N-(2-Cyanoethoxycarbonyloxy)succinimide: A New Reagent for Protection of Amino Groups in Oligonucleotides

Full text of DOI:10.1021/jo982299y

6468
J . Org. Chem. 1999, 64, 6468-6472
bonyl (alloc) group3,4 as a protecting group for amino-
linkers. This group can be removed by the use of
zerovalent palladium (Pd(0)) in either solution phase or
solid phase.4-7
N-(2-Cya n oeth oxyca r bon yloxy)su ccin im id e:
A New Rea gen t for P r otection of Am in o
Gr ou p s in Oligon u cleotid es
Muthiah Manoharan,*,† Thazha P. Prakash,
Isabelle Barber-Peoc’h, Balkrishen Bhat,
Guillermo Vasquez, Bruce S. Ross, and P. Dan Cook
Because it is conveniently removed with ammonium
hydroxide by â-elimination, the â-cyanoethyl ester group
has been successfully used as a phosphate protecting
group for decades.8 If we could use the â-cyanoethoxy-
carbonyl group as a protecting group for amines (on
nucleobases and/or on tethers) as well, both phosphate
and amino group deprotection could be achieved in one
step using the standard ammonium hydroxide treatment.
If successful, one could envision using the â-cyanoethoxy-
carbonyl group for protection of the exocyclic amine of
all nucleobases as well.
In the peptide literature, a related group, the cyano-
tert-butoxycarbonyl group, has been used to protect the
amino acid glycine. This group is cleaved by aqueous
potassium carbonate or triethylamine by â-elimination
at pH 10.9 Recently, a related group, (2-cyano-1-phenyl)-
ethoxycarbonyl (the CPEOC group), has been used as the
base-labile protecting group for the 5′-OH in RNA
synthesis.10 The same research group suggests the pro-
tection of exocyclic amines of nucleobases using the
chloroformate of â-cyanoethanol (Cl(CdO)OCH2CH2CN)
and in situ activation of this reagent with N-methylimi-
dazole in a patent application.11 Surprisingly, this group
and other analogues have not been widely used as a
protecting group6 for amino groups in nucleic acid
synthesis, which may be related to the lack of a simple
reagent to introduce this group.
Department of Medicinal Chemistry, Isis Pharmaceuticals,
2292 Faraday Avenue, Carlsbad, California 92008
Received November 20, 1998
In tr od u ction
During oligonucleotide synthesis, convenient amino
group protection methodology is important not only for
exocyclic amines but also useful for side-chain amino
groups (“aminolinkers” or “aminotethers”). Conjugation
of different ligands to improve the properties of oligo-
nucleotides requires placement of protected amino groups
with appropriate tethers in the building blocks and using
them to synthesize oligonucleotides. The ligand of inter-
est is attached postsynthetically either in the solution
phase or in the solid phase. The amino group present in
tethers can be conveniently deprotected and used for
attaching various functionalities to modify the biological
or chemical properties of oligonucleotides. The conjugated
ligands can improve the uptake of antisense oligonucle-
otides by living cells. For example, attachment of
polyamines and lipophilic molecules such as cholesterol
improves cellular uptake of antisense oligonucleotides.2
The ligands can serve as reporter groups (e.g., fluorescein
or biotin), which are extensively used in DNA-based
diagnostics and also used for following cellular trafficking
of antisense oligonucleotides.1 The attached moieties can
act also as chemical nucleases to degrade the target
pathogenic genes. Despite their widespread use, the
conventional protecting groups used in oligonucleotide
chemistry for aminotethers are either too labile during
the monomer synthesis [e.g., trifluoroacetyl (CF3CO-)
and fluorenylmethoxycarbonyl (Fmoc)] or somewhat in-
ert, requiring harsh conditions during oligonucleotide
deprotection (e.g., phthalimido protecting group, which
requires addition of methylamine to the standard am-
monium hydroxide and heating at 55 °C for 12 h). The
acid-labile monomethoxytrityl (MMTr-) group is another
possibility, but it limits the use of the aminolinker to the
5′-end of the oligonucleotide. To overcome these problems,
we and others have adopted the well-known allyloxycar-
Our desire to make a convenient reagent for cyanoet-
hoxycarbonylation led us to prepare N-(2-cyanoethoxy-
carbonyloxy)succinimide (CEOC-O-succinimide), a stable,
crystalline compound that can be synthesized from
readily available commercial chemicals.
Resu lts a n d Discu ssion
CEOC-O-succinimide 1 was generated by reacting
2-cyanoethanol with disuccinimidyl carbonate (DSC)12-14
in acetonitrile in the presence of pyridine (Scheme 1).
Pyridine was used as the base in place of triethylamine,12
which is commonly used for the conversion of other
(3) Kunz, H.; Unverzagt, C. Angew. Chem. 1984, 96, 426.
(4) (a) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J . Am.
