Practical Synthesis of Cap-4 RNA

Article abstract of DOI:10.1002/cbic.201900590

Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap-0 (m⁷GpppN) shows

Full text of DOI:10.1002/cbic.201900590

Accepted Article
Title: Practical Synthesis of Cap-4 RNA
Authors: Josef Leiter, Dennis Reichert, Andrea Rentmeister, and
Ronald Micura
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201900590
Link to VoR: http://dx.doi.org/10.1002/cbic.201900590
A Journal of
www.chembiochem.org

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
Practical Synthesis of Cap-4 RNA
Josef Leiter,[a] Dennis Reichert,[b] Andrea Rentmeister[b] and Ronald Micura*[a]
Abstract: Eukaryotic mRNAs possess 5’ caps that are determining
for their functions. A structural characteristic of 5’ caps is methylation,
with this feature already present in early eukaryotes such as
trypanosoma. While the common cap-0 (m7GpppN) shows a rather
simple methylation pattern, the trypanosoma cap-4 displays seven
distinguished additional methylations within the first four nucleosides.
The study of essential biological functions mediated by these unique
structural features of the cap-4 and thereby of the metabolism of an
important class of human pathogenic parasites is hindered by the lack
of reliable preparation methods. Here, we describe the synthesis of
component for the translational machinery and which itself is
directed to the 5’-end of the mRNA.[10] Furthermore, the 5’-cap
acts as a physical hindrance to 5’-3’ exoribonucleases so that
decapping enzymes are necessary to start the controlled
decomposition of mRNA.[11] This increases the lifetime of
mRNA[12] and enables an additional level of regulation.[13]
Our growing understanding of the functionality of 5’-caps
and the consequences of the disruption of cap–dependent
biochemical processes that result in severe medical disorders
make 5’-caps attractive for the development of novel
pharmaceutic and therapeutic approaches.[14] Potential
applications for cap analogs are foreseeable in antiviral therapy,
cancer treatment (including eIF4E targeting and mRNA-based
immunotherapy), spinal muscular atrophy treatment, and
treatment of genetic diseases by mRNA based protein
replacement therapy.[15] Essential for the success of RNA-based
therapeutics is the development of methods to introduce mRNA
into cells and the improvement of stability and translation
efficiency of the therapeutic itself.[15,16] The high potential of
mRNA cap–based approaches has been demonstrated in the first
application of 5’-capped RNAs in preclinical studies.[15]
custom-made nucleoside phosphoramidite building blocks for m6 Am
2
and m3Um, their incorporation into short RNAs, the efficient
construction of the 5’ to 5’ triphosphate bridge to guanosine using a
solid phase approach, the selective enzymatic methylation on position
N7 of the inverted guanosine, and enzymatic ligation to generate
trypanosomatid mRNA of up to 40 nt in length. This study introduces
a reliable synthetic strategy to the much-needed cap-4 RNA probes
for integrated structural biology studies, using a combination of
chemical and enzymatic steps.
Introduction
Nature invented posttranscriptional processing to add another
layer of information to RNA. This affects mRNA splicing,[1] nuclear
export,[2] initiation of translation[3] and the stability.[4,5] So called 5’-
caps, with the characteristic and unique feature of a N7-methyl
guanosine connected over a 5’ to 5’ triphosphate bridge to the first
nucleoside of nascent transcripts are hallmarks of eukaryotic
mRNA processing.[6,7] For higher eukaryotes it is known that 5’-
caps are involved in all the above-mentioned processes. For
example, m7G-caps are responsible for the recruitment of a
nuclear cap-binding complex, which plays a key role for the
splicing process and is involved in the packaging process of RNA
into ribonucleoprotein particles. This is an essential step for the
export out of the nucleus.[8,9] Moreover, the 5’-cap binds to the
eukaryotic translation initiation factor 4E (elF4E) which is a key
[a]
J. Leiter, Prof. R. Micura
University of Innsbruck
Institute of Organic Chemistry and Center for Molecular Biosciences
Innrain 80-82, 6020 Innsbruck, Austria
E-mail: ronald.micura@uibk.ac.at
Figure 1. Structure of the hypermethylated cap-4 of trypanosoma mRNAs.
[b]
D. Reichert, Prof. Dr. A. Rentmeister
University of Münster
Department of Chemistry, Institute of Biochemistry
Wilhelm-Klemm-Str. 2, 48149 Münster, Germany
Significant structural diversity is encountered for mRNA 5’-
caps. The most common caps in eukaryotes are cap-0
(m7GpppN), which is typically for lower eukaryotes, cap-1
(m7GpppNm) and cap-2 (m7GpppNmNm), which are typically for
higher eukaryotes.[11] They differ in the complexity of their
Supporting information for this article is given via a link at the end of
the document.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
methylation pattern and are named after how many of the first
nucleosides have a 2’-O methyl group. Recent studies showed
that 2’-O methylations play a key role in cellular discrimination of
endogenous from pathogenic RNA.[11] While various
modifications of these caps have been discovered,[11] surprisingly
the most complex methylation pattern known in nature was found
in early eukaryotes,[17] more precisely, within the family of
trypanosomatidae from the order of trypanosomatida and the
class of Kinetoplastida.[18-22] The relevant cap4 shows seven
additional methylations in the first 4 nucleotides compared to cap-
0 (Figure 1).[17]
phosphitylated with CEP-Cl in the presence of DIPEA. Starting
from nucleoside 4, our route provides phosphoramidite 9 with
46% overall yield in five steps involving five chromatographic
purifications; in total, 0.75 g of compound 9 was prepared during
the course of this study.
The study of essential biological functions mediated by
these unique structural features of the cap-4 and thereby of the
metabolism of an important class of human pathogenic parasites
is hindered by the lack of reliable preparation methods. To the
best of our knowledge, only the chemical synthesis of the terminal
four nucleotide fragment of cap-4 has been published thus far.
