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interatomic non-covalent bonds. Neither free donors nor acceptors
are available for any additional interactions in the vicinity of the
(CH3)2N group, thus no larger substituents are preferred.
5.1.1.1. P1-(N2,N2-diethyl-7-methylguanosine-50-yl)-P3-guano-
sin-50-yl triphosphate, et22,2m7GpppG (2). 24 mg (0.026 mmol),
44%, 1H NMR (400 MHz, D2O): d 8.90 (s, 1H, H-8 et22,2m7G), 7.97
(s, 1H, H-8 G), 5.92 (d, J = 3.04 Hz, 1H, H-10 et22,2m7G), 5.76 (d,
J = 6.03 Hz, 1H, H-10 G), 4.60 (t, J = 5.28 Hz, 1H, H-20 G), 4.56 (t,
J = 4.72 Hz, 1H, H-20 et22,2m7G), 4.45–4.42 (m, 1H, H-30 et22,2m7G),
4.41–4.35 (m, 3H, H-40 et22,2m7G, H-30 and H-40 G), 4.33–4.30 (m,
1H, H-50 et22,2m7G), 4.27–4.21 (m, 3H, H-500 et22,2m7G, H-50 and H-
500 G), 4.02 (s, 3H, N7-CH3), 3.61–3.50 (m, 4H, N-CH2), 1.21 (t,
J = 7.16 Hz, 6H, CH3 etyl); 31P NMR (162 MHz, D2O) d ꢀ11.73 (2P,
Even though such groups are favourable in relation to water
expulsion and self-stacking, the most important condition turned
out to be the steric complementarity of the self-stacked cap confor-
mation to the stiff and compact cap-binding site. Thus, human snur-
portin follow the ‘lock and key’ model, contrary to the ‘induced-fit’
mechanism, accompanied by ordering of the protein structure upon
cap binding, which is characteristic for interactions with eIF4E.16
The eIF4E protein family, although specific for the MMG-cap, can
accept the TMG-cap to some extent, depending on the species,15a,17
while snurportin—which is specific for the TMG-cap—accepts
neither the MMG-cap nor more bulky TMG-cap analogues. This is
essential structural dynamic difference between the eIF4E and
snurportin selectivity towards different forms of the 50 RNA cap.
P
a,c
), ꢀ23.29 (1P, Pb); HRMS (ES+) m/z: (M+H)+: 859.1576, calcu-
lated for C25H38N10O18P+3: 859.1573.
5.1.1.2. P1-(N2-butyl-N2,7-dimethylguanosine-50-yl)-P3-guano-
sin-50-yl triphosphate, but2m22,7GpppG (3). 28 mg (0.030 mmol),
51%, 1H NMR (400 MHz, D2O): d 8.90 (s, 1H, H-8 b2m22,7G), 7.92
(s, 1H, H-8 G), 5.87 (d, J = 2.47 Hz, 1H, H-10 b2m22,7G), 5.71 (d,
J = 5.93 Hz, 1H, H-10 G), 4.54–4.50 (m, 2H, H-20 b2m22,7G, H-20 G),
4.40–4.33 (m, 4H, H-30 and H-400 b2m22,7G, H-30 and H-40 G), 4.29–
4.27 (m, 1H, H-50 b2m22,7G), 4.25–4.18 (m, 3H, H-500 b2m22,7G, H-50
and H-500 G), 4.00 (s, 3H, N7-CH3), 3.50–3.43 (m, 2H, N-CH2), 3.10
(s, 1H, N-CH3), 1.61–1.51 (m, 2H, CH2 butyl), 1.31–1.26 (m, 2H,
CH2CH3), 0.88 (t, J = 7.39, 3H, CH3 butyl); 31P NMR (162 MHz,
D2O) d ꢀ11.66 (2P, P ), ꢀ23.21 (1P, Pb); HRMS (ES+) m/z:
4. Conclusions
The studies show that a sensitive and effective tool for investi-
gation of the molecular mechanism of the 50 TMG-capped U snRNA
recognition by proteins has been created. Our results revealed
strict selectivity of snurportin towards the TMG-cap structure that
mainly relied on the protein structural stiffness and compactness.
The cap analogues with larger substituents at the N2 group cannot
be accommodated in the binding site, while protons do not provide
efficient screening from water. The optimum is thus required in the
form of the TMG-cap.
a,c
(M+H)+: 873.1734, calculated for C26H40N10O18P+3: 873.1729.
