Introducing structural diversity in oligonycleotides via photolabile, convertible C5-substituted nucleotides

Article abstract of DOI:10.1021/ja983273r

Chemically synthesized oligonucleotides are functionalized at defined sites while in their protected form on solid-phase supports via the incorporation of nucleotides containing masked alkyl carboxylic acids or alkylamines. The reactive functional groups

Full text of DOI:10.1021/ja983273r

VOLUME 121, NUMBER 4
FEBRUARY 3, 1999
© Copyright 1999 by the
American Chemical Society
Introducing Structural Diversity in Oligonucleotides via Photolabile,
Convertible C5-Substituted Nucleotides
Jeffrey D. Kahl and Marc M. Greenberg*
Contribution from the Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523
ReceiVed September 14, 1998
Abstract: Chemically synthesized oligonucleotides are functionalized at defined sites while in their protected
form on solid-phase supports via the incorporation of nucleotides containing masked alkyl carboxylic acids or
alkylamines. The reactive functional groups are selectively revealed upon 365 nm irradiation of the appropriate
o-nitrobenzyl based protecting group. For the photolabile nucleotide that reveals alkylamines, a combination
of analytical methods revealed that very high yields (g94%) of the oligonucleotide containing a single reactive
functional group were obtained by using photolysis conditions that do not damage the biopolymer. Either
functionalized nucleotide is efficiently coupled to the corresponding reaction partner via PyBOP mediated
amide bond formation. Isolated yields of the conjugated oligonucleotides are g90%. This method is compatible
with introducing multiple modified sites sequentially and is amenable to the synthesis of libraries of
oligonucleotides in a combinatorial fashion containing nonnative nucleotides while bound to their solid supports.
Modified oligonucleotides are finding increasing application
in biological and biophysical studies. These biomolecules have
been equipped with functional molecules including fluorophores,
enzymes, and nuclease mimics.1-3 In addition, RNA and DNA
molecules containing naturally occurring and nonnative nucle-
otides show promise as ligands for nonnucleic acid receptors
and as catalysts.4-8 The C5-position of (deoxy)uridine is a
popular position for introducing structural diversity in oligo-
nucleotides when designing functional biopolymers for applica-
tions as diverse as sensors, sequence selective nucleic acid
binding and/or damaging agents, and aptamers.9-14 Part of the
allure of utilizing C5-substituted pyrimidines is that large
substituents can be incorporated at this position without perturb-
ing the syn/anti equilibrium about the glycosidic bond, or the
overall structure of the biopolymer. The absence of such
perturbations often enables one to incorporate C5-modified
pyrimidine nucleotides enzymatically via their respective triph-
osphates. The compatibility of C5-modified nucleotide triph-
osphates with polymerase enzymes has proven particularly
useful in expanding the diversity of libraries of nucleic acid
ligands.9 Developments in the deconvolution of such libraries
(1) Zuckerman, R. N.; Corey, D. R.; Schultz, P. G. J. Am. Chem. Soc.
1988, 110, 1614.
(2) Truffert, J.-C.; Asseline, U.; Brack, A.; Thuong, N. T. Tetrahedron
1996, 52, 3005.
(3) Prober, J. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F. W.; Robertson,
C. W.; Zagursky, R. J.; Cocuzza, A. J.; Jensen, M. A.; Baumeister, K.
Science 1987, 238, 336.
(4) Carmi, N.; Shultz, L. A.; Breaker, R. R. Chem. Biol. 1996, 3, 1039.
(5) Osborne, S. E.; Ellington, A. D. Chem. ReV. 1997, 97, 349.
(6) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
4262.
(9) Dewey, T. M.; Mundt, A. A.; Crouch, G. J.; Zyzniewski, M. C.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 8474.
(10) (a) Haginoya, N.; Ono, A.; Nomura, Y.; Ueno, Y.; Matsuda, A.
Bioconj. Chem. 1997, 8, 271. (b) Kohgo, S.; Shinozuka, K.; Ozaki, H.;
Sawai, H. Tetrahedron Lett. 1998, 39, 4067.
(11) Colocci, N.; Dervan, P. B. J. Am. Chem. Soc. 1994, 116, 785.
(12) (a) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645. (b) Dreyer,
G. B.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 968.
(13) Hurley, D. J.; Tor, Y. J. Am. Chem. Soc. 1998, 120, 2194.
(14) (a) Bashkin, J. K.; Xie, J.; Daniher, A. T.; Sampath, U.; Kao, L.-F.
J. Org. Chem. 1996, 61, 2314. (b) Bashkin, J. K.; McBeath, R. J.; Modak,
A. S.; Sample, K. R.; Wise, W. B. J. Org. Chem. 1991, 56, 3168.
(7) Eaton, B. E.; Pieken, W. A. Annu. ReV. Biochem. 1995, 64, 837.
(8) Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54.
10.1021/ja983273r CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/16/1999

598 J. Am. Chem. Soc., Vol. 121, No. 4, 1999
Kahl and Greenberg
Scheme 1a
a variety of small organic molecules and tripeptides via amide
bond formation proceeds in no less than 83% isolated yield,
and quite often in yields that are in excess of 90%. Although a
variety of activation methods have been employed, benzotriazol-
1-yloxytripyrrolidinophosphonium hexafluorophosphate (Py-
BOP) is the activating agent of choice. In addition, reactions
are typically carried out in e2 h between 25 and 55 °C with
e10 equiv of activating agent and small molecule reactant
relative to oligonucleotide substrate. More recently, even more
efficient conjugation has been effected between support-bound
protected oligonucleotides containing 5′-alkylamines and small
molecules.20
by mass spectrometry could provide further impetus for the
incorporation of modified nucleotides in oligonucleotide librar-
ies.15
Independent synthesis of the requisite nucleotide triphosphates
and phosphoramidites (for chemical oligonucleotide synthesis)
can be a lengthy process, although a one-step process that is
applicable for some C5-modified pyrimidines has been reported.9
In an alternative method, a single C5-substituted nucleotide is
modified following its incorporation into an oligonucleotide.10
The use of such convertible nucleosides has the advantage that
only a single phosphoramidite (or nucleotide triphosphate) needs
to be synthesized, making the method convergent and potentially
suitable for constructing libraries of modified oligonucleotides.
Convertible nucleosides functionalized at positions other than
C5 have also been employed for introducing structural diversity
in oligonucleotides.16-18 In general, the previously reported
methods utilizing convertible nucleosides require large excesses
of functionalizing agent (e.g., alkylamines) and reaction periods
as long as 3 days.17 We wish to describe an alternative method
for introducing structural diversity in oligonucleotides from
convertible nucleosides in which the functionalization reaction
is rapid, utilizes reagents efficiently, and produces high yields
of modified oligonucleotides. Convertible nucleotides containing
photolabile triggers are incorporated at defined sites in chemi-
cally synthesized oligonucleotides (Scheme 1). The support
bound, protected oligonucleotides are conjugated following
photolysis, and then deprotected and purified in the usual
fashion. A further advantage of carrying out the conjugation
chemistry while the polymer is on the solid support is the ease
of separation of the product from activating agents and other
reactants. In addition to facilitating the synthesis of oligonucle-
otides containing modified nucleotides, the method is compatible
with the chemical synthesis of oligonucleotide libraries contain-
ing nonnative nucleotides.
