Table Of ContentPreface
A decade has passed since Methods in Enzymology addressed methods
and techniques used in RNA processing. As has been evident since its
inception, research in RNA processing progresses at a rapid pace. Its
expansion into new areas of investigation has been phenomenal with
novel discoveries being made in a variety of subspecialty areas. The
subfield of RNA-ligand interactions concerns research problems in RNA
structure, in the molecular recognition of structured RNA by diverse
ligands, and in the mechanistic details of RNA's functional role following
ligand binding. At the beginning of this new millennium, we celebrate
the explosive development of exciting new tools and procedures whereby
investigators explore RNA structure and function from the perspective
of understanding RNA-ligand interactions.
New insights into RNA processing are accompanied with improve-
ments in older techniques as well as the development of entirely new
methods. Previous Methods in Enzymology volumes in RNA processing
have focused on basic methods generally employed in all RNA processing
systems (Volume 180) or on techniques whose applications might be
considerably more specific to a particular system (Volume 181). RNA-
Ligand Interactions, Volumes 317 and 318, showcase many new methods
that ihave led to significant advances in this subfield. The types of ligands
described in these volumes certainly include proteins; however, ligands
composed of RNA, antibiotics, other small molecules, and even chemical
elements are also found in nature and have been the focus of much
research work. Given the great diversity of RNA-ligand interactions
described in these volumes, we have assembled the contributions according
to whether they pertain to structural biology methods (Volume 317) or
to biochemistry and molecular biology techniques (Volume 318). Aside
from the particular systems for which these techniques have been devel-
oped, we consider it likely that the methods described will enjoy uses
that extend beyond RNA-ligand interactions to include other areas of
RNA processing.
This endeavor has been fraught with many difficult decisions regarding
the selection of topics for these volumes. We were delighted with the
number of chapters received. The authors have taken great care and
dedication to present their contributions in clear language. Their willing-
ness to share with others the techniques used in their laboratories is
iiix
xiv ECAFERP
apparent from the quality of their comprehensive contributions. We
thank them for their effort and appreciate their patience as the volumes
were assembled.
LEINAD W. REDNALeC
NHOJ N. NOSLEBA
Contributors to Volume 317
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
ARDNEJAR K. AGRAWAL (18, 19), Howard 1V T. CHU (10), Department of Biochemistry
Hughes Medical Institute, Health ,hcraeseR and Molecular Biophysics, Columbia -inU
Inc., at eht Wadsworth ,retneC and -trapeD ,ytisrev New York, New York 23001
ment of Biomedical ,secneicS State -revinU MARIA ATSOC (29), Centre ed euqitdn~G Mo-
sity of New York, Albany, New York erialuc~l du CNRS, F-91190 Gif-sur-
9050-10221 Yvette, ecnarF
Russ B. NAMTLA (28), Departments of -ideM DLANOD M. SREHTORC (9), Department of
cine and Computer ,ecneicS Stanford -inU ,yrtsimehC Yale ,ytisrevinU New Haven,
,ytisrev Stanford, California 9745-50349 tucitcennoC 7018-02560
LEAHCIM A. BADA (28), Stanford ,ytisrevinU LEAHCIM L. DERAS (22), Department of Bio-
Stanford, California 9745-50349 physics, Johns Hopkins ,ytisrevinU Balti-
RETEP REYAB (14), MRC Laboratory of Mo- more, Maryland 4682-81212
ralucel Biology, Cambridge 2BC 2QH, En-
REFINNEJ A. DOUDNA (12), Department of
dnalg Molecular Biophysics and Biochemistry,