This was accomplished by obtaining the tetranucleotide fragment
(5'-p-(m6 Am)(Am)(Cm)(m3Um) first via the phosphoramidite
2
solid-phase method. The 5'-phosphorylated tetranucleotide was
then chemically coupled with m7GDP to yield the cap-4
structure.[23] Here, we set out to develop a practical synthetic route
towards T. cruzi trans-spliced leader (SL) RNA of a specific 39-nt
sequence that harbors the unique hypermethylated cap-4
Scheme 1. Synthesis of m3Um phosphoramidite for RNA solid-phase synthesis.
structure.[24-27] We obtained this challenging target by
a
combination of chemical and enzymatic methods, and provide a
practical protocol for cap-4 RNAs of any sequence of up to 40 nt
to the research community.
Results and Discussion
Synthesis of m3Um and m6Am building blocks. The
functionalization of commercially available 2'-O methyl uridine 1
into the desired m3Um phosphoramidite 3 involved three reactions,
which are summarized in Scheme 1. Our route began with
alkylation of N3 under basic condition using iodomethane. After
concentration to a dry solid, the crude product was directly
transformed to the dimethoxytritylated compound 2, using 4,4'-
dimethoxytriphenylmethyl chloride (DMTCl) in pyridine. Finally,
phosphitylation
was
executed
with
2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (CEP-Cl) in the presence of
N,N-diisopropylethylamine (DIPEA). Starting from nucleoside 1,
our route provides phosphoramidite 3 with 45% overall yield in
three
transformations
involving
two
chromatographic
purifications; in total, 0.85 g of compound 3 was prepared during
the course of this study.
Scheme 2. Synthesis of m6Am phosphoramidite for RNA solid-phase synthesis.
The functionalization of commercially available 2'-O methyl
adenosine 4 into the desired m6Am phosphoramidite 9 involved
six reactions, which are summarized in Scheme 2. Our route
began with selective acetylation of the ribose hydroxyl groups
using acetic anhydride. Then, treatment of compound 5 with N,N'-
Synthesis of cap-4 RNA. With the hypermethylated nucleoside
phosphoramidites in hands, we continued with a synthetic
strategy that was originally introduced by Debart and Decroly.[29b]
This approach relies on RNA assembly and introduction of the cap
on the solid support, enabling excess reagents to be removed by
simple washing which makes the synthesis straightforward and
convenient. However, instead of relying on RNA solid-phase
synthesis with the base-labile 2'-O-pivaloyloxymethyl (PivOM)–
bis[(dimethylamino)methylene]-hydrazine
(BDMAMH)
dihy-
drochloride[28] in pyridine gave 9-(2'-methyl-ß-D-ribofuranosyl)-6-
(1,2,4-triazol-4-yl)purine 6, which was converted quantitatively
into N,N,2'-O-trimethyl adenosine 7 with ethanolic dimethylamine.
Compound 7 was tritylated to give compound 8, and finally
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
protected
nucleoside phosphoramidites, we optimized our
converting the free 5'-OH and otherwise protected oligonucleotide
into its 5'-H-phosphonate derivative. This derivative was then
activated as phosphoroimidazolide by amidative oxidation. Then,
coupling of guanosine diphosphate (GDP) as tributylammonium
salt on the RNA bound to the solid support, was followed by
deprotection of acyl groups and release from the solid support,
using methylamine in water/methanol (AMA). Subsequently, silyl
deprotection was achieved with tetrabutylammonium fluoride in
THF, resulted in the desired Gppp-RNA in solution.
protocol for the implementation of standard 2'-O-tert-
butyldimethylsilyl
[(triisopropylsilyl)oxy]methyl (TOM) phosphoramidites (Figure 2).
Attachment of the GDP was derived from earlier protocols for the
synthesis of 5'-triphosphate DNA and RNA on solid support,[30] by
(TBDMS)
and/or
2'-O-
The N7-methyl group was efficiently enzymatically added by
means of the methyltransferase Ecm1 (Encephalitozoon cuniculi
mRNA cap (guanine N7) methyltransferase) following a protocol
previously established by us (Figure 3).[31,32] The enzymes 5′-
methylthioadenosine/S-adenosylhomocysteine
nucleosidase
(MTAN) and S-ribosyl homocysteine lyase (LuxS) were added to
the reaction mixture to enzymatically degrade the by-product S-
adenosylhomocysteine, which is
methyltransferases.
a
strong inhibitor of
Figure 2. Preparation of short cap-4 RNAs on solid phase. a. Schematics of the
individual steps involved. b. Reaction control of the individual steps based on
small portions of RNA assembled on solid-support (I to IV) that were withdrawn
and individually deprotected and analyzed by anion exchange chromatography
(Dionex DNAPac PA-100 (4 x 250 mm) column; temperature: 40 °C; flow rate:
1 mL/min; eluant A: 25 mM Tris.HCl (pH 8.0), 6 M urea; eluant B: 25 mM
Tris.HCl (pH 8.0), 6 M urea, 500 mM NaClO4; gradient: 0–60% B in A within 45
min; UV detection at 260 nm.
Figure 3. Enzymatic methylation of cap-4 RNA using Ecm1 methyltransferase.
a. RNA sequences and reaction scheme; b. Anion exchange HPLC analysis of
a typical methylation reaction (start and after 45 min, for reaction conditions see
experimental section), inset shows methylated product after purification; c. LC-
ESI mass spectrum of Gppp-RNA 10 and purified m7GpppRNA 11.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
Finally, the obtained cap-4 RNAs was readily ligated using
using
a
short m7G-ppp-(m6 Am)(Am)(Cm)(m3Um) synthetic
2
T4 DNA ligase and DNA or 2'-O-methyl RNA splints. Figure 4
illustrates a typical enzymatic ligation of a 39 nt T. cruzi cap-4
spliced leader RNA, using a 1.2–fold excess of the unmodified
RNA fragment and 1.2–fold excess of the splint (Figure 4).
