Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes

6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band (lambda max approximately 600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.     

The synthesis and properties of N6-substituted 2-amino-purine derivatives.

1. The synthesis, ultraviolet absorption spectra, and behaviour in alkali of N6-methoxy-, N6-methyl, hydroxy-, and N6-hydroxy-2-aminopurines have been described 2. N6-Methoxy-2-aminopurine riboside 5'-pyrophosphate has been prepared and used for polymerization with polynucleotide phosphorylase. 3. The copolymer containing N6-methoxy-2-aminopurine riboside and adenosine residues has been obtained; attempts to synthesize the homopolymer have not been successful. 4. All the purine analogues synthesized have been tested and shown to act mutagenically on Salmonella typhimurium TA1530.     

Improved synthesis of antibacterial 3-substituted 6-anilinouracils

3-Substituted 6-anilinouracils, presently the most promising class of inhibitors of the bacterial DNA polymerase in Gram-positive bacteria, have been prepared by a general and straightforward three-step procedure starting from a readily available 1-benzyloxymethyl-protected derivative of 6-chlorouracil.     

Coenzym properties of some Ade-1- and Ade-N6-substituted NAD derivatives (author's transl)]

By reaction of NAD with different oxiranes or with aziridine, derivatives of the coenzyme are obtained with substituents in position 1 or on the amino group in position 6 of the adenine ring. While the Ade-1-substituted derivatives show high Km values with different dehydrogenases and are reduced only very slowly by these enzymes, the coenzyme derivatives substituted at the amino group behave very similarly to NAD. Correlations were found between coenzyme efficiency of the compounds and the lipophilic character of their substituents. The results can be interpreted from the structure of the active site of the dehydrogenases investigated.     

5-substituted arabinofuranosyluracil nucleosides: synthesis and antiviral properties

A number of 5-alkyl (ethyl, propyl, isopropyl, butyl) analogues of araU, their alpha-anomers and N3-isomers have been synthesized by a number of different procedures, based on the catalytic condensation of the appropriate 5-alkyl-2,4-bis-(trimethylsilyloxy)-pyrimidine with 2,3,5-tri-O-benzyl-alpha-D-arabinofuranosyl chloride. The resulting protected nucleosides were deblocked by a new procedure based on the use of BF3 X Et2O in C2H5SH. The chloromercuri derivative of araU, on reaction with allyl chloride in the presence of Li2PdCl4, gave the 5-allyl derivative, which was catalytically reduced to the corresponding 5-propyl analogue. The antiviral activities of these compounds have been evaluated. 5-Allyl-araU showed moderate specific activity (MIC 20 micrograms/ml) against herpes simplex type 1 virus in PRK cell cultures. Structure-activity relationships are discussed for the 5-alkyl deoxy- and arabino- uracil nucleoside series.     

Fluorescence properties of reduced flavins and flavoproteins

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

Chemical synthesis and properties of (6-4) photoproduct and its analogs.

We report the preparation of a deoxyribooligonucleotide containing a new thymine (6-4) photoproduct analog. The (6-4) photoproduct is one of the major forms of DNA lesions, and leads to mutation in DNA. An antibody (64M5) that binds the (6-4) photoproduct has been described. To investigate the interaction of the photoproduct with the 64M5 antibody, we prepared a (6-4) photoproduct analog in which the two thymines were connected with a formacetal linkage. With UV-irradiation, the thymine dimer with the formacetal linkage reacted to the (6-4) photoproduct faster than the phosphodiesterified dimer, and the yields of the analog was higher than those of the natural thymine dimer. The 64M5 antibody exhibited sufficient binding to a tetranucleotide containing the (6-4) photoproduct analog with a formacetal linkage, although the association constant was slightly lower than that for the natural lesion. This (6-4) photoproduct analog may be useful for investigation of other proteins that recognize the (6-4) photoproduct.     

The synthesis of N2,N6-substituted diaminopurine ribosides

A series of new 2,6-substituted diaminopurine riboside derivatives were synthesized by activation of protected xantosine with sulfonyl chlorides followed by treatment with various amines. The relationship between the reactivity of intermediates and the nature of the activating agents was studied.