A novel approach to oligonucleotide synthesis using an imidazolium ion tag as a soluble support

Article abstract of DOI:10.1021/jo061279q

(Chemical Equation Presented) The synthesis of oligonucleotides in solution using a soluble, ionic liquid based support is described. Short oligomers of varying base composition were synthesized using this method in high yields and high purity, requiring

Full text of DOI:10.1021/jo061279q

A Novel Approach to Oligonucleotide Synthesis
Using an Imidazolium Ion Tag as a Soluble
Support
oligonucleotides, the use of insoluble supports has become an
important tool for organic synthesis, especially in the synthesis
of biopolymers, such as oligonucleotides, peptides, and, more
9
-11
recently, carbohydrates.
Although extremely successful,
because of the heterogeneous nature of the insoluble polymers,
solid-phase synthesis has all the problems generally associated
with heterogeneous reaction conditions. In addition, the high
cost of the supports themselves must be considered, as in the
case of the synthesis of oligonucleotides for therapeutic ap-
plications where 30-40% of total raw materials cost come from
Robert A. Donga, Syed M. Khaliq-Uz-Zaman,
Tak-Hang Chan,* and Masad J. Damha*
Department of Chemistry, McGill UniVersity, Montreal, Quebec,
Canada H3A 2K6
masad.damha@mcgill.ca; tak-hang.chan@mcgill.ca
1
2
the solid support.
ReceiVed June 21, 2006
In recent years, the use of soluble polymer supports has
received considerable attention because such “solution-phase”
syntheses retain many of the advantages of conventional solution
chemistry, while still permitting facile purification of the
product. Thus, soluble poly(ethylene glycol) (PEG) and other
9
polymers have been used for the synthesis of oligopeptides,
nucleotides, saccharides, and small molecules.
1
0
11
13,14
Some
limitations experienced when using soluble polymer supports
include low loading capacity, limited solubility during the
synthesis of longer peptides, and often low aqueous solubil-
1
3,14
ity,
purification.
The idea of using ionic liquid based supports for organic
as well as an energy intensive cooling step required for
1
0
The synthesis of oligonucleotides in solution using a soluble,
ionic liquid based support is described. Short oligomers of
varying base composition were synthesized using this method
in high yields and high purity, requiring no chromatography
for purification prior to cleavage from the support. The
solution-phase-synthesized oligomers were compared to the
same sequences prepared using standard gene machine
techniques by LCMS. This methodology may provide a
cheaper route for the large-scale synthesis of oligonucle-
otides.
1
5,16
synthesis
of small molecules,
has been recently demonstrated for the synthesis
17,18
19
20
oligopeptides, and oligosaccharides.
We demonstrate here, for the first time, that ionic liquid
supported synthesis (ILSS) can be applied to oligonucleotide
synthesis, using simple precipitation and phase separation
methods without the need for chromatography. This method
provides reasonably high product purity at each step and should
be much more amenable to large-scale manufacturing.
Solid-phase synthesis of oligonucleotides using the phos-
1
-5
phoramidite method
is an iterative process in which a solid
support with an attached nucleoside is deblocked at the terminus
by removing an acid labile protecting group, thus liberating a
nucleophilic 5′-hydroxyl group. This terminal nucleophile is then
allowed to couple to a protected 3′-O-phosphoramidite monomer
in the presence of an activator. The newly created phosphite
triester 3′,5′-linkage is then oxidized to provide the desired and
more stable phosphate triester. This process is repeated until
an oligomer of the desired length and composition is obtained.
The demands of the scientific community for synthetic
oligonucleotides have grown exponentially over the past de-
cades. The ready availability of DNA oligonucleotide primers
has satisfied the tremendous needs of the genome sequencing
efforts, research into functional genomics, and polymerase chain
reaction (PCR)-based detection methods, but significant ad-
vances in structural biology and biochemistry have only been
achieved through concomitant advances in DNA and RNA
chemical synthesis.1-5 Oligonucleotides have begun to see
widespread use in the development of therapeutics and in
diagnostic applications, and large quantities are now required.