Ligation yields were about 65% and the product was purified by
anion exchange chromatography. Mass spectrometric analysis
using LC-electrospray ionization confirmed the integrity of the
cap-4 RNA product.
oligonucleotide.[33] With the here presented access to larger cap-
4 RNAs, structure elucidation of ribosomal complexes bound to
cap-4 RNAs appears accomplishable in the near future.
Experimental Section
5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-3-N-methyluridine (2). 2'-O-
Methyluridine (1) (0.2 g, 0.77 mmol) was dissolved in dry dimethyl
sulfoxide (2 ml). Sodium hydride (18.6 mg, 0.77 mmol) was added to the
reaction mixture and stirred at room temperature under argon atmosphere
until bubbling stopped. Then, methyl iodide (0.05 ml, 0.77 mmol) was
added dropwise. The solution was stirred for another 5 hours. All volatiles
were evaporated under high vacuum. The residue was coevaporated once
with methanol and twice with pyridine before it was solved in anhydrous
pyridine (1 ml). Dimethoxytrityl chloride (0.32 g, 0.93 mmol) and 4-
dimethylaminopyridine (14.2 mg, 0.12 mmol) were added. The reaction
solution was stirred under argon at room temperature over night before the
reaction was quenched by adding methanol. All volatiles were evaporated,
the residue was diluted in methylene chloride and was washed three times
with a solution of 5% citric acid, once with a saturated sodium bicarbonate
solution and once with a saturated sodium chloride solution. The product
was isolated after column chromatography (100:0.1 to 100:0.5 methylene
chloride:methanol). Yield: 0.24 g (54%) of 2 as white foam. HR-ESI-MS
(m/z): [M+Na]+ calcd. 597.2207; found: 597.2219; 1H-NMR (300 MHz,
DMSO-d6): δ 3.15 (s, 3H,H3C-N); 3.24-3.36 (m, 2H, Ha,b-C(5’)); 3.44 (s,
3H, H3C-O(2’)); 3.75 (s, 6H, 2x H3C-O(DMT)); 3.80-3.58 (m, 1H, H-C(2’));
3.94-4.00 (m, 1H, H-C(4’)); 4.19-4.255 (dd, 1H, J=12 Hz, J=7,0 Hz, H-
C(3’)) ; 5.22 (d, 1H, J=5.1 Hz, H-O(3’)); 5.39 (d, 1H, J=8.1 Hz, H-C(5’));
5.84 (d, 1H, J=2,7 Hz, H-C(1’)); 6.90 (d, 4H, Har-C(DMT)); 7.21-737(m, 9H,
Har-C (DMT)); 7.80 (d, 1H, J=8.1 Hz, H-C(6)). 13C-NMR (75 MHz, DMSO-
d6): δ 27.3 (1C, H3C-N); 55.0 (1C, H3C-O (DMT)); 58.0 (1C, H3C-O(2’));
62.3 (C(5’)); 68.4 (1C, C(2’)); 82.4 (1C, C(4’)); 82.8 (1C, C(3’)); 88.7 (1C,
C(1’)); 100.5 (1C, C(5)); 113.4 (4C, Car(DMT)); 127.0 (1C, Car(DMT));
127.61 (2C, Car(DMT)); 127.80 (2C, Car(DMT)); 129.9 (4C, Car); 138.4 (1C,
C(6)).
5'-O-Dimethoxytrityl-2'-O-methyl-3-N-methyluridine-3'-(2-cyanoethyl-
diisopropylphosporamidite) (3). Compound 2 (0.24 g, 0.41 mmol) was
dried under high vacuum and vented with argon. The solid was dissolved
in dry methylene chloride (6 ml) and treated with N,N-
diisopropylethylamine (0.28 ml, 1.66 mmol) and 2-cyanoethyl-N,N-
diisopropyl-chlorophosphoramidite (0.20 g, 0.83 mmol). The reaction
solution was stirred for 4 hours at room temperature. Afterwards, the
reaction was quenched by adding 1 ml of methanol. It then was diluted
with methylene chloride (1:10), washed with saturated sodium bicarbonate
solution and with a saturated sodium chloride solution. The product was
isolated by column chromatography (1% triethylamine, 35% ethylacetate
und 64% n-hexane). Yield: 0.27 g (84%) of 3 as white foam. HR-ESI-MS
(m/z): [M+H]+ calcd. 775.3466; found: 775.3472. 1H-NMR (700 MHz,
CDCl3): δ 0.96 (d, 3H, J=6.8 Hz, CH-NCH3); 1.08-1.12 (m, 9H, 3.15 CH-
NCH3); 2.33 (t, 1H, J=6.38 Hz, J=7.08 Hz, NC-CH); 2.57 (m, 1H, J=6.35
Figure 4. Enzymatic ligation of T. cruzi cap-4 spliced leader RNA using T4 DNA
ligase. a. RNA sequences and sequence of the 20 nt DNA splint; b. HPLC
analysis of a typical ligation reaction after 3h reaction time; reaction conditions:
10 μM RNA 10, 12,5 μM RNA 11, 12,5 μM splint; 0.5 mM ATP, 40 mM Tris-HCl
(pH 7.8), 10 mM MgCl2, 10 mM DTT, 5% (w/v) PEG 4000, 0.5 U/μL T4 DNA
ligase; c. LC-ESI mass spectrum of the purified 39 nt cap-4 RNA ligation product.
