A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy

Article abstract of DOI:10.1002/chem.201800167

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

Full text of DOI:10.1002/chem.201800167

A Journal of
Accepted Article
Title: A Cytidine Phosphoramidite with Protected Nitroxide Spin Label:
Synthesis of a Full-Length TAR RNA and Investigation by in-line
Probing and EPR Spectroscopy.
Authors: Timo Weinrich, Eva A. Jaumann, Ute Scheffer, Thomas F.
Prisner, and Michael W. Göbel
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To be cited as: Chem. Eur. J. 10.1002/chem.201800167
Link to VoR: http://dx.doi.org/10.1002/chem.201800167
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A Cytidine Phosphoramidite with Protected Nitroxide Spin Label:
Synthesis of a Full-Length TAR RNA and Investigation by in-line
Probing and EPR Spectroscopy.
Timo Weinrich,[a] Eva A. Jaumann,[b] Ute Scheffer,[a] Thomas F. Prisner,[b] and Michael W. Göbel*[a]
Abstract: EPR studies on RNA are complicated by three major
obstacles related to the chemical nature of nitroxide spin labels:
Decomposition while oligonucleotides are chemically synthesized,
further decay during enzymatic strand ligation and undetected
changes in conformational equilibria by the steric demand of the
label. Here we present possible solutions for all three problems: A 2-
nitrobenzyloxymethyl protective group for nitroxides, stable under all
conditions of chemical RNA synthesis that can be removed
photochemically. By careful selection of ligation sites and splint
oligonucleotides high yields are achieved in the assembly of a full-
length HIV-1 TAR RNA labeled with two protected nitroxides.
PELDOR measurements of spin labeled TAR in the absence and
presence of arginine amide indicate an arrest of interhelical motions
upon ligand binding. Finally, even minor changes in sample
conformation due to the presence of spin labels are detected with
high sensitivity by in-line probing.
pulsed electron-electron double resonance experiments
(PELDOR or DEER, Figure 1). The nitroxide part is introduced
postsynthetically by Sonogashira coupling.[21] Sigurdsson’s
[41]
nitroxides 2
and 3,[12] even more rigid than 1, can be
incorporated into DNA or RNA as phosphoramidites when
modified synthetic conditions are used to limit decomposition of
the radical.
Introduction
The analysis of RNA structure and dynamics by EPR
[1-6]
spectroscopy
requires labeling of the samples with
paramagnetic species. This is normally achieved by covalent
[7-34]
attachment of persistent nitroxide radicals
although
noncovalent labeling has become an interesting alternative.[35,36]
Due to the limited stability of nitroxides, postsynthetic
attachment has some advantages. Alkylation of 2’-amines,[7-11,20]
[13,15-19,32]
of 4-thiouridine,[14,30-32] and of thio-phosphates
are
typical approaches. Other methods include Sonogashira
[26,27]
reactions,[21-24] substitutions at convertible nucleotides
and
1,3-dipolar cycloadditions.[29] Most elegant in terms of synthesis
[37-48]
[12]
is the direct introduction by DNA
and RNA
phosphor-
Figure 1. Some nitroxide labeled ribonucleotides used for EPR studies of RNA.
amidites already containing the nitroxide moiety. However,
decomposition of nitroxides may occur during oligonucleotide
assembly or strand ligation unless specific precautions are
taken.[41,42] For spectroscopic reasons, rigid linker structures are
desirable. Cytidine analog 1 [21,37] for example is well suited for
PELDOR experiments [49,50] on oligonucleotides labeled with 2
or 3 not only detect the distance between the nitroxides but also
their relative orientation.[51] Starting from convertible nucleotides,
Höbartner has introduced a method for the postsynthetic
generation of nitroxides 4 and 5.[26] She also suggested a
ligation technique based on deoxyribozymes thus avoiding
nitroxide reduction by thiols.[27] Addition of thiols normally is
required to keep ligase proteins active. Although spin labels 4
and 5 are less rigid when compared to 2 or 3 and also cause
some duplex destabilization, successful PELDOR studies have
been published.[5,26] Furthermore, if some decomposition is
accepted, deoxynucleoside analogs of nitroxides 4 and 5 can be
incorporated into DNA strands by specifically adapted
phosphoramidite chemistry.[40,43,46]
[a]
[b]
T. Weinrich, Dr. U. Scheffer, Prof. M. W. Göbel
Institute for Organic Chemistry and Chemical Biology
Goethe University Frankfurt
Max-von-Laue-Str. 7, D-60438 Frankfurt am Main, Germany.