Chem. Soc. 1990, 112, 1691. (b) Leonard, N. J .; Neelima Nucleosides
Nucleotides, 1996, 15, 1369. (c) Bhat, B.; Leonard, N. J .; Robinson,
H.; Wang, A. H.-J . J . Am. Chem. Soc. 1996, 118, 10744.
(5) Barber-Peoc’h, I.; Manoharan, M.; Cook, P. D. Nucleosides
Nucleotides 1997, 16, 1407.
(6) Bogdan, F. M.; Chow, C. S. Tetrahedron Lett. 1998, 39, 1897.
(7) Hayakawa, Y.; Hirose, M.; Noyari, R. J . Org. Chem. 1993, 58,
5551.
† To whom correspondence should be addressed. E-mail:
mmanoharan@isisph.com.
(1) Manoharan, M. In Antisense Research and Applications; Crooke,
S. T., Lebleu, B., Eds.; CRC Press: Boca Raton, FL, 1993; pp 303-
349.
(2) (a) Manoharan, M.; Tivel, K. L.; Condon, T. P.; Andrade, L. K.;
Barber-Peoch, I.; Inamati, G.; Shah, S.; Mohan, V.; Graham, M. J .;
Bennett, C. F.; Crooke, S. T.; Cook, P. D. Nucleosides Nucleotides 1997,
16, 1129. (b) Alahari, S. K.; Dean, N. M.; Fisher, M. H.; Delong, R.;
Manoharan, M.; Tivel, K. L.; J uliano, R. L. Mol. Pharmacol. 1996, 50,
808. (c) Manoharan, M.; Tivel, K. L.; Condon, T. P.; Graham, M. J .;
Bennett, C. F.; Cook, P. D. Book of Abstracts, 211th ACS National
Meeting, New Orleans, LA, March 24-28 1996; American Chemical
Society: Washington, DC, 1996. (d) Manoharan, M.; Tivel, K. L.;
Andrade, L. K.; Mohan, V.; Condon, T. P.; Bennett, C. F.; Cook, P. D.
Nucleosides Nucleotides 1995, 14, 969. (e) Manoharan, M.; Tivel, K.
L.; Cook, P. D. Tetrahedron Lett. 1995, 36, 3651.
(8) Sinha, N. D.; Biernat, J .: Koster, H. Tetrahedron Lett. 1983, 24,
5843.
(9) Wueusch, E.; Spangenberg, R. Chem. Ber. 1971, 104, 2427.
(10) Munch, U.; Pfleiderer, W. Nucleosides Nucleotides 1997, 16, 801.
(11) Pfleiderer, W.; Merk, C., German Patent DE97-19745708
971016; US 96-738062. Cyanoethyl chloroformate is described also in:
Kondratenko, V. I.; Khaskin, I. G. Zh. Org. Khim. 1970, 6, 2223.
(12) Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W. J .
Tetrahedron Lett. 1992, 33, 2781.
(13) Ghosh, A. K.; Duong, T. McKee, S. P. Tetrahedron Lett. 1991,
32, 4251.
(14) Ogura, H.; Kobayashi, T.; Kawabe, K.; Takeda, K. Tetrahedron
Lett. 1975, 4745.
10.1021/jo982299y CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/29/1999

Notes
J . Org. Chem., Vol. 64, No. 17, 1999 6469
Sch em e 1
tection conditions (concentrated ammonium hydroxide,
12 h, 55 °C) were used to deprotect the oligomers. The
CEOC groups were removed without any side products
(as detected by HPLC, NMR, and mass spectral analysis)
in oligomers containing all four nucleobases. These bases
contained standard protecting groups for exocyclic amines
(benzoyl for deoxyadenosine and deoxycytidine; isobutyryl
for deoxyguanosine). Oligomers synthesized using the
CEOC strategy are shown in Table 1 along with their
mass spectral and HPLC characteristics. The purified
oligonucleotides with aminolinkers were used to conju-
gate other functionalities. They were also evaluated for
their antisense properties in terms of oligonucleotide
RNA target binding. The 2′-O-aminolinker-containing
oligonucleotides were hybridized against RNA (Table 2).
The modified oligonucleotides showed nearly a +1.0 °C
increase in Tm (the midpoint for helix to coil transition)
for each substitution compared to unmodified DNA.19
This translates to nearly 1.8 °C increase per modification,
when compared to 2′-deoxyphosphorothioates that are
used as first-generation antisense oligonucleotide drugs.20
This increase may be a result of overall reduction of
negative charges in the oligomer-RNA duplex, as these
amino groups are expected to be protonated. As noted
previously, these zwitterionic oligonucleotides also im-
prove the nuclease resistance of the antisense oligo-
mers.15
alcohols to their carbonates to avoid any possible â-elim-
ination during workup. The synthesis has been scaled
up easily, and compound 1 has been prepared in multi-
gram quantities several times. It is a stable compound
that can be stored at room temperature for an extended
period of time (more than a year) in a desiccator.