Conclusions
We developed a practical route towards hypermethylated cap-4
RNAs. This included the 3- and 5-step syntheses of m3Um and
m6 Am phosphoramidites. Assembly of the G-5'-ppp-5'-RNA was
2
entirely conducted on solid-phase along the lines of a previously
established protocol, however, with the difference of using 2'-O
silyl protected building blocks which required adaption of the RNA
deprotection steps. The final N7 methylation was performed
enzymatically using Ecm1 methyltransferase. We exemplified the
chemo-enzymatic approach by the synthesis of a 39 nt T. cruzi
trans-spliced leader RNA that awaits applications in structural
biology studies. Very recently, first insights by X-ray
crystallography into how cap-4 is recognized by a Trypanosoma
cruzi eIF4E5 translation factor homologue has become available
Hz, J=7.08 Hz, NC-CH); 3.25 (s, 3H, H3C-N); 3.35-3.6 (m, 4H, H-C(5’) (a,b)
,
NCH-(CH3)2, PO-CH); a: 3.53 (s,s, 3H, H3C-O(2‘)); 3.73 (s,s, 6H, 2x H3C-
O(DMT)) 3.68-3.71 (m, 2H, PO-CH, H-C(2’)); 4.15-4.19 (m, 1H, H-C(4’));
4.36-4.42 (m, 1H, H-C(3’)); 4.51-4.56 (m, 1H, H-C(3’)), 5.21-5.26 (dd, 1H,
J=8.02, J=8.30 Hz, H-C(5’)), 5.93-5.97 (d,d, 1H, J=1.7 Hz, 2.7 Hz, H-C(1’));
6.74-6.79 (m, 4H, H-Car(DMT)); 7.16-7.35 (m, 9H, H-Car(DMT)); 7.88-7.99
(d, d, 1H, J=8.07, J=8.07 Hz, H-C(6)). 31P-NMR (162 MHz, CDCl3, 25 °C):
δ 150.17; 150.73 ppm.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
3',5'-Diacetyl-2'-O-methyladenosine (5). 2'-O-Methyladenosine (4) (0.50
g, 1.78 mmol) was coevaporated with pyridine, before dimethylformamide
(1.4 ml), dry pyridine (0.7 ml), acetic anhydride (0.7 ml, 7.41 mol) and 4-
dimethylaminopyridine (8 mg, 0.065 mmol) were added. The reaction
solution was stirred at 0°C for 3 hours. The reaction progress was
controlled by TLC (5 % methanol in methylene chloride). Methanol (0.5 ml)
was added to quench the reaction. Then all volatiles were removed by
distillation under high vacuum (70 °C) before the residue was
coevaporated once with pyridine and three times with toluene. This crude
product was directly used for the next reaction. For analytical analysis the
product was further purified by column chromatography (1% methanol in
methylene chloride). Yield: 0.64 g (98%) of 5 as white solid. HR-ESI-MS
(m/z): [M+H]+ calcd. 366.1408; found: 366.1414. 1H-NMR (300 MHz,
DMSO-d6): δ 2.04, 2.14 (ss, 6H, O(5’)CO-CH3, O(3’)CO-CH3)); 3.28 (s,
3H, H3C-O(2’)); 4.21-4.38 (m, 3H, C(4’)-H, Ha,b-C(5’)); 4.88 (dd(t), 1H,
J=6Hz, J=5.7 Hz, H-C(2’)); 5.49-5.51 (dd, 1H, J=2.4 Hz, J=4.8 Hz, H-
C(3’)); 6.03 (d, 1H, J=6 Hz, H-C(1‘)); 7.34 (s (broadened), 2H, NH2); 8.16
(s, 1H, H-C(8) or H-C(2)); 8.38 (s, 1H, H-C(2) or H-C(8)). 13C-NMR (75
MHz,CDCl3): δ 20.9, 21.0 (2C, O(5’)-CO-CH3, O(3’)-CO-CH3); 59.5 (1C,
C(5’)); 63.2 (1C, C(4’)); 80.3 (1H, C(2’)); 81.2 (1H, C(3’)); 87.7 (1C, C(1’));
139.5 (1C, C(8) or C(2)); 153.5 (1C, C(2) or C(8)).
85.6 (1C, C(1’)); 86.3 (1C, C(4’)); 138.3 (1C, C(2) or C(8)); 151.9 (1C, C(8)
or C(2)).
5'-O-Dimethoxytrityl-6-(N,N-dimethyl)-2'-O-methyladenosine
(8).
Compound 7 (0.24 g, 0.77 mmol) together with 4-(dimethylamino)-pyridine
(14 mg, 0.12 mmol) were coevaporated with pyridine. The residue was
diluted in dry pyridine (8 ml). 4,4’-O-dimethoxytrityl chloride (0.32 g, 0.92
mmol) was dried for 2 hours on high vacuum before it was added to the
reaction solution and was stirred overnight at room temperature together
with molecular sieves under argon atmosphere. The reaction was
quenched with methanol (0.5 ml) before all volatiles were removed under
reduced pressure. The residue was coevaporated three times with toluene.
Afterwards the product was purified by column chromatography (silica, 0-
3% methanol in methylene chloride). Another column chromatography (12-
25% acetone in toluene) can be necessary to further improve purity. Yield:
0.382 g (81%) of 8 as white foam. HR-ESI-MS (m/z): [M+H]+ calcd.
612.2817; found: 612.2822. 1H-NMR (300 MHz, DMSO-d6): δ (s, 3H,
C(4’)-H, C(5’)-Ha,b); 3.31-3.59 (s broadened, 6H, H3C-N) 3.73 (s, 6H, 2x
H3C-O-(DMT)); 4.06 (m(s), 1H, H-C(4’)); 4.41 ((s), 2H, H-C(3’), H-C(2’));
5.25 (s, 1H, H-O(3’)); 6.06 (s, 1H, H-C(1‘)); 6.78-6.88 (m, 4H, HCar(DMT));
7.16-7.40 (m, 9H, Char(DMT)); 8.18 (s, 1H, H-C(8) or H-C(2)); 8.26 (s, 1H,
H-C(2) or H-C(8)). 13C-NMR (75 MHz, DMSO-d6): δ 38.8 (1C, N-CH3);
55.5 (2C, O-CH3(DMT));59.3 (1C, H3C-O(2’)); 63.4 (1C, C(5’)); 70.1 (1H,
C(3’)); 83.5 (1H, C(2’)); 83.9 (1H, C(4’)); 86.6 (1C, C(1’)); 113.4 (4C,
Car(DMT)); 127.0 (1C, Car(DMT)); 128.1 (2C, Car(DMT)); 128.4 (2C,
Car(DMT)); 130.4 (4C, Car(DMT)); 136.0 (1C, C(2) or C(8)); 152.8 (1C,
C(8) or C(2)).