)rINOEL NAMLEGIEB (3), Ribozyme Pharma- Yale ,ytisrevinU New Haven, Connecticut
,slacituec Inc., Boulder, Colorado 10308 4118-02560
ROGERG BLAHA (19), AG Ribosomen, Max- FmTZ NIETSKCE (5), Max-Planck-Institut far
Planck-Institut far Molekulare Genetik, D- Experimentelle Medizin, D-37075 -t~iG
59141 Berlin, Germany ,negnit Germany
MARC NIALLIVDUOB (10), Howard Hughes GNAWONIX FANO (24), Department of Bio-
Medical Institute, Columbia ,ytisrevinU chemistry and Molecular Biology, -revinU
New York, New York 23001 sity of ,ogacihC ,ogacihC Illinois 73606
LEAHCIM ZT1WONERB (22), Department of MIHCAOJ FRANK (18, 19), Howard Hughes
,yrtsimehcoiB Center for Synchrotron -DiB Medical Institute, Health ,hcraeseR Inc., at
,secneics Albert Einstein College of Medi- eht Wadsworth ,retneC and Department of
,enic Bronx, New York 16401 Biomedical Sciences, State University of
NHOJ M. EKRUB (25), ytisrevinU of ,tnomreV New York, Albany, New York 9050-10221
Burlington, Vermont 50450 ARABRAB L. NEDLOG (8), Department of -DiB
NILS BURKHARDT (17), Gebi~ude 405, ,yrtsimehc Purdue ,ytisrevinU West Lafa-
BAYER AG-Wuppertal, Abteilung MST, ,ettey Indiana 3511-60974
69024-D Wuppertal, Germany TREBOR A. ICCUSSARG (18), Howard Hughes
JAMIE H. CATE (12), Whitehead ,etutitsnI Medical Institute, Health ,hcraeseR Inc., at
,egdirbmaC sttesuhcassaM 9741-24120 eht Wadsworth ,retneC Albany, New York
TREBOR NERGREDEC (27), D~partement ed 9050-10221
Biochimie, ~tisrevinU ed Montreal, Mon- RETEP ILREBEAH (3), Ribozyme -uecamrahP
,laert Quebec H3C 3J7, adanaC ,slacit Inc., Boulder, Colorado 10308
KRAM R. ECNAHC (22), Department of Physi- PAUL J. NAMREGAH (26), Department of -DiB
yKolo and Biophysics, Center for Synchro- yrtsimehc and Molecular ,sciteneG -revinU
tron ,secneicsoiB Albert Einstein egelloC of sity of Colorado Health Sciences ,retneC
,enicideM Bronx, New York 16401 Denver, Colorado 26208
ix
X CONTRIBUTORS TO VOLUME 317
MARK R. HANSEN (15), Department of -mehC MELISSA J. MOORE (7), Department of Bio-
yrtsi and Biochemistry, ytisrevinU of -oloC ,yrtsimehc .W .M Keck Institute for ralulleC
,odar Boulder, Colorado 5120-90308 ,noitazilausiV Brandeis University, Wal-
PAUL HANSON (15), Department of yrtsimehC tham, sttesuhcassaM 45420
and Biochemistry, University of ,odaroloC JAMES B. MURRAY (13), Department of
Boulder, Colorado 5120-90308 yrtsimehC and ,yrtsimehcoiB and retneC for
AMY B. HEAGLE (18), Howard Hughes Medi- eht Molecular Biology of RNA, ytisrevinU
lac Institute, Health Research, Inc., at eht of ,ainrofilaC Santa Cruz, California 46059
Wadsworth Center, Albany, New York KNUD H. NIERHAUS (17, 19), AG Ribosomen,
9050-10221 Max-Planck-Institut far Molekulare Gen-
ECKHARD YKSWOKNAJ (10), Department of etik, D-14195 Berlin, Germany
Biochemistry and Molecular Biophysics, LORI ORTOLEVA-DONNELLY (6), Depart-
Columbia University, New York, New ments of Molecular Biophysics and Bio-
York 23001 ,yrtsimehc and Chemistry, Yale ,ytisrevinU
ALEXANDER KARPEISKY (3), Ribozyme Phar- New Haven, Connecticut 4118-02560
,slacituecam Inc., Boulder, Colorado 10308 TAD PAN (20), Department of Biochemistry
ANNE I. KOSEK (6), Departments of -uceloM and Molecular Biology, University of Chi-
ral Biophysics and Biochemistry, dna ,ogac ,ogacihC Illinois 73606
,yrtsimehC Yale ,ytisrevinU New Haven, ARTHUR PARDI (15), Department of -simehC
tucitcennoC 4118-02560 yrt and Biochemistry, University of Colo-
JON LAPHAM (9), Department of ,yrtsimehC ,odar Boulder, Colorado 5120-90308
Yale ,ytisrevinU New Haven, Connecticut PAWEL PENCZEK (18), Howard Hughes Medi-
7018-02560
lac Institute, Health Research, Inc., at eht
FABRICE LECLERC (27), Department of -mehC Wadsworth ,retneC and Department of -DiB
istry and Chemical Biology, Harvard -inU medical ,secneicS State University of New
,ytisrev ,egdirbmaC sttesuhcassaM 83120 York, Albany, New York 9050-10221
DAVID M. J. LILLEY (23), Department of -DiB JOSEPH D. PUGLISI (16), Department of -curtS
,yrtsimehc University of Dundee, Dundee larut Biology, Stanford ytisrevinU School of
DD1 4HN, United Kingdom ,enicideM Stanford, California 6215-50349
BELSIS LLORENTE (27), Centro ed Quimica ANNA MARIE PYLE (10), Department of -DiB
,acitu~camraF Atabey, Habana, Cuba chemistry and Molecular Biophysics, and
NEHPETS R. LYNCH (16), Department of -curtS Howard Hughes Medical Institute, Colum-
tural Biology, Stanford ytisrevinU School of aib ,ytisrevinU New York, New York 23001
,enicideM Stanford, California 6215-50349 CHARLES C. QUERY (7), Department of lleC
CHRISTIAN MASSIRE (29), Institut ed Biologie Biology, Albert Einstein College of Medi-
erialuc~loM et erialulleC du CNRS, 48076-F ,enic Bronx, New York 16401
,gruobsartS ecnarF CORIE Y. RALSTON (22), Department of Physi-
JASENKA MATULIC-ADAMIC (3), Ribozyme ology and Biophysics, Center for Synchro-
,slacituecamrahP Inc., Boulder, Colorado tron ,secneicsoiB Albert Einstein College of
1O3O8 ,enicideM Bronx, New York 16401
DAVID B. McKAY (11), Department of -curtS MICHAEL I. RECI-rr (16), Department of -curtS
tural Biology, Stanford University School tural Biology, Stanford ytisrevinU School of
of ,enicideM Stanford, California 50349 ,enicideM Stanford, California 6215-50349
FRANCOIS MICHEL (29), Centre de euqit~ndG BEATRIX ROHRDANZ (17), AG Ribosomen,
erialucdloM ud CNRS, F-91190 Gif-sur- Max-Planck-lnstitut far Molekulare Gen-
,ettevY ecnarF etik, D-14195 Berlin, Germany
CONTRIBUTORS TO VOLUME 317 xi
NAES P. RYDER (6), Department of Molecular DIVAD RELDEEWS (3), Ribozyme Pharmaceu-
Biophysics and Biochemistry, Yale Univer- ticals, Inc., Boulder, Colorado 10308
sity, New Haven, Connecticut 4118-02560
SAMOHT J. TREBLOT (2), The Scripps Re-
NEHPETS A. EGNIRACS (1), Dharmacon Re- search Institute, La Jolla, California
search, Inc., Boulder, Colorado 10308 73029
ACNAIB IVALCS (22), Department of Physiol- LEINAD K. TREIBER (21), The Scripps Re-
ogy and Biophysics, Center for Synchrotron search Institute, La Jolla, California
Biosciences, Albert Einstein College of 73029
Medicine, Bronx, New York 16401
OCSICNARF J. OSNOLA-ANAIRT (17), Centro
NLOCNIL G. TTOCS (2), The Scripps Research de Investigaciones ,sacidOmoiB Universidad
Institute, La Jolla, California 92037
de Carabobo, LaMorita, Maracay, Vene-
MAILLIW G. SCOTT (13), Department of zuela
Chemistry and Biochemistry, and Center for
ELEIRBAG VARANI (14), MRC Laboratory
the Molecular Biology of RNA, University
of Molecular Biology, Cambridge CB2
of California, Santa Cruz, California 46059
2QH, England
EIRELAV M. NOTLEHS (24), Department of
ACUL INARAV (14), MRC Laboratory of Mo-
Chemistry, University of Chicago, Chicago,
lecular Biology, Cambridge CB2 2QH, En-
Illinois 60637
gland
NIBOT R. KCINSOS (24), Department of Bio-
L. SUALC S. RELTROV (5), Max-Planck-lnsti-
chemistry and Molecular Biology, Univer-
tut fiir Experimentelle Medizin, D-37075
sity of Chicago, Chicago, Illinois 60637
,negnittOG Germany
RuI ASUOS (4), Department of Biochemistry,
University of Texas Health Science Center, SLIN G. RETLAW (25), Department of Chemis-
San Antonio, Texas 0677-48287 try, University of Michigan, Ann Arbor,
Michigan 5501-90184
NAITSIRHC M. T. NHAPS (17, 19), Wadsworth
Center, New York State Department of HPESOJ E. DNIKEDEW (11), Department of
Health, New York, New York 9050-10221 Structural Biology, Stanford University
School of Medicine, Stanford, California
HCIRLU LZLETS (19), AG Ribosomen, Max-
50349
Planck-lnstitut fiir Molekulare Genetik, D-
59141 Berlin, Germany ERIC FOHTSEW (29), Institut de Biologie Mo-