In this paper, we demonstrate the use of this compound
to protect pendant alkylamines in oligonucleotides. These
alkylamines are present either in the 2′-position of RNA
modifications or in nonnucleosidic tethers. In addition
to helping functionalization of oligonucleotides by other
compounds via amino group mediated conjugation chem-
istry, the nucleosidic aminolinkers can enhance affinity
in binding to the target RNA (vide infra) and improve
their biostability. Oligonucleotides containing short
stretches of alkylamino tethers at their 3′-ends display
extraordinary resistance against degradation by 3′-exo-
nucleases.15 The improvement in nuclease resistance is
presumably due to the zwitterionic nature of the modified
oligonucleotide. The amino group in the 2′-O substituent
has a pKa in the range of 9-10 and can therefore be
expected to be protonated at physiological pH. The
cationic group decreases susceptibility of oligonucleotides
to nucleases.
The CEOC group was used to protect amino groups
present in both in nucleosides and nonnucleosidic build-
ing blocks. Nucleosides16,17 4 and 7, containing amino-
linkers of different lengths (2′-O-CH2CH2NH2 and 2′-O-
CH2CH2CH2CH2CH2CH2NH2), were reacted with the
CEOC reagent to protect the amino groups. The synthesis
of the 2′-O-(hexylamino) compound 7 was carried out
according to a reported procedure.16 Synthesis of com-
pound 4 is shown in Scheme 2. This involves ring opening
of 2,2′-anhydro-5-methyluridine by the borate ester gen-
erated from N-(2-hydroxyethyl)phthalimide and borane
in THF.17 The ring-opening reaction proceeded in 21%
yield. Considering the cost of the inexpensive starting
materials18 and regioselective 2′-alkylation, this simple
one-step process is convenient and economical, despite
its poor yield. The nucleoside was protected at the 5′-
end with dimethoxytrityl chloride and then treated with
hydrazine to liberate the free amine. The amines 4 and
7 were protected by the CEOC reagent 1 to yield
compounds 5 and 8, which were converted to the corre-
sponding â-cyanoethyl phosphoramidite compounds 6
and 9 and used under standard oligonucleotide auto-
mated synthesis conditions. Subsequently, standard depro-
A nonnucleosidic amino alcohol (6-aminohexanol) was
also protected by CEOC (compound 10) and converted
to its phosphoramidite derivative (compound 11; Scheme
4). This was coupled to the 5′-end of the oligonucleotides.
The purified oligonucleotides with aminolinkers (both
nucleosidic and nonnucleosidic) were used for conjugation
chemistry (i.e., attachment of other functionalities such
as fluorescein), in excellent yields starting with an
isothiocyanate to give the product via a thiourea linker
(Table 3).
Su m m a r y
A convenient crystalline reagent to protect aminolink-
ers (CEOC-O-succinimide) has been developed and used
to protect nucleoside-based 2′-O-alkyl aminolinkers and
nonnucleosidic aminolinkers. After oligonucleotide syn-
thesis incorporating these aminolinkers, standard NH4-
OH treatment removes the CEOC group by â-elimination.
The resultant oligonucleotides modified with 2′-O-alky-
lamines stabilize antisense oligomers toward RNA bind-
ing. Aminolinkers generated by this new method are also
useful for conjugation chemistry.
In addition to the potential applications in the nucleic
acid field, the CEOC reagent is of general use for amino
groups in all classes of compounds. Our present efforts
are directed toward this goal and will be reported in due
course.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents and solvents were purchased
from commercial sources unless otherwise noted. 2-Cyanoetha-
nol, N,N′-disuccinimidyl carbonate (DSC), N-(2-hydroxy)phthal-
imide, 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamid-
ite, and 6-aminohexanol were obtained from Aldrich Chemical
(15) Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S. M.;
Greig, M. J .; Guinosso, C. J .; Lesnik, E.; Manalili, S. M.; Mohan, V.;
Owens, S. R.; Ross, B. R.; Sasmor, H.; Wancewicz, E.; Weiler, K.;
Wheeler, P. D.; Cook, P. D. J . Med. Chem. 1996, 39, 5100.
(16) Manoharan, M.; Tivel, K. L.; Andrade, L. K.; Cook, P. D.
Tetrahedron Lett. 1995, 36, 3647.
(17) Ross, B. S.; Springer, R. H.; Tortorici, Z.; Dimock, S. Nucleosides
Nucleotides 1997, 16, 1641.
(18) The cost of 2,2′-anhydro-5-methyluridine is based on a bulk
order from Ajinomoto, Tokyo, J apan.
(19) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429.
The difference between phosphodiester DNA and phosphorothioate
DNA in Tm for an RNA target is nearly 0.8 °C/modification.
(20) Cook, P. D. Annu. Rep. Med. Chem. 1998, 33, 313.

