Synthesis of Leishmania cap-4 intermediates, cap-2 and cap-3

Article abstract of DOI:10.1080/15257770701533446

Synthesis of Leishmania mRNA 5′-cap analogs, m7Gpppm26AmpAm (cap-2), and m7Gpppm26AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4). Copyright Taylor & Francis Group, LLC.

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Synthesis of Leishmania Cap-4
Intermediates, Cap-2 and Cap-3
Magdalena Lewdorowicz a , Janusz Stepinski a , Ryszard Kierzek b ,
Jacek Jemielity a , Joanna Zuberek a , Yael Yoffe c , Michal Shapira c
, Ryszard Stolarski a & Edward Darzynkiewicz a
a Department of Biophysics, Institute of Experimental Physics,
Warsaw University, Warsaw, Poland
b Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Poznan, Poland
c Department of Life Sciences, Ben-Gurion University, Beer Sheva,
Israel
Version of record first published: 05 Dec 2007.
To cite this article: Magdalena Lewdorowicz, Janusz Stepinski, Ryszard Kierzek, Jacek Jemielity,
Joanna Zuberek, Yael Yoffe, Michal Shapira, Ryszard Stolarski & Edward Darzynkiewicz (2007):
Synthesis of Leishmania Cap-4 Intermediates, Cap-2 and Cap-3, Nucleosides, Nucleotides and Nucleic
Acids, 26:10-12, 1339-1348
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1339–1348, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701533446
SYNTHESIS OF LEISHMANIA CAP-4 INTERMEDIATES,
CAP-2 AND CAP-3
Magdalena Lewdorowicz and Janusz Stepinski
Department of Biophysics,
2
Institute of Experimental Physics, Warsaw University, Warsaw, Poland
Ryszard Kierzek
Institute of Bioorganic Chemistry, Polish Academy of Sciences,
2
Poznan, Poland
Jacek Jemielity and Joanna Zuberek
Department of Biophysics, Institute of
2
Experimental Physics, Warsaw University, Warsaw, Poland
Yael Yoffe and Michal Shapira
Department of Life Sciences, Ben-Gurion
2
University, Beer Sheva, Israel
Ryszard Stolarski and Edward Darzynkiewicz
Department of Biophysics,
2
Institute of Experimental Physics, Warsaw University, Warsaw, Poland
Synthesis of Leishmania mRNA 5ꢁ-cap analogs, m7Gpppm26AmpAm (cap-2), and
2
m7Gpppm26AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E
proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to
LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).
Keywords Leishmania; cap analogs; trypanosomatids
INTRODUCTION
Leishmania are trypanosomatids parasites that belong to the Kineto-
plastida order. In trypanosomatids, the 5ꢁ-cap of mRNAs has a unique
structure,[1] which includes base methylation on the first (A) and fourth (U)
nucleotides, and 2ꢁ-O-methylations on the ribose moieties of the first four
6
transcribed nucleotides, resulting in m7G(5ꢁ)ppp(5ꢁ)m2 AmpAmpCmpm3
Um. . ., where m preceding nucleobase denotes methyl group at the
given positions of base and m following nucleobase denotes 2ꢁ-O-methyl
Supported by the Polish Ministry of Science and Higher Education, Grants No. 3 T09A 153 29 and
2 P04A 006 28 and Howard Hughes Medical Institute Grant (to E.D.) No. 55 005604.
Address correspondence to Janusz Stepinski, Department of Biophysics, Institute of Experimental
Physics, Warsaw University, 02 089 Warsaw, Poland. E-mail: jastep@biogeo.uw.edu.pl
1339

1340
M. Lewdorowicz et al.
FIGURE 1 Structures of Leishamnia cap-2 and cap-3 intermediates.
group. This capped tetranucleotide fragment, denoted cap-4, has been re-
cently synthesized and its binding affinity to Leishmania eIF4E homologue
(LeishIF4E-1) was determined using fluorescence spectroscopy.[2,3] Here
6
we report the synthesis of two cap-4 intermediates, m7Gpppm2 AmpAm (1)
6
and m7Gpppm2 AmpAmpCm (2), assigned as “cap-2” and “cap-3”, respec-
tively (Figure 1). Our goal was to determine the binding affinities of those
cap analogs to LeishIF4E proteins compared to the mature trypanosomatids
cap structure.
RESULTS AND DISCUSSION
The synthesis was achieved by coupling of 5ꢁ-phosphorylated dimer
6
6
(pm2 AmpAm, 4) or trimer (pm2 AmpAmpCm, 5) with an imidazole
derivative of 7-methylguanosine diphosphate(3) in the presence of anhy-
drous zinc chloride as a promoter in dimethylformamide as a solvent[2,4]
resulting in cap-2 and cap-3, respectively (Figure 2).
The dimer 4 was prepared by a multistep method “in solution”
starting from 5ꢁ-O-dimethoxytrityl-N6,N6,2ꢁ-O-trimethyladenosine[2] and
FIGURE 2 The final step for the synthesis of cap-2 and cap-3 by coupling of 7-methylguanosine
5ꢁ-diphosphate P2-imidazolide with 4 and 5, respectively.

