel[b], Martin Hengesbach[a] and
Harald Schwalbe*[a]
Abstract: Nature relies on reading and synthesizing the genetic code
with high fidelity. Nucleic acid building blocks that are orthogonal to
the canonical A-T and G-C basepairs are therefore uniquely suitable
to facilitate position-specific labeling of nucleic acids. Here, we employ
the orthogonal kappa-xanthosine-base-pair for in vitro transcription of
labeled RNA. We devised an improved synthetic route to obtain the
phosphoramidite of the deoxy-version of the kappa nucleoside in solid
phase synthesis. From this DNA template, we demonstrate the
reliable incorporation of xanthosine during in vitro transcription. Using
NMR spectroscopy, we show that xanthosine introduces only minor
structural changes in an RNA helix. We furthermore synthesized a
clickable 7-deaza-xanthosine, which allows to site-specifically modify
transcribed RNA molecules with fluorophores or other labels.
Several strategies to circumvent these limitations have been
devised and successfully established in recent years. Many of
these techniques use the standard approach of in vitro
transcription to generate RNA, and employ an additional base pair
that is orthogonal to the two Watson-Crick-like basepairs that
establish sequence specificity during transcription[1–4]
.
H
O
H
N
N
N
H
N
N
N
R
N
N
R
H
O
H
isoC
isoG
H
O2N
R
H3C
N
N
H
O
H
H
O
N
N
N
N
N
H
H
N
N
N
N
R
O
N
R
N
N
R
H
H
Introduction
N
O
H
H
Z
P
S
B
Over the last decade, an increasing number of functional roles
has been identified for ribonucleic acids and their biophysical
investigation is increasingly pursued. For a number of reasons,
methods developed for proteins cannot easily be transferred for
studies of RNAs. Thus, novel methods have to be developed, for
example to attach labels that allow for detailed spectroscopic
studies of RNA. Structural studies additionally require labels to be
inserted in a site-selective manner to obtain position-specific
readouts.
By chemical solid-phase synthesis, RNA with such position-
specific label can be obtained. A variety of chemically modifiable
building blocks has been made available over the last years.
However, the use of solid phase synthesis brings along the well-
known limitations and drawbacks of this technique; most
importantly the limited product yield that decreases with the length
of the RNA and the concomitant increase of by-products make
purification of the target RNA difficult.
O
O
N
S
H
S
N
R
S
R
N
R
O
S
O
R
TPT3CP
NaM
TPT3
NaM
H
N
H
O
N
O
N
7
8
N
6
5
9
N
1
4
N
N
H
3
2
R
NH
R
H
H
Kappa (
Xanthosine (X)
Figure 1. Examples of unnatural base pairs. (top) The isoC-isoG base pair
which was the first developed unnatural base pair [5]. Z and P as well as S and
B are two unnatural base pairs which were developed by Benner et al.[6]
(middle) Hydrophobic base pairs (NaM and TPT3) which was developed by
Romesberg et al.[2] and the TPT3CP and NaM base pair which was developed
by Kath-Schorr et al.[8] (bottom) The kappa (κ) xanthosine (X) base pair used in
this paper.
[7]
[a]
A. Hegelein, D. Müller, Dr. M. Hengesbach, Prof. Dr. H. Schwalbe
Institute for Organic Chemistry and Chemical Biology
Center for Biomolecular Magnetic Resonance
Goethe University Frankfurt
Two main strategies have been previously reported: either larger
hydrophobic moieties establish an entirely novel complementary
system or the hydrogen bonding pattern of the standard purine
and pyrimidine-based scaffolds are expanded. Both these
strategies have generated additional, orthogonal basepairs that
can be employed to different degrees in in vitro applications or for
genetic code expansion in vivo. Some of the designed bases have
also been elegantly used for the incorporation of modifiers, i.e. for
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany)
E-mail: schwalbe@nmr.uni-frankfurt.de
S. Größl, Prof. Dr. M. Göbel
Institute for Organic Chemistry and Chemical Biology
Goethe University Frankfurt
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany)
These authors contributed equally to this work
[b]
+
nitroxide spin labels or fluorophores [9–11]
.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201904623
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Our focus for the current study was to use the capabilities of a
labeling strategy employing one orthogonal DNA nucleotide
together with the complementary ribonucleoside triphosphate to
generate site-specifically modified RNA by in vitro transcription.
To introduce only minimal structural perturbation of the target
RNA, we therefore opted to follow the strategy of modifying the
hydrogen bond arrangement of the near canonical kappa
nucleobase, which would also be sterically similar to the natural
nucleobases and provide comparable stacking interfaces. Based
on the work initiated by Piccirilli et al [4], we first developed a novel
synthesis route for the kappa nucleoside, and incorporated the
kappa nucleoside phosphoramidite into a DNA used as a template
for RNA transcription. We successfully incorporated xanthosine at
this position with high specificity and good efficiency. We
introduced xanthosine into a sizeable RNA riboswitch and could
site-specifically detect the introduced NMR-active label. We
furthermore implemented novel covalent labeling options by
attaching a modifier to the xanthosine-based nucleobase,
focusing on a click chemistry compatible derivative.
4 which acted as a cyclic alkene are coupled to yield the protected
nucleoside 5. The β-anomer was obtained as a single product.
O
N
O
N
TBDMSO
HO
NH
NH
b
c
a
O
O
HO
TBDMSO
O
O
O
O
TBDMSO
TBDMSO
OH
TBDMSO
1
2
3
4
Cl
Cl
Cl
N
N
N
N
N
N
HO
d + e
f
g
HO
HO
HO
O
Cl
Cl
Cl
O
O
O
TBDMSO
TBDMSO
O
OH
4
5
6
7
Cl
NHBz
N
NHBz
N
N
O
N
N
N
j
k
h
i
TBDMSO
TBDMSO
HO
Cl
NHBz
NHBz
O
O
OH
OH
OH
10
8
9
NHBz
NHBz
N
N
N
N
DMTrO
l
NHBz
DMTrO
O
NHBz
O
O
CN
OH
P
O
N
11
Results and Discussion
12
Figure 2. Synthetic route to the benzoyl protected 2,4-diaminopyrimidine
phosphoramidite 12. Novel synthesis options were implemented for compounds
6, which followed a Heck reaction, and compound 9, where we opted for the Pd-
catalyzed Buchwald-Hartwig coupling. Reaction conditions: a. imidazole,
TBDMS-Cl, DMF, RT, 16 h, 99%; b. HMDS, (NH4)2SO4, 140°C, 3,5 h, 88%; c. 1
M TBAF in THF solution, THF, 0°C, 2 h, 45%; d. 2,4-dichloro-5-iodopyrimidine,
Pd(OAc)2, P(PhF5)3, Ag2CO3, CHCl3, 70°C, 10 h; e. 1.0 M TBAF in THF solution,
AcOH, THF, 0°C, 2h, 37% over two steps; f. sodium triacetoxyborohydride,
AcOH, MeCN, 0°C, 15 min, 70%; g. imidazole, TBDMS-Cl, DMF, RT, 16 h, 76%;
h. Pd2(dba)3, xantphos, Cs2CO3, 100°C, 24 h, 37%; i. NEt3·3HF, THF, RT, 2,5
h, 62%; j. DMTr-Cl, DMAP, pyridine, RT, 12 h, 77%; k. N,N-
diisopropylethylamine, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite,
DCM, RT, 2 h, 74%
The steps required here to obtain a site-specifically labeled RNA
can be described as follows: (i) Synthesis of the kappa base (and
DNA synthesis), (ii) transcription, (iii) quality control, and (iv)
labeling.
Synthesis of kappa base phosphoramidite
Our synthetic strategy to obtain the kappa deoxynucleoside
(Figure 1) incorporated a number of novel synthetic developments.
Most importantly, we opted to synthesize the C-C glycosidic bond
using a Heck coupling, which required generation of the glycal
intermediate 4 (Figure 2).[12,13] In addition, formation of the
protected amine ring substitutents 9 was achieved using a
palladium-catalyzed Buchwald-Hartwig coupling.[14]
However, as compound 5 was unstable, we opted for an in situ
deprotection with 1M TBAF to yield the ketoriboside 6 with 37%.
Reduction of the ketone to the desoxyribose 7 was achieved using
sodium triacetoxyborohydride (62% yield). The Buchwald-Hartwig
coupling to obtain the benzoyl-protected pyrimidine diamine,
which was first applied by Wagenknecht et al.[14] shows increased
selectivity when the C5 of the ribose is silylated. We therefore
coupled a TBDMS protection group yielding compound 8. Here
the selectivity was high due to the slightly more reactive C5’-
hydroxyl group, and the yield was 76%. The Buchwald–Hartwig
coupling is catalyzed by palladium, and we increased the amount
of benzamide to three equivalents to obtain the double protected
compound 9 in sufficient yield (37%). However, purification of 9 at
this point proved difficult due to the slow migration on the column
and the poor separation from the starting benzamide. Removal of
TBDMS group using trimethylamine trihydrofluoride yielded
compound 10 in 62%. From here, the protocol for protection to
form the phosphoramidite suitable for solid phase synthesis
followed standard procedures: The DMTr protection of C5 to form
11 was achieved by DMAP-catalyzed coupling of 4,4’-
dimethoxytritylchloride in dry pyridine. Formation of the
To this end, we started with desoxythymidine 1, which was
protected using TBDMSCl to yield 2. The elimination of the
nucleobase to obtain the double silylated glycal 3 turned out to be
experimentally demanding due to the instability of 3 and the
required purification. The following deprotection was achieved
with 1.0 M tetrabutylammonium fluoride (TBAF) in THF solution.
Here, both reaction monitoring and column purification had to be
extensively optimized to reach a yield of 88%. We assume that
selectivity of the deprotection of C5 was limited, the yield of 4
therefore could not be optimized beyond 45%; we could, however,
not fully characterize the side products. As the double silylated
glycal presumably cannot undergo the following Heck reaction[15]
,
it is necessary to block the bottom of the glycal 4 by the sterically
demanding TBDMS-group to avoid the attack from this side which
would result in formation of the wrong α-anomer.
