Convenient large-scale synthesis of universal solid support

Article abstract of DOI:10.1080/00397910600639638

A simple, convenient large-scale synthesis of a universal solid support useful for the synthesis of oligonucleotides is described. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910600639638

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Convenient Large‐Scale Synthesis of
Universal Solid Support
Vasulinga T. Ravikumar a , R. Krishna Kumar a & Xuefeng Zhu a
a Isis Pharmaceuticals , Carlsbad, California, USA
Published online: 22 Sep 2006.
To cite this article: Vasulinga T. Ravikumar , R. Krishna Kumar & Xuefeng Zhu (2006) Convenient Large‐Scale
Synthesis of Universal Solid Support, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 36:16, 2269-2274, DOI: 10.1080/00397910600639638
To link to this article: http://dx.doi.org/10.1080/00397910600639638
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Synthetic Communicationsw, 36: 2269–2274, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910600639638
Convenient Large-Scale Synthesis of
Universal Solid Support
Vasulinga T. Ravikumar, R. Krishna Kumar,
and Xuefeng Zhu
Isis Pharmaceuticals, Carlsbad, California, USA
Abstract: A simple, convenient large-scale synthesis of a universal solid support
useful for the synthesis of oligonucleotides is described.
Keywords: Oligonucleotide, universal solid support, synthesis, antisense
Standard assembly of oligonucleotides of defined sequence and length
involves use of a solid support that contains the first nucleoside covalently
attached to a support via a cleavable linker such as a succinyl or oxalyl
group.[1–3] This support-bound nucleoside becomes the 30-terminal residue
of the synthesized oligonucleotide after cleavage and purification steps.
This methodology suffers from the fact that at least four solid supports for
DNA synthesis and an additional four supports for RNA synthesis are
needed. For therapeutic application of oligonucleotides containing sugar,
backbones and base-modified versions are investigated. This necessitates
making many different solid supports.[4] Recently, we have developed a
versatile nonnucleoside molecule that functions as a universal linker and,
when attached to a solid support, is capable of synthesizing various oligonu-
cleotides and their analogs.[5] This eliminates the need to store different
nucleoside-loaded solid supports.
We report here an easy and convenient method for the large-scale
synthesis of this universal linker solid support. Diels–Alder reaction of
N-phenylmaleimide with furan in acetonitrile at reflux for 5 h gave the
adduct 1 in 78% yield. Cis-dihydroxylation of the olefin was achieved using
Received in the USA January 26, 2006
Address correspondence to Vasulinga T. Ravikumar, Isis Pharmaceuticals, 2282
Faraday Avenue, Carlsbad, CA 92008, USA. E-mail: vravikumar@isisph.com.
2269

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V. T. Ravikumar, R. K. Kumar, and X. Zhu
osmium tetroxide and hydrogen peroxide in acetone to give the diol 2 in 82%
yield. Dimethoxytritylation was achieved using 4,40-dimethoxytrityl chloride
in anhydrous pyridine at room temperature for 2 h to afford product 3 in 83%
yield. Treatment of compound 3 with succinic anhydride in the presence
of triethylamine in anhydrous methylene chloride afforded the linker mole-
cule 4 as the triethylammonium salt in 91% yield. Loading of the succinate
linker molecule to controlled pore glass solid support was achieved
using O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate
(HBTU) as activating agent to give 5. The calculated molar equivalent of
molecule 4 was taken to obtain a loading of 45 mmol/g.

