2-(Phenylthio)ethyl as a novel two-stage base protecting group for thymidine analogues

Article abstract of DOI:10.1055/s-2006-933147

2-(Phenylthio)ethyl is here proposed for temporary masking the thymine residue in the synthesis of sugar modified thymidine derivatives. This protection has been devised as a 'two-stage' system. The 2-(phenylthio)ethyl residue can be easily and regiospecifically inserted at the N3-position of the pyrimidine by a Mitsunobu reaction with 2-(phenylthio)ethanol and is perfectly stable also to strongly basic conditions. This allowed us to selectively achieve O-alkylation of the ribose moieties in satisfactory yields, avoiding undesired base alkylations. After oxidation of the thioether to sulfone, the thymine protecting group can be totally removed, by a β-elimination mechanism, upon the same basic treatment required for the final deprotection and detachment of oligonucleotides from the support in solid-phase synthesis protocols. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-933147

LETTER
845
2-(Phenylthio)ethyl as a Novel Two-Stage Base Protecting Group for
Thymidine Analogues
A
Novel
T
e
w
o-Stage
B
n
ase Protecti
n
ng
G
roup for
i
Thym
f
idine
A
n
e
alogues r D’Onofrio, Lorenzo De Napoli, Giovanni Di Fabio,* Daniela Montesarchio
Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli ‘Federico II’,
Complesso Universitario di Monte S. Angelo, via Cynthia 4, 80126 Napoli, Italy
E-mail: difabio@unina.it
Received 6 December 2005
amples of thymine protections have been described. The
most commonly used protecting group is the phenyl,4 in-
troduced in the thymidine O4-position by reaction of phe-
nol with the activated 4-triazolyl intermediate. Mild basic
Abstract: 2-(Phenylthio)ethyl is here proposed for temporary
masking the thymine residue in the synthesis of sugar modified thy-
midine derivatives. This protection has been devised as a ‘two-
stage’ system. The 2-(phenylthio)ethyl residue can be easily and re-
giospecifically inserted at the N3-position of the pyrimidine by a conditions (typically an overnight oximate ions treatment)
Mitsunobu reaction with 2-(phenylthio)ethanol and is perfectly sta-
ensure their complete removal. To the best of our knowl-
ble also to strongly basic conditions. This allowed us to selectively
edge, there is only one example reported in the literature
achieve O-alkylation of the ribose moieties in satisfactory yields,
of alternative protection for thymine residues in oligonu-
avoiding undesired base alkylations. After oxidation of the thioether
cleotide synthesis, involving N-acylation of the heterocy-
to sulfone, the thymine protecting group can be totally removed, by
cle with p-anisoyl chloride.5 More stringent is the choice
a b-elimination mechanism, upon the same basic treatment required
of the protecting groups for thymine and uracil nucleo-
sides, in case a selective manipulation of the ribose func-
tions is desired to obtain sugar-modified analogues.
Benzyl, p-methoxybenzyl and benzyloxymethyl groups
have been preferentially adopted to this purpose.6
for the final deprotection and detachment of oligonucleotides from
the support in solid-phase synthesis protocols.
Key words: protecting groups, Mitsunobu reactions, thymidine,
oligonucleotides, solid-phase synthesis
In the course of our studies7 on modified oligonucleotides,
and particularly aiming at preparing the 5¢TGGGAG3¢ se-
quence carrying the 3,4-dibenzyloxybenzyl (3,4-DBB)
group at its 5¢-end (Figure 1), discovered by Hotoda and
his group to be endowed with a potent anti-HIV-1 activi-
ty,8 we encountered serious synthetic problems in the
alkylation of the thymidine monomer to be inserted in Ho-
toda’s hexamer as the 5¢-terminal nucleoside.
Crucial issue in a convergent, multistep synthesis is a
careful choice of the protecting groups to transiently mask
potentially interfering functions. Since Khorana’s pre-
cious pioneering work, several protocols have been devel-
oped and optimized in the last decades for the sequential
addition of nucleotides to give an oligonucleotide se-
quence.1 The most efficient methodologies for the oligo-
nucleotide synthesis, i.e. the phosphoramidite2 and the H-
phosphonate3 chemistry, routinely used in fully automat-
ed solid-phase synthetic processes, are both based on a
unique protection strategy of the nucleotide monomers.