Leishmania cap-4 Intermediates
1341
FIGURE 3 Synthesis of dinucleotide m62 AmpAm.
using 4-chlorophenyl dichlorophosphate as 3ꢁ-O-phosphorylating agent[5]
(Figure 3). Next steps of the synthesis included: (1) the reaction of 5ꢁ-
ꢁ
O-dimethoxytrityl-N6,N6,O2 -trimethyladenosine 3ꢁ-(p-chlorophenyl)phos-
phate with 3ꢁ-O-acetyl-N6-benzoyl-2ꢁ-O-methyladenosine; (2) deprotection
reaction with 3% trichloroacetic acid in dichloromethane; (3) deprotection
with 25% ammonia in water; and (4) 5ꢁ-O-phosphorylation with phosphorus
oxychloride in trimethylphosphate.
The trimer 5 was prepared by the phosphoramidite solid-phase
method. Commercial 2ꢁ-O-methylcytidine linked to CPG solid support
was the starting material, and subsequent couplings of adenosine,

1342
M. Lewdorowicz et al.
TABLE 1 The association constants (Kas) for complexes of Leishmania eIF4Es and cap analogs[9]
Kas (µM−1
LeishIF4E-1 LeishIF4E-2 LeishIF4E-2 delta LeishIF4E-3 LeishIF4E-4
)
Cap Analog
m7Gpppm62 Am (cap-1)
0.08 0.01 0.02 0.01
0.07 0.01 0.09 0.01
0.03 0.01
0.09 0.01
0.19 0.03
0.22 0.03
0.03 0.01 0.08 0.03
0.06 0.01 0.23 0.06
m7Gpppm62 AmpAm (cap-2)
m7Gpppm62 AmpAmpCm (cap-3) 0.19 0.03 0.24 0.02
cap-4
0.25 0.01 0.28 0.04
0.05 0.02 0.77 0.08
N6,N6,2ꢁ-O-trimethyladenosine and 2-cyanoethyl-3-(4,4ꢁ-dimetoxytrityloxy)-
2,2-di(ethoxycarbonyl)propyl-1-N,N-diisopropyl phosphoramidite building
blocks were done according to known procedures.[2,6,7]
The final products (cap-2 and cap-3) were isolated by preparative
1
HPLC and their structures were confirmed by H, 31P NMR, and ESI mass
spectrometry.
The new cap analogs were tested for their affinities for LeishIF4E-1 and
other recently identified Leishmania eIF4E isoforms[8,9] by a fast and accu-
rate time-synchronized fluorescence titration method, which is based on
measuring the quenching of intrinsic Trp fluorescence of the proteins in
response to binding of cap analog.[10] The association constants (Kas) for
the complexes of Leishmania eIF4Es and the different cap analogs were de-
termined. The binding affinities of cap-2 and cap-3 were compared to the
affinities of cap-1 and cap-4 to Leishmania eIF4E isoforms.[2] Cap-3 showed
similar although slightly lower affinity to LeishIF4Es compared to cap-4,
whereas cap-2 bound to LeishIF4Es significantly weaker than cap-4. The re-
sults are shown in Table 1.
EXPERIMENTAL
Monitoring of the Chemical Synthesis
TLC analysis was performed on TLC plates coated with silicagel 60
F-254 (Merck, Darmstadt, Germany), developed by chloroform-methanol
9:1 v/v (A); chloroform-ethanol 9:1 v/v (B); chloroform-methanol 9.7:0.3
v/v (C).
Analytical HPLC was performed on a Spectra-Physics SP8800 apparatus,
using a Supelcosil LC-18-T reverse phase column (4.6 × 250 mm, flow rate
1.3 mL/minute) with buffer A (0.05 M ammonium acetate at pH 5.9) and
buffer B (methanol and buffer A, 1:1 v/v). Linear 0–100% gradient of buffer
B in buffer A over 20 minutes.
1H NMR and 13C NMR spectra were run on a Varian UNITY-plus spec-
trometer at 400 and 100.56 MHz, respectively, at ambient temperatures
and at concentrations of ca. 2 mg/mL (1H NMR), and ca. 20 mg/mL