One of the key reactions of our scheme was the palladium-
catalysed Heck reaction. The Heck reaction was applied to
nucleic acid chemistry by Hocek et al. and Wagenknecht et al.[14]
[15] [16] [17] To perform this reaction, the commercially available 2,4-
dichloro-5-iodopyrimidine as the heterocyclic halide and the glycal
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phosphoramidite 12 was performed with 2-cyanoethyl N,N-
diisopropylethylamine in dry DCM.
a 2:1 ratio of copper/palladium [26]. The methyl protection group at
O6 was only then removed using TMS chloride and sodium iodide
at room temperature with a 61% yield of 20.
As synthesis of the longer DNA especially for transcription of a
riboswitch RNA with more than 80 nucleotides is demanding, we
purchased DNA from IBA (Göttingen), providing phosphoramidite
12 to be site-specifically incorporated into the desired
transcription templates.
Synthesis of functionalised 7-deazaxanthosine
Several options are available to provide a modifiable xanthosine
derivative that maintains its Watson-Crick-face base-pairing
interactions. Introduction of chemical modifications at C8 would
be the most immediate choice. However, introduction of sterically
demanding modification at C8 introduces steric clashes in the
major groove of an A-form RNA helix [18]. Thus, attachment of
modifications at N7 promises to be structurally less perturbing, but
their syntheses are difficult. However, several nucleoside
derivatives have been reported that introduce a 7-deazapurine
[19–22]
backbone and a number of synthetic strategies
as well as
suitable starting compounds are available. Major advantages of
this approach are the conservation of the hydrogen bonding
pattern at the Watson-Crick side, the advantageous substrate
properties of such modified nucleotides for RNA polymerases [23]
as well as the possibility to specifically couple extended linkers
using cross-coupling reactions.
Figure 3. Synthetic route to the 7-deazaxanthosine with a C-8 linker 21.
Attachment of the clickable linker was achieved by Sonogashira coupling to 19,
and only after that the methyl ether deprotection to 20 was performed. Reaction
conditions: a. pivaloyl chloride, pyridine, RT, 1 h, 92%; b. N-iodosuccinimide,
THF, RT,
1 h, 93%, c. N,O-Bis(trimethylsilyl) acetamide, RT, 10 min,
trimethylsilyl triflate, 1-acetyl-2,3,5-tribenzoyl-1-ribose, MeCN, 50°C, 24 h, 61%;
d. 0,5 M sodium methoxide solution in MeOH, 80°C, 10 h, 61%; e. sodium nitrite
in H2O, glacial acetic acid, H2O, RT, 1 h, 67%; f. Pd(PPh3)4, copper(I)iodide,
NEt3, 1,7-octadiyne, DMF, RT, 24 h, 96%; g. Trimethylsilyl chloride, sodium
iodide, MeCN, RT, 1 h, 61%; h. dist. POCl3, n(Bu3)N, proton sponge, Me3PO4, -
7,5 °C, 5 h, TBA PPi, RT, 30 min, 0,1 M TEAB buffer, RP-HPLC, 16%.
The synthesis starts with the commercially available 4-chloro-7H-
pyrrolo[2,3-d]pyrimidin-2-amine 13. 13 was protected with pivaloyl
chloride to obtain 14 at the exocyclic amino group in excellent
yield (90%). Without the pivaloyl group, the electrophilic
substitution exclusively places the bromine or iodine at the
undesired C8-position [24]. It was also reported [21] that without the
pivaloyl protection glycosylation under Vorbrüggen conditions
Triphosphorylation of the modified xanthosine derivative is
necessary to make it amenable for T7 RNA polymerase-based
transcription. Therefore, the final step is the coupling of the
triphosphate specifically to C5 of the ribose. Here, protecting
strategies have been used previously [27]. We found that the
conditions used by Yoshikawa and Ludwig [28–30] were compatible
with our modification, and provided the advantage that protection
of the other two ribose hydroxyl moieties was not required to
obtain the desired specificity for C5. We adapted the protocol and
extended the hydrolysis step with 0,1 M TEAB buffer to eight
hours. After purification by RP-HPLC, the final nucleoside
triphosphate 21 was obtained in 16% yield. With this, both
orthogonal base-pairing building blocks were ready to be tested
in transcription and labeling.
failed. Iodination to 15 was achieved with NIS (93%)[25]
.
Initial attempts for glycosylation were made with HMDS as the
silylating agent. As a result, the glycosylation reaction yielded one
single product (16). NMR spectra, however, showed impurities
and the yield was not satisfying (<40%). To improve both purity
and
yield,
glycosylation
was
done
with
N,O-
bis(trimethylsilyl)acetamide (BSA). Under these conditions, the
glycosylation product was pure and the yields varied between 53-
61%. After the following removal of the protecting groups with
0.5M NaOMe at 80°C (17) and deamination using NaNO3, the
desired compound 18 was obtained.
Site-specific incorporation of xanthosine by in vitro
transcription
In a previously published synthesis [21], the methyl ether at the C4-
position was removed with trimethylsilyl (TMS) chloride and
sodium iodide. Under these conditions, it was reported that a 3:2
ratio of the 7-deazaxanthosin without iodine and 7-
deazaxanthosin with iodine was recovered and attempts to
remove the methyl ether using sodium hydroxide were
unsuccessful. In our case, the terminal alkyne linker is sufficiently
stable during Sonogashira coupling conditions. We therefore
directly conducted the Sonogashira coupling of the 1,7-octadiyne
linker to obtain 19. We used 10 equivalents of 1,7-octadiyne and
We first tested incorporation of a xanthosine (X) to be decoded by
a DNA nucleotide containing the kappa nucleobase by standard
T7 run-off transcription. The kappa nucleobase was incorporated
into two DNA-templates, encoding a 14mer RNA hairpin with a
UUCG tetraloop motif, and the aptamer domain of the guanine-
riboswitch-aptamer RNA (Gsw73) from Bacillus subtilis. Run-off
transcription can utilize single-stranded DNA where only the T7
primer region of the template DNA is double stranded. Using this
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strategy, it is sufficient to synthesize the kappa DNA
phosphoramidite [31]
As a proof of concept, a G9X mutation was introduced into the
14mer UUCG-tetraloop hairpin RNA, as guanosine has high
structural similarity to xanthosine. Transcription yielded a
optimized the transcription conditions with regard to the
concentration of XTP (Figure S3). Under optimized conditions, the
yield of the full-length G79X transcript was 71.4% in comparison
with transcription of the unmodified Gsw73-transcript. Not
surprisingly, we observed abortion products at the site of the
mutation, with a ratio of 77:23 full-length RNA to abortion fragment.
In absence of the xanthosine triphosphate, we also observed 22%
full-length product (Figure 5B, Table S2), which presumably
.
arises from misincorporation of adenosine [4]
.
Figure 4. Overlaid Imino-amino- and imino-sugar region of 1H, 1H-NOESY
spectra of the 14mer (grey) and 14merX (red) RNA displayed on the left. The
assignment of the 14mer has been transferred from Fürtig et al.[32] The signals
of the G9X modification are significantly shifted and no crosspeaks can be
detected for the X9, whereas the cross signals of G2, G10 and G12 are mostly
retained. This finding hints towards a restructuring in the loop region by
conservation of the hairpin structure. Experimental condition: 1 mM RNA in
25 mM potassium phosphate buffer pH 6.2 and 10% D2O were measured at
278 K and 600 MHz.
highly pure RNA. The yield upon switching from G to X dropped
by 87% (Figure S1, Table S1). We further investigated the impact
of introducing xanthosine on RNA structure using NMR
1
Figure 5. A secondary structure of the Gsw73X B denaturing PAA-Gel for
comparison of the transcription efficiency of unmutated Gsw73 versus G79X in
absence and presence of XTP. C Overlay of CD melting curves recorded with
Gsw73 (grey) and G79X (black) showing minimal changes in stability of 0.25 K.
D Overlay of the imino-imino- and imino-cross peak region of 1H, 1H-NOESY
spectra of Gsw73 (grey) and G79X (red). Again, a strong shift for the iminos of
the mutated position and minor shifts for the imino signals of the nearby
nucleotides can be observed. Here, the cross signals for the X79 are detectable
showing complete structural conservation. Experimental condition: G79X
(100 µM) was recorded at 950 MHz in 25 mM potassium phosphate pH 6.2
containing 50 mM KCl and 10% D2O Gsw73 was recorded by Janina Buck
(unpublished data) in the same buffer at 900 MHz. The partial assignment has
been transferred from Buck et al.[36] E Imino and aromatic region of 13C, 15N
A,C,G,U-labeled G79X. Bottom: spectra without x-filter, middle: 15N-/13C-edited
spectra, only hydrogens bound to 15N or 13C are visible, top: 15N-/13C-filtered
spectra , only hydrogens bound to the unlabeled xanthosine are visible.
Experimental condition: The RNA was in a 25 mM potassium phosphate pH 6.2
containing 50 mM KCl and 10% D2O for the imino spectra and 100% D2O for
the spectra of the aromatic region. Spectra were recorded at 600 MHz.
spectroscopy. 1D H NMR showed that folding into the hairpin
structure is retained and introduction of xanthosine leads to only
small structural deviations (Figure S2). The UUCG tetraloop is
usually highly ordered[33] and was destabilized by the mutation
which is visible in both 1D H and H, H-NOESY NMR spectra
(Figure 4 and Figure S2). These results show that preparative
transcription using the modified constructs is feasible, and that
replacing a guanosine with a xanthosine yields an interesting
atomic mutagenesis approach to investigate local structural
effects by NMR.