9-(3’,5’-Diacetyl-2’-O-methylfuranosyl)-6-(1,2,4-triazol-4-yl)purine (6).
Compound 5 (0.54 g, 1.48 mmol) was coevaporated with pyridine and
treated with N,N-bis[(dimethylamino)methylene]hydrazine dihydrochloride
(5.16 g, 2.15 mmol; prepared as described below following ref. [28]) which
was previously dried over phosphorpentoxide under high vacuum for 2
hours. This solid was diluted in dry pyridine (8 ml) and stirred under argon
atmosphere in the dark at 85°C for 48 hours. Then all volatiles were
removed under vacuum and the residue was coevaporated twice with
toluene. The residue was filtrated and washed with methylene chloride.
The solid N,N-dimethylformamide-azine dihydrochloride can be recycled.
The filtrate was washed with a solution of 5% citric acid, a saturated
solution of sodium bicarbonate and a saturated solution of sodium chloride.
The organic fractions were united and the volatiles were removed under
vacuum. The residue was then purified by column chromatography (silica,
0.5 to 1.5% methanol in methylene chloride). Yield: 0.47 g (76 %) of 6 as
white foam. HR-ESI-MS (m/z): [M+H]+ calcd. 418.1470; found: 418.1475.
1H-NMR (300 MHz, DMSO-d6): δ 2.05, 2.16 (s, 3H, H3C-CO-O(5’), H3C-
CO-OO(3’)); 3.31 (s, 3H, H3C-O(2’)); 4.28-4.44 (m, 3H, H-C(4’), Ha,b-C(5’));
4.92 (dd(t), 1H, J=6Hz, J=5.4 Hz, H-C(2’)); 5.53-5.56 (dd, 1H, J=3.9 Hz,
J=4.8 Hz, H-C(3’)); 6.24 (d, 1H, J=6 Hz, H-C(1‘)); 7.34 (s broaden, 2H,
NH2); 8.96 (s, 1H, H-C(8) or H-C(2)); 9.03(s, 1H, H-C(2) or H-C(8)); 9.63(s,
2H, N=CH-N). 13C-NMR (75 MHz, DMSO-d6): δ 21.16, 21.19 (2C, O(5’)-
CO-CH3, O(3’)-CO-CH3); 59.0 (1C, H3C-O(2’)); 63.7 (1C, C(5’)); 71.3 (1H,
C(3’)); 80.4 (1H, C(2’)); 80.8(1C, C(4’));86.8 (1C, C(1’)); 141.9 (2C, N=CH-
N); 146.8 (1C, C(2) or C(8)); 152.8 (1C, C(8) or C(2)).
5'-O-Dimethoxytrityl-6-(N,N-dimethyl)-2'-O-methyladenosine-3'-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite) (9). Compound 8 (0.54
g, 0.88 mmol) was dried under high vacuum for several hours and
ventilated with argon. The solid was dissolved in dry methylene chloride
(10 ml), and treated with N,N-diisopropylethylamine (0.61 ml, 3.53 mmol)
and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.42 g, 1.76
mmol). The reaction solution was stirred under argon atmosphere for 3.5
hours at room temperature. Then methanol (2 ml) was added and the
solution was stirred for another 20 minutes. The solution was diluted with
methylene chloride (1:10) and washed with saturated sodium bicarbonate
solution and saturated sodium chloride solution. The product was isolated
by column chromatography (1% triethylamine, 35% ethylacetate und 64%
n-hexane). Yield: 551 mg (77%) of white foam. HR-ESI-MS (m/z): [M+H]+
calcd. 812.3895; found: 812.3901. 1H-NMR (600 MHz, CDCl3): δ 1.06-1.07
(d, 3H,J=6.6 Hz, N-CH-(CH3)2); 1.19-1.22 (m, 9H, N-CH-(CH3)2); 2.38-2.40
(tt(t), 1H, H-C-CN); 2.65-2.68 (tt(t), 1H, H-C-CN); 3.31-3.35 (m, H-C(5’));
3.48-3.69 (m, 12H, N-CH; H2-C(5’), H3C-O(2’), N-(CH3)2, N-CH, P-O-CH2);
3.80-3.79 (ss(d), O-CH3(DMT)); 3.87-3.98 (mP-O-CH2-); 4.33-4.39 (m, 1H,
H-C(4’)); 4.55-4.67 (m, 2H, H-C(2’)), H-C(3’)); 6.13-6.15 (m, 1H, H-C(1‘));
6.80-6.84 (m, 4H, CH(ar, DMT)); 7.20-7.35 (m, 7H, HC(ar, DMT)); 7.44-
7.47 (m, 2H, HC(ar, DMT)); 7.92-7.97 (ss, 1H, H-C(8) or H-C(2)); 8.30-
8.28 (ss, 1H, H-C(2) or H-C(8)). 31P-NMR (162 MHz, CDCl3, 25 °C): δ
150.21; 150.91 ppm.
6-(N,N-Dimethyl)-2'-O-methyladenosine (7). Compound 6 (0.47 g, 1.12
mmol) was suspended in 33% dimethylamine solution in ethanol (3 ml) and
stirred under argon atmosphere at room temperature for 48h. During that
time, the white suspension turned into a clear solution. All volatiles were
removed under vacuum. Further processing was done without further
purification. For analytical reasons the crude product was purified by
column chromatography (silica, 1-3% methanol in methylene chloride).
Yield: 0.34 g (98%) of 7 as white foam. HR-ESI-MS (m/z): [M+H]+ calcd.
