6
M. Laine et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
The residue was suspended in aqueous NaOH (3 M, 50 mL) and the
aqueous phase was extracted with CHCl
(3 ꢁ 50 mL). The com-
bined extracts were dried with K CO and evaporated to dryness
phosphoramidite building blocks (Glenn Research) by conven-
tional phosphoramidite strategy using the standard RNA-coupling
protocol of ABI-3400 DNA/RNA synthesizer and a 1.0 lmol L
3
ꢀ1
2
3
to give 1,7-bis(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodode-
cane (10) in quantitative yield.
scale. The oligonucleotides were released from support by treating
with concentrated aqueous ammonia (1.5 mL) for 2 h at RT and
0
1
,3,5-Tris(bromomethyl)benzene (7.65 g, 21.62 mmol) was dis-
solved in CHCl (150 mL) and Na CO (1.71 g, 16.15 mmol) was
added. Compound 10 (1.08 g, 2.90 mmol) in CHCl (50 mL) was
added dropwise into the reaction mixture during 11 hours at
2 °C. The reaction mixture was refluxed for 3 days at 62 °C,
then 4 hours at 55 °C. 2 -O-TOM protecting groups were removed
3
2
3
with triethylamine trihydrofluoride (160
by heating the mixture for 30 min at 65 °C. Aqueous sodium acet-
ate (0.1 M, 600 L) was added and the heating was continued for
lL) in DMSO (600 lL)
3
l
5
30 min at 65 °C to complete the removal of the hydroxymethyl
remnants of the TOM groups. The reaction mixture was cooled
on an ice bath to RT and the crude oligoribonucleotides were puri-
fied by RP-HPLC and desalted. The identity of the oligonucleotide
filtrated, and evaporated to dryness. The residue was purified by
silica gel chromatography (40–70% EtOAc in hexane), giving 11 in
1
2
7% yield (0.722 g). H NMR (500 MHz, CDCl
3
) d 7.33 (s, 6H), 4.45
0
(
(
s, 8H), 3.71 (s, 4H), 3.21–3.57 (m, 8H), 2.55–2.68 (m, 8H), 1.27
sequences was verified by MS analysis. 5 -CUUUC-3: m/z obsd.
1
3
2ꢀ
3ꢀ
3
s, 18H). C NMR (100 MHz, CDCl ) dppm 155.8, 140.8, 138.5,
(Calcd.) 732.1 (732.4) [M–2H] , 487.7 (488.0) [M–3H] , 365.5
4ꢀ
0
0
1
9
29.9, 128.3, 79.3, 59.5, 55.2, 46.1, 32.9, 28.4. HRMS(ESI): obsd.
21.0840 [M+H] , Calcd. 921.0795 [M+H] .
(365.7) [M–4H] . 5 -CUCUC-3 : m/z obsd. (Calcd.) 731.6 (732.0)
+
+
2ꢀ
3ꢀ
4ꢀ
0
[M–2H] , 487.4 (487.6) [M–3H] , 365.3 (365.5) [M–4H] . 5 -
CCUUC-3 : m/z obsd. (Calcd.) 731.6 (732.0) [M–2H] , 487.4
(487.6) [M–3H] , 365.3 (365.5) [M–4H] . 5 -CUUCC-3 : m/z obsd.
(Calcd.) 731.6 (732.0) [M–2H]
5 -ACUUUAC-3 : m/z obsd. (Calcd.)
07.1 (707.4) [M–3H] , 530.1 (530.3) [M–4H] , 423.8 (424.1)
0
2ꢀ
3ꢀ
4ꢀ
0
0
4.4. 1,7-Bis{3,5-bis[1,5,9-tri(tert-butoxycarbonyl)-1,5,9-triazacyclodo-
2ꢀ
3ꢀ
decan-3-yloxymethyl]benzyl}-4,10-di(tert-butoxycarbonyl)-1,4,7,10-
tetraazacyclododecane (13)
,
487.4 (487.6) [M–3H]
,
4
ꢀ
0
0
365.3 (365.5) [M–4H]
.
3
ꢀ
4ꢀ
7
5ꢀ
Preparation of 3-hydroxy-1,5,9-tri(tert-butoxycarbonyl)-1,5,9-
triazacyclododecane (12) has been reported previously [4a]. This
compound (1.47 g, 3.02 mmol) was dissolved in dry DMF (10 mL)
and added into a solution of 11 (0.56 g, 0.61 mmol) in DMF
[M–5H] .
