Synthesis of the first double-functionalized dinucleotide mRNA cap analogue for its specific labeling

Article abstract of DOI:10.1016/j.tetlet.2017.06.060

The modification of various important nucleotide-based molecules (such as nucleotides, RNA, DNA, oligonucleotides) with fluorophores, affinity tags and reactive moieties is of enormous utility for studying their localization, structure and dynamics, as well as diverse biological functions involving their interacting partners. Herein, we report chemical methodology in which the dinucleotide mRNA cap analogue is doubly modified within its second nucleotide. The prepared dinucleotide contains an alkyne at the N2-position of guanine, and levulinic acid within the ribose moiety. Such modifications may be further used for specific labeling of the cap, for instance with a fluorophore that will allow the molecule to be tracked inside the cell and an attachment cell-penetrating peptide that will help to deliver it to the area of interest. Exemplar molecules were attached in order to demonstrate the utility of the newly synthesized cap analogue.

Full text of DOI:10.1016/j.tetlet.2017.06.060

Accepted Manuscript
Synthesis of the first double-functionalized dinucleotide mRNA cap analogue
for its specific labeling
Karolina Piecyk, Paulina Krynska, Justyna Kaluzna, Marzena Jankowska-
Anyszka
PII:
S0040-4039(17)30791-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.06.060
TETL 49052
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 May 2017
12 June 2017
19 June 2017
Please cite this article as: Piecyk, K., Krynska, P., Kaluzna, J., Jankowska-Anyszka, M., Synthesis of the first double-
functionalized dinucleotide mRNA cap analogue for its specific labeling, Tetrahedron Letters (2017), doi: http://
dx.doi.org/10.1016/j.tetlet.2017.06.060
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Synthesis of the first double-functionalized dinucleotide mRNA cap analogue
for its specific labeling
Karolina Piecyk, Paulina Krynska, Justyna Kaluzna, Marzena Jankowska-Anyszka*
University of Warsaw, Faculty of Chemistry, Pasteura 1, 02-093, Warsaw, Poland
Abstract
The modification of various important nucleotide-based molecules (such as
nucleotides, RNA, DNA, oligonucleotides) with fluorophores, affinity tags and reactive
moieties is of enormous utility for studying their localization, structure and dynamics,
as well as diverse biological functions involving their interacting partners. Herein, we
report chemical methodology in which the dinucleotide mRNA cap analogue is doubly
modified within its second nucleotide. The prepared dinucleotide contains an alkyne
at the N2-position of guanine, and levulinic acid within the ribose moiety. Such
modifications may be further used for specific labeling of the cap, for instance with a
fluorophore that will allow the molecule to be tracked inside the cell and an
attachment cell-penetrating peptide that will help to deliver it to the area of interest.
Exemplar molecules were attached in order to demonstrate the utility of the newly
synthesized cap analogue.
Keywords: cap analogues, dinucleotide, 5’ mRNA cap, molecular probe, labelling
Introduction
Interest in the study of RNA, DNA and their fragments have grown steadily over the
previous decades.1 Chemical modifications of nucleotides are often indispensable,
not only for localization, structure and dynamics studies, but also for research on the
biomolecular interactions of parent compounds with protein partners.2 General
approaches to obtain nucleotides bearing diverse “reporters” at specific locations are
based on the selective insertion of functional groups that are responsible for the
characteristic chemical reactions of those molecules. For this reason, it is mandatory

that the functional groups have different chemical properties. Additionally, labels
should not influence the biological properties of the (oligo)nucleotide or nucleic acid,
in other words it is important that both are placed at the correct distance from each
other and are attached to the appropriate parts of the molecule.
Among widely studied nucleotide-like compounds are analogues of the 5’
end of mRNA, the cap, that in its basic form consists of a 7-methylguanosine
connected via a 5’,5’-triphosphate bridge with the second nucleotide (usually G).3 The
5′-end of RNA plays an essential role in several processes during gene expression
and serves as a signal for engaging proteins required for those processes.4 To date
numerous modified cap analogues have been tested and used in various biological
and biophysical studies to elucidate processes in which it is involved.5 Apart from
analogues with modifications that altered their biological properties,6 these studies
have employed derivatives containing additional single functionalization with an
attached probe or other specific molecule.7
Besides the undeniable role of cap analogues in explaining the molecular processes
associated with gene expression, these compounds have been intensively examined
as effective inhibitors of translation that could be used as anti-cancer drugs.8 The
idea of applying such nucleotide derivatives emerged with reports that numerous
cancer cells exhibit elevated level of eukaryotic initiation factor 4E (eIF4E).9 The
interaction of eIF4E with the mRNA cap structure is crucial and a rate-limiting step of
protein synthesis, and for that reason it can be exploited as a target for discovery.10
In this context, the need for covalent attachment to therapeutic molecules, e.g.
biophysical probes that will allow to follow them inside the cell as well as other
molecules that will increase, for example, their cell membrane permeability is
desirable. To obtain such molecules and to open up new research opportunities,
herein, we describe the first methodology to synthesize double functionalized
dinucleotide mRNA cap analogues. In addition, the work demonstrates the use of
such a functionalized cap analogue for the attachment of two compounds - a
fluorescent dye and a model peptide.
If a particular biologically active compound is used for biophysical and
biological studies, labels should be introduced in a manner that does not influence
the functionality of the compound. For this reason, in our case, it was highly important
to consider which parts of the 5’ end of mRNA are involved in protein interaction. The
cap structure binds to the eIF4E protein mainly by charge-related interactions inside

the cap-binding center, and are accomplished by: sandwich cation-π stacking of
methylated guanine with protein aromatic side-chains, hydrogen bonds between
nitrogen atoms N1 and/or N2 of the m7G base and acidic residues, and salt bridges
between the negatively charged phosphate chain of the cap and basic residues of
eIF4E.5d-g Knowing this, the ribose moiety of the second nucleoside and the second
base of the cap structure are the most favorable sites to conjugate with the two
different labels. To make this possible, the cap analogue was modified with functional
groups complementary to the functionality of the probes. The proposed strategy
included insertion of functional groups that were compatible with "click chemistry" and
the carbodiimide reaction of an activated carboxyl group with primary amines or
alcohols. Click chemistry was chosen as it is a widely used reaction for various
applications, due to the fact that it usually proceeds rapidly and selectively in a
o
neutral environment (pH 6-8) at ambient temperature (20-30 C).11 For the first
alkyne/azide functionalization, the exocyclic NH2 group present within the second
base of the cap was selected. This type of derivative (N2-modified guanosine) has
already been synthesized for modification of the first methylated nucleotide in the cap
structure and evaluated as potential translation inhibitors, whereas now this
methodology was used to modify the second non-methylated base in the dinucleotide
cap mRNA.12
The second modification to the carboxylic group was also introduced within the non-
methylated nucleotide, but in a site that does not cause congestion with the N2
position. Among different possibilities we proposed to functionalize the cap analogue
by derivatization of the 2’,3’–cis diol of the second ribose with levulinic acid. We
chose this functionalization since: a) the introduced reactive group is not present
within the natural compound, which provides high selectivity, b) the coupling reaction
can be successfully carried out under aqueous conditions, and c) such modifications
had been previously used for the preparation of ligands for affinity resins and
employed to isolate the cap specific eIF4E protein.13
The main difficulty in obtaining a double modified molecule is that it requires
attachment of two different fragments that can strongly influence the properties (e.g.
solubility) of the molecule which may lead to the need for alteration or adaptation of
procedures that were previously developed. Therefore, it should be considered which
probe is introduced first, and how it might affect the following step(s) of the synthesis.

The guanosine bearing alkyne modification was chosen to be performed first
according to a procedure that was previously reported by our group.12b This method
is based on the transformation of guanosine into its fully protected form, then
subsequent fluorination and substitution of the obtained derivative to give the N2
modified guanosine analogue (Scheme 1).
Scheme 1. Synthesis of double functionalize mononucleotide 4; Reagents and conditions: i)
Ac2O (3.1 equiv.), DMAP (0.1 equiv.), TEA (7.8 equiv.), MeCN, 0° C, 98%; ii) 2-(4-
nitrophenyl)ethanol (1.5 equiv.), PPh3 (1.5 equiv.), DIAD (1.2 equiv.), anhydrous toluene, 48
h, RT, 70%; iii) 70% HF in pyridine (235 equiv.), anhydrous pyridine, t-butyl nitrite (2.5
equiv.), -35 °C, 85%; iv) a) 2-propyn-1-amine (2 equiv.), DMSO; b) 33% dimethylamine
solution in absolute EtOH (0.05 equiv.), 70%; v) ethyl levulinate (16 equiv.), triethyl
orthoformate (2.4 equiv.), p-toluenesulfonic acid (0.6 equiv.), 5 h, RT, 97%; vi) a) POCl3 (2.5
equiv.), trimethyl phosphate (42 equiv.), 5 h, 4 °C; b) 1 M NaOH 35%.
Briefly, dried guanosine was subjected to triacetylation with acetic anhydride (Ac2O)
in the presence of triethylamine (TEA) and N,N-(dimethylamino)pyridine (DMAP),
followed by protection of the O6 group of guanosine with a p-nitrophenylethyl (NPE)
group using the Mitsunobu reaction as previously described.12 Obtained in this
manner, 2’,3’,5’-tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine was subjected to
diazotization and fluorination reactions under anhydrous conditions with t-butyl nitrite

and HF in pyridine. N2-fluoro-2’,3’,5’-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]inosine (1)
was then converted to alkyne derivative (2) by nucleophilic substitution with
commercially available 2-propyn-1-amine. In the next step the N2 modified guanosine
was treated with ethyl levulinate in the presence of p-toluenesulfonic acid and triethyl
orthoformate to afford 2’,3’-O-[1-[2-(ethoxycarbonyl)ethyl]ethylidene]guanosine with a
propargyl substituent at the N2 position (3) in high yield. Compound 3 was not
isolated from the reaction mixture but directly converted to derivative 4 by 5’
phosphorylation using phosphorus trichloride oxide (POCl3) in trimethyl phosphate at
o
4 C and alkaline hydrolysis. The resulting mononucleotide 4 was purified by ion-
exchange chromatography (DEAE-Sephadex). Notably, prior to ion-exchange
chromatography all steps did not require sophisticated purification methods, only
simple precipitation of the product, washing and drying.
The next step included coupling the functionalized mononucleotide with 7-
methyguanosine-5’-diphosphate (m7GDP). The most popular synthetic pathway
leading to formation of the 5’,5’-triphosphate bridge in the cap structure is based on a
strategy in which two mononucleotide units, one of which has been previously
converted to a reactive imidazolide derivative, are coupled (Scheme 2).14

Scheme 2. Synthesis of double functionalized dinucleotide cap analogue 6; Reagents and
conditions: i) ZnCl2 (10 equiv.), DMF, RT, overnight.
At this point we decided that most rational approach would be conversion of the
nucleoside 5‘-diphosphate into its imidazolide derivative (5) and coupling with the
double modified monophosphate (4). m7GDP was activated via reaction with
imidazole in the presence of 2,2’-dithiodipyridine and triphenylphosphine and used as
a sodium salt in the coupling reaction,7a which was carried out under anhydrous
conditions and led to the formation of compound 6 which was isolated by ion-
exchange chromatography.
To demonstrate the usefulness of the newly obtained cap analogue as a tool
for further functionalization, we decided to connect two chosen probes containing an
amine group that is suitable to react with the carboxyl group of levulinic acid –
fluorescence probe (NH2-dansyl) and an azide group that is appropriate for a “click”
reaction – short N3-peptide (N3-Ala-Ala-Ala-NH2) (Scheme 3).

Scheme 3. Synthesis of dinucleotide cap analogue containing exemplary fluorescence probe
and peptide; Reagents and conditions: i) N-dansylethylenediamine (2 equiv.), EDC/H2O (4
equiv./17 equiv.), 4 h; ii) N3-Ala-Ala-Ala (2 equiv.), CuSO4 (0.13 equiv.), sodium ascorbate
(0.16 equiv.), TBTA (0.19 equiv.), DMF, overnight.
The first conjugation was performed using a water-soluble carbodiimide reagent (1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC) to yield amide containing

compound 7.15 After reaction completion the product was isolated as the TEA salt
using an ion-exchange chromatography. The second coupling of N3-Ala-Ala-Ala to 7
was performed under mild conditions using copper sulphate in the presence of
sodium ascorbate with catalytic tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(TBTA) in DMF and a small amount of H2O as solvent to ensure complete dissolution
of the reagents.16 After completion of the click reaction, product 8 was isolated as the
TEA salt by ion-exchange chromatography on a DEAE-Sephadex column and
additionally purified using HPLC on a C18 column to give the ammonium salt.
In summary, the first double-functionalized dinucleotide cap analogue was
synthesized. The usefulness of such a derivative was shown by attaching two
different molecules - a fluorescent label and a short peptide. The cap analogue can
possibly be employed in the case of other probes dedicated to specific biochemical
and biophysical methods, making it a powerful tool for a broad range applications.
Acknowledgement
This work was supported by grant from the National Science Centre, Poland (DEC-
2013/11/B/ST5/02226 and DEC-2016/21/D/ST5/01654.
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Highlights
• Double functionalized dinucleotide cap analogue for subsequent labelling was
synthesized
• Introduced groups can be linked with probes containing azide and amine
moieties
• Simple and convenient synthesis

Synthesis of the first double-functionalized dinucleotide mRNA cap analogue for its
specific labeling
Karolina Piecyk, Paulina Krynska, Justyna Kaluzna, Marzena Jankowska-Anyszka*
CH3
G
G
.