(2-Acetoxyphenoxy)ethyl (APOe) as a phosphate protecting group in solid-phase synthesis of oligonucleotides via the phosphoramidite approach

Article abstract of DOI:10.1021/op0502147

The (2-Acetoxyphenoxy)ethyl (APOe) group could be an alternative to the conventional 2-cyanoethyl group for phosphate protection in solid-phase oligonucleotide synthesis to circumvent DNA alkylation by acrylonitrile generated under basic condition. This g

Full text of DOI:10.1021/op0502147

Organic Process Research & Development 2006, 10, 251−256
(2-Acetoxyphenoxy)ethyl (APOE) as a Phosphate Protecting Group in
Solid-Phase Synthesis of Oligonucleotides via the Phosphoramidite Approach
Zacharia S. Cheruvallath, Alessandra Eleuteri, Brett Turney, and Vasulinga T. Ravikumar*
Isis Pharmaceuticals, Inc., 2282 Faraday AVenue, Carlsbad, California 92008, U.S.A.
Abstract:
addition of acrylonitrile formed by â-elimination of cyano-
ethyl group to nucleobases. The potent DNA alkylating
properties of acrylonitrile have been investigated by many
laboratories.4 In addition, storage of solutions of cyanoethyl-
protected phosphoramidites for extended periods of time
undergo an Arbuzov-type of rearrangement leading to
depletion of the desired concentration. Even though methods
have been taken to circumvent the DNA-alkylation issue, it
is desirable to have a phosphate protecting group which will
not have such side reactions. Several phosphate protecting
groups have been reported by many laboratories in the last
several years.5 Still there is a need to develop a group that
will meet all of the requirements of a good phosphate
protecting group, viz. stability and compatibility with various
reagents, scalability, absence of side reactions, low cost, and
versatility. Here we report (2-acetoxyphenoxy)ethyl (APOE)
as an alternative to the 2-cyanoethyl group for the efficient
synthesis of oligonucleotides.
The (2-acetoxyphenoxy)ethyl (APOE) group could be an alter-
native to the conventional 2-cyanoethyl group for phosphate
protection in solid-phase oligonucleotide synthesis to circumvent
DNA alkylation by acrylonitrile generated under basic condi-
tion. This group is stable during oligonucleotide synthesis and
can be removed under mild conditions using aqueous am-
monium hydroxide. Multiple phosphorothioate oligodeoxy-
ribonucleotides and 2′-O-methoxyethyl-modified oligoribo-
nucleotide chimera were synthesized and characterized exten-
sively. The deprotection of this group follows an intramolecular
attack on the r-carbon adjacent to phosphate oxygen to liberate
the oligonucleotide and an innocuous cyclic ether as side
product. No modification of nucleobases was observed during
deprotection of this group.
Introduction
With the advent of 2-cyanoethyl-protected nucleoside
phosphoramidites,1 rapid and highly efficient solid-phase
syntheses of oligodeoxyribonucleotides and their analogues
are now available and have, in the past two decades,
revolutionized the therapeutic field as shown by numerous
drugs being evaluated in the clinic.2 Synthetic scales range
from nanomole to almost a mole, and currently it takes less
than 8 h to synthesize a phosphorothioate oligonucleotide
of 20-mer in length at 750 mmol scale on GE Amersham
OligoProcess synthesizer. Synthesis involves monomeric
building blocks containing a 2-cyanoethyl group for phos-
phate protection. This group is eventually removed using an
amine or other conditions.3 Care must be taken to avoid
(4) (a) Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J. Org. Chem.
1999, 64, 7515-7522. (b) Wilk, A.; Grajkowski, A.; Srinivasachar, K.;
Beaucage, S. L. Antisense Nucleic Acid Drug DeV. 1999, 9, 361-366. (c)
Crippa, S.; Di Gennaro, P.; Lucini, R.; Orlandi, M.; Rindone, B. Gazz. Chim.
Ital. 1998, 123, 197-203. (d) Prokopczyk, B.; Bertinato, P.; Hoffman, D.
Carcinogenesis 1988, 9, 2125-2128. (e) Mag, M.; Engels, J. W. Nucleic
Acids Res. 1988, 16, 3525-3543. (f) Chambers, R. W. Biochemistry 1965,
4, 219-226.
