Solid-supported synthesis of 5′-mRNA CAP-4 from trypanosomatids

Article abstract of DOI:10.1080/15257770701533065

The unique structure of 5′ mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m ⁷ Gpppm₃^6,6,2′ Apm^2′ Apm^2′ Cpm₂^3,2′ Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5′-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn²⁺ as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl₂. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701533065

Nucleosides, Nucleotides, and Nucleic Acids, 26:1329–1333, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701533065
SOLID-SUPPORTED SYNTHESIS OF 5ꢀ-mRNA CAP-4 FROM
TRYPANOSOMATIDS
Magdalena Lewdorowicz and Jacek Jemielity
Department of Biophysics,
2
Institute of Experimental Physics, Warsaw University, Warsaw, Poland
Ryszard Kierzek
Institute of Bioorganic Chemistry, Polish Academy of Sciences,
2
Poznan, Poland
Michal Shapira
Department of Life Sciences, The Ben Gurion University at the
2
Negev, Beer Sheva, Israel
Janusz Stepinski and Edward Darzynkiewicz
Department of Biophysics,
2
Institute of Experimental Physics, Warsaw University, Warsaw, Poland
The unique structure of 5ꢁ mRNA cap from Trypanosomatidsꢁ is the most modified cap found
2
ꢁ
ꢁ
Apm2 Apm2 Cpm2 ꢁ Up) on
6,6,2
3,2
in nature. Here we present the synthesis of cap-4 (m7 Gpppm3
a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then pre-
viously described. Moreover such modified resin could be a useful tool for affinity purification of
Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of
phosphorylated tetranucleotide with activated 7-methylguanosine 5ꢁ-diphosphate two systems were
compared. Surprisingly, the coupling in water with Mn2+ as a catalyst, gave better results than
usually more effective coupling in DMF with ZnCl2 .
Keywords 5ꢁ mRNA Cap-4; disulfide linker; Leishmania; oligonucleotide synthesis;
solid support
INTRODUCTION
Cap structure, that is, m7GpppN plays important role in various
biochemical processes and is used as a tool to study translation, splicing
and RNA intercellular transport mechanisms. The recognition of the
cap by initiation factor eIF4E is a crucial step in the initiation of protein
biosynthesis process. The interactions between cap analogues and eIF4E
can be monitored by measuring intrinsic fluorescence quenching of the
This work was supported by grant from Polish Ministry of Science and Higher Education (No. 2
P04A 006 28) and grant from HHMI (No. 55005604).
Address correspondence to Jacek Jemielity, Departament of Biophysics, Institute of Experimental
Physics, Warsaw University, Zwirki i Wigury 93, Warsaw 02-089, Poland. E-mail: jacekj@biogeo.uw.edu.pl
1329