1
1
1
We then turned to a large structured RNA to test whether these
findings also hold true for the analysis of functional/ biologically
relevant RNA. Here, we used the guanine sensing riboswitch RNA
from B. subtilis [34–37]. For this construct, we replaced G79 forming
a G·U wobble base pair in the P1 stem with X79 (Figure 5A). We
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CD melting experiments showed a small difference of 0.25 K in
the melting points (∆Tm) between the Gsw73 and G79X riboswitch,
indicating that the structure is retained (Figure 5C). We further
purified the full-length G79X RNA and obtained about 22 nmol of
RNA, which was sufficient for NMR studies. The NMR data fully
supported our proposed strategy that introduction of xanthosine
induces minimal structural perturbation. Furthermore, the
previous G79 H1` cross peaks are now visible as X79 H1` cross
peaks, indicating that the structure is retained (Figure 5D).
condition for both transcripts. The modified 73mer RNA was
purified by extraction from a polyacrylamide gel. For labeling, a
click reaction with Cy3-Azide was performed, and the resulting
RNA analyzed by PAGE. The signals shown in the gel lanes for
RNA staining showed that the RNA remained intact during
labeling, and no significant shift was observed. RNA that
contained the alkyne-modified xanthosine, a fluorescent signal
could be observed comigrating with the unlabeled RNA,
demonstrating a highly specific labeling (Figure 6B). The
fluorescently labeled RNA was further excised from the gel, and
analyzed using UV-Vis spectroscopy. Based on the intensities at
the absorption maxima (DNA: 260nm, Cy3: 555nm), we
calculated the labeling yield of the RNA to be 11% (Figure 6C).
We prepared a reversed labeled riboswitch with 13C, 15N-labeled
ATP, CTP, GTP and UTP together with unlabeled XTP. With this
isotopically labeled RNA sample, we performed 13C and 15N-
filtered 1D 1H-NMR experiments that suppress signals from
hydrogen atoms bound to 13C and 15N. In this experimental setup,
we detected the single 12C,14N-“labeled” xanthosine, in particular
the signals annotated XH1 and XH3 imino- and the XH8 aromatic
signal in the background of the other 73 nucleotides of the
riboswitch RNA (Figure 5E). Thus, transcribing RNA from a
ssDNA template that contains the kappa nucleotide can yield
large functional RNAs, as both selectivity and specificity are very
high. Purification yields a highly homogenous RNA sample
suitable for NMR spectroscopy.
Conclusions
We prepared two well-structured RNAs – a model 14mer as well
as a 73 nt aptamer of a riboswitch – via run-off transcription and
introduced a single guanosine-to xanthosine mutation, in each
case yielding a 100 µM NMR sample. We show that the structural
perturbations introduced into two different RNA constructs by the
unmodified xanthosine are minor. We furthermore devised a
modifiable 7-deazaxanthosine triphosphate carrying a modifiable
linker for posttranscriptional attachments of spectroscopic labels
e.g. for FRET-, EPR- or IR-measurements. Due to the demanding
synthesis of this compound, the final triphosphorylation currently
limits the obtainable yield, motivating the urgent need for novel
synthetic routes for triphosphorylation [38]. We show that our
approach facilitates labeling of biologically relevant RNAs (i.e.
riboswitches) in a position-selective manner. With the synthesis
of both the kappa DNA phosphoramidite and a modifiable RNA-
xanthosinetriphosphate derivative, we provide a proof of concept
to employ non-natural, Watson-Crick-like base pairs for site-
specific bioorthogonal labeling of RNA.
Post-transcriptional functionalization of RNA via click
reaction
Site-specific introduction of a non-natural nucleobase can be
exploited for labeling purposes [4]. We therefore used the 7-
deazaxanthosine (7dX) derivative containing a terminal alkyne in
the form of a triphosphate during in vitro transcription to introduce
a position-selective spectroscopic label.
Experimental Section
Solid-phase synthesis of the kappa (κ)-containing DNA template
The phosphoramidite containing the κ-nucleobase has been incorporated
into the DNA via solid-phase synthesis by commercial DNA synthesis (IBA
Lifesciences; Göttingen, Germany).
Figure 6. A denaturing PAA-Gel for comparison of the transcription efficiency
of unmutated Gsw73 versus G79-7dX in absence and presence of 7-
deazaxanthosinetriphosphate (7dX). B Fluorescence scan (upper gel) and gel
red staining (lower gel) of denaturing PAA-Gel after Click-reaction. C UV-
spectrum of fluorophore-labeled RNA after further gel purification to determine
the yield of labeling reaction.
Expression and purification of P266L T7 RNA Polymerase
The protein was expressed in BL21(DE3)cells carrying a polyhistidine
(His6-Tag). Expression was induced at OD600=0.6–0.8 with 0.5 mM
isopropyl-D-1-thiogalactopyranoside (IPTG). T7 RNA Polymerase was
expressed over night at 37◦°C and were purified via HisTrap HP(GE
Healthcare) columns using 50 mM Tris/HCl (pH 8.1),400 mM NaCl, 20 mM
imidazol and 5 mM β-mercaptoethanol as lysis buffer. The protein was
further purified via size exclusionchromatography with a buffer containing
20 mM sodium phosphate (pH 7.7) 150 mM NaCl, 1 mM EDTA and 5 mM
DTT.
Transcription of the kappa-modified Gsw73 DNA-template with the
alkyne-XTP 13 resulted in a 1:1 ratio of full-length G79-7dX to
abortion fragment (67 nt), with a total yield of 34.5% full-length
RNA compared to the unmodified riboswitch (Figure 6A, Figure
S4, Table S3). As expected, the rate of misincorporation in
absence of 7dXTP remains comparable to the misincorporation
observed in absence of XTP under the respective optimized
DNA templates
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14mer-template (template 1): GGC ACC GAA GTG CCT ATA GTG AGT
CGT ATT A
was dried and dissolved in an appropriate volume of ddH2O. For removal
of PAA impurities, the RNA was HPLC purified, lyophilized, desalted using
centrifuge concentrators (5 kDa MWCO cut-off, Satorius) and first
precipitated with EtOH (see above), then with 5V 2% LiClO4 in Acetone
(w/v) for 3 times to remove residual salt from HPLC-buffer. In the last step,
the sample was desalted again and concentrated to 100-300 µM.
14mer: GGCACUUCGGUGCC
14merX- template (template 2): GGC ACκ GAA GTG CCT ATA GTG AGT
CGT ATT A
Click-reaction
14mer: GGCACUUCXGUGCC
2 µM RNA, 15X excess of Cy-3-azide, 50 % DMSO, 0.3 mM CuSO4, 2 mM
TBTA and 5 mM sodium ascorbate were mixed and incubated at 20 °C for
1 h. A sample was separated using PAGE and analysis of the fluorescence
signal was carried out with a Typhoon9400. After further PAGE purification
of the RNA, the click efficiency could be determined via UV absorption
measurement using ε260 of 796.5 L*mmol-1*cm-1 for the RNA and ε555 of
150.0 L*mmol-1*cm-1 for Cy3.
Gsw73-template (template 3): GGG ACC CAT AGT CGG ACA TTT ACG
GTG CCC GGT AGA AAC CTG CGT GCC ATA TCC ACG CAG TTA TAT
GAG TCC CTA TAG TGA GTC GTA TTA
Gsw73: GGG ACU CAU AUA ACU GCG UGG AUA UGG CAC GCA GGU
UUC UAC CGG GCA CCG UAA AUG UCC GAC UAU GGG UCC C
CD melting analysis
G79X-template (template 4): GGG ACκ CAT AGT CGG ACA TTT ACG
GTG CCC GGT AGA AAC CTG CGT GCC ATA TCC ACG CAG TTA TAT
GAG TCC CTA TAG TGA GTC GTA TTA
16 µM RNA in 25 mM potassium phosphate buffer pH 6.2 and 50 mM KCl
was provided. 400 µM Mg2+ was added. The melting curves were recorded
between 5-95 °C and reverse at 264.2 nm. SigmaPlot 12.5 was used for
the analysis. A bisigmoidal fit was used to determine the melting points.
G79X: GGG ACU CAU AUA ACU GCG UGG AUA UGG CAC GCA GGU
UUC UAC CGG GCA CCG UAA AUG UCC GAC UAU GXG UCC C
NMR-spectroscopy
G79-7dX: GGG ACU CAU AUA ACU GCG UGG AUA UGG CAC GCA
GGU UUC UAC CGG GCA CCG UAA AUG UCC GAC UAU G7dXG UCC
C
14mer and 14merX were measured at 278 K and 600 MHz at 1 mM RNA
concentration in 25 mM potassium phosphate buffer pH 6.2 and 10% D2O.
The underlined regions correspond to the inverse complementary T7
promoter sequence.
The 1H,1H-NOESY of G79X was recorded at 950 MHz on an unlabeled
sample (100 µM) in 25 mM potassium phosphate pH 6.2 containing 50 mM
KCl and 10% D2O.
A
sequence with jump-return-echo water
In vitro transcription
suppression[41] was used. For the x-filter experiments [42], a 13C, 15N-A, C,
G, U labeled sample was prepared. For the imino region, jump-return-echo
water suppression was used as well. The concentration was 160 µM in the
same buffer as described above and 10% D2O. The aromatic region was
40 mM tris glutamate (pH 8.1), 2 mM spermidine, 20 mM DTT and
10 ng/µL P266L T7 RNA polymerase were used for all the transcription
reactions. The other reaction components have been optimised for the
14mer to 45 mM Mg2+, 1 mM NTPs (each) and 300 nM DNA template 1
and for the 14merX to 10 mM Mg2+, 2 mM NTPs (each), 1 mM XTP, and
200 nM DNA template 2. The transcription reactions of Gsw73, G79X and
G79-7dX were performed with the same concentration of tris glutamate,
spermidine, DTT and RNA polymerase. Gsw73 was transcribed from
300 nM DNA template 3 in presence of 35 mM Mg2+ and 6 mM NTPs
(each). For the in vitro transcription of G79X 200 nM DNA template 4 were
used and 35 mM Mg2+, 6 mM NTPs (each) and 3 mM XTP (Jena
Bioscience) were added. The transcription of G79-7dX was performed in
presence of 200 nM DNA template 4, 35 mM Mg2+, 6 mM NTPs (each),
15% DMSO and 2.5 mM 7-deazaxanthosine derivative. The relative yield
was determined with ImageJ[39] or ImageLab software after polyacrylamide
gel electrophoresis (PAGE).
recorded with transfer using DIPSI2 sequence for mixing with 13C- and 15N-
[42–46]
filter
and watergate sequences for water suppression [47]. The
concentration was 60 µM in the same buffer as described above in 100%
D2O. The spectra were recorded at 600 MHz at 308 K.
