Synthesis of 2'-modified oligodeoxynucleotides via on-column conjugation.

Article abstract of DOI:10.1021/jo000917g

Oligodeoxynucleotides modified at the 2'-position of 2'-amino-2'-deoxyuridine or uridine were prepared in high yield and purity using phosphoramidites 2 and 3, respectively. Oligodeoxynucleotide conjugates were prepared on the solid-phase synthesis support following selective unmasking of the nucleophile incorporated in these phosphoramidites. Synthesis of oligodeoxynucleotides modified at the 2'-position of an internal nucleotide provides molecules that are complementary to those previously prepared via a similar approach using C5-substituted pyrimidines. The efficiency of functionalization of the 2'-O-alkylamino-uridine derived from 3 in a protected oligodeoxynucleotide was less susceptible to steric hindrance than the 2'-amino-2'-deoxyuridine in the same polymeric substrate. However, the greater reactivity of the 2'-O-alkylamine containing nucleotide gave rise to undesired acetamide formation resulting from nucleophilic attack on the 5'-terminal acetate in capped failure sequences. This problem was overcome by using 2,2,2-trimethylacetyl anhydride as a capping agent during the automated synthesis cycles. Finally, the efficiency of the photochemical unmasking of the support bound alkylamine on a 1 mumole scale was improved by using two 20 min photolysis cycles, coupled with removing reaction byproducts between cycles.

Full text of DOI:10.1021/jo000917g

VOLUME 66, NUMBER 2
J ANUARY 26, 2001
© Copyright 2001 by the American Chemical Society
Articles
Syn th esis of 2′-Mod ified Oligod eoxyn u cleotid es via On -Colu m n
Con ju ga tion
J ae-Taeg Hwang and Marc M. Greenberg*
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
mgreenbe@lamar.colostate.edu
Received J une 17, 2000
Oligodeoxynucleotides modified at the 2′-position of 2′-amino-2′-deoxyuridine or uridine were
prepared in high yield and purity using phosphoramidites 2 and 3, respectively. Oligodeoxynucleo-
tide conjugates were prepared on the solid-phase synthesis support following selective unmasking
of the nucleophile incorporated in these phosphoramidites. Synthesis of oligodeoxynucleotides
modified at the 2′-position of an internal nucleotide provides molecules that are complementary to
those previously prepared via a similar approach using C5-substituted pyrimidines. The efficiency
of functionalization of the 2′-O-alkylamino-uridine derived from 3 in a protected oligodeoxynucleotide
was less susceptible to steric hindrance than the 2′-amino-2′-deoxyuridine in the same polymeric
substrate. However, the greater reactivity of the 2′-O-alkylamine containing nucleotide gave rise
to undesired acetamide formation resulting from nucleophilic attack on the 5′-terminal acetate in
capped failure sequences. This problem was overcome by using 2,2,2-trimethylacetyl anhydride as
a capping agent during the automated synthesis cycles. Finally, the efficiency of the photochemical
unmasking of the support bound alkylamine on a 1 µmole scale was improved by using two 20 min
photolysis cycles, coupled with removing reaction byproducts between cycles.
The incorporation of suitably functionalized nucleotides
at defined sites of oligonucleotides facilitates the parallel
synthesis of modified nucleic acids containing diverse
structural features. Modified oligonucleotides are finding
increasing uses that include structural/diagnostic probes,
therapeutic candidates, and artificial enzymes.1-3 Struc-
tural diversity can be incorporated via individually
prepared phosphoramidites, using the fully deprotected
polymer as a substrate in solution, or in chemically
synthesized oligonucleotides that retain their exocyclic
amine, phosphate diester, and 5′-terminal hydroxyl pro-
tecting groups.4-7 Postsynthetic modification of protected
(1) O’Donnell, M. J .; McLaughlin, L. W. In Bioorganic Chemistry
Nucleic Acids; Hecht, S. M., Ed.; Oxford University Press: Oxford,
1996.
(2) (a) Reed, M. W.; Wald, A.; Meyer, R. B. J . Am. Chem. Soc. 1998,
120, 9729. (b) Robles, J .; McLaughlin, L. W. J . Am. Chem. Soc. 1997,
119, 6014.
(4) Beaucage, S. L. In Comprehensive Natural Products Chemistry,
Volume 7 DNA and Aspects of Molecular Biology; Kool, E. T., Ed.;
Elsevier: Amsterdam, 1999.
(5) (a) Ozaki, H.; Ogawa, Y.; Mine, M.; Sawai, H. Nucleosides
Nucleotides 1998, 17, 911. (b) Nomura, Y.; Ueno, Y.; Matsuda, A.
Nucleic Acids Res. 1997, 25, 6253.
(3) (a) Santoro, S. W.; J oyce, G. F.; Sakthivel, K.; Gramatikova, S.;
Barbas, C. F., III J . Am. Chem. Soc. 2000, 122, 2433. (b) Wiegand, T.
W.; J anssen, R. C.; Eaton, B. E. Chem. Biol. 1997, 4, 675. (c) Tarasow,
T. W.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54.
(6) (a) Ghosh, S. S.; Musso, G. F. Nucleic Acids Res. 1987, 15, 5353.
(b) Bischoff, R.; Coull, J . M.; Regnier, F. E. Anal. Biochem. 1987, 164,
336. (c) Erout, M.-N.; Troesch, A.; Pichot, C.; Cros, P. Bioconjugate
Chem. 1996, 7, 568.