Phosphoramidite and Oligonucleotide Synthesis. We an-
ticipated that incorporation of C5-substituted molecules contain-
ing alkylamines (1) or alkyl carboxylic acids (3), protected with
photolabile groups as their respective phosphoramidites (12, 17),
would facilitate the postsynthetic introduction of functional
groups at defined sites in chemically synthesized biopolymers
and would enjoy the benefits of the convertible nucleoside
approach mentioned above (eqs 1 and 2). While one can
envision a variety of tethers between the C5-position of the
nucleoside and the conjugatable functional group(s), we chose
an alkyl alkynyl linker to demonstrate this method. The selection
of this linker is based upon its ease of synthesis via Pd(0)
chemistry, and the sufficient length of the alkyl chain which
was expected to provide a sterically unhindered environment
in the region of the reactive functional group.24
The silylated alkynol (5) served as a common intermediate
for the preparation of 12 and 17. However, one should note
that the resulting electrophilic (4) monomer contains one less
methylene unit in its alkyl tether than its nucleophilic comple-
ment (2). Conversion of 5, which was readily prepared from
5-iododeoxyuridine, to the respective alkylamine was achieved
via reduction of the azide (7, Scheme 2). Due to difficulties in
purifying the alkylamine, it was carried on in crude form to the
bis-silyl protected photolabile carbamate (9). Synthesis of the
Results and Discussion
The strategy presented below capitalizes on developments
for selectively unmasking a single functional group within a
protected oligonucleotide, and for conjugating oligonucleotides
to small organic molecules in solution or while anchored to a
solid-phase support.19-22 Oligonucleotides containing 3′-alky-
lamines or 3′-alkyl carboxylic acids which retain their nucleo-
base, phosphate, and 5′-hydroxyl protecting groups are obtained
from photolabile, orthogonal solid-phase synthesis supports that
are based upon the o-nitrobenzyl photoredox process.21,23
Solution-phase coupling of these protected oligonucleotides to
(15) Pomerantz, S. C.; McCloskey, J. A.; Tarasow, T. M.; Eaton, B. E.
J. Am. Chem. Soc. 1997, 119, 3861.
(16) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J. Am. Chem. Soc.
1997, 119, 7423.
(17) Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T. M. J. Am. Chem.
Soc. 1991, 113, 4328.
(18) (a) Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J. Org. Chem. 1992, 57,
3839. (b) Xu, Y.-Z. Tetrahedron 1996, 52, 10737.
(19) (a) McMinn, D. L.; Matray, T. J.; Greenberg, M. M. J. Org. Chem.
1997, 62, 7074. (b) McMinn, D. L.; Greenberg, M. M. J. Am. Chem. Soc.
1998, 120, 3289.
(20) Kahl, J. D.; McMinn, D. L.; Greenberg, M. M. J. Org. Chem. 1998,
63, 4870.
(22) (a) Haralambidis, J.; Angus, K.; Pownall, S.; Duncan, L.; Chai, M.;
Tregear, G. W. Nucleic Acids Res. 1990, 18, 501. (b) Kremsky, J. N.;
Pluskal, M.; Casey, S.; Perry-O’Keefe, H.; Kates, S. A.; Sinha, N. D.
Tetrahedron Lett. 1996, 37, 4313.
(23) McMinn, D. L.; Greenberg, M. M. Tetrahedron 1996, 52, 3827.
(24) (a) Hobbs, F. W. J. Org. Chem. 1989, 54, 3420. (b) Casalnuovo,
A. L.; Calabrese, J. C. J. Am. Chem. Soc. 1990, 112, 4324. (c) Osborne, S.
E.; Cain, R. J.; Glick, G. D. J. Am. Chem. Soc. 1997, 119, 1172.
(21) Kahl, J. D.; Greenberg, M. M. J. Org. Chem. In press.

Introducing Structural DiVersity in Oligonucleotides
J. Am. Chem. Soc., Vol. 121, No. 4, 1999 599
Scheme 2a
chemically synthesized oligonucleotide (18) by a variety of
methods. The deprotected alkylamine (21b) and acetylated
alkylamine (21c, produced via capping and/or acetylation during
detritylation) oligonucleotides isolated following photolysis and
deprotection/cleavage were separable by gel electrophoresis and
anion exchange HPLC from carbamate (21a) containing oligo-
nucleotide. Irradiation of the support-bound, protected biopoly-
mer (18) was carried out for 1 h with use of a transilluminator
at 365 nm, as previously described for the photochemical
cleavage of protected oligonucleotides from solid-phase sup-
ports.23,26 Subsequent anion exchange HPLC analysis of 21
following deprotection indicated that photoconversion of 1 to
2 was complete. Similarly, complete conversion of 1 to 2 was
observed upon photolysis of 19 for 1 h. In contrast, gel
electrophoretic analysis of photodeprotected oligonucleotide
containing 1 at position 17 (20) revealed that a small amount
of carbamate remained after photolysis for 1 h, but none after
2 h of irradiation. Isolated yields of oligonucleotides (obtained
as a mixture of 23b and 23c), which were 92% and 98%
following irradiation for 1 and 2 h, respectively, were consistent
with these observations. The less efficient photolysis of 1 when
it is positioned closer to the 3′-terminus (and consequently the
solid-phase support) is consistent with the observations that
photolabile solid-phase supports containing o-nitrobenzyl linkers
require longer irradiation times than do 1 to achieve comparable
photoconversion, and that oligonucleotide array synthesis
becomes more efficient as the photolabile nucleotides are
incorporated further from the solid support.23,26,27
Scheme 3a
carboxylated analogue was accomplished via oxidation of 5,
and esterification of the bis-silylated carboxylic acid (13,
Scheme 3). The respective photolabile bis-silyl compounds (9,
14) were carried on to 12 and 17 via standard dimethoxytrity-
lation and phosphitylation, following desilylation.
On the basis of previous observations with respect to
photostability and propensity for phenoxyacetyl protected exo-
cyclic amines of nucleobases to undergo transamidation, the
respective N-isobutyryl protected phosphoramidites of dA, dC,
and dG were employed during the synthesis of oligonucleotides
containing 1 and 3.23,25,26 The oligonucleotides containing the
photolabile monomers at defined sites were prepared via
standard automated cycles on either 0.2 or 1.0 mmol scale. No
differences were observed in the coupling of 12 and 17
compared to the standard, commercially obtained phosphora-
midites.
Characterization of the Photodeprotection of 1 in Oligo-
nucleotides. Treatment of carbamate 10 with concentrated
aqueous ammonia at 55 °C for 24 h revealed that this functional
group was stable to the oligonucleotide deprotection conditions.