Chemical synthesis
Synthesis of 3′,5′-bis-O-(tert-butyldimethylsilyl) thymidine (2)
In a round flask tert-butyldimethylsilylchloride (TBDMS-Cl) (3 equiv.,
65.1 g, 0.432 mol) and imidazole (6 equiv., 59.01 g, 0.867 mol) was
dissolved in 1085 ml N,N-dimethylformamide (DMF). After that, thymidine
(1 equiv., 35 g, 0.144 mol) was added. The reaction mixture was stirred
over night at room temperature. The mixture was quenched with 75 ml
methanol (MeOH) and was diluted with 750 ml ethyl acetate (EtOAc). The
organic phase was washed with distilled water (2 x 600 ml), with saturated
sodium bicarbonate (300 ml, NaHCO3) and with saturated sodium chloride
(300 ml, NaCl). After that it was dried over sodium sulfate (Na2SO4). The
solvent was evaporated under reduced pressure to afford a white solid.
This solid was purified by column chromatography on silica gel, elution
with cyclohexane (Cy) / ethyl acetate (EE) 2:1 afforded 2 as a white solid.
RNA purification
The 14mer transcription reaction was purified using the protocol described
by Helmling et al.[40]
.
The G79X/-7dX transcription reaction was desalted with ddH2O using
centrifuge concentrators (5 kDa MWCO cut-off, Satorius), subsequently
gel purified with a 15% denaturing polyacrylamide gel. The desired fraction
were extracted from gel by incubating in 0.3 M NaOAc over night at 37 °C.
The supernatant was separated and the dissolved RNA was precipitated
by addition of 2.5V 99.5% EtOH (Carl Roth) and storage at -20 °C for 2 h.
The RNA was collected by centrifugation at 8500g for 30 min and the pellet
TLC (cyclohexane/ethylacetate 2:1): Rf = 0,44
Yield: 67,1 g (99%) (Figures S5-S7)
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1H-NMR (500 MHz, CDCl3) δ [ppm] 8.43 (br s, 1H, NH), 7.47 (m, 1H, H-6),
6.33 (dd, J = 5.7, 7.9 Hz, 1H, H-1’), 4.40 (m, 1H, H-3’), 3.93 (m, 1H, H-4’),
3.88-3.85 (m, 1H, H-5’a), 3.77-3.74 (m, 1H, H-5’b), 2.26-2.22 (m, 1H, H-
2’a), 2.02-1.97 (m, 1H, H-2’b), 1.91 (m, 3H, CH3), 0.93 (s, 9H, C(CH3)3),
0.89 (s, 9H, C(CH3)3), 0.11 (d, J = 1.3 Hz, 6H, Si(CH3)2), 0.08 (s ,3H,
Si(CH3)2), 0.07 (s, 3H, Si(CH3)2)
Yield: 12.8 g (45%) (Figures S11-S13)
1H-NMR (500 MHz, DMSO-d6) δ [ppm] 6.61 (dd, JH2 = 2.7 Hz, JH3 = 0.8 Hz,
1H, H-1’), 5.02 (t, JH1 = 2.8 Hz, JH3 = 2.8 Hz, 1H, H-2’), 4.93 (t, JH5 = 5.8
Hz, 1H, OH), 4.80 (td, JH3 = JH4 = 2.7 Hz, JH1 = 0.93 Hz, 1H, H-3’), 4.11 (td,
JH5 = 6.2 Hz, JH3 = 2.8 Hz, 1H, H-4’), 3.43 and 3.28 (2 x td, Jgem = 11.6 Hz,
JH4 = 6 Hz, JOH = 5.8 Hz, 2 x 1H, H-5’), 0.85 (s, 9H, C(CH3)3), 0.06 und
0.05 (2 x s, 2 x 3H, (Si(CH3)2)
13C-NMR (125 MHz, CDCl3) δ [ppm] 163.6 (C-4), 150.3 (C-2), 135.6 (C-6),
110.9 (C-5), 87.9 (C-4’), 84.9 (C-1’), 72.4 (C-3’), 63.1 (C-5’), 41.5 (C-2’),
26.0 (CCH3a), 25.9 (CCH3b), 18.5 (CCH3a), 18.1 (CCH3b), 12.7 (CH3), -4.5
(SiCH3a), -4.7 (SiCH3b), -5.2 (SiCH3c), -5.3 (SiCH3d)
13C-NMR (125 MHz, DMSO-d6) δ [ppm] 149.2 (C-1‘), 103.2 (C-2‘), 89.0 (C-
3‘), 75.7 (C-4‘), 60.9 (C-5‘), 25.8 (C(CH3)3), 17.7 (C(CH3)3), -4.4 (SiCH3), -
4.5 (SiCH3)
ESI-MS: m/z calcd for C22H42N2O5Si2 [M+H+]+: 470,75; found: 471,25
MALDI-MS: m/z calcd for C11H22O3Si2 [M+H+]+: 230,38; found: 230,98
Synthesis of 1,4-anhydro-3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-
D-erythro-pent-1-enitol (3)
Synthesis of 1β-(2,4-dichloropyrimidin-5-yl)-1,2,3-trideoxy-3-oxo-D-
ribofuranose (6)
Compound 2 (1 equiv., 67.07 g, 0.142 mol) was placed into a dried Schlenk
flask under an argon atmosphere. Hexamethyldisilazane (HMDS) (744 ml,
3.60 mol) and ammonium sulfate (0.25 equiv., 4.7 g, 0.036 mol) was added,
and the solution was stirred until dissolved. The solution was refluxed at
140°C for 3.5 hours. HMDS was evaporated under reduced pressure.
MeOH (600 ml) was slowly added at room temperature. Potassium
carbonate (K2CO3) (1.1 equiv., 21.59 g, 0.156 mol) was added in portions
and stirred for 45 min at 0°C.
Palladium(II)acetate (0.1 equiv., 1.26 g, 0.0056 mol) and
tris(pentafluorophenyl)phosphine (0.2 equiv., 5.96 g, 0.011 mol) was
dissolved in a dried Schlenk flask in dry chloroform (194 ml). The mixture
was stirred for one hour at room temperature under an argon atmosphere.
After 1 h compound 4 (1 equiv., 12.8 g, 0.056 mol), 2,4-dichloro-5-
iodopyrimidine (1.2 equiv., 18.4 g, 0.067 mol) and silver carbonate
(1 equiv., 15.4 g, 0.056 mol) were added. The reaction mixture was stirred
for 10 h at 70°C. After being cooled to room temperature, the reaction
mixture was filtered on pad of celite and eluted with chloroform.
This solution was filtered over a pad celite and the solvent was evaporated
under reduced pressure. The brown residue was purified by column
chromatography on silica gel, elution with dichloromethane/ethyl acetate
(EE) 1:2 afforded 2 a yellow oil.
The solvent was removed under reduced pressure, and the remaining
brown oil was dissolved in THF (357 ml). At 0°C, 1.09 ml acetic acid and
4.35 ml of 1M TBAF solution in THF were added. After 2 h, the solvent was
removed under reduced pressure. The residue was purified by column
chromatography on silica gel, elution with n-hexane/ethyl acetate 1:1
afforded 6 as a yellow oil.
TLC (ethylacetate/dichloromethane 2:1) Rf = 0,91
Yield: 42,9 g (88%) (Figures S8-S10)
1H-NMR (500 MHz, CDCl3) δ [ppm] 6.46 (d, JH2 = 2.5 Hz, 1H, H-1’), 5.00
TLC (n-hexane/ethyl acetate 1:1): Rf = 0,44
(t, J = 2.66 Hz, 1H, H-2’), 4.89 (m, 1H, H-3’), 4.28 (dt, JH3 = 2.7 Hz, JH5
=
6.2 Hz, 1H, H-4’), 3.69 (dd, JH4 = 5.7 Hz, JH5 = 10.7 Hz, 1H, H-5’), 3.50 (dd,
JH4 = 6.3 Hz, JH5 = 10.7 Hz, 1H, H-5’), 0.89 (2s, 18 H, C(CH3)3), 0.09, 0.08,
0.07, 0.06 (4s, 12H, Si(CH3)2)
Yield: 5,5 g (37%) (Figures S14-S16)
1H-NMR (500 MHz, CDCl3) δ [ppm] 8.97 (d, J = 0.6 Hz, 1H, H-6), 5.46 (ddd,
J = 10.4, 6.3, 0.5 Hz, 1H, H-1’), 4.12 (t, J = 3.1 Hz, 1H, H-4’), 4.04 (dd, J =
12.1, 3.1 Hz, 1H, H-5’), 4.00 (dd, J = 12.1, 3.3 Hz, 1H, H-5’), 3.17 (dd, J =
18.2, 6.4 Hz, 1H, H-2’), 2.37 (dd, J = 18.2, 10.6 Hz, 1H, H-2’)
13C-NMR (100 MHz, CDCl3) δ [ppm] 149.1 (C-1’), 103.5 (C-2’), 89.0 (C-3’),
76.1 (C-4’), 62.9 (C-5’), 26.0, 25.9 (C(CH3)3), 18.5, 18.2 (C(CH3)3), -4.2
(SiCH3), -4.3 (SiCH3), -5.2 (SiCH3), -5.3 (SiCH3)
13C-NMR (125 MHz, CDCl3): δ [ppm] 211.5 (C-3’), 160.1 (C-2/C-4), 159.9
(C-2/C-4), 158.5 (C-4), 158.4, 132.0 (C-5’), 82.2 (C-4’), 72.3 (C-1’), 61.6
(C-5’), 43.5 (C-2’)
MALDI-MS: m/z calcd for C17H36O3Si2 [M+H+]+: 344,64; found: 344,91
Synthesis of 3-O-(tert-butyldimethylsilyl)-1,2-dideoxy-2,3-didehydro-
D-ribofuranose (4)
MALDI-MS: m/z calcd for C9H8Cl2N2O3 [M+2H+]+: 263,07; found: 264,92
To a solution of compound 3 (1 equiv., 42.89 g, 0.124 mol) in THF (445 ml)
at 0°C was added in portions a solution of TBAF 1M in THF (1 equiv.,
124.45 ml, 0.124 mol), and the reaction mixture was stirred for 2 hours.