310.1510; found: 310.1515. 1H-NMR (300 MHz, DMSO-d6): δ 3.31 (s, 3H,
H3C-O(2’)); 3.46 (s (broadened), 6H, (CH3)2-N); 3.53-3.71 (m, 3H, H-C(4’),
Ha,b-C(5’)); 3.98 (s, 1H, O(3’)); 4.33-4.34 (ss(d), 1H, H-C(2’), H-O(5’)) ; 5.24
(d, 1H, J=4.2 Hz, H-C(3’)); 5.33-537 (dd(t), 1H, J=4.8 Hz, J=5.0 Hz, H-
C(4’)); 6.03 (d, 1H, J=4,2 Hz, H-C(1‘)); 8.22 (s, 1H, H-C(8) or H-C(2)); 8.40
(s, 1H, H-C(2) or H-C(8)). 13C-NMR (75 MHz, DMSO-d6): δ 37.8 (1C, H3C-
N); 57.4 (1C, H3C-O(2’)); 61.3 (1C, C(5’)); 68.7 (1H, C(3’)); 82.4 (1H, C(2’));
N,N-bis[(Dimethylamino)methylene]hydrazine dihydrochloride.[28] To
dry DMF (7 ml), thionyl chloride (1.4 ml, 19.3 mmol) was added dropwise
under cooling with ice water. This transparent, slightly yellow solution was
stirred for 20 hours under argon atmosphere. A solution of hydrazine
monohydrate (0.24 ml, 1 eq, 5.0 mmol) in dry DMF (7 ml) was added in
drops and under cooling. This slightly yellow suspension was stirred for
another 24 hours under argon atmosphere. After the filtration the filter cake
was washed with 10 ml DMF and 15 ml ethyl acetate and dried under high
vacuum. Yield: 1.94 g (99%) of white powder. 1H-NMR (300MHz, DMSO):
δ 3.00 (s, 12H, 4x NCH3); 8.36 (s, 2H, 2 x N=CH-N) ppm.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
Guanosine 5’-diphosphate di-tributylammonium salt. DOWEX-50WX8
200-400 resin (H+ form) was filtered with nanopure water using a glass frit
(pore size 16-40 µm) to remove small particles. Afterwards, guanosine 5’-
diphosphate disodium salt (0.5 g, 1 mmol) was dissolved in water (4 ml)
and eluted through a column (1,5 cm) packed with 2 cm of wet DOWEX-
50WX8 200-400 for 10 minutes. After filtration into a flask with 25 ml
ethanol at 0°C, tributylamine (0.5 ml, 2 mmol) was added. The solution
was stirred while the column was rinsed with water until pH was about 7-8
before all volatiles were removed under high vacuum. To preserve a dry
powder further coevaporations (3 times with ethanol and 2 times with
dioxane) were performed. The resulting white solid was directly used
suspensions were filtered and the filtrates dried. Removal of 2'-O silyl
protecting groups was carried out with of 1 M tetrabutylammonium fluoride
trihydrate in tetrahydrofuran (1 ml) for 6 hours at 40°C. The reaction was
quenched by the addition of 1 M triethylammonium acetate solution (pH
7.4, 1 ml) and then concentrated to approximately 1 ml. This viscous
solution was desalted by size exclusion chromatography on a HiPrep
26/10 Sephadex G25 column (GE Healthcare). The crude products were
evaporated and redissolved in water (1 ml). Quality assessment of the
crude Gppp-capped RNAs via anion-exchange HPLC on a Dionex
DNAPac PA-100 column (4 x 250 mm); conditions: flow 1 mL min-1; eluent
A: 25 mM Tris hydrochloride, 6 M urea, pH 8.0, eluent B: 500 mM sodium
perchlorate, 25 mM Tris hydrochloride, 6 M urea, pH 8.0; gradient: 0 - 60%
B in 45 minutes; temperature: 40 °C, UV detection at 260 nm. The Gppp-
capped RNAs were isolated via anion-exchange HPLC on a Dionex
DNAPac PA-100 column (4 x 250 mm); Conditions: flow 1 mL min-1;
eluents: see above; temperature 40°C, UV detection at 260 nm. The
product fractions were diluted with an equal amount of triethylammonium
bicarbonate buffer (100 mM, pH 7.4) and loaded onto equilibrated C18
Sep-Pak (Waters Corporation). The cartridge was washed with water and
the RNA was eluted with acetonitrile/water 1/1. All volatiles were
evaporated and the residue redissolved in water (1 mL). Yields were
determined UV-photometrically. The quality of the product was analyzed
via anion-exchange HPLC as described above and via reversed-phase
LC-ESI-MS.
1
without further purifications. Yield: 0.70 g (86%) of a white solid. H-NMR
(400 MHz, DMSO-d6): δ 0.89 (t, 9H, J = 7.4 Hz, CH3); 1.29 (m, 6H, CH2-
CH3); 1.55 (m, 6H, N-CH2-CH2); 2.86 (s (broadened), 6H, J= 7.6 Hz, N-
CH2); 3.92-4.05 (m, 3H, H-C(4’), H2-C(5’)); 4.27 (t, 1H, J = 4 Hz, H-C(3’);
4.47 (t, 1H, J = 4.95 Hz, H-C(2’)); 5.68 (d, 1H, J= 5.3 Hz, H-C(1’)) 6.62 (s
(broadened), NH2); 7.89 (s, 1H, H-C(8); 10.6 (s (broadened), NH). 31P-
NMR (162 MHz, DMSO, 25 °C): δ –10.20 (d), –10.85 (d) ppm.
RNA solid-phase synthesis. All RNAs were assembled on an ABI392
synthesizer using 2'-O-TOM nucleoside phosphoramidites (ChemGenes
Corporation) and CPG supports (2'-tBDSilyl Guanosine (n-PAC) 3'-lcaa
CPG 1000Å, 2'-tBDSilyl Adenosine (n-PAC) 3'-lcaa CPG 1000Å, ~40
µmol/g Chem Genes corporation). Standard RNA synthesis cycle:
detritylation (120 s) with dichloroacetic acid/1,2-dichloroethane (4/96);
coupling (2.0 min) with phosphoramidites/acetonitrile (0.1 M)
andbenzylthiotetrazole/acetonitrile (0.3 M); capping (2 x 0.25 min, Cap
A/Cap B= 1/1) with Cap A: 4-(dimethylamino)pyridine in acetonitrile (0.5
M) and Cap B: acetic anhydride/sym-collidine/acetonitrile (2/3/5); oxidation
(1.0 min) with iodine (20 mM) in tetrahydrofuran (THF)/pyridine/H2O
(35/10/5). Commercially available modified nucleoside building blocks: 2'-
O-methyl adenosine and 2'-O-methyl cytidine (ChemGenes Corporation),
were incorporated using modified synthesis cycles with longer coupling
times (six minutes).
