New protected protecting groups for the 5′-hydroxy group of deoxynucleosides by use of 2-(hydroxymethyl)- and 2-[(methylamino)methyl]benzoyl skeletons and oxidatively cleavable tritylthio and (4-methoxytrityl)thio groups

Article abstract of DOI:10.1002/hlca.200490207

The new protecting groups 1a,b and 2a,b were developed for the 5′-OH group of deoxynucleosides by utilizing the unique characters of the sulfenate and sulfenamide linkage. These new protecting groups have a 2-(hydroxymethyl) benzoyl or 2-[(methylamino)met

Full text of DOI:10.1002/hlca.200490207

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Helvetica Chimica Acta Vol. 87 (2004)
New Protected Protecting Groups for the 5'-Hydroxy Group of
Deoxynucleosides by Use of 2-(Hydroxymethyl)- and 2-
[(Methylamino)methyl]benzoyl Skeletons and Oxidatively Cleavable
Tritylthio and (4-Methoxytrityl)thio Groups
by Kohji Seioa)c), Eri Utagawab)c), and Mitsuo Sekine*b)c)
a) Frontier Collaborative Research Center, Tokyo Institute of Technology
b) Department of Life Science, Tokyo Institute of Technology
c) CREST, JST (Japan Science and Technology Corporation) 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501,
Japan
(phone: ‡81-45-924-5706; fax: ‡81-45-924-5772; e-mail: msekine@bio.titech.ac.jp)
The new protecting groups 1a,b and 2a,b were developed for the 5'-OH group of deoxynucleosides by
utilizing the unique characters of the sulfenate and sulfenamide linkage. These new protecting groups have a 2-
(hydroxymethyl)benzoyl or 2-[(methylamino)methyl]benzoyl skeleton whose hydroxy O-atom or amino N-
atom was blocked with a tritylthio-type substituent. They are removable by intramolecular cyclization following
the oxidative hydrolysis of the tritylthio-type substituents under mildly oxidative conditions (Schemes 3 and 6).
Among them, 2-{{[(4-methoxytrityl)sulfenyl]oxy}methyl}benzoyl (MOB; 2b) was found to be the most
preferable for protection of the 5'-OH function of deoxynucleosides. MOB can be introduced at the 5'-OH
groups of various deoxynucleosides without the protection of the 3'-OH functions (Scheme 5). The applicability
of the MOB group to a new oligodeoxynucleotide synthesis protocol without acid treatment was demonstrated
by the solid-phase synthesis of a tetrathymidylate (Scheme 8).
Introduction. Oxidatively cleavable protecting groups in place of the acid-labile
4,4'-dimethoxytrityl ((MeO)2Tr) group for the 5'-hydroxy function [1][2] have been
developed aiming at new oligonucleotide-synthesis protocols without acid treatment.
These new protocols suppress the unfavorable depurination promoted by the acid
treatment during the detritylation step in the current DNA synthesis [3 6]. Moreover,
if appropriately designed, use of such protecting groups can reduce the chemical steps
included in a single-chain-elongation cycle from the conventional four (coupling,
capping, oxidation, and detritylation) to three (coupling, capping, and oxidation/
deprotection) steps [1][2]. To date, there have been reported only two classes of
protecting groups that can be used for this strategy. One is phenoxycarbonyl-type
protecting groups reported by Caruthers×s group [1], and the other is the (4-
methoxytrityl)thio (MeOTrS) group [2] reported by us.
We have been interested in the MeOTrS group because of the unique property of
the single bond between the O- and divalent S-atom, which are cleavable under mild
oxidation conditions such as I2 solution in H2O/pyridine [2][7]. Moreover, the
MeOTrS-protected nucleoside could be converted to the corresponding nucleoside
phosphoramidite unit, which was successfully applied to the three-step oligodeoxynu-
cleotide synthesis without acid treatment [2]. The MeOTrS group was superior in this
respect to the (2,4-dinitrophenyl)thio [8][9] group (DNPS) reported previously as a
¹ 2004Verlag Helvetica Chimica Acta AG, Z¸rich

Helvetica Chimica Acta Vol. 87 (2004)
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sulfenate-type protecting group, because the 5'-O-DNPS-nucleoside phosphoramidite
units were reported to be unstable due to the inter- or intramolecular nucleophilic
attack of the trivalent P-atom of the 3'-phosphoramidite to the electron-deficient S-
atom [9].
In spite of these advantages of the MeOTrS group in the oligodeoxynucleotide
synthesis, the following problems still remained unsolved, impeding full benefit from
the fascinating nature of the sulfenate-type protecting group in the oligonucleotide
synthesis [2]. The first problem is that the introduction of the MeOTrS group at the 5'-
OH group requires the abstraction of the OH proton by a strong base such as lithium
hexamethyldisilazide. The second is that the selective activation of the 5'-OH group
requires the protection of the 3'-OH group by a base-stable protecting group such as
tBuMe2Si. Because of the former problem, it is difficult to introduce the MeOTrS group
at the 5'-position of nucleoside derivatives having a base-labile acyl-type protecting
group at their amino group. The latter leads to the time-consuming preparation of the
phosphoramidite via a multi-step procedure. Therefore, it is necessary to design new
protecting groups that can keep the favorable nature of the MeOTrS group unchanged
and allow its easy and selective introduction at the 5'-OH group.
To overcome these problems, we designed new protecting groups, 2-{[methyl(tri-
tylthio)amino]methyl}benzoyl (TAB); (1a), 2-{{[(4-methoxytrityl)thio]methylamino}-
methyl}benzoyl (MAB) (1b), 2-{[(tritylthio)oxy]methyl}benzoyl (TOB) (2a), and 2-
{{[(4-methoxytrityl)thio]oxy}methyl}benzoyl (MOB) (2b), in which a tritylthio (TrS)
or MeOTrS group was introduced to their amino N-atom or alcoholic O-atom.
These protecting groups were expected to be easily introduced at the 5'-OH
function as carboxylic acid esters and removable under mild conditions triggered by the
oxidative cleavage of the TrS or MeOTrS group with aqueous I2 solution followed by
the intramolecular cyclization, as shown in Scheme 1.
Scheme 1