10.1021/jo000917g CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/05/2001

364 J . Org. Chem., Vol. 66, No. 2, 2001
Hwang and Greenberg
oligonucleotides has been carried out in solution or while
the biopolymer substrate is still covalently linked to the
support on which it was synthesized.7-9 We have reported
on a general strategy that utilizes photolabile protecting
groups to selectively release electrophiles or nucleophiles
at the 3′-termini of oligodeoxynucleotides, as well as at
internal positions.10-12 In the latter, masked reactive sites
are introduced using modified phosphoramidites (e.g., 1,
2), and the photochemical deprotection is orthogonal with
respect to the other families of protecting groups present
throughout the oligonucleotide. o-Nitrobenzyl-protected
monomers 1 and 2 are complementary to one another in
the sense that hybridization of oligonucleotides contain-
ing them via Watson-Crick base pairing allows one to
introduce structural modifications in the major and minor
grooves of the duplexes, respectively. We now wish to
report further on the scope and limitations of 2 and
related phosphoramidite 3 for the synthesis of oligode-
oxynucleotides that are modified at the 2′-position of a
pyrimidine nucleoside (eq 1).
the chemically synthesized biopolymer is still bound to
its solid support. The general strategy has utilized the
o-nitrobenzyl photochemical reaction to selectively reveal
an electrophile or nucleophile in an otherwise completely
protected oligonucleotide.10-13 Compared to methods for
postsynthetic modification of deprotected oligonucleo-
tides, this approach often proceeds in higher yield and
uses shorter reaction times and relatively modest ex-
cesses of reagents. Furthermore, the use of a single
phosphoramidite or solid phase support as a precursor
for the preparation of multiple modified oligonucleotides
is also more efficient than individually synthesizing
phosphoramidites which are then incorporated into oli-
gonucleotides during the linear synthesis process.
Resu lts a n d Discu ssion
P r ep a r a tion of Oligod eoxyn u cleotid es Con ta in -
in g Am in e Nu cleop h iles a t th e 2′-P osition of a
Nu cleotid e Usin g 2′-Am in o-2′-d eoxyu r id in e. Phos-
phoramidites 2 and 3 were designed to facilitate the
functionalization of oligonucleotides at the 2′-position of
(2′-deoxy)uridine, such that Watson-Crick complexation
of these biopolymers would introduce the modification
into the minor groove of the duplex.14 Although one could
envision a plethora of derivatives to prepare, we chose
two molecules that would produce oligonucleotide con-
jugates differing in their degrees of freedom (and conse-
quently steric hindrance) at the site where the modifi-
cation is introduced (eq 1). Phosphoramidites 2 and 3
were readily prepared from known precursors. Although
2 was previously reported by us, details of its preparation
from 6 are provided in the Experimental Section.12,15
Similarly, 2′-O-alkylamine derivatives of uridine, such
as the cyanoethyl carbamate phosphoramidites (7), have
been reported.14b Hence, the synthesis of 3 was also
straightforward from 8 (Scheme 1).16 The greater steric
hindrance at the phosphorus center in 2 compared to 3
was reflected in the required coupling conditions during
the synthesis of protected oligonucleotides 10 and 14.
With the exception of 2 and 3, N-isobutyrylated phos-
phoramidites were used for preparing the oligonucleo-
As mentioned above, we have developed methods for
preparing modified oligonucleotides in solution, or while
(7) (a) Kremsky, J . N.; Pluskal, M.; Casey, S.; Perry-O’Keefe, H.;
Kates, S. A.; Sinha, N. D. Tetrahedron Lett. 1996, 37, 4313. (b) Habus,
I.; Xie, J .; Iyer, R. P.; Zhou, W.-Q.; Shen, L. X.; Agrawal, S. Bioconjugate
Chem. 1998, 9, 283. (c) Khan, S. I.; Grinstaff, M. W. J . Am. Chem.
Soc. 1999, 121, 4704.
(8) Hovinen, J .; Guzaev, A.; Azhayeva, E.; Azhayev, A.; Lo¨nnberg,
H. J . Org. Chem. 1995, 60, 2205.
(9) (a) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J . Am. Chem. Soc.
1997, 119, 7423. (b) Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T.
M. J . Am. Chem. Soc. 1991, 113, 4328. (c) Xu, Y.-Z.; Zheng, Q.; Swann,
P. F. J . Org. Chem. 1992, 57, 3839.
(10) (a) McMinn, D. L.; Greenberg, M. M. J . Am. Chem. Soc. 1998,
120, 3289. (b) Kahl, J . D.; McMinn, D. L.; Greenberg, M. M. J . Org.
Chem. 1998, 63, 4870. (c) Kahl, J . D.; Greenberg, M. M. J . Org. Chem.
1999, 64, 507.
(13) (a) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R.
J . Am. Chem. Soc. 1988, 110, 7170. (b) Pillai, V. N. R. Synthesis 1980,
1. (c) Patchornik, A.; Amit, B.; Woodward, R. B. J . Am. Chem. Soc.
1970, 92, 6333.
(14) For other examples of introducing modifications at the C2′-
position of oligonucleotides, see ref 4 and: (a) Salo, H.; Virta, P.;
Hakala, H.; Prakash, T. P.; Kawasaki, A. M.; Manoharan, M.; Lo¨nn-
berg, H. Bioconjugate Chem. 1999, 10, 815. (b) Manoharan, M.;
Prakash, T. P.; Barber-Peoc’h, I.; Bhat, B.; Vasquez, G.; Ross, B. S.;
Cook, P. D. J . Org. Chem. 1999, 64, 6468. (c) Sigurdsson, S. T.;
Eckstein, F. Nucleic Acids Res. 1996, 24, 3129.
