Efficient solid phase synthesis of cleavable oligodeoxynucleotides based on a novel strategy for the synthesis of 5′-S-(4,4′-dimethoxytrityl)- 2′-deoxy-5′-thionucleoside phosphoramidites

Article abstract of DOI:10.1002/hlca.200490252

The incorporation of a specific cleavage site into an oligodeoxynucleotide can be achieved by utilizing the four 5′-S-(4,4′-dimethoxytrityl)- 2′-deoxy-5′-thionucleoside 3′-(2-cyanoethyl diisopropylphosphoramidites) 5 and 15a-c (Fig.1). Based on the silver

Full text of DOI:10.1002/hlca.200490252

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Helvetica Chimica Acta Vol. 87 (2004)
Efficient Solid Phase Synthesis of Cleavable Oligodeoxynucleotides Based on
a Novel Strategy for the Synthesis of 5'-S-(4,4'-Dimethoxytrityl)-2'-deoxy-5'-
thionucleoside Phosphoramidites
by Kerstin Jahn-Hofmann and Joachim W. Engels*
Institut f¸r Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe-Universit‰t,
Marie-Curie-Strasse 11, D-60439 Frankfurt am Main
(e-mail: joachim.engels@chemie.uni-frankfurt.de)
The incorporation of a specific cleavage site into an oligodeoxynucleotide can be achieved by utilizing the
four 5'-S-(4,4'-dimethoxytrityl)-2'-deoxy-5'-thionucleoside 3'-(2-cyanoethyl diisopropylphosphoramidites) 5
and 15a c (Fig. 1). Based on the silver ion assisted cleavage of PÀS and CÀS bonds, we synthesized
oligodeoxynucleotides with an achiral 5'-phosphorothioate linkage 3'
O P S 5' by the solid-phase
phosphoramidite procedure. The efficient cleavage of these modified oligodeoxynucleotides can be detected
by HPLC, PAGE, and surface plasmon resonance (SPR) spectrometry. The liberated 5'-thiol moiety can be used
directly for post-reaction labeling with appropriately functionalized reporter groups.
1. Introduction. Modified oligodeoxynucleotides are versatile tools to solve a
variety of problems in molecular biology. The cleavage of oligodeoxynucleotides with
restriction enzymes is selective and quantitative. For an equivalent chemical cleavage,
the oligodeoxynucleotides must be modified beforehand. We selected the thio-
modification because of its electronic and steric similarity with the natural congener
and the possibility for later derivatization. These backbone modified oligodeoxynu-
cleotides can be cleaved selectively and quantitatively at the PÀS or CÀS bonds by
‡
Ag and Hg2‡ ions or by a concentrated I2 solution under mild conditions [1]. The
cleavage with I2 however, does not occur on relatively low functionalized CPG material
but fortunately has been shown to cause no problem during the oxidation step in DNA
synthesis [1b]. Furthermore, oligodeoxynucleotides containing a free SH group can be
derivatized with thiol-specific probes [1a][2a d][3]. Thus, the thiol group is
particularly useful for post-synthetic modifications and can also be used to reversibly
form disulfide bonds.
Currently, we are developing a new concept for the detection and construction of
DNA arrays using 5'-thio-modified phosphoramidites 5 and 15a c based on the
incorporation of a selective cleavage site [4].
For the synthesis of a phosphorothioate linkage, most references [1][5] describe a
5'-S-trityl-2'-deoxy-5'-thionucleoside 3'-phosphoramidite building block. Since we
intended to use the 4,4'-dimethoxytrityl group ((MeO)2Tr-group) for the 5'-protection
of all four 2'-deoxynucleosides, the aim of this work was to develop an easy procedure
for incorporating a 3' O P S 5' internucleotide linkage that can be adapted to a
fully automated synthesis via phosphoramidite chemistry. Originally, for the coupling
‡
of the 5'-S-trityl building block, the S-trityl group had to be removed by Ag ions. This
¹ 2004 Verlag Helvetica Chimica Acta AG, Z¸rich

Helvetica Chimica Acta Vol. 87 (2004)
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heavy-metal-ion cleavage is necessary because the S-trityl function cannot easily be
cleaved by mild acids (used in the standard solid-phase phosphoramidite procedure
[6]). The advantage of the S-[(MeO)2Tr] function as 5'-protecting group over the S-
trityl function is its higher lability against acid. Thus, for the 5'-S-[(MeO)2Tr]-protected
amidites during oligodeoxynucleotide synthesis, we can now apply the standard
detritylation solution also for the 5'-S-[(MeO)2Tr] cleavage. With aqueous heavy-
metal-ion solutions, we observed some problems during the oligodeoxynucleotide
synthesis in the synthesizer. Oxidation by an I2 solution and several CH2Cl2 washes used
during the synthesis gave rise to small amounts of free halogen resulting in a
detrimental precipitate in the tubings. In addition, working with aqueous AgNO3
solutions on CPG material made further reaction under anhydrous conditions more
difficult to proceed. For these reasons, most of the users do not carry out this cleavage
in the synthesizer but remove the column from the synthesizer, place a syringe on each
side of the column, and flush the aqueous heavy metal ion solutions between two
syringes. After this S-trityl cleavage, the resin (CPG material) is treated with
dithiothreitol solution (reduction) to ensure that no disulfide formation has occurred.
Then the column is washed, dried by several washes with CH2Cl2 and MeCN, and
placed back in the machine, and the synthesis can be continued.
The four modified amidites 5 and 15a c described here now allow the synthesis of
modified oligodeoxynucleotides by the solid-phase phosphoramidite procedure
completely in the synthesizer.
Results and Discussion. 1. Phosphoramidite Synthesis. To incorporate the 3' O
P S 5' linkage into 2'-deoxyoligonucleotides, the phosphoramidite solid-phase syn-
thesis was used. Therefore, we synthesized the four 5'-S-(4,4'-dimethoxytrityl)-2'-
deoxy-5'-thionucleoside 3'-(2-cyanoethyl diisopropylphosphoramidites) 5 and 15a c
(Fig. 1). For the 5'-S-(4,4'-dimethoxytrityl)-5'-thiothymidine 3'-(2-cyanoethyl diisopro-
pylphosphoramidite) (ˆ T amidite; 5), we found two different synthetic approaches
(see below, Schemes 1 and 2). But these two strategies were possible only for the Td
amidite 5, we were unable to synthesize the Ad amidite by these strategies, which
yielded a large amount of by-products, the major one being the depurinated nucleoside.
Fig. 1. Modified monomer building blocks 5 and 15a
c

