Efficient synthesis of D-[1′-13c]-ribonucleosides for incorporation into oligo-RNA

Article abstract of DOI:10.1081/NCN-100002463

Syntheses of the monomer building blocks used for the solid-phase synthesis of specifically 1′-¹³C-labeled oligoribonucleotides from the D-[1-¹³C]ribose is presented. Procedure has been used for the selective formation of 2′-O-silylated ribonucleosides. After 5′-O-dimethoxytritylation, the synthesis of D-[1′-¹³C] ribonucleoside phosphoramidites has been achieved.

Full text of DOI:10.1081/NCN-100002463

This article was downloaded by: [The UC Irvine Libraries]
On: 02 November 2014, At: 05:34
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
EFFICIENT SYNTHESIS OF D-[1′-13C]-
RIBONUCLEOSIDES FOR INCORPORATION INTO OLIGO-
RNA
Y. Saito a , A. Nyilas a & L. A. Agrofoglio a
a I.C.O.A. associé CNRS , Faculté des Sciences, Orléans, 45100, France
Published online: 07 Feb 2007.
To cite this article: Y. Saito , A. Nyilas & L. A. Agrofoglio (2001) EFFICIENT SYNTHESIS OF D-[1′-13C]-RIBONUCLEOSIDES
FOR INCORPORATION INTO OLIGO-RNA, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 937-940, DOI: 10.1081/
NCN-100002463
To link to this article: http://dx.doi.org/10.1081/NCN-100002463
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 937–940 (2001)
EFFICIENT SYNTHESIS OF
D-[1ꢀ-13C]-RIBONUCLEOSIDES FOR
INCORPORATION INTO OLIGO-RNA
Y. Saito, A. Nyilas, and L. A. Agrofoglio∗
I.C.O.A. associe´ CNRS, Faculte´ des Sciences, 45100 Orle´ans-France
ABSTRACT
Syntheses of the monomer building blocks used for the solid-phase synthesis
of specifically 1ꢀ-13C-labeled oligoribonucleotides from the D-[1-13C]ribose is
presented. Procedure has been used for the selective formation of 2ꢀ-O-silylated
ribonucleosides. After 5ꢀ-O-dimethoxytritylation, the synthesis of D-[1ꢀ-13C]
ribonucleoside phosphoramidites has been achieved.
The utility of 13C-labeled RNAs and DNAs for the heteronuclear multidi-
mensional NMR studies for nucleic acids has been shown in several reports (1).
One of the major problems in the chemical synthesis of 13C enriched nucleoside
derivatives is the price of the starting compound that requires optimized reac-
tions. Standard procedures for preparation of nucleoside synthons used in solid-
phase synthesis of oligoribonucleotides include protection of the amino groups on
the nucleobases, protection of the 5ꢀ- and 2ꢀ-hydroxyl groups as dimethoxytrityl
(DMTr) and TBDMS ether, respectively, and phosphitylation of the 3ꢀ-oxygen
to the β-cyanoethyl N,N-diisopropylphosphoramidite (Ogilvie’s approach) (2). In
designing the synthesis of monomer for labeled oligo-RNA, we considered a re-
gioselective protection of the ribose hydroxyls in such a way as to obtain solely
the 2ꢀ-O- tert-butyldimethylsilyl derivative and to circumvent the ever aggravating
problemofthe2ꢀ-vs. 3ꢀ-silylation. Thispaperdescribesthesynthesesofthe[1ꢀ-13C]-
labeled building blocks for the preparation of oligo-RNA. A selective and effici-
ent procedure for the 2ꢀ-O-silylation has been applied for the synthesis
of [1ꢀ-13C]-ribonucleoside phosphoramidites. The D-[1-13C]ribose was converted
into the D-[1ꢀ-13C]ribonucleosides following well known procedure (Scheme 1).
937
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
REPRINTS
938
SAITO, NYILAS, AND AGROFOGLIO
Scheme 1. Reagents and Conditions: (a) MeOH, H2SO4; (b) BzCl, pyridine, rt; (c) Ac2O, AcOH,
conc. H2SO4, 0◦C to rt; (d) Base, TMSOTf, DCE, reflux; (e) 1N NaOH pyridine/EtOH (for 4a and
4c) or NH3/MeOH (for 4b) then amberlite H+.
Compound 3 was subjected to a coupling reaction (3) with persilylated nucleobases
(persilylated N6-benzoyladenine, uracil, and N4-benzoylcytosine) to give 2,3,5-O-
tri-benzoyl-[1ꢀ-13C]ribonucleoside derivatives 4a–c with fairly good yields (86%,
57% and 73% respectively). In case of the synthesis of protected uridine 4b, the
temperatures must be kept under 45◦C to avoid undesired side product. Finally, the
selective deprotection of O-acyl groups from 4a–c was achieved with aqueous 1N
NaOH-ethanol-pyridine (for 4a and 4c) or NH3/MeOH (for 4b).
In all cases, deacylated nucleosides 5a–c were directly engaged into the next
reaction. A principal consideration in the chemical syntheses of the ribonucleoside
phosphorimidates is contamination of the desired 2ꢀ-O-silyl-3-ꢀO-phosphoramidite
with undesired 3ꢀ-O-silyl-2ꢀ-O-phosphoramidite. Thus, the classical procedure to
introduce tert-butyldimethylsilyl as a 2ꢀ-OH protecting group presents the problem
of optimizing the yield of the desired 2ꢀ-O-tBDMS isomer, consuming valuable
labeled ribonucleosides and lowering the yield of desired labeled ribonucleoside
phosphoramidites. This is mainly due to the fact that during the silylation reaction,
the 2ꢀ- and 3ꢀ-hydroxyl groups are not markedly distinguishable; these intercon-
versions are base catalyzed. To circumvent this problem, we applied a three steps
procedure involving the initial and regioselective protection of 3ꢀ,5ꢀ-hydroxyl using
the Markiewicz disiloxane reagent (4). As a result, we obtain the desired com-
pounds avoiding by-products as obtained by the classical approach (5). Thus treat-
ment of D-[1ꢀ-13C]ribonucleosides 5a,b with di-tert-butyldichlorosilane in the pres-
ence of AgNO3 in DMF afforded the corresponding 3ꢀ,5ꢀ-di-O-tert-butylsilanediyl
derivatives (6) 6a (72%) and 6b (97%), respectively, (Scheme 2). Treatment with
tert-butyldimethylsilyl chloride (TBDMSiCl) and silver nitrate in DMF led 2ꢀ-O-
tBDMS ether 7a and 7b in 74% and 90% yield, respectively.
The di-tert-butylsilylene group was then removed by treatment with pyri-
dinium poly(hydrogen fluoride) at −20◦C to afford 8a and 8b with 96% and
95% yield, respectively. One hour in the presence of DMTrCl and AgNO3 was