Consequently, it was possible to analyze the efficiency of the
photochemical deprotection following incorporation of 12 in a
An alternative method for the quantitative analysis of the
photochemical deprotection process (eq 1) involved examination
of the free nucleosides released upon enzymatic digestion of
the polymer obtained starting from 24. For this method of
analysis, the oligonucleotides containing photochemically re-
(25) McMinn, D. L.; Greenberg, M. M. Tetrahedron Lett. 1997, 38, 3123.
(26) Venkatesan, H.; Greenberg, M. M. J. Org. Chem. 1996, 61, 525.
(27) McGall, G. H.; Barone, A. D.; Diggelmann, M.; Fodor, S. P. A.;
Gentalen, E.; Ngo, N. J. Am. Chem. Soc. 1997, 119, 5081.

600 J. Am. Chem. Soc., Vol. 121, No. 4, 1999
Kahl and Greenberg
Table 1. Coupling Yields of Oligodeoxynucleotide Conjugates
Prepared by On-Column Derivatization
leased alkylamine groups were capped with acetic anhydride
(to facilitate subsequent nucleoside analysis), and the biopolymer
was deprotected with concentrated aqueous ammonia. Following
enzymatic digestion, the free nucleosides were analyzed by
reverse-phase HPLC.28 On the basis of detection limits estab-
lished by using an internal standard and measured response
factors of independently synthesized 10 and 25, it was deter-
mined that a 2 h photolysis yielded g94% conversion of the
carbamate to the reactive alkylamine. The conclusion drawn
from these experiments was that regardless of the position where
1 was incorporated in an oligonucleotide, the carbamate could
be completely converted to the conjugatable alkylamine (2) via
a 2 h photolysis at 365 nm by using a readily available UV-
light source. One should note that these photolysis conditions
have been shown previously to not damage the oligonucle-
otides.23,26
On-Column Conjugation of C5-Substituted Deoxyuridines.
C5′-Terminal primary amines in oligonucleotides, which are
bound to their solid supports, serve as suitable substrates for
bioconjugate formation.20,22 We recently reported that oligo-
nucleotide conjugates can be efficiently prepared from alky-
lamines via PyBOP mediated on-column coupling in 15 min at
25 °C, using as few as 2 equiv (typically 5 equiv) of the
appropriate carboxylic acid. This method was extended to
conjugating 2 to a series of alkyl carboxylic acids (eq 1, Table
1). In all cases, average coupling yields exceeded 90% when
carried out for 1 h at 25 °C with 5 equiv of the respective
carboxylic acid and activating agent relative to oligonucleotide.
Conjugate 21f was subjected to enzymatic digestion. Quantita-
tive analysis of the nucleosides released indicated that dA, dC,
dG, T, and 26 were present in the expected ratios.28
The dependency of the efficiency of the coupling process on
the position of the convertible nucleotide in the biopolymer was
investigated by using two eicosameric oligonucleotides which
differed from 18 with respect to which thymidine was substituted
by 1. By using the conjugation conditions described above,
biotin was coupled to 2, generated at position 5 (from 19), in
essentially quantitative yield (22e, 100 ( 2%).29 However, the
isolated yield of 23e, in which 1 is incorporated at position 17
(20), was consistently slightly lower (84 ( 5%) than at other
a Isolated yields were determined via comparing the amount of
oligonucleotide obtained from a comparable amount of unconjugated
material subjected to the identical deprotection, purification, and
isolation conditions. b Yields represent an average of separate reactions
( standard deviation from this value. The number of reactions run are
noted in parentheses.
positions in comparable oligonucleotides.29 Increasing the
number of equivalents of activating agent and biotin relative to
oligonucleotide to 10 did not lead to any improvement in the
isolated yield of 23e (80 ( 1%).29 Three possible explanations
were considered for the lower isolated yield of conjugate
compared to that obtained at other sites of incorporation of 1.
Analysis of the crude conjugate by gel electrophoresis confirmed
that the lower coupling yield at the fourth position was not due
to the presence of unconverted carbamate (23a). However, this
method could not distinguish between unreacted alkylamine (or
the respective acetate which would be formed upon detritylation,
23b and 23c) and isobutyryl amide (23d), as the respective
products were inseparable by gel electrophoresis. Although it
had not been observed previously during conjugation reactions
with this approach, the isobutyrylated product (23d) could arise
via transamidation following photolysis.25 The third explanation
was that a minor population of the alkylamine at the fourth
position from the 3′-terminus in a protected eicosamer bound
to its solid support is inaccessible, and unreactive. To distinguish
(28) See Supporting Information.
(29) The yields were determined as described in the footnote to Table 1
and in refs 19a and 19b.
(30) Eaton, B. E.; Gold, L.; Hicke, B. J.; Janji’c, N.; Jucker, F. M.;
Sebesta, D. P.; Tarasow, T. M.; Willis, M. C.; Zichi, D. A. Bioorganic
Med. Chem. 1997, 5, 1087.

Introducing Structural DiVersity in Oligonucleotides
J. Am. Chem. Soc., Vol. 121, No. 4, 1999 601
between the latter two explanations, an unpurified sample of
23, which had been photolyzed, conjugated to biotin, and
deprotected, was digested. Provided that photolyses of 23 were
cooled with a fan, no isobutyrylated nucleoside (27) was
observed by reverse-phase HPLC analysis. However, a small
amount of the N-acetylated nucleoside (25) was detected. On
the basis of this observation, we hypothesize that 25 is produced
from oligonucleotide containing unreacted free amine upon
detritylation. The decreased reactivity at the 17th position in
an eicosamer may be attributable to lower accessibility when it
is buried closer to the support as part of a longer oligonucleotide.
Scheme 4a
1 at position 2 (position 17 in the ultimate eicosamer, 33) was
synthesized. Following photolytic unmasking of the primary
alkylamine, an equimolar mixture of biotin and N-acetyl
phenylalanine was conjugated to the oligonucleotide as described
above. After washing away the excess reagents, the pentamer
was manually acetylated by using commercially available
automated DNA synthesis capping reagents. An aliquot of the
resin was removed, and the oligonucleotide was enzymatically
digested following standard deprotection/detritylation. HPLC
analysis revealed that the expected amide coupling products (30,
31) were present in equimolar ratio. Carbamate (10), acetate
(25), and isobutyrylated (27) nucleosides were not observed.
The remainder of the resin was placed back on the oligonucle-
otide synthesizer, and the preparation of the protected eicosamer
containing 1 at the fifth position of the eicosamer was completed.
The second site was coupled to pyrene butyric acid and 9-(5-
pentanoic acid)acridine, followed by capping. The crude mixture
of deprotected oligonucleotides (33) was enzymatically digested
and analyzed by reverse-phase HPLC.28 In addition to 30 and
3l being present in a 1:1 ratio, the second set of deoxyuridine
derivatives (26, 32) were also present in equal amounts to one
another. In addition to the conjugated products, the digest of
the crude eicosameric mixture contained a small amount of 25,
but carbamate (10) and isobutyrylated (27) nucleosides were
not observed. The presence of small amounts of unreacted
molecules is inconsequential for preparing a library. The mixture
of oligonucleotides was further characterized by electrospray
mass spectrometry, following their separation from the typical
deletions produced during automated synthesis.