E-mail: M.Goebel@chemie.uni-frankfurt.de
E. A. Jaumann, Prof. T. F. Prisner
Institute for Physical and Theoretical Chemistry
Goethe University Frankfurt
Max-von-Laue-Str. 7, D-60438 Frankfurt am Main, Germany.
Supporting information for this article is given via a link at the end of
the document.
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To provide a more general solution to the problem of nitroxide
instability we have developed a protection strategy that is based
on the alkylation of hydroxylamines with light sensitive groups
(Figure 2). A phosphoramidite building block related to 6 could
be incorporated into DNA using standard conditions without
destruction of the protected label. After photolysis of the
coumarin group, the nitroxide radical was formed by
spontaneous air oxidation.[52] More recently, we have optimized
the synthesis and adapted it to ribonucleotides. Using
phosphoramidite 6 we prepared a short palindromic RNA
labeled with two protected hydroxylamines. Irradiation of the
sample under air then recovered the nitroxides. No further
purification was required for subsequent PELDOR analysis.[53]
However, the conversion of protected hydroxylamines into
nitroxides depends on pH. In neutral or slightly acidic media N-O
cleavage is the dominating process leading to amines instead of
nitroxides. At pH 8.5 the radical prevails but still 15 % of the
material is permanently reduced.[53] Our strategy for minimizing
the loss of nitroxides was then to separate the light sensitive
group and the N-O bond by a short additional spacer. First trials
with 2-nitrobenzyloxycarbonyl failed. This group is cleaved off
during nucleobase deprotection by ammonia. We therefore
synthesized amidite 7. The acetal linker should be resistant
against nucleophiles but not against strong acids. However, 2-
nitrobenzyloxymethyl (NBOM) has been already used to protect
2’-hydroxy groups in RNA chemistry.[54,55] It withstands the
conditions of solid phase synthesis and can be removed without
causing photochemical damage to the RNA strands.[55,56]
acid. Compound 11 proved to be sufficiently stable (see page
S6).
Scheme 1. Preparation of model compound 11. a) 8, SO2Cl2, 1 h, rt; b) 9, 10,
Cu, Cu(OTf)2, 4,4´-dimethyl-2,2´-bipyridyl, toluene, 20 h, reflux.
Trifluoroacetylation of 12 to give 13, followed by oxidation (14)
and reductive alkylation led to intermediate 15. After removal of
trifluoroacetyl with KOH, amine 16 reacted with the O-activated
uridine derivative 17 [27,61] in the presence of an unprotected 3’-
hydroxy group. Phosphitylation of product 17 then led to multi-
gram amounts of amidite 7 in good yield (Scheme 2). The 2’-
TOM protection has been chosen to eliminate the risk of 2’,3’-
silyl shifts and to enable the synthesis of longer RNA strands in
future work.
Scheme 2. Synthesis of phosphoramidite 7. a) Trifluoroacetic anhydride,
pyridine; b) Na2WO4, H2O2; c) 9, Cu, Cu(OTf)2, 4,4´-dimethyl-2,2´-bipyridyl,
toluene, 20 h, reflux; d) KOH, MeOH; e) 16, diisopropylethylamine, DMF,
80 °C f) Et3N, N,N-diisopropylaminocyanoethylphosphoramidic chloride.
Figure 2. Cytidine phosphoramidites with a protected TEMPO spin label.
The self complementary RNA oligonucleotides 19 and 22
were prepared from phosphoramidite 7 and commercial 2’-O-
TBS protected amidites by standard solid phase synthesis
(Figure 3). According to the trityl assay, compound 7 reacted
well and normal yields of RNA could be isolated by HPLC. The
integrity of the protected TEMPO groups was verified by mass
spectrometry. Photochemical removal at 365 nm in aqueous
buffer (at pH 4.2, 7.4, 8.0, 8.5) was complete after 20 min. At
any pH value tested, only traces of the reduction products 21
and 24 could be detected (see page S10). However, a strong
new peak appeared in each case in addition to the nitroxides 20
and 23. This peak did not occur when 20 was synthesized from
Results and Discussion
To test the stability of the NBOM protected hydroxylamine,
TEMPO derivative 10 was coupled with 9 in a copper mediated
reduction.[57,58] In this process, radicals obtained from
9
recombine with the nitroxide forming the O-alkylated product 11.