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Such protecting groups cleavable by a two-step mechanism are called −protected
protecting groups [10][11] and the mechanisms are called −assisted cleavage× [12].
Some examples of protected protecting groups have been reported for the protection of
hydroxy or amino functions of nucleosides [10][11][13 15].
In this paper, we report the preparation and the properties of 1a,b and 2a,b as new
−protected protecting groups×, which can be cleaved by the intramolecular cyclization
triggered by the oxidative hydrolysis of the sulfenate or the sulfenamide linkage. Their
application to the oligoDNA synthesis without acid treatment was also examined by the
solid-phase synthesis of tetrathymidylate.
To the best of our knowledge, TAB (1a) is the first example of a protected
protecting group with a 2-[(methylamino)methyl]benzoyl skeleton that can be
successfully introduced at the 5'-OH group of nucleosides. Therefore, it is also
interesting to study the intrinsic properties of TAB as a model of 2-[(methylamino)-
methyl]benzoyl-containing protecting groups.
Results and Discussion. 1. Synthesis of TAB-OH (4a) and MAB-OH (4b). For the
synthesis of 4a and 4b, o-phthalaldehydic acid (ˆ2-formylbenzoic acid) was
quantitatively converted to the amino acid 3 (Scheme 2). This product had a
zwitterionic structure (IR: 1373 and 1553 cmÀ1 (COOÀ) and 2295 2818 cmÀ1
‡
(NH2Me ). Compound 3 was further converted to TAB-OH (4a) in 69% yield by
the reaction with TrSCl [7][16 22]. Since 3 was highly polar and insoluble in organic
solvents, this conversion was only successful when the reaction was carried out in
aqueous solvent systems such as acetone/THF/H2O 1:1:1 (v/v/v), which were slightly
modified systems compared to those reported by Branchaud [16][17].
Scheme 2
i) 1. MeNH2, MeOH/H2O, r.t., 1 h; 2. NaBH4, r.t., 30 min. ii) R1SCl, Na2CO3, acetone/THF/H2O 1:1:1.
Interestingly, TAB-OH (4a) had not a zwitterionic but a neutral structure (IR:
1690 cmÀ1 (CˆO, characteristic of COOH), no ammonium-ion absorption). This
difference in the structures of 3 and 4a suggested that the basicity of the N-atom of the
sulfenamide moiety in 4a is lower than that of the corresponding amine moiety in 3.
Bayfield and Cole [23] reported a pKa value of 2.82 for N-(phenylthio)aniline, which is
smaller by two orders than that for aniline (pKa ˆ 4.69).
The lower basicity could be due to the back-donation effect arising from the
molecular-orbital interaction between the vacant d-orbital of the S-atom and the lone-
pair of the amino N-atom. The effect of the tritylthio-type substituents (see 4a) to

Helvetica Chimica Acta Vol. 87 (2004)
2321
reduce the basicity of the N-atom might be weaker than that of the phenylthio groups
because of the absence of the delocalization of p-electrons over the aromatic ring.
However, it is possible that the steric hindrance of the trityl group provides an
additional contribution to lower the basicity of 4a to such an extent as to prohibit the
intramolecular salt formation at least in the solid state.
Next, we attempted to convert compound 3 to MAB-OH (4b) by treatment with
MeOTrSCl [2] according to a procedure similar to that described in the case of TAB-
OH (4a). Although the presence of 4b was detected in the reaction mixture by TLC, it
could not be isolated, probably because of the decomposition during the evaporation
and purification by column chromatography (CC; silica gel). These observations
indicated that the more acid-labile MeOTrS group was incompatible with the
neighboring carboxy group in MAB-OH (4b). Therefore, the chemically stable TAB-
OH (4a) was chosen as a reagent for the protection of the 5'-OH function of
nucleosides.
2. TAB Group for Protection of the 5'-Hydroxy Function. The introduction of TAB-
OH (4a) at the 5'-OH group of nucleosides was examined with 3'-O-[(tert-
butyl)dimethylsilyl]thymidine (TBDMS-T) as a model compound. Various combina-
tions of condensing agents, nucleophilic catalysts, and solvents were examined. As the
result, compound 5 was obtained in 64% yield by reaction of TBDMS-T with 4a in
pyridine in the presence of BOPCl (ˆ bis(2-oxooxazolidin-3-yl)phosphinic chloride)
and DMAP (ˆ N,N-dimethylpyridin-4-amine) (Scheme 3). It should be noted that our
protecting group TAB is the first example having a 2-[(methylamino)methyl]benzoyl
skeleton which could be successfully introduced at the 5'-OH group of nucleosides.
Previously, Christodoulou et al. [15] proposed two 2-(aminomethyl)benzoyl-type
protecting groups in which the reactivity of the N-atom was suppressed by the
introduction of a (2,4-dinitrophenyl)thio group, but the successful introduction at the
5'-OH groups of nucleosides has not been reported.
Scheme 3
In the preparation of 5, we observed the competitive self-cyclization of TAB-OH
(4a) during the reaction. Actually, 4a underwent cyclization when treated with BOPCl

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Helvetica Chimica Acta Vol. 87 (2004)
and DMAP in pyridine to give 2,3-dihydro-2-methyl-1H-isoindol-1-one in 63% yield
after 5 h. Although the basicity of the amino N-atom of 4a was lowered significantly by
the introduction of the TrS group as described above, the suppression of the
nucleophilicity of the N-atom of 4a was not efficient enough to prevent its
intramolecular cyclization when the neighboring carbonyl group was strongly activated
by the condensing agent. However, in contrast to the instability of 4a under the
coupling conditions, the condensation product 5 was stable during the purification.
Therefore, the deprotection of TAB was examined with 5.
First, removal of the TrS group of 5 was examined. When 5 was treated with 1.0m I2
in pyridine/H2O 9 :1 [2][7], compound 5 was immediately converted to a more-polar
material (TLC). Stirring the polar material in pyridine gave TBDMS-T in 82% yield
after 19 h (Scheme 3; see Exper. Part for details). This result suggested that TAB has a
potential as a new protecting group for the OH group removable under mild oxidation
conditions. The detailed mechanism of the oxidative cleavage of the sulfenamide
linkage in 5 is not evaluated yet. However, the generation of a trityl cation might be
involved as suggested by the fact that the MeOTrS group, which can generate the more-
stable MeOTr cation, was more labile than the TrS group towards I2 in MeCN/pyridine/
H2O [2]. The rather slow kinetics of the second-step reaction to liberate the 5'-OH
group suggested that the polar material detected as an intermediate was the ammonium
species 6, which is weakly nucleophilic toward the neighboring carbonyl group. If so,
the nucleophilic attack must be accelerated by neutralization of this ammonium
intermediate by addition of a base. As expected, when 1.0 equiv. of Et3N was added to
intermediate 6, the second-step cyclization reaction yielding TBDMS-Twas completed
within a minute.
Next, the stability of TAB was examined with 5 as a model compound. Compound 5
was stable for hours when dissolved and stirred at room temperature in MeCN. However,
5 gradually decomposed to give several materials when dissolved in pyridine so that the
intact 5 was recovered in only 39% yield after 17 h; the formation of TBDMS-T, 2,3-
dihydro-2-methyl-1H-isoindol-1-one and trityl alcohol was detected by TLC.
On the basis of these observations, we concluded that a protecting group having a 2-
[(methylamino)methyl]benzoyl skeleton like TAB has interesting characteristics as a
new protecting group for an OH group that can be deprotected with aqueous I2 solution
and Et3N. However, TAB itself was too labile for further applications because it was
unstable when dissolved in pyridine.
3. Synthesis of TOB-OH (8a) and MOB-OH (8b). To improve the above-
mentioned instability of TAB, we designed new protecting groups, 2-{[(tritylthio)oxy]-
methyl}benzoyl (2a; TOB) and 2-{{[(4-methoxytrityl)thio]oxy}methyl}benzoyl (2b;
MOB). These protecting groups have a hydroxymethyl substituent in place of the
(methylamino)methyl group of TAB at the position ortho to the carbonyl group. The
lower nucleophilicity of the hydroxy O-atom than that of the amino N-atom would
make TOB and MOB chemically more stable than TAB. Several 5'-OH protecting
groups with a 2-(hydroxymethyl)benzoyl skeleton have been reported, and the
possibility to use these protecting groups for the oligonucleotide synthesis without acid
treatment has also been suggested [9][13][14].
MOB-OH (8b) was synthesized by hydrolysis of phthalide with Bu4NOH according
‡
to a known procedure [13], and the resulting Bu4N salt 7 of 2-(hydroxymethyl)ben-

