Synthesis and application of fluorous-tagged oligonucleotides

Article abstract of DOI:10.1246/cl.2006.1184

Nucleoside phosphoramidites bearing a adsorbent tert-butylphenyl-1H,1H,2H, 2H-heptafluorodecyloxysilyl (BPFOS) group were used to synthesize fluorous-tagged oligonucleotides, viz. 5-mer, 10-mer, 13-mer, 17-mer, and 19-mer, which were subjected to liquid-p

Full text of DOI:10.1246/cl.2006.1184

1184
Chemistry Letters Vol.35, No.10 (2006)
Synthesis and Application of Fluorous-tagged Oligonucleotides
Roli Mishra,ꢀ Satyendra Mishra, and Krishna Misra
N A R Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211002, India
(Received June 20, 2006; CL-060698)
Nucleoside phosphoramidites bearing a adsorbent tert-
butylphenyl-1H,1H,2H,2H-heptafluorodecyloxysilyl (BPFOS)
group were used to synthesize fluorous-tagged oligonucleotides,
viz. 5-mer, 10-mer, 13-mer, 17-mer, and 19-mer, which were
subjected to liquid-phase extraction. The labeled oligomers were
found to be highly hydrophobic in nature, become precipitated
in water, therefore allowing easy purification from the failure
sequences. The silyl fluorous group was deprotected by TBAF
treatment and oligomers were easily purified by CH3CN/
FC-72 liquid–liquid extraction. Fluorous-tagged oligonucleoti-
des have high selectivity for the removal of failure sequences,
high recoveries (typically 70–100%).
and water or via solid-phase extraction with fluorous reverse
phase silica gel.13,14 (C6F13CH2CH2)3Si group, the classical
fluorous silyl tag is quite acid sensitive. Since, the bis-alkoxy-
silyl ethers have shown improved acid stability therefore, Wipf
et al.15 have tried fluorous alkoxysilyl groups and they found it
to be more acid stable and with primary and secondary alcohols
the yields were found to be fair to excellent. They reported tert-
butylphenyl-1H,1H,2H,2H-heptafluorodecyloxysilyl (BPFOS)
to be more acid stable than (C6F13CH2CH2)3Si group. Here,
the BPFOS group is used for protection of 50-hydroxy group
of nucleosides, an example of primary hydroxy group.
A general method for the preparation of 50-protected
deoxyribonucleosides has been out in Schemes 1 and 2. The
alkoxysilyl ethers (BPFOS) were obtained by the reaction of
decanol with 1.1–1.3 equiv. of the appropriate silyl chloride
and 1.5 equiv imidazole at rt. in CH2Cl2 as solvent. Excellent
yields of alkoxysilyl ethers results in all cases, except for the hy-
drolytically. This alkoxysilyl ether (2)16 was brominated in 1,2-
dichloroethane at 0 ꢂC to yield (BPFOSiBr) (3)17 (Scheme 1).
Therefore, the tert-butylphenyl-1H,1H,2H,2H-heptafluoro-
decyloxysilyl bromide (BPFOSiBr) group was used for protec-
tion of 50-hydroxy group of nucleosides, an example of primary
hydroxy group, the 50-O-protected nucleosides (4a–4d)18 were
phosphitylated using 2-cyanoethyl-N,N-diisopropylchlorophos-
phoramidite (bis-reagent) and activator pyridinium trifluoroace-
tate (Py-TFA) in dry DCM (Scheme 2).19
The purification and yield improvement are the two much
challenged jobs in the field of oligonucleotide synthesis. To
avoid chromatographic purification, reactions should be planed
such that the phase of the desired product is different from phas-
es of all the other reaction components and undesired products.
The ‘‘fluorous phase’’1–5 has been recently used to advantage in
traditional organic synthesis. The new techniques rely on the
ability of a ‘‘fluorous’’ (highly fluorinated) molecule to partition
into the fluorous phase in a liquid–liquid extraction between an
organic solvent and a fluorous solvent.6,7 One or more reactions
are then conducted and the fluorous components of the reaction
are subsequently separated from all non-fluorous (organic,
inorganic, solid or volatile) components by an appropriate
phase-separation technique. At the desired stage, the fluorous la-
bels are cleaved and the product rendered organic. Furthermore,
fluorous solvents are much robust than most polymers and link-
ers in current use for solid-phase synthesis.
The fluorous-tagged sequences were readily precipitated out
when dissolved in deionized water. This may be due to enhance-
ment in hydrophobicity. Fluorous tag from 50-OH of oligonu-
cleotides sequences was removed by treating fluorous-tagged
sequences with tetrabutylammonium fluoride (TBAF).