6470 J . Org. Chem., Vol. 64, No. 17, 1999
Notes
Sch em e 2
Ta ble 1. Am in olin k er -Con ta in in g Oligon u cleotid es Syn th esized Usin g CEOC Str a tegya
mass
oligonucleotide
no.
modification
2′-O(CH2)nNH2, n
oligonucleotide sequence
GAT*CTe
T*CCAGGT*GT*CCGCAT*C
CTCGTACT*T*T*T*CCGGTCC
CTCGTACCT*TTCCGGTCC
GAT*CT
calcd
found
HPLC tR,d min
1
2
3
4
5
6
7
6
6
6
6
2
2
1895.00b
5599.00b
5853.83c
5493.21c
1839.65b
5368.00b
6161.68
1895.57
5597.24
5854.56
5493.91
1839.60
5370.40
6160.72
20.05
24.01
20.25
18.22
19.92
23.88
17.86f
T*CCAGGT*GT*CCGGCAT*C
L*-GCATCCCCCAGGCCACCAT
nonnucleosidic
The asterisked position indicates the site of modification. DMT-on. c DMT-off. HPLC conditions: Waters C-18 Delta-Pak reversed-
a
b
d
phase column, 3.9 × 300 mm; solvent A ) 100 mM triethylammonium acetate (TEAAc) pH 7; solvent B ) CH3CN; gradient of 8-18% B
e
in 30 min; flow rate 1.5 mL/min, λ ) 260 nm. 31P NMR (in D2O, ppm) -0.05 (one), -0.38 (two), -0.46 (one). f Waters C-4, 3.9 × 300
mm, solvent A ) 50 mm TEAAc, pH 7; solvent B ) CH3CN; gradient 5-60% B in 55 min; flow rate 1.5 mL/min, λ ) 260 nm.
Ta ble 2. Tm Da ta for Oligon u cleotid esa w ith 2′-O-(2-Am in oeth yl) a n d 2′-O-(6-Am in oh exyl) Mod ifica tion sb
Tm (°C)
oligonucleotide
no.
modification
-O(CH2)nNH2
Tm (°C) (against
RNA target)
(unmodified
parent DNA)
∆Tm/
modification (°C)
sequence
6
2
3
T*CCAGGT*GT*CCGGCAT*C
T*CCAGGT*GT*CCGCAT*C
CTCGTACT*T*T*T*CCGGTCC
2
6
6
66.42
65.54
66.2
62.3
62.3
61.8
1.03
0.81
1.1
a
b
All nucleosides with asterisk contain 2′-O-(aminotether). Tm conditions as described by Freier and Altmann.19
Co., Inc. (Milwaukee, WI). Reagents for the DNA synthesizer
were purchased from PerSeptive Biosystems, Inc. (Framingham,
MA). 2,2′-Anhydro-5-methyluridine was purchased from Ajino-
moto (Tokyo, J apan). Flash chromatography was performed on
silica gel (Baker, 40 µm). Thin-layer chromatography was
performed on Kieselgel glass plates from E. Merck and visualized
with UV light and p-anisaldehyde/sulfuric acid/acetic acid spray
followed by charring.
N-(2-Cya n oet h oxyca r b on yloxy)su ccin im id e (CE OC-O-
su ccin im id e) (1). To a stirred solution of 2-cyanoethanol (7.23
g, 102 mmol) in 300 mL of anhydrous CH3CN, under argon
atmosphere, was added N,N′-disuccinimidyl carbonate (34.0 g,
133 mmol) followed by pyridine (11.3 mL, 140 mmol). The
suspension became a clear solution after about 1 h. The solution
was stirred for an additional 6 h and then concentrated in vacuo.
It was redissolved in dichloromethane (200 mL) and extracted
with saturated NaHCO3 solution (3 × 50 mL) followed by
saturated NaCl solution (3 × 50 mL). The organic layer was
dried (anhydrous Na2SO4) and concentrated to give a white solid.
Traces of pyridine were removed by coevaporation with dry
acetonitrile. The white solid was dried overnight in vacuo and
then triturated with ether (150 mL) to yield 20.23 g of a colorless
amorphous powder of 1 (94%). This material is stable at room
temperature in a desiccator for an extended period (1-2 years).
For all derivatizations described in this paper, the material can
be used as is. The proton and carbon NMR spectra revealed a
homogeneous material even at this stage. The material was
further purified by chromatography on silica gel using CH2Cl2/

Notes
J . Org. Chem., Vol. 64, No. 17, 1999 6471
was purified by chromatography on silica gel starting with ethyl
acetate to remove the excess phthalimide reagent followed by
ethyl acetate-methanol 95/5 to elute 2 (22.2 g, 20.6%): 1H NMR
(200 MHz, DMSO-d6); δ 1.8 (s, 3H), 3.4-4.2 (m, 6H), 5.0-5.2
(m, 2H), 5.8 (d, J ) 5.1 Hz, 1H), 7.65 (s, 1H), 7.8-8.0 (m, 4H),
11.2 (s, 1H).
Sch em e 3
2′-O-(2-P h th a lim id oeth yl)-5′-O-(4,4′-d im eth oxytr ityl)-5-
m eth ylu r id in e (3). 2′-O-Phthalimidoethyl-5-methyluridine (2,
22.2 g, 0.053 mol) was coevaporated with pyridine (2 × 75 mL)
and then dissolved in 100 mL of pyridine. Dimethoxytrityl
chloride (27 g, 0.080 mol) was added in one portion with stirring.