(15) McGee, D. P. C.; Vaughn-Settle, A.; Vargeese, C.; Zhai, Y. J .
Org. Chem. 1996, 61, 781.
(11) Kahl, J . D.; Greenberg, M. M. J . Am. Chem. Soc. 1999, 121,
597.
(12) Hwang, J .-T.; Greenberg, M. M. Org. Lett. 1999, 1, 2021.
(16) (a) Wagner, D.; Verheyden, J . P. H.; Moffat, J . G. J . Org. Chem.
1974, 39, 24. (b) Manoharran, M.; Tivel, K. L.; Andrade, L. K.; Cook,
P. D. Tetrahedron Lett. 1995, 36, 3647.

Synthesis of 2′-Modified Oligodeoxynucleotides
J . Org. Chem., Vol. 66, No. 2, 2001 365
Sch em e 1a
a
Key: (a) dibutyltin oxide, MeOH; (b) N-4-bromobutylphthalimide, NaI, DMF; (c) DMTCl, pyridine; (d) hydrazine, MeOH; (e) 4,5-
dimethoxy-2-nitrobenzylchloroformate, Hu¨nig’s base, THF; (f) Hu¨nig’s base, N,N-diisopropylaminocyanoethyl phosphonamidic chloride,
CH2Cl2.
tides.17 Standard oligonucleotide synthesis cycles were
employed for coupling the phosphoramidites of native
nucleotides. However, it was necessary to double-couple
3 (0.05 M) for 5 min each cycle, resulting in a 95% yield
(as determined via dimethoxytrityl cation release); whereas
only an 87% yield was obtained upon double-coupling 2
(0.1 M, 10 min/cycle).12
Similarly, reaction of 11 with alkyl isocyanate 13 only
yielded the respective conjugate (12b) on average in 43%
yield, even when 10 equiv of the isocyanate was allowed
to react with 11 for 3 h.
It was assumed that oligodeoxynucleotide 14 prepared
from 3 would yield a conjugation substrate (15) that is
more amenable to reaction with less reactive electro-
philes. Initial attempts at conjugating 15 to pyrene-
butyric acid (5 equiv, 3 h) produced 16c in only modest
yields, which did not increase significantly when reac-
tions were run in the presence of a greater number of
equivalents (10) of acid and activating agent (PyBOP).
Analysis of the crude mixture by anion exchange HPLC
revealed the presence of a previously unobserved product
that did not coelute with the carbamate (16a ), or conju-
gate (16c), and eluted slightly later than the free amine
(16b).18 Isolation and characterization of this product by
ESI-MS indicated that it was the acetamide (16d ).18
Based upon the observations that the photolabile car-
bamate is stable to concentrated aqueous ammonia
deprotection conditions, and that the acetamide was only
Moreover, the steric hindrance at the amino position
of 2′-amino-2′-deoxyuridine (4, eq 1) manifested itself
during conjugation reactions of 11, and provided further
impetus for the synthesis of 3. High yields of conjugates
were obtained when 11 was reacted with aryl isocyan-
ates, or alkyl carboxylic acids containing methylene
groups at their R-carbons.12 However, attempted coupling
of 11 with carboxylic acids containing tertiary R-carbons,
such as N-acetylphenylalanine, under the identical con-
ditions employed previously (10 equiv of PyBOP and
carboxylic acid, 3 h, 25 °C) produced 12a in only an
average yield of 23%. Increasing the number of equiva-
lents of reagents relative to 11 to 50 and the reaction
time to 5 h increased the yield of 12a to only 37%.
(17) McMinn, D. L.; Greenberg, M. M. Tetrahedron Lett. 1997, 39,
1103.
(18) See the Supporting Information.

366 J . Org. Chem., Vol. 66, No. 2, 2001
Hwang and Greenberg
observed in oligodeoxynucleotides that were photolyzed
prior to deprotection, we hypothesized that this product
was derived from nucleophilic attack by the photochemi-
cally revealed amine while the otherwise protected
substrate (15) was covalently bound to the long chain
alkylamine-controlled pore glass (LCAA-CPG) support
on which it was synthesized. Attempts at eliminating this
undesired product by changing the solvent conditions
during the photochemical release of the alkylamine met
with limited success. Removing water from the solvent
mixture routinely used during photolyses (CH3CN:H2O,
9:1), or changing to the less polar solvent, benzene
increased the ratio of 16a :16d from 1:1 to as much as
2.5:1 (benzene).
tected. (The HPLC chromatograms and ESI-MS spectra
can be found in the Supporting Information.) The only
possible source of isobutyrylated material was the exo-
cyclic amine protecting groups of 2′-deoxyadenosine, 2′-
deoxycytidine, and 2′-deoxyguanosine. These protecting
groups have been observed to undergo transamidation
to a small extent in other studies.11
Having solved this unexpected problem, we briefly
turned our attention to optimizing the photochemical
unmasking of the alkylamine in 17. Through several
previous studies involving photolabile solid-phase syn-
thesis supports and phosphoramidites (including 1 and
2), we had established irradiation conditions (365 nm,
2-3 h) that did not impart detectable damage on the
oligodeoxynucleotides.10-12 We were able to improve the
efficiency of the photochemical conversion of 17 by taking
advantage of the covalent linkage of the product (and
substrate) to the solid support. Conversion of the car-
bamate in 17 was determined by measuring the amount
of carbamate present in fully deprotected material (19n )
by anion exchange HPLC. However, none of this product
was observed following two 20 min irradiations (CH3CN)
in which the resin was filtered and washed between
photolysis periods on scales as large as 1 µmole. Increased
photochemical efficiency is attributed to removal of the
nitroso substituted aromatic aldehyde byproduct released
during the o-nitrobenzyl photoredox reaction, which may
be acting as a filter for the process.