Silicon-based methodology for the protection of alcohols
has made a major contribution in organic synthesis.8 The ability
to modulate selectivity and reactivity by varying the steric
and electronic requirements of the substituents on silicon has
been demonstrated by such reagents as tert-butyldimethylsilyl
chloride9 and tert-butyldiphenylsilyl chloride.10 Therapeutic
uses of antisense oligonucleotides warrant efficient synthesis
and purification. But on solid-support synthesis, however, the
final yield of the desired sequence is greatly affected by purifica-
tion cycles.
The idea nurtured in this communication was to carry out the
solid-support synthesis in such a way that the desired sequences
after cleavage from the support may be easily separated from the
failed (n ꢁ 1) sequences by liquid–liquid extraction or by simple
precipitation, thus tedious chromatographic separations may be
avoided. To implement the idea, the fluorous alkoxy silyl ethers
were tried. These are reported to impart acid stability to the
silane protecting groups.11,12 The fluorous synthesis schemes
allow for easy purification or removal of highly fluorinated
intermediates or reagents e.g. via liquid–liquid extraction (3
phase extraction) between organic solvents, fluorinated solvents
C8F17
Ph
ii.
C8F17
Cl
Ph
C8F17
O
O
Ph
i.
Si
HO
+
Si
Si
Ph
t-Bu
t-Bu
t-Bu
Ph
Br
2
3
Scheme 1. Synthesis of fluorous alkoxysilyl ether: Reagent and
conditions; i). DCM, DMAP, imidazole, rt. ii) Br2 DCM, 0 ꢂC, rt.
HO
BPFOS-O
BPFOS-O
Base
Base
Base
O
O
O
i
ii
OH
(3)
O
OH
4a - 4d
N(i-Pr)2
P
O
5a - 5d
1a - 1d
Series Base
NC
Yield of 4
Yield of 5
a
b
c
d
Thymine
75%
89%
91%
94%
79%
68%
65%
81%
N6-benzoyladenine
N4-benzoylcytidine
N2-isobutyrylguanine
Scheme 2. Synthesis of 50-O-BPFOS nucleoside phosphorami-
dites; Reagent and conditions; bis-reagent /DCM, Py-trifluoro-
acetate.
Copyright Ó 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.10 (2006)
1185
88%, 2.472 g; 1H NMR (CDCl3) 7.69–7.66 (m, 4H), 7.45–7.38
(m, 6H), 3.96 (t, 2H), 2.45–2.25 (m, 2H), 1.07 (s, 9H).
O
N
N
N
NH
NH2
17 3: tert-Butylphenyl-1H,1H,2H,2H-perfluorodecyloxysilyl bro-
mide. Bromine (3.0 mmol, 1.43 mL) was added drop-wise to a
solution of the TBDPS ether (2.5 mmol, 1.755 g) in 1,2-dichloro-
ethane (15 mL) at 0 ꢂC. The reaction mixture was stirred at rt for
overnight. Distillation at reduced pressure yielded the product as
colorless oil. Yield: 71% (2.003 g); 1H NMR (CDCl3): 7.69–7.65
(m, 2H), 7.48–7.39 (m, 3H), 4.11–4.06 (m, 2H), 2.47–2.35 (m,
2H), 1.01 (s, 9H).
Ph
t-Bu
F
F
F
F F
F
F
Si
F
O
O
F
BPFOS-G =
O
F F F FF F
F
F
5'-BPFOS-G-AC GT-3'
5'-BPFOS-G-AC GTA CGT T-3'
5'-BPFOS-G-AA TGG AGC CAG T-3'
5'-BPFOS-G-TT CAG TCC CAC CTC CC-3'
5'-BPFOS-G-GC TAT GTC AGT TCC CCT T-3'
O
O
P O
O
18 4a–4d: Synthesis of 50-O-tert-butylphenyl-1H,1H,2H,2H-per-
fluorodecyloxysilyl deoxyribonucleosides. Above compound
(3) (1.1 mmol) was dissolved in CH2Cl2 (15 mL). The four
deoxyribonucleosides viz. (dA/dC/dG/T; 1.42 mmol) and
DMAP (0.05 mmol) were added and the reaction mixture was
stirred at rt. overnight. CH2Cl2(15 mL) was added and the mix-
ture was washed with NaHCO3 solution. The organic phase
was dried over Na2SO4, and DCM was evaporated in vacuo.