Synthesis of Universal Solid Support
EXPERIMENTAL
2271
Furan, N-phenylmaleimide, pyridine, diisopropylethylamine, triethylamine,
succinic anhydride, hydrogen peroxide, tert-butyl alcohol, 4-dimethylamino-
pyridine, N-methylimidazole, anhydrous acetonitrile, methylene chloride, and
toluene were purchased from Aldrich Chemical Company, Milwaukee,
Wisconsin, and used as received. Osmium tetroxide was purchased from
Sterm Company, Newburyport, MA. 4,40-Dimethoxytriphenylmethyl
chloride was purchased from ChemGenes, Wilmington, MA. HBTU reagent
1
was purchased from SENN chemicals, San Diego, CA. H and 13C NMR
spectra were recorded on a Varian Gemini 200-MHz or Bruker AM 300-
MHz spectrometer. Chemical shifts are reported in parts per million (ppm)
using tetramethylsilane as an internal standard with DMSO-d6 or CDCl3 as
solvent.
N-Phenyl-tetrahydro-4,7-epoxyisobenzopyrrole-1,3-dione (1)
A three-necked, round-bottomed flask (5 L), equipped with a magnetic
stirring bar, a heating mantle, a reflux condenser, and a dropping funnel,
was charged with a solution of N-phenylmaleimide (0.5 kg, 2.89 mol) in
acetonitrile (1.6 L). Following the addition of furan (0.5 L), the stirred
solution was heated at reflux for 5 h. Then the reaction mixture was
cooled to room temperature, when a colorless solid precipitated out. The
material was filtered and washed with acetonitrile (0.5 L). The filtrate
solution was concentrated to afford more of product, which was also
filtered and washed with acetonitrile (0.3 L). The combined solid portions
were dried under high vacuum at room temperature overnight to afford
1
0.54 kg (78%) of product. H NMR (DMSO-d6, 300 MHz) d: 3.06 (s, 2H),
5.22 (s, 2H), 6.58 (s, 2H), 7.18–7.58 (m, 5H). 13C NMR (DMSO-d6,
75.5 MHz) d: 47.41, 80.75, 126.76, 128.38, 128.92, 132.09, 136.58,
175.66. MS (ESI, m/z): 241. Anal. calcd. (%) for C14H11NO3: C, 69.70;
H, 4.60; N, 5.81. Found: C, 69.74; H, 4.69; N, 5.88.
N-Phenyl-5,6-dihydroxy-hexahydro-4,7-epoxy-isobenzopyrrole-1,3-
dione (2)
The content of a 1-g sealed vial of osmium tetroxide was dissolved in purified
tert-butyl alcohol (0.2 L). The pale green solution was treated with 3–5 drops
of 30% hydrogen peroxide and allowed to remain at room temperature for 1
day. If the solution became dark, the dropwise addition of 30% hydrogen
peroxide was repeated until the pale green color persisted. This solution
is stable for at least 1 year at room temperature. Each mL contains
2 ꢀ 1025 mol of osmium tetroxide.

2272
V. T. Ravikumar, R. K. Kumar, and X. Zhu
A 5-L three-necked flask, fitted with a mechanical stirrer, reflux
condenser with ice water cooling, and a heating mantle, was charged with a
solution of olefin 1 (0.23 kg, 0.93 mol) in acetone (2.5 L). A 30% hydrogen
peroxide solution (0.5 L) was added, followed by an osmium tetroxide
solution prepared earlier (0.18 L). Warning: For large scales, the reaction
could be exothermic! Slow addition (1–2 h) of osmium tetroxide solution is
recommended. Gentle refluxing of reaction mixture with stirring was main-
tained for 7–8 h. During this period, reaction color changed from brown to
pale brown to colorless, and the solid started crashing out. Vigorous stirring
was maintained throughout the period. TLC indicated the disappearance of
the starting material. The reaction mixture was cooled to room temperature,
and the precipitated solid was filtered. The solid was washed with ether
(2 L) and dried in vacuum oven at room temperature overnight. The filtrate
solution was concentrated, and ether (1 L) was added when an additional
solid precipitated out, which was filtered and washed with ether (0.3 L). The
combined solid product was dried in an oven at 458C for 2 days to afford a
1
total of 211 g (82%) of colorless product 2. H NMR (DMSO-d6, 300 MHz)
d: 3.14 (s, 2H), 3.88 (d, 2H), 4.39 (s, 2H), 5.1 (d, 2H), 7.18–7.58 (m, 5H).
13C NMR (DMSO-d6, 75.5 MHz) d: 45.47, 71.82, 84.08, 126.72, 128.40,
128.90, 132.16, 176.26. MS (ESI, m/z): 275. Anal. calcd. (%) for
C14H13NO5: C, 61.09; H, 4.76; N, 5.09. Found: C, 61.21; H, 4.84; N, 5.17.
N-Phenyl-5-(4,40-dimethoxytriphenylmethyloxy)-6-hydroxy-
hexahydro-4,7-epoxyisobenzopyrrole-1,3-dione (3)
The dihydroxy compound 2 (0.28 g, 1 mol) was taken in a 5-L round-bottomed
flask and co-evaporated with anhydrous pyridine (1.2 L). This step was
repeated one more time to render the material anhydrous. Pyridine (3 L)
was added and stirred using a magnetic stirrer at room temperature.
4,40-Dimethoxytrityl chloride (0.5 kg, 1.5 mol, 1.5 equiv) was slowly added
as solid over a period of 2 h. The solution was stirred overnight. TLC
indicated the disappearance of the starting material. All volatiles were
removed; toluene (2 L) was added and concentrated under vacuum using a
rotary evaporator. This step was repeated one more time. The remaining
crude material was purified by flash silica-gel chromatography using
hexane–ethyl acetate. Triethylamine (1%) was used throughout purification
1
to afford 0.47 kg (83%) of product 3. H NMR (CDCl3, 300 MHz) d: 2.42
(d, 1H), 3.78 (d, 1H), 3.45 (s, 1H), 3.62 (d, 1H), 3.81 (s, 6H), 3.85 (s, 2H),
4.72 (s, 1H), 6.82 (d, 4H), 7.1–7.55 (m, 14H). 13C NMR (DMSO-d6,
75.5 MHz) d: 20.99, 45.27, 45.68, 55.06, 73.92, 75.45, 81.96, 84.40, 87.40,
113.48, 113.60, 125.28, 126.73, 127.72, 127.98, 128.16, 128.34, 128.85,
129.79, 129.89, 132.11, 135.84, 136.34, 145.44, 158.47, 175.30, 176.03. MS
(ESI, m/z): 577.5. Anal. calcd. (%) for C35H31NO7: C, 72.78; H, 5.41; N,
2.42. Found: C, 72.93; H, 5.54; N, 2.47.