Con ju ga tion Rea ction s of Oligod eoxyn u cleotid e
Con ta in in g 5 (18). Under the best circumstances,
satisfactory conjugation of 2′-amino-2′-deoxyuridine (4)
required 10 equivalents of reagents and a 3 h reaction
time.12 Using pyrene butyric acid as a standard, we found
that excellent isolated yields of 19e could be obtained
using 5 equivalents of acid and PyBOP relative to
oligonucleotide for 2 h at room temperature (Table 1).
As expected, based upon previous conjugation experi-
ments utilizing protected oligonucleotides as substrates,
ESI-MS, as well as enzymatic digestion (followed by
reverse phase HPLC analysis) indicated that the conju-
gate was of high purity.18 Comparable yields and purity
(as determined by ESI-MS analysis) of oligonucleotide
conjugate products were also obtained using carboxylic
acids such as biotin (19f), the tripeptide N-Fmoc-gly‚gly‚
gly-CO2H (19h ), and a terpyridine (19g) (Table 1).
Interestingly, the ESI-MS of 19g revealed that the
oligonucleotide conjugate was isolated as a mixture of the
free ligand and its copper complex.18 Isolated yields of
conjugates in the 80-90% range (Table 2) were also
obtained from reaction of 18 with more hindered sub-
strates such as N-acetylphenylalanine (19i), which did
not couple efficiently with the oligonucleotide containing
the more hindered 2′-amino substituted nucleotide (11).
The isolated yield of 19i improved slightly (90 ( 1%) by
increasing the number of equivalents of reagents to 10,
and the reaction time to 3 h. These latter conditions
yielded very good isolated yields of the respective oligo-
nucleotide conjugates of diphenyl acetic acid (19j) and a
tripeptide containing proline at its C-terminus (19k ).
Finally, coupling of the aryl isocyanate, m-nitrophenyl
isocyanate (19l) to 18 also required shorter reaction time
(2 h) and fewer equivalents (5 equiv) of isocyanate than
did the more hindered substrate (11). However, only
It was determined that the acetates introduced upon
capping of failure sequences produced during oligo-
nucleotide synthesis gave rise to the acetamide product
described above (16d ). This information was gleaned from
experiments in which LCAA-CPG support containing 5′-
O-dimethoxytritylthymidine was prepared using 2,2,2-
trimethylacetyl chloride as capping agent for the un-
derivatized alkylamines. Isobutyryl anhydride was sub-
stituted for acetic anhydride (maintaining the same
concentration, 1 M) in the capping step of the automated
synthesis cycle during the synthesis of 17. Anion ex-
change HPLC analysis of the crude mixture obtained
upon photolysis and subsequent ammonia deprotection
still revealed the presence of an additional product that
eluted slightly later than 19a . The intensity of this
product relative to that of 19a was approximately 50%.
Isolation and analysis by ESI-MS revealed that this
product was the isobutyryl amide (19c), indicating that
the acetate esters introduced during the capping of
failure sequences were the predominant source of the
acetyl group. The respective acetamide product (19b) was
not observed. Formation of the undesired product(s) were
subsequently reduced to <5% by synthesizing the oligo-
deoxynucleotides on commercially available LCAA-CPG
support using 2,2,2-trimethylacetyl anhydride (1 M) as
capping agent for the primary hydroxyls that failed to
couple during the automated synthesis cycles. The iden-
tity of the minor impurity remaining was ascertained via
ESI-MS, and was still found to be the isobutyrylated
product (19c). No pivaloylated material (19d ) was de-

Synthesis of 2′-Modified Oligodeoxynucleotides
J . Org. Chem., Vol. 66, No. 2, 2001 367
Ta ble 1. Yield of Oligon u cleotid e Con ju ga tes fr om th e
Rea ction of 18 w ith Un h in d er ed Ca r boxylic Acid s
Ta ble 2. Con ju ga tion of Isocya n a tes a n d Hin d er ed
Ca r boxylic Acid s to 18
a
Isolated yields were determined via comparing the amount of
oligonucleotide obtained from a comparable amount of unconju-
gated material subjected to the identical deprotection, purification,
and isolation conditions. Yields represent an average of separate
b
a
Isolated yields were determined via comparing the amount of
reactions ( standard deviation from this value. The number of
reactions run are noted in parentheses.
oligonucleotide obtained from a comparable amount of unconju-
gated material subjected to the identical deprotection, purification,
b
and isolation conditions. Yields represent an average of separate
reactions ( standard deviation from this value. The number of
reactions run are noted in parentheses. c For amide bond forma-
tion, equal number of equivalents of PyBOP are added.
modest yields of 19m were obtained when coupling 18
to the alkyl isocyanate (13), even when as many as 10
equiv were employed for 3 h at 55 °C. The reasons for
the poor coupling of 13 to 18 are unknown at this time.
Su m m a r y. Oligonucleotides containing modifications
at the 2′-position of (2′-deoxy)uridine can be prepared in
high yields via conjugation to protected nucleic acids
containing a single unmasked nucleophile. Conjugation
of hindered electrophiles requires the use of a 2′-O-
alkylamine substituted uridine. These phosphoramidite
reagents provide a simple, efficient method for introduc-
ing structural diversity in the minor groove of duplexes
composed of such chemically prepared modified oligo-
nucleotides.
were prepared by precipitating from NH4OAc. Preparative
oligonucleotide separations was carried out using 20% poly-
acrylamide denaturing gels (5% cross-link, 45% urea (by
weight)).