Helvetica Chimica Acta Vol. 87 (2004)
2323
‡
zoic acid was treated with NaH and then MeOTrSCl to give 8b as the Et3NH salt in
70% yield after CC (silica gel, eluent containing Et3N (Scheme 4). As expected, MOB-
OH (8b) was much more stable during the purification by CC (silica gel) than TAB-
OH, probably because of the lower nucleophilicity of the sulfenate-type O-atom than
the sulfenamide-type N-atom. Similarly, compound TOB-OH (8a) having a TrS group
instead of the MeOTrS group of MOB-OH (8b) was synthesized in 54% yield
(Scheme 4).
Scheme 4
i) Bu4NOH (1.0 equiv.), 30 min, 98%. ii) 1. NaH (1.5 equiv.), TrSCl or MeOTrSCl (1.5 equiv.), THF, 20 min; 2.
column chromatography (CHCl3/MeOH/Et3N 50 :1 :5); 54 % of 8a ¥ Et3N, 70% of 8b ¥ Et3N.
In contrast to the clear difference in stability between the sulfenamide derivatives
TAB-OH (4a) and MAB-OH (4b), it turned out that both 8a and 8b were chemically
stable enough during CC (silica gel) purification and on storage, independent of the
presence of a MeO group at the trityl moiety. Since the MeOTrS group was superior to
the TrS group as a OH-protecting group in terms of the ease of deprotection by dilute
aqueous I2 solution, we chose MOB for the further development as a new protecting
group.
Next, the condensation of MOB-OH (8b) with the 5'-OH function of TBDMS-T
was examined. In the case of 8b, the use of 4-methoxypyridine 1-oxide (MPO) [24
26], in place of DMAP, in combination with BOPCl proved to be the best and gave the
desired product 9a in 89% yield (Scheme 5, Table 1).
Scheme 5

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Helvetica Chimica Acta Vol. 87 (2004)
Table 1. Incorporation of MOB-OH (8b) at the 5'-Hydroxy Group of Nucleosides in the Presence of Condensing
Agents and Nucleophilic Catalysts
Product
Condensing agent
Catalyst (equiv.)
Time [h]
Yield [%]
9a
9b
9b
9b
9c
9d
9e
BOPCl
HBTU
HBTU
BOPCl
BOPCl
BOPCl
BOPCl
MPO (0.75)
DMAP (1.0)
DABCO (1.0)
MPO (0.75)
MPO (0.75)
MPO (0.75)
MPO (0.75)
15
36
48
15
13
13
13
89
39
31
71
76
66
69
We also examined the selective reaction of 8b with the nucleoside derivatives
where both the 5'- and 3'-OH groups were unprotected. The selectivity for the primary
over the secondary OH group is a quite important property of a protecting group for
OH functions. As expected, the condensation of unprotected thymidine with 8b in the
presence of BOPCl and MPO gave the desired product 9b in 71% yield with good
selectivity for the 5'-OH group (Table 1). The attempts to improve the selectivity and
reactivity by using other condensing agents such as HBTU [27 30] or other
nucleophilic catalysts such as DMAP or 1,4-diazabicyclo[2.2.2]octane (DABCO) [31]
gave less-satisfactory results because of the formation of an acid anhydride
intermediate having poor reactivity. Therefore, the combination of MPO and BOPCl
was applied to other deoxynucleoside derivatives such as N6-benzoyl-2'-deoxyadeno-
sine, N4-benzoyl-2'-deoxycytidine, and 2'-deoxy-N2-isobutyrylguanosine to give 9c
(76%), 9d, (66%), and 9e (69%), respectively (Scheme 5)
4. Stability and Deprotection Conditions of the MOB Group. The stability and
deprotection conditions of the MOB group were examined with 9a as a model
compound (Table 2). Treatment of 9a with 3% CCl3COOH in CH2Cl2 resulted in the
facile removal of the MeOTrS group and the formation of 5'-O-[2-(hydroxymethyl)-
benzoyl]thymidine. On the other hand, the protecting group showed considerable
stability to other reagents listed in Table 2. For example, Ac2O acetylated the base
moiety of 9a within 2.5 h; the intact starting material 9a was recovered quantitatively
after the hydrolysis of the acetyl group followed by CC (silica gel) purification. The
protecting group was also stable for hours towards several oxidizing agents, 0.5 0.6m
tBuOOH [32] and sulfurizing agents [33][34] (Table 2).
Table 2. Stability of the MOB Group under Various Conditions
Reagents 3% CCl3CO2H Ac2O/pyridine
0.5 0.6m
0.2m 1,2-benzodithiol- 0.5m N,N,N',N'-
in CH2Cl2
1 : 9, 0.1m DMAP tBuOOH/MeCN 3-one 1,1-dioxide
tetramethylthiouram
disulfide
Stability dec. < 1 min
stable (> 2.5 h)
stable (11 h)
stable (2.5 h)
stable (> 18 h)
In contrast, the MeOTrS group of 9a could be removed quite rapidly (<1 min) by
treatment with 0.1m I2 in pyridine/H2O 9 :1 (v/v) (Scheme 6). An attempt to purify the
partially deprotected product 10 failed because further cyclization proceeded on CC
(silica gel) purification to give a 7:2 mixture 10/TBDMS-T.