(
9) Bayer, E.; Mutter, M. Nature 1972, 237, 512-513.
(10) Bonora, G. M.; Scremin, C. L.; Colonna, F. P.; Garbesi, A. Nucleic
Acids Res. 1990, 18, 3155-3159.
6
7,8
(11) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem.
Soc. 1991, 113, 5095-5097.
Since Merrifield and Letsinger et al. introduced the use
of polymer supports for the synthesis of oligopeptides and
(12) (a) Pon, R. T.; Yu, S.; Guo, Z.; Deshmukh, R.; Sanghvi, Y. S. J.
Chem. Soc., Perkin Trans. 1 2001, 2638-2643. (b) Sanghvi, Y. S. Personal
communication.
(1) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y.
(13) Gravert, D. J.; Janda, K. D. Chem. ReV. 1997, 97, 489-509.
(14) Toy, P. H.; Janda, K. D. Acc. Chem. Res. 2000, 33, 546-554.
(15) Chan, T.-H.; Li, L.-H.; Yang, Y.; Lu, W. Clean SolVents; Abraham,
M. A., Moens, L., Eds.; ACS Symposium Series 819; American Chemical
Society: Washington, DC, 2002.
(16) Law, M. C.; Wong, K.-Y.; Chan, T. H. J. Org. Chem. 2005, 70,
10434-10439.
(17) Miao, W.; Chan, T.-H. Org. Lett. 2003, 5, 5003-5005.
(18) Fraga-Dubreuil, J.; Bazureau, J. P. Tetrahedron 2003, 59, 6121-
6130.
Methods Enzymol. 1987, 154, 287-313.
(2) Alvarado-Urbina, G.; Sathe, G. M.; Liu, W. C.; Gillen, M. F.; Duck,
P. D.; Bender, R.; Ogilvie, K. K. Science 1981, 214, 270-274.
3) Ogilvie, K. K.; Usman, N.; Nicoghosian, K.; Cedergren, R. J. Proc.
Natl. Acad. Sci. U.S.A. 1988, 85, 5764-5768.
(
(
(
(
(
4) Damha, M. J.; Ogilvie, K. K. Methods Mol. Biol. 1993, 20, 81-114.
5) Bellon, L.; Wincott, F. Solid-Phase Synth. 2000, 475-528.
6) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.
7) Letsinger, R. L.; Mahadevan, V. J. Am. Chem. Soc. 1965, 87, 3526-
3
3
527.
(19) Miao, W.; Chan, T.-H. J. Org. Chem. 2005, 70, 3251-3255.
(20) (a) He, X.; Chan, T.-H Synthesis 2006, 10, 1645-1651. (b) Huang,
J.-Y.; Lei, M.; Wang, Y.-G. Tetrahedron Lett. 2006, 47, 3047-3050.
(
8) Letsinger, R. L.; Hamilton, S. B. J. Am. Chem. Soc. 1959, 81, 3009-
012.
1
0.1021/jo061279q CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/02/2006
J. Org. Chem. 2006, 71, 7907-7910
7907

SCHEME 1. Derivatization of Ionic Liquida
a
Reagents and conditions: (a) DCC, DMAP, CH3CN, 3 days, rt; (b) 3% TFA in CH2Cl2 or CH3CN.
TABLE 1. Recovery and Physical Data for Ionic Liquid
Supported Oligomersa
The same iterative methodology may be used when soluble
supports are employed.
m/z
m/z
The synthesis of the ionic liquid supported oligonucleotide
began with the ionic liquid 1 (Scheme 1), which was readily
prepared from N-methylimidazole and 2-bromoethanol followed
compound sequence % recovery 31P NMR (ppm) (exptl) (calcd)
DMT
6
6
6
6
a
b
c
ApT
CpT
GpT
TpT
89
91
90
91
93
95
96
78
92
98
89
100
-1.324, -1.477 1223.4 1223.4
-1.545, -1.754 1199.4 1199.4
-1.149, -1.194 1205.6 1205.4
-1.494, -1.584 1110.4 1110.4
DMT
DMT
DMT
HO
1
9
by anion exchange in accordance with literature procedures.