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Helvetica Chimica Acta Vol. 87 (2004)
Thus, we needed a general method for the conversion of nucleosides into the
correspondending thiols (see below, Scheme 3).
The starting material for the synthesis of 5 by the strategy of Scheme 1 is thymidine
(1a). Treatment of 1a with TsCl gave the p-toluenesulfonate 2 (in 68% yield [7]),
which was converted in acceptable yield (58%) with potassium thioacetate in acetone
to compound 3 [8]. Using basic medium (NaOH, NaOMe, NH3 in MeOH) for the 5'-
deprotection of 3, we isolated the symmetric disulfide that was formed in high yield.
But when we used an acidic medium for deprotection (HCl in MeOH), the free 5'-thiol
was liberated [7], which reacted with 4,4'-dimethoxytrityl chloride ((MeO)2Tr-Cl) in
H2O and AcOH to give compound 4 in 68% yield, after purification by flash
chromatography [9]. The transformation of the 5'-S-acetyl-5'-thiothymidine 3 to the 5'-
S-(4,4'-dimethoxytrityl)-5'-thiothymidine (4) was achieved under conditions usually
applied for standard deprotection of the 5'-O-(4,4'-dimethoxytrityl) group. The greater
nucleophilicity of the SH in contrast to the OH group is the reason why we were
successful under these conditions. Treatment of 4 with 2-cyanoethyl diisopropylphos-
i
phoramidochloridite in the presence of Pr2NH at room temperature gave the
corresponding modified phosphoramidite 5 in 73% yield [1b].
The second method for the synthesis of Td amidite 5 (Scheme 2) started with the
same material, thymidine (1a). Reese and co-workers introduced 9-(4-methoxyphenyl)-
9H-xanthene-9-thiol (AXT) for inserting the thio modification preferentially in the
synthesis of 2'-thioadenosine [10]. We followed his method for the synthesis of our 2'-
deoxy-5'-thionucleosides 4 and 14 a c. Treatment of the 5'-OH group with CCl4 and
triphenylphosphine gave 6 in 73% yield [8]. The 5'-chloro-thymidine (6) reacted with 9-
(4-methoxyphenyl)-9H-xanthene-9-thiol (AXT) and 1,1,3,3-tetramethylguanidine in
70 95% yield to 5'-S-[9-(4-methoxyphenyl)-9H-xanthen-9-yl]-5'-thiothymidine (7)
[10]. The easiest way would be to phosphitylate this compound and to use it in an
automated synthesis via phosphoramidite chemistry. But we could not apply the
standard solution for detritylation (3% CHCl2COOH in CH2Cl2); the reactivity
towards this acid was too low. So we needed the 5'-S-[(MeO)2Tr]-protected amidites
for an automated synthesis. We converted the 5'-S-[9(4'-methoxyphenyl)-9H-xanthen-
9-yl]-5-thiothymidine (7) into the 5'-S-(4,4'-dimethoxytrityl)-5'-thiothymidine (4) [11]
by treatment with AgNO3 in MeOH and pyridine, trituration of the obtained Ag salt
with MeOH/AcOH prior to H2S bubbling ( ! 8; yield 68% after workup), and
treatment of the intermediate thiol 8 with (MeO)2TrCl in pyridine. The final
phosphitylation of compound 4 with 2-cyanoethyl diisopropylphosphoramidochloridite
in the presence of iPr2NEt at 48 gave the corresponding modified phosphoramidite 5 in
73% yield [1b].
The synthetic procedures according to Schemes 1 and 2 could not be applied to the
other amidites. Thus, we envisaged to prepare the four natural 5'-thionucleosides by
introducing the (MeO)2Tr group with the thiol reagent 12 according to Scheme 3. Since
we did not find thiol 12 in the literature, we synthesized it from 4,4'-dimethoxytriphen-
ylmethanol (11)1) which was obtained in turn by a Grignard reaction of p-bromoanisole
(9) and methyl benzoate (10) in THF. We converted 11 to methanethiol 12 under the
conditions used by Reese and co-workers for the synthesis of the AXT reagent [10].
1
)
First step: reaction to (MeO)2TrCl.

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Scheme 1. Synthesis of 5'-S-(4,4'-Dimethoxytrityl)-5'-thiothymidine 3'-(2-Cyanoethyl Diisopropylphosphor-
amidite) (5)

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Scheme 2. Synthesis of 5'-S-(4,4'-Dimethoxytrityl)-5'-thiothymidine 3'-(2-Cyanoethyl Diisopropylphosphor-
amidite) (5)

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 3. Synthesis of 5'-S-(4,4'-Dimethoxytrityl)-2'-deoxy-5'-thionucleoside 3'-(2-Cyanoethyl Diisopropyl-
phosphoramidites) 5 and 15a
c