After two hours the reaction mixture was diluted with distilled water
(500 ml) and with dichloromethane (400 ml). The organic phase was
washed with distilled water (200ml), brine (200 ml) and dried over sodium
sulfate (Na2SO4). The solvent was evaporated under reduced pressure.
The residue was purified by column chromatography on silica gel, elution
with n-hexane/ethyl acetate 4:1 afforded 4 as a pale yellow oil.
Synthesis
ribofuranose (7)
of
1β-(2,4-dichloropyrimidin-5-yl)-1,2-dideoxy-D-
Compound 6 (1 equiv., 5.46 g, 0.013 mol) was dissolved in acetonitrile
(438 ml) and acetic acid (72 ml). At 0°C, sodium triacetoxyborohydride
(2.52 equiv., 11.1 g, 0.052 mol) was slowly added. The reaction mixture
was stirred at 0°C for 15 min. 72 ml of 50% aqueous ethanol was added.
All solvents were removed under reduced pressure, and the residue was
purified by column chromatography on silica gel, elution with
dichloromethane/methanol 95:5 afforded 7 as a white foam.
TLC (n-hexane/ethylacetate 4:1) Rf = 0,39
TLC (dichloromethan/methanol: 95/5): Rf = 0,21
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Yield: 3,9 g (70%) (Figures S17-S19)
5.4 Hz, 1H, H-1’), 4.39 (dt, J = 5.4, 1.4 Hz, 1H, H-3’), 4.11 (td, J = 3.7, 2.1
Hz, 1H, H-4’), 3.75 (s, 1H, H-5’), 3.74 (s, 1H, H-5’), 2.43 (ddd, J = 12.8, 5.4,
1.2 Hz, 1H, H-2’), 2.24-2.19 (m, 1H, H-2’), 0.80 (s, 9H, C(CH3)3), -0.02 (s,
3H, Si(CH3)2), -0.01 (s, 3H, Si(CH3)2)
1H-NMR (500 MHz, MeOD-d4) δ [ppm] 8.85 (d, J = 0.6 Hz, 1H, H-6), 5.30
(dd, J = 10.0, 5.9 Hz, 1H, H-1’), 4.32 (ddd, J = 5.9, 3.0, 2.2 Hz, 1H, H-3’),
3.95 (td, J = 4.5, 2.7 Hz, 1H, H-4’), 3.67 (dd, J = 11.9, 4.3 Hz, 2H, 2 x H-
5’),2.44 (ddd, J = 13.0, 11.0, 5.8, 1H, H-2’), 1.85 (ddd, J = 13.0, 10.0, 5.8
Hz, 1H, H-2’)
13C-NMR (125 MHz, MeOD-d4) δ [ppm] 167.5 (Camide), 167.2 (Camide),
158.2 (C-2/C-4), 158.1 (C-2/C-4), 157.9 (C-6), 135.4 (Cquart.), 135.1 (Cquart.),
134.2, 133.7, 130.1, 129.8, 128.9, 128.9 (CHar.), 120.3 (C-5), 89.8 (C-4’),
77.4 (C-1’), 73.8 (C-3’), 64.8 (C-5’), 42.3 (C-2’), 26.3 (CCH3), 19.1 (CCH3),
-5.4 (SiCH3), -5.5 (SiCH3)
13C-NMR (125 MHz, MeOD-d4) δ [ppm] 160.8 (C-2/C-4), 159.96 (C-6),
159.94 (C-2/C-4), 135.1 (C-5), 89.4 (C-4‘), 76.0 (C-1‘), 74.0 (C-3‘), 63.5
(C-5‘), 42.7 (C-2‘)
MALDI-MS: m/z calcd for C29H36N4O5Si [M+H+]+: 549.61; found: 548.61
MALDI-MS: m/z calcd for C9H10Cl2N2O3 [M+H+]+: 265,09; found: 266,74
Synthesis of 1β-[2,4-bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-
D-ribofuranose (10)
Synthesis of 1β-(2,4-dichloropyrimidin-5-yl)-1,2-dideoxy-5-O-(tert-
butyl-dimethylsilyl)-D-ribofuranose (8)
Compound 9 (1 equiv., 2.31 g, 4.21 mmol) was dissolved in 59 ml THF.
NEt3·3HF (2.6 equiv., 1.78 ml, 10.93 mmol) was added dropwise. After two
hours, volatiles were removed under reduced pressure, and the remaining
solid was purified by column chromatography on silica gel. Elution with
dichloromethane/methanol 9:1 afforded 10 as a white solid.
In a dried Schlenk flask compound 7 (1equiv., 3.67 g, 0.014 mol) and
imidazole (2.5 equiv., 2.36 g, 0.035 mol) were dissolved in dry DMF. Tert-
butyl-dimethylsilyl chloride (1.2 equiv., 2.5 g, 0.017 mol) was slowly added
to the reaction mixture. The mixture was stirred for 16 h at room
temperature, and 150 ml of water were added. The aqueous phase was
washed three times with 100 ml of ethyl acetate and dried over magnesium
sulfate. The solvent was removed under reduced pressure. The remaining
yellowish oil was purified by column chromatography on silica gel. Elution
with n-hexane/ethyl acetate 3:1 afforded 8 as a colourless oil.
TLC (dichloromethane/methanol 9:1): Rf = 0,25
Yield: 1,2 g (62%) (Figures S26-S28)
1H-NMR (500 MHz, DMSO-d6) δ [ppm] 11.05 (s, 1H, NH), 10.86 (s, 1H,
NH), 8.91 (s, 1H, H-6), 8.01-7.96 (m, 4H), 7.66-7.49 (m, 6H), 5.13 (dd, J =
10.1, 5.6 Hz, 1H, H-1’), 5.03 (d, J = 4.1 Hz, 1H), 4.83 (t, J = 5.9 Hz, 1H),
4.18-4.17 (m, 1H), 3.75 (td, J = 4.8, 2.5 Hz, 1H), 3.49 (t, J = 5.2 Hz, 2H, H-
5’), 2.21 (ddd, J = 12.9, 5.6, 1.7 Hz, 1H, H-2’), 1.89 (ddd, J = 12.9, 10.3,
5.9 Hz, 1H, H-2’)
TLC (n-hexane/ethyl acetate 3:1): Rf = 0,30
Yield: 3,99 g (76%) (Figures S20-S22)
1H-NMR (500 MHz, CDCl3) δ [ppm] 8.79 (d, 1H, H-6), 5.35 (dd, J = 5.9 Hz,
1H, H-1’), 4.46 (ddt, J = 5.6, 2.2, 1.8 Hz, 1H, H-3’), 4.09 (ddd, J = 4.2, 3.1,
2.3 Hz, 1H, H-4’), 3.79 (dd, J = 10.8, 3.4 Hz, 1H, H-5’), 3.75 (dd, J = 10.7,
4.3 Hz, 1H, H-5’), 2.50 (ddd, J = 12.9, 5.9, 2.0 Hz, 1H, H-2’), 1.90 (ddd, J
= 13.0, 9.9, 5.7 Hz, 1H, H-2’), 0.82 (s, 9H, C(CH3)3), 0.04 (s, 6H, Si(CH3)2)
13C-NMR (125 MHz, DMSO-d6) δ [ppm] 166.1 (Camide), 165.6 (Camide),
158.3 (C-6), 157.1 (C-2/C-4), 156.4 (C-2/C-4), 134.1 (Cquart.), 133.1 (Cquart.),
132.5, 132.1 (CHar.), 128.6, 128.4, 128.2, 128.1 (CHar.), 126.2 (CHquart.),
115.2 (C-5), 87.6 (C-4’), 74.0 (C-1’), 72.1 (C-3’), 62.1 (C-5’), 41.6 (C-2’)
13C-NMR (125 MHz, CDCl3) δ [ppm] 159.6 (C-2/C-4), 159.3 (C-2/C-4),
158.7 (C-6), 133.6 (C-5), 87.7 (C-4’), 75.0 (C-1’), 74.4 (C-3’), 63.8 (C-5’),
42.2 (C-2’), 26.0 (CCH3), 18.4 (CCH3), -5.28 (SiCH3), -5.41 (SiCH3)
MALDI-MS: m/z calcd for C23H22N4O5 [M+H+]+: 434,44; found: 434,72
Synthesis of 1β-[2,4-bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-
O-(4,4’-dimethoxytriphenylmethyl)-D-ribofuranose (11)
MALDI-MS: m/z calcd for C15H24Cl2N2O3Si [M+H+]+: 379,35; found: 380,58
Synthesis of 1β-[2,4-bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-
O-(tert-butyl-dimethylsilyl)-D-ribofuranose (9)
In an argon purged dried Schlenk flask compound 10 (1 equiv., 1.15 g,
2.65 mmol) and DMAP (0.05 equiv., 16 mg, 0.133 mmol) were dissolved
in 32 dry pyridine. 4,4’-dimethoxytrityl chloride (1.5 equiv., 1.35 g,
4.0 mmol) was added slowly and the mixture was stirred at room
temperature for 12 h. The reaction was stopped with 13 ml of MeOH and
all solvents were removed under reduced pressure. The remaining crude
product was purified by column chromatography on silica gel. Elution with
n-hexane/acetone 2:3 + 0.1% NEt3 afforded 11 as a white foam.
In an argon purged dry Schlenk flask were dissolved in dry 1,4-dioxane
Pd2(dba)3 (0.04 equiv., 0.39 g, 0.42 mmol), xantphos (0.12 equiv., 0.73 g,
1.26 mmol), benzamide (3 equiv., 3.83 g, 0.032 mol), Cs2CO3 (3 equiv.,
10.3 g, 0.032 mol) and compound 8 (1 equiv., 3.99 g, 0.011 mol). The
reaction mixture was stirred for 24 hours at 100°C. After being cooled to
room temperature, the mixture was filtered over pad of celite and eluted
with MeOH. The solvent was removed under reduced pressure and the
remaining crude product was purified by column chromatography on silica
gel. Elution with n-hexane/ethyl acetate 1:3 afforded 9 as a colourless solid.