Having established the general reactivity of alkylamine-
containing nucleotides in solid-phase supported oligonucleotides,
attention was turned to reversing the polarity of the bond formed
during the conjugation reaction. It had previously been dem-
onstrated that there is no statistical difference in the yields of
conjugates when protected oligonucleotides containing either
3′-alkylamines or 3′-alkyl carboxylic acids are coupled in
solution.21 We anticipated that support bound oligonucleotides
would exhibit similar reactivity patterns. Consequently, 3 was
incorporated into an oligonucleotide at the 11th position of an
eicosamer (28) that was otherwise identical to 18. Indeed,
conjugation to pyrene butylamine (5 equiv), following photolysis
of the protected oligonucleotide on its solid-phase support for
2 h, yielded 29 in 97 ( 3%.29
Using Photoconvertible Nucleotides To Introduce Struc-
tural Diversity in Oligonucleotides. The efficiency of the
photolysis/coupling process presented above suggested that this
method would be useful for preparing libraries of chemically
synthesized oligonucleotides containing nonnative nucleosides.
For example, this method could be useful for expanding the
diversity of a nucleic acid ligand that had been selected via
enzymatic synthesis for which one or more nucleotides have
been identified as being desirable for modification.30 The
advantage of this chemical process over enzymatic methods is
that one can readily control the site(s) where the biopolymer is
diversified. A further advantage of employing a convertible
nucleotide is that one does not need to chemically synthesize
and purify multiple phosphoramidites. In addition, since cou-
pling is achieved “on-column” without affecting other functional
(protecting) groups in the biopolymer, this method is compatible
with introducing diversity at multiple sites in the oligonucleotide
sequentially (Scheme 4). Other convertible nucleosides are not
amenable to this, because these molecules typically involve
introducing chemical diversity via nucleophilic aromatic
substitution.16-18 The nucleophiles can be expected to at least
partially deprotect the biopolymer, making subsequent chemical
oligonucleotide synthesis impossible.
The principle was demonstrated by preparing a two-site, four-
component library. A pentameric oligonucleotide (24) containing
Summary. Oligonucleotides containing modified C5-deox-
yuridines can be prepared in high yields via “on-column”

602 J. Am. Chem. Soc., Vol. 121, No. 4, 1999
Kahl and Greenberg
Enzymatic Digestion of Modified Oligonucleotides. The oligo-
nucleotide substrate (1 O.D. unit) was evaporated to dryness in a
sterilized Eppendorf tube. The residue was resuspended in 1 M MgCl2
(10 µL), (10×) calf intestine alkaline phosphatase buffer (10 µL),
nuclease P1 (0.3 U/µL, 10 µL), snake venom phosphodiesterase (3 ×
10-3 U/µL, 2 µL), and calf intestine alkaline phosphatase (10 U/µL, 2
µL) and diluted to a total volume of 100 µL with autoclaved, doubly
deionized H2O. The digest was incubated at 37 °C for 36 h. An aliquot
of nuclease P1 (5 µL) was added after each 12 h incubation period.
The digest was filtered through a 0.45 µM Nylon filter. The Eppendorf
tube was rinsed with MeOH (5 × 100 µL), which was passed through
the Nylon filter and combined with the original digest material. The
mixture was evaporated to dryness, resuspended in MeOH (80 µL),
and analyzed by reverse-phase HPLC. HPLC conditions: solvent A,
2.5% CH3CN in KH2PO4 (10 mM, pH 6.0); solvent B, 72% CH3CN in
KH2PO4 (10 mM, pH 6.0); time 0, 100% A; linear gradient to 28% B
over 15 min; hold at 28% B for 20 min; linear gradient to 100% B
over 5 min; hold 100% B for 15 min.
conjugation of protected oligonucleotides containing a single
reactive functional group. Oligonucleotide substrates containing
alkylamines or alkyl carboxylic acids at defined sites are
obtained by utilizing the appropriate photolabile phosphora-
midite. The reactive functional groups are revealed in an
orthogonal manner under photolysis conditions that do not
damage the oligonucleotides. In addition to simplifying the
synthesis of modified oligonucleotides, this method is directly
applicable to expanding the diversity of oligonucleotide libraries.
Experimental Section
General Methods. All reactions were carried out in oven-dried
glassware under an argon atmosphere, unless noted otherwise. Diiso-
propylethylamine, CH2Cl2, pyridine, and DMF (aspirator pressure) were
distilled from CaH2. THF was distilled from Na°/benzophenone ketyl.
Oligonucleotides were synthesized by using an Applied Biosystems
Incorporated 380B automated synthesizer on standard succinato long
chain alkylamine controlled pore glass support purchased from Glen
Research. Isobutyryl protected phosphoramidites were purchased from
Glen Research and Pharmacia Biotech. Reverse-phase HPLC was
carried out on a Rainin C18-Microsorb column (4.6 × 25 mm). Anion
exchange HPLC was carried out on Vydac No. 301VHP575. Anion
exchange HPLC conditions: solvent A, H2O; solvent B, aqueous NaCl
(1 M); 100% A to 100% B linearly over 40 min.
General Procedure for Photolytic Deprotection of Solid-Phase
Supported Oligonucleotides Containing Modified Nucleotides (1,
2). Oligonucleotide bound to support (5-8 mg) was added to a Pyrex
tube containing a stir bar constructed from a standard pipe cleaner (in
order to not crush the CPG containing the oligonucleotide) and CH3-
CN/H2O (12 mL, 9:1 by volume). The tube was fitted with a rubber
septum and sparged with N2 for 20 min, after which the needle was
raised well above the surface of the solvent. Photolyses were carried
out with a VWR Chromato-Vue transilluminator (λmax ) 365 nm) for
2 h. It is important to maintain the temperature at e25 °C with a fan.
The resin was filtered, washed with EtOAc (15 mL), followed by Et2O
(15 mL), collected, and placed in a screw-capped vial for storage.
Sample General Procedure for Conjugation of Protected Resin-
Bound Oligonucleotides. A solution (33 mM) of the coupling reagents
(11.2 mg of PyBOP and 8 µL of diisopropylethylamine (2 molar equiv))
in DMF (653 µL) was prepared in an oven-dried 1 dram vial equipped
with a septum. A solution (33 mM) of biotin (7.1 mg in 746 µL of
DMF) was prepared in a second oven-dried vial. The resin-bound DNA
(2 mg, containing ∼66 nmol of DNA, based on trityl response) was
treated with 20 µL (5 molar equiv) of a 1:1 mixture (by volume) of
the PyBOP and biotin solutions. The reaction was capped and shaken
at room temperature for 1 h. The resin was washed with DMF (3 × 50
µL) and dried in vacuo. The free-flowing resin was treated with 28%
aqueous ammonia (600 µL) for 6 h at 55 °C and concentrated under
vacuum. Detritylation was effected by adding 80% aqueous acetic acid
(100 µL) to the resin for 20 min, at which time the reaction was
quenched with absolute EtOH (100 µL) and concentrated to dryness.