2′-N-(4,5-Dim eth oxy-2-n itr oben zylca r bon yl)a m in o-5′-
O-(4,4′-d im eth oxytr ityl)-2′-d eoxyu r id in e. To a solution of
4,5-dimethoxy-2-nitrobenzylchloroformate (0.16 g, 0.6 mmol)
in THF (15 mL) was added diisopropylethylamine (0.12 g, 0.92
mmol), followed by addition of 2′-deoxy-2′-amino-5′-O-dimeth-
oxytrityluridine (6)15 (0.25 g, 0.46 mmol) at room temperature.
After being stirred for 30 min, the reaction was diluted with
dichloromethane, washed with saturated NaHCO3 and brine,
and dried over Na2SO4. The solvent was removed to give crude
product as a brownish foam which was purified on silica gel
(CH2Cl2/EtOAc, 4:1 to 2:1, containing 1% Et3N) to yield the
carbamate as white loaves (0.28 g, 78%): 1H NMR (CDCl3) δ
9.45 (bs, 1H), 7.70 (d, 1H, J ) 8.1 Hz), 7.66 (s, 1H), 7.50-7.20
(m, 9H), 7.02 (s, 1H), 6.87 (d, 4H, J ) 8.4 Hz), 6.29 (d, 1H, J
) 7.5 Hz), 6.20 (d, 1H, J ) 8.1 Hz), 5.46 (m, 3H), 4.40 (m, 2H),
4.24 (s, 1H), 3.96 (s, 6H), 3.92 (s, 3H), 3.81 (s, 6H), 3.46 (m,
2H); IR (film) 3650 (br), 3316, 3064, 2359, 2341, 1693, 1607,
1520, 1509 cm-1; 13C NMR (CDCl3) δ 163.5, 158.7, 156.1, 153.7,
151.6, 148.1, 144.2, 140.3, 139.5, 135.3, 135.1, 130.2, 128.7,
128.1, 127.7, 127.2, 113.4, 110.4, 108.1, 103.2, 87.3, 86.0, 85.7,
76.8, 71.9, 64.2, 63.9, 58.4, 56.6, 56.4, 55.4; HRMS (FAB) calcd
for C40H40N4O13 784.2592 (M+), found 784.2600.
Exp er im en ta l Section
Oligonucleotides were synthesized using standard 1µmol
cycles, except where noted, and â-cyanoethyl protected phos-
phoramidites. The exocyclic amines of deoxyadenosine, deoxy-
cytidine, and deoxyguanosine were protected as their iso-
butyryl amides. With the exception of polystyrene solid-phase
support and N-isobutyryl phosphoramidite for deoxycytidine
(Pharmacia), all DNA synthesis reagents were obtained from
Glen Research, Inc. Benzotriazole-1-yloxy-tri-pyrrolidinophos-
phonium hexafluorophosphate (PyBOP) was obtained from
NovaBiochem. Calf intestine alkaline phosphatase was ob-
tained from New England Biolabs. Nuclease P1 and snake
venom phosphodiesterase were from Boehringer Mannheim.
Dimethylformamide (DMF), diisopropylethylamine (DIEA),
and CH3CN were freshly distilled from CaH2. THF was
distilled from sodium/benzophenone ketyl. Electrospray samples
2′-N-(4,5-Dim eth oxy-2-n itr oben zylca r bon yl)a m in o-5′-
O-(4,4′-d im eth oxytr ityl)-2′-d eoxyu r id in e 3′-O-[(2-Cya n o-
eth yl)-N,N-d iisop r op yl]p h osp h or a m id ite (2). To a cloudy
solution of 2′-N-4,5-dimethoxy-2-nitrobenzylcarbonylamino-5′-
O-(4,4′-dimethoxytrityl)-2′-deoxyuridine (0.1 g, 0.13 mmol) in

368 J . Org. Chem., Vol. 66, No. 2, 2001
Hwang and Greenberg
dichloromethane (10 mL) was added diisopropylethylamine (67
mg, 0.52 mmol), followed by addition of N,N-diisopropyl-
aminocyanoethyl phosphonamidic chloride (48 mg, 0.2 mmol)
at 0 °C. After stirring for 2 h at room temperature, additional
phosphitylating reagent (16 mg, 0.07 mmol) was added to the
reaction mixture. After being stirred for an additional 2 h at
room temperature, the reaction was quenched with propan-
2-ol (0.24 g, 3.9 mmol). The solution was stirred for 1 h at room
temperature, whereupon a solution of 5% Na2CO3 (5 mL) was
added. The aqueous layer was extracted with CH2Cl2. The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The crude phosphoramid-
ite was purified on silica gel (CH2Cl2/EtOAc, 10:1 to 1:1,
containing 1% Et3N) to yield a mixture of diastereomers of 2
as a pale brownish foam (0.1 g, 79%): 1H NMR (CDCl3) δ 7.70
(m, 2H), 7.42-7.25 (m, 9H), 7.06 (s, 1H), 6.86 (d, 4H, J ) 8.7
Hz), 6.14 (t, 1H, J ) 9.2 Hz), 5.97 (d, 1H, J ) 7.5 Hz, minor),
5.62 (dd, 1H, J ) 11.1, 25.5 Hz), 5.45 (m, 2H), 4.80-4.35 (m,
2H), 4.30 (d, 1H, J ) 54.3 Hz), 4.01 (s, 3H), 3.96 (s, 3H), 3.85
(m, 1H), 3.81 (s, 6H), 3.81-3.54 (m, 3H) 3.43 (m, 2H), 2.63
(m, 1H), 2.51 (m, 1H), 1.21 (m, 12H); 31P NMR (CDCl3) δ
152.44, 150.31; IR (film) 3232, 3065, 2967, 2252, 1694, 1607,
1519 cm-1; HRMS (FAB) calcd for C49H57N6O14P 985.3749 (M+
+ H), found 985.3766.
