A base-stable dithiomethyl linker for solid-phase synthesis of oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2006.11.045

A novel linkage, useful for the synthesis of oligonucleotides is described. The linking function is compatible with all conditions used for oligonucleotide synthesis, orthogonal to all other protecting groups, but regenerates 3′-OH rapidly upon mild reduc

Full text of DOI:10.1016/j.tetlet.2006.11.045

Tetrahedron Letters 48 (2007) 469–472
A base-stable dithiomethyl linker for solid-phase
synthesis of oligonucleotides
Andrey Semenyuka and Marek Kwiatkowskia,b,
*
aDepartment of Genetics and Pathology, Uppsala University, 75185 Uppsala, Sweden
bQuiatech AB, 75108 Uppsala, Sweden
Received 14 September 2006; revised 1 November 2006; accepted 8 November 2006
Available online 4 December 2006
Abstract—A novel linkage, useful for the synthesis of oligonucleotides is described. The linking function is compatible with all
conditions used for oligonucleotide synthesis, orthogonal to all other protecting groups, but regenerates 30-OH rapidly upon mild
reduction under aqueous conditions. This method is employed in the removal of depurinated fragments during the synthesis of
oligonucleotides.
Ó 2006 Elsevier Ltd. All rights reserved.
Oligonucleotides conjugated to other oligonucleotides,
proteins, labels, haptens or separation tags by means of
a linker that can be cleaved with liberation of the
native oligonucleotide, have potential to find application
in all fields of molecular biology. Solid-phase based
methods for chemical synthesis are the most attractive
routes for the synthesis of such conjugates. This requires,
however, an access to a linkage that can withstand all
reaction conditions for oligonucleotide synthesis and
deprotection, being at the same time cleavable under
mild and preferably aqueous conditions. Preferentially,
the cleavage of the conjugate should liberate 30-OH of
the oligonucleotide, since it is often needed as a starting
point for template-based DNA extension. These highly
demanding conditions are not easy to realize. It is thus
not surprising that amongst all existing linkages only
photolabile1 and silyl-based linkers2 tend to approach
these demands. However, photolabile functions often
demand a long deprotection time, and are inadequate
for application in sites which are nonaccessible by light.
The cleavage of silyl-based functions proceeds under
nonaqueous conditions, thus it is cumbersome and
incompatible with most biological systems.
described and applied for the removal of depurinated
oligonucleotides during the synthesis and purification
of synthetic DNA.
Introduction of the dithiomethyl group onto a nucleo-
side was achieved via 50-O-MMTr-30-O-(methylthio-
methyl)thymidine
1
obtained according to the
published procedure4 from 50-O-MMTr-thymidine. This
synthon, upon activation, reacted with 6-mercapto-
1-hexanol, 3-mercapto-3-methyl-1-butanol5 or 6-mer-
capto-6-methyl-1-heptanol (prepared according to the
modified procedure)6 to produce several nucleoside
derivatives (2, 4, 5, and 6) (Scheme 1). These nucleosides
were converted to respective phosphoramidites (3, 7, 8,
and 9) bearing the dithiomethyl group. The stability of
both nucleosides and nucleotide amidites was examined.
It was found that tertiary alkyl substituents had pro-
nounced stabilizing effects on the disulfide bond toward
aqueous ammonia and iodine compared to the less sta-
ble primary analogues. Additionally, the stability of
the disulfide bond in the obtained phosphoramidites
was dependent on the presence of a bulky, tertiary car-
bon atom, flanking the dithio function. Thus, nucleoside
2 was not completely stable in 0.02 M iodine oxidation
solution and phosphoramidite 3 had limited stability
when dissolved in acetonitrile.
Recently, we reported the use of tert-butyldithiomethyl
as a 20-OH protecting group for solid-phase RNA
synthesis.3 Herein, a strategy for the conjugation of
oligonucleotides via the 30-O-dithiomethyl linkage is
It was found that phosphoramidite 7 was prone to
degradation. After isolation, derivative 7 underwent
decomposition within few hours at room temperature
in dry acetonitrile. HPLC studies showed the formation
of intermediate 10, which was further converted to 11
*
Corresponding author. Tel.: +46 18 4714809; fax: +46 18 47
14808; e-mail: marek.kwiatkowski@genpat.uu.se
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.11.045