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Helvetica Chimica Acta Vol. 87 (2004)
Then, the methanethiol 12 reacted with the different activated 5'-modified nucleosides
2, 6, or 13a e and 1,1,3,3-tetramethylguanidine to the 5'-S-(4,4'-dimethoxytrityl)-5'-
thionucleosides 4 and 14a c [10] (see Table 1). Subsequent phosphitylation (described
for compound 5) afforded the desired 3'-phosphoramidite building blocks 5 and 15a c
[1b]. The kind of leaving group (activation) to be used depended on the nucleoside and
the base-protecting group. The base protecting group has an effect on the solubility
and, therefore, on the reactivity too. In conclusion, we demonstrate that we can insert a
selective cleavage site at every position in an oligonucleotide.
Table 1. Yields of Reactions in Scheme 3
Nucleoside
5'-Activated nucleoside
5'-Thionucleoside
5'-Thionucleoside phosphoramidite
No.
B
No. 5'-leaving group X Yield [%] No.
Yield [%]
No.
Yield [%]
1a
1a
1a
1b
1b
1c
1d
T
T
T
6
2
Cl
TsO
72
66
64
39
79
48
93
4
4
4
14a
14a
14b
14c
63
94
97
94
41
82
45
5
5
5
15a
15a
15b
15c
73
72
13a MsO
13b TsO
13c MsO
13d MsO
13e MsO
bzA
bzA
bzC
ibuG
72
76
2. Oligonucleotides. 2.1. Synthesis. The oligonucleotides were synthesized on an
Expedite (PerSeptive Biosystems) or an ABI-392 synthesizer (Applied Biosystems) by
the phosphoramidite chemistry. Coupling times for modified amidites 5 and 15a c, 5'-
biotin amidite (from Glen Research), and 5'-Amino-Modifier C6 (from Glen Research)
were enhanced to 300 s. For 5 and 15a c, we used further modifications in the synthesis
cycle, described in Table 2. We changed the time for detritylation, and we needed an
additional reduction step (DTT (dithiothreitol) solution) after the detritylation. We
used tert-butyl hydroperoxide [12] or a lower concentrated I2 solution as oxidation
reagent for improved yields. Thiols, especially on highly loaded supports are very
reactive, so it is useful to minimize the time between the removal of the DTT solution
and the addition of the next phosphoramidite. The [5'(MeO)2Tr-on]-synthesized
oligonucleotides were deprotected (base protecting groups) and cleaved from the
controlled-pore-glass (CPG) support with ammonia at room temperature for 24 h.
¾
Crude DNA oligomers were purified by reversed-phase HPLC (Poros -R3 column, see
Exper. Part) with an eluting gradient of 0 25% MeCN in 0.1m (Et3NH)OAc (pH 7.0)
within 12 min (flow rate: 4 ml/min). Then the 5'-[(MeO)2Tr group was removed with
80% aqueous AcOH within 60 min. After evaporation of the acid, the residue was
diluted in 2m NH4OAc and precipitated with EtOH under ice cooling (2 h). The
precipitate was filtered off and dried. Fig. 2 shows the purity-control HPLC of
oligodeoxynucleotide 16 (modified model oligodeoxynucleotide). Pure oligonucleo-
tides were characterized by mass spectroscopy, and the detected masses were in good
agreement with the calculated values. Table 3 shows the characteristic data of the
oligodeoxynucleotides 16 23, 26, and 29 34.
The model oligodeoxynucleotide 16 (24mer) was synthesized to examine the
cleavage of the CÀS bond, where 5'-S-(4,4'-dimethoxytrityl)-5'-thiothymidine 3'-(2-

Helvetica Chimica Acta Vol. 87 (2004)
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Table 2. Standard Cycle and Modified Cycle for the Oligonucleotide Synthesis (ABI-394 synthesizer)
Step
Reagent
Standard cycle
Modified cycle
t [s]a)
wait time [s]
t [s]a)
wait time [s]
Detritylation
Reduction
Washing
3% CCl3COOH in CH2Cl2
50 mm DTT (dithiothreitol)
MeCN
125
20
375
60
60
60
12
10
8
60
600
23
CH2Cl2
Coupling
Capping
Oxidation
amidite ‡ 1H-tetrazole
Ac2O/NMI (1-methyl-1H-imidazole)/THF
I2/H2O/pyridine
12
10
8
25
30
15
300
30
15
a) Time reagent to column.
Fig. 2. Reversed-phase HPLC profile of modified oligodeoxynuleotide 16. Conditions, see Exper. Part.
cyanoethyl-diisopropylphosphoramidite (Td) 5 was inserted as a modified building
block. The sequence of the model oligodeoxynucleotide (24mer) 16 is:
5'-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
(16)
We carried out several tests for the cleavage, and the cleavage was detected by gel
chromatography for the oligodeoxynucleotides 17 and 21 23, by analytical HPLC for
oligodeoxynucleotide 16, on a chip for the oligodeoxynucleotides 21 and 22, and by
surface plasmon resonance spectroscopy (SPR) for the oligodeoxynucleotides 17 20.
The oligodeoxynucleotides 23, 30, and 32 34 were synthesized as controls for the
following experiments. Table 4 shows the synthesized oligodeoxynuleotides.
2.2. Cleavages. 2.2.1 Cleavage of Modified Oligonucleotides in Solution. First, we
evaluated specific chemical conditions for the cleavage of the C S internucleotide
bond by using heavy-metal ions. Therefore, the modified oligodeoxynucleotide was
diluted in H2O and was treated with aqueous 50 mm AgNO3. After 15 min at room