4.7. Kinetic experiments
(
10 mL). Sodium hydride (76.2 mg, 3.18 mmol) was added and
The reactions were carried out in Eppendorf tubes immersed in
a water bath, the temperature of which was kept at 35 ± 0.1 °C. The
pH was adjusted to 7.5 with a HEPES buffer (0.1 M). The concentra-
the mixture was stirred for 3 h at room temperature under
nitrogen. MeOH (3 mL) was added and the volatiles were
removed under reduced pressure. The residue was dissolved in
tion of the target oligonucleotide was 50
-nitrobenzenesulfonate (0.1 mM) was used as an internal stan-
dard and the ionic strength was adjusted to 0.1 M with sodium
lM, potassium
water (60 mL) and extracted with CH
The combined extracts were dried with Na
to dryness. The residue was purified by silica gel chromatography
EtOAc/CH Cl , 25:75, v/v, containing 1% Et N). The yield was
.570 g (36%). H NMR (500 MHz, CDCl ) dppm 7.13 (s, 6H), 4.57
s, 8H), 3.89 (br. s, 4H), 3.65–3.05 (m, 60H), 2.61 (br.s, 8H), 2.09–
2
Cl
2
(1 ꢁ 60 mL, 5 ꢁ 30 mL).
4
2
SO and evaporated
4
2
+
nitrate. The solution of the Zn complex was prepared by mixing
stoichiometric amounts of compound 5a and Zn(NO . In other
(
0
(
1
2
2
3
1
3 2
)
3
2
+
words, the Zn ion concentration was 0.25 mM, 0.5 mM, 1.0 mM
or 2.5 mM and the concentration of 5a 0.05 mM, 0.1 mM, 0.2 mM
or 0.5 mM, respectively. The total volume of the reaction mixture
13
.99 (m, 8H), 1.83-1.73 (m, 8H), 1.45 (s, 126H).
C NMR
(
3
126 MHz, CDCl ) dppm 156.3, 156.0, 155.6, 139.2, 138.6, 127.3,
was 200
intervals. The reaction was quenched by adding aqueous hydrogen
chloride (1 L of 1.0 M solution). The aliquots were then cooled to
°C and analyzed immediately by capillary zone electrophoresis
lL and aliquots of 20 lL were withdrawn at suitable time
1
4
2
25.0, 79.8, 79.6, 79.0, 75.9, 72.0, 58.9, 54.6, 49.5, 47.1, 46.0,
+
4.7, 29.5, 28.5, 28.3. HRMS(ESI): obsd. 2550.6926 [M+H] , Calcd.
+
l
550.6779 [M+H] .
0
using a fused silica capillary. The temperature of the capillary
was kept at 25 °C. The samples were injected using hydrodynamic
injection with 2 psi for 8 s. The capillary was flushed for 3 min with
water, 10 mM aqueous hydrogen chloride and the background
electrolyte buffer (0.2 M citrate buffer, pH = 3.1) between every
analytical run.
First-order rate constants were calculated by applying the inte-
grated first-order rate law to the diminution of the signal of the
starting material. The peak area was first normalized by dividing
the area by the migration time and then by the similarly normal-
ized area of the internal standard.
0
0
4
.5. 1,7-Bis{[3 ,5 -bis[(1,5,9-triazacyclododecan-3-yloxy)methyl]
benzyl}-1,4,7,10-tetraazacyclododecane (5a)
Compound 13 (0.18 g, 0.07 mmol) was dissolved in MeOH
(
8 mL). Aqueous HCl (6 M, 1.6 mL) was added and the mixture
was stirred for 5 h at 42 °C. The volatiles were removed under
reduced pressure and the residue was coevaporated with H
2
O
(
5 ꢁ 10 mL). The residue was dissolved in water (40 mL) and
ꢀ
Dowex 1X2 resin (100–200 mesh, OH form) was added and the
resulting mixture was stirred for 3 h. The resin was collected by fil-
tration and washed with H
evaporated to dryness. The yield was 78 mg (98%). H NMR
500 MHz, D O/MeOD, 3:2, v/v) dppm 7.21 (s, 4H), 7.17 (s, 2H),
.42 (s, 8H), 3.55 (s, 4H), 3.50–3.45 (m, 4H), 2.81–2.73 (m, 16H),
2
O (3 ꢁ 15 mL) and the filtrate was
1
(
4
2
2
References
1
3
[
.66–2.56 (m, 32H), 2.46 (br.s, 16H), 1.55–1.48 (m, 16H).
O/MeOD, 3:2, v/v) dppm 139.8, 138.3, 128.2,
26.8, 76.1, 70.8, 58.3, 50.7, 49.5, 48.0, 47.8, 43.3, 24.6. HRMS
C
NMR (126 MHz, D
2
1
(
2
+
+
ESI): obsd. 1149.9405 [M+H] , Calcd. 1149.9438 [M+H] . UV: kmax
65 nm and 275 nm.
[
4
.6. Synthesis of oligonucleotides
Oligoribonucleotides were synthesized from commercially
0
0
available 2 -O-triisopropylsilyloxymethyl (2 -O-TOM) protected