4
290
Helvetica Chimica Acta ± Vol. 86 (2003)
Table 3 (cont.)
Entry p-RNA
Deprotectionb)
Analytical HPLC
Reverse phasec) Ion Exchanged)
MALDI-TOF-MSe)
À
À
b-d-pr-Oligonucleotide
Methods
[M À H]
[M À H]
a
system )
gradient, t
R
[min]
gradient, t
R
[min]
(obs.)
(calc.)
264
265
266
267
268
269
270
271
272
273
274
275
276
278
279
280
281
282
283
284
285
286
TAT
TAT
2
g)
g)
g)
1567.3
1564.0
2526.6
2544.0
1534.0
1574.0
2535.7
5133.3
5437.3
1331.8
1313.8
1331.8
1313.8
2498.4
3780.4
±
4
A
2
C, G
C, G
B, F
B, F
A, F
A, F
A, F
B, D, F
0 ! 100% B in 30, 21.8 2527.0
0 ! 100% B in 30, 18.9 2544.7
0 ! 100% B in 30, 17.4 1539.4
0 ! 100% B in 30, 19.9 1572.4
T
2
T
2
T
2
T
4
T
4
4
A TA
CGC
GCG
A
A
4
0 ! 40% B in 30, 20.7 0 ! 100% B in 30, 18.4 2533.2
3
TA
2
TA
2
T
0 ! 40% B in 30, 17.5
0 ! 40% B in 30, 17.9
5133.8
5438.1
d(T)-pr(T
TDCG-2'-phosphate
TDCG-2',3'-cyclophosphate
TDGC-2'-phosphate
TDGC-2',3'-cyclophosphate
4 3 3 2
A TA TA T)
0 ! 100% B in 30, 17.1 1331.6
0 ! 100% B in 30, 19.9 1315.0
0 ! 100% B in 30, 17.1 1331.7
0 ! 100% B in 30, 19.3 1312.7
B, D, F
T
T
8
A, F
C, G
A, E
A, E
A, E
A, E
A, E
A, E
A, E
A, E
0 ! 40% B in 30, 19.6 0 ! 100% B in 30, 14.0 2497.6
0 ! 100% B in 30, 26.5 3782.0
12
UA
3
UAU
2
0 ! 40% B in 30, 18.6 0 ! 100% B in 30, 16.2
±
(UA)
4
0 ! 40% B in 30, 18.4 0 ! 100% B in 30, 16.0 2481.4
2478.6
549.4
±
U
U
U
U
U
U
2
2
3
4
7
8
0 ! 40% B in 30, 9.6
0 ! 100% B in 30, 2.9
549.0
±
855.7
2480.0
2080.8
AUA
3
U
0 ! 40% B in 30, 18.9 0 ! 100% B in 30, 16.4
0 ! 40% B in 30, 11.9 0 ! 100% B in 30, 6.2
0 ! 40% B in 30, 19.0
856.6
A
4
2478.6
2081.2
2386.4
0 ! 40% B in 30, 18.9
0 ! 40% B in 30, 15.7 0 ! 100% B in 30, 13.4 2386.1
a) All sequences refer to d-p-RNA (d-pr) unless indicated otherwise; ꢀl) l-pr l-p-RNA; pl d-pyranosyl-
lyxoNA; d(T) Thymidine. All cyclophosphate containing sequences were prepared from their respective 2'-
b
or 3'- or 4'-phosphate precursor sequences by treatment with DEC ¥ HCl. ) Method A: Allyl deprotection for
0
−
(
Trityl-off× sequences: 360 ml (3.7 mmol) of BuNH
225 mmol) of Ph P, 140 ml (3.7 ml) of HCO H in 4.5 ml of THF, 558, 1.5 h; 4.5 ml of 0.1m aq. NaCS
B: Allyl deprotection for −Trityl-on× sequences: 20 mg (148 mmol) of Et NH CO [54] in 0.75 ml of CH
Cl , room temperature, 2 h (12 ± 24 h
. Method C: Allyl deprotection for −Trityl-off× sequences:
2
, 20 mg (22 mmol) of (dibenzylideneacetone)
3
Pd , 60 mg
2
3
2
2
NEt
. Method
Cl
2
2
2
3
2
0
1
1.6 mg (10 mmol) of (Ph
for isoG sequences); 3.5 ml of 0.1m aq. NaCS
60 ml (4.5 mmol) of Et NH , 177 mg (171 mmol) of (dibenzylideneacetone)
2.7 mmol) of Ph P, 360 ml (9.6 mmol) of HCO H in 6.5 ml of THF, 508, 3 ± 4 h; 4.5 ml of 0.1m aq. NaCS
Method D: Detachment from CPG/acyl deprotection: sat. aq. NH /EtOH, 3 : 1, room temperature, 3 ± 4 h.