TLC (ethyl acetate-hexanes 50:50) after 1 h indicated complete
reaction. Methanol (10 mL) was added to quench the reaction.
The mixture was concentrated and the residue was partitioned
between ethyl acetate and saturated sodium bicarbonate solution
(150 mL each). The organic layer was concentrated, and the
residue was dissolved in a minimum amount of dichloromethane
and applied to a silica gel column. The compound was eluted
with ethyl acetate-hexanes-triethylamine (50:50:1 to 80:20:1)
to give 3 (26.1 g, 68%) as a white foam: 1H NMR (200 MHz,
CDCl3) δ 1.33 (s, 3H), 3.05 (d, J ) 8.4 Hz, 1H), 3.49 (m, 2H), 3.8
(s, 6H), 3.9-4.1 (m, 4H), 4.18-4.26 (m, 1H), 4.47 (m, 1H), 5.88
(s, 1H), 6.84 (d, J ) 8.78 Hz, 4H), 7.22-7.43 (m, 9H), 7.69-7.88
(m, 5H), 8.26 (s, 1H); HRMS(FAB) calcd for C41H39N3O10Na+
756.2533, found 756.2553.
2′-O-(2-Am in oeth yl)-5′-O-(4,4′-d im eth oxytr ityl)-5-m eth -
ylu r id in e (4). 2′-O-Phthalimidoethyl-5′-O-DMT-5-methyluri-
dine (3, 21.1 g, 0.029 mol) was dissolved in methanol (500 mL).
Anhydrous hydrazine (4.9 mL, 0.15 mol) was added, and the
solution was heated to reflux. TLC after 3 h indicated a complete
reaction. The residue was purified by chromatography on silica
gel using methanol and then methanol-ammonium hydroxide
(98:2) to give 4 (12.4 g, 70%). The material was completely
soluble in methylene chloride, and any traces of silica from the
leaching of the column were removed by filtration at this stage
and reevaporation of the solution: 1H NMR (200 MHz, CDCl3)
δ 1.39 (s, 3H), 2.98 (t, J ) 3.48 Hz, 2H), 3.45 (d, J ) 2.56 Hz,
1H), 3.53 (d, J ) 1.96 Hz, 1H), 3.56-3.68 (m, 2H), 3.81 (s, 6H),
3.99 (m, 1H), 4.1 (t, J ) 4.56 Hz, 1H), 4.17 (m, 1H), 4.45 (t, J )
5.06 Hz, 1H), 6.06 (d, J ) 4.12 Hz, 1H), 6.86 (d, J ) 8.9 Hz, 4H),
7.25-7.46 (m, 9H), 7.67 (s, 1H); 13C (50 MHz, CDCl3) δ 11.71,
40.55, 45.76, 55.03, 62.47, 69.15, 70.65, 82.64, 83.49, 86.62, 87.10,
110.98, 113.09, 126.91, 127.77, 127.97, 129.95, 135.35, 144.25,
151.27, 158.46, 164.97; HRMS (FAB) calcd for C33H37O8N3Na+
626.2478, found 626.2501.
Sch em e 4
2′-O-[N-(2-Cya n oet h oxyca r b on yl)-2-a m in oet h yl]-5′-O-
(4,4′-d im eth oxytr ityl)-5-m eth ylu r id in e (5). To compound 1
(0.6 g, 2.8 mmol) in 10 mL of CH2Cl2 was added 0.5 mL of
pyridine followed by 2′-O-(aminoethyl)-5′-O-DMT-5-methyluri-
dine (4, 1.43 g, 2.35 mmol), and the mixture was stirred for 1 h.
TLC (CH2Cl2/CH3OH 9:1; Rf ) 0.48) indicated the complete
conversion of amine into the carbamate derivative. The mixture
was diluted with CH2Cl2 (50 mL), washed successively with
aqueous NaHCO3 solution and saturated NaCl solution, and
dried over MgSO4. Chromatography over silica and elution with
CH2Cl2/EtOAc gave the desired nucleoside 5 (1.2 g, 73%): 1H
NMR (400 MHz, DMSO-d6) δ 1.41 (s, 3H), 2.82 (t, J ) 6 Hz,
2H), 3.18-3.3 (m, 4H), 3.56-3.64 (m, 2H), 3.94-4.08 (m, 2H),
4.11 (t, J ) 6 Hz, 2H), 4.20-4.28 (m, 1H), 5.14 (d, J ) 6.83 Hz,
1H), 5.82 (d, J ) 4.06 Hz, 1H), 6.90 (d, J ) 8.97 Hz, 4H), 7.24-
7.42 (m, 9H), 7.48 (s, 1H), 11.38 (s, 1H); 13C NMR (50 MHz,
CDCl3) δ 11.70, 18.21, 40.77, 55.08, 59.07, 61.88, 68.87, 70.04,
82.59, 83.28, 86.63, 87.42, 111.03, 113.3, 117.3, 126.97, 127.87,
127.98, 129.95, 135.12, 136.21, 144.19, 150.77, 156.01, 158.50,
164,24; HRMS (FAB) calcd for C37H40O10N4Cs+ 833.1799, found
833.1824.