Compound is accessible from 2-nitrobenzyl alcohol by
9
Pummerer rearrangement (8) and chlorination (Scheme 1).[59,60]
Subsequently compound 11 was challenged by reagents typical
for oligonucleotide synthesis and ligation: ammonia, iodine,
triethylamine trihydrofluoride, dithiothreitol and trifluoroacetic
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amidite 6.[53] The products are slowly converted into 20 and 23 at
pH 7.0, faster at pH 8.5 and disappeared almost completely
upon heating to 90 °C for 60 min (see page S11). We assign
them to the hemiacetals 19a and 22a.
samples of long RNAs which can be studied by NMR [63] or EPR
[31,64] spectroscopy or by a combination of both.[30]
Figure 5. Structure of TAR oligonucleotides 25 – 27. Spin labels are in
positions 19 and 29. Arrows point to ligation sites. The fluorescent dye enables
in-line probing by a DNA sequencer.
After chemical synthesis of RNA fragment 30a from
phosphoramidite 7 and successful ligation (see below), the
59mer RNA 26b was photochemically deprotected to yield the
twofold spin labeled TAR RNA 27b, finally studied by EPR in the
absence and presence of arginine amide 28.
Figure 3. Spin labeled RNA palindromes 20 and 23 bearing a nitroxide on
position 9 or 10.
For PELDOR experiments, samples of RNAs 19 or 22 were
irradiated at pH 7.4, annealed to form the palindromic duplexes
20 or 23 and used directly for EPR spectroscopy without any
further purification (see page S12). The labeling efficiency of 20
was 95 % according to HPLC and 90 % as determined by EPR.
Consistent with our previous finding, both experiments showed a
main distance of 1.8 nm.[53] In case of RNA 20, end-to-end
stacking of the duplexes leads to a small population of longer
distances.[53] To preclude this effect, an extra uridine residue
was attached to RNA 22. The PELDOR data after
photochemical deprotection (23) now shows a single and well
defined distance (Figure 4).
Figure 6. Sequences of RNA and of splint oligonucleotides used to assemble
RNAs 25 - 27. p stands for a 5’ phosphate. Positions of the protected TEMPO
label are shown in bold letters.
For ligation of RNA 25 we used T4 RNA ligase 2 [64] in a buffer
containing dithiothreitol, the dye-labeled RNA 29 and the two 5’
phosphorylated fragments 30 and 31 (molar ratio 1:1:1). Ligation
sites between two purines were chosen for optimized stacking of
the fragments (see arrows in Figure 5). To guide the ligation and
to prevent unfavorable backfolding of RNA, we also added DNA
splint oligonucleotides (see page S17). Best results were
obtained in the presence of three splints 32, 33, and 34 (1.5
equiv. in each case) leading to about 80 % ligation yield of 25
that was purified further by gel electrophoresis. When RNA 29,
the TEMPO modified fragment 30a (1.5 eq.), and 31 were
ligated by the three-splint approach, up to 80 % analytical yield
of the full-length RNA 26a could be observed. The steric
demand of the protected TEMPO moieties seems not to impede
Figure 4. PELDOR measurement of RNA 23 (black). a) PELDOR time trace
after background correction (original data see page S20) and fit with Tikhonov
regularization (red) by using DeerAnalysis.[62] b) PELDOR distance distribution
with a dominant intramolecular value ~1.8 nm.
To demonstrate the stability of the protective group under
conditions of enzymatic RNA ligation, we assembled a 59mer
full-length TAR RNA (Figure 5) from three RNA fragments
(Figure 6). The non-labeled analog 25 was also prepared for
comparison. Covalent coupling of fragments by ligase proteins
[27]
or by deoxyribozymes
is a successful strategy to prepare
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the reaction. Dye labeling helps to optimize the ligation protocol.
However, it interferes with photochemical deprotection. For
PELDOR experiments we therefore chose RNA 26b lacking the
fluorescent dye. It was assembled in the three-splint ligation by
replacing RNA fragment 29 by 29a. Material used for PELDOR
was purified by gel electrophoresis (ligation scale: 40 nmol; 27-
46 % isolated yield) and RP-HPLC. Light induced removal of
nitrobenzyl then occurred without problems. However, when
hemiacetal cleavage was enforced by heating to 90 °C for 60
min, product 27b partially hydrolyzed. This problem could be
minimized by heating to 70 °C for 5 h and to 90 °C for only 10
min (pH 5.5-6.0). To check the conformational integrity of the
RNA sample by in-line probing,65,66 an aliquot of 27b was
converted into dye-labeled 27c by reaction of the amino linker
with an active ester of Cy5.