The succinylated 5′-DMT-thymidine derivative 22 was then
1
d
coupled to the ionic liquid 1 using dicyclohexylcarbodiimide
7a
ApT
CpT
-1.176, -1.516
-1.381, -1.613
-1.047, -1.064
-1.188, -1.284
921.4 921.3
897.3 897.3
903.4 903.3
808.3 808.3
HO
HO
HO
7
7
7
8
9
1
1
b
c
d
(
(
DCC) and catalytic amounts of 4-(dimethylamino)pyridine
DMAP) in acetonitrile (CH3CN) to give the ionic liquid
GpT
TpT
supported nucleoside 3. Compound 3 was isolated and purified
by precipitation from an ethyl ether-ethyl acetate solution. The
resulting precipitate was then taken up in chloroform and
extracted with water. The excess ionic liquid 1 was removed
with the aqueous phase, while the desired product 3 remained
in the organic phase due to the hydrophobicity of the dimethox-
ytrityl protecting group attached at the 5′-position of thymidine.
Exposure of 3 to acidic conditions yielded compound 4 as a
light brown foam in 96% yield. Structure and purity of 3 and 4
DMT
HO
TpTpT
-1.081 to -1.477 1467.5 1467.8
-1.157 to -1.531 1165.2 1165.3
-1.169 to -1.859 1824.2 1824.5
-1.142 to -1.51 1522.4 1522.4
TpTpT
DMT
0
1
TpTpTpT
TpTpTpT
HO
a
Ade N6 and Cyt N4 benzoyl protected, Gua N2 isobutyryl pro-
tected; 3′-terminal thymidine linked through succinyl ester to 1 as in 3 of
Scheme 1.
aqueous layer removed the resultant salts (NaI, Na2SO4, excess
bisulfite, pyridinium iodide, etc.) in addition to any uncoupled
ionic liquid supported nucleoside(tide) since it lacked a terminal
trityl group, rendering it water soluble. The organic layer
contained, in principle, only the desired product. Removal of
the organic solvent under reduced pressure yielded the products
1
were verified by H NMR and ESI-MS.
The dinucleoside phosphotriesters TpT, ApT, CpT, and GpT
2
2
(
6a-d) were prepared at the 250 µmol scale by reacting the
ionic liquid supported nucleoside 4 with a 1.5-fold excess of
the appropriate phosphoramidite derivatives (5a-d) using 4,5-
dicyanoimidazole (DCI) as the activating agent in THF or CH3-
CN and stirring for 1-2 h. After the reaction had come to
completion, the excess activated phosphoramidite was quenched
through the addition of anhydrous ethanol and a further 10 min
of stirring. Quenching the phosphoramidites facilitates their
removal during purification. The dinucleoside phosphite triester
intermediates were then isolated by simply precipitating from
(
6a-d) as light brown foams in good yields (Table 1) and high
purity, eliminating the need for a capping step. Detritylation of
a-d was achieved by the addition of 3% v/v trifluoroacetic
6
acid (TFA) in dichloromethane or CH3CN, stirring for 20 min,
and precipitation in 1:9 ethyl acetate:ethyl ether. The material
obtained at this point may contain 5% or more of tritylated
starting material, resulting from the equilibrium established
+
+
during this step (ROH + DMT f DMT - OR + H ).