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Table 3. Characteristic Data of the Oligonucleotides 16 23, 26, and 29 31
Synthesis cyclea) Reversed-phase HPLC
gradient
MALDI- or ESI-MS
Thermal stability
No.
Tm [8]c)
yieldg)
calc.
exper.
16 modified
17 modified
18 standard
19 standard
20 standard
21 modified
22 standard
23 standard
26 modified
29 modified
30 standard
31 standard
32 standard
33 standard
34 standard
0
0
0
0
0
0
0
0
30% B in 10 minb) 66.1 OD (61.2%) 7309.8
30% B in 10 minb) 40.1 OD (64.4%) 7715.3
25% B in 10 minb) 60.7 OD (89.5%) 6110.0
25% B in 10 minb) 63.4 OD (52.3%) 6125.1
7331.94
7717.97
6112.35
6126.1
6175.71
7491.46
7475.64
7702.02
4672.8
4601.55
7702.02
7491.35
7475.64
4267.03
2961.42
17 ‡ 18 49.9
17 ‡ 19 55.0
17 ‡ 20 58.0
25% B 10 minb)
61.6 OD (48.5%) 6174.1
30 % B in 10 minb) 71.5 OD (84.3%) 7489.0
30% B in 10 minb) 72.7 OD (66%)
7475.94
d
d
)
)
30% B in 10 minb) 72.4 OD (58.2%) 7699.24
e
)
)
)
)
)
10 50% B in 25 minf) 32.6 OD (43%)
10 50% B in 25 minf) 46.9 OD (58%)
4672.24
4600.18
e
d
d
d
0
0
0
0
0
30% B in 10 minb) 72.4 OD (58.2%) 7699.24
30% B in 10 minb) 72.7 OD (84.3%) 7489.00
30% B in 10 minb) 72.7 OD (66%)
7472.94
30% B in 10 minb) 66.1 OD (75.5%) 4268.8
30% B in 10 minb) 35 OD (55.6%)
2963.0
a) See Table 1. b) Column PorosR3. B ˆ MeCN, in 0.1m (Et3NH)OAc. c) Buffer 0.2m NaCl, 10 mm NaH2PO4,
pH 7.4. d) Wait time for 5'-modification was 300 s. e) Post-labeling on CPG material. f) Column RP18; B ˆ
MeCN, in 0.1m (Et3NH)OAc. g) Yield of crude; OD ˆ optical density.
Table 4. Synthesized Oligodeoxynucleotides 16 25, and 27 34. Td ˆ modified 5'-thiothymidine unit; CPG ˆ
controlled-pore class linkage; AF ˆ 5-(acetamido)fluorescein
Oligodeoxynucleotide
16
17
18
19
20
21
22
23
24
25
27
28
29
30
31
32
33
34
5'-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-Biotin-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-d(GCA GCT AGA TAT ACC GTC AA)-3'
5'-d(GCT AGA TAT ACC GTC AAA GT)-3'
5'-d(GAT ATA CCG TCA AAG TAA GG)-3'
5'-Amino-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-Amino-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-d(ACG CCT TAC TTT GAC GGT ATA TCT)-3'
5'-[(MeO)2Tr]-S-d(TT GAC GGT ATA TCT-CPG)d-3'
5'-HS-d(TT GAC GGT ATA TCT-CPG)-3'
5'-[(MeO)2Tr]-S-d(TT TTT TTT TTT TTT-CPG)-3'
5'-HS-d(TT TTT TTT TTT TTT-CPG)-3'
5'-AF-d(TT TTT TTT TTT TTT)-3'
5'-Biotin-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-Cholesterol-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-Cholesterol-d(AGC CCT TAC TTT GAC GGT ATA TCT)-3'
5'-d(TT GAC GGT ATA TCT)-3'
5'-d(AGC CCT TAC T)-3'
temperature, 220 mm dithiothreitol in H2O was added, and the precipitated silver salt
was removed after 15 min by centrifugation. The supernatant was analyzed by
polyacrylamide-gel electrophorese (PAGE; 1 mm, 15% PAA, 7m urea, 200 V) for the
oligodeoxynucleotides 17 21, 31, and 32 (Fig. 3), and by reversed-phase HPLC (Gen-

Helvetica Chimica Acta Vol. 87 (2004)
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Fig. 3. Cleavage of the modified model oligodeoxynucleotide 17 and of 21 23 detected by PAGE. Conditions,
see Exper. Part. Lane 1: 33 (14mer) ‡ two markers; Lane 2: 34 (10mer); Lane 3: 32 (control); Lane 4: empty;
Lanes 5 and 6: 32 after treatment with AgNO3; Lane 7: 30 (control); Lanes 8 and 9: 30 after treatment with
AgNO3; Lane 10: 31 (control); Lanes 11 and 12: 31 after treatment with AgNO3; Lane 13: 17 (control); Lanes
14 and 15: 17 after treatment with AgNO3.
Pak Fax (column; eluent; see Exper. Part)) for the oligodeoxynucleotide 16 (Fig. 4).
We can show that 50 mm aqueous AgNO3 cleaves the model oligodeoxynucleotide
within 5 min completely.
2.2.2 Cleavage of Modified Oligonucleotides on a Chip. We synthesized the two
oligodeoxynucleotides 21 and 22 for immobilization on the surface of epoxy-modified
glass slides (Quantifoil Micro Tools GmbH, Jena, Germany). For selective cleavage of
the scissile bond, the oligodeoxynucleotides 21 and 22 were incubated for 30 min at
room temperature in 50 mm aqueous AgNO3. In experiments where a double-stranded
DNA hybrid was formed prior to the cleavage reaction, the ionic strength of the
cleaving solution was raised to keep the hybrid stable during the cleavage reaction. This
was done by adding aqueous NaNO3 solution (soluble Ag salt!) to a final concentration
of 1m.
2.2.3 Cleavage of Modified Oligonucleotides on a Biacore Chip. Biacore (Pharmacia
Biosensor, Sweden) takes advantage of surface plasmon resonance spectroscopy (SPR)
to measure reactions as a function of mass changes [13]; we used the unmodified CM5
sensor chip [14]. First we coupled streptavidin as ligand to the derivatized dextran
matrix located on the sensor-chip surface. Fig. 5 shows the construct for this
experiment. Streptavidin immobilized on the sensor-chip surface can be used to

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Helvetica Chimica Acta Vol. 87 (2004)
Fig. 4. Reversed-phase HPLC profile of the cleavage (modified model oligodeoxynucleotide 16). Conditions,
see Exper. Part.
Fig. 5. Principle of the experiment by Biacore measurement. See text. The nucleoside units A, C, G, and T stand
for Ad, Cd, Gd, and Td. Conditions, see Exper. Part.
capture biotinylated ligands. For that, we synthesized the biotinylated oligodeoxynu-
cleotide 17 (which contained the amidite 5, and biotin at the 5'-end).
The streptavidin biotin affinity is very high and serves to immobilize the modified
oligodexynucleotide 17 for the following hybridization and the subsequent cleavage.
The partly complementary oligodeoxynucleotides 18 20 were added in three different
experiments, and the successful hybridization with the biotinylated probe 17 at room
temperature was detected by mass change. Scheme 4 exemplifies the hybridization of
the oligodeoxynucleotides 17 and 18 and the following cleavage of the internucleotide
SÀC bond by AgNO3, and Table 5 shows the yield of hybridization of the
oligodeoxynucleotide 17 with 18 20 followed by cleavage with AgNO3. Here we
can demonstrate a successful hybridization and cleavage detected by Biacore measure-
ment at room temperature. Silver ions on the chip surface can be removed by an EDTA
solution.

