Synthesis of novel tetrazole C5-linked C0- and C 2-ribonucleoside phosphoramidites using MePOM and POM groups for probing RNA catalysis

Article abstract of DOI:10.1016/j.tetlet.2012.08.082

Novel C5-linked C₀- and C₂-tetrazole ribonucleoside phosphoramidites were designed and synthesized via tetrazole C-nucleosides. Pivaloyloxymethyl (POM) and methyl-substituted POM (MePOM) groups were introduced as N-protecting groups in the tetrazole ring that can be readily removed under mild basic conditions. The phosphoramidites were successfully incorporated into the VS ribozyme substrate and hence providing a chemogenetic approach to determine which nucleobases of ribozymes function as the acid or base, in the studies of ribozyme general acid and base catalysis.

Full text of DOI:10.1016/j.tetlet.2012.08.082

Tetrahedron Letters 53 (2012) 5891–5894
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of novel tetrazole C5-linked C0- and C2-ribonucleoside
phosphoramidites using MePOM and POM groups for probing RNA catalysis
Shinya Harusawa a, , Hiroki Yoneyama a, Daiki Fujisue a, Masayoshi Nishiura a, Mihoyo Fujitake a,
⇑
Yoshihide Usami a, Zheng-yun Zhao b, Scott A. McPhee c, Timothy J. Wilson c, David M. J. Lilley c
a Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
b School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
c CR-UK Nucleic Acid Structure Group, MSI/WTB Complex, University of Dundee, Dundee DDI 5EH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel C5-linked C0- and C2-tetrazole ribonucleoside phosphoramidites were designed and synthesized
via tetrazole C-nucleosides. Pivaloyloxymethyl (POM) and methyl-substituted POM (MePOM) groups
were introduced as N-protecting groups in the tetrazole ring that can be readily removed under mild
basic conditions. The phosphoramidites were successfully incorporated into the VS ribozyme substrate
and hence providing a chemogenetic approach to determine which nucleobases of ribozymes function
as the acid or base, in the studies of ribozyme general acid and base catalysis.
Received 1 June 2012
Revised 14 August 2012
Accepted 22 August 2012
Available online 29 August 2012
Keywords:
Tetrazole
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphoramidite
Protecting group
Chemogenetic approach
Ribozyme
RNA catalysis occurs in cells for the processing of RNA molecules,
the control of gene expression, and even the synthesis of proteins;
yet the catalysis mechanisms are still unclear in most cases.1 The
Varkud satellite (VS) ribozymes are the largest of a group of nucle-
olytic ribozymes that include hammerhead, hairpin, HDV, and GlmS
ribozymes.2 While no crystal structure of VS ribozymes has been
determined yet, a small-angle X-ray scattering-derived model
places G638 and A756 in proximity to the scissile phosphate.2d
We have recently developed a novel chemogenetic approach for
the analysis of general acid–base catalysis by nucleobases in ribo-
zymes.3 This involves substitution of a C4 linked-ribonucleoside
with imidazole (pKa: 7.1)4 being a good donor and acceptor of
proton in place of an important nucleobase in VS and hairpin
ribozymes.5 In this study, N-pivaloyloxymethyl (POM) 20-O-
tBDMS-imidazole C0-phosphoramidite (PA) 25a,b was first
employed to yield an imidazole-substituted VS ribozyme by
covalently placing imidazole as a pseudonucleobase at position
756 (replacing adenine) of the VS ribozyme (Fig. 1).3a The imidaz-
ole ribozyme (A756Imz) catalyzed the almost complete cleavage of
substrate stem-loops at the correct position; this confirms the di-
rect role of the nucleobase at position 756 in the chemistry of
the natural VS ribozyme.3 The overall yield of 2 tends to be rather
limited due to the lack of selective introduction of the tBDMS
group for 20-hydroxy group as well as due to its acid-labile charac-
ter.5a,b Therefore, a new combination of protecting groups was
introduced for the carbon-elongated homologs PAs 3 (n = 0–3):
POM for imidazole-N and cyanoethyl (CE) groups for the 20-hydro-
xy group (Fig. 1).5c,e In addition, PAs 3a–d are much more stable
than PA 2, making the purification steps easier. Further, it is of par-
ticular interest that the C2-imidazole VS ribozyme (G638C2Imz)
incorporated from PA 3c (n = 2) showed a 15-fold greater catalytic
5c
activity than the C0-imidazole ribozyme (G638C0Imz) These re-
sults definitely demonstrated that the key functionalities in the
catalytic mechanism of VS ribozyme are G638 and A756 that con-
certedly act in general acid–base catalysis. However, it is impossi-
ble by this approach to determine which nucleobases specifically
function as the acid or base when imidazole is employed as a
nucleobase.