TLC (n-hexane/acetone 2:3): Rf = 0,23
Yield: 1,49 g (77%) (Figures S29-S31)
1H-NMR (500 MHz, CDCl3) δ [ppm] 10.49 (s, 1H, NH), 9.74 (s, 1H, NH),
8.43 (s, 1H, H-6), 7.99 (d, J = 7.4 Hz, 2H), 7.82 (d, J = 7.8 Hz, 2H), 7.52-
7.26 (m, 8H), 7.18-7.14 (m, 4H), 6.70-6.68 (m, 4H), 5.28 (dd, J = 10.3, 4.9
Hz, 1H, H-1’), 4.55 (br s, 1H, OH), 4.30 (br s, 1H, H-3’), 3.79 (t, 1H, H-4’),
3.70 (s, 6H, 2 x OCH3), 3.27 (dd, J = 10.3, 5.1 Hz, 1H, H-5’), 3.19 (dd, J =
TLC (ethyl acetate/n-hexane 3:1): Rf = 0,08
Yield: 2,31 g (37%) (Figures S23-S25)
1H-NMR (500 MHz, MeOD-d4) δ [ppm] 8.56 (s, 1H, H-6), 8.07-8.05 (m, 2H),
8.02-8.00 (m, 2H), 7.69-7.62 (m, 2H), 7.60-7.53 (m, 4H), 5.40 (dd, J = 10.6,
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10.1002/chem.201904623
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10.1, 4.5 Hz, 1H, H-5’), 2.51-2.49 (m, 1H, H-2’), 2.30 (dd, J = 12.4, 5.6 Hz,
1H, H-2’)
1H-NMR (500 MHz, DMSO-d6) δ [ppm] 12.33 (s, 1H, NH), 10.03 (s, 1H,
NH), 7.54 (d, J = 3.6 Hz, 1H, H-7), 6.52 (d, J = 3.5 Hz, 1H, H-8), 1.23 (s,
9H, t-Butyl)
13C-NMR (125 MHz, CDCl3) δ [ppm] 165.3 (Camide), 164.9 (Camide), 158.6
(Car.-O), 157.5 (C-2/C-4), 157.2 (C-2/C-4), 156.4 (C-6), 144.5, 135.7, 135.7,
133.8 (Cquart.), 132.9, 132.4, 130.0, 129.1, 128.7, 128.1, 127.9, 127.8,
127.6, 113.2 (CHar.), 87.4 (C-4’), 86.5 (Cquart.), 76.1 (C-1’), 73.6 (C-3’), 64.3
(C-5’), 55.3 (OCH3), 40.0 (C-2’)
13C-NMR (126 MHz, DMSO-d6) δ [ppm] 175.9, 152.8, 151.4, 150.2, 127.4,
113.2, 98.9, 39.4, 26.9
ESI-MS: m/z calcd for C11H13ClN4O [M+H+]+ 252,70; found: 253,11
MALDI-MS: m/z calcd for C44H40N4O7 [M-H+]+: 736,81; found: 736,45
Synthesis of N‐(4‐chloro‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐2‐yl)‐2,2‐
dimethyl‐propionamide (15)
Synthesis of 1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-
O-(4,4’-dimethoxytriphenylmethyl)-D-ribofuranose-3-[(2-
cyanoethyl)(N,N-diisopropyl)]phosphoramidite (12)
Compound 14 (1 eq., 18 g, 71.2 mmol) was dissolved under argon
atmosphere in 360 mL dry tetrahydrofuran. To this solution was then
added N-iodosuccinimide (1.1 eq., 17.6 g, 78.4 mmol). The reaction
mixture was stirred for one hour at room temperature. Then the solvent
was removed in vacuo and the residue was taken up in 1.5 L
dichloromethane. It was then washed first with a saturated sodium sulfate
solution (Na2S 4), then with a saturated sodium chloride solution and then
dried over magnesium sulfate. The solvent was removed in vacuo and the
residue was purified by column chromatography. The eluent used was
dichloromethane/methanol in the ratio 20: 1. Compound 15 was isolated
as a colorless solid.
In an argon purged dried Schlenk flask compound 11 (1 equiv., 253 mg,
0.343 mmol) and N,N-diisopropylethylamine (3 equiv., 0.18 ml, 1.03 mmol)
were dissolved in 8 ml of dry dichloromethane. 2-cyanoethyl N,N-
diisopropylethylamine (1.5 equiv., 0.115 ml, 0.515 mmol) was added and
the reaction mixture was stirred for two hours at room temperature. All
volatiles were removed under reduced pressure and crude product was
purified by column chromatography on silica gel. Elution with
cyclohexane/acetone 2:1 + 0.1% triethylamine afforded 12 as a colourless
solid.
TLC (dichloromethane/methanol 20:1): Rf = 0,77
Yield: 25,1 g (93%) (Figures S38-S40)
TLC (cyclohexane/acetone 2:1): Rf = 0,31
Yield: 238 mg (74%) (Figures S32-S34)
1H-NMR (600 MHz, DMSO-d6) δ [ppm] 12.67 (s, 1H, NH), 10.09 (s, 1H,
NH), 7.76 (d, J = 2.2 Hz, 1H, H-8), 1.22 (s, 9H, t-Butyl)
31P-NMR (202 MHz, DMSO-d6) δ [ppm] 147.1, 146.5
1H-NMR (400 MHz, DMSO-d6) δ [ppm] 11.10 (s, 1H, NH), 11,01 (s, 1H,
NH), 8.88 (d, J = 2.4 Hz, 1H, H-6), 8.04-7.97 (m, 4H, Phenyl), 7.67-7.50
(m, 7H, Phenyl), 7.34-7.21 (m, 8H, Phenyl), 6.89 (dd, J = 3.8, 8.8 Hz, 4H,
Phenyl), 5.14 (m, 1H, H-1’), 4.40 (m, 1H, H-4’), 4.02 (m, 1H, H-3’), 3.73 (s,
6H, 2xOCH3), 3.57 (m, 1H, H-5’), 3.46 (m, 1H, H-5’), 3.42 (m, 2H, Alkan),
3.19 (m, 2H, H-2’), 2.56 (m, 2H, Isopropyl), 2.48 (m, 2H, Alkan), 1.04 (m,
6H, 2xCH3), 0.90 (m, 6H, 2xCH3)
13C-NMR (151 MHz, DMSO-d6) δ [ppm] 175.8, 152.5, 151.4, 150.7, 132.7,
112.3, 51.5, 26.8
ESI-MS: m/z calcd for C11H12ClIN4O [M+Na+]+ 378,60; found: 400,90
Synthesis
of
4-chloro-5-iodo-2-pivaloylamino-7-[(2,3,5-tri-O-
benzoyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (16)
MALDI-MS: m/z calcd for C53H57N6O8P [M-H+]: 937,03; found: 936,36.
Compound 15 (1 eq., 15 g, 39.6 mmol) was suspended under an argon
atmosphere in 350 mL of dry acetonitrile. To this was added dropwise N,O-
bis(trimethylsilyl) acetamide (1.24 eq., 12 mL, 49.1 mmol) and the solid
went completely into solution. The reaction mixture was stirred for ten
minutes at room temperature. Then, trimethylsilyl trifluoro-
methanesulfonate (1.4 eq., 10 mL, 55.5 mmol) and 1'-acetate-2 ', 3',
5'-tribenzoate-β-D-ribofuranose (0.67 eq., 13 , 4 g, 26.6 mmol) were added
and heated to 50 °C. 1'-acetate-2', 3', 5'-tribenzoate-β-D-ribofuranose
(0.67 eq., 13.4 g, 26.6 mmol) was then added again after six and twelve
hours. After stirring for 24 hours, 800 mL of dichloromethane were added.
The organic phase was washed with 150 mL of a saturated aqueous
sodium bicarbonate solution, with 150 mL of a saturated sodium chloride
solution (evolution of gas) and dried over magnesium sulfate. The solvent
was removed in vacuo and a column chromatographic purification was
performed. The eluent used was cyclohexane / ethyl acetate in the ratio
3:1. The product 16 was isolated as a yellow/colorless foam.
Synthesis
of
N‐(4‐chloro‐7H‐pyrrolo[2,3‐d]pyrimidin‐2‐yl)‐2,2‐
dimethyl‐propionamide (14)
Compound 13 (1 eq., 14 g, 83 mmol) was dissolved in 196 mL of dry
pyridine under an argon atmosphere. Pivaloyl chloride (1.8 eq., 18.5 mL,
151 mmol) was then slowly added dropwise to this solution at room
temperature. The reaction mixture was stirred for one hour at room
temperature. During the reaction, a colorless solid precipitated (pyridine
hydrochloride). By adding 100 ml of methanol, the reaction was stopped.
The solvent was removed in an oil pump vacuum leaving a yellow solid.
The yellow solid was taken up in 200 ml of ethyl acetate and washed three
times with 100 ml of a 0.1 M hydrochloric acid solution. The organic phase
was dried over magnesium sulfate (MgSO4) and the solvent removed in
vacuo. The crude product was purified by column chromatography. The
eluent used was dichloromethane/methanol in a ratio of 10: 1. Compound
14 was obtained as a yellowish solid.