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 4. Cleavage of the Modified Oligonucleotide 17 Detected by a Biacore Measurement. The nucleoside
units A, C, G, and T stand for Ad, Cd, Gd, and Td. Conditions, see Exper. Part.
Table 5. Data from the Biacore Experimenta). Conditions, see Exper. Part.
Streptavidin labeling Reaction with 17
Hybridization
Cleavage
RU abs.
RU abs.
RU rel.
No. RU abs. RU rel.
RU abs. RU rel.
22237.4
21684.5
19562.9
23943.8
23429.1
20578.0
1706.4
1674.6
1015.1
18
19
20
25007.3
24492.8
21327.0
2769.9 (78.7%) 23233.8
2738.3 (80.0%) 22766.0
1764.1 (92.2%) 20206.8
996.4 (85.9%)
1011.5 (83.4%)
643.9 (83.7%)
a) RU abs. ˆ response unit absorption.
2.3. Post-Labeling. For the post-labeling with 5-(iodoacetamido)-fluorescein (IAF)
(Scheme 5), we synthesized the two modified oligodeoxynucleotides 24 and 27. The
[(MeO)2Tr]-S group at the 5'-end of the oligodeoxynucleotides could be detritylated
with 3% CHCl2COOH in CH2Cl2. The oligodeoxythionucleotides 25 and 28 dimerized
quickly and had to be reduced prior to use. We used a 220 mm aq. DTT solution for this
reduction step. The DTT solution had to be fully removed before the next coupling
reaction (side reactions). This is the reason why we washed the CPG material with
H2O, MeOH, and MeCN. The free SH group was very reactive; thus, we minimized the
time between the removal of the DTT and the addition of 5-(iodoacetamido)-
fluorescein (IAF). The post-labeling on the CPG material saved time because the
washing steps (filtration) were fast and easy to perform. Thus, the CPG-oligonucleotide
was treated with the fluorescein derivate IAF at room temperature for 24 h. Then, we
washed the CPG material with MeOH and MeCN and cleaved the oligodeoxynucleo-

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Scheme 5. Post-Reaction Labeling of Oligonucleotide 25. CPG ˆ controlled-pore glass.
tides from the CPG support with ammonia at room temperature, for 1 h in the case of
29 and for 24 h in the case of 26 (cleavage and base deprotection). The crude modified
oligodeoxynucleotides were purified by reversed-phase HPLC (RP-18 column). Fig. 6
shows the HPLC profile for the post labeling of oligodeoxynucleotide 25 with 5-
(iodoacetamido)fluorescein (IAF). The free SH group of the oligodeoxynucleotides 25
and 28 reacted in acceptable yields to the oligodeoxynucleotides 26 (43%) and 29
(58%).
K. Jahn-Hofmann would like to thank the Fonds der Chemischen Industrie for a Ph.D. fellowship and
financial support, Dr. Eugen Uhlmann, Silvia Hein, and Lothar Hornung (Aventis) for laboratory space and a
nice working atmosphere, and the company Clondiag Chip Technologies (Jena) for a fruitful cooperation.
Experimental Part
General. All starting materials were obtained from Fluka or Aldrich, and were used without further
purification. Moisture-sensitive reactions were conducted in dried glassware under a positive pressure of dry Ar.
Dry MeCN (H2O < 30 ppm) for the phosphitylation reaction was purchased from PerSeptive Biosystems or
Biosolve. Oligodeoxynucleotide synthesis: Expedite-8909 synthesizer from PerSeptive Biosystems or ABI-392
synthesizer from Applied Biosystems. Oligodeoxynucleotide reagents: PerSeptive Biosystems and Glen
Research. Flash column chromatography (FC): silica gel 60 (40 63 mm) from Merck. TLC: silica gel 60 F254

Helvetica Chimica Acta Vol. 87 (2004)
2825
Fig. 6. HPLC Profile of the product of post labeling of oligodeoxynucleotide 25. Conditions, see Exper. Part.
¾
plates from Merck. Prep. HPLC: Poros OligoTM-R3 column (4.6 mm  100 mm) from Applied Biosystems
¾
(order-No. material 1-1339-03) or LiChroCart 250-10, RP-18e (5 mm) from Merck (order-no. 50257); eluent:
¾
buffer A ˆ 0.1m (Et3NH)OAc, buffer B ˆ MeCN. Anal. HPLC: EcoCART 125-3-RP-18 column (5 mm) from
Merck (order-no. 51232); eluent: buffer A ˆ 0.1m (Et3NH)OAc, buffer B ˆ MeCN; and Gen-Pak Fax column
(4.6 mm  100 mm) from Waters (order-no. 15490); eluent: buffer A ˆ 10 mm NaH2PO4 in MeCN/H2O 1:4
(v/v), buffer B ˆ 1.5m NaCl, 10 mm NaH2PO4 in MeCN/H2O 1:4 (v/v). Gel chromatography (PAGE): 1 mm,
15 cm, 15% PAA, 7m urea, 220 V. UV (for OD measurements): Varian Cary-1-UV/VIS spectrophotometer or
Hitachi U-1100 spectrophotometer: 10-mm cuvette. NMR: Bruker AMX-250 (1H) and Bruker AMX-400
(1H,31P) spectrometers; d in ppm, J in Hz; primed locants are given to the monosaccharide moiety and unprimed
ones to the nucleobase moiety. MS: PerSeptive Biosystems MALDI-TOF spectrometer Voyager DE; ESI ˆ
electrospray ionization.
5'-S-Acetyl-5'-thiothymidine (3). To MeCOSK (18 g, 124.8 mmol) and 2 (12 g, 30.3 mmol) was added
acetone (250 ml). The suspension was stirred under Ar at 508 for 6 h and then r.t. overnight. The suspension was
cooled (ice-water bath) before filtration, and the filtrate was evaporated. The residual oil was subjected to FC
(silica gel, CH2Cl2/MeOH (9 :1, v/v)): 3 (8.6 g, 94.5%). Colorless foam. TLC (CH2Cl2/MeOH 9 :1): Rf 0.37.
1H-NMR ((D6)DMSO): 11.03 (s, NH); 7.45 (d, HÀC(6)); 6.15 (t, HÀC(1')); 5.41 (d, OHÀC(3')); 4.1 (m,
HÀC(3')); 3.75 (m, HÀC(4')); 3.23 (m, 1 HÀC(5')); 3.1 (m, 1 HÀC(5')); 2.37 (s, Ac); 2.23 (m, 1 HÀC(2')); 2.07
‡
‡
(m, 1 HÀC(2')); 1.81 (s, M eÀC(5)). ESI-MS (pos.) 301.2 ([M ‡ H] , C12H17N2O5S ; calc. 301.33).
5'-S-(4,4'-Dimethoxytrityl)-5'-thiothymidine (4). a) A soln. of 3 (6 g, 20 mmol) in 1m HCl/MeOH (145 ml)
under Ar was stirred for 2 h at 458. The soln. was concentrated to 1/2 the volume and added to a soln. of AcOH
(40 ml), H2O (20 ml), and (MeO)2TrCl (3 g, 35.4 mmol). The soln. was stirred at r.t. for 3 h and then
concentrated to nearly 100 ml. After addition of H2O (200 ml), the pH was adjusted to 10 with 2m NaOH. The
mixture was extracted with CH2Cl2 (3 Â 150 ml), the combined org. phase washed with 5% aq. NaHCO3 soln.
(150 ml) and brine (200 ml), dried (Na2SO4), and evaporated, and the residual oil subjected to FC (silica gel,
CH2Cl2/MeOH 95 :5 ‡ 0.5% Et3N). 4 (7.61 g, 68%). Foam.
b) To a soln. of 8 (1.29 g, 5 mmol) in pyridine (20 ml) was added (MeO)2TrCl (2.03 g, 6 mmol) in pyridine
(5 ml) at r.t. The soln. was stirred overnight, and the reaction was quenched with MeOH. The mixture was
diluted with CH2Cl2 (200 ml) and washed with 5% aq. NaHCO3 soln. (100 ml), the aq. phase extracted with
CH2Cl2 (100 ml), the combined org. phase washed with brine (100 ml), dried (Na2SO4), and evaporated, and the
residual oil subjected to FC (silica gel, CH2Cl2/MeOH 95 :5 ‡ 0.5% Et3N): 4 (2.21 g, 79%). Foam.
c) Dry 1,1,3,3-tetramethylguanidine (0.14 ml, 1.11 mmol) was added dropwise to a stirred soln. of a 5'-X-
thymidine 2, 6, or 13a (X ˆ leaving group; 1 mmol; 2: 0.396 g; 6: 0.261 g; 13a: 0.320 g) and 4,4'-
dimethoxytriphenylmethanethiol (0.504 g, 1.5 mmol) in dry DMSO (10 ml) under Ar at r.t. After 3 h, cooled
CH2Cl2 (300 ml) was added, and the mixture was washed with sat. aq. NaHCO3 soln. (150 ml). The aq. layer was
extracted with CH2Cl2 (100 ml), the combined org. layer washed with H2O (4 Â 100 ml), dried (MgSO4), and