Helvetica Chimica Acta Vol. 87 (2004)
2325
Scheme 6
The crude mixture 10/TBDMS-T thus obtained was still useful to study the second-
step cyclization of 10 releasing TBDMS-T because the presence of TBDMS-T does not
affect the kinetics. The second-step deprotection was studied by the reaction of crude
10/TBDMS-T with 30 equiv. of various bases as catalysts (Table 3).
Previously, Kamaike et al. reported the cyclization of 2-(hydroxymethyl)-5-nitro-
benzoate derivatives, which were formed by the deprotection of the 5'-O-[2-
{(levulinyloxy)methyl]-5-nitrobenzoyl}ribonucleoside derivatives [14] by the action
of 0.5m 1H-imidazole in MeCN. Therefore, these conditions were tested first (Table 3).
When the crude product 10 was dissolved in 0.5m 1H-imidazole/MeCN, the cyclization
was accelerated, and 10 was hydrolyzed completely to TBDMS-T and phthalide within
3 h. Interestingly, the cyclization was accelerated significantly by adding a small amount
(10%, v/v) of H2O to this reaction mixture (Tcomp 40 min). A similar acceleration was
also observed when more-basic amines were used instead of 1H-imidazole. Interest-
ingly, while Et3N was a poorer catalyst than 1H-imidazole in MeCN, Et3N was more
efficient than 1H-imidazole when H2O was added (see Table 3). Among the amines
i
tested in this study, Pr2NEt was efficient and comparable to a much stronger base,
N,N,N',N'-tetramethylnaphthalene-1,8-diamine which is known as a proton sponge [35]
(Table 3).
Table 3. Kinetics of the Cyclization of Intermediate 10
i
Base
0.5m 1H- 0.5m 1H-
0.2m Et3N 0.2m Et3N
0.2m Pr2NEt
0.2m N,N,N',N'-
imidazole imidazole
tetramethylnaph-
thalene-1,8-diamine
Solvent MeCN
Tcomp 3 h
MeCN/H2O 9 : 1 MeCN
40 min 6.5 h
MeCN/H2O 9 : 1 MeCN/H2O 9 : 1 MeCN
< 2 min < 1 min < 1 min
5. MOB in Solid-Phase DNA Synthesis. As described above, the rate of the
i
deprotection achieved by Pr2NEt was so fast and comparable to the deprotection
kinetics of the (MeO)2Tr group which is the most-popular protecting group in current
DNA synthesis and removable within a minute by the acid treatment. Therefore, it was
important to test the possibility to use the MOB group in the solid-phase synthesis of
DNA. For this purpose, the synthesis of tetrathymidylate (T4) on CPG supports was
carried out with MOB phosphoramidite 11, which was synthesized from 9b (Scheme 7).

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Helvetica Chimica Acta Vol. 87 (2004)
Scheme 7
Initially, the time required for the cyclization (Step 3b in Scheme 8) on CPG solid
supports was checked by the T4 synthesis in which the thymidine residues at the second
and the last positions from the 3'-end were incorporated by using the conventional
(MeO)2Tr phosphoramidite building block and acidic deprotection of the (MeO)2Tr
group, and only the third residue was incorporated by using the MOB phosphoramidite.
In the chain elongation with the conventional (MeO)2Tr phosphoramidite building
block, the times for the coupling, the capping with Ac2O, and the treatment with
aqueous I2 solution for the oxidation of the trivalent P-intermediates and the removal
of the MeOTrS group, were set to 1, 2, and 2 min, respectively.
On the other hand, the reaction time for Step 3b in Scheme 8 was optimized by
performing the synthesis under several conditions. At first, on the basis of the liquid-
phase data shown in Table 3, the reaction time for Step 3b was set to 1 min. The time
periods for coupling, capping, and oxidation were identical to those for the (MeO)2Tr
phosphoramidite described above. The T4 thus synthesized on the CPG support was
deprotected and cleaved from the solid support by treatment with aqueous ammonia.
Unexpectedly, the reversed-phase HPLC analysis of the products revealed consid-
erable amounts of trithymidylate (T3), T4, and an unidentified product with a slower
retention time than T4 (Fig. 1,a). The unidentified product gave T3 after an additional
treatment with aqueous ammonia for 21 h (data not shown). This observation indicated
that the unidentified product was a modified tetrathymidylate 12 in which the 5'-OH
group of T3 and the phosphate group of Tp were linked via a 2-(hydroxymethyl)ben-
zoyl linker (see Fig. 1,a). When the time period for Step 3b was prolonged to 5 min, no
trace of 12 could be detected by reversed-phase HPLC (Fig. 1,b).
However, the same HPLC profile also revealed that the crude product contained
95% (HPLC) of T4 and 5% of T3. Assuming that the coupling yield of the conventional
(MeO)2Tr phophoramidite building block was usually more than 99%, this result
suggested that the coupling yield of the MOB phosphoramidite was ca. 95%, which is
too low when DNA is synthesized by use of only the MOB phosphoramidite.
Therefore, we prolonged the coupling time to 2 min, which was long enough to
achieve a coupling yield of more than 99%, as judged from the HPLC profiles of
thymidylyl(3'-5')thymidine (TpT) synthesized by the reaction with CPG-bounded
thymidine and MOB phosphoramidite (data not shown), and carried out the chemical
synthesis of T4 on the CPG support with the MOB phosphoramidite without using the
(MeO)Tr phosphoramidite unit and the acid detritylation step.
Fig. 2 shows the HPLC profile of T4 which was synthesized by using only the MOB
phosphoramidite. The desired product T4 was successfully obtained as the main

Helvetica Chimica Acta Vol. 87 (2004)
2327
Scheme 8
Step 1: 11, 1H-tetrazole/MeCN, 2 min. Step 2: Ac2O/pyridine 1:9, 0.1m DMAP, 2 min. Step 3a: 0.1m I2, pyridine/
i
H2O 9 :1, 2 min. Step 3b: 0.2m Pr2NEt, MeCN/H2O 9 :1
Fig. 1. Reversed-phase HPLC profiles of T4 synthesized by a combination of conventional (MeO)2Tr-
phosphoramidite and MOB-phosphoramidite 11 for a time period of a) 1 min and b) 5 min for Step 3b in
Scheme 8, resp.
product. No detectable by-products like 12 were observed so that the complete removal
of the MOB group and chain elongation without acid treatment could be performed on
the solid support.
The coupling yield of the MOB phosphoramidite was estimated to be 96%. The
yield was comparable to the coupling yields reported by Guzaev and Manoharan [36]
for the elongation of the nucleotide chain in the presence of unprotected internucleo-

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Helvetica Chimica Acta Vol. 87 (2004)
Fig. 2. Reversed-Phase HPLC profiles of T4 synthesized without acid treatment by use of MOB-phosphoramidite
11
tidic phosphates. Although tertiary amines such as iPr2NEt have been recognized to be
less reactive towards the 2-cyanoethyl group at the phosphate moiety than primary
i
amines [37], use of excess Pr2NEt in the solid-phase synthesis might cause partial or
full deprotection of the 2-cyanoethyl group.
Conclusions. The new protecting groups TAB (1a) and MOB (2b) reported in this
study possess several unique characteristics that are potentially useful for oligonucleo-
tide synthesis and other organic synthesis.
To the best of our knowledge, TAB is the first example with a 2-[(methylamino)-
methyl]benzoyl skeleton and can be introduced successfully at the 5'-OH group of
thymidine. The use of the 2-[(methylamino)methyl]benzoyl group became possible
due to the inactivation of the nucleophilicity of the amino group by the introduction of
a tritylthio group at its N-atom. Although the suppression of the nucleophilicity was not
sufficient for the general application as described above, further structural modification
on either the substituent at the N-atom or the tritylthio group might overcome this
problem.
MOB (2b) was more promising because of the stability and the facile removal by
the 0.1m I2/pyridine/H2O treatment followed by the cyclization catalyzed by weak base.
These properties must be preferable for a protecting group in the synthesis of organic
molecules having OH functions such as sugar and nucleic acid derivatives. As an
example, we applied MOB protection to oligonucleotide synthesis without acid
treatment and achieved the synthesis of tetrathymidylate avoiding any acid treatment.
Currently, complete and facile removal of the MOB group requires treatment with
i
trialkylamines such as Pr2NEt in the presence of a small amount of H2O. The
conditions are somewhat problematic when MOB protection is applied to longer
oligodeoxynucleotides, probably because of the partial elimination of the 2-cyanoethyl
groups from the internucleotidic phosphate units.
We previously observed the different rate of the acid-catalyzed hydrolysis of the
(MeO)2Tr groups on oligoDNA terminal depending on the surface conditions and
chemicals of the solid support [38]. If this is also the case for the elimination of 2-