TLC (cyclohexane/ethyl acetate 3:1): Rf = 0,33
Yield: 19 g (58%) (Figures S41-S43)
TLC (dichloromethane/methanol 98:2): Rf = 0,37
Yield: 19 g (90%) (Figures S35-S37)
1H-NMR (600 MHz, DMSO-d6) δ [ppm] 10.32 (s, 1H, NH), 8.06 (s, 1H, H-
8), 7.93-7.89 (m, 6H, Aromat), 7.66-7.62 (m, 4H, Aromat), 7.48-7.43 (m,
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10.1002/chem.201904623
Chemistry - A European Journal
FULL PAPER
6H, Aromat), 6.52 (d, J = 3.8 Hz, 1H, H-1’), 6.44 (t, J = 5.9, 6.2 Hz, 1H, H-
3’), 6.36 (dd, J = 3.8, 6.0 Hz, 1H, H-2’), 4.84 (dd, J = 5.3, 10.5 Hz, 1H, H-
4’), 4.79 (dd, J = 4.3, 11.7 Hz, 1H, H-5’), 4.66 (dd, J = 5.6, 11.5 Hz, H-5’)
ESI-MS: m/z calcd for C12H14IN3O6 [M+H+]+ 423,16; found: 423,98
Synthesis of 1,7-dihydro-5-(octa-1,7-diynyl)-4-methoxy-7-(β-D-
ribofuranosyl)-2H-pyrrolo[2,3-d]pyrimidin-2-amine (19)
13C-NMR (151 MHz, DMSO-d6) δ [ppm] 175.8, 165.3, 164.5, 164.4, 151.8,
151.5, 151.3, 133.9, 133.7, 133.67, 133.4, 129.4, 129.24, 129.22, 129.1,
128.8, 128.72, 128.70, 128.6, 128.4, 113.2, 87.6, 79.1, 73.9, 71.2, 63.7,
53.97, 26.6
Compound 18 (1 eq., 1.2 g, 2.84 mmol) was first dried in vacuo at 65 °C
for one hour. Then, tetrakis(triphenylphosphine)palladium(0) (Pd (PPh3) 4,
0.1 Eq., 328 mg, 284 µmol) and copper iodide (CuI, 0.2 Eq., 108 mg, 567
µmol) were added under argon atmosphere. The solids were again dried
in vacuo for 20 minutes. To this was then added 10 mL of N,N-
dimethylformamide and triethylamine (NEt3, 2.84 eq., 1.12 mL, 8.05 mmol).
The reaction solution was then placed under argon and vacuum 20 times
to remove any residual oxygen. Finally, 1,7-octadiyne (10 eq., 3.76 mL,
28.4 mmol) was added. The reaction was stirred at room temperature
overnight. Thereafter, the solvent was removed in an oil pump vacuum at
65°C to 90%. Subsequently, a column chromatographic purification was
carried out. The eluent used was dichloromethane / methanol in the ratio
9:1. The product 19 was isolated as an orange solid.
ESI-MS: m/z calcd for C37H32ClIN4O8 [M+H+]+ 823,03; found: 823,19
Synthesis of 2-amino-5-iodo-3,7-dihydro-7-(β-D-ribofuranosyl)-4H-
pyrrolo-[2,3-d]pyrimidin-4-one (17)
Compound 16 (1 eq., 18.95 g, 23.02 mmol) was dissolved in 345 mL of a
0.5 M sodium methoxide solution and heated at reflux for 12 hours. It was
then neutralized with glacial acetic acid and the solvent was removed in
vacuo. The remaining residue was purified by column chromatography.
The eluent used was dichloromethane/methanol in the ratio 9:1. The
product 17 was isolated as a colorless solid.
Yield: 1,09 g (96%) (Figures S50-S52)
The product still contains residues of triethylamine, which could not be
removed by the column-chromatographic purification.
TLC (dichloromethane/methanol 9:1): Rf = 0,29
Yield: 9,7 g (61%) (Figures S44-S46)
TLC (dichloromethane/methanol 9:1): Rf = 0,39
1H-NMR (600 MHz, DMSO-d6) δ [ppm] 7.31 (s, 1H, H-8), 6.36 (s, 2H, NH2),
5.94 (d, J = 6.6 Hz, 1H, H-1’), 5.25 (d, J = 4.7 Hz, 1H, OH-2’), 5.05 (s, 1H,
OH-3’), 4.99 (t, J = 4.9, 5.2 Hz, OH-5’), 4.27 (dd, J = 4.6, 6.2 Hz, 1H, H-2’),
4.02 (s, 1H, H-3’), 3.92 (s, 3H, CH3), 3.81 (dd, J = 3.9, 6.8 Hz, 1H, H-4’),
3.58-3.56 (m, 1H, H-5’), 3.50-3.49 (m, 1H, H-5’)
1H-NMR (600 MHz, DMSO-d6) δ [ppm] 11.45 (s, 1H, NH), 7.53 (s, 1H, H-
8), 5.97 (s, 1H, OH-2’), 5.32 (d, J = 6.2 Hz, 1H, H-1’), 5.10 (s, 1H, OH-3’),
5.03 (s, 1H, OH-5’), 4.29 (s, 1H, H-2’), 4.05 (dd, J = 4.5, 7.6, 12.1 Hz, 1H,
H-3’), 3.97 (s, 3H, CH3), 3.86 (s, 1H, H-4’), 3.61-3.59 (m, 1H, H-5’), 3.54-
3.51 (m, 1H, H-5’), 2.79 (t, J = 2.6 Hz, 1H, H-a), 2.43 (t, J = 6.5 Hz, 2H, H-
b), 2.25-2.22 (m, 2H, H-e), 1.68-1.60 (m, 4H, H-c,d)
13C-NMR (151 MHz, DMSO-d6) δ [ppm] 162.8, 159.4, 154.7, 124.4, 98.7,
85.7, 84.7, 73.5, 70.5, 61.5, 52.96, 51.59
13C-NMR (126 MHz, DMSO-d6) δ [ppm] 164.2, 134.9, 133.8, 133.7, 130.1,
130.0, 125.2, 90.3, 86.3, 85.1, 84.4, 73.8, 71.3, 70.5, 61.4, 53.6, 27.3, 27.0,
18.4, 17.3
ESI-MS: m/z calcd for C12H15IN4O5 [M+H+]+ 422,18; found: 423,00
Synthesis of 1,7-dihydro-5-iodo-4-methoxy-7-(β-D-ribofuranosyl)-2H-
pyrrolo[2,3-d]pyrimidin-2-amine (18)
ESI-MS: m/z calcd for C20H23N3O6 [M+Na+]+ 401,41; found: 424,13
Synthesis of 5-(octa-1,7-diynyl)-7-(β-D-ribofuranosyl)-1,3,7-trihydro-
2H,4H-pyrrolo-[2,3-d]pyrimidin-2,4-dione (20)
Compound 17 (1 eq., 2.5 g, 5.92 mmol) was suspended in 505 mL of a 7:1
mixture of water (433 mL) and glacial acetic acid (72 mL). To this was then
slowly added dropwise sodium nitrite (2.46 eq., 1.01 g, 14.57 mmol)
dissolved in 11.2 ml of water. After about ten minutes, a yellow-orange
solution formed. The reaction mixture was stirred for one hour at room
temperature. It was then neutralized with a 25% aqueous ammonia
solution. The solvent was removed in vacuo and the residue was purified
by column chromatography. The eluent used was dichloromethane /
methanol in the ratio 5:1. Compound 18 was isolated as a yellowish solid.
Compound 19 (1 eq., 750 mg, 1.87 mmol) and sodium iodide (1.97 eq.,
197 mg, 1.32 mmol) were suspended under an argon atmosphere in 28 mL
of dry acetonitrile. To this suspension trimethylsilyl chloride (TMS-Cl, 2.28
eq., 193 µL, 1.52 mmol) was added. A greenish discoloration of the
reaction solution was observed. The reaction was stirred for one hour at
room temperature. The reaction solution was filtered and washed with
acetonitrile. The solvent was removed in vacuo and
a column
chromatographic purification followed. The eluent used was
dichloromethane / methanol in the ratio 5:1. The product 20 was isolated
as a colorless solid.
TLC (dichloromethane/methanol 5:1): Rf = 0,52
Yield: 1,5 g (61%) (Figures S47-S49)
TLC (dichloromethane/methanol 5:1): Rf = 0,56
Yield: 453 mg (63%) (Figures S53-S55)
1H-NMR (600 MHz, DMSO-d6) δ [ppm] 11.60 (s, 1H, NH), 7.48 (s, 1H, H-
8), 5.94 (d, J = 6.1 Hz, 1H, H-1’), 5.31 (s, 1H, OH-2’), 5.10 (s, 2H, OH-3’+
OH-5’), 4.28 (dd, J = 5.6, 6.0 Hz, 1H, H-2’), 4.04 (s, 1H, H-3’), 3.97 (s, 3H,
CH3), 3.87 (m, 1H, H-4’), 3.61-3.58 (ddd, J = 3.5, 7.8, 11.2 Hz, 1H, H-5’),
3.54-3.52 (ddd, J = 3.5, 8.2, 11.7 Hz, 1H, H-5’)
1H-NMR (500 MHz, DMSO-d6) δ [ppm] 11.52 (s, 1H, NH), 10.64 (s, 1H,
NH), 6.90 (s, 1H, H-8), 5.77 (m, 1H, OH), 5.66 (d, J = 6.9 Hz, 1H, H-1’),
5.37 (d, J = 6.6 Hz, OH), 5.21 (d, J = 3.9 Hz, 1H, OH), 4.16-4.15 (m, 1H,
H-2’), 4.01 (m, 1H, H-3’), 3.96-3.95 (m, 1H, H-4’), 3.62 (m, 2H, H-5’), 2.70
13C-NMR (151 MHz, DMSO-d6) δ [ppm] 172.2, 163.9, 160.1, 126.0, 100.5,
86.6, 85.2, 73.8, 70.5, 61.5, 53.6, 52.1
This article is protected by copyright. All rights reserved.

10.1002/chem.201904623
Chemistry - A European Journal
FULL PAPER
[4]
J. Piccirilli, T. Krauch, S. E. Moroney, S. Benner, Nature 1990, 343,
33–37.
A. Rich, On the Problems of Evolution and Biochemical Information
Transfer, 1962.
(t, J = 2.6, 5.3 Hz, 1H, H-a), 2.08-2.06 (m, 2H, H-b), 1.95-1.93 (m, 2H, H-
e), 1.53-137 (m, 4H, H-c,d)
[5]
13C-NMR (126 MHz, DMSO-d6) δ [ppm] 158.9, 151.1, 138.9, 120.7, 117.4,
97.8, 94.9, 89.7, 86.0, 84.9, 84.8, 74.5, 74.4, 71.8, 71.2, 61.8, 27.9, 27.7,
27.5, 18.1, 17.9
[6]
Z. Yang, D. Hutter, P. Sheng, A. M. Sismour, S. A. Benner, Nucleic
Acids Res. 2006, 34, 6095–6101.
H. J. Kim, N. A. Leal, S. A. Benner, Bioorganic Med. Chem. 2009,
17, 3728–3732.
[7]
[8]
F. Eggert, S. Kath-Schorr, Chem. Commun. 2016, 7284, 7284–
7287.