U23 and C24 of the UCU bulge in the ligand-free TAR RNA
are known from NMR studies to form a continuous stack with
their neighbors.[67] The two additional bases in one half of the
duplex cause a bend (~50 °) between the upper and lower
[67-69]
helical axes
and allow for large inter-helical motions.[70]
Upon binding of arginine amide or Tat-derived peptides, the
unpaired bulge nucleotides are expelled from the stack and an
almost straight and rigid helix results,[ 68-71] where U23 is brought
into close proximity to A27.[72] Consistent with these findings, the
PELDOR experiment with ligand-free RNA 27b shows a broad
distance distribution around 2.7 ± 0.4 nm, reflecting a dynamic
structure (Figure 7a). In the presence of arginine amide,
however, the structure becomes more ordered and shows a
narrow distance distribution of 2.3 ± 0.2 nm (Figure 7b). The
corresponding data taken from the NMR structure of the TAR-
arginine complex (1ARJ) [72] agrees well with the PELDOR result.
Figure 7. PELDOR measurements of TAR RNA 27b a) without and b) with
arginine amide. PELDOR time traces after background correction (original
data see page S21) and fit with Tikhonov regularization (red) by using
DeerAnalysis.[62] a) TAR RNA 27b without any ligand bound shows a broad
distance distribution of 2.7 ± 0.4 nm. b) In presence of arginine amide, the
distance distribution of TAR RNA 27b becomes narrow and the mean distance
slightly shorter with 2.3 ± 0.2 nm. The distance compilation based on the
arginine-bound TAR RNA NMR structure (1ARJ) [72] is shown in grey.
The influence of the spin labels on local conformation has
been studied by in-line probing,[65] a method leading to partial
RNA cleavage specifically in sites of enhanced conformational
mobility. When applied to the Cy5 labeled RNA 25,[66] the single
nucleotide bulges at C5, A17 and the hexaloop (C30-A35) show
the expected number of signals (Figure 8a). However, four
peaks are visible for the bulge U23-U25. The additional peak is
caused by the well known lability of the base pair A22/U40.[67,73]
Figure 8b shows in-line probing of RNA 26c containing two
protected spin labels on C19 and C29. One of the former single
nucleotide bulges now opens up to a three nucleotide bulge
A17, C18, and C19 (black arrows in Figure 8b). Note that the
spacing between C18 and C19 is twice as usual due to the
reduced mobility of fragments containing the spin label.
Furthermore, four extra peaks appear between U25 and C29
(red arrows) giving direct evidence for a destabilization of the
upper stem region. Much less structural perturbation is seen in
the deprotected RNA 27c (Figure 8c). The pattern is very similar
to that of the unmodified RNA. Only the base pair C18/G44
seems to be slightly destabilized.
Figure 8. Secondary structure analysis by in-line probing. This technique is
based on Mg2+ induced partial hydrolysis of 5’ dye-labeled RNA in positions of
unrestricted conformational mobility such as single strands, loops and bulges.
The resulting fragments are separated and visualized by gel electrophoresis in
a DNA sequencer (see page S19). a) Unmodified TAR RNA 25. b) RNA 26c
modified by protected spin labels on C19 and C29. c) Deprotected RNA 27c.
Conclusions
Phosphoramidite 7 is accessible in good yield and in gram
quantities. In oligonucleotide solid phase synthesis, it behaves
like a normal 2’-O-TOM protected building block. No special
reaction conditions are required because the NBOM group is
stable during all steps of chain assembly, RNA deprotection and
enzymatic ligation. In contrast to coumarin groups we have used
earlier in amidite 6, photochemical removal of NBOM does not
lead to N-O cleavage, not even at acidic pH. Short RNA samples
such as 20 and 23 are ready for PELDOR experiments after
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Keywords: enzymatic ligation of RNA • PELDOR spectroscopy •
photolabile protection • secondary structure mapping • TEMPO
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10.1002/chem.201800167
Chemistry - A European Journal
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Nitroxides are important spin labels for
EPR studies of biopolymers. However,
sample preparation can be severely
restricted by their limited chemical
stability. The 2-nitrobenzyloxymethyl
group protects the TEMPO label
against all critical conditions of
chemical RNA synthesis and
enzymatic ligation. After photo-
chemical removal, the nitroxide radical
is recovered by spontaneous air
oxidation. Assembly of a spin labeled
59mer RNA is shown as example.
Timo Weinrich, Eva A. Jaumann, Dr. Ute
Scheffer, Prof. Dr. Thomas F. Prisner,
Prof. Dr. Michael W. Göbel*
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A Cytidine Phosphoramidite with
Protected Nitroxide Spin Label:
Synthesis of a Full-Length TAR RNA
and Investigation by in-line Probing
and EPR Spectroscopy.
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