T
G
A
G
G
G
O
P
O
P
P
P
OH
P
In order to obtain the regioselective formation of 3¢→5¢-
phosphodiester linkages in the oligonucleotide chain, or-
thogonal protections are required for the 5¢-OH groups,
typically transiently blocked as acid-labile 5¢-O-(4,4¢-
dimethoxytriphenylmethyl)ethers (DMT-ethers), and for
the exocyclic amino groups of purines and pyrimidines.
These nucleophiles are normally rendered innocuous by
conversion into amides, then hydrolyzed at the end of the
synthesis by the final basic treatment. No explicit protec-
tion is usually adopted for the thymine base, where the po-
tential nucleophilic centers, i.e. the N3- and O4-positions,
under normal conditions, interfere to a very marginal ex-
tent with the phosphodiester bond formation, either using
phosphoramidite or H-phosphonate reactive intermedi-
ates. A survey of the literature revealed that only a few ex-
O
P = 3'→5' phosphodiester linkage
Figure 1
In the procedures described by Hotoda and co-workers,9
the formation of the 5¢-DBB-ether was achieved by reac-
tion of DBB-bromide with 3¢-O-TBDMS-thymidine in
THF in the presence of NaH. The target molecule was fi-
nally obtained, even if in modest yields (23%) and, after
TBDMS removal, phosphitylated to give the correspond-
ing 3¢-phosphoramidite derivative, used as building block
in the automated oligonucleotide synthesis. When we re-
peated this procedure, with the sole difference of using the
commercially available DBB-chloride in lieu of the bro-
mide adopted by Hotoda, we always observed exclusively
the formation of the N-alkylation product over the desired
5¢-O-alkylation one, with the N3-linked DBB adduct ob-
tained also in high yields. Several conditions were tested,
SYNLETT 2006, No. 6, pp 0845–0848
0
4.
0
4.
2
0
0
6
Advanced online publication: 14.03.2006
DOI: 10.1055/s-2006-933147; Art ID: G39205ST
© Georg Thieme Verlag Stuttgart · New York

846
J. D’Onofrio et al.
LETTER
varying the solvent, temperature, activation and excess of
the reagents, but in all cases we could isolate from the re-
action mixtures only the N-alkylated thymidine or, when
further forcing the reaction conditions, N3,O-bis-alkylat-
ed derivatives. In fact, an additional side reaction was ob-
served in the DBB-ether formation starting from 3¢-O-
TBDMS-thymidine: the concomitant formation, in almost
1:1 ratio, of both the N3,O-5¢- and the N3,O-3¢-bis-alkyl-
ated derivative, as a result of the hydride-mediated migra-
tion of the TBDMS group from the 3¢- to the 5¢-position,
which was also reported by Kobe et al.10
Si
O
O
HN
N
N
N
O
O
HO
DMTO
a–c
O
O
OH
OTBDMS
1
2
Scheme 1 Reagents and conditions: a) DMTCl, DMAP, pyridine,
5 h, r.t. (97%); b) TBDMSCl, imidazole, DMF, 12 h, r.t. (95%);
c) (CH3)3SiCH2CH2OH, PBu3-ADDP in benzene, 18 h, 0 °C to r.t.
(65%).
In order to get a deeper insight into the structure–activity
relationships of this G-rich, quadruplex-forming hexamer
and related analogues, the large scale preparation of these
oligonucleotides was a prerequisite. In this frame, protec-
tion of the thymine base of the nucleoside was the neces-
sary next step. However, all base-labile protecting groups
for thymine seemed not appropriate in our case, nor in all
those cases where specific transformations of the ribose
moieties requiring quite strong basic media are desired.
Therefore we have undertaken a more detailed study
aimed at regiospecifically protecting thymidine on the
heterocycle with a group satisfying the following criteria:
i) facile, high-yielding and highly reproducible installa-
tion; ii) elevated stability to a wide range of different con-
ditions, including strongly basic conditions, so allowing
to differently manipulate the sugar functions; iii) high
compatibility with the standard oligonucleotide synthesis
conditions; iv) facile and total removal, in conditions not
affecting the integrity of the 5¢-DBB-ether function. The
latter requirement led us to discard benzyl-type protecting
groups, for their incomplete orthogonality with respect to
the DBB group. Candidate protecting groups could there-
fore be ‘safety catch’ or ’two-stage’ systems,11 designed
to be very stable in their native structure, but, after a sim-
ple and quantitative conversion into an ‘activated’ form,
easily removable under mild conditions when required.
from the support, a suitable bifunctional linker was de-
signed, bearing a 2-mercapto-ethanol moiety. After nucle-
oside incorporation and oligonucleotide chain assembly,
the cleavage from this support involved two steps: oxida-
tion with 3-chloroperbenzoic acid (MCPBA) of the sulfur
atom to sulfone, followed by a standard aqueous ammonia
treatment (Figure 2). We reckoned that a similar system
could be profitably introduced as a new ‘two-stage’ pro-
tecting group for thymine base in nucleoside and oligonu-
cleotide analogues synthesis.