470
A. Semenyuk, M. Kwiatkowski / Tetrahedron Letters 48 (2007) 469–472
MMTr
O
T
S
MMTr
O
T
S
O
O
iii
N
O
O
O
OH
O P
S
S
CE
2
3
43%
i, thiol = 6-mercapto-1-hexanol
MMTr
O
T
S
O
O
=
3-mercapto-3-methyl-1-butanol
i, thiol
i, thiol = 6-mercapto-6-methyl-1-heptanol
1
MMTr
O
T
S
O
MMTr
O
T
MMTr
O
T
S
ii
O
O
O
O
S
O
N
O
OH
S
O
OH
S
OH
H
S
4
5
6
64%
33%
82%
iii
iii
iii
MMTr
O
T
S
MMTr
O
T
O
MMTr
O
T
O
O
O
N
O
N
P
O
N
O
O
S
S
O
O
S
S
N
O
P
O
O P
S
H
CE
CE
O
CE
7
9
8
81%
71%
Scheme 1. Synthesis of phosphoramidites used in the present study: (i) a. SO2Cl2 (1.1 equiv), TEA (1.2 equiv) in 1,2-dichloroethane; b. p-MeC6H4SK
(3.0 equiv) in DMF; c. thiol (5.0 equiv), DIEA (5.0 equiv); (ii) a. CDI (2.0 equiv) in Py; b. 5-aminopentanol (5.0 equiv); (iii) (iPr)2NP-
(Cl)OCH2CH2CN (1.5 equiv), TEA (4.5 equiv) in CH2Cl2.
after the addition of pH 8.0 aqueous buffer. A possible
explanation of such rapid conversion of 7 could be made
by assuming neighboring attack of the trivalent phos-
phorus atom on the disulfide bond, favored by a six-
membered ring intramolecular conformation (Scheme
2). This hypothesis was supported by the observation
that phosphoramidites 8 and 9 were much more stable
in acetonitrile at room temperature.
compound 4 degraded completely after 14 h incubation
in 32% aq NH3–EtOH (1:1) at 55 °C. In contrast, deriv-
ative 5 obtained from 4, and nucleoside 6 did not show
any sign of disulfide bond cleavage under identical con-
ditions. The fast decomposition of 4 cannot be explained
only by the electronegativity of the hydroxyl group
destabilizing the dithio bond, since the presence of the
carbamido group in compound 5 did not result in a
similar rate of decomposition. The stability of oligo-
nucleotides bearing dithiomethyl linkers toward ammo-
nia was verified by the synthesis of oligonucleotide 12
using the standard conditions for DNA synthesis and
deprotection.
The destabilizing effect of the hydroxyl group located in
close proximity to the disulfide bond was also observed,
similar to the described effect of the neighboring amino
group on the stability of the disulfide bond.7,8 Thus,
O
5'
O
S
HO-
3'
TTTTTTTTTT
S
O
N
O
-OH
TTTTT
H
12
O
5'
HO-
3'
5'
O
3'
-OH
+
O
SH
TTTTTTTTTT
HS
O
N
TTTTT
H
13
14

A. Semenyuk, M. Kwiatkowski / Tetrahedron Letters 48 (2007) 469–472
471
MMTr
O
T
S
MMTr
O
T
MMTr
O
T
O
O
O
O
O
OH
11
SH
S
P
10
CE O
O
N(iPr)2
7
Scheme 2. Putative mechanism of amidite 7 decomposition.
Figure 1. Deprotection of 12 studied by RP HPLC: (A) crude oligonucleotide 12; (B) 0.2 mM 12 incubated with 50 mM DTT, 5 min, 20 °C, pH 10;
(C) 0.2 mM 12 incubated with 50 mM DTT, 120 min, 20 °C, pH 10; (D) 0.2 mM 12 incubated with 2 mM DTT, 30 min, 20 °C, pH 8.5.
Cleavage of the dithiomethyl linker in 12 with 1,4-di-
thiothreitol (DTT) proceeded through the formation of
intermediate 13 (Fig. 1), yielding finally oligonucleotide
T10 and 50-modified T5 fragment 14. The rate of linker
cleavage was dependent both on DTT concentration
and on the pH of reaction. The above example illus-
trates the general possibility for the synthesis of oligo-
nucleotide conjugates linked by cleavable units. The
pentathymidilic acid (T5) present in oligonucleotide 12
can be easily substituted by any existing functional
or labeling group to form the desired conjugate. When
necessary these functions can be cleaved releasing free
oligonucleotide with intact 30-OH.
abasic sites.2 The latter linkage has some disadvantages
as it demands an extra drying step since the cleavage of
the siloxyl bond has to be maintained under anhydrous
conditions. Additionally, cleavage of the disiloxyl
group, linked to the polystyrene, is not very efficient,
limiting this chemistry only to controlled pore glass
(CPG) supports only. In contrast, the dithiomethyl lin-
ker can be easily applied for oligonucleotide synthesis
on a polystyrene support. It can be cleaved fast in aque-
ous media, withstanding the harsh conditions of depro-
tection in ammonia, but the shorter fragments formed
due to the cleavage of abasic sites, under these basic con-
ditions, can be easily washed away from the support-
bound product.
We realized early that the present dithiomethyl linker
could be used as an alternative to the disiloxyl linker
employed during oligonucleotide synthesis in order to
remove failure fragments arising from the cleavage of
To demonstrate the applicability of this novel linker,
the oligonucleotide 50-d(AG)30T, was synthesized on
an amino modified polystyrene-grafted poly(tetrafluoro-
0.38
0.38
0.28
0.18
0.08
-0.02
A
B
0.28
0.18
0.08
-0.02
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
Figure 2. RP HPLC analysis of 50-d(AG)30T released from the polystyrene support: (A) oligonucleotide was attached to the support via a succinyl
linker; (B) oligonucleotide was attached via a dithiomethyl linker and released after the ammonia deprotection step according to the described
procedure.

