Nelson J. Leonard

Fluorescent adenosine and cytidine derivatives.

Summary The reaction of chloroacetaldehyde with adenosine and cytidine produces a fluorescent product in each case. These products are easily distinguishable spectroscopically by their fluorescence emission maxima. Since the reaction is carried out in aqueous media under mild conditions of pH and temperature, it should prove extremely useful in nucleic acid chemistry. For example, reaction occurs between chloroacetaldehyde and E. coli tRNA, and the reaction can be followed by monitoring the increase in the fluorescence emission. Fluorescence lifetimes observed for the adenosine and cytidine products are close to 20 and 7 nsec, respectively.

Cytokinins: Structure/activity relationships☆

Abstract Sixty-nine compounds, mostly purine derivatives and closely related substances, were tested for promotion of growth and regulation of organ formation in the tobacco bioassay to determine relationships between chemical structure and cytokinin activity. Forty-three substances were synthesized in this study, and 13 of these were reported for the first time. N 6 -Alkyladenines (I) varied in activity over a wide concentration range depending on the length of the alkyl chain. Starting with adenine, detectable at ⩾200 μM, activity increased with the chain length to an optimum for 6-pentylaminopurine detectable at ca. 0–001 μM, and then decreased to reach a barely detectable level for 6-decylaminopurine. The result of the incorporation of polar groups in the side chain was not necessarily reduction in activity. One hydroxyl group, as in zeatin (Id), improved the activity of 6-(γ,γ-dimethylallylamino)purine (Ib) if it affected it at all; two hydroxyl groups, as in 6-(2,3-dihydroxy-3-methylbutylamino)purine strongly reduced activity. Comparisons of 6-isoamylaminopurine with 6-(γ,γ-dimethylallylamino)purine and of other closely related pairs of compounds showed that a double bond in the side chain greatly increased cytokinin activity. Adenine derivatives with cyclic substituents in the N 6 -position (benzyl-Ic), cyclohexyl-, etc.) showed the same general range of activity, potentiation by unsaturation, and variation in activity with substituent size, etc. as did the alkyl derivatives. Heteroatoms in or on the substituent groups decreased activity (in the case of N or Cl) or had little effect (S for O in furfuryl). Of the mono-substituted adenines only the N 6 -derivatives definitely possessed cytokinin activity. The 1-(III), 3-(II), or 9-substituted adenines probably are inactive but could be activated by conversion to the N 6 -isomers. Except for slight activity in tests of high concentrations, which could be ascribed to contaminants, 7-substituted adenines were completely inactive. Modification in the adenine moiety lowered the cytokinin activity, often by 95 per cent or more. Substitution of N for the 8-C atom in kinetin and in 6-benzylaminopurine or S for the 6-amino N atom in 6-(γ,γ-dimethylallylamino)purine did not eliminate but drastically reduced activity in the tobacco bioassay. Elimination of the 6-amino group without substituting another group completely removed activity; thus, the purine derivatives, 1-benzylpurine and 1-(γ,γ-dimethylallyl)purine, were inactive in tests where the 1-adenine derivatives could be activated to give a positive response. Addition of a second substituent on the 1-or 3-position of N 6 -substituted adenines drastically reduced or eliminated cytokinin activity. It is suggested that the 1-position and possibly also the 3-position must be free. A second substituent in the N 6 -, 7-, or 9-position of N 6 -substituted adenine derivatives lowered but did not eliminate activity. Also, the disubstituted 1-adenine derivatives, 1,9-dibenzyladenine and 1,7-dibenzyladenine were active, presumably after rearrangement to the corresponding N 6 -substituted isomers.

A Fluorescent Modification of Adenosine Triphosphate with Activity in Enzyme Systems: 1,N6-Ethenoadenosine Triphosphate

A new, highly fluorescent adenosine triphophate (ATP) analog, 1,N6 ethenoadenosine triphosphate, has been synthesized. Its fluorescence properties, including the long fluorescence lifetime and the possibility of detection at very low concentrations, in conjunction with its activity in the representative enzyme systems here reported, make it a valuable probe of enzymic mechanism and structure.

Etheno-Substituted Nucleotides and Coenzymes: Fluorescence and Biological Activity

AbstractDefinition of the individual adenine binding sites of enzymes is important for many reasons. Rendering an adenine moiety fluorescent while retaining the biological activity of the molecule of which it is a component can provide useful binding information because of the fluorescence properties. The 1,N6-ethenoadenosine phosphates and, similarly, e-modified polynucleotides, RNA, and DNA have been synthesized, and their spectroscopic properties and interactions have been studied in depth. The use of e-substituted nucleotides has helped to clarify numerous enzyme reactions, including those of ATPase and energy transfer, ATP transphosphorylase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphorylase b, protein kinase, pyruvate kinase, ribonucleases, RNA ligase, and others. The binding of e-substituted nucleotides or polynucleotides to proteins has been determined for F- and G-actin, heavy meromyosin, tobacco mosaic virus protein, gene 32 protein of bacteriophage T4, and chloroplast...