Quenching with ethanol to trap the DMT cation did not seem
to eliminate this problem. However, purity is significantly
enhanced (no tritylated product observed by ESI-MS in the bulk
product) if the TFA treatment is repeated (i.e., redissolution of
the precipitate in 3% v/v TFA solution followed with a second
precipitation by dropwise addition to 1:9 ethyl acetate:ethyl
ether). The purified products are simply filtered off, yielding
off white to light yellow powdery solids in high yields (Table
1
:9 ethyl acetate:ethyl ether at room temperature, prior to
oxidation. Precipitation of the dinucleoside phosphite triester
at this stage is critical as the quenched excess mononucleoside
3
′-O-phosphoramidite is much more soluble in ethyl acetate-
ethyl ether than the coupled product, thus making its removal
from the dinucleoside phosphite triester possible. Generally, the
precipitation was repeated in order to enhance the purity of the
desired product. While we recognize that dinucleoside phosphite
triesters (P-III) are less stable than the corresponding phosphate
triesters (P-V), no decomposition was observed at this step.
To carry out the oxidation of the phosphite triester intermedi-
ates, the collected precipitate was again dissolved in a small
amount of CH3CN, a small excess of pyridine, or 2,4,6-collidine,
and a large excess (2-5 equiv) of a 0.1 M solution of iodine in
1). The collected solids (7a-d) were ready for further coupling
or, in cases where the desired length of oligo had been achieved,
deprotection.
In addition to the dimers 7a-d described above, a thymidine
trimer (8, 9) and a tetramer (10, 11) were also synthesized at
the 50-100 µmol scale. The identities of the ionic liquid
supported compounds have been confirmed with both low- and
2
:1 THF:water was added. Once the persistence of color due to
iodine was established (5 min), 5% w/v aqueous sodium bisulfite
was added to reduce the excess iodine. This reaction mixture is
then diluted with chloroform and extracted with water. The
31
high-resolution ESI-MS as well as P NMR in conjunction with
3
1
23
the use of a P CIGAR experiment, allowing the observation
of the expected 3′-5′ connectivity through the diastereomeric
phosphotriester linkages (Table 1). The yields are expressed as
recoveries since traditional solution-phase phosphoramidite
chemistry is known to go to 98-100% completion, but for the
(
21) Usman, N.; Ogilvie, K. K.; Jiang, M. Y.; Cedergren, R. J. J. Am.
Chem. Soc. 1987, 109, 7845-7854.
22) Damha, M. J.; Ogilvie, K. K. J. Org. Chem. 1988, 53, 3710-3722.
(
7
908 J. Org. Chem., Vol. 71, No. 20, 2006

TABLE 2. HPLC/MS Analysis of Deprotected Oligonucleotidesa
oxidation extraction largely removes this material from the IL
mediated synthesis.
synthetic
support
retention
time (min)
% total
area
low resolution
MS (-ve mode)
sequence
ApT
In this study, a new strategy to synthesize oligonucleotides
using an ionic liquid as a soluble support has been demonstrated.
Oligonucleotides up to tetrameric species have been synthesized
and have been shown to be comparable to products generated
using standard automated DNA synthesis techniques. Overall,
the solution based ILSS method provides a novel route to
IL
CPG
IL
CPG
IL
CPG
IL
CPG
IL
CPG
IL
26.3
26.3
16.7
16.8
19.0
18.9
25.3
25.3
29.9
29.5
32.4
31.9
99.3
96.1
93.3
89.5
97.1
95.1
91.7
92.5
92.1
94.0
87.3
90.5
554.2
554.2
530.2
530.2
570.2
570.2
545.2
545.2
849.1
849.2
1153.2
1153.2
CpT
GpT
1
31
TpT
construct and characterize (by TLC, H NMR, P NMR, and
MS techniques) oligonucleotides during chain assembly as well
as facile, precipitation based purifications of the intermediates
during chain elongation. At this point, the method has been
demonstrated at the 100-250 µmol scale and should be scalable.
Compared to the solid phase method, it offers the potential
advantage of reduced cost of the relatively expensive solid
supports (CPG, OligoPrep, NittoPhase, etc.). Compared to solu-
tion-phase synthesis using soluble polymer supports, such as
poly(ethylene glycol) (PEG, MW ) 5000-20 000), the ILSS
method with compound 1 (MW ) 214) as the support has a
much better loading capacity. Of course, while we have
demonstrated that the ILSS approach is compatible with standard
oligonucleotide synthesis chemistries, it remains to be deter-
mined if the approach is applicable to longer oligomers.
