SYNTHESIS AND PROPERTIES OF THE NEW ACYCLIC DERIVATIVE OF LUMINAROSINE J. Nowak, J. Milecki, B. Skalski Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
Pyridinium derivatives of nucleosides are interesting objects of spectral,
photophysical and photochemical research . Pyridinium salt derived from 2',3', 5'-tri-O-acetylinosine, when irradiated with UV-light ( λ>300nm) undergoes transformation to 2',3', 5'-tri-O-acetyl-luminarosine- the strongly fluorescing nucleoside, being characterized with the long-wave absorption (λ Amax= 425 nm) and the intensive
fluorescence (λ Fmax= 528 nm) [2,3]. Because of its large chemical and photochemical
stability and very profitable absorption-emission properties , 2',3', 5'-tri-O-acetyl-luminarosine and analogues can serve as fluorescent probes in biological system . Luminarosine can also be used to fluorescent marking of oligonucleotides in the automatic synthesis on the solid support. But there is certain difficulty because of the high susceptibility of this nucleoside to spontaneous anomerisation leading in the final result to the mixture of oligonucleotides containing λ and β anomers of luminarosine .Good alternative for luminarosine, as the fluorescent mark of DNA, can be its acyclonucleoside analogues, which don’t undergo the anomerisation reaction. The attempt of the synthesis of such analogue, according to procedures worked out previously, basing on the photochemical transformation of the suitable N-(purin-6-yl)-pyridinium chloride is presented in this paper.
1) Synthesis of the N[(9-tert-butoxycarbonylmethyl)purin-6-yl]pyridinium chloride (2).
In the nucleophic substitution of compound (1) - 6-chloro-9-(tert-butoxy-
carbonylmethyl)purine, according to the well-known procedure, consisting on the treatment of the chloroderivative of purine or nucleoside with the pyridine at 60oC during 2-5 hours, pyridium salt (2) has been obtained with 20% yield (Scheme 1).
The product 2 has UV spectrum characteristic for pyridinium salts (Fig.1) with two maximum of the absorption at λ=272nm and λ=295 nm. Figure 1. The spectrum of the absorption of compound 2.
2) Synthesis of 9-N-(tert-butoxycarbonylmethyl)luminarine (4).
The obtained pyridinium salt (2) has been exposed to photochemical reaction in
In the first step of reaction, deoxidized aqueous solution of the N-[(9-tert-
butoxycarbonylmethyl)purin-6-yl]pyridinium chloride (2) was subjected to irradiation with near UV light (λ> 300 nm) using the reactor equipped with dip mercuric lamp TQ -150 Original Hanau stocked with the cylindrical pyrex filter. The course of the reaction was followed spectrophotometrically (Fig. 2A). The disappearance of pyridinium salt and the formation of the product with maximum of the absorption at λ=242 nm was noted. The analysis of irradiated solution (Fig. 2B and 2C) showed the formation of the desirable intermediate product 3 in the synthesis of the acyclic derivative of luminarosine.
In the second step of reaction the aqueous solution of the compound 3 was
alkalized to pH~7,5, 1,5 eq. of N-(9-methylpurin-6-yl)pyridium chloride (5) was added as the photosensitizer and mixture was exposed to sunlight in aerobic conditions. Based on analyses of the solution after the irradiation (Fig. 3B and 3C) and spectrophotometrical data (Fig. 3A) it was found that under this conditions of irradiation the compound 3 has been transformed into 9-N-(tert- butoxycarbonylmethyl)luminarine (4). The yield of the photochemical reaction was 18%. In order to isolate the product the aqueous solution of the compound 4 was extracted by CHCl3. Obtained product 4 shows band of absorption in the visible range
with λmax = 426 nm characteristic for luminarosine.
Figure 2. (A) Changes in the absorption spectra during the irradiation of deoxidized aqueous solution of the compound 1, (B) the HPLC analysis of the solution after the irradiation and (C) the absorption spectrum of compound 3. Figure 3. (A) Changes in the absorption spectra during the irradiation of aqueous solution of the compound 3, (B) the HPLC analysis of the solution after the irradiation and (C) the absorption spectrum of compound 4.
Structure of the compound was confirmed by H-NMR, ESI-MS and UV-VIS.
ESI MSm/z 328 (M+H+), calculated m/z 327,13 for C16H17N5O3 UV-VIS λmax (H2O) 264 nm, 426 nm 1H NMR (CDCl3), δ 1,48 (s, 9, H-8) 4,28 (d, J=6,0 Hz, 2, H-7), 7,62 (t, J=6,0 Hz, 1, H-1), 8,08 (m, J=7,0 Hz, 1, H-4), 8,26 (s, 1, H-2), 8,42 (m, J=7,6 Hz, 1, H-5), 9,10 (d, J=8,0 Hz, 1, H-6), 10,17 (d, J=6,6 Hz, 1, H-3)
The received new acyclic derivative of luminarosine - 9-N-(tert-
butoxycarbonylmethyl)luminarine (4) after suitable modification of ester part can replace luminarosine and be used to the marking of any sequence of oligonucleotides on 3′ or 5′ end.
 B. Skalski, S. Paszyc, R. W. Adamiak, R. P. Steer, R. E. Verrall, Canadian Journal of Chemistry-Revue Canadienne de Chimie, 68 (1990) 2164.  B. Skalski, J. Bartoszewicz, S. Paszyc, Z Gdaniec, R. W. Adamiak, Tetrahedron, 43 (1987) 3955.  B. Skalski,R. P. Steer, R. E. Verrall, J.Am.Chem.Soc., 113 (1991) 1756. B. Skalski, S. Paszyc, R. W. Adamiak, R. P. Steer, R. E. Verrall, Journal of The Chemical Society-Perkin Transactions 2 (1989) 1691.  S. Jayaraman, L. Teitler, B. Skalski, A., S. Verkman, American Journal of Physiology-Cell Physiology, 277 (1999) C1008.  A. Burdzy, B. Skalski, S. Paszyc, M. Popenda, R. W. Adamiak, Acta Biochim.Pol., 45 (1998) 941.  A. Burdzy, B. Skalski, S. Paszyc, Z Gdaniec, R. W. Adamiak, Nucleosides Nucleotides, 17 (1998) 143.
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