Leishmania cap-4 Intermediates
1343
(13C NMR) using tetramethylsilane (TMS) as the internal standard in CDCl3
and sodium 3-trimethylsilyl-[2,2,3,3-D4]-propinonate (TSP) in D2O.
Mass spectra were recorded on a Micromass QToF 1 MS spectrometer
using electrospray negative ionization.
Preparative HPLC
Preparative HPLC was performed on a Waters 600E Multisolvent De-
livery System apparatus, using a Waters HR-C-18 reverse phase column
(19 × 300 mm, flow rate 5.0 mL/minute). The mobile phase for this method
was the same as for analytical HPLC (see above). Linear 0–100% gradient of
buffer B in buffer A over 40 minutes.
Synthesis
ꢀ
ꢁ
N6-Benzoyl-O2 -methyladenosine.[11]
O2 -Adenosine (1 g, 3.5 mmol)
was benzoylated in dry pyridine (10 mL) with 1.75 mL (2.1 g, 15 mmol)
of benzoyl chloride for 1 hour at room temperature. The progress of the
reaction was monitored by TLC (Rfsubstrate = 0.1, Rfproduct = 0.6 in A). The
resulting solution was added into 100 mL of cold water and the insoluble
product extracted with chloroform (3 × 100 mL). The chloroform extract
was evaporated under reduced pressure to a gum and dissolved in a mix-
ture of ethyl alcohol (45 mL) and pyridine (30 mL). The solution was
treated with 15 mL of 2 M sodium hydroxide at room temperature for
5 minutes. An excess of pyridinium Dowex-50 ion exchange resin was
then added to remove sodium ions, the resin was removed by filtra-
tion and the filtrate was concentrated under reduced pressure. Then
water (50 mL) was added and resulting solution was extracted with di-
ethyl ether (3 × 50 mL). Preparative HPLC purification gave N6-benzoyl-
ꢁ
O2 -methyladenosine (Rtsubstrate = 10.95 minutes, Rtproduct = 17.27 minutes,
0.7 g, 1.8 mmol, yield 51%, m/z = 386.14 by ESI-MS).
ꢀ
N6-Benzoyl-5ꢀ-dimethoxytrityl-O2 -methyladenosine.
A
mixture of
ꢁ
N6-benzoyl-O2-methyladenosine (0.47 g, 1.2 mmol), 4,4ꢁ-dimethoxytrityl
chloride (1.15 g, 3.4 mmol) and triethylamine (0.404 mL, 0.29 g,
2.9 mmol) in anhydrous pyridine (5 mL) was stirred for 3 hours
at room temperature, and the progress of the reaction was mon-
itored by TLC (B). The mixture was quenched with methanol
(3 mL) and evaporated under reduced pressure at room temper-
ature. The residual syrup was dissolved in chloroform (30 mL)
and washed with two 23-mL portions of 1 M aqueous sodium hydro-
gen carbonate solution. The chloroform layer was dried over anhydrous
sodium sulphate, filtered, and evaporated under reduced pressure. The
residual pyridine was removed by coevaporation with three 3-mL portions

