Synthesis of pentathymidylate using a 4-monomethoxytritylthio (MMTrS) group as a 5′-hydroxyl protecting group: Toward oligonucleotide synthesis without acid treatment

Article abstract of DOI:10.1016/S0040-4039(01)01873-1

A phosphoramidite unit having 4-monomethoxytritylthio as a new 5′-hydroxyl protecting group was prepared and employed in oligonucleotide synthesis. The new phosphoramidite enabled the synthesis of oligonucleotides without the use of acids such as TFA or DCA.

Full text of DOI:10.1016/S0040-4039(01)01873-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8657–8660
Synthesis of pentathymidylate using a 4-monomethoxytritylthio
(MMTrS) group as a 5%-hydroxyl protecting group: toward
oligonucleotide synthesis without acid treatment
Kohji Seio and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan
Received 2 July 2001; revised 1 October 2001; accepted 5 October 2001
Abstract—A phosphoramidite unit having 4-monomethoxytritylthio as a new 5%-hydroxyl protecting group was prepared and
employed in oligonucleotide synthesis. The new phosphoramidite enabled the synthesis of oligonucleotides without the use of acids
such as TFA or DCA. © 2001 Elsevier Science Ltd. All rights reserved.
Several alkyl- or arylthio groups have been used for
sulfenamide-type protection of various amino com-
pounds.1 In particular, protection of nucleic acid bases
by the tritylthio (TrS)2 group has been studied widely
because it can be removed by treatment with mild
neutral reagents such as tributyltin hydride2a and
iodine.2b,c Although the property of the TrS group as
an amino protecting group has been well studied, little
is known about this protecting group as a hydroxyl
protecting group in oligonulcleotide chemistry. For
example, Bazin et al. reported the introduction of a TrS
group to the 5%-hydroxyl group of an adenosine deriva-
tive, but the application to the oligonucleotide synthesis
was not described.3 In this paper we report 4-
monomethoxytritylthio (MMTrS) as a new hydroxyl
protecting group in oligonucleotide synthesis. The
MMTrS group has an electron-donating methoxy
group which is expected to accelerate the deprotection
as compared to the conventional TrS group.
MMTrSH. The MMTrS group was successfully intro-
duced to the 5%-hydroxyl group of 3%-O-tert-butyl-
dimethylsilylthymidine (2) in 76% yield by the selective
sulfenylation of a 5%-alkoxy species generated by lithium
hexamethyldisilazide (2.2 equiv.) with MMTrSCl (1.6
equiv.). Interestingly, the use of 1.1 equiv. of hexa-
methyldisilazane lithium in this reaction gave 3-N-
MMTrS-3%-O-tert-butyldimethylsilylthymidine in 26%
as a single product even after the prolonged reaction
(15 h). The TBDMS group of 37 thus obtained was
removed by TBAF·H2O and then the resulting 3%-
hydroxyl group of 48 was phosphitylated in 82% yield
by treatment with chloro(2-cyanoethoxy)(N,N-diiso-
propylamino)phosphine9 (1.5 equiv.) in the presence of
2.3 equiv. of N,N-diisopropylethylamine (Scheme 1).
Previously, Christodoulou et al. reported that 2,4-dini-
trobenzenesulfenyl (DNBS) esters were not compatible
with phosphoramidite functional groups because of the
intramolecular nucleophilic reaction between the sulfur
and the tervalent phosphorus atom.10 However, it was
found that the phophoramidite (5) was quite stable
during the isolation process and in storage for months
regardless of the presence of both the sulfenyl ester and
the tervalent phosphorus functions. The stability of the
MMTrS ester must be attributed to the steric bulk and
the lower electron-withdrawing property of the MMTr
moiety both of which reduce the electrophilicity of the
sulfur atom of MMTrS as compared to that of the
corresponding DNBS ester. The stability of the
MMTrS group of 4 was evaluated under various condi-
tions. The MMTrS group was stable in both aq NH3–
EtOH (3:1, v/v, 24 h, rt) and 1 M tert-BuOOH/CH3CN
4-Monomethoxytritylsulfenyl chloride (1: MMTrSCl)
used in this study was synthesized in 76% yield by
treatment of 4-monomethoxytriphenylmethanethiol
(MMTrSH)4 with 1,3-dichloro-5,5-dimethylhydantoin
(0.5 equiv.) in 1,4-dioxane at room temperature for 30
min.5 Sulfuryl chloride used in the TrSCl synthesis6 was
not effective probably because use of the acidic chlori-
nating agent led to decomposition of the acid-labile
Keywords: oligonucleotide synthesis; protective group; sulfenic acid
esters.