erialucOl et Cellulaire du CNRS, F-67084
ScoTt A. LEBORr'S (6), Departments of Mo-
Strasbourg, France
lecular Biophysics and Biochemistry, and
Chemistry, Yale University, New Haven, SEMAJ R. NOSMAILLIW (2, 21), The Scripps
Connecticut 4118-02560 Research Institute, La Jolla, California
LEAHCIM NAVILLUS (22), Department of 73029
Physiology and Biophysics, Center for Syn- HARAS A. NOSDOOW (22), Department of Bio-
chrotron Biosciences, Albert Einstein Col- physics, Johns Hopkins University, Balti-
lege of Medicine, Bronx, New York 16401 more, Maryland 4682-81212
[ 11 R5N'A- SILYL-2'-ACE EDITOELCUNOGILO SISEHTNYS 3
11[ Advanced 5'-Sflyl-2'-Orthoester Approach to
RNA O1igonucleotide Synthesis
By STEPHEN A. SCARINGE
Introduction
The need for routine syntheses of RNA oligonucleotides has grown
rapidly during the 1990s as research reveals the increasing breadth of
RNA's biological functions. 1 RNA synthesis can be accomplished for most
applications via either biochemical methods, e.g., T7 transcription, or chemi-
cal methods. Transcription and chemical methodologies complement each
other well and enable a wide range of RNAs to be synthesized. Current
chemical synthetic methods 2 enable the synthesis of RNA in acceptable
yields and quality, but none are as routine and dependable as DNA. The
need for an improved oligoribonucleotide synthesis technology has contin-
ued to persist. This article describes a recently developed technological
advance in the chemical synthesis of RNA oligonucleotides utilizing a novel
5'-O-silyl ether protecting group in conjunction with an acid-labile 2'-0-
orthoester. 3 Using this technology, numerous RNA oligonucleotides have
been routinely synthesized in high yields and of unprecedented quality.
The advantageous properties of 5'-silyl-2'-orthoester RNA chemistry
make it possible to synthesize RNA oligonucleotides with a quality only
previously observed in DNA. The ribonucleoside phosphoramidites couple
in >99% stepwise yields in less than 90 sec. Consequently, yields are rou-
tinely 1.5-3 times that observed with older RNA synthesis chemistries.
At the same time, the overall purity is significantly increased. For some
applications, the RNA is of sufficient purity to use without further pro-
cessing. After synthesis of an RNA oligonucleotide, the 2'-orthoester pro-
t S. Altman, Proc. Natl. Acad. Sci. U.S.A. 90, 10898 (1993); B. A. Sullenger and T. R. Cech,
Science 262, 1566 (1993); T. Cech, Curr. Opin. Struct. Biol. 2, 605 (1992); N. Usman and
R. Cedergren, Trends Biochem. Sci. 17, 334 (1992).
2 F. Wincott, A. DiRenzo, C. Shaffer, S. Grimm, D. Tracz, C. Workman, D. Sweedler, C.
Gonzalez, S. Scaringe, and N. Usman, Nucleic Acids Res. 23, 2677 (1995); N. Usman,
K. I(. Ogilvie, M.-Y. Jiang, and R. J. Cedergren, J. Am. Chem. Soc. 109, 7845 (1987); T.
Wu, K. K. Ogilvie, and R. T. Pon, Nucleic Acids Res. 17, 3501 (1989); T. Tanaka, S.
Tamatsukuri, and M. Ikehara, Nucleic Acids Res. 14, 6265 (1986); J. A. Hayes, M.J . Brunden,
P. T. Gilham, and G. R. Gough, Tetrahedron Lett. 26, 2407 (1985); M. V. Rao, C. B. Reese,
V. Schehlman, and P. S. Yu, .J Chem. Soc. Perkin Trans. I, 43 (1993).
3 .S A. Scaringe, F. E. Wincott, and M. H. Caruthers, J. Am. Chem. Soc. 129, 11820
(1998).
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4 CITEHTNYSIMES METHODOLOGIES 1
o ~ , v , O ~ , ~ N . ~ O e M ' ~ / ~
Uridine Cytidine
'1 H
Adenosine Guanosine
FIG. .1 Protected ribonucleoside phosphoramidites for 5'-silyl-2'-orthoester RNA synthe-
sis chemistry.
tected RNA is water soluble and significantly more stable to degradation
than the final fully deprotected RNA product. These features of the 2'-
orthoester group enable the RNA to be easily handled in aqueous solutions.