Helvetica Chimica Acta Vol. 87 (2004)
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cyanoethyl groups under basic conditions, the use of another solid support such as
highly cross-linked polystyrene [39][40] might suppress the unfavorable side reactions.
More-straightforward approaches to avoid the above-mentioned problem are to
design new protecting groups which can be removed under milder nonaqueous basic or
neutral conditions. For example, Kamaike et al. [14] reported the 2-(hydroxymethyl)-5-
nitrobenzoyl skeleton as a component of their 5'-OH protecting group for RNA
synthesis, and Shafer et al. [41] reported the 2-(1-hydroxy-1-phenylethyl)benzoyl
skeleton which cyclized 2700 times faster than the unsubstituted 2-(hydroxymethyl)-
benzoyl skeleton under certain conditions. These substituted 2-(hydroxymethyl)ben-
zoyl groups are easily combined with our tritylthio-type protecting groups. Such
molecular designs considering both the electronic and steric factors should be pursued
in the future. Further studies in this direction are now under way and will be reported
elsewhere.
This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (C) −Medical Genome
Science× from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by CREST of
JST (Japan Science and Technology Corporation).
Experimental Part
General. Abbreviations: BOPCl, bis(2-oxooxazolidin-3-yl)phosphinic chloride; DMAP, N,N-dimethylpyr-
idin-4-amine; MPO, 4-methoxypyridine 1-oxide. Pyridine was distilled after being refluxed for several hours,
redistilled from CaH2, and stored over 4-ä molecular sieves. HPLC: mBondapak column (Waters, C18-100),
linear gradient of 0 30% MeCN in 0.1m NH4OAc (pH 7.0) for 30 min at 508C, flow rate 1.0 ml/min. Column
chromatography (CC): NH silica gel from Fuji Silysia Chemical Ltd. 1H- and 13C-NMR Spectra: at 500 and
67.8 MHz, resp.; SiMe4 as an internal standard in CDCl3; d in ppm, J in Hz. ESI-TOF-MS: positive-ion mode;
calibration with reserpine and dioctyl phthalate.
2-[(Methylamino)methyl]benzoic Acid (3). To a soln. of O-phthalaldehydic acid (50 g, 0.33 mol) in MeOH
(670 ml) was added 40% MeNH2 in H2O (67 ml, 0.67 mol), and the resulting soln. was stirred at r.t. After 1 h,
NaBH4 (5.0 g, 0.18 mol) was added portionwise, and the soln. was stirred for an additional 30 min. The reaction
was quenched by addition of acetone (30 ml), and the solvent was evaporated. The residue was trituated with
acetone, and the precipitation was collected by filtration to give 3 (54g, 98%). 1H-NMR (500 MHz, D2O): 2.48
‡
(s, Me), 3.99 (s, 1 H, CHs); 7.37 7.28 (m, 3 arom. H); 7.57 (m, 1 arom. H). ESI-MS: 166.0581 (C9H12NO2 , [M ‡
‡
H] ; calc. 166.0868).
2-{[Methyl(tritylthio)amino]methyl}benzoic Acid (4a). To a soln. of 3 (165 mg, 1 mmol) and Na2CO3
(212 mg, 2 mmol) in H2O acetone 2 :1 (6 ml) was added tritylsulfenyl chloride (311 mg, 1 mmol) in THF/
acetone 2 :1 (6 ml) within 10 min, and the resulting mixture was stirred at r.t. for an additional 10 min. Na2CO3
was removed by filtration, and the filtrate was diluted with CHCl3 (50 ml). The CHCl3 soln. was washed twice
with sat. Na2HPO4 soln. (50 ml) and evaporated, the residual H2O was removed by repeated co-evaporation
with acetone and CHCl3. and the residue subjected to CC (silica gel): 4a (300 mg, 69%). 1H-NMR (500 MHz,
CDCl3): 2.46 (s, Me); 4 .03 (s, CH2); 7.46 7.13 (m, 18 arom. H); 7.94( m, 1 arom. H (Bz)). 13C-NMR (500 MHz,
CDCl3): 46.6; 63.0; 66.9; 72.0; 126.4 131.8; 140.6; 144.1; 146.9; 146.9; 172.1. Anal. calc. for C28H25NO2S ¥ 7/
5 H2O: C 72.36, H 6.03, N 3.01, S 6.90; found: C 72.23, H 5.59, N 3.08, S, 6.58.
3'-O-[(tert-Butyl)dimethylsilyl]-5'-O-{2-{[methyl(tritylthio)amino]methyl}benzoyl}thymidine (5). 3'-O-
[(tert-Butyl)dimethylsilyl]thymidine (356 mg, 1.0 mmol) and 4a (440 mg, 1.0 mmol) were rendered anhydrous
by repeated co-evaporation with dry pyridine. To the nucleoside soln. in 1,4-dioxane (2 ml) were added
successively the anh. 4a, DMAP (122 mg, 2.0 mmol), and BOPCl (255 mg, 1.0 mmol). After 2 h stirring at r.t.,
the mixture was quenched by addition of sat. NaHCO3 soln. (5 ml). The mixture was extracted with AcOEt
(15 ml), the org. layer washed with sat. aq. NaHCO3 soln. (3 Â 15 ml), the combined org. extract evaporated,
and the residue subjected to CC (silica gel, hexane/AcOEt 8 :2): 5 (500 mg, 64%). 1H-NMR (500 MHz, CDCl3):
t
0.07 (s, Me2Si); 0.88 (s, BuSi); 1.60 (s, MeÀC(5)); 2.07 (m, 1 HÀC(2')); 2.31 (m, 1 HÀC(2'')); 4.13 (m,
HÀC(3')); 4.23 (s, CH2N); 4.36 4.46 (m, HÀC(4'), 2 HÀC(5')); 6.26 (t, J ˆ 6.5, HÀC(1')); 7.18 7.43 (m, 18
arom. H, HÀC(6)); 7.58 (d, J ˆ 6.5, 1 arom. H (Bz)); 8.38 (s, NH(3)). 13C-NMR (500 MHz, CDCl3): À 4.9;