Purification of the conjugated oligonucleotide was carried out via 20%
polyacrylamide denaturing gel electrophoresis. Isolated yields were
obtained (O.D. 260 nm) by comparing the amount of conjugated
oligonucleotide to the amount of unconjugated material subjected to
identical deprotection, purification, and isolation conditions.
Synthesis of an Oligonucleotide Library. The pentameric oligo-
nucleotide (24) was synthesized on a 1 µmol scale with standard DNA
synthesis cycles. Resin corresponding to 0.2 µmol of the original loading
was subjected to the photolysis and conjugation conditions described
above, with the exception that biotin and N-acetyl phenylalanine were
each present in 5 molar equiv relative to oligonucleotide. Following
conjugation and washing of the support with DMF (3 × 100 µL) and
CH3CN (3 × 100 µL) sequentially, the support was placed back on
the DNA synthesizer and extended to the fully protected eicosamer
containing 1 at position 5. The resin was then photolyzed and conjugated
to pyrene butyric acid and the acridine carboxylic acid as described.
Deprotection was carried out via standard methods following removal
of the excess reagents from the resin and washing, as described above.
Preparation of 5. To a solution of 3′,5′-O-bis-(tert-butyldimethyl-
silyloxy)-5-iododeoxyuridine (701 mg, 1.20 mmol) in degassed DMF
(10 mL) were added with stirring (PhP3)4Pd (139 mg, 0.12 mmol), CuI
(46 mg, 0.24 mmol), Et3N (243 mg, 2.4 mmol), and 5-hexyn-1-ol (353
mg, 3.6 mmol). The reaction mixture was allowed to stir for 18 h at
25 °C, at which time it was diluted with EtOAc (50 mL) and poured
into a separatory funnel containing saturated NaHCO3 (25 mL). The
aqueous layer was extracted with EtOAc (2 × 25 mL). The combined
organic layers were washed with brine (20 mL), dried over MgSO4,
filtered, and concentrated under vacuum. Chromatography (1:4, EtOAc:
Hex to 2:3, EtOAc:Hex) yielded 5 (461 mg, 70%) as a light yellow
foam. 1H NMR (CDCl3): δ 8.99 (s, 1H), 7.89 (s, 1H), 6.27 (dd, 1H, J
) 6.6, 6.3 Hz), 4.38 (m, 1H), 3.89 (m, 2H), 3.71 (m, 3H), 2.39 (t, 2H,
J ) 6.3 Hz), 2.27 (m, 2H), 1.99 (p, 2H, J ) 7.5 Hz), 1.68 (m, 4H),
0.91 (s, 9H), 0.86 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H), 0.05 (s, 3H), 0.05
(s, 3H). 13C NMR (CDCl3): δ 162.1, 149.2, 141.5, 100.7, 95.1, 88.3,
85.6, 72.2, 62.9, 62.2, 41.9, 31.9, 26.0, 25.7, 24.6, 19.4, 18.4, 18.0,
-4.7, -4.9, -5.4, -5.5. IR (film): 3423, 3189, 3069, 2929, 2857,
1698, 1626, 1462, 1279, 1255, 1104 cm-1. Anal. Calcd for C27H48N2O6-
Si2: C, 58.66; H, 8.76; N, 5.07. Found: C, 58.64; H, 8.68; N, 4.91.
Preparation of the Mesylate of 5 (6). To a solution of 5 (462 mg,
0.84 mmol) in CH2Cl2 (8 mL) at 0 °C was added with stirring
diisopropylethylamine (152 mg, 1.18 mmol) and methanesulfonyl
chloride (115 mg, 1.0 mmol) dropwise over 5 min. The mixture was
warmed to room temperature over a 20 min period, at which time TLC
analysis (4:1, EtOAc:Hex) indicated that the starting material had been
completely consumed. The reaction was diluted with Et2O (15 mL)
and poured into a separatory funnel containing saturated NaHCO3 (10
mL). The aqueous layer was extracted with Et2O (2 × 10 mL). The
combined organic layers were washed with brine (10 mL), dried over
MgSO4, filtered, and concentrated under vacuum. Chromatography (3:
7, EtOAc:Hex to 3:2, EtOAc:Hex) yielded the mesylate 6 (520 mg,
1
99%) as a white foam. H NMR (CDCl3): δ 8.81 (s, 1H), 7.90 (s,
1H), 6.26 (dd, 1H, J ) 7.2, 6.3 Hz), 4.37 (m, 1H), 4.25 (t, 2H, J ) 6.0
Hz), 3.94 (m, 1H), 3.87 (dd, 1H, J ) 11.4, 2.1 Hz), 3.73 (dd, 1H, J )
11.4, 2.1 Hz), 2.98 (s, 3H), 2.42 (t, 2H, J ) 6.6 Hz), 2.27 (m, 1H),
3.01-1.84 (m, 4H), 1.67 (m, 3H), 0.91 (s, 9H), 0.96 (s, 9H), 0.11 (s,
3H), 0.10 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H). 13C NMR (CDCl3): δ
161.8, 149.2, 141.8, 100.5, 93.8, 88.3, 85.6, 72.3, 69.5, 63.0, 42.0, 37.4,
28.3, 26.0, 25.7, 24.3, 19.1, 18.4, 18.0, -4.7, -4.9, -5.4, -5.5. IR
(film): 3186, 3065, 2952, 2929, 2856, 1712, 1461, 1354, 1279, 1255,
1173, 1104 cm-1
.
Preparation of 7. To a solution of 6 (340 mg, 0.54 mmol) in DMF
(5 mL) was added NaN3 (207 mg, 2.7 mmol). The reaction mixture
was heated to 35 °C for 2 h, after which it was diluted with EtOAc (15
mL) and poured into a separatory funnel containing saturated NaHCO3
(10 mL). The aqueous layer was extracted with EtOAc (2 × 10 mL).