S
S
O
O
.
.
N
N
N
N
O
O
DMTO
DMTO
O
O
TBDMSO
TBDMSO
N3
Figure 2
We therefore synthesized 5¢-O-TBDMS-3¢-O-DMT-thy-
midine as the starting material and reacted it with 2-(phe-
nylthio)ethanol under standard Mitsunobu conditions.
The 2-(phenylthio)ethyl group had been previously tested
only for protection of phosphodiester functions in oligo-
Our first attempts were carried out using as starting mate-
rial 5¢-O-DMT-3¢-O-TBDMS-thymidine, as in Hotoda’s nucleotide synthesis through the phosphotriester chemis-
synthetic scheme. We tested the 2-(trimethylsilyl)ethyl try.16 To the best of our knowledge, its use for pyrimidine
group as the protecting group for the thymine base, protection is unprecedented. The desired N3-alkylated nu-
previously reported in the literature as a suitable protec- cleoside 317,18 (Scheme 2) was obtained in 90% yields.
tion for carboxyl and phosphate moieties,12 which could Subsequent standard triethylammonium fluoride treat-
be cleaved upon the same fluoride treatment required for ment furnished the 5¢-OH derivative in almost quantita-
TBDMS removal (Scheme 1). In our experiments, tive yields, which was then reacted with DBBCl and NaH
with cat. NaI, to give the desired 5¢-DBB adduct 4 in 70%
yields. In no case were side reactions observed and the un-
reacted nucleoside could be recovered intact after chro-
matography and recycled. Next, the thioether function
was quantitatively oxidized with MCPBA to sulfone. This
reaction, which in principle can be conveniently carried
out also in a successive step, at the level of the already as-
sembled oligonucleotide, was here realized on the mono-
mer in solution in order to more directly control the ‘two-
stage’ removal process: the efficiency of the crucial ‘acti-
vation’ of the protecting group and, then, of its base-in-
duced scission from the heterocycle. Successively,
addition of 3% TFA in CH2Cl2 cleanly liberated the 3¢-OH
TBDMS proved to be unsuitable to protect the 3¢-OH
function, for its above-mentioned tendency to migrate to
the 5¢-OH under basic conditions. In addition, the 2-(tri-
methylsilyl)ethyl group in 213,14 was found to be not
completely cleaved even after repeated treatments with
fluoride ions and was therefore finally discarded.
We then moved to a different protection strategy. Our pre-
vious results on the solid-phase immobilization of pyrim-
idine nucleosides through the base showed that
regioselective N3-alkylation could be easily achieved un-
der Mitsunobu conditions, allowing high incorporation
yields of the nucleoside onto the solid matrix.15 In order to
ensure a facile and complete detachment of the nucleotide
Synlett 2006, No. 6, 845–848 © Thieme Stuttgart · New York

LETTER
A Novel Two-Stage Base Protecting Group for Thymidine Analogues
847
moiety, not affecting the 5¢-DBB-ether function. The protecting group is stable to a large variety of conditions,
obtained nucleoside could then be phosphitylated by a including strongly basic media. Only after oxidation of
standard reaction with 2-cyanoethyl, N,N-diisopropyl- the thioether to sulfone, the system can fragment via a b-
chlorophosphoramidite and DIPEA in anhydrous CH2Cl2, elimination mechanism, induced by 0.1 M NaOH treat-
to give 5.19 Starting from 5¢-O-TBDMS-3¢-O-DMT-thy- ment, basic conditions required also for the final deprotec-
midine, 3¢-phosphoramidite building block 5 was pre- tion and detachment of oligonucleotides from the solid
pared in five, easy synthetic steps and 48% overall yields support. The utility of this new protection was demon-
(46% starting from thymidine).
strated through a more efficient synthesis of 5¢-DBB-thy-
midine, which, after standard phosphitylation, was then
used for the solid phase synthesis of anti-HIV-1 active
Hotoda’s hexamer. Application of this two-stage protect-
ing group can offer easy and large scale synthetic access
to a variety of ribose-modified thymidine derivatives and
be profitably extended also to uridine nucleosides, allow-
ing a wide set of O-alkylated analogues, including benzyl
ether derivatives. These modified nucleosides can be of
interest per se, for their biological activity, or as suitable
building blocks for the synthesis of a number of sugar-
modified oligonucleotides, attractive tools as potential an-
tisense and/or antigene agents.