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Helvetica Chimica Acta Vol. 87 (2004)
À 4.7; 12.3; 17.9; 25.7; 41.0; 46.5; 62.6; 63.7; 71.9; 72.1; 84.7; 85.1; 111.1; 126.7; 130.3; 132.4; 135.0; 141.0; 144.1;
150.0; 163.3; 166.7. Anal. calc. for C44H51N3O6SSi ¥ H2O: C 66.39, H 6.71, N 5.27, S 4.02; found: C 66.57, H 6.42,
N 5.33, S 4.00.
Deprotection of 5 without Adding Et3N. Compound 5 (155 mg, 0.2 mmol) was dissolved in 2 ml of 1m I2 in
pyridine/H2O 9 :1 (v/v). After 1 min, the reaction was quenched by addition of 1m aq. Na2S2O3 (2.0 ml). The
soln. was diluted with H2O (10 ml) and extracted with CH2Cl2 (15 ml), the org. layer concentrated at r.t., and the
resulting pyridine soln. stirred at r.t. for 19 h. Then the remaining pyridine was evaporated and the residue
subjected to CC (silica gel, hexane/AcOEt 1 :1): TBDMS-T (61.8 mg, 82%).
Deprotection of 5 in the Presence of Et3N. Compound 5 (163 mg, 0.21 mmol) was dissolved in 2.1 ml of 1m I2
in pyridine/H2O 9 :1. After 1 min, the reaction was quenched with 1m aq. Na2S2O3 (4.0 ml). The soln. was diluted
with H2O (10 ml) and extracted with CH2Cl2 (15 ml) and the org. layer concentrated at r.t.. To the resulting
pyridine soln. was added Et3N (0.21 mmol), and the mixture was stirred at r.t. for 1 min. After evaporation, the
residue was subjected to CC (silica gel, hexane/AcOEt 1 :1): TBDMS-T (59 mg, 79%).
Triethylammonium 2-{[(Tritylthio)oxy]methyl}benzoate (8a). Tetrabutylammonium 2-(hydroxymethyl)-
benzoate (394mg, 1.0 mmol) was suspended in THF (5 ml), and the mixture was heated to 47 8 to give a clear
soln. NaH (60 mg, 1.5 mmol; 60% in oil) was added, and the resulting suspension was stirred at 478 for 15 min.
The mixture was cooled to r.t., and to this soln. was added tritylsulfenyl chloride (443 mg, 1.5 mmol). After
20 min stirring, the mixture was quenched by addition of conc. ammonia (2 ml), and the soln. was partitioned
between CH2Cl2 (100 ml) and aq. NaHCO3 soln. (100 ml). The org. layer was washed twice with aq. NaHCO3
soln. (100 ml), dried (MgSO4), and evaporated, and the residue purified by CC with a two-layer column (silica
gel (30 g, upper layer), NH silica gel (15 g, lower layer), CHCl/MeOH/Et3N 100 :2 :10): 8a (268.4mg, 54%).
1H-NMR (500 MHz, CDCl3): 1.27 (t, J ˆ 7.0, 3 MeCH2); 3.03 (m, 3 MeCH2); 4.78 (s, CH2O); 7.14 7.38 ( m, 18
arom. H); 7.80 (m, 1 arom. H (Bz)). 13C-NMR (126 MHz, CDCl3): 8.4; 44.9; 71.9; 78.6; 127.0; 127.1; 127.7; 127.7;
129.8; 129.9; 130.0; 133.4; 138.2; 142.8; 172.1. Anal. calc. for C 27H22O3S ¥ Et3N ¥ H2O: C 72.63, H 7.20, N 2.57,
S 5.88; found: C 72.69, H 6.66, N 2.35, S 6.13.
Triethylammonium 2-{{[(4-Methoxytrityl)thio]oxy}methyl}benzoate (8b). As described for 8a, with (4-
methoxytrityl)sulfenyl chloride (510 mg, 1.5 mmol) instead of tritylsulfenyl chloride: 8b (391 mg, 70%).
1H-NMR (500 MHz, CDCl3): 1.27 (t, J ˆ 7.0, 3 MeCH2); 3.05 (q, J ˆ 7.0, 3 MeCH2); 3.72 (s, MeO); 4.76 (s, CH2);
6.77 (dd, 3J(H,H) ˆ 2.5, 3.8, 2 H, MeOTr); 7.20 7.37 (m, 15 arom. H); 7.84( dd, 1 arom. H (Bz)). 13C-NMR
(500 MHz, CDCl3): 8.5; 45.3; 55.1; 71.6; 78.6; 113.2; 125.2 131.2; 134.4; 139.4; 143.3; 158.6; 171.8. Anal. calc. for
C28H24O4S ¥ Et3N ¥ 4 H2O: C 66.08, H 7.56, N 2.34, S 5.35; found: C 65.91, H 6.03, N 2.29, S 5.16.
General Procedure for 9a e. 3'-O-[(tert-Butyl)dimethylsilyl]-5'-O-{2-{{[(4-methoxytrityl)thio]oxy}me-
thyl}benzoyl}thymidine (9a). The 3'-O-[(tert-butyl)dimethylsilyl]thymidine (254mg, 0.71 mmol), MOB-OH
(8b; 596 mg, 1.07 mmol), and MPO hydrate (100.2 mg, 0.801 mmol) were rendered anhydrous by repeated co-
evaporation with dry pyridine, and each material was dissolved in 2, 5, and 5 ml of pyridine, resp. To the soln. of
3'-O-[(tert-butyl)dimethylsilyl]thymidine were added successively the pyridine soln. of 8b and MPO, and finally
BOPCl (227 mg, 1.07 mmol) was added. After 15 h stirring at r.t., the mixture was quenched by addition of H2O
(5 ml). The soln. was diluted with AcOEt (50 ml) and washed with aq. NaHCO3 soln. (3Â). The org. layer was
dried (MgSO4) and evaporated and the residue subjected to CC (NH silica gel, hexane/AcOEt 3 :7): 9a
1
t
(366 mg, 89%). H-NMR (500 MHz, CDCl3): 0.069 (s, Me2Si); 0.88 (s, BuSi); 1.57 (s, MeÀC(5)); 2.08 (m, 1
HÀC(2')); 2.31 (m, 1 HÀC(2')); 3.77 (s, MeO); 4.15 (dd, J ˆ 3.5, 7.5, HÀC(3')); 4.39 4.47 (m, HÀC(4')
2 HÀC(5')); 4.66 (s, CH2); 6.27 (t, J ˆ 6.5, HÀC(1')); 6.80, (m, 2 H MeOTr); 7.16 7.45 (16 H, m, 15 arom.
HÀC(6)); 7.76 (d, J ˆ 7.0, 1 arom. H (Bz)); 8.21 (s, NH(3)). 13C-NMR (500 MHz, CDCl3): À 4.9; À 4.7; 12.2;
17.9; 25.7; 41.0; 55.2; 63.8; 71.8; 72.0; 77.9; 84.7; 85.1; 111.1; 113.2; 127.2 135.0; 140.3; 143.1; 149.9; 158.7; 163.2;
166.1. Anal. calc. for C44H50N2O8SSi ¥ H2O: C 65.00, H 6.45, N 3.45, S 3.94; found: C 65.10, H 6.35, N 3.58, S 3.76.
5'-O-{2-{{[(4-Methoxytrityl)thio]oxy}methyl}benzoyl}thymidine (9b). With thymidine (259 mg,
1.07 mmol), 8b (900 mg, 1.61 mmol), and MPO hydrate (100.7 mg, 0.81 mmol): 9b (516 mg, 71%). 1H-NMR
(500 MHz, CDCl3): 1.53 (s, MeÀC(5)); 2.09 (m, 1 HÀC(2')); 2.44 (m, 1 HÀC(2')); 3.74( s, MeO); 4.22 (dd, J ˆ
3.5, 7.0 HÀC(3')); 4.57 (m, HÀC(4'), 2 HÀC(5'), CH2); 6.3 (t, J ˆ 6.5 Hz, HÀC(1')); 6.79 (d, J ˆ 8.5, 2 H,
MeOTr); 7.18 7.4( m, 15 arom. H, HÀC(6)); 7.74( d, J ˆ 26.5, 1 arom. H (Bz)); 10.00 (s, NH(3)). 13C-NMR
(500 MHz, CDCl3): 12.0; 40.2; 55.1; 64.0; 71.2; 71.6; 77.8; 84.3; 84.9; 111.2; 113.2; 127.2 135.1; 139.9; 143.0;
150.6; 164.0; 166.3. Anal. calc. for C38H36N2O8S ¥ H2O: C 65.32, H 5.48, N 4.01, S 4.59; found: C 65.62, H 5.60,
N 3.70, S 4.47.
N6-Benzoyl-2'-deoxy-5'-O-{2-{{[(4-methoxytrityl)thio]oxy}methyl}benzoyl}adenosine (9c): With-N6-benzo-
yl-2-deoxyadenosine (710 mg, 2.0 mmol), 8b (1.7 g, 3.0 mmol), and MPO hydrate (188 mg, 1.5 mmol): 9c (1.3 g,
76%). 1H-NMR (500 MHz, CDCl3): 2.57 (m, 1 HÀC(2')); 2.93 (m, 1 HÀC(2')); 3.76 (s, MeO); 4.28 (m,