5-Aliphatic-1H-tetrazoles with pKa of 4.5–4.9 are often used as
metabolism-resistant isomeric replacements for carboxylic acids in
medicinal chemistry.6 Given that, the synthesis of tetrazole C-
nucleoside PAs may allow the incorporation of an acid surrogate
at the G638 or A756 position to specify the role of the nucleobase
as a general acid in the chemistry of natural VS ribozyme. However,
the tetrazole-related C-nucleosides have not been extensively
studied so far.7 We herein describe the synthesis of a novel
C5-linked C0- and C2-tetrazole ribonucleoside PAs 1a and 1b
(Fig. 1). The latter, with a flexible linker, may lead to greater cata-
lytic activity at the G638 position of VS ribozyme. In this study,
⇑
Corresponding author. Tel./fax: +81 72 690 1086.
E-mail address: harusawa@gly.oups.ac.jp (S. Harusawa).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.08.082

5892
S. Harusawa et al. / Tetrahedron Letters 53 (2012) 5891–5894
O
Me
O
N
O
N
N
N
N
O
N
N
(POM)
N
DMTO
DMTO
iPr2N
(CH2)2
Si
O
O
(MePOM)
iPr2N
NC
O
O
O
O
P
P
(TBDMS)
CH2CH2CN
(CE)
O
O
NC
1a
1b
N
N-POM
2 : R = TBDMS, n = 0
DMTO
(CH2)n
R
O
3
3a
3b
: R = CH2CH2CN, n = 0 (
n = 1 (
)
)
iPr2N
NC
O
O
n = 2 (3c)
n = 3 (3d)
P
O
Figure 1. Structures of imidazole and tetrazole Cn-ribonucleoside PAs employed for solid-phase RNA synthesis.
POM and a novel methyl-substituted POM (MePOM) groups were
used as protecting groups for the tetrazole-N. In addition, we dem-
onstrate initial results about the efficient syntheses of RNA oligo-
nucleotides using PAs 1a and 1b.
N-MePOM-tetrazole C-nucleoside 7 (98%). It should be noted that
MePOM group tolerates the debenzylation-condition. In addition,
to our knowledge, the MePOM group has not been employed as
an N-protecting group. 30,50-O-TIPDS-protection (1,1,3,3-tetra-
isopropyldisiloxanediyl) of 7 gave 8 (86%), and this allowed selec-
tive introduction of the 20-hydroxy protecting group.
Cyanoethylation of 8 with acrylonitrile gave fully protected inter-
mediate 9 (62%).9 The TIPDS group of 9 was selectively removed
by treatment with Et3Nꢀ3HF to give 30,50-O-unprotected ribonu-
cleoside derivative 10 (95%). Standard 50-O-dimethoxytritylation
(80%) of 10 followed by phosphitylation of 30 hydroxy afforded
tetrazole C0-PA 1a as a mixture of four diastereomers (67%).10
C2-tetrazole ribonucleoside PA 1b was synthesized from alde-
We previously reported an efficient synthesis of b-
anosyl cyanide 4, in 56% overall yield from commercially avail-
able 2,3,5-tri-O-benzyl-
-ribose in two steps (Scheme 1).5c
D-ribofur-
D
Tetrazole C-ribonucleoside 5 (n = 0) was prepared in quantitative
yield by the treatment of nitrile 4 with sodium azide and trieth-
ylammonium chloride in DMF. This reaction proceeded suffi-
ciently rapidly due to the activation of the neighboring
electron-withdrawing oxygen in the sugar moiety. We first
examined the use of the POM group as a protecting group for
tetrazole-N of 5. The POM group was used in the synthesis of
imidazole Cn-PAs and was removed in the final step of RNA
synthesis by a mixture of ammonia and ethanol with other
base-protecting groups.5a,b However, the introduction of the
POM group into tetrazole 5 resulted in a 1:1 inseparable mixture
11ab (74%) of N-1 and N-2 POM-protected tetrazoles (Scheme 1,
the bottom left). The subsequent synthetic processes from 11ab
suffered a setback owing to the formation of further complicated
mixtures. We then tried MePOM protection via 1-chloroethyl
pivaloate (MePOMCl),8 with expectation of N-2 protection caused
preferentially by stereo hindrance of the methyl group. Indeed,
the reaction of tetrazole C-nucleoside 5 with MePOMCl in the
presence of DMAP in DMF afforded N-1 and N-2 MePOM deriva-
tives 6a (24%) and 6b (60%), which could be isolated as diastere-
oisomers by column chromatography because of the additional
stereogenic center present in the MePOM group (which, of course,
could easily be identified from NMR spectra). The location of the
MePOM group on the tetrazole ring was assigned, since the 13C
NMR signal from the only carbon in tetrazole was generally about
10 ppm deshielded in 2,5-disubstituted derivatives relative to the
corresponding 1,5-disubstituted isomers [13C NMR shift of d 5-C
(ppm) in CDCl3: 6a, 153.4; 6b, 165.0].6d MePOM groups of 6a
and 6b could be removed quantitatively by aqueous ammonia/
EtOH (MeOH) (1:3, v/v) at room temperature (rt) in 17 h to con-
vert back into 5. Removal of the MePOM group from tetrazole-N
proceeded somewhat slowly compared to the removal of the
POM group at imidazole-N (rt, 3 h).5a,b In contrast, stirring of 6b
in 2N HCl–EtOH (1:3, v/v) at rt for 17 h resulted in recovery of
6b in 94% yield, indicating the stability of the N-MePOM group
under an aqueous acidic condition. Subsequent treatment of 6b
with Pd(OH)2-C/cyclohexene in refluxing ethanol produced
hyde 12 that was prepared in two steps (92%) from
4
(Scheme 1).5c Wittig olefination of 12 using (cyanomethyl)triphen-
ylphosphonium chloride gave the vinylnitrile (73%) and then
reduction of the formed double bond yielded the two-carbon-elon-
gated nitrile 13 (94%). Conversion of the inactive alkylcyanide 13
into tetrazole 14 in the presence of sodium azide and triethylam-
monium chloride proceeded slowly (40 h, 130 °C, DMF) in a mod-
erate yield (57%), while microwave (MW) irradiation (130 °C,
DMF) led to both a shorter reaction time (2 h) and an improved
yield (95%). The treatment of 14 with POMCl (DBU, 100 °C, 0.5 h,
DMF, MW) yielded N-1 and N-2 POM derivatives 15a (40%) and
15b (55%), respectively, which could be easily separated by column
chromatography. Meanwhile, MePOM-protection of 14 also gave
almost the similar result. The N-2 POM isomer 15b was used for
the subsequent synthetic process because it showed a clearer 1H
NMR peak pattern than 15a did. The base-labile POM 15a could
be transformed back to tetrazole 14 quantitatively for reuse, while
15a remained intact under 2N HCl/EtOH (1:2, v/v; rt, 5 h). There is
little information regarding N-POM tetrazoles in the literature,11
and their role as a protecting group has not yet been fully clarified.
Debenzylation of 15b produced N-2-POM-tetrazole C2-ribonucleo-
side 16 (91%) and subsequent 30,50-O-DTBS-protection (88%,
DTBS = di-tert-butylsilanediyl) afforded 17 (88%). Although 20-O-
TBDMS protection of 17 provided 20-O-silylated derivative 18
(98%), cyanoethylation of 17 afforded only a POM removed tetra-
zole 20 (5%) (Scheme 1). Alternatively, 30,50-O-TIPDS protection of
16 gave 21 (quant), but the cyanoethylation of 21 to the corre-
sponding 22 could not be obtained at all (Scheme 1). Selective re-
moval (90%) of the DTBS group in 18, DM-tritylation (quant), and
phosphitylation (87%) proceeded successfully to give the final 20-
12
O-TBDMS tetrazole C2-PA 1b.