2826
Helvetica Chimica Acta Vol. 87 (2004)
evaporated, and the residue subjected to FC (silica gel, CH2Cl2/MeOH 97 :3): 4 (from 2, 0.52 g (94%); from 6,
0.356 g (63.6%); from 13a; 0.546 g (97.5%)). Foam. TLC (CH2Cl2/MeOH 9 :1): Rf 0.51. 1H-NMR (CDCl3):
7.22 7.43 (m, 9 arom. H); 6.83 (m, HÀC(6), 4 arom. H); 6.16 (t, HÀC(1')); 4.1 (m, HÀC(3')); 3.78 (m,
HÀC(4'), 2 MeO); 2.54 (m, 1 HÀC(5')); 2.5 (m, 1 HÀC(5')); 2.29 (m, 1 HÀC(2')); 2.06 (m, 1 HÀC(2')); 1.86 (s,
MeÀC(5)). ESI-MS (neg.): 560.2 ([M ‡ H]À C31H33N2O6SÀ; calc. 560.66).
5'-S-(4,4'-Dimethoxytrityl)-5'-thiothymidine 3'-(2-Cyanoethyl-Diisopropylphosphoramidite) (5). A soln. of
4 (0.56 g, 1 mmol) in MeCN (4 ml) and CH2Cl2 (4 ml) in the presence of iPr2NEt (0.424 ml, 3 mmol) was cooled
with ice, and 2-cyanoethyl diisopropylphosphoramidochloridite (0.219 g, 1.2 mmol) was added dropwise under
Ar. The reaction was quenched after 1 h at r.t. by adding BuOH (0.5 ml). The soln. was diluted with CH2Cl2
(100 ml), washed with 5% aq. NaHCO3 soln. (100 ml) and brine (100 ml), dried (Na2SO4), and evaporated and
the residual oil subjected to FC (short column of silica gel, CH2Cl2/MeOH 97:3): 5 (0.56 g, 73%). Foam. TLC
i
(CH2Cl2/MeOH 9 :1): Rf 0.65. 31P-NMR (CDCl3 ‡ 0.1% Pr2NEt) 149.55; 149.35. ESI-MS (pos.): 761.2 ([M ‡
‡
‡
H] , C40H50N4O7PS ; calc. 761.86).
5'-Chloro-thymidine (6). A soln. of thymidine (1a; 9.6 g, 40 mmol), PPh3 (14 g, 54 mmol), and CCl4 (20 ml,
200 mmol) in DMF (200 ml) was kept at r.t. for 24 h and then quenched with MeOH. The soln. was evaporated,
and the residue was crystallized from MeOH: pure 6 (7.56 g, 72.6%). TLC (CH2Cl2/MeOH 9 :1): Rf 0.38.
1H-NMR ((D6)DMSO): 11.38 (s, NH); 7.55 (d, HÀC(6)); 6.22 (t, HÀC(1')); 4.25 (m, HÀC(3')); 4.24 (m,
HÀC(4'), 2 HÀC(5')); 3.34 (d, OHÀC(3')); 2.32 2.21 (m, 1 HÀC(2')); 2.14 2.06 (m, 1 HÀC(2')); 1.8 (s,
‡
‡
MeÀC(5)). ESI-MS (pos.): 261.9 ([M ‡ H] ; C17H21N2O7S ; calc. 261.68).
5'-S-[9-(4-Methoxyphenyl)-9H-xanthen-9-yl]-5'-thiothymidine (7). Anh. 1,1,3,3-tetramethylguanidine
(2.42 ml, 18.92 mmol) was added dropwise to a stirred soln. of 6 (4.4 g, 17 mmol) and 9-(4-methoxyphenyl)-
9H-xanthene-9-thiol (AXT; 8.17 g, 25.5 mmol) in dry DMSO (150 ml) under Ar at r.t. After 3 h, cooled CH2Cl2
(1 l) was added, and the mixture was washed with sat. aq. NaHCO3 soln. (800 ml) (Caution!), the aq. layer
extracted with CH2Cl2 (200 ml), and the combined org. layer washed with H2O (4 Â 400 ml), dried (MgSO4),
and evaporated, and the residue subjected to FC (silica gel, CH2Cl2/MeOH 97:3): 7 (7.1 g, 95%). Foam. TLC
(CH2Cl2/MeOH 95 :5): Rf 0.183. TLC (CH2Cl2/MeOH 9 :1): Rf 0.46. 1H-NMR (CDCl3): 8.82 (s, NH); 7.38 7.35
(m, 2 arom. H); 7.2 7.02 (m, 7 arom. H); 6.93 6.9 (m, 2 arom. H); 6.81 6.78 (m, 2 arom. H); 6.06 6.01 (dd, 1