2′-O-(4-P h th a lim id obu tyl)-5′-O-(4,4′-d im eth oxytr ityl)-
u r id in e (8). A suspension of uridine (2.5 g, 10.3 mmol) and
dibutyltin oxide (2.86 g, 11.2 mmol) in methanol (500 mL) was
heated under reflux for 1 h, and the resulting clear solution
was evaporated to yield 2′,3′-O-dibutylstannyleneuridine as a
white solid.16 The crude product was dried further under high
vacuum. To a solution of 2′,3′-O-dibutylstannyleneuridine (4.92
g, 10.3 mmol) in DMF (60 mL) were added N-4-bromobutyl-
phthalimide (8.10 g, 28.7 mmol) and NaI (1.1 g, 7.3 mmol).
The reaction mixture was heated at 130-140 °C for 24 h, after
which time it was poured into a mixture of ether (100 mL)
and water (100 mL). The aqueous layer was extracted with
ether. The combined organic layers were washed with brine
and dried over Na2SO4. The solvent was removed in vacuo to
give a crude product mixture (55:45 ratio of 2′- and 3′-isomers
as determined by 1H NMR) as an oil which was used in the
next step without purification. To a solution of the mixture of
2′ and 3′-alkylated isomers, which was coevaporated with
pyridine, in pyridine (30 mL) in an ice-water bath was added
4,4′-dimethoxytrityl chloride (3.56 g, 10.5 mmol). The reaction
mixture was warmed to room temperature. After the mixture
was stirred overnight, the pyridine was removed under
reduced pressure to give a syrup, which was purified by flash
column chromatography (hexanes/EtOAc, 1:2 to 1:1, containing
1% Et3N). The 2′-alkylated product (8) eluted first as a pale
brownish foam (1.94 g, 25%), followed by the 3′-alkylated
product as a white foam (1.34 g, 18%). 2′-Alkylated product:
1H NMR (CDCl3) δ 9.70 (br, 1H), 8.09 (d, 1H, J ) 8.1 Hz),
7.86 (dd, 2H, J ) 3.0, 5.7 Hz), 7.73 (dd, 2H, J ) 3.3, 5.4 Hz),
7.45-7.20 (m, 9H), 6.85 (d, 4H, J ) 8.7 Hz), 5.99 (s, 1H), 5.31
(d, 1H, J ) 8.7 Hz), 4.52 (br, 1H), 4.09 (m, 2H), 3.94 (d, 1H, J
) 5.1 Hz), 3.83 (s, 6H), 3.78 (m, 3H), 3.60 (s, 2H), 3.03 (br,
1H), 1.86-1.66 (m, 4H); 13C NMR (CDCl3) δ 168.5, 163.7, 158.7,
150.2, 144.5, 140.1, 135.4, 135.2, 134.0, 132.1, 130.3, 130.2,
128.2, 128.1, 127.2, 123.3, 113.4, 102.1, 87.7, 87.1, 83.3, 82.7,
70.6, 68.6, 61.3, 55.4, 37.8, 26.6, 25.5; IR (film) 3467 (br), 3193
(br), 3058, 1770, 1712, 1608, 1509 cm-1; HRMS (FAB) calcd
for C42H41N3O10 747.2792 (M+), found 747.2677. 3′-Alkylated
product: 1H NMR (CDCl3) δ 9.65 (br, 1H), 7.90 (d, 1H, J )
8.1 Hz), 7.86 (dd, 2H, J ) 2.7, 5.4 Hz), 7.72 (dd, 2H, J ) 3.0,
5.4 Hz), 7.45-7.20 (m, 9H), 6.88 (d, 4H, J ) 9.0 Hz), 5.97 (d,
1H, J ) 3.9 Hz), 5.44 (d, 1H, J ) 8.1 Hz), 4.39 (t, 1H, J ) 4.2
Hz), 4.23 (m, 1H), 4.10 (m, 1H), 3.81 (s, 6H), 3.75-3.50 (m,
5H), 3.42 (m, 1H), 1.86-1.60 (m, 4H); 13C NMR (CDCl3) δ
168.5, 163.4, 158.7, 150.7, 144.3, 140.2, 135.4, 135.2, 134.0,
132.1, 130.1, 128.2, 128.1, 127.2, 123.3, 113.4, 102.5, 90.0, 87.1,
81.4, 77.4, 74.0, 70.3, 62.4, 55.4, 37.6, 26.9, 25.4
the reaction mixture was heated to reflux. After 5 h, the
solvent was removed to give a crude product as a foam, which
was purified on silica gel (CH2Cl2/MeOH, 10:1, containing 1%
1
Et3N) to yield a brownish foam (1 g, 83%). H NMR (CDCl3) δ
8.15 (d, 1H, J ) 7.8 Hz), 7.44-7.26 (m, 9H), 6.87 (d, 4H, J )
9.3 Hz), 6.00 (d, 1H, J ) 1.5 Hz), 5.33 (t, 1H, J ) 4.1 Hz), 4.07
(m, 1H), 3.93 (m, 2H), 3.82-3.64 (m, 8H), 3.56 (m, 2H), 2.79
(br, 2H), 1.80-1.54 (m, 4H); 13C NMR (CDCl3) δ 164.2, 158.8,
150.8, 144.5, 140.0, 135.4, 135.2, 130.3, 130.2, 128.3, 128.1,
127.2, 113.4, 102.4, 87.6, 87.2, 83.6, 82.9, 71.0, 68.7, 61.7, 55.4,
41.5, 29.5, 27.2; IR (film) 3357 (br), 3160, 3058, 2933, 1694,
1633, 1509 cm-1; HRMS (FAB) calcd for C34H39N3O8 618.2815
(M++ H), found 618.2814.