The combined organic layers were washed with brine (15 mL), dried
over MgSO4, filtered, and concentrated under vacuum. Chromatography
(1:4, EtOAc:Hex) yielded 7 (303 mg, 95%) as a white foam. 1H NMR
(CDCl3): δ 8.35 (s, 1H), 7.89 (s, 1H), 6.27 (dd, 1H, J ) 7.5, 6.0 Hz),
4.38 (m, 1H), 3.95 (m, 1H), 3.88 (dd, 1H, J ) 11.4, 2.4 Hz), 3.74 (dd,
1H, J ) 11.4, 2.1 Hz), 3.30 (t, 2H, J ) 6.6 Hz), 2.40 (t, 2H, J ) 6.6

Introducing Structural DiVersity in Oligonucleotides
J. Am. Chem. Soc., Vol. 121, No. 4, 1999 603
Hz), 2.27 (m, 1H), 1.74-1.58 (m, 5H), 0.92 (s, 9H), 0.86 (s, 9H), 0.11
(s, 3H), 0.10 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H). 13C NMR (CDCl3): δ
161.7, 149.2, 141.6, 100.5, 94.0, 88.2, 85.6, 72.3, 72.1, 62.9, 51.0, 41.9,
28.1, 25.9, 25.6, 25.5, 19.2, 18.4, 18.0, -4.7, -4.9, -5.4, -5.6. IR
(film): 3186, 3066, 2953, 2929, 2857, 2359, 2341, 2096, 1715, 1624,
1576, 1558, 1546, 1506, 1458, 1404, 1360, 1322, 1279, 1255, 1196,
1104, 1068, 1030 cm-1. Anal. Calcd for C27H47N5O5Si2: C, 56.12; H,
8.20; N, 12.13. Found: C, 55.96; H, 7.96; N, 11.84.
(dd, 1H, J ) 8.1, 2.9 Hz), 3.25 (dd, 1H, J ) 8.1, 2.9 Hz), 3.07 (m,
2H), 2.50 (m, 1H, J ) 6.3 Hz), 2.26 (m, 1H), 2.10 (t, 1H, J ) 6.9 Hz),
1.42 (m, 2H), 1.27 (m, 2H). 13C NMR (CDCl3): δ 166.7, 157.5, 154.0,
151.5, 149.0, 144.7, 143.5, 140.0, 137.1, 135.2, 131.1, 125.5, 124.0,
123.6, 122.5, 108.8, 105.7, 103.6, 96.3, 91.6, 82.5, 81.9, 81.0, 67.8,
66.7, 58.9, 58.8, 55.9, 51.9, 50.7, 37.0, 35.9, 24.3, 20.6, 16.6, 14.6,
9.7. IR (film) 3374, 2936, 2359, 1698, 1607, 1581, 1509, 1461, 1328,
1277, 1249, 1220, 1176, 1066 cm-1
.
Preparation of 12. To a solution of 11 (241 mg, 0.28 mmol) in
CH2Cl2 (10 mL) protected from light was added diisopropylethylamine
(180 mg, 1.4 mmol). The reaction was cooled to 0 °C and 2-cyanoethyl
diisopropylchlorophosphoramidite (69.3 mg, 0.29 mmol) was added
dropwise over 15 min. After 30 min, the reaction was quenched with
methanol (12 mL, 0.32 mmol). The mixture was diluted with freshly
distilled EtOAc (15 mL) and poured into a separatory funnel containing
saturated Na2CO3 (15 mL). The organic layer was washed with brine
(15 mL), dried over Na2SO4, filtered, and concentrated under vacuum.
Chromatography (9:1, EtOAc:Hex) yielded 12 (286 mg, 78%) as a light
yellow foam. 1H NMR (CDCl3): δ 8.94 (br, 1H), 8.16 (s, 0.5H), 8.11
(s, 0.5H), 7.72 (s, 1H), 7.72-7.24 (m, 9H), 7.07 (s, 1H), 6.86 (m, 4H),
6.11 (m, 1H), 5.51 (s, 2H), 5.12 (m, 1H), 4.60 (m, 1H), 4.22-4.12 (m,
1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.89-3.45 (m, 8H), 3.31-3.28 (m,
5H), 3.05 (p, 1H, J ) 6.6 Hz), 2.67-2.5 (m, 2H), 2.33 (p, 2H, J ) 6.1
Hz), 2.08 (p, 2H, J ) 6.1 Hz), 1.45-1.35 (m, 2H), 1.31-1.18 (m,
10H), 1.06 (t, 3H, J ) 6.6 Hz). 31P NMR (MeOH-d4, H3PO4
reference): δ 149.6, 149.2. HRMS (FAB) calcd 1065.4375 (M + H),
found 1065.4373.
Preparation of 9. To a solution of 7 (302 mg, 0.51 mmol) in THF
(5 mL) was added triphenylphosphine (141 mg, 0.53 mmol). After 6 h
water (14 mL, 0.77 mmol) was added. The reaction was stirred for an
additional 6 h, after which the volatiles were removed under vacuum
to afford a white foam. Due to difficulties with purification, the crude
amine (8) was carried on as is. To a solution of 8 (287 mg, 0.51 mmol)
in THF (10 mL) was added diisopropylethylamine (92 mg, 0.77 mmol),
followed by neat 4,5-dimethoxy-2-nitrobenzyl chloroformate (155 mg,
0.56 mmol). The reaction was protected from light and stirred at 25
°C for 1 h. The reaction mixture was diluted with Et2O (15 mL) and
poured into a separatory funnel containing saturated NaHCO3 (15 mL).
The aqueous layer was extracted with Et2O (2 × 10 mL). The combined
organic layers were washed with brine (15 mL), dried over MgSO4,
filtered, and concentrated under vacuum. Chromatography (2:3, EtOAc:
Hex) afforded the carbamate 9 (309 mg, 75% yield over two steps) as
1
a light yellow foam. H NMR (CDCl3): δ 8.46 (s, 1H), 7.91 (s, 1H),
7.68 (s, 1H), 7.02 (s, 1H), 6.26 (t, 1H, J ) 6.3 Hz), 5.48 (s, 2H), 5.30
(m, 1H), 4.38 (m, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.88 (dd, 1H, J )
11.4, 1.8 Hz), 3.24 (m, 2H), 2.38 (t, 2H, J ) 6.3 Hz), 2.27 (m, 1H),
1.99 (m, 1H), 1.64 (m, 6H), 0.91 (s, 9H), 0.86 (s, 9H), 0.11 (s, 3H),
0.10 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H). 13C NMR (CDCl3): δ 162.1,
155.9, 153.5, 149.1, 147.9, 141.6, 139.6, 128.7, 109.9, 108.0, 100.5,
94.6, 88.3, 85.6, 72.2, 72.0, 63.3, 62.9, 56.4, 56.3, 41.9, 40.4, 28.9,
25.9, 25.7, 25.2, 19.2, 18.4, 17.9, -4.7, -4.9, -5.4, -5.6. IR (film):
3343, 3183, 3067, 2952, 2930, 2856, 2359, 2340, 1713, 1621, 1581,
1521, 1461, 1327, 1277, 1255, 1220, 1124, 1067 cm-1. Anal. Calcd
for C37H58N4O11Si2: C, 56.18; H, 7.39; N, 7.08. Found: C, 56.24; H,
7.17; N, 6.92.