EtOAc (50:50) to give a white crystalline compound (18.72 g,
1
87%): mp 105.5 °C. (Rf ) 0.21 in the same solvent system); H
NMR (400 MHz, CDCl3) δ 2.85 (t, J ) 6.62 Hz, 2H), 2.86 (s, 4H)
4.45 (t, J ) 5.96 Hz); 13C NMR (80 MHz, DMSO-d6) δ 17.33,
25.39, 65.86, 117.91, 150.91, 169.82; HRMS(FAB) calcd for
+
C8H9N2O5
213.0511, found 213.0509. Anal. Calcd for
C8H8N2O5: C, 45.29; H, 3.80; N, 13.20. Found: C, 45.19; H, 3.45;
N, 13.02.
P r otection of Nu cleosid ic Am in olin k er s. 2′-O-P h th a l-
im id oeth yl-5-m eth ylu r id in e (2). N-(2-Hydroxyethyl)phthal-
imide (277 g, 1.45 mol) was slowly added to a solution of borane
in tetrahydrofuran (1 M, 600 mL) with stirring. Hydrogen gas
evolved as the solid dissolved. Once the rate of gas evolution
subsided, the solution was placed in a 2 L stainless steel bomb.
2,2′-Anhydro-5-methyluridine (60 g, 0.25 mol) and sodium
bicarbonate (120 mg) were added, and the bomb was sealed.
After 30 min, the bomb was vented and placed in an oil bath
and heated to 150 °C internal temperature for 24 h. The bomb
was cooled to room temperature and opened. TLC (ethyl
acetate-methanol 95/5) revealed the disappearance of starting
material. The crude solution was concentrated, and the residue
2′-O-[N-(2-Cya n oet h oxyca r b on yl)-2-a m in oet h yl]-5′-O-
(4,4′-dim eth oxytr ityl)-5-m eth ylu r idin e 3′-O-[(2-cyan oeth yl)-
N,N-d iisop r op yl]p h osp h or a m id ite (6). Compound 5 (0.7 g,
1 mmol) was dissolved in dry CH2Cl2 (15 mL), and N,N-
diisopropylammonium tetrazolide (0.085 g, 0.5 mmol) followed
by 2-cyanoethyl-N,N,N′N′-tetraisopropyl phosphorodiamidite
(0.420 mL, 1.1 mmol) was added slowly using a syringe under
argon. The mixture was stirred at room temperature overnight,
after which TLC (CH2Cl2/EtOAc 50:50) indicated almost com-
plete reaction. An additional aliquot of the phosphitylation
reagent (0.045 mL, 0.12 mmol) was added and the mixture
stirred for an additional 2 h. TLC then indicated complete
conversion of the starting material to the phosphoramidite (CH2-

6472 J . Org. Chem., Vol. 64, No. 17, 1999
Ta ble 3. Con ju ga tion Ch em istr y of Oligon u cleotid es Der ived fr om CEOC Str a tegy
Notes
mass
oligonucleotide no.
sequence (5′-3′)
HPLCa tR (min)
calcd
found
4
8
7
9
CTCGTACCT*TTCCGGTCC
CTCGTACCTFLTTCCGGTCC
L*-TGCATCCCCCAGGCCACCAT
18.22
22.08
17.96
23.74
5493.21
5881.60
6161.68
6550.68
5493.91
5880.89
6160.72
6550.01
L
FL-TGCATCCCCCAGGCCACCAT
a
C-4 Waters, Delta-Pak C4 column 3.9 × 300 mm, sSolvent A ) 50 mM triethylammonium acetate pH 7; solvent B ) acetonitrile,
5-60% B in 55 min; flow 1.5 mL/min, λ ) 260 nm. L* ) 2′-O-(CH2)6NH2. LFL ) fluorescein-NHC(dS)NH(CH2)6O-. T* ) 2′-O-(6-aminohexyl)
5MeU. TFL ) 2′-O-(fluorescein-NHC(dS)NH(CH2)6-) 5MeU.
+
Cl2/EtOAc 50:50; Rf ) 0.33). The reaction mixture was diluted
with 50 mL of CH2Cl2 and washed with saturated NaHCO3
solution followed by saturated NaCl solution. The organic layer
was dried over MgSO4 and evaporated to dryness. The crude
foam was purified by chromatography on silica gel and eluted
with 50:50 EtOAc/CH2Cl2 to give 6 (0.72 g, 81%): 1H NMR (200
MHz, CDCl3) δ 1.02 (d, J ) 5.18 Hz, 3H), 1.14-1.2 (m, 9H), 2.44
(t, J ) 6.14, 2H), 2.654-2.78 (m, 4H), 3.21-3.47 (m, 4H), 3.51-
3.71 (m, 4H), 3.75-3.95 (m, 4H), 3.81 (s, 6H), 4.1-4.16 (m, 2H),
4.28 (t, J ) 5.96 Hz, 4H), 4.51-4.61 (m, 1H), 5.97-6.01 (m, 1H),
6.83-6.88 (m, 4H), 7.2-7.47 (m, 9H), 7.69 (s, 1H), 7.73(s,1H);
31P NMR (80 MHz, CDCl3) δ 149.5 and 150.5; MS (ES) m/z 923
[M + Na]+.