2′-O-[N-((4,5-Dim eth oxy-2-n itr oben zyl)car bon yl)-4-am i-
n obu tyl]-5′-O-(4,4′-d im eth oxytr ityl)u r id in e (9). To a solu-
tion of 4,5-dimethoxy-2-nitrobenzylchloroformate (0.20 g, 0.73
mmol) in THF (15 mL) was added diisopropylethylamine (0.14
g, 1.1 mmol), followed by addition of 2′-O-(4-aminobutyl)-5′-
O-(4,4′-dimethoxytrityl)uridine (0.34 g, 0.55 mmol) at room
temperature. After being stirred for 30 min, the reaction was
diluted with dichloromethane, washed with saturated NaHCO3
and brine, and dried over Na2SO4. The solvent was removed
to give a crude product as a brownish powder which was
purified on silica gel (CH2Cl2/EtOAc, 4:1 to 2:1, containing 1%
Et3N) to yield a white foam (0.46 g, 97%): 1H NMR (CDCl3) δ
9.98 (br s, 1H), 8.07 (d, 1H, J ) 8.4 Hz), 7.68 (s, 1H), 7.20-
7.48 (m, 9H), 7.02 (s, 1H), 6.87 (d, 4H, J ) 9.0 Hz), 5.95 (s,
1H), 5.58-5.40 (m, 3H), 5.32 (d, 1H, J ) 5.7 Hz), 4.50 (m, 1H),
4.10-3.88 (m, 9H), 3.82 (s, 6H), 3.73 (m, 1H), 3.57 (m, 2H),
3.29 (m, 2H), 3.06 (br, 1H), 1.71 (m, 4H); 13C NMR (CDCl3,) δ
163.6, 158.71, 158.68, 156.1, 153.5, 150.5, 148.1, 144.4, 140.1,
139.9, 135.3, 135.1, 130.3, 130.2, 128.2, 128.1, 127.2, 113.3,
110.5, 108.2, 102.2, 87.7, 87.1, 83.3, 82.7, 77.4, 70.9, 68.5, 63.6,
61.3, 56.52, 56.49, 55.4, 53.6, 40.9, 26.8, 26.6; IR (film) 3314
(br), 2939, 2865, 1702, 1697, 1618, 1581 cm-1
.
2′-O-[N-((4,5-Dim eth oxy-2-n itr oben zyl)car bon yl)-4-am i-
n ob u t yl]-5′-O-(4,4′-d im et h oxyt r it yl)u r id in e 3′-O-[(2-Cy-
a n oet h yl)-N,N-d iisop r op yl]p h osp h or a m id it e (3). To a
cloudy solution of 2′-O-[N-((4,5-dimethoxy-2-nitrobenzyl)car-
bonyl)-4-aminobutyl]-5′-O-(4,4′-dimethoxytrityl)uridine (0.4 g,
0.47 mmol) in dichloromethane (15 mL) was added diisopropyl-
ethylamine (0.24 g, 1.88 mmol), followed by the addition of
N,N-diisopropylaminocyanoethyl phosphonamidic chloride (0.14
g, 0.7 mmol) at 0 °C. After being stirred overnight at room
temperature, the reaction was quenched with propan-2-ol (0.79
g, 14.1 mmol) and stirred for 1 h at room temperature, at which
time a solution of 5% Na2CO3 (5 mL) was added to the reaction
mixture. The aqueous layer was extracted with CH2Cl2. The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The crude phosphoramid-
ite was purified on silica gel (CH2Cl2/EtOAc, 10:1 to 1:1,
containing 1% Et3N) to yield a pale brownish foam (0.4 g, 82
mmol): 1H NMR (CDCl3) δ 8.06 (m, 1H), 7.71(s, 1H), 7.20-
7.50 (m, 9H), 7.04 (m, 1H), 6.87 (m, 4H), 5.95 (m, 1H), 5.60-
5.2 (m, 4H), 4.70-4.40 (m, 1H), 4.25 (m, 1H), 3.88-4.08 (m,
7H), 3.88-3.65 (m, 8H), 3.65-3.20 (m, 8H), 2.68 (t, 1H, J )
6.3 Hz), 2.46 (t, 1H, J ) 6.3 Hz), 1.70(br, 4H), 1.05-1.33 (m,
13H); 31P NMR (CDCl3) δ 150.67, 150.06; IR (film) 3325, 3198,
3057, 2966, 2935, 2872, 2368, 2342, 1693, 1682, 1519, 1277,
1252 cm-1; HRMS (FAB) calcd for C53H65N6O15P 1057.4324
(M++ H), found 1057.4355.