Helvetica Chimica Acta Vol. 87 (2004)
2331
HÀC(3')); 4.49 4.79 (m, HÀC(4'), 2 HÀC(5'), CH2); 6.42 (t, J ˆ 6.0, HÀC(1'); 6.80 (d, J ˆ 9.0, 2 arom. H);
7.21 7.59 (m, 18 arom. H, 2 H); 7.71 (d, J ˆ 8, 1 arom. H); 7.99 (d, J ˆ 9.5, 2 arom. H); 8.91 (s, HÀC(2)); 8.64( s,
HÀC(8)). 13C-NMR): 14.2; 21.0; 39.6; 55.2; 60.4; 63.8; 71.5; 71.7; 78.2; 84.7; 84.8; 113.3; 123.4; 127.3 134.3;
139.8; 141.7; 143.1; 143.1; 149.5; 151.2; 152.5; 158.7; 166.5. Anal. calc. for C45H39N5O7S ¥ 2 H2O: C 65.13, H 5.22,
N 8.44, S 3.86; found: C 64.74, H 4.99, N 8.04, S 3.74.
N4-Benzoyl-2'-deoxy-5'-O-{2-{{[(4-methoxytrityl)thio]oxy}methyl}benzoyl}cytidine (9d). With N4-benzoyl-
2'-deoxycytidine (662 mg, 2.0 mmol), 8b (1.7 g, 3.0 mmol), and MPO hydrate (188 mg, 1.5 mmol): 9d (1.0 g,
66%). 1H-NMR (500 MHz, CDCl3): 2.16 (m, 1 HÀC(2')); 2.72 (m, 1 HÀC(2')); 3.76 (s, MeO); 4.29 4.71 (m,
HÀC(3'), HÀC(4'), 2 HÀC(5'), CH2); 6.246 (t, J ˆ 6.0, HÀC(1')); 6.794( d, J ˆ 9.0, 2 arom. H); 7.21 7.58
(19 H, m, 17 arom. H, HÀC(5), HÀC(6)); 7.76 (d, J ˆ 7.0, 1 arom. H (Bz)); 7.87 (d, J ˆ 7.5, 2 arom. H (Bz)); 8.06
(d, J ˆ 7.0, 1 arom. H (Bz)). 13C-NMR (500 MHz, CDCl3): 14.2; 21.0; 41.4; 55.2; 60.4; 63.5; 70.5; 71.7; 78.1; 84.9;
87.2; 96.7; 113.3; 127.3 134.2; 139.9; 143.1; 143.1; 144.0; 158.7; 162.3; 166.5. Anal. calc. for C44H39N3O8S ¥
1.5 H2O: C 66.32, H 5.31, N 5.27, S 4.02; found: C 66.41, H 5.07, N 5.17, S 3.95.
2'-Deoxy-N2-isobutyryl-5'-O-{2-{{[(4-methoxytrityl)thio]oxy}methyl}benzoyl}guanosine (9e). With 2'-de-
oxy-N2-isobutyrylguanosine (675 mg, 2.0 mmol), 8b (1.7 g, 3.0 mmol), and MPO hydrate (188 mg, 1.5 mmol): 9e
(1.1 g, 69%). 1H-NMR (500 MHz, CDCl3): 1.24( d, J ˆ 6.3, 3 H, Me2CHCO); 1.27 (3 H, d, J ˆ 6.3, 3 H,
Me2CHCO); 2.52 (m, 1 HÀC(2')); 2.79 (m, 1 HÀC(2')); 2.86 (m, Me2CHCO); 3.76 (s, MeO); 4.35 (m,
HÀC(4')); 4.45 (m, 1 HÀC(5')); 4.62 4.69 (m, 1 HÀC(5'), CH2); 4.96 (m, 1 HÀC(3')); 6.21 (t, J ˆ 6.5,
HÀC(1')); 6.79 (d, J ˆ 9.0, 2 H, MeOTr); 7.18 7.40 (m, 15 arom. H); 7.72 (d, J ˆ 8.0, 1 arom. H (Bz)); 7.82 (s,
HÀC(8)); 10.14( s, 1 H, NH2ÀC(2)); 12.34( s, NH(1)). 13C (500 MHz, CDCl3): 14.1; 18.9; 19.0; 21.0; 36.1; 39.5;
55.2; 60.4; 64.4; 71.3; 71.7; 78.0; 84.7; 85.2; 113.2; 121.5; 127.2 134.1; 138.6; 139.8; 143.1; 147.8; 148.0; 155.80;
158.6; 166.8; 171.2; 180.0. Anal. calc. for C42H41N5O8S ¥ 3/5 H2O: C 64.12, H 5.41, N 8.90, S 4.08; found: C 64.42,
H 5.58, N 8.45, S 3.92.
Crude Mixture of 3'-O-[(tert-Butyl)dimethylsilyl]-5'-O-[2-(hydroxymethyl)benzoyl]thymidine (10) and 3'-
O-[(tert-Butyl)dimethylsilyl]thymidine. Compound 9a (163 mg, 0.2 mmol) was dissolved in 10 ml of 0.6m I2 in
pyridine/H2O 9 :1 (v/v). After 1 min, the reaction was quenched by adding 30 ml of 5% Na2S2O3 soln. The
mixture was extracted with CH2Cl2 (50 ml), the org. phase dried (MgSO4) and evaporated, and the residue
subjected to CC (silica gel, hexane/AcOEt 4:6): 10 TBDMS-T 7:2 (97% yield). 1H-NMR (500 MHz, CDCl3):
0.10 (s, Me2Si); 0.91 (s, tBuSi); 1.69 (s, MeÀC(5)); 2.15 2.21 (m, 1 HÀC(2')); 2.34 2.39 ( m, 1 HÀC(2')); 4.19
(m, 1 HÀC(3')); 4.50 4.59 (m, 1 HÀC(3'), 1 HÀC(4'), CH2); 4.82 (m, 2 HÀC(5')); 6.27 (d, J ˆ 7, H ÀC(1'));
7.19 (s, 6 H); 7.36 7.60 (m, 3 arom. H (Bz)); 7.97 (d, 1 arom. H (Bz)); 8.53 (s, HÀC(6)). 13C-NMR (126 MHz,
CDCl3): À 4.6; 12.3; 25.7; 40.9; 64.1; 64.6; 71.8; 84.5; 85.3; 128.0; 128.2; 130.6; 130.7; 133.6; 135.1; 143.5; 167.4.
Anal. calc. for a 7:2 mixture of C24H34N2O7Si and C18H28N2O5Si: C 57.92, H 7.15, N 6.08; found: C 57.55, H 6.87,
N 5.77.
5'-O-{2-{{[(4-Methoxytrityl)thio]oxy}methyl}benzoyl}thymidine 3'-(2-Cyanoethyl Diisopropylphosphor-
amidite) (ˆ MOB Phosphoramidite; 11). Compound 9b (480 mg, 0.71 mmol) was rendered anhydrous by
repeated co-evaporation with dry pyridine, toluene, and CH2Cl2, and finally dissolved in dry CH2Cl2. To this
soln. was added 2-cyanoethyl tetraisopropylphosphoramidite (269 ml, 0.85 mmol) and diisopropylammonium
tetrazolide (48 mg, 0.28 mmol), and the resulting soln. was stirred at r.t. for 15 h. The reaction was quenched by
adding H2O (5 ml). The mixure was diluted with AcOEt/Et2O/iPr2O 1:1:1 (30 ml), washed with 5% Na2CO3
soln. (7 Â 30 ml), dried (MgSO4), and evaporated and the residue subjected to CC (NH silica gel (15 g), hexane/
AcOEt 1:3 containing 2% of Et3N): 11 (416 mg, 67%). 1H-NMR (500 MHz, CDCl3): 1.19 (m, 2 Me2CH); 2.29
(m, 1 HÀC(2')); 2.55 (m, 1 HÀC(2')); 2.66 (m, NCOH2CH2O); 3.61 (m, 2 Me2CH); 3.71 3.79 (m, 1 H of
NCCH2CH2O, MeO); 3.88 (m, 1 H, NCCH2CH2O); 4.32 (m, HÀC(4')); 4.56 (m, HÀC(3'), 2 HÀC(5')); 4.81
4.82 (m, CH2); 6.27 (t, J ˆ 6.75 HÀC(1')); 6.80 (d, J ˆ 8.5, 2 H, MeOTr); 7.13 7.37 (m, 13 arom. H); 7.57 (m, 1
arom. H (Bz)); 8.24( m, 1 arom. H (Bz)); 8.67 (m, 1 arom. H (Bz)); 9.14( s, NH(3)). 13C-NMR (500 MHz,
CDCl3): 12.3; 20.4; 20.5; 24.5; 24.6; 39.1; 39.2; 43.3; 43.3; 43.4; 43.4; 55.2; 57.9; 57.9; 58.1; 64.5; 64.7; 72.1; 72.8;
72.9; 73.1; 73.2; 77.6; 82.7; 82.8; 83.2; 85.4; 111.4; 113.3; 117.6; 125.0; 126.7; 127.4 131.2; 133.8; 135.2; 135.3;
142.8; 146.5; 148.0; 150.0; 158.8; 163.3; 164.0. Anal. calc. for C47H53N4O9PS ¥ H2O: C 62.79, H 6.17, N 6.23, S 3.57;
found: C 62.35; H 6.03; N 6.21; S 3.35.
Solid-Phase Synthesis. The chain-elongation cycle with commercially available 5'-O-(4,4'-dimethoxy-
trityl)thymidine (2-cyanoethyl diisopropylphosphoramidite) (MeO)2Tr phosphoramidite), consists of four
chemical steps, 1) coupling, 2) capping, 3) oxidation, 4) detritylation. The conditions for each step are the
following.
1) Coupling: Chain elongation was performed by the reaction of the free OH group on the solid support and
40 equiv. of(MeO)2Tr phosphoramidite in the presence of 80 equiv. of 1H-tetrazole in dry MeCN (125 ml) for

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Helvetica Chimica Acta Vol. 87 (2004)
2 min. 2) Capping: Failure sequence was capped by the reaction with 50 ml of Ac2O for 2 min in pyridine (450 ml)
in the presence of 0.1m DMAP. 3) Oxidation: The oxidation of the phosphite intermediate was performed by the
reaction with 0.1m I2 for 2 min in pyridine/H2O 9 :1 (250 ml). 4) Detritylation: The MeO2Tr group was removed
by treating several times with 3% CCl3COOH in CH2Cl2.
In the case of the chain elongation with MOB phosphoramidite 11, the chain elongation cycle was changed
to 1) coupling, 2) capping, 3) oxidation/deprotection. The conditions for coupling and capping were identical to
those in the case of conventional phosphoramidite. The conditions for the oxidation/deprotection are the
following.
Oxidation/deprotection: The oxidation of the phosphite intermediates and oxidative cleavage of the
MeOTrS groups were performed by the reaction with 0.1m I2 for 2 min in pyridine/H2O 9 :1 (250 ml). After the
oxidation, the 5'-OH group of the oligonucleotides were liberated by the treatment with 0.2m iPr2EtN in MeCN/
H2O 9 :1 for 5 min.
After the chain elongation was completed, the oligoDNA was treated with 28% ammonia for 30 min to
perform the deprotection of the 2-cyanoethyl group and cleavage from the solid support.
The whole experiment was carried out on a 0.5-mmol scale commercially available CPG solid support
(11.36 mg, 44 mmol/g) bearing thymidine residues.
The structure of the tetrathymidylate was confirmed by the comparison of the retention time and UV with
those of an authentic sample by means of reversed-phase HPLC.
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Received March 18, 2004