S
O
O
HN
O
N
N
O
HO
N
TBDMSO
a–c
O
O
HO
ODMT
1
3
d–e
O
O
S
S
O
O
N
N
N
N
Acknowledgment
O
O
DBBO
DBBO
We acknowledge MIUR (PRIN) and Regione Campania (L5) for
grants in support of this investigation. We also thank C.I.M.C.F.,
Università degli Studi di Napoli ‘Federico II’, for the NMR and MS
facilities.
f–h
O
O
ODMT
O
P
5
4
N
OCE
References and Notes
Scheme 2 Reagents and conditions: a) TBDMSCl, imidazole,
DMF, 5 h, r.t. (quant.); b) DMTCl, DMAP, pyridine, 12 h, r.t. (95%);
c) PhSCH2CH2OH, PBu3-ADDP in benzene, 18 h, 0 °C to r.t. (90%);
d) 1 M TBAF in THF, 30 min, r.t. (98%); e) NaH, DBBCl, NaI, DMF,
18 h, r.t. (70%); f) MCPBA, CH2Cl2, 1 h, r.t. (98%); g) 3% TFA in
CH2Cl2, 30 min, r.t. (94%); h) 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite, TEA, CH2Cl2, 30 min, r.t. (85%).
(1) For a recent review, see for example: Reese, C. B. Org.
Biomol. Chem. 2005, 3, 3851.
(2) For a recent review, see for example: Natarajan, V.; Jeong,
K. S.; Byeang, K. Curr. Med. Chem. 2003, 10, 1973.
(3) For a recent review, see for example: Stawinski, J.;
Kraszewski, A. Acc. Chem. Res. 2002, 35, 952.
(4) (a) Reese, C. B.; Yan, H. J. Chem. Soc., Perkin Trans. 1
2002, 23, 2619. (b) Reese, C. B.; Skone, P. A. J. Chem. Soc.,
Perkin Trans. 1 1984, 1263.
Assembly of the 5¢DBBTGGGAG3¢ oligomer could then be
achieved on an automated DNA synthesizer by exploiting
standard phosphoramidite chemistry protocols. In all
cases, DMT tests, monitoring the efficiency of the
oligodeoxyribonucleotide (ODN) chain elongation, indi-
cated yields not inferior to 98% for each coupling cycle.
The last coupling was carried out using DBB-thymidine
phosphoramidite 5 as the building block, in a standard
DMT-on protocol.
(5) Reese, C. B.; Song, Q.; Rao, M. V.; Beckett, I. Nucleosides
Nucleotides 1998, 17, 451.
(6) (a) Kozai, S.; Maruyama, T.; Kimura, T.; Yamamoto, I.
Chem. Pharm. Bull. 2001, 49, 1185. (b) Robles, R.;
Rodrìguez, C.; Izquierdo, I.; Plaza, M. T.; Mota, A.;
Cienfuegos, L. A. Tetrahedron: Asymmetry 2000, 11, 3069.
(c) Luzzio, F. A.; Menes, M. E. J. Org. Chem. 1994, 59,
7267. (d) Dobson, N.; McDowell, D. G.; French, D. J.;
Brown, L. J.; Mellor, J. M.; Brown, T. Chem. Commun.
2003, 1234.
(7) D’Onofrio, J.; de Champdoré, M.; De Napoli, L.;
Montesarchio, D.; Di Fabio, G. Bioconjugate Chem. 2005,
16, 1299.
(8) Hotoda, H.; Koizumi, M.; Koga, R.; Kaneko, M.; Momota,
K.; Ohmine, T.; Furukawa, H.; Agatsuma, T.; Nishigaki, T.;
Sone, J.; Tsutsumi, S.; Kosaka, T.; Abe, K.; Kimura, S.;
Shimada, K. J. Med. Chem. 1998, 41, 3655.
(9) Hotoda, H.; Momota, K.; Furukawa, H.; Nakamura, T.;
Kaneko, M. Nucleosides Nucleotides 1994, 13, 1375.