TpTpT
TpTpTpT
CPG
a
IL ) prepared with ionic liquid support; CPG ) prepared with
controlled pore glass with gene machine; % total area for IL based
oligonucleotides excludes peak area due to IL peaks.
methodology employed, there are small losses associated with
the manipulations involved in the purifications of the com-
pounds. These losses are likely fixed, and larger-scale synthesis
should show higher proportional recoveries.
The oligonucleotides synthesized above in solution have been
compared to the same sequences synthesized on controlled pore
glass (CPG; 1 µmol scale), a commonly used solid support, and
the two systems have been deprotected in parallel. Complete
deprotection of the desired oligonucleotides was achieved by
treating them with concentrated ammonium hydroxide/ethanol
for 48 h at room temperature or 16 h at 60 °C. These conditions
ensure complete cleavage of the cyanoethyl protecting group,
the ionic liquid moiety, any protection of the exocyclic amines
of the bases (Ade, Cyt, and Gua), and the monosuccinate linker.
The oligonucleotides were isolated by removal of the ethanol
and ammonium hydroxide solution under vacuum, redissolution
in water (the solid support is simply settled by centrifugation
for the CPG supported oligomers), and then chromatographic
purification by ion-pairing reverse phase HPLC, anion-exchange
HPLC, or polyacrylamide gel electrophoresis.
Experimental Section
DMTTSucc-IL (3). Compound 2 (1 g, 1.63 mmol), substituted
imidazolium tetrafluoroborate 1 (0.38 g, 1.76 mmol), and DMAP
(
0.052 g, 0.41 mmol) were placed in a dry, nitrogen-purged 100
mL round-bottom flask. To this mixture was added dicyclohexyl-
carbodiimide (DCC) (0.68 g, 3.3 mmol), followed by dry CH CN
20 mL). The reaction mixture was stirred for 3 days at room
3
(
temperature. TLC analysis in 9:1 chloroform:methanol showed the
formation of a more polar product. When the reaction was stopped,
the insoluble DCU byproduct was allowed to settle, and the reaction
mixture was filtered and washed with CH CN several times. The
3
solvent was evaporated, and the residue was again washed with
ether to remove any unreacted DCC and finally collected and dried
under vacuum. The product 3 was obtained as a light brown foam
The products of the ILSS procedure have been compared via
LCMS to those obtained through automated solid-phase syn-
1
(
9
1.1 g, 83% yield). H NMR (acetone-d
6
): δ 10.03 (1H, s, NH),
.10 (1H, s, CH), 7.83 (1H, s, CH), 7.71 (1H, s, CH), 7.60 (1H, s,
CH), 7.50-6.92 (14H, m, Ar-H), 6.33 (1H, t, J ) 8 Hz, CH),
.50 (1H, d, J ) 6 Hz, CH), 4.55-4.51 (2H, m, CH ), 4.70-4.66
1
thesis techniques (Table 2). In all cases, the retention times of
the IL generated material correlate well with the CPG generated
oligomer. The purities of the oligomers prepared by the two
methods are comparable. For the dimers, the IL method gave
products of better purities, whereas for the trimers and tetramers,
the CPG method gave slightly purer products. The origin of
impurities appears to be different in the two methods. In the
ILSS generated trimer and tetramer, the impurities are due to
the presence of small amounts of “n - 1” peaks, which are
likely due to the incomplete detritylation at the stage prior to
coupling. This tritylated material would not be removed during
the extraction and would be deblocked in the detritylation after
coupling giving the n - l oligomer. Thus it is extremely
important to ensure complete detritylation at each step. On the
other hand, thymidine nucleoside is visible in several of the
HPLC traces of oligomers synthesized on CPG. Its presence
5
2
(2H, m, CH ), 4.15 (1H, br s, CH), 4.05 (3, s, CH ), 3.80 (3H, s,
2
3
CH
3
), 3.50-3.40 (2H, m, CH
2
), 2.70 (4H, br s, 2 CH ), 2.61-2.40
2
+
2 3 43 4 9
(2H, m, CH ), 1.42 (3H, s, CH ). ESI-MS for C40H N O : calcd
7
23.3, found 723.4.