117.34, 155.64; HRMS (FAB) calcd for C10H19N2O3 215.1396,
found 215.1395.
N-(2-Cya n oeth oxyca r bon yl)-6-a m in oh exyl O-(2-cya n o-
eth yl N,N-d iisop r op ylp h osp h or a m id ite) (11). Compound 10
(0.72 g, 3.36 mmol) was mixed with N,N-diisopropylammonium
tetrazolide (0.29 g, 1.68 mmol). The mixture was then dried over
P2O5 in vacuo overnight at 40 °C. The reaction flask was flushed
with argon. Anhydrous acetonitrile (17 mL) was added, followed
by dropwise addition of 2-cyanoethyl N,N,N′,N′-tetraisopropy-
lphosphorodiamidite (1.52 mL, 5.04 mmol). The reaction mixture
was stirred at room temperature for 4 h under argon. Solvent
was removed in vacuo. The residue was placed on a flash column
and eluted with ethyl acetate/hexane 1:1 to get 11 as an oil (0.62
g, 44% yield): Rf (0.05, EtOAc/hexane 50:50); 1H NMR (200 MHz,
CDCl3) δ 1.22 (s, 6H), 1.22 (s, 6H), 1.48-1.7 (m, 8H), 2.62-2.75
(m, 4H), 3.21 (q, J ) 6.46 Hz, 2H), 3.53-3.95 (m, 6H), 4.29 (t, J
) 6.24 Hz, 2H); 31P NMR (80 MHz, CDCl3) δ 147.78; MS (FAB)
m/z 437 [M + Na]+.
Oligon u cleotid e Syn th esis. Compound 6, 8, or 11 was
dissolved in anhydrous acetonitrile to give 0.1 M solution and
loaded onto an Expedite Nucleic Acid Synthesis system (Milli-
pore 8909) to synthesize the oligonucleotides. The coupling
efficiencies were more than 98%. For the coupling of the modified
amidites (6, 8, or 11), coupling time was extended to 10 min,
and this step was carried out twice. All other steps in the protocol
supplied by Millipore were used as such. The oligomers were
cleaved from the controlled pore glass (CPG) supports and
deprotected under standard conditions (12 h) using concentrated
aqueous NH4OH (30%) at 55 °C. 5′-O-DMT-containing oligomers
were then purified by reversed-phase high-performance liquid
chromatography [(C-4 column, Waters, 7.8 × 300 mm, A ) 50
mM triethylammonium acetate, pH ) 7, B)acetonitrile, 5-60%
of B in 55 min, flow 2.5 mL/min), λ ) 260 nm]. Detritylation of
the purified full-length oligonucleotide with aqueous 80% acetic
acid and evaporation, followed by desalting in a Sephadex G-25
column, gave modified oligonucleotides. Oligonucleotides were
analyzed by HPLC, capillary gel electrophoresis and mass
spectrometry.
2′-O-[N-(2-Cya n oet h oxyca r b on yl)-6-a m in oh exyl]-5′-O-
(4,4′-d im eth oxytr ityl)-5-m eth ylu r id in e (8). To a solution of
7 (3.3 g, 5 mmol)13 in anhydrous CH2Cl2 (20 mL) was added
anhydrous pyridine (1 mL) followed by compound 1 (1.2 g, 5.6
mmol). The reaction mixture was stirred for 2 h and tested by
TLC (CH2Cl2/CH3OH 9:1). The reaction was complete, and the
reaction mixture was applied to silica gel equilibrated with CH2-
Cl2/CH3OH 9:1 and eluted with the same solvent system (Rf )
0.51) to give 8 (3.13 g, 82%): 1H NMR (200 MHz, CDCl3) δ 1.28-
1.6 (m, 11H), 2.6-2.78 (m, 3H), 3.21 (q, J ) 6.24, 2H), 3.43 (d,
J ) 8.4 Hz, 1H), 3.57 (d, J ) 10.74 Hz, 1H), 3.66-3.95 (m, 2H),
3.81 (s, 6H), 4.03-4.11 (m, 2H), 4.14-4.5 (m, 1H), 4.25-4.35
(m, 2H), 5.18 (br. s, 1H), 6.01 (d, J ) 2.94 Hz, 1H), 6.85 (d, J )
8.8 Hz, 4H), 7.22-7.44 (m, 9H), 7.68 (s, 1H), 8.62 (br s, 1H); 13
C
NMR (CDCl3) δ 11.59, 18.2, 25.24, 26.03, 29.11, 29.37, 40.68,
55.05, 58.83, 62.10, 68.97, 70.66, 82.59, 83.38, 86.66, 87.1, 110.94,
113.09, 117.17, 126.94, 127.80, 127.97, 129.93, 135.09, 135.22,
144.14, 150.45, 155.44, 158.48, 164.14. HRMS(FAB) calcd for
C41H48N4O10Na+ 779.3268, found 779.3259.