Method E: Detachment from CPG/acyl deprotection: 4.5 ml of 0.2m MeONH ¥ HCl in aq. conc. NH /EtOH,
room temperature, 20 h. Method F: Detachment from CPG/acyl deprotection: 1.5 ml of 15 ± 25% aq. NH NH
O, 48, 20 ± 30 h (room temperature, 48 h for D-containing sequences). Method G: Detachment from CPG/
acyl deprotection: 5 ml of 40% (w/w) aq. MeNH /conc. aq. NH
1 : 1, room temperature, 5 ± 6 h. c) Aquapore
3
P)
4
Pd ,1.3 mg (5 mmol) of Ph
3
P in 1.0 ml of CH
2
2
2
NEt
2
0
4
(
2
2
3
Pd ÀCHCl
3
adduct [61], 432 mg
2
NEt .
3
2
2
3
2
3
2
2
¥
H
2
2
3
RP-300 C-8 Brownlee, 220 Â 4.6 mm, 7 mm, flow 1 ml/min. Mobile phase: eluant A: 0.1m Et
3 2
N, 0.1m AcOH, H O,
d
pH 7.0; eluant B: 0.1m Et
3
N, 0.1m AcOH, H
2
O/MeCN 1 : 4. ) MONO-Q HR 5/5 Pharmacia, 10 Â 0.5 cm, flow
1
ml/min. Mobile phase: eluant A: 10 mm Na HPO , H O, pH 11.5; eluant B: 10 mm Na HPO , 1m NaCl, H O,
2
4
2
2
4
2
e
pH 11.5. ) Matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy; matrix: 3-hydro-
picolinic acid or a-cyanohydroxycinnamic acid or 2,4,6-trihydroxyacetophenone and ammonium citrate buffer.
f
)
Nucleogen-DEAE 60-7 Macherey-Nagel, 125 Â 4 mm, flow 1 ml/min eluant A: 10 mm Na
2 4 2
HPO , H O/MeCN
g
1
: 4, pH 11.5; eluant B: 10 mm Na
2
HPO
4
, 1m NaCl, H
2
O/MeCN 1 : 4, pH 11.5. ) From Hoechst, Z¸rich.
amidites 9a ± 9g and the suitably derivatized CPG solid supports 11a ± 11g, utilizing the
protocols that were developed earlier for the homo-DNA-oligonucleotide synthesis
and adapting them to the specific demands of the p-RNA synthesis [28]. The following
were the conditions used: a) detritylation with 6% Cl CHCO H in ClCH CH Cl for a
2
2
2
2
period of 5 ± 7 min, b) coupling for 45 ± 60 min with 10% (w/v) soln. of phosphor-
amidites in MeCN (160 ± 400 ml per coupling) in the presence of a mixture of 0.15m
5-(4-nitrophenyl)-1H-tetrazole and 0.35m 1H-tetrazole in MeCN (180 ± 600 ml per
coupling), c) capping for 1.5 min with 0.45m DMAP in MeCN plus a mixture of