* Corresponding author. Fax: +81-45-924-5772; e-mail: msekine@
bio.titech.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01873-1

8658
K. Seio, M. Sekine / Tetrahedron Letters 42 (2001) 8657–8660
O
Cl
N
N Cl
O
MeO
SH
MeO
SCl
O
O
r. t., 30 min
1
O
N
O
N
O
N
O
N
NH
NH
NH
NH
MMTrSO
MMTrSO
O
O
O
O
MMTrSO
HO
i)
ii)
O
O
iii)
O
O
iPr2N
P
O
O
O
TBDMS
O
TBDMS
OH
2
3
4
5
CN
Scheme 1. Reagents and conditions: (i) lithium hexamethyldisilazide (2.2 equiv.), THF, rt, 25 min then 1 (1.6 equiv.), THF, rt,
1 h; (ii) TBAF·H2O (1.2 equiv.), THF, rt, 1 h; (iii) N,N-diisopropylethylamine (2.3 equiv.), chloro(2-cyanoethoxy)-
(diisopropylamino)phosphine (1.5 equiv.), CH2Cl2, rt, 1 h.
(20 min, rt), while it was removed rapidly to give
thymidine by treatment with 0.1 M I2/CH3CN–pyr-
idine–H2O (10:9:1, v/v/v, Tcomp=1 min).
was also prepared according to the procedure almost
identical to that of 6a. The dimers 6a and 6b were
treated with 0.1 M I2/CH3CN–pyridine–H2O (10:9:1,
v/v/v) for 2 min and then with aqueous ammonia for 30
min. As shown in Fig. 1a, it turned out that the
MMTrS group could be removed completely to give
thymidylyl(3%–5%)thymidine (TpT) in 76% yield after
reversed-phase HPLC purification in the case of 6a. On
the other hand, the TrS group of 6b was not removed
completely under the conditions examined here (Fig.
The deprotection using iodine was further examined in
the solid-phase oligonucleotide synthesis. The protected
thymdine dimer 6a was prepared on solid support as
shown in Scheme 2 using commercially available 5%-
DMTr-thymidine CPG support having a long chain
alkylamino (LCAA) linker. The TrS-protected dimer 6b
Scheme 2. Reagents: (i) 0.1 M I2/CH3CN–pyridine–H2O (9:10:1, v/v/v); (ii) aq. NH3.
Figure 1. Reversed-phase HPLC profile of the deprotection of (a) 6a, and (b) 6b (Scheme 2); (c) reversed-phase HPLC profile of
the pentathymidylate synthesized according to the procedure depicted in Scheme 3.

K. Seio, M. Sekine / Tetrahedron Letters 42 (2001) 8657–8660
8659
1) 5 (20 equiv.), 1H-tetrazole (80 equiv.)
CH3CN, 1 min
O
N
2) Ac2O : 0.1 M DMAP-pyridine (1:9, v/v)
x4
2 min
NH
3) 0.1 M I2 /pyridine-CH3CN-H20 (9:10:1, v/v/v)
RO
O
2min
aq. NH3
30 min
O
TpTpTpTpT
O
R= DMTr
R= H
1% TFA /CH2Cl2
Scheme 3. Pentathymidylate synthesis by acid free chain elongation using phosphramidite 5.
1b). Because the order of the reaction rate observed
here is in good correlation with that of the stability of
the corresponding trityl cations, we can hypothesize
that the deprotection reaction proceeded via the trityl
cation intermediate. While the direct detection of the
trityl cation and reaction intermediates is necessary to
confirm this reaction mechanism, it was difficult
because the deprotection was carried out in aqueous
media. Other kinetic study and model reactions in
organic solvents are now under way to clarify the
detailed reaction mechanism.
References
1. (a) Craine, L.; Raban, M. Chem. Rev. 1989, 89, 689–712
and references cited therein; (b) Netscher, T.; Wellar, T.
Tetrahedron 1991, 47, 8145–8154; (c) Ueki, M.; Honda, M.;
Kazama, Y.; Katoh, T. Synthesis 1994, 21–22.
2. (a) Seio, K.; Sekine, M. J. Chem. Soc., Perkin Trans. 1
1993, 3087–3093; (b) Takaku, H.; Imai, K.; Nagai, M.
Chem. Lett. 1988, 857–860; (c) Alvarez, K.; Tworkowski,
I.; Vasseur, J.-J.; Imbach, J.-L.; Rayner, B. Nucleosides
Nucleotides 1998, 17, 365–378.
3. Bazin, H.; Heikkila¨, J.; Chattopadhyaya, J. Acta Chem.
Scand. B 1985, 39, 391–400.
It should be noted that in the oligonucleotide synthesis
depicted in Scheme 2, the deprotection of the MMTrS
group and the oxidation of the phosphite intermediate
could be carried out simultaneously. This simultaneous
deprotection–oxidation reduced the reaction steps
required for the correct oligonucleotide synthesis in
addition to avoiding any acid treatment which may
cause unfavorable side reactions during longer
oligoDNA and RNA synthesis. The usefulness of phos-
phoramidite 5 was further evaluated by the pen-
tathymidylate synthesis according to our acid-free
procedure (Scheme 3). The reversed-phase HPLC
profile of the pentathymidylate (Fig. 1c) clearly shows
that the MMTrS-protected phosphoramidite 5 is appli-
cable to oligonucleotide synthesis. The pentathymidy-
late was obtained in 48% yield after reversed-phase
HPLC purification, and the structure was confirmed by
MALDI-TOF mass spectroscopy.11 An application of
the MMTrS group to synthesize oligoDNA containing
all four common nucleotides is now under way and will
be reported in due course.
4. MMTrSH was synthesized as follows: A suspension of
4-monomethoxytrityl alcohol (124 g, 0.43 mol) in toluene
(2 L) was added Lawesson’s reagent (104 g, 0.24 mol), and
the resulting suspension was heated to give a dark-colored
solution (ca. 75°C). The solution was cooled to room
temperature and the precipitation was removed by filtra-
tion. The filtrate was washed with water (1 L) and the
organic layer was dried with magnesium sulfate, filtered
and evaporated under reduced pressure. The residue was
chromatographed on silica gel column with hexane and the
fraction was concentrated under reduced pressure. The
residue was added to isopropyl ether and the resulting
precipitation was filtered to give MMTrSH (80 g, 57%). 1H
NMR (270 MHz, CDCl3): l 3.06 (1H, s), 3.78 (3H, s), 6.80
(2H, d, J=7.6 Hz), 7.16 (2H, d, J=7.6 Hz), 7.26 (10H, m);
13C NMR (67.8 MHz, CDCl3): l 55.27, 62.46, 112.95,
126.67, 127.67, 129.15, 130.39, 139.16, 147.31, 158.11.
5. The detailed synthetic procedure of MMTrSCl (1) is as
follows: A solution of MMTrSH (10 g, 32.5 mmol) in
dioxane (100 mL) was added 1,3-dichloro-5, 5-dimethyl-
hydantoin (3.2 g, 16 mmol). The solution was stirred for
30 min, diluted with diethyl ether (200 mL), washed with
water (100 mL) and then washed three times with saturated
sodium bicarbonate (100 mL each). The organic layer was
dried over magnesium sulfate, filtered, and evaporated
under reduced pressure. The residue was treated with ethyl
acetate, and the precipitate was collected by filtration to
Acknowledgements
This work was supported by a Grant from ‘Research
for the Future’ Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301) and a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan.
1
give 1 (7.2 g, 65%). H NMR (270 MHz, CDCl3): l 3.81
(3H, s), 6.85 (2H, d, J=8.9 Hz), 7.23 (2H, d, J=10.2 Hz),
7.31–7.35 (10H, m); 13C NMR (67.8 MHz, CDCl3): l
55.29, 71.82, 113.43, 127.70, 128.03, 129.74, 131.08, 133.24,
142.02, 159.05.

8660
K. Seio, M. Sekine / Tetrahedron Letters 42 (2001) 8657–8660
6. (a) Vorlander, D.; Mittag, E. Chem. Ber. B 1919, 52,
(1H, d, J=11.7 Hz), 3.62 (1H, dd, J=11.7 Hz, J=2.3
Hz), 3.73 (1H, m), 3.82 (3H, s), 4.07 (1H, m), 6.26 (1H, t,
J=6.6 Hz), 6.85 (2H, d, J=8.6 Hz), 7.24–7.35 (12H, m),
7.51 (1H, s,), 8.33 (1H, br); 13C NMR (67.8 MHz,
CDCl3): l 12.79, 40.77, 55.33, 71.47, 72.11, 84.69, 85.76,
110.93, 113.37, 127.45, 128.00, 129.81, 130.97, 133.69,
135.42, 142.62, 142.71, 149.93, 158.79. Anal. calcd for
C30H30N2O6S·1/4H2O: C, 65.37; H, 5.58; N, 5.08; S, 5.82.
Found: C, 65.29; H, 5.17; N, 4.70; S, 5.08.
413–423; (b) Woulfe, S. R.; Miller, M. J. J. Org. Chem.
1986, 51, 3133–3139.
1
7. Spectral and analytical data for 3: H NMR (270 MHz,
CDCl3): l 0.01 (6H, s), 0.83 (9H, s), 1.91 (4H, m, CH3),
2.14 (1H, m), 3.40 (1H, d, J=11.7 Hz), 3.52 (1H, d,
J=11.7 Hz), 3.69 (1H, m), 3.81 (3H, s), 4.08 (1H, m),
6.25 (1H, t, J=8.2 Hz), 6.83 (2H, d, J=8.2 Hz), 7.23–
7.35 (12H, m), 7.48 (1H, s), 8.33 (1H, br); 13C NMR (67.8
MHz, CDCl3): l −4.74, −4.62, 12.72, 17.93, 25.73, 41.09,
55.21, 71.83, 71.93, 84.81, 86.36, 110.88, 113.27, 127.33,
127.90, 129.69, 129.74, 130.94, 133.69, 135.43, 142.67,
142.70, 150.21, 158.66, 163.78. Anal. calcd for
C36H44N2O6SSi·1/4H2O: C, 64.98; H, 6.74; N, 4.21; S,
4.82. Found: C, 64.75; H, 6.15; N, 3.96; S, 4.16.
9. Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H.
Nucleic Acids Res. 1984, 12, 4539–4557.
10. (a) Christodoulou, C.; Agrawal, S.; Gait, M. J.
Nucleosides Nucleotides 1987, 6, 341–344; (b) Beaucage,
S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
11. The structure of the pentathymidylate was confirmed by
MALDI-TOF mass spectroscopy. MALDI-MS [M–H]−
calcd for C50H65N10O33P4 1457.27, found 1457.98.
1
8. Spectral and analytical data for 4: H NMR (270 MHz,
CDCl3): l 1.95 (3H, s), 2.00 (1H, m), 2.25 (1H, m), 3.42