ESI-MS: m/z calcd for C19H21N3O6 [M-H+]- 387,39; found: 386,23
[9]
C. Domnick, F. Eggert, S. Kath-Schorr, Chem. Commun. 2015, 51,
8253–8256.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
F. Eggert, K. Kulikov, C. Domnick, P. Leifels, S. Kath-Schorr,
Methods 2017, 120, 17–27.
Synthesis of 5-(octa-1,7-diynyl)-7-(β-D-ribofuranosyl)-1,3,7-trihydro-
2H,4H-pyrrolo-[2,3-d]pyrimidin-2,4-dione TP (21)
C. Domnick, G. Hagelueken, F. Eggert, O. Schiemann, S. Kath-
Schorr, Org. Biomol. Chem. 2019, 17, 1805–1808.
E. Larsen, P. T. Jorgensen, M. A. Sofan, E. B. Pedersen, Synthesis
(Stuttg). 1994, DOI 10.1055/s-1994-25633.
M. A. Cameron, S. B. Cush, R. P. Hammer, J. Org. Chem. 1997, 62,
9065–9069.
M. Weinberger, F. Berndt, R. Mahrwald, N. P. Ernsting, H. A.
Wagenknecht, J. Org. Chem. 2013, 78, 2589–2599.
T. Kubelka, L. Slavětínská, B. Klepetářová, M. Hocek, European J.
Org. Chem. 2010, 2666–2669.
N. Joubert, R. Pohl, B. Klepetářová, M. Hocek, J. Org. Chem. 2007,
72, 6797–6805.
A. Häberli, C. J. Leumann, Org. Lett. 2001, 3, 489–492.
W. Saenger, Principles of Nucleic Acid Structure, p.21, Springer-
Verlag New York Inc., 1984.
Compound 20 (1 eq., 50 mg, 129 µmol) and freshly triturated proton
sponge (1.5 eq., 41.5 mg, 194 µmol) were dried in a predried Schlenk flask
overnight in an oil pump vacuum. It was the dissolved under an argon
atmosphere in 1,6 mL trimethyl phosphate (dried over a 4Å molecular
sieve for 4 days). The reaction solution was cooled to -7.5°C and freshly
distilled phosphorus oxychloride (1.05 eq., 12.7 µL, 136 µmol) was rapidly
added. After five hours, phosphorus oxychloride (0.4 eq., 4.8 µL,
51.6 µmol) was added again, and the reaction mixture was further stirred
for one hour. Then tributylamine (4.4 eq., 135 µL, 568 µmol) and 2.5 mL
of 0.4 M tributylamine pyrophosphate solution in dry DMF were added. The
mixture was further stirred at room temperature for 30 minutes. Next, the
reaction mixture was slowly added dropwise to a rapidly stirring solution of
0.1 M TEAB buffer (pH 8, 9.2 mL). The solution was hydrolyzed for eight
hours and the product 21 was isolated by RP-HPLC. The fractions
containing the desired product were lyophilized. 21 was isolated as a
colorless solid.
[17]
[18]
[19]
[20]
M. Singer, A. Nierth, A. Jäschke, European J. Org. Chem. 2013,
2766–2769.
F. Seela, X. Peng, Collect. Czechoslov. Chem. Commun. 2006, 71,
956–977.
F. Seela, X. Peng, J. Org. Chem. 2005, 81–90.
X. Peng, F. Seela, Nucleosides. Nucleotides Nucleic Acids 2007,
26, 603–606.
[21]
[22]
[23]
C. Weldon, I. Behm-Ansmant, L. H. Hurley, G. A. Burley, C.
Branlant, I. C. Eperon, C. Dominguez, Nat. Chem. Biol. 2017, 13,
18–20.
Yield: 13 mg (16%) (Figures S56-S59)
[24]
[25]
[26]
F. Seela, N. Ramzaeva, Helv. Chim. Acta 1995, 78, DOI
10.1002/hlca.19950780505.
F. Klepper, E. M. Jahn, V. Hickmann, T. Carell, Angew. Chemie -
Int. Ed. 2007, 46, 2325–2327.
F. Seela, M. Zulauf, Synthesis (Stuttg). 1995, 1, DOI 10.1055/s-
1996-4282.
J. Ludwig, F. Eckstein, J. Org. Chem. 1989, 19, 631–635.
M. Yoshikawa, T. Kato, T. Takenishi, Tetrahedron Lett. 1967, 5065–
5068.
J. Ludwig, Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131–
3.
M. Merkel, S. Arndt, D. Ploschik, G. B. Cserép, U. Wenge, P. Kele,
H. A. Wagenknecht, J. Org. Chem. 2016, 81, 7527–7538.
J. F. Milligan, D. R. Groebe, G. W. Whherell, O. C. Uhlenbeck,
Nucleic Acids Res. 1987, 15, 8783–8798.
B. Fürtig, C. Richter, W. Bermel, H. Schwalbe, J. Biomol. NMR
2004, 28, 69–79.
S. Nozinovic, B. Fürtig, H. R. A. Jonker, C. Richter, H. Schwalbe,
Nucleic Acids Res. 2009, 38, 683–694.
H. Steinert, F. Sochor, A. Wacker, J. Buck, C. Helmling, F. Hiller, S.
Keyhani, J. Noeske, S. Grimm, M. M. Rudolph, et al., Elife 2017, 6,
1–18.
J. Buck, J. Noeske, J. Wöhnert, H. Schwalbe, Nucleic Acids Res.
2010, 38, 4143–4153.
J. Buck, B. Fürtig, J. Noeske, J. Wönert, H. Schwalbe, Proc. Natl.
Acad. Sci. U. S. A. 2007, 104, 15699–15704.
J. Noeske, J. Buck, B. Fürtig, H. R. Nasiri, H. Schwalbe, J. Wöhnert,
Nucleic Acids Res. 2007, 35, 572–583.
J. Singh, N. Steck, D. De, A. Hofer, A. Ripp, I. Captain, M. Keller, P.
A. Wender, R. Bhandari, H. J. Jessen, Angew. Chemie - Int. Ed.
2019, 58, 3928–3933.
J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair,
T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, et al.,
Nat. Methods 2012, 9, 676–682.
1H-NMR (600 MHz, D2O) δ [ppm] 7.89 (s, 1H, H-8), 6.08 (d, J = 6.5 Hz, 1H,
H-1’), 4.59 (t, J = 5.9 Hz, 1H, H-2’), 4.48 (dd, J = 3.3, 5.5 Hz, 1H, H-3’),
4.33-4.32 (m, 1H, H-4’), 4.28-4.24 (m, 2H, H-5’), 2.45 (t, J = 6.6 Hz, 1H, H-
a), 2.27-2.26 (m, 2H, H-b), 2.19-2.16 (m, 2H, H-e), 1.69-1.65 (m, 4H, H-
c,d)
[27]
[28]
31P-NMR (162 MHz, D2O) δ [ppm] -5.48 (d, 1P, γ-PO3 2-), -10.62 (d, 1P, α-
[29]
[30]
[31]
[32]
[33]
[34]
2-
PO3 2-), -18.88 (t, 1P, β-PO3
)
MALDI-MS: m/z calcd for C19H24N3O15P3 [M+Na+]+ 627,33; found:
650,0450.
Acknowledgements
We thank Oliver Binas and Dr. Christian Richter for support with
NMR experiments. The work was supported by DFG in
collaborative research center 902. The BMRZ is supported by
state of Hesse.
[35]
[36]
[37]
[38]
Keywords: Genetic code expansion • RNA • site-specific
labeling • NMR • fluorescence
[39]
[40]
[1]
[2]
[3]
Y. Zhang, J. L. Ptacin, E. C. Fischer, H. R. Aerni, C. E. Caffaro, K.
San Jose, A. W. Feldman, C. R. Turner, F. E. Romesberg, Nature
2017, 551, 644–647.
A. W. Feldman, V. T. Dien, R. J. Karadeema, E. C. Fischer, Y. You,
B. A. Anderson, R. Krishnamurthy, J. S. Chen, L. Li, F. E.
Romesberg, J. Am. Chem. Soc. 2019, 141, 10644–10653.
V. T. Dien, M. Holcomb, F. E. Romesberg, Biochemistry 2019, 58,
2581–2583.
C. Helmling, S. Keyhani, F. Sochor, B. Fürtig, M. Hengesbach, H.
Schwalbe, J. Biomol. NMR 2015, 63, 67–76.
V. Sklenář, A. Bax, J. Magn. Reson. 1987, 74, 469–479.
A. L. Breeze, Prog. Nucl. Magn. Reson. Spectrosc. 2000, 36, 323–
372.
[41]
[42]
[43]
J. Noeske, C. Richter, M. a Grundl, H. R. Nasiri, H. Schwalbe, J.
Wöhnert, Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 1372–1377.
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Chemistry - A European Journal
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[44]
[45]
[46]
[47]
K. Ogura, H. Terasawa, F. Inagaki, J. Biomol. NMR 1996, 8, 492–
498.
C. Zwahlen, P. Legault, S. J. F. Vincent, J. Greenblatt, R. Konrat, L.
E. Kay, J. Am. Chem. Soc. 1997, 119, 6711–6721.
J. Iwahara, J. M. Wojciak, R. T. Clubb, J. Biomol. NMR 2001, 19,
231–241.
M. Liu, X. Mao, C. Ye, H. Huang, J. K. Nicholson, J. C. Lindon, J.
Magn. Reson. 1998, 132, 125–129.
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Entry for the Table of Contents
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Orthogonal nucleic acid building
blocks to the canonical A-T and G-C
basepairs are uniquely suitable to
facilitate position-specific labeling.
Here, we employ the orthogonal
kappa-xanthosine-base-pair for in
vitro transcription. Using NMR
spectroscopy, we show that
Andreas Hegelein+[a], Diana Müller+[a]
,
Sylvester Größl[b], Michael Göbel[b],
Martin Hengesbach[a] and Harald
Schwalbe*[a]*
Page No. – Page No.
Genetic code expansion facilitates
position-selective labeling of RNA
for biophysical studies
xanthosine introduces only minor
structural changes. We also
synthesized a clickable 7-deaza-
xanthosine, which allows to site-
specifically modify transcribed RNA.
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