S. Harusawa et al. / Tetrahedron Letters 53 (2012) 5891–5894
5893
N
N
N
N
H
(CH2)n
(CH2)n
d
CN
BnO
BnO
O
O
c
e
O
BnO OBn
BnO OBn
(quant)
BnO
O
H
Ref. 5c
12
4
(n = 0)
5: n = 0 (quant)
14: n = 2 (95%)
BnO
OBn
13 (n = 2)
a (73%), b (94%)
N
N
N
N
N
N
N
N
N R
N
N
R
N
(CH2)n
(CH2)n
(CH2)n
BnO
BnO
HO
O
O
O
R
f
+
(6b, 15b)
BnO OBn
BnO OBn
HO OH
6a
6b: n = 0, R = MePOM (60%)
15b: n = 2, R = POM (55%)
: n = 0, R = MePOM (24%)
: n = 2, R = POM (40%)
7: n = 0, R = MePOM (98%)
16: n = 2, R = POM (91%)
15a
N
N
N
N
N
R1
N
R
N
(CH2)n
O
P
N
(CH2)n
O
O
O
i
g
h
P
O
OR2
O
OH
9: n = 0, R1 = MePOM, R2 = CE, P = TIPDS (62%)
18: n = 2, R1 = POM, R2 = TBDMS, P = DTBS (98%)
8: n = 0, R = MePOM, P = TIPDS (86%)
17: n = 2, R = POM, P = DTBS (88%)
N
N
N
R1
j, k
N
(CH2)n
HO
O
1a (54% from 9)
1b (87% from 18)
HO OR2
10: n = 0, R1 = MePOM, R2 = CE (95%)
19: n = 2, R1 = POM, R2 = TBDMS (90%)
N
POM
N
N
N
N
NH
N
N
N
N(2)
N
O
N
O
O
Si
POM
BnO
O
Si
O
21: R = H (quant from 16)
( 22: R = CE)
(1)
O
Si
O
R
O
O
OCE
BnO
OBn
11ab (74% from 5)
20: (5% from 17)
Scheme 1. Synthesis of tetrazole C0 and C2-PAs 1 and related intermediates. Reagents and conditions: (a) BuLi, (cyanomethyl)triphenylphosphonium chloride, toluene,
ꢁ14 °C, 30 min, then rt, 2 h; (b) 10% Pd/C (cat.), H2, 2Kg/cm2, AcOEt, 1 h; (c) for 4: NaN3 (3.0 equiv), Et3NꢀHCl (3.0 equiv), DMF, 130 °C, 2 h; for 13: NaN3 (3.0 equiv), Et3NꢀHCl
(3.0 equiv), DMF, MW, 130 °C, 2 h; (d) for 5: MePOMCl (4 equiv), DMAP (4 equiv), DMF, 50 °C, 10 h; for 14: POMCl (2 equiv), DBU (2 equiv), DMF, MW, 100 °C, 0.5 h; (e) 28%
aq. NH3/EtOH (MeOH) (1:3, v/v), rt, 14–17 h; (f) for 6b: 20% Pd(OH)2-C, cyclohexene , EtOH, reflux, 25 h; for 15b: 20% Pd(OH)2-C, cyclohexene, EtOH, MW, 80 °C, 0.5 h; (g) for
7: TIPDSCI2, py, 0 °C then rt, 2 h; for 16: tBu2Si(OTf)2, py., rt, 10 min; (h) for 8: CH2@CHCN, Cs2CO3, tBuOH, 40 °C, 10 h; for 17: TBDMSCI, imidazole, DMF, 50 °C 5 h; (i) for 9:
Et3N.3HF, Et3N, THF, rt, 1.5 h; for 18: py.HF, py, CH2Cl2, 0oC, 1h; (j) DMTCl, Et3N, DMAP, py, rt, 2.5 - 3.0 h; (k) Et3Nꢀ3HF, (iPr2N)2POCH2CH2CN, 4,5-DCI, ClCH2CH2Cl, 40 °C, 20–
30 min.