(5) Phosphate protecting groups: 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-
(2-methyl)]aminoethyl: Cleslak, J.; Beaucage, S. L. J. Org. Chem. 2003,
68, 10123-10129. 3-(N-tert-Butylcarboxamido)-1-propyl: Wilk, A.;
Chmielewski, M. K.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J.
Org. Chem. 2002, 67, 6430-6438. 4-[N-Methyl-N-(2,2,2-trifluoroacetyl)-
amino]butyl: Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J.
Org. Chem. 1999, 64, 7515-7522. 4-Cyano-2-butenyl: Ravikumar, V. T.;
Cheruvallath, Z. S.; Cole, D. L. Tetrahedron Lett. 1996, 37, 6643-6646.
Allyl: (a) Hayakawa, Y.; Uchiyama, M.; Kato, H.; Noyori, R. Tetrahedron
Lett. 1985, 26, 6505-6508. (b) Hayakawa, Y.; Kato, H.; Uchiyama, M.;
Kajino, H.; Noyori, R. J. Org. Chem. 1986, 51, 2400-2402. (c) Hayakawa,
Y.; Kato, H.; Nobori, T.; Noyori, R.; Imai, J. Nucleic Acids Res. Ser. 1986,
17, 97-100. (d) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J.
Am. Chem. Soc. 1990, 112, 1691-1696. (e) Hayakawa, Y.; Hirose, M.;
Noyori, R. J. Org. Chem. 1993, 58, 5551-5555. (f) Hayakawa, Y.; Hirose,
M.; Noyori, R. Nucleosides Nucleotides 1994, 13, 1337-1345. (g) Berg-
mann, F.; Kueng, E.; Laiza, P.; Bannwarth, W. Tetrahedron Lett. 1995, 51,
6971-6976. 2-(Trimethylsilyl)ethyl: Wada, T.; Sekine, M. Tetrahedron Lett.
1994, 35, 757-760. (2-Diphenylmethylsilyl)ethyl: (a) Ravikumar, V. T.;
Sasmor, H.; Cole, D. L. Bioorg. Med. Chem. Lett. 1993, 3, 2637-2640.
(b) Ravikumar, V. T.; Wyrzykiewicz, T. K.; Cole, D. L. Tetrahedron 1994,
50, 9255-9266. (c) Ravikumar, V. T.; Cole, D. L. Gene 1994, 149, 157-
161. (d) Krotz, A. H.; Cole, D. L.; Ravikumar, V. T. Tetrahedron Lett.
1996, 37, 1999-2002. (e) Krotz, A. H.; Wheeler, P.; Ravikumar, V. T.
Angew. Chem., Int. Ed. Engl. 1995, 34, 2406-2409. (f) Krotz, A. H.;
Cheruvallath, Z. S.; Cole, D. L.; Ravikumar, V. T. Nucleosides Nucleotides
1998, 17, 2335-2338. 2,2,2-Trichloro-1,1-dimethylethyl: (a) Letsinger, R.
L.; Groody, E. P.; Tanaka, T. J. Am. Chem. Soc. 1982, 104, 6805-6806.
(b) Letsinger, R. L.; Groody, E. P.; Lander, N.; Tanaka, T. Tetrahedron
1984, 40, 137-143. p-Nitrophenylethyl: (a) Schwarz, M. W.; Pfleiderer,
W. Tetrahedron Lett. 1984, 25, 5513-5516. (b) Himmelsbach, F.; Schulz,
B. S.; Trichtinger, T.; Charubala, R.; Pfleiderer, W. Tetrahedron 1984, 40,
59-72. (c) Schwarz, M. W.; Pfleiderer, W. Nucleosides Nucleotides 1985,
4, 291-292. (d) Hamamoto, S.; Takaku, H. Chem. Lett. 1986, 1401-1404.
* To whom correspondence should be addressed. Telephone: (760) 603-
2412. Fax: (760) 603-4655. E-mail: vravikumar@isisph.com.
(1) (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-
1862. (b) McBridge, L. J.; Caruthers, M. H. Tetrahedron Lett. 1983, 24,
245-248. (c) Sinha, N. D.; Biemat, J.; Koster, H. Tetrahedron Lett. 1983,
24, 5843-5846. (d) Sinha, N. D.; Biemat, J.; McManus, J.; Koster, H.
Nucleic Acids Res. 1984, 12, 4539-4557. (e) Gasparutto, D.; Molko, D.;
Teoule, R. Nucleosides Nucleotides 1990, 9, 1087-1098. (f) Lyttle, M. H.;
Wright, P. B.; Sinha, N. D.; Bain, J. D.; Chamberlin, A. R. J. Org. Chem.
1991, 56, 4608-4615. (g) Beaucage, S. L.; Caruthers, M. H. In Current
Protocols in Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E.,
Glick, G. D., Jones, R. A., Eds.; John Wiley & Sons: New York, 1999; pp
3.3.1-3.3.20.
(2) A number of second-generation phosphorothioate oligonucleotides are in
various stages of preclinical and clinical trials against ApoB-100, PTP-1B,
VLA4, TRPM2, survivin, STAT-3, eIF-4E, etc. for the treatment of a variety
of diseases such as cancer, psoriasis, diabetes, asthma, arthritis, multiple
sclerosis, etc. Refer to website: www.isispharm.com for additional informa-
tion.
(3) (a) Capaldi, D. C.; Gaus, H.; Krotz, A. H.; Arnold, J.; Carty, R. L.; Moore,
M. N.; Scozzari, A. N.; Lowery, K.; Cole, D. L.; Ravikumar, V. T. Org.
Process Res. DeV. 2003, 7, 832-838. (b) Umemoto, T.; Wada, T.
Tetahedron Lett. 2005, 46, 4251-4253.
10.1021/op0502147 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/31/2006
Vol. 10, No. 2, 2006 / Organic Process Research & Development
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251

Figure 1. Various APOE-protected phosphoramidites synthesized.
Scheme 1. Synthesis of 2-(2-acetoxyphenoxy)ethanol
Earlier, we reported the use of deoxyribonucleotide
phosphoramidites in the solid-phase synthesis of oligonucleo-
tides having the 2-diphenylmethylsilylethyl (DPSE) group
as a phosphate protecting group.5 This group is easily
removed from oligonucleotides by simple treatment with
tetrabutylammonium fluoride (TBAF). DPSE had many of
the favorable properties of a good phosphate protecting
group; however, the starting material, 2-diphenylmethyl-
silylethanol is relatively expensive even in bulk quantities.
Because of this these phosphoramidites might not cost-
effectively compete with conventional 2-cyanoethyl amidites.
In an effort to reduce the cost of therapeutic oligonucleo-
tides, we initiated a search for an inexpensive molecule which
could serve as an efficient phosphate protecting group. We
now propose the use of (2-acetoxyphenoxy)ethyl (APOE)
for enhanced phosphate protection and report its incorpora-
tion into oligonucleotides via the nucleoside phosphorami-
dites 1a-h (Figure 1). The usefulness of this group is
demonstrated in the solid-phase synthesis of several first-
and second-generation phosphorothioate oligonucleotides.
The rapid and facile removal of the APOE group under
standard ammonia deprotection conditions is shown, and the
rates of deprotection kinetics under different conditions are
addressed.
Chemical Synthesis. Direct acetylation of 2-(2-hydroxy-
phenoxy)ethanol with acetic anhydride under various condi-
tions gave different selectivities. A key to obtaining high
yield and quality is by slowly adding acetic anhydride to a
vigorously stirred mixture of 2-(2-hydroxyphenoxy)ethanol
in acetone and powdered potassium carbonate. The reaction
is fast and complete within 4 h. Slow addition of anhydride
and fast mechanical stirring is very important for achieving
best results. The desired product was obtained as a pale-
yellow viscous oil in >80% chemical yield and quantitative
chemoselectivity after purification (based on HPLC analysis)
(Scheme 1). The other regioisomeric acetate 4 and bis-acetate
5 were obtained as colorless solids.
An alternative choice to obtaining monoprotection is
formation of an acetal with triethyl orthoformate and
chemoselective ring opening with a Lewis acid such as AlCl3
or DIBALH. Multiple attempts at optimizing the reaction
conditions did not afford any better yield and chemoselec-
tivity as compared to those from direct acetylation (Scheme
1).
Enzymatic Synthesis. Monoacetylation was also achieved
via the use of lipase enzymes. However, after screening
several enzymes, it was not found to be better than direct
chemical acylation and was not pursued further (Scheme 1).
Synthesis of (2-Acetoxyphenoxy)ethanol
Synthesis of APOE-Protected Phosphoramidites
2-(2-Hydroxyphenoxy)ethanol is an inexpensive starting
material ($70/kg) and is available in bulk from multiple
vendors. Protection of the phenolic hydroxyl group could
be achieved by different routes. The key to choosing the best
route is the one which will afford the maximum yield with
chemospecific protection of the phenoxy hydroxyl group and
not the aliphatic hydroxyl group.
Using Nucleoside Bisamidite Approach. Treatment of
a DMT-protected nucleoside with bis(diisopropylamino)-
chlorophosphine in the presence of triethylamine as base in
anhydrous acetonitrile or dichloromethane gave the bis-
amidite which, upon further reaction with 2-(2-acetoxyphe-
noxy)ethanol, gave after workup and purification the corre-
sponding phosphoramidites as colorless, foamy solids. 31P
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analyzed by CGE.7 Subsequently, the material was purified
by C-18 reversed-phase HPLC; all DMT-on fractions were
collected, detritylated, and lyophilized to a colorless hygro-
scopic powder. The purified oligonucleotide was analyzed
by CGE, anion-exchange HPLC, and 31P NMR spectros-
copy.8 These data clearly indicate that APOE is a suitable
phosphate protecting group. This excellent performance of
the APOE group was exemplified with another sequence (5′-
GCC-CAA-GCT-GGC-ATC-CGT-CA) synthesized under
identical conditions.
Scheme 2. Various syntheses of APOE-protected
nucleoside phosphoramidites
Second-Generation Oligonucleotides. To extend the
utility of the APOE protecting group to the synthesis of 2′-
O-methoxyethyl-modified oligoribonucleotide phosphorothio-
ates, a 20-nucleotide hemi-mer [5′-d(GmeCmeCmeCAAGme-
CTGGmeC)-r(AmeUmeCmeCGmeU)] [ISIS 15839] (where medC
and MOE meC are methylated at the 5-position of the
nucleobase, r ) 2′-O-methoxyethylribonucleoside) was
chosen as an example. Synthetic conditions similar to those
for first-generation phosphorothioate oligonucleotides were
followed. The oligonucleotide was purified similarly to afford
the desired colorless hygroscopic powder. The purified
oligonucleotide was analyzed by CGE, anion-exchange
HPLC, and 31P NMR spectroscopy. The oligonucleotide was
further confirmed by mass spectroscopy, demonstrating that
APOE is a suitable phosphate protecting group for modified
sequences also.
NMR of deoxyribonucleoside phosphoramidites synthesized
through this approach showed the characteristic signals
(∼146 ppm) corresponding to diastereoisomeric mixtures,
and no 3′,3′-dinucleoside or bis-APOE phosphites could be
detected. The phosphoramidites 1 a-d were prepared in 65-
80% yields (Scheme 2).
Using APOE Phosphitylating Reagent 6. Alternatively,
treatment of 2-(2-acetoxyphenoxy)ethanol with bis(diiso-
propylamino)chlorophosphine in the presence of triethyl-
amine under anhydrous conditiond gave the phosphitylating
reagent 6 as a colorless solid which, on reaction with DMT-
protected nucleosides in the presence of 1H-tetrazole, gave
(after work up and column chromatography purification) the
desired phosphoramidites in 60-82% overall yield (Scheme
3).6
Oligonucleotide Synthesis. The applicability of phos-
phoramidites 1 a-d were demonstrated by the synthesis of
four heterodimers d(TpsC), d(CpsT), d(GpsA), and d(ApsG)
on solid support (yield >99%). The integrity and authenticity
of these compounds were confirmed by comparing with
identical hetrodimers independently synthesized using 2-cya-
noethyl protection of the phosphorothioate backbone.
Experimental Section
Materials and Methods. 1H-Tetrazole was purchased
from American International Co. Standard 5′-O-4,4′-
dimethoxytrityl-protected deoxyribonucleosides were pur-
chased from Yamasa, Japan, and 2′-O-methoxyethylribonu-
cleosides (benzoyl dA and MOE A, benzoyl dC and MOE
meC, isobutyryl dG and MOE G, T and 5meU) were
purchased from customs manufacturers, viz. Innovasynth
Chemical Co, India, Sai Dru Syn, India, and Shasun, India.
Phenylacetyl disulfide (PADS) was purchased from Acharya
Chemicals, Dombivili, India, and dichloroacetic acid, from
Clariant Life Sciences, Germany. Derivatized polystyrene
supports loaded with the corresponding nucleoside were
obtained from GE Amersham Biosciences, Uppsala, Sweden.
Acetylation of 2-(2-Hydroxyphenoxy)ethanol. 2-(2-
Hydroxyphenoxy)ethanol (308 g; 2 mol) was taken up in a
5-L Erlenmeyer flask fitted with mechanical stirrer. Anhy-
drous acetone (dried with K2CO3) (4 L) and potassium
carbonate powder (290 g; 2.1 mol) were added to it and
stirred vigorously. Acetic anhydride (207 mL; 2.2 mol) was
taken up in an additional funnel and added slowly over a
period of 1 h. Stirring was continued for 3-4 h. TLC (CH2-
Cl2/MeOH, 9:1, v/v) showed the disappearance of the starting
material. The reaction mixture was filtered, the solid was
washed thoroughly with acetone (1 L). The combined
fractions were concentrated and purified by silica gel
chromatography, eluting with hexane and ethyl acetate (0%
Synthesis of 20-mer Phosphorothioates on Solid Support
First-Generation Oligonucleotides. To evaluate the
coupling efficiency of the APOE group, a 20-mer phospho-
rothioate oligodeoxyribonucleotide (5′-TCC-CGC-CTG-
TGA-CAT-GCA-TT) (ISIS 5132) was chosen as an example.
The synthesis was carried out on a 160 µmole scale using
Pharmacia OligoPilot II synthesizer. Pharmacia’s HL30
primary support (90 µmol/g loading) with 2.5 molar excess
of amidites was used. Phenylacetyl disulfide (0.2 M, CH3-
CN/3-picoline, 1:1 v/v) was used for sulfurization. Following
the synthesis, the oligonucleotide was deprotected with
concentrated aqueous ammonium hydroxide for 12 h at 55
°C. The sample was cooled, filtered, concentrated, and
(7) (a) Srivatsa, G. S.; Batt, M.; Schuette, J.; Carlson, R. H.; Fitchett, J.; Lee,
C.; Cole, D. L. J. Chromatogr. 1994, 680, 469-477. (b) Cummins, L. L.;
Winniman, M.; Gaus, H. J. Bioorg. Med. Chem. Lett. 1997, 7, 1225-1230.
(8) Srivatsa, G. S.; Klopchin, P.; Batt, M.; Feldman, M.; Carlson, R. H.; Cole,
D. L. J. Pharm. Biomed. Anal. 1997, 16, 619-630.
(6) Gukathasan, R.; Massoudipour, M.; Gupta, I.; Chowdhury, A.; Pulst, S.;
Ratnam, S.; Sanghvi, Y. S.; Laneman, S. A. J. Organmet. Chem. 2005,
690, 2603-2607.
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253

Scheme 3. Synthesis of APOE-protected nucleoside phosphoramidites
Table 1. Synthesis parameters of cycle used on Pharmacia
Scheme 4. Mechanism of APOE group deprotection
OligoPilot II DNA/RNA synthesizer
volume time
(mL) (min)
step
reagent
detritylation 10% dichloroacetic acid/
toluene
18
3
3
3
coupling
phosphoramidite (0.2M) and
1H-tetrazole (0.4 M) in CH3CN
2.2, 2.2
11
sulfurization PADS (0.2M) in
3-picoline-CH3CN (1:1, v/v)
Ac2O/pyridine/
CH3CN, NMI/CH3CN
capping
7.5, 7.5 2.5
Scheme 5. Attempted deprotection of
2-(4-acetoxyphenoxy)ethyl-protected TpsT dimer
to 35% EtOAc; v/v). The product was obtained as a colorless,
viscous oil. Yield 295-302 g (80-82%). Purification: 180
cm (length) × 100 cm (diameter) column was packed with
silica gel (ca. 900 g) made into a slurry with 100% hexanes.
The crude reaction material was loaded onto the column
gently without disturbing the top bed, eluting started with
10% EtOAc/hexanes (6 L), increased gradually to 15%
EtOAc/hexanes (6 L), and then to 20% EtOAc/hexanes when
the diacetate started to come off. The same 20% EtOAc/
hexanes gradient was used until all the diacetate came off.
Then the polarity was increased to 35% EtOAc/hexanes to
get the product out. The product fractions were collected in
1000 mL sizes, checked by TLC (10% MeOH/methylene
chloride), pooled, and concentrated to afford the product as
a colorless liquid. A total of ca. 30 L of solvent was used
for the column purification. These are not optimized condi-
tions, and the amount of silica gel as well as solvent volume
consumption could be reduced further on larger scale.
5′-O-DMT-N4-Benzoyl-2′-deoxycytidine-3′-O-(2-ace-
toxy phenoxyethyl)-N,N-diisopropyl Phosphoramidite.
Bis(diisopropylamino)chlorophosphine (46.96 g; 0.176 mol)
was dissolved in anhydrous dichloromethane (700 mL) under
nitrogen at room temperature. Triethylamine (22.26 g; 30.67
mL) was added, followed by 5′-O-DMT-N4-benzoyl-2′-
deoxycytidine (92.52 g; 0.146 mol) as a solid. After 60 min,
the solution was evaporated in a vacuum to remove dichlo-
romethane and excess triethylamine. The residue was dis-
solved in anhydrous acetonitrile (1200 mL) and stirred at
room temperature. 2-Acetoxyphenoxyethanol (31.36 g; 0.16
mol) was dissolved in acetonitrile (300 mL) and added,
followed immediately by 1H-tetrazole (5.6 g; 0.08 mol).
After 2 h, the reaction mixture was evaporated to foam and
redissolved in dichloromethane (1000 mL). The solution was
washed with cold, saturated sodium bicarbonate (250 mL)
and cold, saturated sodium chloride (4 × 200 mL) and was
dried over magnesium sulfate for 3 h. After filtration and
evaporation, the residue was purified by silica gel chroma-
tography. Purification: 2.75 kg silica gel; column dimen-
sions: 15 cm diameter × 95 cm height; bed height 36 cm.
The crude product was mixed with 120 g of silica gel and
200 mL of dichloromethane for loading onto column;
eluant: hexane/ethyl acetate (1:1) (packed with 0.5% tri-
ethylamine); run column with 0.25% triethylamine; void
volume: 7 L; upper impurities 4 L, no product; collected 4
L product. Solutions were evaporated and azeotroped with
dry acetonitrile to give 86 g (70%) of the desired product.
Oligonucleotide Synthesis. Syntheses were performed on
a Amersham Pharmacia Biotech OligoPilot II DNA/RNA
synthesizer. The support was tightly packed in a stainless
steel column (volume 6.33 mL). Details of the synthesis cycle
are given in Table 1. Dichloroacetic acid (10%) in toluene
was used for deblocking of the dimethoxytrityl (DMT)
groups from 5′-hydroxyl group of the nucleotide.9
1H-Tetrazole (0.4 M) in acetonitrile was used as activator
during the coupling step. Low-water acetonitrile (water
content <10 ppm) was used for preparing phosphoramidite
and activator solutions. 2.5 equiv of amidites (both deoxy-
and 2′-O-methoxyethylribonucleosides) and a ratio of 40:
60 (v/v) of amidite to activator solution was used during the
coupling step. Phenylacetyl disulfide (PADS) (0.2 M in
3-picoline/CH3CN, 1:1, v/v) was used as the sulfur transfer
reagent.10 Capping reagents were made to the recommended
GE Amersham Bioscience receipe: Cap A: N-methylimi-
dazole-CH3CN (1:4 v/v), Cap B: acetic anhydride/pyridine/
CH3CN (2:3:5, v/v/v). Subsequently, the solid support
(9) (a) Krotz, A. H.; Cole, D. L.; Ravikumar, V. T. Bioorg. Med. Chem. 1999,
7, 435-439. (b) Krotz, A. H.; Cart, R. L.; Moore, M. N.; Scozzari, A. N.;
Cole, D. L.; Ravikumar, V. T. Green Chem. 1999, 277-281. (c) Krotz, A.
H.; Carty, R. L.; Scozzari, A. N.; Cole, D. L.; Ravikumar, V. T. Org. Process
Res. DeV. 2000, 4, 190-193. (d) Gaus, H.; Olsen, P.; Van Sooy, K.; Rentel,
C.; Turney, B.; Walker, K. L.; McArdle, J. V.; Capaldi, D. C. Bioorg. Med.
Chem. Lett. 2005, 15, 4118-4124. (e) Capaldi, D. C.; Gaus, H. J.; Carty,
R. L.; Moore, M. N.; Turney, B. J.; Decottignies, S. D.; MaArdle, J. V.;
Scozzari, A. N.; Ravikumar, V. T.; Krotz, A. H. Bioorg. Med. Chem. Lett.
2004, 14, 4683-4690. (f) Krishna Kumar, R.; Ravikumar, V. T. Bioorg.
Med. Chem. Lett. 2005, 15, 3426-3429. (g) Cieslak, J.; Austin, C.;
Chmielewski, M. K.; Kauffman, J. S.; Snyder, J.; Del-Grosso, A.; Beaucage,
S. L. J. Org. Chem. 2005, 70, 3303-3306.
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Chart 1. Rates of deprotection of APOE-protected dimers under different conditions
Scheme 6. Probable mechanism of deprotection of APOE group
containing oligonucleotide was incubated with concentrated
aqueous ammonium hydroxide at 55 °C for 14 h to complete
cleavage from the support and deprotection of the base-
protecting groups. The crude DMT-on oligonucleotide was
concentrated to remove ammonia and taken up in a known
volume by addition of water with the final concentration
checked and analyzed by CGE. The crude material was
purified by reversed-phase C18 silica HPLC chromatography,
and all DMT-on fractions were collected, detritylated, and
lyophilized to a colorless, amorphous, hygroscopic solid.
At 55 °C, the deprotection of all APOE-protected dimers
was complete within 10 min.
Mechanism of Deprotection. Initially, we thought the
mechanism involves a nucleophilic attack of the base on the
acetyl group to liberate the phenolic hydroxyl group which
undergoes subsequent fragmentation to liberate the nucleo-
tide, ethylene, and o-quinone (Scheme 4).
To investigate this proposed mechanism, synthesis of an
analogue of APOE protecting group, viz. 2-(4-acetoxyphe-
noxy)ethyl-protected TpsT dimer in solution, was carried out
and treated with ammonium hydroxide under identical
conditions (55 °C). No measurable deprotection occurred as
Results and Discussion
judged by the absence of change in the chemical shift in 31
NMR spectroscopy (Scheme 5).
P
Investigation on the Deprotection of APOE Protecting
Group. To check the stability of this new protecting group
in the presence of aqueous ammonium hydroxide, the APOE-
protected phosphate and phosphorothioate heterodimers
d(TC), d(CT), d(GA), and d(AG) were synthesized in
solution, purified, and subsequently treated with aqueous
ammonium hydroxide dissolved in ethanol (1:1, v/v) as well
as in aqueous methylamine. The deprotection rates were
studied at 25 °C and 55 °C by 31P NMR spectroscopy. Chart
1 shows the rates of deprotection under various conditions.
Hence, a probable mechanistic pathway could be a direct
attack on the R-carbon adjacent to the phosphate oxygen,
leading to the formation of unreactive bicyclic ether as a
side product (Scheme 6).
Conclusion
In summary, the APOE is a suitable protecting group for
internucleotidic phosphate protection. Our initial experiments
comparing the APOE with the cyanoethyl group indicated
that the two groups had equal coupling efficiency (based on
CGE analyses and isolated yields) besides the obvious
elimination of cyanoethyl adduct formation (data not shown).
The APOE phosphitylating reagent as well as the phosphor-
amidites 1e-h were scaled up to multi-hundreds of grams
(10) (a) Cheruvallath, Z. S.; Wheeler, P. D.; Cole, D. L.; Ravikumar, V. T.
Nucleosides Nucleotides 1999, 18, 485-492. (b) Cheruvallath, Z. S.; Carty,
R. L.; Moore, M. N.; Capaldi, D. C.; Krotz, A. H.; Wheeler, P. D.; Turney,
B. J.; Craig, S. R.; Gaus, H. J.; Scozzari, A. N.; Cole, D. L.; Ravikumar, V.
T. Org. Process Res. DeV. 2000, 4, 199-204. (c) Krotz, A. H.; Gorman,
D.; Mataruse, P.; Foster, C.; Godbout, J. D.; Coffin, C. C.; Scozzari, A. N.
Org. Process Res. DeV. 2004, 8, 852-858.
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with no major hurdles. Several synthetic applications of this
versatile protecting group in oligonucleotide synthesis are
currently being developed, including the scale-up of phos-
phorothioate oligonucleotides for potential therapeutic ap-
plications.
Supporting Information Available
1H NMR and 31P NMR spectra, HPLC analyses, CGE
analyses. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment
Received for review October 23, 2005.
OP0502147
We thank Robert Day at R.I. Chemicals, California, S.
N. Acharya at Hebert Brown Laboratories, India, and
Douglas L. Cole for their valuable help.
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