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Z. Cui et al. / Bioorg. Med. Chem. 12 (2004) 927–933
be synthesized. These results suggested that the leaving
group of the substrate is not the binding site for R180
ribozyme and the biotin of the substrate is the binding
site (data not shown).
desired fractions were evaporated quickly within 10 min
by vacuum pump and the aqueous solution was lyophi-
lized to yield a white solid. H NMR (D2O): d 8.45 (m,
1
1H), 8.26 (m, 1H), 6.12 (d, J=6.87 Hz, 1H), 4.74 (m,
1H), 4.57–4.46 (m, 3H), 4.36 (m, 2H), 4.21 (m, 2H), 3.25
(m, 1H), 3.09 (m, 2H), 2.96 (m, 1H), 2.72 (m, 1H), 2.52-
2.39 (m, 2H), 2.25–1.89 (m, 9H), 1.68–1.22 (m, 12H); 31P
NMR (D2O): d-7.52; ESI-MS (M-H): calculated 816.3,
found 816.3. Other biotin-aminoacyl-AMP anhydrides
were prepared as above. The compounds were char-
acterized by NMR, HPLC, and mass spectrometry.
3. Conclusion
This study implicates that the catalytic RNA may have
the potential application in the peptide synthesis. The
ribozyme can function as the peptidyl transferase for the
protein synthesis. Our selected ribozyme is capable of
catalyzing random dipeptide synthesis, thus further sup-
porting the powerful RNA catalysis. As proposed that
the polypeptide may be synthesized by multiple ribozymes
mimicking the modular polyketide and non-ribosomal
polypeptide synthesis.19 Such, the study implied that the
ribozyme might be able to make randomized polypeptide
library. The ribozyme may function as a general peptide-
synthesizing enzyme to make the peptide.
4.3. Synthesis of L-aminoacyl-pyridyldithioethylamide
Pyridyldithioethylamine (PDA) was prepared by the
procedures described in the literature.21 The l-amino-
acyl-PDA derivatives (Phe-PDA, Leu-PDA, Gln-PDA,
Lys-PDA, and Trp-PDA) were synthesized as follows.
A solution of PDA (0.74 g, 3.3 mmol) and 1.0 mL of
diisopropylethylamine (DIEA) in 10 mL of anhydrous
dimethylformamide (DMF) at room temperature was
added into a solution of N-Boc-aminoacyl-N-hydroxy-
succinimidyl (NHS) ester (3.0 mmol) in 10 mL of anhy-
drous DMF. The mixture was stirred for two days at
room temperature. After removing the DMF solvent,
the solid was dissolved in 100 mL of chloroform and
then washed with water three times. The organic layer
was dried over anhydrous Na2SO4. The crude product
was purified by a silica gel column eluted with metha-
nol/chloroform (0–5%). The pure Boc-aminoacyl-PDA
compounds were dissolved in 20 mL of methylene chlo-
ride and 2.0 mL of trifluoroacetic acid (TFA) was
dropwise added at 0 ꢀC. The mixture was stirred for one
h at 0–5 ꢀC and 2 h at room temperature. The reactions
were monitored by thin layer chromatography (TLC)
of silica gel plate run in chloroform/methanol, 8:2
(vol/vol). After removing TFA and solvent, the material
was applied to flash column chromatography (silica
gel) and eluted with chloroform/methanol (0–20%). The
final products were confirmed by NMR and mass
spectrometry.
4. Experimental
4.1. Ribozyme preparation
The double stranded DNA as the template for in vitro
transcription was made by PCR amplification in 10 mM
Tris–HCl; pH 8.3; 50 mM KCl; 0.01% (W/V) gelatin;
0.05% Tween 20; 0.2 mM dNTPs; 1.0 mM primers and
3.0 mM MgCl2, in 30 cycles (94 ꢀC, 1 min; 52 ꢀC, 1 min;
72 ꢀC, 2 min). In vitro transcription was performed with
10 mg of PCR-amplified 196-mer DNA in a 250 mL
reaction mixture (40 mM Tris–HCl, pH 7.5; 10 mM
DTT; 4 mM spermidine; 0.05% Triton X-100; 12 mM
MgCl2; 1.0 mM each ATP, CTP, UTP; 8 mM GMPS;
1.0 mM GTP; and T7 RNA polymerase) for 3 h at
37 ꢀC. For 32P-labaled RNA was transcribed in the pre-
sence of 10 mCi [a-32P]-ATP. 50-GSMP-RNA was purified
by electrophoresis on a denaturing 8% polyacrylamide
gel. 50-GMPS-RNA was reacted with N-bromoacetyl-
N’-phenylalanyl-cystamine (10 mM) in the chemical
linkage buffer (40 mM HEPES, pH 8.0; 150 mM NaCl,
and 1 mM EDTA) for 2 h at room temperature. The
reaction mixture was extracted with chloroform/phenol
once, chloroform once, and precipitated with ethanol to
give 50-Phe-SS-GMPS-RNA.
4.4. Activity assay
The reactions were performed with 0.5 mM 50-Phe-SS-
GMS-RNA (R180) and 50 mM biotin-Met-AMP in the
presence of 300 mM KCl, 100 mM MgCl2, and 50 mM
HEPES buffer (pH 7.4) at 25 ꢀC. The RNA was pre-
incubated for 10 min at 50 ꢀC and then slowly cooled to
room temperature. Reactions were initiated after addi-
tion of biotin-aminoacyl-AMP substrate. Aliquots of 2
mL were removed from a 20 mL reaction mixture at
specific time points, quenched with equal volumes of
stop buffer [100 mM HEPES (pH 7.4), 100 mM EDTA,
90% formamide, 0.01% bromophenol blue, and
0.025% xylene cyanol] and frozen on dry ice. Thawed
samples were incubated with 7.5 mg of streptavidin for
20 min. The biotinylated RNA products were resolved
by electrophoresis on 7.5 M urea/8% polyacrylamide
gels with 1ꢂTBE buffer at 800 V at 4 ꢀC. The fraction of
product formation relative to total substrate and
product at each time point was quantitated using a
Molecular Dynamics PhosphorImager.
4.2. Biotin-L-aminoacyl-adenylate synthesis
The described20 synthesis of biotin-Met-AMP anhy-
dride was modified as follows. 0.8 M HCl (138 mL) was
added to a mixture of N-biotinylamidocaproyl-l-amino
acid (0.1 mmol) and nucleoside-50-monophophate (0.11
mmol) in water (51 mL) and pyridine (572 mL) at 0 ꢀC. A
solution of dicyclohexylcarbodiimide (DCC) (2.6 mmol)
in pyridine (600 mL) was added. The mixture was stirred
in an ice-water bath for 3.5 h. After removing the pre-
cipitation, the solution was precipitated with acetone at
ꢁ70 ꢀC and collected by centrifugation. The pellet was
washed with ethyl ether (30 mL), dissolved in water at
0 ꢀC, and immediately applied to a C18 reverse-phase
column and eluted with water/methanol (0–50%). The
Cui, Zhiyong