Purification of crude PAs 1a and 1b could be easily carried out
by medium-pressure flash column chromatography (MPLC) using
a standard silica gel column (Fuji Silysia Chemical FL60D, pH
6.6). This suggests that 1a and 1b are considerably more stable
than the acid-sensitive imidazole PAs. Base-protecting groups of
RNA are conventionally removed in the final step of RNA synthesis
by ammonia and ethanol mixture [28% aq NH3/EtOH (3:1, v/v)] at
55 °C for 16 h. As the POM and MePOM groups can be removed un-
der milder basic conditions at rt, it is particularly attractive to sen-
sitive RNA such as Cy5 labeled RNA where deprotection should
preferably be carried out at rt in order to minimize the destruction
of cyanine dye. In addition, MS measurements of tetrazole Cn-PAs
1a and 1b were problematic owing to their labile properties, but
we recently reported that the molecular weight of PAs may be
accurately determined by using a novel matrix system [triethanol-
amine (TEOA)–NaCl] on FABMS equipped with a double-focusing
mass spectrometer.13
The present method successfully revealed the sodium adduct ions
[M+Na]+ of 1a and 1b, and thus, their composition formulas could be
determined.10,12 The overall yields of 1a is 16.0% in 8 steps from ni-
trile 4, and that of 1b is 22.0% in 10 steps from aldehyde 12; making
them feasible approaches for the scaling up of their synthesis.
Preliminary incorporation of these modified tetrazole PAs 1a
and 1b into VS ribozyme substrate stands (25nt) was carried out

5894
S. Harusawa et al. / Tetrahedron Letters 53 (2012) 5891–5894
312, 663; (c) Lafontaine, D. A.; Wilson, T. J.; Zhao, Z.; Lilley, D. M. J. J. Mol. Biol.
2002, 323, 23; (d) Lipfert, J.; Ouellet, J.; Norman, D. G.; Doniach, S.; Lilley, D. M.
J. Structure 2008, 16, 1357.
3. (a) Zhao, Z.; McLeod, A.; Harusawa, S.; Araki, L.; Yamaguchi, M.; Kurihara, T.;
Lilley, D. M. J. J. Am. Chem. Soc. 2005, 127, 5026; (b) Wilson, T. J.; Ouellet, J.;
Zhao, Z.; Harusawa, S.; Araki, L.; Kurihara, T.; Lilley, D. M. J. RNA 2006, 12, 980;
(c) Lilley, D. M. J. Biol. Chem. 2007, 388, 699.
4. Perrotta, A. T.; Shih, I.; Been, M. D. Science 1999, 286, 123.
5. (a) Araki, L.; Harusawa, S.; Yamaguchi, M.; Yonezawa, S.; Taniguchi, N.; Lilley,
D. M. J.; Zhao, Z.; Kurihara, T. Tetrahedron Lett. 2004, 45, 2657; (b) Araki, L.;
Harusawa, S.; Yamaguchi, M.; Yonezawa, S.; Taniguchi, N.; Lilley, D. M. J.; Zhao,
Z.; Kurihara, T. Tetrahedron 2005, 61, 11976; (c) Araki, L.; Morita, K.;
Yamaguchi, M.; Zhao, Z.; Wilson, T. J.; Lilley, D. M. J.; Harusawa, S. J. Org.
Chem. 2009, 74, 2350; (d) Araki, L.; Zhao, Z.; Lilley, D. M. J.; Harusawa, S.
Heterocycles 2010, 81, 1861; (e) Harusawa, S.; Fujii, K.; Nishiura, M.; Araki, L.;
Usami, Y.; Zhao, Z.; Lilley, D. M. J. Heterocycles 2011, 83, 2041.
6. For recent reviews on tetrazoles, see: (a) Bhatt, U. Five-membered Heterocycles
with Four Heteroatoms Tetrazoles In Modern Heterocyclic Chemistry; Alvarez-
Builla, J., Vaquero, J. J., Barluenga, J., Eds.; Wiley-VCH: Weinheim, 2011; Vol. 3,
pp 1401–1430; (b) Herr, R. J. Bioorg. Med. Chem. 2002, 10, 3379; (c) Bräse, S.;
Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem., Int. Ed. 2005, 44, 5188; (d)
Butler, R. N. Tetrazoles In Comprehensive Heterocyclic Chemistry II; Katritzky, A.