(10) Jaksa, S.; Kralj, B.; Pannecouque, C.; Balzarini, J.; De
Clercq, E.; Kobe, J. Nucleosides, Nucleotides Nucleic Acids
2004, 23, 77.
Once the ODN chain assembly was complete, detachment
from the solid support and full deprotection of the 5¢-con-
jugated oligomer, including thymine protecting group re-
moval, were achieved by treatment with 0.1 M NaOH.20
After HPLC purification and desalting by gel filtration
chromatography, the synthesized ODN was characterized
by mass analysis.21
In conclusion, highly selective alkylation of the ribose
moiety of thymidine could be finally achieved by intro-
ducing a new, two-stage protecting group on thymine,
namely the 2-(phenylthio)ethyl group, which prevented
otherwise predominant N-alkylation of the base. This
(11) Schelhaas, M.; Waldmann, H. Angew. Chem., Int. Ed. Engl.
1996, 35, 2056.
Synlett 2006, No. 6, 845–848 © Thieme Stuttgart · New York

848
J. D’Onofrio et al.
LETTER
(12) (a) Kocienski, P. J. In Protecting Groups, 3rd ed.; Thieme:
Stuttgart, 2003. (b) Greene, T. W.; Wuts, P. G. M. In
Protective Groups in Organic Synthesis, 3rd ed.; Wiley:
New York, 1999.
(17) Selected Data for Compound 3.
1H NMR (400 MHz, CDCl3): d = 7.47–6.85 (18 H, complex
signals, arom. protons), 6.48 (1 H, dd, H-1¢, J = 5.6, 9.6 Hz),
4.27 (1 H, d, H-3¢, J3¢,2¢b = 12.0 Hz), 4.22 (2 H, m, CH2N),
4.01 (1 H, br s, H-4¢), 3.82 (6 H, s, OCH3), 3.68 (1 H, dd, H-
5¢a, J = 4.0, 12.0 Hz), 3.29 (1 H, dd, H-5¢b, J = 4.0, 12.0 Hz),
3.19 (2 H, t, CH2S, J = 8.0, 8.0 Hz), 1.87 (3 H, s, CH3 T),
1.76 (1 H, dd, H-2¢a, J = 4.0, 12.0 Hz), 1.57 (1 H, complex
signal, H-2¢b), 0.83 {9 H, s, [(CH3)3C]Si}, –0.02 and –0.08
[3 H each, s, Si(CH3)2]. 13C NMR (100 MHz, CDCl3): d =
162.83 (C-4), 150.37 (C-2), 135.86 (C-6), 158.29, 150.37,
144.70, 135.95, 135.28, 133.27, 129.87, 129.75, 128.51,
127.99, 127.86, 127.51, 126.63, 125.35, 112.83 (arom.
carbons), 109.62 (C-5), 86.81 (C of DMT group), 86.16 (C-
1¢), 85.12 (C-4¢), 74.55 (C-3¢), 63.12 (C-5¢), 54.80 (OCH3),
40.59 (CH2N), 39.55 (C-2¢), 29.22 (CH2S), 25.34
{[(CH3)3C]Si}, 17.77 [(CH3)3C], 12.68 (CH3 T), –5.86 and
–6.18 [Si(CH3)2]. MS: m/z calcd for C45H54N2O7SSi: 794.34.
ESI-MS (positive ions) found: m/z 833.75 [M + K+].
(18) Typical Procedure for the Insertion of 2-(Phenylthio)-
ethyl Group: Synthesis of 3
(13) Selected Data for Compound 2.
1H NMR (400 MHz, CDCl3): d = 7.61–6.82 (13 H, complex
signals, arom. protons), 6.38 (1 H, dd, H-1¢, J = 6.2, 6.8 Hz),
4.49 (1 H, m, H-3¢), 4.01–3.96 (3 H, overlapped signals,
CH2N and H-4¢), 3.79 (6 H, s, OCH3), 3.45 (1 H, dd, H-5¢a,
J = 4.0, 12.0 Hz), 3.25 (1 H, dd, H-5¢b, J = 4.0, 12.0 Hz),
2.33–2.17 (2 H, complex signal, H2-2¢), 1.54 (3 H, s, CH3 T),
0.97 (2 H, m, CH2Si), 0.83 {9 H, s, [(CH3)3C]Si of TBDMS
group}, 0.07 [9 H, s, (CH3)3Si], –0.02 and –0.04 [3 H each,
s, Si(CH3)2 of TBDMS group]. 13C NMR (100 MHz,
CDCl3): d = 163.22 (C-4), 150.57 (C-2), 135.40 (C-6),
158.61, 144.28, 133.19, 129.95, 126.98, 113.15 (arom.