Enzymatic N7 methylation of Gppp-RNA. The enzymatic transformation
was conducted in analogy to references [31] and [32]. In short, lyophilized
Gppp-RNA 10 (4 nmol) was dissolved in buffer (8.0 µl, 1.5 M NaCl, 200
mM Na2HPO4, pH 7.4) followed by the addition of an aqueous solution of
S-adenosylmethionine (2.0 µl, 12 nmol), and the addition of water to obtain
a total volume of 68.8 µl. To the mixed solution, enzymes were added
successively (1.6 µl of 50 µM MTAN, 1.6 µl of 50 µM LuxS, 8 µl of 50 µM
Ecm1 (Supporting Figures S1 and S2)). The mixture was incubated for 45
min at 37 °C. The solution was extracted twice with an equal volume of
chloroform/isoamyl alcohol solution (24/1, v/v). The organic layers were
rewashed twice with an equal volume of water. The aqueous layers were
combined and lyophilized, to eliminate remaining organic solvents.
Analysis of the methylation reaction and purification of the product was
performed by anion exchange chromatography (conditions see above).
The integrity of the product was confirmed by LC-ESI mass spectrometry
(conditions see below).
RNA phosphitylation, hydrolysis, oxidative amidation was performed
in analogy to reference [29a], while Gpp attachment was performed in
analogy in reference [29b]. The construction of the triphosphate bridged
inverted guanosine to the RNA on the solid phase involved four steps, all
of them performed under argon atmosphere. RNA phosphitylation was
carried out by applying 0.5 ml of phosphitylation solution (2 ml
diphenylphosphite, 8 ml anhydrous pyridine) over 300 seconds. This
treatment was repeated twice. After washing the beads with acetonitrile,
hydrolysis was performed using 0.5 ml of hydrolysis solution (1.0 mL of 1
Enzymatic ligation of cap-4 RNA. The 38 nt T.cruzi cap-4 RNA was
prepared by splinted enzymatic ligation of a 11 nt cap-4 RNA and a
chemically synthesized 5'-phosphorylated 27 nt RNA using T4 DNA ligase
(Thermo Fisher) in analogy to reference [34]. Briefly, 10 μM of RNA
fragment 10 (8 nmol), 12,5 μM of RNA fragment 11 (10 nmol), and 12,5
μM of a 20 nt DNA splint oligonucleotide (IDT, 10 nmol) were heated at
70 °C for 2 min and passively cooled to room temperature. Afterwards
water was added up to a total volume of 560 μl before 10× ligation buffer
(80 μl, Thermo Fisher) and PEG (80 μl, Thermo Fisher) was added. T4
DNA ligase (80 μl; Thermo Fisher, 5 U/μl) was added to a final
concentration of 0.5 U/μl in a total volume of 0.8 mL before the mixtre was
incubated for 2 h at 37°C. Ligation was stopped by chloroform/ isoamyl
alcohol (24/1, v/v) extraction. After lyophilisation and redissolving the
analysis of the ligation reaction and purification of the ligation products
were performed by anion exchange chromatography (conditions see
above). The integrity of product was confirmed by LC-ESI mass
spectrometry (conditions see below).
M
aqueous triethylammonium bicarbonate, 5 ml water and 4 ml
acetonitrile) over a period of 5 minutes. This treatment was repeated four
times. The solid support was then rinsed with 20 ml of acetonitrile before
being dried under vaccuum for several hours. Oxidative amidation was
done by incubation with 0.5 ml of a solution containing 600 mg imidazole,
2 ml N,O-bis(trimethylsilyl)acetamide, 4 ml anhydrous acetonitrile, 4 ml
bromotrichloromethane and 0.4 ml trimethylamine for 30 minutes. This
treatment was repeated four times. The solid support was intensively
rinsed with dry acetonitrile. Finally, Gpp attachment was achieved by
application of 0.5 ml of coupling solution (0.28 M guanosine 5’-diphosphate
di-tributylammonium salt [preparation see above] in dry DMF, 500 mM zinc
chloride) over a time period of 8 hours at room temperature. This treatment
was repeated three times.
Gppp-capped RNA deprotection. The solid supports with the Gppp-
capped and otherwise fully protected oligos were rinsed with DBU in
acetonitrile (6 ml; 1 M), then washed with acetonitrile and dried. Cleavage
from the support and base deprotection was effected by treating the solid
support with a 1/1 mixture of 40% aqueous methylamine and 30% aqueous
ammonia (AMA) in a screw-cap vial for 2 hours at 40 °C. The resulting
Mass spectrometry of cap-4 RNA. All experiments were performed on a
Finnigan LCQ Advantage MAX ion trap instrument connected to a Thermo
Fisher Ultimate 3000 system. RNA sequences were analyzed in negative-
ion mode with a potential of −4 kV applied to the spray needle. LC: sample
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
(200 pmol RNA dissolved in 30 μl of 20 mM EDTA solution; average
injection volume: 30 μl), column (Waters XTerraMS, C18 2.5 μm; 2.1 × 50
mm) at 21 °C; flow rate: 0.1 ml/min; eluent A: Et3N (8.6 mM), 1,1,1,3,3,3-
hexafluoroisopropanol (100 mM) in H2O (pH 8.0); eluent B: MeOH;
gradient: 0−100% B in A within 20 min; UV detection at 254 nm.