R., Rees, C. W., Scriven, E. F. V., Eds.; Pergamon: Oxford, 1996; Vol. 4, pp 621–
678.
7. (a) Popsavin, M.; Torovic´, L.; Spaic´, S.; Stankov, S.; Kapor, A.; Tomic´, Z.;
Popsavin, V. Tetrahedron 2002, 58, 569; (b) Kobe, J.; Prhavc, M.; Hohnjec, M.;
Townsend, L. B. Nucleosides Nucleotides 1994, 13, 2209.
8. Yoshimura, Y.; Hamaguchi, N.; Yashiki, T. J. Antibiot. 1986, 39, 1329.
9. Saneyoshi, H.; Seio, K.; Sekine, M. J. Org. Chem. 2005, 70, 10453.
10. 1a: oil; 1H NMR (500 MHz, CDCl3) d 1.01–1.26 (21H, m), 1.84 (1.05H, d,
J = 6.5 Hz), 1.85 (0.45H, d, J = 6.5 Hz), 1.88 (1.05H, d, J = 6.5 Hz), 1.90 (0.45H, d,
J = 6.5 Hz), 2.35 (0.6H, t, J = 6.6 Hz), 2.60 (1.4H, t, J = 6.3 Hz), 2.56–2.68 (2H, m),
3.06–3.11 (1H, m), 3.37–3.47 (1H, m), 3.54–3.62 (2H, m), 3.64–3.86 (2H, m),
3.78 (4.2H, s), 3.79 (1.8H, s), 3.88–4.00 (2H, m), 4.28–4.30 (0.7H, m), 4.33–4.35
(0.3H, m), 4.45–4.52 (1H, m), 4.56–4.62 (1H, m), 5.26–5.30 (1H, m), 6.78–6.82
(4H, m), 7.16–7.44 (10H, m); 31P NMR (202 MHz, CDCl3) d 149.8, 149.9, 150.4,
150.5; HRMS (FABMS:TEOA+NaCl) m/z calcd for C46H60N7O9P+Na [M+Na]+
908.4088, found 908.4085.
11. (a) Heerding, J. M.; Lampe, J. W.; Darges, J. W.; Stamper, M. L. Bioorg. Med. Chem.
1995, 5, 1839; (b) Mcdonald, I. M.; Black, J. M.; Buck, I. M.; Dunstone, D. J.;
Griffin, E. P.; Harper, E. A.; Hull, R. A. D.; Kalindjian, S. B.; Lilley, E. J.; Linney, I.
D.; Pether, M. J.; Roberts, S. P.; Shaxted, M. E.; Spencer, J.; Steel, K. I. M.; Sykes,
D. A.; Walker, M. K.; Watt, G. F.; Wright, L. W.; Wright, P. T.; Xun, W. J. Med.
Chem. 2007, 50, 3101.
12. 1b: oil; 1H NMR (400 MHz, CDCl3) d 0.11 (1.8H, s), 0.13 (2.1H, s), 0.14 (2.1H, s),
0.92 (8.4H, d, J = 7.2 Hz), 0.98 (3.6H, d, J = 6.8 Hz), 1.08–1.18 (9H, m), 1.19 (9H,
s), 1.92–2.04 (1H, m), 2.18–2.30 (2H, m), 2.53–2.67 (1H, m), 2.98–3.14 (2H, m),
3.19–3.38 (2H, m); 3.48–3.64 (3H, m), 3.74–4.22 (5H, m), 3.78 (4.2H, s), 3.79
(1.8H, s), 6.40 (0.6H, s), 6.41 (1.4H, s), 6.80–6.86 (4H, m), 7.16–7.48 (9H, m); 31
P
NMR (120 MHz, CDCl3) d 148.6, 151.2; HRMS (FABMS: TEOA+NaCl) m/z calcd
for C50H73N6O9PSi+Na [M+Na]+ 983.4844, found 983.4841.