2′-O-[N-(2-Cya n oet h oxyca r b on yl)-6-a m in oh exyl]-5′-O-
(4,4′d im eth oxytr ityl)-5-m eth ylu r id in e 3′-O-[(2-Cya n oeth -
yl)N,N-d iisop r op yl) P h osp h or a m id ite (9). Nucleoside 8 (1.51
g, 2 mmol) was dissolved in 30 mL of anhydrous CH2Cl2. To this
solution was added N,N-diisopropylammonium tetrazolide (0.17
g, 1 mmol) followed by 2-cyanoethyl N,N,N′N′-tetraisopropy-
lphosphorodiamidite (0.99 mL, 2.6 mmol) under argon atmo-
sphere. The reaction mixture was stirred for 16 h. TLC analysis
(50:50 CH2Cl2/EtOAc) indicated completion of the reaction. The
reaction mixture was then diluted with CH2Cl2 (100 mL),
extracted with saturated NaHCO3 solution (2 × 50 mL), washed
with saturated NaCl solution (50 mL), and dried over MgSO4.
Evaporation to dryness yielded a white foam. This white foam
was dissolved in CH2Cl2 and applied to a silica gel column
equilibrated with CH2Cl2 containing 0.1% pyridine. Elution with
40:60 EtOAc/CH2Cl2 yielded 9 (1.3 g, 68%) as a foam: 1H NMR
(200 MHz, CDCl3) δ 1.00 (d, J ) 6.74 Hz, 3H), 1.11-1.4 (m, 12H),
1.46-1.78 (m, 8H), 2.62-2.77 (m, 4H), 3.13-3.22 (m, 2H), 3.4-
3.72 (m, 4H), 3.79 (s, 6H), 3.84-3.98 (m, 2H), 4.14-4.5 (m, 5H),
4-4.12 (m, 2H), 5.98-6.09 (m, 1H), 6.78-6.9 (m, 4H), 7.21-
7.48 (m, 9H), 7.59 (s, 1H), 7.71 (s, 1H); 31P NMR (80 MHz,
CDCl3) δ 150.5, 151 ppm; MS (ES) m/z 979 (M + Na]+.
Oligon u cleot id e Con ju ga t ion Ch em ist r y. Oligonucle-
otides 4 and 7 (Table 1) containing the tethered amino func-
tionality were used to conjugate fluorescein to the oligonucle-
otide. Purified oligonucleotides (15 OD/mL) in water were
evaporated, and the residue was dissolved in 100 µL of 1 M
NaHCO3/Na2CO3 buffer (pH 9.2). A solution of fluorescein
isothiocyanate (100 µL, 1 M solution in DMF) was added to the
solution of oligonucleotides and kept at room temperature for
24 h. Unreacted fluorescein isothiocyanate was removed by
passing the reaction mixture through a column of Sephadex G-25
and eluting with water. Conjugated oligonucleotides were then
purified by reversed-phase HPLC and characterized by electro-
spray mass spectrometry, analytical HPLC, and CGE (Table 3).
Ack n ow led gm en t. We thank Professor Michael E.
J ung (UCLA) for helpful discussions, Dr. Elena Lesnik
for Tm studies, and Mr. Ask Pu¨schl (University of
Copenhagen) for checking the large-scale synthesis
protocol of CEOC-O-succinimide.
P r ot ect ion of
a Non n u cleosid ic Am in olin k er . N-(2-
Cya n oeth oxyca r bon yl)-6-a m in oh exa n ol (10). 6-Aminohex-
anol (0.5 g, 4.23 mmol) was dissolved in anhydrous CH2Cl2 (10
mL). Compound 1 (1.08 g, 5.09 mmol) was added, and the
mixture was stirred for 2 h. The reaction was followed by TLC
(5% MeOH in CH2Cl2). Solvent was removed in vacuo, and the
residue was placed on a flash column and eluted with 5% MeOH
in CH2Cl2 to get 10 as a white powder (0.883 g, 96% yield): Rf
) 0.28, 5% MeOH in CH2Cl2; 1H NMR (200 MHz, CDCl3) δ 1.6-
1.39 (m, 8H), 2.73 (t, J ) 6.12 Hz, 2H,), 3.68 (t, J ) 3.71 Hz,
3H), 4.3 (t, J ) 6.12 Hz, 2H,), 4.9 (br, 1H); 13C NMR (50 MHz,
CDCl3) δ 18.49, 25.30, 26.35, 29.73, 32.47, 40.92, 59.01, 62.49,
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
and high-resolution mass spectra of compounds 1, 4, 5, 8, and
10. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O982299Y