5.1.1.3. P1-(N2-isobutyl-N2,7-dimethylguanosine-50-yl)-P3-gua-
nosin-50-yl
triphosphate,
ibut2m22,7GpppG
(4). 25 mg
(0.027 mmol), 46%, 1H NMR (400 MHz, D2O): d 8.92 (s, 1H, H-8
ib2m22,7G), 7.95 (s, 1H, H-8 G), 5.88 (d, J = 2.76 Hz, 1H, H-10
ib2m22,7G), 5.73 (d, J = 5.93 Hz, 1H, H-10 G), 4.56 (t, J = 5.38 Hz, 1H,
H-20 ib2m22,7G), 4.51 (t, J = 4.28 Hz, 1H, H-20 G), 4.43–4.39 (m, 2H,
H-ib2m22,7G, H-30 G), 4.38–4.33 (m, 2H, H-40 ib2m22,7G, H-40 G),
4.32–4.29 (m, 1H, H-50 ib2m22,7G), 4.26–4.18 (m, 3H, H-500
ib2m22,7G, H-50 and H-500 G), 4.01 (s, 3H, N7-CH3), 3.30–3.25 (m,
2H, N-CH2), 3.14 (s, 3H, N-CH3), 2.08–2.02 (m, 1H, CH isobutyl),
0.90–0.86 (m, 6H, CH3 isobutyl); 31P NMR (162 MHz, D2O) d
ꢀ11.66 (2P, P ), ꢀ23.24 (1P, Pb); HRMS (ES+) m/z: (M+H)+:
5. Experimental section
5.1. Chemistry
All reagents were the highest available purity and purchased
from Sigma–Aldrich Chemical Co. Triethylammonium bicarbonate
(TEAB) buffer was prepared by bubbling CO2 through an ice-cold
aqueous solution of redistilled triethylamine. Intermediate nucleo-
tides were separated by ion-exchange chromatography on a DEAE-
Sephadex A-25 (HCOꢀ3 form) using a linear gradient of TEAB buffer,
pH 7.6. Fractions containing products were combined and evapo-
rated under reduced pressure with several additions of ethanol
and isolated as triethylammonium salts (TEA salts). Homogeneity
of the final dinucleotide analogues was checked by reversed-phase
analytical HPLC. HPLC was performed using a Supelcosil LC-18-T RP
column (4.6 ꢃ 250 mm, flow rate 1.0 mL/min) with a linear gradient
of methanol from 0% to 50% (v/v) in 0.05 M ammonium acetate (pH
5.9) on a Knauer instrument, with UV detection at 254 nm. MS spec-
tra were acquired using Waters Micromass Q-TOF Premier spec-
trometer with positive electrospray ionisation source. 1H and 31P
NMR spectra of the final compounds were recorded on a Varian
INOVA 700 MHz spectrometer.
a,c
873.1740, calculated for C26H40N10O18P+3: 873.1729.
5.1.1.4.
P1-(N2,7-dimethylguanosine-50-yl)-P3-guanosin-50-yl
triphosphate, m22,7GpppG (5). 31 mg (0.035 mmol), 58%, 1H NMR
(400 MHz, D2O) m22,7G: d 5.93 (d, 1H, H-10), 4.55 (dd, 1H, H-20),
4.43–4.39 (m, 1H, H-30), 4.43–4.40 (m, 1H, H-50), 4.39–4.36 (m,
1H, H-40), 4.31–4.25 (m, 1H, H-500), 4.04 (s, 3H, 7-CH3), 2.93 (s,
3H, N2-CH3); G: d 7.98 (s, 1H, H-8), 5.77 (d, 1H, H-10), 4.61 (dd,
1H, H-20), 4.48–4.42 (m, 1H, H-30), 4.36–4.32 (m, 1H, H-40), 4.31–
4.26 (m, 1H, H-50), 4.24–4.20 (m. 1H, H-500); 31P NMR (162 MHz,
D2O) d ꢀ11.2 (2P, P ), ꢀ22.8 (1P, Pb); HRMS (ES+) m/z: (M+H):
a,c
817.1106, calculated for C22H32N10O18P+3: 817.1103.
5.1.1.5. P1-(N2-butyl-7-methylguanosine-50-yl)-P3-guanosin-50-
yl triphosphate, but2m7GpppG (6). 32 mg (0.036 mmol), 60%, 1H
NMR (400 MHz, D2O): d 8.94 (s, 1H, H-8 b2m7G), 7.96 (s, 1H, H-8
G), 5.90 (d, J = 3.04 Hz, 1H, H-10 b2m7G), 5.75 (d, J = 6.14 Hz, 1H,
H-10 G), 4.59 (t, J = 4.57 Hz, 1H, H-20 G), 4.52 (t, J = 4.72 Hz, 1H,
H-20 b2m7G), 4.44–4.39 (m, 2H, H-30 b2m7G, H-30 G), 4.38–4.34
(m, 2H, H-40 b2m7G, H-40 G), 4.31–4.29 (m, 1H, H-50 b2m7G),
4.27–4.19 (m, 3H, H-500 b2m7G, H-50 G, H-500 G), 4.00 (s, 3H, N7-
CH3), 3.35–3.25 (m, 2H, N-CH2), 1.59–1.53 (m, 2H, CH2 butyl),
1.39–1.30 (m, 2H, CH2CH3), 0.89 (t, J = 7.38, 3H, CH3 butyl); 31P
NMR (162 MHz, D2O) d ꢀ11.64 (2P, P ), ꢀ23.23 (1P, Pb); HRMS
5.1.1. General procedure for the preparation of N2-substituted
dinucleotide cap analogues (2–10)
Compounds R21,R22m7GMP (0.059 mmol TEA salt) and imGDP
(30 mg, 0.059 mmol sodium salt) were suspended in 1.2 mL of
DMF. Subsequently, anhydrous ZnCl2 (80 mg, 0.59 mmol) was
added. The resulting solution was maintained for 2 days at room
temperature. The reaction was quenched by addition of 280 mg
(0.74 mmol) of EDTA in 4.0 mL of water and neutralized with solid
sodium bicarbonate. Products were separated by semipreparative
RP HPLC using a linear gradient of methanol in 0.05 M ammonium
acetate, pH 5.9, from 0% to 50% within 60 min. Yield (as NH+4 salts).
a,c
(ES+) m/z: (M+H)+: 859.1585, calculated for C25H38N10O18P+3:
859.1572.