Oligonucleotides incorporating 7-(aminoalkyn-1-yl)-7-deaza-2'-deoxyguanosines: duplex stability and phosphodiester hydrolysis by exonucleases.

Self-complementary [[5'-d(G-C)4]2] and non-selfcomplementary oligonucleotides [5'-d(TAG GTC AAT ACT) x 3'-d(ATC CAG TTA TGA)] containing 7-(omega-aminoalkyn-1-yl)-7-deaza-2'-deoxyguanosines (1a-c) (1) and 7-deaza-2'-deoxyguanosine instead of dG were studied regarding their thermal stability as well as their phosphodiester hydrolysis by either 3' --> 5'- or 5' --> 3'-phosphodiesterase studied by MALDI-TOF MS.

Phosphoramidites and oligonucleotides containing 7-deazapurines and pyrimidines carrying aminopropargyl side chains.

The synthesis of phosphoramidites containing 7-deazaguanine, 7-deazaadenine, uracil and cytosine carrying aminopropargyl chains is described. The corresponding oligonucleotides are stabilized in duplexes thermally as well as against degradation by exonucleases.

Fluorescent DNA: the development of 7-deazapurine nucleoside triphosphates applicable for sequencing at the single molecule level.

7-Deaza-2'-deoxyadenosine and -guanosine phosphoramidite building blocks as well as corresponding 5'-triphosphate derivatives are described carrying in position 7 substituents such as iodo, hexyn-1-yl or 5-aminopentyn-1-yl residues. The phosphoramidites were used to synthesize a series of modified oligodeoxynucleotides. A systematic study of the thermal stabilities of these oligonucleotide duplexes demonstrated that the 7-substituents are well accommodated in the major groove of B-DNA. The 7-(aminoalkyn-1-yl)-7-deazapurine 2'-deoxynucleoside triphosphates were labeled with bulky fluorophores such as Rhodamine Green(R) or tetramethylrhodamine.

2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides.

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.

Photochemical conversion of oligonucleotides containing 2-chloro-2'-deoxyadenosine: enhanced UV sensitivity of a modified purine base induced by the nearest neighbor.

Irradiation of alternating dodecamers containing 2-chloro-2'-deoxyadenosine (Cl2dAdo) with ultraviolet light (254 nm) has been investigated. The photoconversion of Cl2dAdo into 2'-deoxyisoguanosine (isodGuo) and the completeness of the reaction were studied by reversed-phase HPLC and UV absorption spectra. The photosensitivity of Cl2dAdo within an oligonucleotide was found to be sequence-specific and depends on the nearest neighbor; 2'-deoxyguanosine residues accelerate the rate of photoconversion by a factor of 3.

Purine nucleoside phosphorylase: inhibition by purine N(7)- and N(9)-acyclonucleosides; and substrate properties of 7-beta-D-ribofuranosylguanine and 7-beta-D-ribofuranosylhypoxanthine.

A series of 10 N(7)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8-SH and 8-NH2), and two N(7)-acyclonucleosides of hypoxanthine, were tested for their ability to inhibit purine nucleoside phosphorylase (PNP) (E.C. 2.4.2.1) from human erythrocytes and rabbit kidney. The acyclic chains contained a nitrogen in place of a carbon at the 3', 4' or 5' position and, in one case, an ether oxygen at the 2' position. Most striking was the finding that one of the N(7)-acyclonucleoside analogues, 7-[(1,3-dihydroxypropyl-2)amino]ethylguanine, proved to be a 3-fold more effective inhibitor than its corresponding N(9) counterpart, with Ki = 5 vs 14 microM for the human enzyme and 0.7 vs 2.3 microM for the rabbit enzyme. Both analogues, as well as the others examined, inhibited phosphorolysis competitively with respect to nucleoside substrates (inosine with the human enzyme and guanosine with the rabbit enzyme). The foregoing logically led to the finding that the 7-beta-D-ribosides of guanine (N7Guo) and hypoxanthine (N7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E. coli. With the human enzyme the pseudo-first-order rate constants (Vmax/Km) for phosphorolysis of N7Guo and N7Ino were 0.08 and 0.02% that for Ino. The Michaelis constants (Km) for N7Guo were 27 (calf PNP), 108 (human PNP) and 450 microM (E. coli PNP). For N7Ino the corresponding Km values were 1.52, 1.26 and 0.64 mM. Four previously well-characterized N(9)-acyclonucleoside inhibitors of calf spleen PNP were found to inhibit phosphorolysis of N7Ino by the same enzyme 2-10-fold more effectively than the parent Ino. The overall results, along with the known excellent substrate properties of N(7)-alkyl- Guo and Ino (Bzowska et al. J Biol Chem 263, 9212-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes.