2-Methyl-5-tert-butylthiophenol - An odorless deprotecting reagent useful in synthesis of oligonucleotides and their analogs

Article abstract of DOI:10.1081/NCN-120022038

A solution of 2-methyl-5-tert-butylthiophenol and triethylamine in acetonitrile efficiently removed the methyl protecting groups from phosphate and phosphorothioate triesters and yielded oligonucleotides of high quality. This inexpensive odorless liquid i

Full text of DOI:10.1081/NCN-120022038

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
2-Methyl-5-tert-butylthiophenol—An Odorless
Deprotecting Reagent Useful in Synthesis of
Oligonucleotides and Their Analogs
R. Krishna Kumar , Douglas L. Cole & Vasulinga T. Ravikumar
To cite this article: R. Krishna Kumar , Douglas L. Cole & Vasulinga T. Ravikumar (2003) 2-
Methyl-5-tert-butylthiophenol—An Odorless Deprotecting Reagent Useful in Synthesis of
Oligonucleotides and Their Analogs, Nucleosides, Nucleotides and Nucleic Acids, 22:4, 453-460,
DOI: 10.1081/NCN-120022038
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Date: 05 November 2015, At: 19:36

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 4, pp. 453–460, 2003
2-Methyl-5-tert-butylthiophenol—An Odorless Deprotecting
Reagent Useful in Synthesis of Oligonucleotides
and Their Analogs
*
R. Krishna Kumar, Douglas L. Cole, and Vasulinga T. Ravikumar
Isis Pharmaceuticals, Carlsbad, California, USA
ABSTRACT
A solution of 2-methyl-5-tert-butylthiophenol and triethylamine in acetonitrile
efficiently removed the methyl protecting groups from phosphate and phosphoro-
thioate triesters and yielded oligonucleotides of high quality. This inexpensive
odorless liquid is not toxic and is a suitable replacement for hazardous thiophenol
and other reagents often used for this purpose.
*Correspondence: Vasulinga T. Ravikumar, Isis Pharmaceuticals, 2292 Faraday Avenue,
Carlsbad, CA 92008, USA; Fax: (760) 603-4655; E-mail: vravikumar@isisph.com.
453
DOI: 10.1081/NCN-120022038
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

454
Kumar, Cole, and Ravikumar
Key Words: Oligonucleotides; Phosphorothioates; Deprotection; Odorless
reagent; Phosphoramidite.
Antisense oligonucleotides as modulators of gene expression represent an excit-
ing new drug technology.[1–7] Phosphorothioate oligonucleotides are among the most
intensively investigated nuclease-resistant antisense analogs, as evidenced by a num-
ber of ongoing clinical trials and one FDA approved drug. With the first antisense
drug (VitraveneTM) being approved by the US Food and Drug Administration
and potentially several systemic drugs to reach market in next several years, the
development of economical and environmentally safe methods for synthesis of high
quality oligonucleotides has become a major focus of our research. Currently, synthe-
sis of phosphorothioate oligodeoxyribonucleotides of 20-mer in length are routinely
performed on scales up to 300–600 mmole in a cyclic manner on automated solid-
phase synthesizers using b-cyanoethyl protected phosphoramidite monomers.
However, this commonly used protecting group has some undesirable properties. The
by-product formed during deprotection of oligonucleotide viz., acrylonitrile adds to
N3 of thymine to form an irreversible adduct.[8] In addition, the manufacturing scale
up of high quality phosphitylating reagent (b-cyanoethyl-N,N,N0,N0-tetraisopropyl
phosphorodiamidite) to make phosphoramidites of high grade is an issue faced by
many vendors. For example, the starting alcohol viz., 3-hydroxypropionitrile is con-
taminated with other reactive alcohols such as methanol or ethylene glycol and also
react to form corresponding amidites that get incorporated into oligonucleotide.[9]
In addition, the phosphitylating reagent during distillation undergoes Arbuzov re-
arrangement to form undesired products. Thus, there is an urgent need to eliminate
the above problems or identify an alternative protecting group.
Besides b-cyanoethyl, the most extensively investigated protecting group that
does not possess the above limitations is the O-methyl group0.[10–15] Several labora-
tories have reported use of the methyl group as a phosphate protecting group during
solid-phase synthesis of oligodeoxynucleotides,[16–24] oligoribonucleotides,[25–27] and
a-anomeric oligodeoxyribonucleotides.[28] This group can be selectively removed by
a mixture of thiophenol and triethylamine.[29] However, the reagent has an obnox-
ious smell and has led virtually every researcher to switch to b-cyanoethyl group.
The higher reactivity of methyl compared to b-cyanoethyl phosphoramidites and a
relatively low boiling point (75–78 ꢀC=0.5 mm Hg) of corresponding phosphitylating
reagent (methyl-N,N,N0,N0-tetraisopropylphosphorodiamidite) makes it highly desir-
able to have as an alternative to current b-cyanoethyl protection. To alleviate this
problem other reagents such as 2-mercaptobenzothiazole 1 and disodium 2-carba-
moyl-2-cyanoethylene-1, 1-dithiolate 2 have also been reported.[30,31] We report here
that commercially available 2-methyl-5-tert-butylthiophenol 3 meets all of our

2-Methyl-5-tert-butylthiophenol
455
requirements and the reagent is odorless, inexpensive, non-toxic and removes methyl
groups from phosphate and phosphorothioate triesters fast and clean.
2-methyl-5-tert-butylthiophenol is commercially available from Clariant LSM as
a colorless, odorless liquid.[32] It is miscible in organic solvents such as acetonitrile,
dioxane, DMF, toluene and the solutions are stable at least for two weeks when stored
under proper conditions. To investigate the deprotection reaction, a fully-protected
20-O-methoxyethyl substituted phosphorothioate triester dimer 7 was synthesized in
solution by coupling 50-O-dimethoxytrityl-20-O-methoxyethyl-N 4-benzoyl-5-methyl-
cytidine-30-O-methyl N, N0-diisopropylphosphoramidite 4 with 30-O-levulinyl-20-
O-methoxyethyl-N2-isobutyrylguanosine 5 in anhydrous acetonitrile in presence
of 1H-tetrazole as activator (Sch. 1). The phosphite triester intermediate 6 was sul-
furized using diethyldithiocarbonate disulfide (DDD)[33] (82% combined coupling
and sulfurization yield based on 5). Sulfurization could also be efficiently per-
formed using phenylacetyl disulfide (PADS).[34,35]
The dealkylation reactions were monitored by 31P NMR spectroscopy on a Var-
ian 200 spectrometer at 28ꢀC in 5 mm tubes. A CD3CN solution of 7 (0.4 M, 0.2 mL)
was mixed with a CH3CN solution of 2-methyl-5-tert-butylthiophenol 3 and
triethylamine (1:1 mol=mol; 0.4 M, 0.2 mL) at zero time, and 1H-decoupled 31P
NMR spectra were obtained at different time intervals. Complete deprotection of
dimer was observed in 2 h. Effect of base on rate of demethylation was examined
by repeating above experiment in N, N-diisopropylethylamine under identical condi-
tions. No significant increase in the rate of deprotection was observed (Sch. 2).
Influence of solvent on rate of demethylation was also investigated. Acetonitrile
was found to be the best among a variety of solvents (DMF, 1,4-dioxane, 1,2-
dimethoxythane, 1-methyl-2-pyrrolidinone) evaluated. A critical requirement for
removal of methyl protecting group from oligonucleotide phosphate and phosphor-
othioate triesters by thiolate anion is stability of the internucleotidic linkage under
deprotecting conditions. The dinucleotide phosphate triester 9 (X ¼ O) and phos-
phorothioate triester 9 (X ¼ S) (Sch. 3) immobilized on Pharmacia Primer support
were treated with an equimolar (1.0M) solution of 3 and triethylamine in acetonitrile
at ambient temperature for 2 h. The solid support was then thoroughly washed with
acetonitrile and the dimer cleaved from the support according to standard proce-
dures. HPLC analysis of the reaction mixture indicated only the presence of product
along with traces of unreacted monomer. There was no evidence of 50-S-(2-methyl-
Scheme 1. Preparation of methyl protected dimer.

456
Kumar, Cole, and Ravikumar
Scheme 2. Rate of deprotection with different bases.
5-tert-butyl)-thiophenylthymidine that could have been produced from nucleophilic
attack of thiolate anion at 50-carbon of the internucleotidic linkage. In addition, the
HPLC profile of the dimer was superimposable with that obtained using
b-cyanoethyl as protecting group. This indicates that deprotection of the methyl
group by 2-methyl-5-tert-butylthiophenol is clean and fast relative to internucleotidic
cleavage. Further attack after formation of the phosphate=phosphorothioate diester
is prohibited, presumably due to significant electrostatic repulsion toward the
incoming thiolate anion.
Having established the effectiveness of 3 as a fast and clean deprotecting reagent
for dimers, applicability was extended to solid-supported synthesis of oligonucleo-
tides using methoxy phosphoramidites. A homothymidine 20-mer DNA was
Scheme 3. Synthesis of methyl protected dimers on solid support.

2-Methyl-5-tert-butylthiophenol
Table 1. Synthesis of different oligonucleotides for investigation.
Mass
457
HPLC
Oligonucleotide
Retention timea (min)
Calculated
Found
T20
50-[meCmeUG]-AGTCTGTTT-
17.679
21.076
6021.9
6910.9
6021.2
6910.8
[
meUmeCmeCAmeUmeUmeCmeU]
aAnalysis and purification of oligonucleotides by reversed phase high performance liquid chro-
matography (RP-HPLC) was performed on a Phenomenex Luna C18 column (250 ꢁ 4.6 mm,
5 m size) using a Waters HPLC system (600E System Controller, 996 Photodiode Array Detec-
tor, 717 Autosampler). For analysis an acetonitrile (B)=0.1 M triethylammonium acetate (A)
(pH 7.0) (flow rate ¼ 1 mL=min) gradient was used: 0% to 40% B from 0 to 25 min, 40% B
from 25 to 30 min, 100% B from 30 to 39 min, 100% A from 39 min to 45 min. The DMT-
on fraction was collected and was evaporated in vacuum, redissolved in 50 mL water and
DMT group removed as follows: An aliquot (50–100 OD) was transferred into an Eppendorff
tube (1.5 mL), and 0.01 M sodium acetate (pH 3, 175 mL) was added. After 30 to 60 min at
room temperature, sodium acetate (3 M, 10 mL) was added, centrifuged to remove trityl and
to the supernatent was added cold ethanol (1.2 mL). The mixture was vortexed and cooled
in dry ice for 20 min. The precipitate was spun down with a centrifuge, the supernatant was
discarded and the precipitate was rinsed with ethanol and dried under vacuum.
synthesized on an ABI DNA=RNA synthesizer using controlled-pore glass support
(loading 47 mmole=g). Detritylation was effected using 3% trichloroacetic acid in
dichloromethane and oxidation was performed using iodine=water under standard
conditions. The crude oligonucleotide was demethylated on support by treatment
with a solution of 3:triethylamine:acetonitrile (1:1:3; v=v=v; 5 ml=g of support) at
ambient temperature for 2 h. The partially deprotected oligonucleotide was cleaved
from the support using concentrated aqueous ammonium hydroxide under standard
conditions. Purification by reversed phase HPLC followed by LC-MS analysis
indicated purity comparable to that obtained using b-cyanoethyl phosphoramidite
(Table 1).
The scope of this methodology was further extended to synthesis of 20-O-alky-
oligoribonucleotides. A chimeric 20-mer oligonucleotide containing 20-O-meth-
oxyethyl wings [50-(meCmeUG)-AGTCTGTTT-(meUmeCmeCAmeUmeUmeCmeU)] was
synthesized on an ABI DNA=RNA synthesizer using controlled-pore glass support
(loading 74 mmole=g). Similar oligomerization and deprotection conditions were
followed as above for synthesis of homothymidine 20-mer. Purification by reversed
phase HPLC followed by LC-MS analysis indicated purity comparable to those
obtained using b-cyanoethyl phosphoramidite (Table 1).
In conclusion, we have demonstrated that 3 is an excellent alternative
reagent for deprotection of methyl phosphotriester groups. Because of the
odorless and inexpensive nature of this reagent and its rapid deprotection
kinetics, it could be applicable to synthesis of RNA and other oligomeric ana-
logs as well as in general synthetic organic chemistry where odiferous thiols have
been employed.

458
Kumar, Cole, and Ravikumar
ACKNOWLEDGMENTS
The authors thank Clariant LSM for a generous gift of reagent and also Claus
Rentel for IP-LC-MS analysis.
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Received December 23, 2002
Accepted February 19, 2003