P r ep a r a tion of th e 5′-O-Dim eth oxytr ityl-3′-su ccin a to-
th ym id in e LCAA-CP G Ca p p ed by Tr im eth yla cetic An -
h yd r id e. LCAA-CPG (0.1 g), PyBOP (2.9 mg, 5.5 µmol),
diisopropylethylamine (2 mL, 12 µmol), DMAP (0.7 mg, 5.7
µmol), and 5′-O-dimethoxytrityl-3′-succinatothymidine (3.5 mg,
5.4 µmol) were combined in a screw-capped vial with aceto-
nitrile (2 mL). The reaction mixture was shaken at room
temperature for 20 min. The resin was filtered, washed with
MeOH and CH2Cl2, and then dried under high vacuum. The
amount of nucleoside loading on the support (∼30 µmol/g) was
determined by trityl analysis.19 Unreacted alkylamines were
2′-O-(4-Am in ob u t yl)-5′-O-(4,4′-d im e t h oxyt r it yl)u r i-
d in e. 2′-O-(4-Phthalimidobutyl)-5′-O-(4,4′-dimethoxytrityl)-
uridine (1.46 g, 1.95 mmol) was dissolved in methanol (30 mL).
Hydrazine monohydrate (0.50 g, 9.96 mmol) was added, and
(19) Applied Biosystems User Bulletin #13 1987, 11.

Synthesis of 2′-Modified Oligodeoxynucleotides
J . Org. Chem., Vol. 66, No. 2, 2001 369
capped by reacting the DMT-thymidine-CPG (0.1 g), trimethyl-
acetyl chloride (14 µL, 113 µmol), DMAP (6.7 mg, 55 µmol),
pyridine (18 µL, 220 µmol), and acetonitrile (2 mL) in a screw-
capped vial. The reaction mixture was shaken at room tem-
perature for 2 h. The resin was filtered, washed with MeOH
followed by CH2Cl2, and dried under high vacuum.
P h otolytic Dep r otection . Oligodeoxyribonucleotide bound
to support (30 mg) was added to a Pyrex tube containing a
stir bar constructed from a standard (white) pipe cleaner and
CH3CN (20 mL). The tube was fitted with a rubber septum
and the solution was sparged with Ar for 20 min, after which
the needle was raised well above the surface of the solvent.
vacuum. The free flowing resin was treated with 28% aqueous
ammonia (600 µL) for 6 h at 55 °C and concentrated under
vacuum. Purification of biotin-conjugated oligonucleotide was
carried out via 20% polyacrylamide denaturing gel electro-
phoresis. The others was purified by anion-exchange HPLC
column (Vydac 301VHP575; A, 10 mM Tri, pH 8.0; B, 10 mM
Tri, 0.5 M NH4Cl, pH 8.0; 0-80% B linearly over 24 min; 80%
B over 10 min for pyrenebutyryl and terpyridinyl conjugated
oligonucleotides; 0-52% B linearly over 43 min; 52-80% B
linearly over 3 min for other conjugated oligonucleotides; flow
rate, 1 mL/min). Isolated yields were obtained (o.d. 264 nm;
352 nm for pyrenebutyryl conjugated products) by comparing
the amount of conjugated oligonucleotide to the amount of
unconjugated material.
Photolyses were carried out with
a VWR Chromato-Vue
transilluminator (λmax ) 365 nm) for 20 min. The resin was
filtered, washed with MeOH, followed by CH2Cl2, collected, and
dried under high vacuum. The resin was transferred to a tube
in CH3CN (20 mL), and the sparging and photolysis procedures
were repeated. The resin was filtered, washed with MeOH,
followed by CH2Cl2, dried under vacuum, and placed in a screw
capped vial for storage. Note: It is important to maintain the
temperature during photolysis at e25 °C using a fan.
Gen er al P r ocedu r e for Con ju gation of P r otected Resin -
Bou n d Oligod eoxyr ibon u cleotid es. A solution (12 mM) of
the coupling reagents (3.2 mg of PyBOP and 2.1 µL of
diisopropylethylamine (2 molar equiv) in DMF (500 mL) was
prepared in an oven-dried 1 dram vial equipped with a septum.
A solution (12 mM) of carboxylic acid (6 µmol in 500 mL of
DMF) was prepared in a second oven-dried vial. The resin-
bound DNA (2 mg, containing ∼60 nmol of DNA, based on
trityl response) was treated with 50 µL (5 molar equiv) of a
1:1 mixture (by volume) of the PyBOP and carboxylic acid
solutions. The reaction was capped and shaken at room
temperature for 2 h. The resin was washed with MeOH and
dried in vacuo. Detritylation was effected by transferring the
resin to a standard oligonucleotide synthesis column and
passing the standard trichloroacetic acid solution (3 × 10 s)
through the column, followed by CH3CN and drying under
Ack n ow led gm en t. We are grateful for financial
support of this research from the National Science
Foundation (CHE-9732843) and the Colorado Commis-
sion on Higher Education. 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
GM49631. We thank Professor Alan Kennan for provid-
ing us with the terpyridine carboxylic acid.
Su p p or tin g In for m a tion Ava ila ble: Electrospray mass
spectra of 12b, 16a -d , 19a ,c,e-n . Anion-exchange HPLC
chromatograms of oligonucleotides. Experimental procedure
for the synthesis of 2′-O-[(1-pyrenebutyryl)-4-aminobutyl)]-
uridine. Procedure for, and reversed-phase HPLC chromato-
gram of, enzyme digest of 19e. Spectra (1H, 13C, 31P) of the
molecules prepared in this study. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O000917G