Furthermore, the 2'-orthoester groups interrupt secondary structure. This
property has made it possible to analyze and purify RNA oligonucleotides
of every sequence to date regardless of secondary structure. This includes
10- to 15-base-long homopolymers of guanosine. Finally, when the RNA
is ready for use, the 2'-orthoester groups are completely removed in less
than 30 min under extremely mild conditions in common aqueous buffers.
These unique properties of the 5'-silyl ether and 2'-orthoester protecting
groups have made it possible to routinely synthesize high-quality RNA
oligonucleotides.
1 5'-SlLYL-2'-ACE RNA EDITOELCUNOGILO SISEHTNYS 5
H~
ess 20--1 esaB H% esaB
, - ,, ^
"y'( _5
.GIF .2 General sisehtnys emehcs from detcetorp-SPIT edisoelcun to fully protected -oelcun
side ,etidimarohpsohp where R si lycedodolcyc and Base si either ,eninedalyrytubosi-N -N
,enisotyclyteca ,eninauglyrytubosi-N or licaru noitcaer ;)i( ,etamrofohtro)yxohteyxoteca-2(sirt
muinidiryp toluene ;etanoflus reaction ;)ii( ,FH-DEMET ;elirtinoteca reaction ;)iii( ,1C-DOD
,elozadimi THF; reaction (iv); ,enihpsohpyxohtem)enimalyporposiid-N,N(sib tetrazole,
.MCD
Synthesis of Nucleoside Phosphoramidites
The Y-hydroxyl, 2'-hydroxyl, and amine protecting groups used in 5'-
silyl-2'-orthoester chemistry continue to be refined and optimized. At this
time the ribonucleoside phosphoramidites use the bis(trimethylsiloxy)-
cyclododecyloxysilyl ether (DOD) protecting group on the Y-hydroxyl and
the bis(2-acetoxyethoxy)methyl (ACE) orthoester protecting group on the
2'-hydroxyl (Fig. i). The exocyclic amines are protected with the following
acyl groups: acetyl for cytidine and isobutyryl for adenosine and guanosine.
Synthesis of these compounds proceeds according to the general outline
in Fig. 2 reactions (i)-(iv).
The N-acyl-5'-O-3'-O-tetraisopropyldisiloxanyl-protected ribonucleo-
side starting materials (1) (N-acyl-TIPS nucleosides) can be synthesized
according to the literature 4 or obtained commercially (Aldrich, Milwaukee,
WI, or Monomer Sciences, Huntsville, AL). The remaining reactions can
be effected utilizing the following generalized protocols.
4 .G .S ,iT .B .L Gaffney, and .R A. Jones, .J Am. Chem. Soc. ,401 6131 ;)2891( .W .T -raM
zciweik dna .M ,iksworoiweiW Nucl. Acids Res. Spec. Pub. ,581,4 ;)8791( .W .T ,zciweikraM
E. Biala, .R .W Adamiak, .K ,kaiwoksezrG R. Kierzek, A. ,ikswezsarK .J ,iksniwatS and
.M ,iksworoweiW NucL Acids Res. Symp. ,7 511 .)0891(
6 CITEHTNYSIMES METHODOLOGIES 1
Synthesis of 2'-O-ACE Protected Nucleoside (2): Reaction )i(
The ACE orthoester is introduced onto the 2'-hydroxyl by reacting
the N-acyI-TIPS nucleoside with the trisorthoformate reagent under acid
catalysis. The 2'-hydroxyl displaces one of the alcohols on the orthoformate
reagent (Fig. 3) to produce the desired product (2). As described later, the
reaction proceeds under high vacuum to remove the 2-acetoxyethanol by-
product and drive the reaction forward. An improved method for introduc-
ing the 2'-O-ACE orthoester is currently being developed and will be
reported shortly.
Procedure. N-acyl-TIPS-nucleoside (1) (1 equivalent, 01 mmol) is re-
acted neat with tris(2-acetoxyethoxy) orthoformate (322.31 g/mol, 5.6
equivalent, 18.04 g) and pyridinium p-toluene sulfonate (251.31 g/mol, 0.2
equivalent, 0.50 g) at 55 ° for 3 hr under high vacuum (<0.015 mm Hg).
The reaction is cooled to room temperature, neutralized with N,N,N',N'-
tetramethylethylenediamine (TEMED) (150 ml/mol, 0.5 equivalent, 0.75
ml), diluted with 50 ml dichloromethane (DCM) and 150 ml hexanes, and
purified on 300 g silica gel (Merck-VWR Scientific) with a hexane/ethyl
acetate gradient. Column chromatography removes the neutralized catalyst
but does not yield pure product because the excess orthoformate reagent
generally eluted with the nucleoside product. However, the excess reagent
does not interfere with the following reaction and it is easily removed
during purification of the next nucleoside intermediate (3). Therefore, the
semipurified product is carried through to the next reaction.
Removal of 5'-Y-TIPS Protecting Group: Reaction (ii)
The 5'-3'-TIPS group is removed with fluoride ions, e.g., tetrabutyl-
ammonium fluoride or amine hydrofluoride salts. These salts can chro-
matograph with the product and complicate purification. (Tetrabutylam-
monium fluoride and triethylammonium hydrofluoride are known to cause
this problem.) Therefore, a very polar amine salt of hydrofluoric acid is
used to ensure that during chromatography these salts do not elute with
the product.
H ase '4"
1
FIG. 3. Reaction of protected nucleoside with tris(2-acetoxyethoxy) orthoformate. Base is
either N-isobutyryladenine, N-acetylcytosine, N-isobutyrylguanine, or uracil.
1 5'-SILYL-2'-ACE RNA EmTOELCUNOGILO SISEHTNYS 7
Procedure. To TEMED (150 ml/mol, 5 equivalent, 7.50 ml) in acetoni-
trile (CH3CN) (100 ml) is slowly added 48% hydrofluoric acid (36 ml/mol,
3.5 equivalent, 1.26 ml) at 0 .° This solution is then added to compound .2
The reaction proceeds at room temperature with mixing. After 6 hr, the
CH3CN is removed under vacuum, but not to dryness. The residue is
resuspended in 100 ml of DCM and purified on 300 g of silica gel with an
ethyl acetate/methanol gradient. The overall yield from 1 reactions (i) and
(ii) was 40-70%. The 2'-ACE uridine nucleoside is a clear oil and the
remaining three nucleosides are white foams.
5'-O-Silylation: Reaction (iii)
The steric hindrance of the bis(trimethylsiloxy)cyclododecyloxysilyl
chloride (DOD-CI) silylating reagent permits the silyl chloride to react
preferentially with the primary 5'-hydroxyl group over the secondary 3'-
hydroxyl. The silyl chloride will react with the 3'-hydroxyl but factors such
as a slow rate of addition and low temperature enhance the selectivity and
increase yields.
Procedure. To a solution of 2'-O-ACE-nucleoside (3) (1 equivalent, 01
retool) and imidazole (68.08 g/mol, 4 equivalent, 2.72 g) in tetrahydro-
furan (50 ml) at 0 ° is added bis(trimethylsiloxy)cyclododecyloxysilyl chlo-
ride (DOD-CI) (424 g/mol, 5.1 equivalent, 6.36 g in 20 ml tetrahydrofuran)
over 30 min with stirring. The reaction is worked up by adding 70 ml of
ethyl acetate, washing with saturated sodium chloride and drying the organic
phase over sodium sulfate. The solvent is removed from the organic phase
and the residue resuspended in 50 ml DCM and 051 ml hexanes. The 5'-
silyl-2'-ACE nucleoside product (4) is purified on 300 g silica gel with a
hexane/ethyl acetate gradient in the presence of 20% acetone. The products
are isolated as oils or oily foams in 75-85% yields.
Y-O-Phosphitylation: Reaction (iv)
The final nucleoside phosphoramidite products are synthesized using
the bis(N,N,-diisopropylamine)methoxyphosphine method. 5
Procedure. To a solution of a 5'-O-silyl-2'-O-ACE-nucleoside
(1 equivalent, 01 mmol) in 25 ml of dry dichloromethane is added bis(N,N-
diisopropylamine)methoxyphosphine (262 g/mol, 5.1 equivalent, 3.93 g)
and then tetrazole (70 g/mol, 0.8 equivalent, 0.56 g) with stirring. After
4 hr the reaction is washed with saturated sodium chloride and the organic
phase dried over sodium sulfate. The nucleoside phosphoramidite product
(5) is purified on 300 g silica gel with a hexane/dichloromethane gradient
5 A. D. Barone, J.-Y. Tang, and M. H. Caruthers, Nucleic Acids Res. 12, 4051 (1984).