472
A. Semenyuk, M. Kwiatkowski / Tetrahedron Letters 48 (2007) 469–472
ethylene) support,9 further functionalized to obtain
hydroxyalkyl functions according to the described pro-
cedure.2 The support with oligonucleotide was treated
with aqueous ammonia at 55 °C overnight (16 h) to
ensure a complete cleavage of all apurinic sites and
aglycone protecting groups, then washed with aqueous
ammonia, and treated again with aqueous ammonia
containing 50 mM DTT at 55 °C for 10 min to cleave
the oligonucleotide from the support. Alternatively,
the dithiomethyl linker cleavage can be performed at
an ambient temperature within 1 h. The collected
ammonia fraction containing the crude oligonucleotide
was analyzed by HPLC and compared to the crude
oligonucleotide of the same sequence synthesized on
the same support, but derivatized with succinyl linker
(Fig. 2). The oligonucleotides were purified using a
cartridge-based separation methodology,10 which in
conjunction with the present method produced a good
quality purine-rich material.
more user friendly by eliminating a time consuming and
laborious step.
References and notes
1. Bai, X.; Li, Z.; Jockusch, S.; Turro, N. J.; Ju, J. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 409–413.
2. Kwiatkowski, M.; Nilsson, M.; Landegren, U. Nucleic
Acids Res. 1996, 24, 4632–4638.
3. Semenyuk, A.; Fo¨ldesi, A.; Johansson, T.; Estmer-Nils-
son, C.; Blomgren, P.; Bra¨nnvall, M.; Kirsebom, L. A.;
Kwiatkowski, M. J. Am. Chem. Soc. 2006, 128, 12356–
12357.
4. Veeneman, G. H.; Vandermarel, G. A.; Vandenelst, H.;
Vanboom, J. H. Tetrahedron 1991, 47, 1547–1562.
5. Ohsugi, S.-I.; Nishide, K.; Node, M. Tetrahedron 2003, 59,
1859–1871.
6. Cao, Z.-B.; Chen, Y.; Guo, S.; Chen, K.-Q. Youji Huaxue
1989, 9, 337–340.
7. Gawron, O.; Mahboob, S.; Fernando, J. J. Am. Chem.
Soc. 1964, 86, 2283–2286.
8. Overman, L. E.; O’Connor, E. M. J. Am. Chem. Soc.
1976, 98, 771–775.
9. The polystyrene support was kindly provided by Dr.
Eckhard Birch-Hirschfeld. The synthesis of the support is
described in Birch-Hirschfeld, E.; Fo¨ldes-Papp, Z.; Guhrs,
K.-H.; Seliger, H. Helv. Chim. Acta 1996, 79, 137–150.
10. Semenyuk, A.; Ahnfelt, M.; Estmer-Nilsson, C.; Hao,
X.-Y.; Fo¨ldesi, A.; Kao, Y.-Sh.; Chen, H.-H.; Kao, W.
Ch.; Peck, K.; Kwiatkowski, M. Anal. Biochem. 2006, 356,
132–141.
Thus, it has been shown that the dithiomethyl linker
construct can be used as a linker between a solid support
and the oligonucleotide synthesized. We have demon-
strated the expected base stability of the novel linker
and the mild conditions for its cleavage. It was proven
that oligonucleotides made according to this proce-
dure have purities similar to oligonucleotides, which
were made on disiloxyl support and purified on RP
cartridges. The present methodology represents an
improvement over the existing one, making purification