13. (a) Fujitake, M.; Harusawa, S.; Araki, L.; Yamaguchi, M.; Lilley, D. M. J.; Zhao, Z.;
Kurihara, T. Tetrahedron 2005, 61, 4689; (b) Fujitake, M.; Harusawa, S.; Zhao, Z.;
Kurihara, T. Bull. Osaka Univ. Pharm. Sci. 2007, 1, 107; (c) Harusawa, S.; Fujitake,
M.; Kurihara, T.; Zhao, Z.; Lilley, D. M. J. Mass Determination of
Phosphoramidites. In Current Protocols in Nucleic Acid Chemistry; Beaucage, S.
L., Bergstrom, D. E., Herdewijn, P., Matsuda, A., Eds.; John Wiley & Sons: New
York, 2006; pp 10.11.1–10.11.16; (d) Fujitake, M.; Harusawa, S. Bull. Osaka
Univ. Pharm. Sci. 2011, 5, 49.
14. (a) Oligoribonucleotides were synthesized on an Applied Biosystems 394
instrument using 20-O-tBDMS PA chemistry on Applied Biosystems LV 200
polystyrene 20-deoxythymidine columns. A coupling time of 12 min was used.
0.5 M 5-ethylthio-1H-tetrazole in acetonitrile was used as an activator and
0.1 M iodine/water was used as an oxidiser.; (b) Oligoribonucleotides
containing tetrazole modification were deprotected using 75% aqueous
ammonia (35%) in ethanol for 17 h at rt and then evaporated to dryness.
Oligoribonucleotides were redissolved in 0.3 ml of 1 M TBAF in THF to remove
t-BDMS, and agitated at rt for 16 h prior to desalting by NAP-10 columns
(GEHealthcare). Following ethanol precipitation, an aliquot (0.5 OD) of
oligoribonucleotides was loaded on 20% denaturing polyacrylamide gel with
7 M urea and 90 mM TBE, and electrophoresed at 25 W for 2.5 h with
bromophenyl blue (BPB) and xylene cyanol (XC) as makers. The gel was
Figure 2. (A) Analysis of the products of RNA synthesis by gel electrophoresis.14
Lane 1 VS substrate wtG638; Lane 2 G638C0Tez; Lane 3 G638C2Tez. (B) HPLC profile
of G638C2Tez. Desired 25mer has a retention-time of 14 min.15
with Applied Biosystems 394 synthesiser with stepwise coupling
yield over 97%.14 Demonstrated here is the denaturing gel image
for wild type substrate and C0- and C2-tetrazole substituted
(G638C0Tez and G638C2Tez) strands at the same position of the se-
quence as well as the HPLC profile15 of G638C2Tez, as shown in
Figure 2.
Further investigation on the application of 1a and 1b in ribo-
zyme studies is under way and will be published in due course.
Acknowledgment
This work was supported in part by a Grant-in-Aid for Scientific
Research [Grant No.2159030 (to S.H.)] from JSPS, 2009–2013.
visualized by UV shadowing over
a F254 TLC plate.; (c) Sequences of
oligoribonucleotides synthesized; Wild type: GCGCGAAGGGCGUCGUCGC
CCCGAdT; C0Tez-strand: GCGCGAAGGGCGUCGUCGCCCC(C0Tet)AdT; C2Tez-
strand: GCGCGAAGGGCGUCGUCGCCCC(C2Tez)AdT.
References and notes
15. It was analyzed on an Ace C18–300 column, eluted (1 mL/min) with buffer A
(0.1 M TEAA, pH 7.0) and then eluted with a linear gradient from buffer A to
100% buffer B (MeCN) over a period of 60 min.
1. Lilley, D. M. J.; Eckstein, F. In Ribozymes and RNA Catalysis; Royal Society of
Chemistry: Cambridge, 2008.
2. (a) Wilson, T. J.; McLeod, A. C.; Lilley, D. M. J. EMBO J. 2007, 26, 2489; (b)
Lafontaine, D. A.; Wilson, T. J.; Norman, D. G.; Lilley, D. M. J. J. Mol. Biol. 2001,