HÀC(1')); 3.86 3.82 (m, 1 HÀC(4')); 3.73 (s, MeO); 3.6 3.52 (m, HÀC3')); 2.21 2.11 (m, 2 HÀC(5')); 1.9
1.79 (m, 2 HÀC(2')); 1.84 (d, M eÀC(5)). ESI-MS (neg.): 543.3 ([M À H]À, C30H27N2O6SÀ; calc. 543.55).
4,4'-Dimethoxytriphenylmethanethiol (12). H2S was bubbled through a cooled (ice-water bath), stirred,
anh. soln. of CHCl2COOH (19.75 ml, 240 mmol) in CH2Cl2 (400 ml), while a soln. of 11 (38.44 g, 120 mmol) in
anh. CH2Cl2 (400 ml) was added dropwise during 1 h. After H2S had been bubbled through the mixture for a
further 15 min, the H2S gas was replaced by N2 gas for 15 min. Then the soln. was washed with sat. aq. NaHCO3
soln. (2 Â 1 l) and H2O (2 Â 1 l), dried (MgSO4), and evaporated and the residue subjected to FC (silica gel,
hexane/AcOEt 4 :1): 12 (36.33 g, 90%). Foam. TLC (hexane/AcOEt 3 :1): Rf 0.66. 1H-NMR (CDCl3): 7.24 7.06
‡
‡
(m, 9 arom. H); 6.75 6.69 (m, 4 arom. H); 3.72 (s, 2 MeO). MALDI-MS: 336.45 (M , C21H20O2S ; calc.
336.45).
5'-S-(4,4'-Dimethoxytrityl)-2'-deoxy-5'-thionucleosides 4 and 14a c). Dry 1,1,3,3-tetramethylguanidine
(0.142 ml, 1.11 mmol) was added dropwise to a stirred soln. of an activated 5'-X-nucleoside 13a d (X ˆ leaving
group, see Table 1; 1 mmol; 0.320 g, 0.509 g, 0.433 g, 0.409 g, and 0.415 g, resp.) and 4,4'-dimethoxytriphenyl-
methanethiol (0.505 g, 1.5 mmol) in anh. DMSO (10 ml) under Ar at r.t. After 3 h, cooled CH2Cl2 (300 ml) was
added, the mixture washed with sat. aq. NaHCO3 soln. (150 ml), the aq. layer extracted with CH2Cl2 (100 ml),
the combined org. layer washed with H2O (4 Â 100 ml), dried (MgSO4), and evaporated, and the residue
subjected to FC (silica gel, CH2Cl2/MeOH 97 :3): Foam.
Data of 14a: 0.63 g (93%) from 13b; 0.26 g (41%) from 13c. TLC (CH2Cl2/MeOH 9 :1): Rf 0.45. 1H-NMR
(CDCl3): 9.08 (s, NH); 8.65, 8.22 (s, HÀC(2), HÀC(8)); 7.93 7.92 (m, 2 Ho (bz)); 7.51 7.11 (m, 12 arom. H);
6.74 6.68 (m, 4 arom. H); 6.27 6.22 (m, HÀC(1')); 4.33 4.29 (m, HÀC(3')); 3.77 3.74 (m, HÀC(4')); 3.69 (s,
2 MeO); 2.74 2.36 (m, 2 HÀC(5'), 2 HÀC(2')). ESI-MS (neg.): 672.6 ([M À H]À, C38H34N5O5SÀ; calc. 672.77).
Data of 14b: 0.54 g (83%) from 13d. TLC (CH2Cl2/MeOH 9 :1): Rf 0.44. 1H-NMR (CDCl3): 8.17 8.15 (m,
HÀC(5), HÀC(6)); 7.86 7.04 (m, 2 Ho (bz), 14 arom. H ((MeO)2Tr); 7.81 6.77 (m, 2 arom. H); 6.06 6.01 (d, 1
HÀC(1')); 3.90 3.83 (m, HÀC(3'), HÀC(4')); 3.73 (s, 2 MeO); 2.57 2.29 (m, 2 HÀC(5'), 2 HÀC(2')). ESI-MS
(neg.): 643.4 ([M À Me]À, C34H34N5O6SÀ; calc. 640.73).
Data of 14c: 0.3 g, (45%) from 13e. TLC (CH2Cl2/MeOH 9 :1): Rf 0.43. 1H-NMR (CDCl3): 10.24 (s, NH);
7.66 (s, HÀC(8)); 7.29 7.02 (m, 9 arom. H); 6.75 6.62 (m, 4 arom. H); 5.98 5.93 (t, HÀC(1')); 4.98 (s,
OHÀC(3')); 4.63 (m, HÀC(3')); 4.09 4.01 (m, HÀC(4')); 3.64 (s, 2 MeO); 2.89 2.84 (m, M eCHCO);
2

