1,2-Ethylene-3,3-bis(4′,4″-dimethoxytrityl chloride) (E-DMT): Synthesis and applications of a novel protecting reagent

Article abstract of DOI:10.1055/s-2004-820033

1,2-Ethylene-3,3-bis(4′,4″-dimethoxytrityl chloride), (E-DMT) was developed as a novel, bifunctional protecting reagent. This new compound was found to have a potential as a multipurpose acid-labile protecting reagent which can afford a 5′,5′-tritylthymidine dimer and a unique 5′,3′-cyclic protected thymidine derivative in modest to good yields.

Full text of DOI:10.1055/s-2004-820033

LETTER
823
1,2-Ethylene-3,3-bis(4¢,4¢¢-dimethoxytrityl Chloride) (E-DMT): Synthesis and
Applications of a Novel Protecting Reagent
1
, 2-Ethylene-3,3-B
a
is(4
¢
,4¢¢
-
Di
t
methox
s
ytrityl Ch
u
loride) hisa Oka,a Yogesh S. Sanghvi,*b Emmanuel A. Theodorakisa
a
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358,
USA
b
Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA
Fax +1(760)9441543; E-mail: rasayan@sbcglobal.net
Received 5 January 2004
improved synthetic protocols5,8 and the development of
new protecting reagents.9
Abstract: 1,2-Ethylene-3,3-bis(4¢,4¢¢-dimethoxytrityl
chloride),
(E-DMT) was developed as a novel, bifunctional protecting re-
agent. This new compound was found to have a potential as a mul-
tipurpose acid-labile protecting reagent which can afford a 5¢,5¢-
tritylthymidine dimer and a unique 5¢,3¢-cyclic protected thymidine
derivative in modest to good yields.
Despite the mentioned advantages, application of com-
pound 1 in nucleic acid chemistry is limited by its suscep-
tibility to base treatment. This limitation is particularly
significant for large-scale solution-phase synthesis of oli-
gonucleotides where extensive treatment with ammonium
hydroxide is required. We envisioned that this issue could
be addressed if the two-trityl groups could be linked to-
gether via an all carbon bridge. This modification would
lead to a chemically stable bis(trityl) group amenable for
oligonucleotide synthesis. Moreover such a protecting
group could be recovered and recycled to make the pro-
cess environmentally friendly.10 Our continued interest in
the development of novel protecting groups11 prompted us
to explore the synthesis of 1,2-ethylene-3,3-bis(4¢,4¢¢-
dimethoxytrityl chloride) (E-DMT, 2; Figure 1). Herein,
we wish to report our results on the expeditious synthesis
of E-DMT and show the preliminary results of potential
applications of 2 as a protecting reagent.
Key words: bis(trityl chloride), bifunctional protecting reagent,
acid-labile protecting group, solution-phase DNA synthesis,
nucleoside, thymidine
Triphenylmethyl (trityl) group and its derivatives have
been widely used for the protection of hydroxyl, amino,
and carboxyl groups. The usefulness and importance of
the trityl group are clearly demonstrated by (i) highly se-
lective protection of less-hindered functional groups, (ii)
stability under neutral or alkaline conditions, (iii) cleav-
age under mild acidic conditions, (iv) remarkable hydro-
phobicity, and (v) sensitive detection of the corresponding
trityl cation by visual or spectrophotometric methods.1
Due to these advantages, the trityl groups and in particular
its 4¢,4¢¢-dimethoxy trityl (DMT) analogue are widely
used for the protection of the 5¢-hydroxyl group during
DNA and RNA synthesis.2
A general route for the synthesis of trityl derivatives is
based on the Grignard reaction between an aryl halide and
a carboxylic ester. This method could be implemented for
In addition to these conventional monofunctional trityl
groups, some bis(trityl chloride)s having two trityl chlo-
rides connected with a bifunctional ester linkage have
been reported by Köster et al. as a scaffold for solution-
phase DNA synthesis (1, Figure 1).3 These bifunctional
trityl chlorides4 can react with 2 equivalents of a thymi-
dine derivative at the 5¢-OH group allowing synthesis of
two strands of DNA via the 3¢-ends. This method signifi-
cantly differentiates the trityl bearing two oligonucleotide
chains from the monomers in molecular size to enable a
facile purification of the trityl bearing oligonucleotide by
size exclusion gel permeation chromatography. Recently,
a solution-phase approach has been evaluated as a scal-
able route for the large-scale synthesis of therapeutic oli-
gonucleotides.5 The growing demand for synthetic DNA
and RNA and its analogues as therapeutics6 and reagents
for diagnostic7 applications has triggered a need for
base-labile
R
R
O
Cl
O
O
Cl
O
R
1: R = OMe
R
stable under basic conditions
OMe
OMe
Cl
Cl
MeO
MeO
2: E-DMT
SYNLETT 2004, No. 5, pp 0823–0826
0
2.
0
4.
2
0
0
4
Advanced online publication: 10.03.2004
DOI: 10.1055/s-2004-820033; Art ID: S00104ST
© Georg Thieme Verlag Stuttgart · New York
Figure 1 Chemical structures of bis(trityl chloride)s 1 and 2.

824
N. Oka et al.
LETTER
the synthesis of bis(trityl chloride)s using the appropriate The latter compound 5b was generated in variable
bis(benzoate ester)s (Scheme 1). We decided to link the amounts (ca. 20–30%) when the reaction was carried out
two benzoate esters at the m-position since substituents at at room temperature. This side reaction could be
o- or p-positions are known to alter significantly the suppressed when the reaction temperature was kept at
stability of the resulting trityl ether. For example, under 5–10 °C.
acidic conditions p-alkyl substituted DMT ethers are
Next, we chose p-anisylmagnesium bromide for the
Grignard reaction, which would give an analogue of 4¢,4¢¢-
dimethoxytrityl chloride (DMTCl) as the final product.
reported to be twice less stable than the parent DMT ether
analogs.12
The above considerations led us to explore esters of the DMTCl is a well-studied and established protecting re-
1,2-ethylene-3,3-bis benzoic acid (4a or 4b) as synthetic agent for oligonucleotide synthesis, and we assumed that
precursors for a new DMT dimer. Such a motif was ex- the stability of the resultant ether E-DMT (2) could
pected to meet the above criteria and could also be easily probably be similar to the DMT protected nucleosides.
accessible from coupling of the corresponding benzyl The addition of a solution of p-anisylmagnesium bromide
bromide monomers (3a or 3b).13
in THF to a solution of 4 in THF under a variety of condi-
tions (–78 °C, 0 °C, r.t., or inverse addition) resulted in
the formation of some unidentified by-products (stained
orange with H2SO4–MeOH on TLC). Gratifyingly, the
desired product 6 was obtained in nearly quantitative yield
when a solution of p-anisylmagnesium bromide in THF
was added drop wise to a solution of 4 in THF under re-
flux to ensure that the Grignard reagent was consumed
immediately. The crude diol 6 was used for next step (iii)
without further purification.
CO2R
CO2R
CO2R
(i)
Br-
+
(52–63%)
CH2Br
+
PPh3
5
3a (R = Et)
3b (R = Me)
CO2R
Treatment of 6 with AcCl under reflux for 1.5 h, followed
by precipitation from cyclohexane at 4 °C afforded E-
DMT (2) as an orange powder.18 E-DMT (2) was stored at
–20 °C for at least six months without noticeable degrada-
tion.
4a (R = Et)
4b (R = Me)
(ii)
OMe
OMe
To evaluate the reactivity of 2 toward nucleosides, com-
pound 2 was treated with 2 molar equivalents of thymi-
dine in pyridine at room temperature (Scheme 2, reaction
A). To ensure its quality prior to use, 2 was treated with
(COCl)2 for 1 hour to rechlorinate any trace amounts of
trityl carbinol 6, that may be formed due to adverse con-
ditions. After removal of (COCl)2 under vacuum, 2 was
suspended in dry pyridine, and to this dry thymidine (co-
evaporated with pyridine) was added in one portion. The
symmetric dimer 7,19 having two thymidines attached to
the E-DMT motif, was obtained in 53% yield. Under these
conditions we also isolated the monosubstituted adduct in
19% yield. Incomplete conversion of the latter compound
to dimer 7 may be due to the residual moisture and a
slower reaction rate for the incorporation of the second
thymidine.
X
X
MeO
MeO
6 (X = OH)
2 (X = Cl)
(iii)
(70–88% from 4)
Scheme 1 Synthesis of E-DMT 2. Reagents and conditions: (i)
CoCl(PPh3)3 (1.2 equiv), degassed benzene, 5–10 °C, 1 h, 52–63%;
(ii) p-anisylmagnesium bromide (5 equiv), THF, reflux, 30 min; (iii)
AcCl, reflux, 1.5 h then precipitated from C6H12, 70–88% from 4.
The synthesis of E-DMT (2) is shown in Scheme 1. 3-
Bromomethylbenzoic acid ethyl ester (3a) has been re-
ported for the synthesis of 4a.13 However, 3a is not com-
mercially available and its reported synthesis is based on
a carefully controlled bromination of ethyl m-toluate.14 To
circumvent this issue, we also used bromomethyl m-tolu-
ate (3b) which is commercially available.15 Both 3a and
3b gave almost identical results in the step i and ii
(Scheme 1).