1344
M. Lewdorowicz et al.
of toluene. The crude product was purified by column chromatogra-
phy over silicagel eluting with a stepwise gradient system (0.25–2%) of
ꢁ
methanol in dichloromethane to give N6-benzoyl-5ꢁ-dimethoxytrityl-O2 -
methyladenosine (0.71 g, 1.03 mmoles, 85%, Rfproduct = 0.46 in B, m/z =
688.3 by ESI-MS).
ꢀ
ꢀ
O3 -Acetyl-N6-benzoyl-O2 -methyladenosine. A mixture of N6-benzoyl-
ꢁ
5ꢁ-dimethoxytrityl-O2 -methyladenosine (0.483 g, 0.7 mmol) and acetic an-
hydride (0.079 mL, 0.84 mmol) in anhydrous pyridine (3.5 mL) was stirred
overnight at room temperature. The progress of the reaction was mon-
itored by TLC (Rfsubstrate = 0.3, Rfproduct = 0.44 in C). The reaction mix-
ture was then added into 20 mL of dichloromethane and resulting solu-
tion was extracted with 1 M NaHCO3 (3 × 10 mL). The organic layer was
dried over anhydrous sodium sulphate, filtered, and evaporated to an oil un-
der reduced pressure. Then 5 mL of 3% solution of trichloroacetic acid in
dichloromethane (20 mL) was added, the mixture was kept at room temper-
ature for 1 hour and extracted with water (3 × 30 mL). The organic layer was
evaporated under reduced pressure. The resulting product was purified by
column chromatography over silicagel and eluting with a stepwiseꢁ gradient
system (0.25–0.2%) of methanol in dichloromethane to give O3-acetyl-N6-
ꢁ
benzoyl-O2 -methyladenosine (0.21 g, 0.49 mmol, yield 70%, m/z = 428.17
by ESI-MS).
ꢀ
5ꢀ-Dimethoxytrityl-N6,N6,O2 -trimethyladenosine
monophosphate (Figure 3).
3ꢀ-(p-chlorophenyl)
ꢁ
The mixture of 5ꢁ-dimethoxytrityl-N6,N6,O2 -
trimethyladenosine[2] (0.1 g, 0.164 mmol) and p -chlorophenyl phospho-
dichloridate (0.133 mL, 0.2 g, 1.4 mmol) in dry pyridine was stirred for
0.5 hours. The progress of the reaction was monitored by TLC (Rfsubstrate
=
0.4, Rfproduct = 0.06 in A). Then 20 mL of 1 M buffer TAEB (triethylammo-
nium hydrogen carbonate) was added and the reaction mixture was cooled
to 0◦C for 10 minutes. After 10 minutes chloroform was added (20 mL)
and the mixture was washed with TEAB buffer (0.1 M). The organic phase
was evaporated under reduced pressure then coevaporated witꢁh toluene
(3 × 5 mL) and dried in vacuo. 5ꢁ-Dimethoxytrityl-N6,N6,O2 -trimethy-
ladenosine 3ꢁ-(p-chlorophenyl)monophosphate was obtained as white solid
(0.123 g, 0.15 mmol, yield 93%).
Protected Dinucleotide m62AprmpAprm (Figure 3) .
5ꢁ-Dimethoxytrityl-
ꢁ
N6, N6,O2-trimethyladenosine 3ꢁ-(p-chlorophenyl)monophosphate (0.123 g,
ꢁ
ꢁ
0.15 mmol) and O3 -acetyl-N6-benzoyl-O2 -methyladenosine (0.047 g, 0.11
mmol) were dried by coevaporation with anhydrous acetonitrile (20 mL).
Then a solution of 2,4,6-triisopropylbenzenesulfonyl chloride (0.104 g,
0.347 mmol) and N-methylimidazole (0.058 g, 0.69 mmol) in 2 mL of