Helvetica Chimica Acta Vol. 87 (2004)
2827
‡
2.53 2.45 (m, 2 HÀC(5')); 2.31 (m, 2 HÀC(2')); 1.21 1.16 (dd, Me2CHCO). ESI-MS (pos.): 656.1 ([M ‡ H] ,
‡
C35H38N5O6S ; calc. 656.76).
5'-S-(4,4'-Dimethoxytrityl)-2'-deoxy-5'-thionucleoside 3'-(2-Cyanoethyl Diisopropyl)phosphoramidite)
15a c. A soln. of 14a c (see Table 1; 1 mmol; 0.674 g, 0.650 g, and 0.655 g, resp.) in MeCN (4 ml) and CH2Cl2
i
(4 ml) in the presence of Pr2NEt (0.424 ml, 3 mmol) was cooled with ice, and 2-cyanoethyl diisopropylphos-
phoramidochloridite (0.219 g, 1.2 mmol) was added dropwise unter Ar. After stirring for 1 h at r.t., the reaction
was quenched by adding BuOH (0.5 ml). The soln. was diluted with CH2Cl2 (100 ml), washed with 5% aq.
NaHCO3 soln. (100 ml) and brine (100 ml), dried (Na2SO4), and evaporated and the residual oil subjected to FC
(short column of silica gel, CH2Cl2/MeOH 97:3): Foam.
Data of 15a: 0.59 g (68%). TLC (CH2Cl2/MeOH 9 :1): Rf 0.90. TLC (CH2Cl2/MeOH 95 :5): Rf 0.81, 0.77.
i
‡
‡
31P-NMR (CDCl3 ‡ 0.1% Pr2NEt): 149.99; 149.74. ESI-MS (pos.): 858.5 ([M À Me] , C46H49N7O6PS ; calc.
858.94).
Data of 15b: 0.65 g (76.4%). TLC (CH2Cl2/MeOH 9 :1): Rf 0.76, 0.67. 31P-NMR (CDCl3 ‡ 0.1% iPr2NEt):
‡
‡
150.43; 149.99. ESI-MS (pos.): 832.5 ([M À Me] , C45H49N5O7PS ; calc. 834.96).
Data of 15c: 0.62 g (72%). TLC (CH2Cl2/MeOH 9 :1): Rf 0.71. 31P-NMR (CDCl3 ‡ 0.1% iPr2NEt): 150.13;
‡
‡
148.93. ESI-MS (pos.): 856.6 ([M ‡ H] , C44H55N7O7PS ; calc. 857.6).
Cleavage of Oligodeoxynucleotide 16. A soln. of 1 A260 unit of oligodeoxynucleotide 16 in H2O (100 ml) was
treated with 50 mm aq. AgNO3 (20 ml) at r.t. for 15 min. Then 220 mm DTT in H2O (5 ml) was added, and the
precipitated silver salt was removed after 15 min by centrifugation. Aliquots (60 ml) were used for HPLC
analysis (Gen-Pak Fax 4.6 mm  100 mm); cf. General: 5% buffer B to 60% B in 30 min (Fig. 4).
Cleavage of Oligodeoxynucleotides 17 and 21 23. As described above for 16, with 1 A260 unit of
oligodeoxynucleotide, H2O (40 ml), 50 mm aq. AgNO3 (10 ml) and 220 mm DTT in H2O (2.5 ml). After
centrifugation, the soln. (10 ml) was analyzed by PAGE (1 mm, 15% PAA, 7m urea, 220 V) after adding 10 ml of
formamide (see Fig. 3).
Biacore Measurement (Biacore 1000). 1) Conditions for streptavidin immobilization (Chip CM 5): a)
Continuous-flow buffer HBS, flow rate 5 ml/min; b) 50 mm EDC/200 mm NHS, inject 35 ml (7 min); c)
streptavidin (50 mg/ml), inject 35 ml (7 min); d) 0.05% SDS, inject 35 ml (7 min).
2) Conditions for binding (a)), hybridization (b)), and cleavage (c) and d)): a) 25 ml of oligodeoxynucleo-
tide 17 (0.1 mg/ml); b) 25 ml of oligodeoxynucleotide 18 20 (500 nM); c) 2 Â 15 ml of 10 mm AgNO3; d) 2 Â 5 ml
of 0.5m EDTA. The results are shown in Table 5.
Immobilization and Cleavage of Oligonucleotides 21 and 22 on the Chip (Clondiag Chip Technology
GmbH; Jena, Germany). The oligonucleotides 21 (phosphorothioate oligonucleotide) and 22 (all-phosphate-
backbone oligonucleotide) were immobilized on the surface of epoxy modified glass slides (Quantifoil Micro
Tools GmbH; Jena, Germany). Both oligonucleotides carried a primary NH2 group at the 5'-terminus that was
introduced during synthesis by using a 5'-Amino-Modifier C6. The oligonucleotides were dissolved at a final
concentration of 10 mm in 0.5 mm sodium phosphate buffer (pH 8.0). The slides were diced in squares (3 Â 3 mm
in size). Three 0.2-ml droplets were applied by using a hand-held pipet (Eppendorf) to each square, representing
either the all-phosphate or the phosphorothioate oligonucleotide. The squares were incubated at r.t. until the
droplets were dried. Then the squares were incubated at 608 for 20 min, washed twice in 1.5-ml reaction vessels
(Eppendorf) with 50 mm aq. KCl and once with dist. H2O, and dried in a Speed Vac concentrator for storage.
For selective cleavage of the scissile linkage, the squares were incubated at r.t. for 30 min in 50 mm AgNO3.
In experiments where a double-stranded DNA hybrid was formed prior to the cleavage reaction, the ion
strength of the cleaving soln. was raised by adding aq. NaNO3 soln. to a final concentration of 1m to keep the
hybrid stable during the cleavage. After cleavage of the scissile bond, the squares were washed 3 times in dist.
H2O (immobilized single-stranded DNA) or in 1m aq. NaNO3 (DNA hybrids).
Post-labeling of Oligodeoxnucleotides 25 and 28. The (MeO)2Tr-on (1 mmol) synthesis by phosphoramidite
chemistry, and the final deblocking were carried out on the synthesizer (Table 2). Then the CPG material was
suspended in 220 mm DTT for 10 min and then washed with H2O and MeCN ( ! 25 or 28). A soln. of 1 mg of 5-
(iodoacetamido)fluorescein (IAF) in DMF, 100 ml of H2O and 10 ml of 1m Tris ¥ HCl (pH 8) were added to 25 (or
28), and the mixture was kept at r.t. for 24 h. The CPG 26 (or 29) was washed with H2O, CH2Cl2, and MeCN.
Conc. aq. ammonia was added for cleavage and deprotection (for 26: 24 h at r.t.; for 29: 1 h at r.t.). The labeled
¾
oligodeoxynucleotides 26 and 29 were purified by HPLC (LiChroCart -250-10 column, RP-18e (5 mm); cf.
General): 15% buffer B to 60% in 25 min (see Fig. 6). The labeled oligodeoxynucleotides were isolated in 43%
(26) and 58% (29) yield and characterized by mass spectra, see Table 3.

2828
Helvetica Chimica Acta Vol. 87 (2004)
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Received August 11, 2004