In addition to the above products, a less-polar by-product
was detected (TLC). Interestingly, the structure of this
product was confirmed as 5¢,3¢-cyclic thymidine 8 (1H
NMR, 13C NMR and MS).20 This unexpected by-product
is very attractive since selective protecting reagents for
5¢,3¢-hydroxyl groups of ribonucleosides are significantly
important for the synthesis of base- and/or 2¢-modified
ribonucleosides derivatives.11,21 Regardless of the impor-
tance, there are only a few bifunctional protecting re-
agents that are selective for the 5¢,3¢-hydroxyl groups of
ribonucleosides. To the best of our knowledge, there is no
cyclic protecting group, which is labile under mild, anhy-
drous acidic conditions, for 5¢,3¢-hydroxyl groups of
The yield of step i was dependent on the purity of
CoCl(PPh3)3.16 We found that reproducible results were
obtained by using freshly prepared CoCl(PPh3)3.17 The
competing by-product of this reaction was [(3-alkoxycar-
bonylphenyl)methyl]triphenylphosphonium bromide (5).
Synlett 2004, No. 5, 823–826 © Thieme Stuttgart · New York

LETTER
1,2-Ethylene-3,3-bis(4¢,4¢¢-dimethoxytrityl Chloride)
825
ribonucleosides. To improve the yield of 8 we tested a
variety of conditions. Best results were obtained by drop
wise addition of a pyridine solution of thymidine to a so-
lution of 2 in pyridine at 65 °C. Using these conditions 8
was obtained in 65% yield. The optimized conditions fur-
nished the best results (Scheme 2, reaction B). We postu-
late that this ‘bending’ property of 2 is due to the flexible
ethylene bridge that may undergo free rotation to react
with the neighboring 3¢-OH group under appropriate
conditions.
Acknowledgment
We gratefully acknowledge ISIS Pharmaceuticals, Inc. for partially
supporting this research.
References
(1) For a review, see: Greene, T. W.; Wuts, P. G. M. Protective
Groups in Organic Synthesis, 3rd Ed.; Wiley: New York,
1999, 102–112.
(2) (a) For a review, see: Seliger, H. In Current Protocols in
Nucleic Acid Chemistry; Beaucage, S. L.; Bergstrom, D. E.;
Glick, G. D.; Jones, R. A., Eds.; Wiley: New York, 2000,
Chap. 2.3.1-2.3.34. (b) Successful use of trityl groups as
acid-labile 5¢-protecting groups was originally reported by
Khorana et al. in: Gilman, P. T.; Khorana, H. G. J. Am.
Chem. Soc. 1958, 80, 6212–6222. (c) See also: Smith, M.;
Rammler, D. H.; Goldberg, I. H.; Khorana, H. G. J. Am.
Chem. Soc. 1962, 84, 430.
To evaluate the lability of this new protecting group, 7 and
8 were treated with 3% dichloroacetic acid–CH2Cl2, a
standard detritylating reagent for DMT groups, at room
temperature.2 In both cases, the protecting group was
completely removed within 3 minutes to give thymidine
quantitatively as in the case of DMT group.
(3) Biernat, J.; Wolter, A.; Köster, H. Tetrahedron Lett. 1983,
24, 751.
(4) For a review of the recent studies on bifunctional trityl
chlorides, see: Shchepinov, M. S.; Korshun, V. A. Chem.
Soc. Rev. 2003, 32, 170.
(5) (a) Bonora, G. M.; Rossin, R.; Zaramella, S.; Cole, D. L.;
Eleuteri, A.; Ravikumar, V. T. Org. Proc. Res. Dev. 2000, 4,
225. (b) Reese, C. B.; Yan, H. J. Chem. Soc., Perkin Trans.
1 2002, 2619.
(6) Sanghvi, Y. S.; Andrade, M.; Deshmukh, R. R.; Holmberg,
L.; Scozzari, A. N.; Cole, D. L. In Manual of Antisense
Methodology; Hartmann, G.; Endres, S., Eds.; Kluwer
Academic Publishers: Norwell, MA, 1999, 3–23.
(7) Pon, R. T. Current Protocols in Nucleic Acid Chemistry;
Beaucage, S. L.; Bergstrom, D. E.; Glick, G. D.; Jones, R.
A., Eds.; Wiley: New York, 2000, Chap. 3.1.1-3.1.28, and
references therein.
In conclusion, a novel 1,2-ethylene-3,3-bis(DMTCl) (E-
DMT, 2) has been synthesized as a multipurpose, flexible
bifunctional protecting reagent. Under optimized condi-
tions, compound 2 reacted with 2 equivalents of thymi-
dine to afford the di(thymidine-5¢-yl)trityl ether 7. This
property of 2 may be useful as a purification handle during
solution-phase oligonucleotide syntheses and is currently
under investigation. We also demonstrated that 2 reacted
with 1 equivalent of thymidine to afford the 5¢,3¢cyclic
thymidine 8. We believe that the unusual ability of E-
DMT group to ‘stretch’ or ‘bend’ is unique and could be
further exploited for more complex synthetic manipula-
tions.22
OMe
(8) Mihaichuk, J. C.; Hurley, T. B.; Vagle, K. E.; Smith, R. S.;
Yegge, J. A.; Pratt, G. M.; Tompkins, C. J.; Sebesta, D. P.;
Pieken, W. A. Org. Proc. Res. Dev. 2000, 4, 214.
(9) (a) Ravikumar, V. T.; Wyrzykiewicz, T. K.; Cole, D. L.
Tetrahedron 1994, 50, 9255. (b) Krotz, A. H.; Cole, D. L.;
Ravikumar, V. T. Tetrahedron Lett. 1996, 37, 1999.
(c) Ravikumar, V. T.; Cheruvallath, Z. S.; Cole, D. L.
Nucleosides Nucleotides 1997, 16, 1709. (d) Bhat, B.;
Sanghvi, Y. S. Tetrahedron Lett. 1997, 38, 8811.
(10) (a) Krotz, A. H.; Carty, R. L.; Scozzari, A. N.; Cole, D. L.;
Ravikumar, V. T. Org. Proc. Res. Dev. 2000, 4, 190.
(b) Pon, R. T.; Yu, S.; Guo, Z.; Deshmukh, R.; Sanghvi, Y.
S. J. Chem. Soc., Perkin Trans. 1 2001, 2638. (c) Sanghvi,
Y. S.; Guo, Z.; Pfundheller, H. M.; Converso, A. Org. Proc.
Res. Dev. 2000, 4, 175.
O
MeO
Me
NH
O
reaction B
N
O
O
thymidine
(1 equiv)
2
O
pyridine
65 °C, 5 h
OMe
reaction A
OMe
thymidine (2 equiv)
/pyridine, r.t., 5 h
8
(11) (a) Wen, K.; Chow, S.; Sanghvi, Y. S.; Theodorakis, E. A. J.
Org. Chem. 2002, 67, 7887. (b) Chow, S.; Wen, K.;
Sanghvi, Y. S.; Theodorakis, E. A. Bioorg. Med. Chem. Lett.
2003, 13, 1631.
(12) Ramage, R.; Wahl, F. O. Tetrahedron Lett. 1993, 34, 7133.
(13) Goswami, S.; Mahapatra, A. K. Tetrahedron Lett. 1998, 39,
1981.
65% yield
O
OMe
OMe
Me
Me
HN
O
N
O
O
N
NH
O
(14) Windisch, B.; Vögtle, F.; Nieger, M.; Lahtinen, T.;
Rissanen, K. J. Prakt. Chem. 2000, 342, 642.
OH
O
O
O
(15) Compound 3b was purchased from TCI
MeO
OH
(www.tciamerica.com).
MeO
(16) The purity of the complex can roughly be checked by the
color of the complex and/or the solution of it since the green
color of Co(I)Cl(PPh3)3 is changed into blue color of Co(II)
species generated by degradation.
7
53% yield
Scheme 2 Reactions of E-DMT (2) with thymidine.
(17) Wakatsuki, Y.; Yamazaki, H. Inorg. Synth. 1989, 26, 189.
Synlett 2004, No. 5, 823–826 © Thieme Stuttgart · New York

826
N. Oka et al.
LETTER
(18) Experimental for Synthesis of 2: Ethyl ester 4a (1.96 g, 6.0
mmol) was dried by repeated coevaporations with dry
toluene and dissolved in dry THF (30 mL) under argon.
Freshly prepared 1 M p-anisyl magnesium bromide solution
in dry THF was added drop wise to the mixture under reflux
and stirred for 30 min. The mixture was cooled to r.t., diluted
with Et2O (150 mL), and washed with sat. NH4Cl aq solution
(150 mL). The aqueous layer was back-extracted with Et2O
(2 × 150 mL). The combined organic layers were washed
with sat. NaCl aq solution (150 mL), dried over MgSO4,
filtered, and concentrated to dryness. The residue was dried
by repeated coevaporations with dry toluene and dissolved
in AcCl (3 mL). The mixture was heated under reflux for 1.5
h, and then cooled to r.t. The mixture was added to
(19) Analytical data for 7: 1H NMR (400 MHz, DMSO-d6): d =
11.3 (brs, 2 H), 7.50 (s, 2 H), 7.19–7.14 (m, 14 H), 6.94–6.93
(m, 2 H), 6.85–6.82 (m, 8 H), 6.20 (dd, J = 6.7, 6.7 Hz, 2 H),
5.32 (d, J = 4.4 Hz, 2 H), 4.33 (m, 2 H), 3.84 (m, 2 H), 3.71
(s, 12 H), 3.12 (m, 4 H), 2.72 (s, 4 H), 2.24 (ddd, J = 13.5,
6.7, 6.7 Hz, 2 H), 2.15 (ddd, J = 13.5, 6.7, 3.2 Hz, 2 H), 1.38
(s, 6 H). 13C NMR (100 MHz, DMSO-d6): d = 163.4, 157.8,
157.8, 150.1, 143.8, 140.4, 135.6, 135.4, 135.2, 129.5,
129.3, 127.8, 127.6, 126.7, 125.3, 113.0, 109.4, 85.7, 85.4,
83.6, 79.1, 70.5, 63.6, 55.0, 37.1, 11.7. HRMS: calcd for
C64H65N4Na2O14+ (M – H+ +2 Na+) 1159.4287; found:
1159.4342.
(20) Analytical data for 8: 1H NMR (400 MHz, CDCl3): d = 8.04
(brs, 1 H), 7.54 (s, 1 H), 7.37–6.69 (m, 24 H), 6.56 (dd,
J = 9.2, 5.2 Hz, 1 H), 4.34 (m, 1 H), 4.04 (m, 1 H), 3.80 (s, 3
H), 3.78 (s, 3 H), 3.76 (s, 3 H), 3.72 (s, 3 H), 3.15–2.57 (m,
6 H), 2.27 (dd, J = 10.8, 3.2 Hz, 1 H), 2.11 (m, 1 H), 1.41 (s,
3 H). 13C NMR (100 MHz, CDCl3): d = 163.3, 158.6, 158.2,
158.1, 157.8, 150.0, 141.6, 141.5, 140.5, 140.1, 139.8,
138.5, 136.9, 135.6, 133.9, 130.9, 130.8, 129.4, 129.3,
129.3, 128.2, 127.7, 127.7, 127.6, 127.4, 127.3, 127.1,
113.2, 113.1, 113.0, 112.9, 110.8, 87.6, 86.6, 86.2, 85.5,
75.5, 55.3, 55.3, 55.3, 55.2, 40.4, 37.6, 36.8, 11.8. HRMS:
calcd for C54H53N2O9 (M + H+) 873.3745; found: 873.3732.
(21) (a) Furusawa, K.; Katsura, T. Tetrahedron Lett. 1985, 26,
887. (b) Beijer, B.; Grøtli, M.; Douglas, M. E.; Sproat, B. S.
Nucleosides Nucleotides 1994, 13, 1905. (c) Wada, T.;
Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M.
Tetrahedron Lett. 1995, 36, 1683.
cyclohexane (30 mL), stirred for 5 min, and kept at 4 °C for
20 h. The mixture was warmed to r.t. and the resultant
precipitate was collected by suction filtration. The collected
solid was washed with dry cyclohexane (5 × 3 mL) and dried
under vacuum to afford 2 (2.97 g, 4.22 mmol, 70%) as an
orange powder. 1H NMR (400 MHz, CDCl3): d = 7.16–7.11
(m, 4 H), 7.08 (d, J = 8.8 Hz, 8 H), 7.01 (d, J = 7.6 Hz, 2 H),
6.94 (d, J = 8.8 Hz, 2 H), 6.78 (d, J = 8.8 Hz, 8 H), 3.80 (s,
12 H), 2.86 (s, 4 H). 13C NMR (100 MHz, CDCl3): d = 158.7,
145.4, 140.6, 137.6, 130.9, 129.9, 127.9, 127.3, 127.1,
112.8, 82.5, 55.3, 37.5. ESI-MS: m/z for C44H41Cl2O4 (M +
H+) 703. Anal. Calcd. for C44H40Cl2O4: C, 75.10; H, 5.73; Cl,
10.08. Found: C, 74.68; H, 6.07; Cl, 9.51.
(22) E-DMT is commercially available from Sai Dru Syn
Laboratories, Hyderabad, India (www.saiintgroup.com).
Synlett 2004, No. 5, 823–826 © Thieme Stuttgart · New York