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M. Lewdorowicz et al.
tryptophan residues.[1] Since affinities of cap analogues to the eIF4E
differ, they are attractive compounds for studying the molecular basis
of cap recognition. Moreover, these differences may have also potential
therapeutic significance.
The 5ꢁ cap structure of Leishmania, denoted cap-4, is a complex struc-
ture that contains unusualꢁ modifications on the first four nucleotides—
ꢁ
ꢁ
ꢁ
m7Gpppm36,6,2 Apm2 Apm2 Cpm23,2 U. The two of four nucleosides:
ꢁ
ꢁ
N6,N6,O2 -trimethyladenosine and N3,O2 -dimethyluridine, were previously
unknown in nature.[2] In previous articles, we showed that a synthetically
prepared cap-4 analogue was efficiently recognized by an LeishIF4E-1
homologue from Leishmania.[3,4] Moreover, this structure is also efficiently
recognized by three of four eIF4E isoforms from Leishmania—LeishIF4E-1,
LeishIF4E-2, and LeishIF4E-4.
Here we describe the chemical synthesis of cap-4 on a disulfide-tethered
solid support (Figure 1). This system can be used for affinity purification
of Leishmania proteins that interact with cap-4. In order to obtain the cap-4
attached to a resin, we synthesized a disulfide-tethered solid support that
would be resistant for ammonolysis.[5,6] The 5ꢁ-phosphorylated tetramer
was prepared by the standard phosphoramidite solid phase method. How-
ever, since two of theꢁ four monomers for solid phase synthesis (phospho-
ꢁ
ramidites of N6,N6,O2 -trimethyladenosine and N3,O2 -dimethyluridine) are
not available commercially, we synthesized them according to the standard
procedures.[3] As a solid support two commercially available resins were cho-
sen: CPG and TentaGel. The 5ꢁ-phosphorylated tetramer was coupled on the
resin with an imidazolide derivative of 7-methylguanosine 5ꢁ-diphosphate in
the presence of 8-fold excess of ZnCl2 (in DMF)[3,7,8] and in the presence
of Mn2+ (in aqueous solution).[9]
RESULTS AND DISCUSSION
The disulfide linker allowed the RNA chain assembly without cleavage
from the support during ammonolysis of base protecting groups; moreover,
such linkers also may be reductively cleaved under mild conditions.[5,6]
The synthesis of disulfide-tethered linker was performed in similar way
like described earlier.[5,6] The amino groups of the standard CPG and
TentaGel supports were acylated with dimeric 11-mercaptoundecanoic acid
in the presence of N-hydroxysuccinimide, N,N-diisopropylcarbodiimide and
4-(dimethylamino)pyridine. The disulfide bond of dimers was cleaved on
solid support by reduction with DTT.
The SH function of resin (1) was treated with 2-[(4,4ꢁ-dimethoxytrityl)
oxy]ethyl 2-pyridyl disulfide and gave modified resin (2) ready to use in
automated synthesis (Figure 2). According to the dimethoxytrityl (DMTr)
cation assay, the loading was 60 µmol/g (for CPG) and 160 µmol/g (for
TentaGel).

Solid-Support Synthesis
1331
FIGURE 1 The structure of cap-4 on a disulfide-tethered solid support.
2-[(4,4ꢁ-Dimethoxytrityl)oxy]ethyl 2-Pyridyl Disulfide was obtained
in a two-step reactions as described earlier involving synthesis of
2-hydroxyethyl 2-pyridyl disulfide from 2,2ꢁ-dithiodipyridine and mer-
captoethanol and protection of OH-group with 4,4ꢁ-dimethoxytrityl
ꢁ
chloride.[5] 5ꢁ-O-dimethoxytrityl-N6,N6,O2 -trimethyladenosine-3ꢁ-O-(2-cya-
noethyl)-N,N-diisopropylphosphoramidite was prepared as described
ꢁ
earlier.[3] 5ꢁ-O-dimethoxytrityl-N3,O2 -dimethyluridine-3ꢁ-O-(2-cyanoethyl)-
N,N-diisopropylphosphoramidite was obtained from 5ꢁ-O-dimethoxytrityl-
ꢁ
N3,O2 -dimethyluridine in the presence of N,N-diisopropylethylamine
and 2-cyanoethoxy N,N-diisopropylaminochlorophosphine in anhydrous
acetonitrile.
After removal of the DMTr group, the tetranucleotide sequence was
assembled by normal phosphoramidite chemistry and the 5ꢁ-hydroxy
function in tetranucleotide was phosphitylated with 3-DMTrO-2,2-

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M. Lewdorowicz et al.
FIGURE 2 Structure of the supports.
bis(ethoxycarbonyl)propyl 2-cyanoethyl N,N-diisopropylphosphoramidite
and oxidized to a phosphate triester.[10] The base and phosphate
protections were removed by standard DMTr-off ammonolysis. The
HPLC control of the ammonolysis of the protective groups have shown
partial lost of tetranucleotide from CPG resin. We finished the ammonolysis
when the concentration of deprotected oligonucleotide was about 90%
(according to the HPLC analysis). In the case of tetranucleotide on Tent-
aGel resin, we haven’t observed the cleavage of the product from the resin
during ammonolysis. The support-anchored oligonucleotide 5ꢁ-phosphate
was then treated with 7-methylguanosine 5ꢁ-diphosphate imidazolide in
DMF in the presence of 8-fold excess of ZnCl2 (Figure 3) or in aqueous
solution in the presence of Mn2+. Surprisingly, the latter method gave
better results in coupling reaction.
We monitored the capping reaction using HPLC (the disulfide bond
was finally cleaved with DTT and the 2-mercaptoethyl group was allowed to
FIGURE 3 The synthesis of cap-4 on disulfide-tethered solid support.