[14] M. Ziemniak, M. Strenkowska, J. Kowalska, J. Jemielity, Future Med.
Chem. 2013, 5, 1141–1172.
[15] Magadum, A., Kaur, K. & Zangi, L. Mol. Ther. 2019, 27, 785–793.
[16] D. Weissman, K. Karikó, Mol. Ther. 2015, 23, 1416–1417.
[17] J. R. Zamudio, et al. Eukaryotic Cell 2006, 5, 905–915.
[18] I. D’Orso, J. G. De Gaudenzi, A. C. C. Frasch, Trends Parasitol. 2003,
19, 151–155.
[19] M. Navarro, C. Gabbiani, L. Messori, D. Gambino, Drug Discov. Today
2010, 15, 1070–1078.
Acknowledgements
[21] C. Preußer, N. Jaé, A. Bindereif, A. Int. J. Med. Microbiol. 2010, 302,
221–224.
We thank Yaser Hashem (IECB Bordeaux) for discussion,
Maximilian Himmelstoß and Christoph Kreutz (University of
Innsbruck) for mass spectrometric and NMR spectroscopic
assistance, and Ann-Marie Lawrence Dörner (University of
Münster) for technical support. Funding from the Austrian Science
Fund FWF (P27947, P31691 to R.M.), the Austrian Research
Promotion Agency FFG (West-Austrian BioNMR 858017 to R.M.),
and the DFG (SFB858) to A.R., is gratefully acknowledged.
[22] G. Gilinger, V. Bellofatto, Nucleic Acids Res. 2001, 29, 1556–1564.
[23] M. Lewdorowicz, Y. Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R.
Kierzek, R. Stolarski, M. Shapira, E. Darzynkiewicz, RNA 2004, 10,
1469–1478.
[24] C. McCarthy-Burke, Z. A. Taylor, G.A. Buck, Gene 1989, 82, 177-189.
[25] A. Günzl, Eukaryot. Cell. 2010, 9, 1159–1170.
[26] J.R. Zamudio, B. Mittra, D. A. Campbell, N. R Sturm Mol. Microbiol. 2009,
72, 1100–1110.
[27] A.V. Ignatochkina, Y. Takagi, Y. Liu, K. Nagata, C. Kiong Ho, Proc. Nat.
Acad. Sci. USA 2015, 112, 6967–6972.
Keywords: mRNA caps • RNA modifications • solid-phase
[28] S. Neuner, R. Micura, Bioorg. Med. Chem. 2014, 22, 6989–6995.
[29] a) I. Zlatev, J. G. Lackey, L. Zhang, A. Dell, K. McRae, S. Shaikh, R. G.
Duncan, K. G. Rajeev, M. Manoharan M, Bioorg. Med. Chem. 2013, 21,
722–732. b) Y. Thillier, E. Decroly, F. Morvan, B. Canard, J.-J. Vasseur,
F. Debart, RNA 2012, 18, 856–868.
synthesis • oligonucleotides • chemoenzymatic synthesis
[1]
[2]
[3]
[4]
[5]
I. Edery, N. Sonenberg, Proc. Nat. Acad. Sci. USA 1985, 82, 7590–7594.
A. Köhler, E. Hurt, Nat. Rev. Mol. Cell Biol. 2007, 8, 761–773.
N. Sonenberg, A. G. Hinnebusch, Cell 2009, 136, 731–745.
Y. Furuichi, A. LaFiandra, A. J. Shatkin, Nature 1977, 266, 235–239.
S. Hocine, R. H. Singer, D. Grünwald, CHS Perspect. Biol. 2010, 2,
a000752.
[30] I. Zlatev, T. Lavergne, F. Debart, J.-J. Vasseur, M. Manoharan, Fr.
Morvan, Org. Lett. 2010, 12, 2190–2193.
[31] a) F. Muttach, N. Muthmann, D. Reichert, L. Anhauser and A.
Rentmeister, Chem. Sci. 2017, 8, 7947-7953; b) N. Muthmann, T. Guez,
J.-J. Vasseur, S. R. Jaffrey, F. Debart A. Rentmeister, ChemBioChem
2019, 20, 1693–1700.
[6]
[7]
A. Ghosh, C. D. Lima, Wiley Interdiscip. Rev. RNA 2010, 1, 152–172.
F. Muttach, N. Muthmann, A. Rentmeister, Beilstein J. Org. Chem. 2017,
13, 2819–2832.
[32] J. M. Holstein, L. Anhäuser, A. Rentmeister, Angew. Chem. Int. Ed. 2016,
55, 10899–10903.
[8]
[9]
J. D. Lewis, E. Izaurralde, A. Jarmolowski, C. McGuigan, I. W. Mattaj,
Genes Develop. 1996, 10, 1683–1698.
[33] L.W. Reolon, S. Vichier-Guerre, B. Moisés de Matos, L. Dugué, T.
Reichert da Silva Assunção, N. I. T. Zanchin, S. Pochet, B. Gomes
Guimarães, Nucleic Acids Res. 2019, 47, 5973–5987.
[34] K. Lang, R. Micura, Nat. Protoc. 2008, 3, 1457–1466.
E. Izaurralde, et al. Nature 1995, 376, 709–712.
[10] G. Varani, Structure 1997, 5, 855–858.
[11] M. Warminski, P. J. Sikorski, J. Kowalska, J. Jemielity, Topics Curr.
Chem. 2017, 375, 16.
[12] H. Liu, M. Kiledjian, Biochem. Soc. Trans. 2006, 34, 35–38.
[13] E. Grudzien-Nogalska, M. Kiledjian, Wiley Interdiscip. Rev. RNA 2017, 8,
e1379.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900590
ChemBioChem
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Highly methylated mRNA
caps are encountered in
trypanosoma. A solid phase–
based, chemoenzymatic
approach makes these RNAs
synthetically accessible.
Josef Leiter, Dennis
Reichert, Andrea
Rentmeister and Ronald
Micura*
Page No. – Page No.
Practical Synthesis of Cap-
4 RNA
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.