carbons), 110.18 (C-5), 86.81 (quat. C of DMT group),
86.56 (C-1¢), 85.35 (C-4¢), 71.97 (C-3¢), 62.82 (C-5¢), 55.14
(OCH3), 41.53 (C-2¢), 37.96 [(CH3)3SiCH2CH2N], 25.59
{[(CH3)3C]Si of TBDMS group}, 17.81 [(CH3)3C of
TBDMS group], 15.87 [(CH3)3SiCH2CH2N], 12.54 (CH3 T),
–1.89 [(CH3)3SiCH2CH2N], –4.98 [Si(CH3)2 of TBDMS
group]. MS: m/z calcd for C42H58N2O7Si2: 758.38. ESI-MS
found: m/z = 759.61 [M + H+]; m/z = 781.53 [M + Na+];
m/z = 797.50 [M + K+].
The amount of 900 mg (1.37 mmol) of 5¢-O-tert-butyl-
dimethylsilyl-3¢-O-(4,4¢-dimethoxytriphenylmethyl)thy-
midine was dissolved in 10 mL of benzene at 0 °C and
treated with 38 mL (1.02 mmol) of 2-(phenylthio)ethanol and
422 mL (1.71 mmol) of tributylphosphine. After 10 min the
reaction mixture was taken to r.t., treated with 431 mg (1.71
mmol) of ADDP and left at r.t. for 18 h. The crude was then
taken to dryness, redissolved in 100 mL of EtOAc and
washed twice with H2O. The organic layer was concentrated
under reduced pressure and purified by silica gel
chromatography [eluent system: 2% acetone in CHCl3–
pyridine (1:0.05)], affording 980 mg (1.23 mmol, 90%) of
pure 3.
(14) Typical Procedure for the Insertion of 2-(Trimethyl-
silyl)ethyl Group: Synthesis of 2
The amount of 1.0 g (1.52 mmol) of 5¢-O-(4,4¢-dimethoxy-
triphenylmethyl)-3¢-O-(tert-butyldimethylsilyl)thymidine
was dissolved in 10 mL of benzene at 0 °C and treated with
160 mL (1.14 mmol) of 2-(trimethylsilyl)ethanol and 470 mL
(1.90 mmol) of tributylphosphine. After 10 min the reaction
mixture was taken to r.t. and treated with 480 mg (1.90
mmol) of 1,1¢-(azodicarbonyl)dipiperidine (ADDP).
After 18 h, the crude was taken to dryness, redissolved in
100 mL of EtOAc and washed twice with H2O. The organic
layer was concentrated under reduced pressure and purified
by silica gel chromatography [eluent system: 2% MeOH in
CHCl3–pyridine (1:0.05)], affording 745 mg (0.98 mmol,
65%) of pure 2.
(19) Selected Data for Compound 5.
31P NMR (161.98 MHz, CDCl3): 149.3. MS: calcd for
C48H57N4O10PS: 912.35. ESI-MS (positive ions) found:
m/z = 935.89 [M + Na+]; m/z = 952.90 [M + K+].
(20) Removal of the 2-(phenylsulfonyl)ethyl group by aq NH3 at
55 °C after an overnight treatment was only partial,
requiring longer reaction times to go to completion (ca.
72 h). On the other hand, a 0.1 M NaOH solution ensured
complete thymine deprotection after an overnight treatment
at 50 °C, i.e. conditions usually adopted also for oligo-
nucleotide deprotection and detachment from the solid
support.
(15) (a) de Champdoré, M.; De Napoli, L.; Di Fabio, G.; Messere,
A.; Montesarchio, D.; Piccialli, G. Chem. Commun. 2001,
2598. (b) De Napoli, L.; Di Fabio, G.; D’Onofrio, J.;
Montesarchio, D. Synlett 2004, 1975.
(16) Smrt, J. Collect. Czech. Chem. Commun. 1974, 39, 972.
(21) Data for 5¢DBBTGGGAG3¢OH
MS: m/z calcd for C81H92N27O36P5: 2173.49; MALDI-TOF
(negative ions) found: 2171.76 [M – H]–.
Synlett 2006, No. 6, 845–848 © Thieme Stuttgart · New York