Solid-Support Synthesis
1333
eliminate in aqueous solution of methanol containing 0.01% triethy-
lamine). On the basis of HPLC the yield of the capping step was 50%, (in
the case of TentaGel resin).
The cap-4 on solid support (the structure shown on Figure 1.) will be
used in affinity chromatography of eIF4E homologues from Leishmania.
REFERENCES
1. Niedzwiecka, A.; Marcotrigiano, J.; Stepinski, J.; Jankowska-Anyszka, M.; Wyslouch-Cieszynska, A.;
Dadlez, M.; Gingras, A.-C.; Mak, P.; Darzynkiewicz, E.; Sonenberg, N. Biophysical studies of eIF4E
cap-binding protein: Recognition of mRNA 5ꢁ cap structure and synthetic fragments of eIF4G and
4E-BP proteins. J. Mol. Biol. 2002, 319, 615–635.
2. Bangs, J.D.; Crain, P.F.; Hashizume, T.; McCloskey, J.A.; Boothroyd, J.C. Mass spectrometry of mRNA
cap 4 from trypanosomatids reveals two novel nucleosides. J. Biol. Chem. 1992, 267, 9805–9815.
3. Lewdorowicz, M.; Yoffe, Y.; Zuberek, J.; Jemielity, J.; Stepinski, J.; Kierzek, R.; Stolarski, R.; Shapira,
M.; Darzynkiewicz, E. Chemical synthesis and binding activity of the trypanosomatid cap-4 structure.
RNA 2004, 10, 1469–1478.
4. Yoffe, Y.; Zuberek, J.; Lewdorowicz, M.; Zeira, Z.; Keasar, C.; Orr-Dahan, I.; Jankowska-Anyszka,
M.; Stepinski, J.; Darzynkiewicz, E.; Shapira, M. Cap-binding activity of an eIF4E homolog from
Leishmania. RNA 2004, 10, 1764–1775.
5. Salo, H.; Guzaev, A.; Lonnberg, H. Disulfide-tethered solid supports for synthesis of photolumines-
cent oligonucleotide conjugates: Hydrolytic stability and labeling on the support. Bioconjugate Chem.
1998, 9, 365–371.
6. Jemielity, J.; Heinonen, P.; Lo¨nnberg, H.; Darzynkiewicz, E. A novel approach to solid phase chem-
ical synthesis of oligonucleotide mRNA cap analogs. Nucleosides, Nucleotides Nucleic Acids 2005, 24,
601–605.
7. Jemielity, J.; Fowler, T.; Zuberek, J.; Stepinski, J.; Lewdorowicz, M.; Niedzwiecka, A.; Stolarski, R.;
Darzynkiewicz, E.; Rhoads, R.E. Novel “anti-reverse” cap analogs with superior translational proper-
ties. RNA 2003, 9, 1108–1122.
8. Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R.E. Synthesis and properties
of mRNAs containing the novel “anti-reverse” cap analogues 7-methyl(3ꢁ-O-methyl)GpppG and 7-
methyl (3ꢁ-deoxy)GpppG. RNA 2001, 7, 1486–1495.
9. Sawai, H.; Wakai, H.; Nakamura-Ozaki, A. Synthesis and reactions of nucleoside 5ꢁ-diphosphate
imidazolide. A nonenzymatic capping agent for 5ꢁ-monophosphorylated oligoribonucleotides in
aqueous solution. J. Org. Chem. 1999, 64, 5836–5840.
10. Guzaev, A.; Salo, H.; Azhayev, A.; Lonnberg, H. A new approach for chemical phosphorylation of
oligonucleotides at the 5ꢁ-terminus. Tetrahedron 1995, 51, 9375–9384.