The product was filtered through silica (DCM:hexane; 60:40)
The desired batches were pooled and evaporated to get product.
19 5a–5d: 50-O-tert-butylphenyl-1H,1H,2H,2H-perfluorodecyloxy-
silyl deoxyribonucleosides-30-O-phosphoramidite. The products
4a–4d (1 mmol) each was suspended in dry DCM and bis-reagent
(20-cyanoethyl-N,N,N0,N0-tetraisopropyl phosphoramidite–phos-
phoramidite) (1.2 mmol) was added to it at ambient temperature.
Pyridinium trifluoroacetate (1.2 mmol) was added to reaction
mixture and the solution was stirred for 4 h. Upon complete
consumption of the starting material (tlc), the entire reaction
mixture was transferred directly on the top of a short silica
column. The product was eluted with DCM:hexane, 60:40. The
appropriate fractions were collected and pooled and the solvent
was evaporated to furnish the product.
Figure 1. Examples of fluorous-tagged oligodeoxyribonucleo-
tides.20,21
The present communication to use fluorous group as
efficient purification tag will be helpful over the existing
purification protocols, which mainly comprise of the tedious
chromatographic techniques. Thus, if a sequence is synthesized
by conventional phosphoramidite chemistry and is tagged at the
end with this fluorous alkoxysilyl-protected nucleoside then after
cleavage from the CPG it is supposed to be separated easily.
Different length of sequences were chosen to prove the viability
of the fluorous protecting groups and to see that how much long
sequence it can separate easily in liquid–liquid extraction. The
maximum length used over here is of 19-mer (18-mer tagged
with the fluorous tagged dG) and it shows easy precipitation,
100% recovery after deprotection in acetonitrile and the ability
to purify small as well as long oligonucleotides.
Thus, the desired sequences may be purified from the rest
of the (n ꢁ 1) failure sequences by suspending the sequence in
water after deprotecting them from the solid support. The desired
sequences precipitated out and were separated by simple vacuum
filtration.
20 Synthesis of fluorous-tagged oligonucletides. All the oligomers
were synthesized at 0.2 mmol scale on an ABI 392 model DNA/
RNA synthesizer using nucleoside analogues (5a–5d) with only
benzoyl and isobutyryl group on exocyclic amino functions. The
last coupling cycle was increased by 10 min. All the oligomers
were mixed type. Five (4-mer, 9-mer, 12-mer, 16-mer, and 18
mer) sequences were synthesized using phosphoramidite chemis-
try Modified 50-O-t-butylphenyl-1H,1H,2H,2H-perfluorodecyl-
oxysilyl deoxyribonucleosides-30-O-phosphoramidite (of thymi-
dine, cytidine, adenosine, and guanosine) were added in last cou-
pling cycle (Figure 1). All the oligomers were treated with 30%
aqueous ammonia for 16 h at 55 ꢂC to remove protecting groups
from the bases and internucleotide phosphates and also to cleave
oligomer from the solid support. The ammoniacal solution was
concentrated under a vacuum in a speed vac, and the residue
was subjected to desalting on a reverse phase silica gel column.
21 Solubility pattern of fluorous tagged oligonucletides. After the
cleavage from solid support the fluorous-tagged oligonucleotides
were readily precipitated when suspended in deionized water
due to enhanced hydrophobicity. They are sparingly soluble in
CH3CN (organic solvent) and insoluble in FC-72 (fluorous sol-
vent). To deprotect the fluorous tag the sequences were added
to a solution of TBAF (0.6 M) in 0.5 mL of THF. After 3 h the
deprotected oligonucleotides was extracted in acetonitrile. The
organic layer was pooled and evaporated and the residue was
partitioned in FC-72 and fresh CH3CN. The sequences are re-
tained in organic solvent layer while the protecting group passes
to FC-72 layer. The organic phase on evaporated in vacuo gave
the desired sequences in better yield. The purity of desired
oligonucleotide were analyzed on reverse phase HPLC with a
Merck lichrosphere RP-18 column using 0.1 M ammonium
acetate buffer at pH 7.1 and acetonitrile solvent.
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16 2: tert-Butyldiphenyl-1H,1H,2H,2H-perfluorodecylsilyl ether. A
solution of tert-butyldiphenylsilyl chloride (4.39 mmol, 1.20 g),
1H,1H,2H,2H-perfluoro-1-decanol (4 mmol, 1.586 g), DMAP
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Published on the web (Advance View) September 27, 2006; doi:10.1246/cl.2006.1184