Cytokinin from Soluble RNA of Escherichia coli: 6-(3-Methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine

We have isolated a compound responsible for the cytokinin activity of soluble RNA from Escherichia coli. The structure, indicated as 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine, C16H23N504S, on the basis of low-and high-reso!ution mass spectrometry, was established by unequivocal synthesis. The mass spectra, chromatographic behavior, and ultraviolet spectra of the compounds from natural and synthetic sources were identical.

The synthesis of ribosyl-cis-zeatin and thin layer chromatographic separation of the cis and trans isomers of ribosylzeatin.

Ribosyl-cis-zeatin has been synthesized. With this authentic ribonucleoside on hand for comparison, it has been shown that the cis and trans isomers of ribosylzeatin can be readily distinguished by thin layer chromatography on silica gel using chloroform-methanol (9:1) as eluant. Using this method, the ribosylzeatin from the tRNA of tobacco callus and wheat germ has been characterized as the cis isomer and that from pea epicotyls has been shown to be a mixture of both ribosyl-cis-zeatin and ribosyl-trans-zeatin.

Tri-s-triazine: synthesis, chemical behavior, and spectroscopic and theoretical probes of valence orbital structure

Synthese du tri-s-triazine, et determination de sa structure par diffraction RX; etude de ses proprietes chimiques, spectroscopiques et physiques. On utilise la spectrometrie photoelectronique ainsi que des calculs ab initio et HAM3

Cytokinins: Synthesis and growth-promoting activity of 2-substituted compounds in the N6-isopentenyladenine and zeatin series

Abstract Fourteen compounds were tested for relative promotion of cell division and growth (cytokinin) activity in the tobacco bioassay. The results suggested that 2-substituted- N 6 -(hydroxy)isopentenylaminopurines were generally less active than their unsubstituted couterparts. Thus, a 2-OH substituent greatly lowered cytokinin activity in both the isopentenyl and hydroxyisopentenyl (zeatin) series, while 2-NH 2 and 2-SCH 3 groups had a lesser effect on activity and a 2-Cl substituent had a negligible effect. Mass spectra were determined for all of the 9-β- d -ribofuranosides and for a number of the purines as well; the fragmentation patterns were consistent and characteristic, providing a possible systematic approach to the identification of new 2-substituted- N 6 -(hydroxy)isopentenyladenines.

FLUORESCENT NUCLEOSIDES AND NUCLEOTIDES

In investigating the three-dimensional structure of several biologically important macromolecules, various spectroscopic methods have been employed in gaining information concerning the correlation between molecular structure and biological function. In this regard, applications of fluorescence spectroscopy in the field of protein chemistry have been overwhelmingly successful, and many valuable structural and dynamic properties of macromolecular proteins have been obtained. Reviews outlining the many uses and advantages of fluorescence spectroscopy in the study of biologically important macromolecules are available.’-4 Although similar experiments using fluorescence techniques in investigating the three-dimensional structure of nucleic acids are feasible, fluorescence spectroscopy has had limited use in nucleic acid chemistry because of the infrequent occurrence in nature of fluorescent n~cleos ides .~ Because of the attractive advantages that fluorescence techniques afford in studying the tertiary structure of molecules, we have endeavored to develop chemical means by which fluorescent probes can be introduced selectively into transfer RNA and into the dinucleotide coenzymes such as NAD’ and FAD so that the versatile and highly sensitive techniques of fluorescent spectroscopy can be used to help in understanding the structure-function relationships operating in these important molecules.

Comparison of cytokinin activities of naturally occurring ribonucleosides and corresponding bases

Abstract Cytokinin activities in the tobacco bioassay have been determined for four adenosine derivatives known to be components of wheat germ t RNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β- d -ribofuranosylpurine, 6-(3-methyl-2-butenylamino)-9-β- d -ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)- 2-methylthio-9-β- d -ribofuranosylpurine, and 6-(3-methyl-2-butenylamino)-2-methylthio-9-β- d -ribofuranosylpurine. Also determined and compared with the four natural components of t RNA were the activities of the four 3-methylbutylamino analogs of the naturally occurring species and the eight substituted purines corresponding to both sets of ribonucleosides. The systematic structural modifications within this group of sixteen compounds were reflected in the variations in cytokinin activity with the level of modification.