Leishmania cap-4 Intermediates
1345
acetonitrile was added, and the mixture was allowed to react for 4 hours.
The progress of the reaction was monitored by TLC (Rfproduct = 0.44 in
A). The crude product was purified by column chromatography (silicagel).
Elution was performed with a step-wise gradient (0.25–2%) of methanol in
chloroform, to give protected dinucleotide (0.09 g, 0.074 mmol, yield 67%,
m/z = 1211.38 by ESI-MS).
6
Dinucleotide m2 AmpAm (Figure 3).
To the protected dinucleotide
(0.02 g, 0.016 mmol) 10 mL of 3% solution of trichloroacetic acid in
dichloromethane was added and the mixture was stirred at room tempara-
ture for 1 hour and then extracted with water (3 × 20 mL). The organic
layer was evaporated under reduced pressure and 25% ammonia (20 mL)
was added. The reaction mixture was kept for 2 days at room tempera-
ture. The crude product was purified by HPLC (Rtproduct = 20.13 minutes.,
5.7 mg, 0.008 mmol, yield 16%, m/z = 651.21).
6
Dinucleotide 5ꢁ-Monophosphate (pm2 AmpAm).
A mixture of dinu-
6
cleotide (m2 AmpAm, 0.017 g, 0.026 mmol) and POCl3 (0.02 mL, 0.033 g,
0.2 mmol) in trimethyl phosphate (0.9 mL) was stirred for 1 hour at 0◦C.
The progress of the reaction was monitored by HPLC (Rtsubstrate = 20.13
minutes, Rtproduct = 16.20 minutes). Then water (3 mL) was added and
mixture was neutralized (to pH = 7) with 0.7 M TEAB buffer. Crude prod-
6
uct was purified by HPLC to give pm2 AmpAm (ammonium salt) (0.011 g,
0.015 mmol, yield 58%, m/z = 731.21 by ESI-MS).
6
Cap-2 (m7Gpppm2 AmpAm; Figures 1 and 2).
A mixture of am-
6
monium salt of dinucleotide 5ꢁ-monophosphate (pm2 AmpAm; 0.011 g,
0.015 mmol), a sodium salt of P2-imidazolide 7-methylguanosine 5ꢁ-
diphosphate [Im(m7GDP)] 0.027 g, 0.053 mmol), and ZnCl2 (0.062 g, 0.46
mmol) in dimethylformamide (0.5 mL) was stirred for 2 days at room tem-
perature. The progress of the reaction was monitored by HPLC (Rtsubstrate
=
16.64 minutes, Rtproduct = 12.83 minutes). Then addition of a few drops of
water quenched the reaction and EDTA was added until the reaction mix-
ture turned clear. The crude product was then purified by HPLC (0.0063
g, 0.005 mmol, yield 37%, m/z = 1169.2 by ESI-MS; calculated molecular
weight for cap-2: 1171.8).
1H NMR (D2O, δppm): 8.24 (1H, H2, s, adenine), 8.20 (1H, H2, s,
N6,N6-dimethyladenine), 8.19 (1H, H8, s, adenine), 7.86 (1H, H8, s, N6,
N6-dimethyladenine), 6.08 (1H, H1ꢁ, d, 7-methylguanosine, J = 3.99 Hz),
5.85 (1H, H1ꢁ, d, adenine), 5.81 (1H, H1ꢁ, d, N6,N6-dimethyladenine),
ꢁ
ꢁ
4.58-4.54 (3H, H2ꢁ, m, 7-methylguanosine, O2 -methyladenosine, N6,N6,O2 -
ꢁ
trimethyladenosine), 4.54-4.40 (3H, H3ꢁ, m, 7-methylguanosine, O2 -
ꢁ
methyladenosine, N6,N6,O2 -trimethyladenosine), 4.40-4.20 (9H, H4ꢁ and

1346
M. Lewdorowicz et al.
ꢁ
ꢁ
H5ꢁ, m, 7-methylguanosine, O2 -methyladenosine, N6,N6,O2 -trimethylade-
nosine), 3.97 (3H, N-CH3, s, 7-methylguanosine), 3.54 (6H, s, O-CH3,
ꢁ
ꢁ
O2 -methyladenosine and N6,N6,O2 -trimethyladenosine), 3.47 (6H, s, N-
CH3, N6,N6-dimethyladenine).
31P NMR (D2O, δppm) 0.709 ApA, -11.207 Pαγ , -22.65 Pβ.
Solid Phase Synthesis[2]
.
The RNA trimer was synthesized on an Ap-
plied Biosystems 392 synthesizer using a 1 µm scale protocol with 10 minutes
coupling for each step. The starting material, 5ꢁ-Dimethoxytrityl-N-benzoyl-
2ꢁ-O-methylcytidine-3ꢁ-succinoyl-long chain alkylamino-CPG (Glen Research,
Sterling, VA, USA) was placed in a standard reaction vessel and, successively,
following building blocks were coupled for elongation of the ribonucleic
chain: (1) 5ꢁ-dimethoxytrityl-2ꢁ-O-methyl-N-phenoxyacetyladenosine-3ꢁ-[(2-
cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research); (2)
ꢁ
5ꢁ -Dimethoxytrityl-N 6,N 6,O 2 -trimethyladenosine-3ꢁ-[(2-cyanoethyl)-(N,N-
diisopropyl)]-phosphoramidite[2]; and (3) 5ꢁ-phosphorylating reagent,
2-cyanoethyl-3-(4,4ꢁ-dimethoxytrityloxy)-2,2-di(ethoxycarbonyl)propyl-1-N,
N-diisopropyl phosphoramidite (Glen Research).
6
Unprotected RNA Fragment (pm2 AmpAmpCm).
A 5ꢁ-blocked RNA
fragment was detritylated for 20 minutes with 3% trichloroacetic acid
in dichloromethane. After 20 minutes the resin was washed with
dichloromethane and 25% ammonia was added. The reaction mixture was
kept overnight at 54◦C. The crude product was purified by preparative
6
HPLC (Rtproduct = 41.5 minutes) to give pm2 AmpAmpCm (ammonium
salt; 1.32 mg, 1.2 µmol, m/z = 1050.29 by ESI-MS).
6
Cap-3 (m7Gpppm2 AmpAmpCm; Figures 1 and 2). A mixture of
6
ammonium salt of trinucleotide 5ꢁ-monophosphate (pm2 AmpAmpCm;
1.06 mg, 1.009 µmol), a sodium salt of P2-imidazolide 7-methylguanosine
5ꢁ-diphosphate (Im-m7GDP; 0.002 g, 3.4 µmol), and ZnCl2 (0.003 g,
22 µmol) in dimethylformamide (0.150 mL) was stirred for 5 days at
room temperature. The progress of the reaction was monitored by HPLC
(Rtsubstrate = 16.73 minutes, Rtproduct = 14.33 minutes). Then EDTA (1,2-
ethylenediaminetetraacetic acid) was added until the reaction mixture
turned clear. The crude product was then purified by preparative HPLC
(Rtproduct = 36 minutes, 0.006 g, 0.4 µmol, yield 39%, m/z = 1489.43
by ESI-MS). ESI-MS spectra (negative ionization): m/z 1489.4 (calculated
molecular weight for cap-3: 1491.0).
1H NMR (D2O, δppm): 8.20 (1H, H8, s, adenine), 8.10 (1H, H8s,
N6,N6-dimethyladenine), 8.08 (1H, H2, s, adenine), 7.78 (1H, H2, s,
N6,N6-dimethyladenine), 7.60 (1H, H6, d, cytosine), 5.83 (1H, H1ꢁ,
ꢁ
s, N6,N6,O2 -trimethyladenosine), 5.99 (1H, H1ꢁ s, 7-methylguanosine)

