readily synthesized from the imidazolide nucleotides and
inorganic phosphate in prebiotic conditions, would require,
unlike the 2-methylimidazole-activated nucleotides, some kind
of catalyst for template directed polymerisation (probably
“inorganic”, like metal ions or mineral) but seem better candi-
dates for the prebiotic job of synthesizing the first polyribo-
nucleotides because of their kinetical stability to nucleophile
substitution.
and/or TRIS (up to 0.2 ) in D2O. Buffer pD was adjusted
with concentrated KOD and/or DNO3 to 7.6 (pD = pHmeasured ϩ
0.4)12 using a Crison Micro pH 2002 pHmeter and an Ingold
U402 microelectrode calibrated with pH concentration buffers
4 and 7. Samples were prepared by dissolving in the NMR tube
the required amount of 2-MeImpN in 0.5 ml of buffer to give a
concentration of 0.020 0.001 . Spectra were taken with 2 to
3 h intervals at 308 K (35 ЊC) until 2-MeImpN 1H signals were
no longer detected. Typically, 10 to 16 spectra were obtained;
the time attributed to each spectrum was its acquisition finish-
ing time. Mole fractions for each time were calculated based on
the relative integrals of each nuclei signal set. Kinetic functions
were fitted to the mole fraction data using linear and non-linear
least-squares analysis.
Experimental
Reagents
Nucleotides, KOD and NaOD (conc.) and reagents for
2-MeImpN synthesis were from Sigma; D2O (99.5% D), NaCl
and buffers were from Merck.
Molecular modelling
Synthesis of 2-MeImpA and 2-MeImpG
Semiempirical PM3 molecular modelling was done with PC
Spartan Plus (Wavefunction, Inc.), ver. 1.4, using H–H repul-
sion correction, running in a 300 MHz Pentium II PC with
Windows NT 4.0 Workstation OS.
Preparation of the activated nucleotides was done by a pro-
cedure previously described.2 The products were dried in a Petri
dish for at least a week under vacuum with desiccant and stored
also under these conditions in a small capped flask; yield was
better than 95% in weight. However, the 1H NMR spectra
revealed unaccounted singlets at 0.68 and 1.48 ppm (chemical
shift reference: tert-butyl alcohol) that do not belong to the
products. The intensity and chemical shifts of these signals
didn’t change with time. The 31P NMR spectra didn’t show
any unexpected signals. The chemical shifts observed for
2-MeImpA and 2-MeImpG spectra taken in the standard con-
ditions for this work (see below) are presented in Fig. 1.
Acknowledgements
The authors would like to thank CEQUP and ICBAS for
financial support and Professor Anake Kijjoa for fruitful
discussions.
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Sample preparation and standard experimental conditions for
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Buffer stock solutions were prepared by dissolving the required
amounts of NaCl (0.5 ), Na2HPO4/NaH2PO4 (up to 0.5 )
Paper 9/00884E
1148
J. Chem. Soc., Perkin Trans. 2, 1999, 1143–1148