Synthesis of Universal Solid Support
2273
Triethylammonium N-Phenyl-5-(4,40-dimethoxytriphenyl)-6-[2-
carboxypropanoyloxy]-hexahydro-4,7-epoxyisobenzopyrrole-1,3-
dione (4)
A 500-mL round-bottomed flask equipped with a magnetic stirrer was
charged with a solution of N-phenyl-5-(4,40-dimethoxytriphenylmethyloxy)-
6-hydroxy-hexahydro-4,7-epoxyisobenzopyrrole-1,3-dione 3 (0.05 kg, 0.09 mol)
in methylene chloride (0.48 L). Triethylamine (0.07 L, 0.51 mol, 6 equiv
with respect to the starting DMT compound) was added at room temperature.
To this clear solution, succinic anhydride (0.03 kg, 0.36 mol, 4 equiv with
respect to the starting DMT compound) was added as a solid all at once.
Stirring was continued overnight. TLC indicated the disappearance of the
starting material. The reaction mixture was diluted with ethyl acetate
(0.3 L), washed with water (2 ꢀ 0.2 L) and brine (0.12 L), and dried with
magnesium sulfate. If colored, the material was passed through a short pad
of silica gel using methylene chloride–methanol to afford 0.061 kg (91%)
1
of product 4 as a pale yellow solid. H NMR (DMSO-d6, 300 MHz) d: 0.96
(t, 9H), 2.58 (q, 4H), 2.59–2.78 (m, 8H), 2.91 (s, 1H), 3.17 (s, 1H), 3.75
(s, 6H), 4.09 (d, 1H), 4.54 (s, 1H), 5.18 (d, 1H), 6.92 (d, 4H), 7.08–7.55
(m, 14H). 13C NMR (DMSO-d6, 75.5 MHz) d: 10.16, 29.87, 30.19, 45.03,
45.15, 45.75, 55.04, 74.39, 75.29, 81.75, 81.90, 87.17, 113.56, 113.64,
126.72, 126.82, 127.51, 128.03, 128.14, 128.40, 128.87, 129.67, 129.76,
132.04, 135.46, 135.93, 145.16, 158.50, 172.55, 174.18, 175.02, 175.52. MS
(ESI, m/z): 778.4. Anal. calcd. (%) for C45H50N2O10: C, 69.39; H, 6.47; N,
3.60. Found: C, 69.61; H, 6.77; N, 3.87.
Loading of Linker Molecule (4) to Controlled Pore Glass
Support (5)
Controlled pore glass (CPG) (100 g), succinate molecule 4 (4.05 g, 5.2 mmol),
HBTU (1.97 g, 5.2 mmol), diisopropylethylamine (1.86 mL, 10.4 mmol), and
anhydrous acetonitrile (500 mL) were successively added to a 1-L round-
bottom flask. The resulting mixture was then tightly capped and shaken
overnight (more than 16 h) at room temperature. The support was then
filtered and washed with acetonitrile.
Capping of Unreacted Hydroxyl Sites on CPG Support
The loaded CPG, 4-dimethylaminopyridine (DMAP) (2.44 g, 20.0 mmol),
CAP A (250 mL), and CAP B (250 mL) solutions were successively added
to a 1-L round-bottom flask. CAP A is a mixture of N-methylimidazole,
pyridine and acetonitrile (20 : 30 : 50, v : v : v); CAP B is a mixture of acetic
anhydride and acetic acetonitrile (20 : 80, v : v). The resulting mixture was

2274
V. T. Ravikumar, R. K. Kumar, and X. Zhu
then tightly capped and shaken overnight at room temperature. The support
was filtered, washed with acetonitrile, and dried thoroughly under high
vacuum. Loading of the linker molecule was determined by UV and found
to be 45 mmol/g.
ACKNOWLEDGMENT
The authors thank Douglas Cole and Phil Olsen for their help.
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