Leishmania cap-4 Intermediates
1347
ꢁ
5.90 (1H, H1ꢁ, d, O2 -methyladenosine, J = 3.6 Hz), 5.83 (1H, H1ꢁ,
ꢁ
d, N6,N6,O2 -trimethyladenosine), 5.71 (1H, H5 s, cytosine), 5.55 (1H,
ꢁ
ꢁ
H1ꢁ d, O2 -methylcytidine), 4.52 (1H, H2ꢁ m, O2 -methyladenosine), 4.46
ꢁ
ꢁ
(1H, H2ꢁ m, N6,N6,O2 -trimethyladenosine), 4.31 (1H, H3ꢁ m, N6,N6,O2 -
ꢁ
trimethyladenosine), 4.26 (1H, H3ꢁ m, O2 -methyladenosine), 4.22 (1H,
ꢁ
H3ꢁ m, O2 -methylcytidine), 4.14 (1H, H2ꢁ m, 7-methylguanosine), 4.40-4.0
ꢁ
ꢁ
(12H, H4ꢁ and H5ꢁ m, N6,N6,O2 -trimethyladenosine, O2 -methylcytidine,
ꢁ
7-methylguanosine, O2 -methyladenosine), 3.99 (3H, N-CH3 s, 7-methyl-
guanosine), 3.80-3.60 (15H m, N-CH3, N6,N6-dimethyladenine and O-CH3
ꢁ
ꢁ
ꢁ
N6,N6,O2 -trimethyladenosine, O2 -methylcytidine, O2 -methyladenosine),
ꢁ
3.73 (1H, H2ꢁ m, O2 -methylcytidine).
31P NMR (D2O, δppm) 10.022 pA, ApC, -0.024 Pαγ , -10.428 Pβ.
Fluorescence Spectroscopy
Fluorescence titration measurements were carried out with LS-50B spec-
trofluorometer (Perkin Elmer Co. Norwalk, CT, USA) in 50 mM Hepes pH
7.2, 100 mM NaCl, 1 mM EDTA, 1 mM DTT at 20◦C using different protein
concentrations, from 0.2 µM to 1 µM. The fluorescence intensity (excited at
295 nm and observed at 320 or 345 nm) was corrected, taking into account
the sample dilution, the inner filter effect and the instability of protein flu-
orescence as described previously.[10]
REFERENCES
1. Bangs, J.D.; Crain, P.F.; Hashizume, T.; McCloskey, J.A.; Boothroyd, J.C. Mass spectrometry of mRNA
cap-4 from Trypanosomatids reveals two novel nucleosides. J. Biol. Chem. 1992, 267, 9805–9815.
2. Lewdorowicz, M.; Yoffe, Y.; Zuberek, J.; Jemielity, J.; Stepinski, J.; Kierzek, R.; Stolarski, R.; Shapira,
M.; Darzynkiewicz, E. Chemical synthesis and binding activity of the trypanosomatid cap-4 structure.
RNA 2004, 10, 1469–1478.
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