ORDER
REPRINTS
SYNTHESIS OF D-[1ꢀ-13C]-RIBONUCLEOSIDES
939
Scheme 2. Reagents and Conditions: (a) DTBSCl, AgNO3, DMF; (b) TBDMSiCl, AgNO3, DMF;
(c) HF-Py, −20◦C; (d) DMTrCl, AgNO3, DMF; (e) 2,4,6-collidine, CH2Cl2, N-methylimidazole.
sufficient to convert 8a,b to 9a,b. Under these conditions, no migration of the
2ꢀ-O-TBDMS group to the 3ꢀ-oxygen was observed. Finally, the synthesis of the
phosphoramidites 10a,b by isomerisation free procedure (7), was accomplished
using (N,N-diisopropylamino)(cyanoethyl)-phosphonamidic chloride as the phos-
phitylating agent. And with 2,4,6-collidine as a base and N-methylimidazole as it
has been reported to not isomerize the 2ꢀ-O-silyl into 3ꢀ-O-silyl derivatives. The
desired monomers for oligo-RNA, the labeled D-[1ꢀ-13C]-ribonucleoside phospho-
ramidites 10a,b (8) were thus obtained.
ACKNOWLEDGMENT
This work was supported by a research grant from the CNRS (Grant ACC
Physique Chimie du Vivant). We gratefully acknowledge the Region Centre and
the Studium for a postdoctoral fellowship.
REFERENCES
1. (a) Sekine, T.; Kawashima, E.; Ishido, Y. Tetrahedron Lett. 1996, 37, 7757. (b) Agro-
foglio, L. A.; Jacquinet, J.-C.; Lancelot, G. Tetrahedron Lett. 1997, 38, 1411.
2. (a) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault, N. Y.; Sadana, K. L.
Can. J. Chem. 1978, 56, 2768. (b) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can.
J. Chem. 1982, 60, 1106.
3. Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber. 1981, 114, 1234.
4. Markiewicz, W. T. J. Chem. Res. (S) 1979, 24.
5. (a) Grasby, J. A.; Jonathan, P.; Butler, G.; Gait M. J. Nucleic Acid. Res. 1993, 21,
4444. (b) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990, 18, 5433.

ORDER
REPRINTS
940
SAITO, NYILAS, AND AGROFOGLIO
(c) Quant, S.; Wechselberger, R. W.; Wolter, M. A.; Wo¨rner, K.-H.; Schell, P.; Engels,
J. W.; Griesinger, C.; Schwalbe, H. Tetrahedron Lett. 1994, 35, 6649.
6. General Procedure for the Sequential Transformation of ribonucleosides to
5ꢀ-DMTr- 2ꢀ-O-Silylribonucleosides: D-[1ꢀ-13C]-3ꢀ,5ꢀ-O-(Di-tert-butylsilanediyl)-N6-
benzoyladenosine (6a): To a solution of 5a (0.58 g, 1.54 mmol) and AgNO3
(0.78 g, 4.6 mmol) in anhydrous DMF (9 mL) at 0◦C was added dropwise di-tert-
butyldichlorosilane (0.5 mL, 2.37 mmol) with vigorous stirring. The mixture was
warmed to room temperature and subsequently stirred for 15 min. Triethylamine
(0.65 mL, 4.64 mmol) was added, and the mixture was stirred for an additional 5 min.
The solvent was removed under reduced pressure. Water (15 mL) was added and the re-
sulting solution extracted with CH2Cl2. The organic layer was separated, washed with
brine, dried over Na2SO4, and evaporated to dryness under reduced pressure. The
residue was purified by silica gel chromatography to give 7a (72%). All NMR analyses
were in agreement with the structure. D-[1ꢀ-13C]-2ꢀ-O-tert-butyldimethylsilyl-3ꢀ,5ꢀ-O-
di-tert-butylsilanediyl-N6-benzoyladenosine (7a): To a solution of 6a (0.41 g, 0.81
mmol) and catalytic amount of DMAP in anhydrous pyridine (10 mL) was added
dropwise tert-butyldimethylsilyl chloride (2 eq, 0.12 g, 0.78 mmol). The mixture was
refluxed overnight. After evaporation under reduced pressure, the resulting mixture was
diluted with CH2Cl2, and the organic phase washed with brine and dried over Na2SO4.
After a column chromatography on silica gel, the desired product 7a was isolated with
74% yield. All NMR analyses were in agreement with the structure. D-[1ꢀ-13c]-2ꢀ-O-
tert-butyldimethylsilyl-N6-benzoyladenosine (8a): A solution of HF.pyridine (60 µL,
2.34 mmol; commercially available) was carefully diluted with anhydrous pyridine
(0.31 mL) and then added dropwise to a solution of 7a (0.36 g, 0.58 mmol) in THF
(3 mL) at 0◦C. The mixture was warmed to room temperature, stirred for 5 min. and
then diluted with pyridine (0.5 mL). Water (5 mL) was added and the extraction was
performed with CH2Cl2. The organic layer was collected, washed with 5% NaHCO3
and dried over Na2SO4. After evaporation under reduced pressure, the residue was pu-
rified by a column chromatography on silica gel to yield the desired compound 8a with
96% yield.
7. (a) Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nucleic Acid Res. 1984, 12, 4539.
(b) Milecki, J.; Dembek, P.; Antkowiak, W. Z.; Gdaniec, Z.; Mielewczyk, S.; Adamiak,
R. W. Nucleosides & Nucleotides 1989, 8, 463.
8. All synthesized products have been fully characterized by 1H, 13C NMR and MS.

Request Permission or Order Reprints Instantly!
Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article’s rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly. Simply click on the "Request
Permission/Reprints Here" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers’
(AAP) website for guidelines on Fair Use in the Classroom.
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as
part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our Website
User Agreement for more details.
Order now!
Reprints of this article can also be ordered at
http://www.dekker.com/servlet/product/DOI/101081NCN100002463