Preparation of 13. Pyridinium dichromate (3.93 g, 10.5 mmol) was
added to a solution of 5 (1.15 g, 2.1 mmol) in DMF (10 mL) with
stirring. The reaction was allowed to stir at room temperature for 3 h,
after which two drops of trifluoroacetic acid were added. TLC analysis
(4:1, EtOAc:Hex) indicated that the starting material was completely
consumed within 15 min. The reaction was diluted with EtOAc (60
mL) and poured into a separatory funnel containing H2O (40 mL). The
aqueous layer was extracted with EtOAc (2 × 30 mL). The combined
organic layers were washed with brine (25 mL), dried over MgSO4,
filtered, and concentrated in vacuo to afford the crude acid as an orange
oil. Typically, the crude product (13) was carried on without purifica-
tion. However, 13 could be purified by chromatography (4:1, EtOAc:
Preparation of Free Carbamate Nucleoside (10). To a solution
of 9 (309 mg, 0.38 mmol) in DMF (1 mL) at 25 °C (protected from
light) was added a solution (0.5 M) of tetrabutylammonium fluoride
(1.68 mL, 0.84 mmol) in DMF.31 After 1 h, the reaction was diluted
with EtOAc (25 mL) and poured into a separatory funnel containing
saturated NaHCO3 (15 mL). The aqueous layer was extracted with
EtOAc (5 × 15 mL). The combined organic layers were washed with
brine (15 mL), dried over MgSO4, filtered, and concentrated under
vacuum. Chromatography (1:19, MeOH:EtOAc) yielded the free
1
Hex to EtOAc) H NMR (CDCl3): δ 9.09 (s, 1H), 7.98 (s, 1H), 6.32
(t, 1H, J ) 6.3 Hz), 4.44 (m, 1H), 4.0 (m, 1H), 3.94 (d, 1H, J ) 11.1
Hz), 3.79 (d, 1H, J ) 11.4 Hz), 2.54 (m, 4H), 2.34 (m, 1H), 2.08 (m,
1H), 1.96 (m, 2H), 0.97 (s, 9H), 0.93 (s, 9H), 0.18 (s, 3H), 0.17 (s,
3H), 0.12 (s, 3H), 0.11 (s, 3H). IR (film): 3188 (bd), 3064, 3033, 2922,
2850, 2358, 1697, 1497, 1449, 1381, 1337, 1309, 1295, 1272, 1214,
1170 cm-1. HRMS (FAB) calcd 567.2922 (M + H), found 567.2921.
Preparation of 14. To a solution of 13 (1.18 g, 2.1 mmol) in CH2-
Cl2 (25 mL) was added DMAP (26 mg, 0.21 mmol), DCC (431 mg,
2.31 mmol), and dimethoxynitrobenzyl alcohol (446 mg, 2.31 mmol).
The reaction mixture was protected from light. After the solution was
stirred for 24 h, the reaction was filtered through a pad of Celite (2
cm), and the filter cake rinsed with additional CH2Cl2 (15 mL). The
filtrate was poured into a separatory funnel containing EtOAc (75 mL)
and saturated NaHCO3 (25 mL). The aqueous layer was extracted with
EtOAc (2 × 25 mL) and the combined organic layers washed with
brine (25 mL), dried over MgSO4, filtered, and concentrated in vacuo
to afford a yellow oil. Chromatography (3:2 EtOAc:Hex) afforded 14
(0.95 g, 60% overall from 5) as a light yellow foam. 1H NMR
(CDCl3): δ 8.43 (s, 1H), 7.93 (s, 1H), 7.73 (s, 1H), 7.04 (s, 1H), 6.29
(t, 1H, J ) 6.2 Hz), 5.53 (s, 2H), 4.42 (m, 1H), 4.0 (s, 3H), 3.99 (m,
1H), 3.97 (s, 3H), 3.91 (dd, 1H, J ) 11.4, 1.2 Hz), 3.77 (dd, 1H, J )
11.7, 1.8 Hz), 2.63 (t, 2H, J ) 7.2 Hz), 2.50 (t, 2H, J ) 7.2 Hz), 2.33
(m, 1H), 2.01 (m, 3H), 0.93 (s, 9H), 0.91 (s, 9H), 0.15 (s, 3H), 0.14 (s,
3H), 0.10 (s, 3H), 0.09 (s, 3H). 13C NMR (CDCl3): δ 172.6, 161.7,
153.8, 149.3, 148.4, 142.1, 140.1, 127.4, 110.5, 108.4, 100.6, 93.7,
88.6, 85.9, 72.6, 63.4, 63.2, 56.7, 56.6, 42.2, 33.2, 26.2, 26.0, 23.8,
19.3, 18.6, 18.2, -4.4, -4.6, -5.2, -5.3. IR (film): 3186, 3067, 2953,
2930, 2856, 2359, 1715, 1622, 1581, 1523, 1462, 1327, 1279, 1256,
1221, 1067 cm-1. HRMS (FAB) calcd (M + H) 762.3455, found
762.3467.
1
nucleoside 10 (200 mg, 80%). H NMR (MeOH-d4): δ 8.14 (s, 1H),
7.72 (s, 1H), 7.14 (s, 1H), 6.18 (t, 1H, J ) 6.3 Hz), 5.44 (s, 2H), 4.36
(m, 1H), 3.94 (s, 3H), 3.89 (s. 3H), 3.79 (dq, 2H, J ) 11.4, 1.8 Hz),
3.17 (m, 2H), 2.41 (t, 2H, J ) 6.3 Hz), 2.20 (m, 2H), 1.61 (m, 4H). IR
(film): 3361, 3066, 2929, 1697, 1521, 1457, 1324, 1270, 1221, 1059
cm-1. Anal. Calcd for C25H30O11N4: C, 53.36; H, 5.38; N, 9.96.
Found: C, 53.50; H, 5.55; N, 9.72.
Preparation of 5′-O-Dimethoxytritylated 10 (11). To a solution
of 10 (200 mg, 0.33 mmol) in pyridine (6 mL) protected from light
was added dimethoxytrityl chloride (134 mg, 0.40 mmol). The reaction
was allowed to stir overnight at 25 °C, after which it was quenched
with excess methanol. The reaction mixture was diluted with EtOAc
(30 mL) and poured into a separatory funnel containing saturated
NaHCO3 (50 mL). The aqueous layer was extracted with EtOAc (2 ×
15 mL). The combined organic layers were washed with brine (15 mL),
dried over MgSO4, filtered, and concentrated under vacuum. Chroma-
tography (EtOAc) afforded 11 (241 mg, 81%), which was contaminated
with a small amount of inseparable pyridinium salts. The material was
used in this form during the phosphoramidite preparation (next step).
1H NMR (CDCl3): δ 8.62 (s, 1H), 8.06 (s, 1H), 7.69 (s, 1H), 7.46 (d,
2H, J ) 1.46 Hz), 7.33 (m, 8H) 7.05 (s, 1H), 6.84 (d, 4H, J ) 8.8 Hz)
6.34 (t, 1H, J ) 7.3 Hz), 5.48 (s, 2H), 5.39 (t, 1H, J ) 5.5 Hz), 4.54
(m, 1H), 4.11 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.79 (s, 6H), 3.43
(31) Majetich, G.; Casares, A.; Chapman, D.; Behnke, M. J. Org. Chem.