HO
T
Succ-IL (4). To a solution of 3 (2.96 g, 3.65 mmol) in
dichloromethane (200 mL) was added 3% v/v TFA in dichlo-
romethane or CH CN (100 mL). During the addition of TFA, the
solution became reddish-orange, and stirring was maintained for
0 min. The product was precipitated from 1:9 ethyl acetate:ethyl
3
2
ether, filtered, redissolved in a minimum amount of the acid
solution, precipitated again, and filtered. The precipitate was rinsed
with 1:9 ethyl acetate:ethyl ether, recovered from the filter by
3
dissolving in CH CN, and evaporated under reduced pressure,
yielding compound 4 as a light brown foam (1.88 g, 96% yield).
1
H NMR (DMSO-d
.10 (1H, s, CH), 7.76 (1H, s, CH), 7.72 and 7.45 (1H, s, CH),
7.70 (1H, s, CH), 6.15 (1H, m, CH), 5.27 and 5.19 (1H, m, CH),
6
): δ 11.40 and 11.39 (total of 1H, 2 s, NH),
9
arises from incomplete coupling or partial detritylation of the
solid support prior to the initial capping step.24 Though this
4
.60 and 3.60 (2H, m, CH
CH
unresolved m, 2 CH
2
), 4.45 (2H, br s, CH
2
), 4.39 (2H, br s,
), 2.61-2.60 (4H,
), 1.77 (3H, s, CH ).
is normally present in materials synthesized on CPG, the post-
2
), 4.24-3.93 (1H, m, CH), 3.86 (3, s, CH
3
2
), 2.40-2.16 (2H, m, CH
2
3
(
23) Hadden, C. E.; Martin, G. E.; Krishnamurthy, V. V. Magn. Reson.
Chem. 2000, 38, 143-147.
24) Pon, R. T.; Usman, N.; Ogilvie, K. K. Biotechniques 1988, 6, 768-
75.
+
ESI-HRMS for C20
H
27
N
4
O
8
: required 451.18289, found 451.18234.
DMT
(
TpTSucc-IL (6a). Compound 4 (0.24 g, 0.45 mmol), thymidine
7
phosphoramidite 5a (0.57 g, 0.77 mmol), and dicyanoimidazole
J. Org. Chem, Vol. 71, No. 20, 2006 7909

(
0.66 g, 5.6 mmol) were transferred to a 50 mL oven-dried,
nitrogen-purged round-bottom flask. To the mixture was added dry
THF or CH CN (5 mL) to the flask, and the resulting solution was
stirred at room temperature for 1-2 h. At this point, a small amount
3
NMR (acetonitrile-d ): δ -1.157 to -1.531 (broad overlap of
+
peaks). ESI-HRMS for C46
found 1165.32752.
59 10 22 2
H N O P : required 1165.32807,
3
DMTTpTpTpTSucc-IL (10). Compound 9 (170 mg, 0.136 mmol)
was mixed with 3′-phosphoramidite 5a (0.160 g, 0.215 mmol) and
DCI (0.21 g, 1.8 mmol) in dry CH CN (5 mL) at room temperature.
3
After being stirred for 2 h and quenched with anhydrous ethanol
for 10 min, the product was precipitated from 1:9 ethyl acetate:
ethyl ether twice, oxidized, and extracted in the same manner as
(
1-2 equiv) of anhydrous ethanol was added and stirring was
continued for a further 10 min. The product was then precipitated
twice from 1:9 ethyl acetate:ethyl ether. At this point, the precipitate
was redissolved in CH CN, and 2,4,6-collidine or pyridine (ap-
3
proximately 300 µL) was added followed by addition of an aqueous
iodine solution (0.1 M in THF/water 2:1, excess), dropwise until
the iodine color persisted, to oxidize the phosphite triester
intermediate. After 5 min, the reaction mixture was quenched with
an aqueous sodium bisulfite solution (9 mL), diluted with 90 mL
for compound 6a, to give compound 10 (231 mg, 89% yield) in
31
high purity. P NMR (acetonitrile-d
3
) -1.169 to -1.859 (broad
+
overlap of peaks). ESI-MS for C80
found 1824.2.
93 13 31 3
H N O P : calcd 1824.5,
of CHCl
obtained in high yield as a foam of 6a (0.492 g, 91%). P NMR
3
, and extracted with 50 mL of water. The product was
HOTpTpTpTSucc-IL (11). To a solution of 10 (190 mg, 0.122
mmol) in CH CN (1-2 mL) was added 3% v/v TFA in CH CN
2-3 mL). The reaction mixture was worked up as that of 4 giving
31
3
3
+
(
acetonitrile-d
calcd 1110.4, found 1110.4.
HOTpTSucc-IL (7a). To a solution of 6a (0.21 g, 0.18 mmol) in
CH CN (1-2 mL) was added 3% v/v TFA in CH CN (2-3 mL).
The reaction mixture was worked up following the same procedure
as that for 4 giving 7a as a foam in good yield (0.123 g, 78%). 31
NMR (acetonitrile-d ): δ -1.188, -1.284. ESI-HRMS for
15P : required 808.25548, found 808.25493.
TpTpTSucc-IL (8). Compound 7a (0.065 g, 0.073 mmol) was
mixed with 3′-phosphoramidite 5a (0.233 g, 0.31 mmol) and
dicyanoimidazole (0.31 g, 2.6 mmol) in dry CH CN (5 mL) at room
3
61 7
): δ -1.494, -1.584. ESI-MS for C54H N O17P :
(
31
compound 11 (160 mg, 99.9% yield). P NMR (acetonitrile-d
3
):
δ -1.142 to -1.51 (broad overlap of peaks). ESI-HRMS for
3
3
+
59 75 13 29 3
C H N O P : required 1522.40066, found 1522.40011.
P
Acknowledgment. We acknowledge financial support from
3
+
the Natural Sciences and Engineering Research Council of
Canada (NSERC) in the form of grants to M.J.D. and T.-H.C.
This work was also supported by a scholarship award from the
Chemical Biology Strategic Training Initiative of the Canadian
Institutes of Health Research to R.A.D. We would like to
33
DMT
43 7
C H N O
3
temperature. After being stirred for 2 h and quenched with
anhydrous ethanol for 10 min, the product was precipitated from
31
acknowledge Zhicheng Xia for developing the P CIGAR
1
31
1
:9 ethyl acetate:ethyl ether twice, oxidized, and extracted in the
same manner as for compound 6a, to give compound 8 (104 mg,
2% yield) in high purity. 31P NMR (acetonitrile-d
experiment used to observe H- P cross coupling.
9
3
): δ -1.081,
1
Supporting Information Available: Representative H NMR
spectra, 31P NMR spectra, comparative HPLC chromatograms of
compounds 12a-f (cleaved and deprotected products of compounds
-
1.194, -1.233, -1.262, -1.381, -1.403, -1.448, -1.477. ESI-
+
MS for C67
H N
77 10
O P
24 2
: calcd 1467.5, found 1467.8.
HO
TpTpTSucc-IL (9)b. To a solution of 8 (97 mg, 0.06 mmol) in
CN (2-3 mL).
The reaction mixture was worked up following the same procedure
as that of 4 giving 9 as a glassy solid (77 mg, 98% yield). 31
7
a-d, 9, and 11), and expanded experimental section. This material
CH
3
CN (1-2 mL) was added 3% v/v TFA in CH
3
is available free of charge via the Internet at http://pubs.acs.org.
P
JO061279Q
7
910 J. Org. Chem., Vol. 71, No. 20, 2006