1986, 51, 1745.
Preparation of 15. To a solution of 14 (0.95 g, 1.7 mmol) in THF

604 J. Am. Chem. Soc., Vol. 121, No. 4, 1999
Kahl and Greenberg
(5 mL) at room temperature (protected from light) was added
triethylamine trihydrofluoride (1 mL, 8.5 mmol). After the mixture was
stirred for 24 h, the reaction was quenched with saturated NaHCO3
(15 mL). The mixture was extracted with EtOAc (3 × 20 mL), and
the combined organic layers were washed with brine (15 mL), dried
over MgSO4, filtered, and concentrated under vacuum. Chromatography
(5% MeOH, EtOAc) afforded 15 (0.51 g, 74%) as a light yellow foam.
1H NMR (MeOH-d4): δ 8.18 (s, 1H), 7.77 (s, 1H), 7.19 (s, 1H), 6.23
(t, 1H, J ) 6.3 Hz), 5.51 (dd, 2H, J ) 25.2, 15 Hz), 4.42 (m, 1H),
3.99 (s, 3H), 3.95 (s, 3H), 3.83 (dd, 1H, J ) 12.0, 3.3 Hz), 3.75 (dd,
1H, J ) 10.2, 3.3 Hz), 2.67 (t, 2H, J ) 6.9 Hz), 2.52 (t, 2H, J ) 6.6
Hz), 2.27 (m, 2H), 1.97 (m, 2H). IR (KBr) 3446, 3217, 3065, 2943,
2857, 2563, 2356, 1698, 1578, 1523, 1458, 1327, 1273, 1218, 1147,
1060 cm-1. HRMS (FAB) calcd (M + H) 534.1724, found 534.1735.
Preparation of 5′-O-Dimethoxytritylated 15 (16). To a solution
of 15 (438 mg, 0.80 mmol) in pyridine (6 mL) protected from light
was added dimethoxytrityl chloride (335 mg, 1.0 mmol). After the
mixture was stirred overnight at room temperature, the reaction was
quenched with MeOH (5 mL). The reaction mixture was poured into
a separatory funnel containing saturated NaHCO3 (50 mL), and the
aqueous layer was extracted with EtOAc (3 × 30 mL). The combined
organic layers were washed with brine, dried over MgSO4, filtered,
and concentrated under vacuum. Chromatography (EtOAc) afforded
(140 mg, 1.1 mmol). The reaction was cooled to 0 °C, and 2-cyanoethyl
diisopropylchlorophosphoramidite (54 mg, 0.23 mmol) was added
dropwise over 15 min. After the mixture was stirred for an additional
30 min, the reaction was quenched with MeOH (9 mL, 0.28 mmol).
The mixture was diluted with freshly distilled EtOAc (15 mL) and
poured into a separatory funnel containing saturated Na2CO3 (15 mL).
The organic layer was washed with brine (15 mL), dried over Na2SO4,
filtered, and concentrated under vacuum. Chromatography (9:1, EtOAc:
1
hexane) afforded 17 (191 g, 85%) as a light yellow foam. H NMR
(CDCl3): δ 8.03 (s, 0.5H), 7.99 (s, 0.5H), 7.67 (s, 1H), 7.40 (m, 2H),
7.26 (m, 7H), 6.95 (s, 0.5H), 6.94 (s, 0.5H), 6.80 (m, 4H), 6.25 (m,
1H), 5.43 (s, 2H), 4.55 (m,1H), 4.13 (m, 1H), 3.93 (s, 3H), 3.91 (s,
3H), 3.74 (s, 3H), 3.73 (s, 3H), 3.71 (m, 1H), 3.54 (m, 1H), 3.38 (m,
1H), 3.26 (m, 1H), 2.58 (t, 1H, J ) 7.0 Hz), 2.50 (m, 1H), 2.39 (t, 1H,
J ) 7.0 Hz), 2.26 (m, 2H), 2.11 (m, 2H), 1.56 (m, 2H), 1.22 (m, 2H),
1.11 (m, 8.5H), 1.03 (d, 3.5H, J ) 6.9 Hz). 31P NMR (MeOH-d4, H3-
PO4 reference): δ 148.8, 148.4. Anal. Calcd for C54H62N5O14P: C,
62.58; H, 6.03; N, 6.76. Found: C, 62.49; H, 6.20; N, 6.52.
Acknowledgment. We are grateful for support of this
research by the National Science Foundation (CHE-9732843),
the Colorado Advanced Technology Institute Center for RNA
Chemistry, and Dupont. M.M.G. thanks the Alfred P. Sloan
Foundation for a fellowship. Mass spectra were obtained on
instruments supported by the National Institutes of Health shared
instrumentation grant GM-49631. We thank Professor Yitzhak
Tor for helpful suggestions and Ms. Stephanie Price for
assistance with the synthesis of 15.
1
16 (562 mg, 82%) as a light yellow foam. H NMR (CDCl3): δ 9.25
(s, 1H), 8.04 (s, 1H), 7.74 (s, 1H), 7.45 (d, 2H, J ) 7.5 Hz), 7.34 (m,
7H), 7.04 (s, 1H), 6.88 (d, 4H, J ) 12 Hz), 6.34 (t, 1H, J ) 6 Hz),
5.50 (s, 2H), 4.56 (m, 1H), 4.13 (m,1H), 4.0 (s, 3H), 3.97 (s, 3H), 3.80
(s, 6H), 3.40 (m, 2H), 2.74 (s, 1H), 2.53 (m, 1H), 2.49 (t, 2H, J ) 7.5
Hz), 2.31 (m, 1H), 2.22 (t, 2H, J ) 7.2 Hz), 1.69 (p, 2H, J ) 6.9 Hz).
13C NMR (CDCl3): δ 172.3, 161.8, 158.5, 153.5, 149.4, 148.1, 144.5,
141.8, 139.8, 135.5, 129.9, 129.0, 127.9, 127.2, 126.9, 112.2, 110.2,
108.1, 100.7, 93.6, 86.9, 85.6, 72.3, 71.8, 63.5, 63.1, 56.5, 56.3, 55.2,
41.4, 32.8, 23.3, 18.8. IR (film) 3482, 3183, 3067, 2941, 2835, 2314,
1713, 1689, 1607, 1583, 1510, 1462, 1327, 1274, 1245, 1221, 1177,
1066, 1028 cm-1. HRMS (FAB) calcd (M + H) 836.3032, found
836.3014.
Supporting Information Available: Table of HPLC re-
sponse factors for nucleosides; experimental procedures for the
preparation of 25-27 and 30-32; reverse-phase HPLC chro-
matograms of enzyme digests; electrospray mass spectra of
oligonucleotides (21a-h, 29). This material is available free
of charge via the Internet at http://pubs.acs.org.
Preparation of 17. To a solution of 16 (182 mg, 0.22 mmol) in
CH2Cl2 (10 mL) protected from light was added diisopropylethylamine
JA983273R