The 12 possible dinucleoside phosphates combining adenosine and cytidine with adenosine, cytidine, guanosine, and uridine were readily converted to the 1,7V6-ethenoadenosine (eA) and 3,TV4-ethenocytidine (eC) analogs by reaction with chloroacetaldehyde. Those dinucleoside phosphates containing 1,7V6-ethenoadenosine are fluorescent in neutral solution, while those containing 3,7V4ethenocytidine are not since 3,7V4-ethenocytidine is fluorescent only in the protonated form. Chloroacetaldehyde modification in general renders the dinucleoside phosphates more resistant to nucleolytic cleavage. Dinucleoside phosphates of the form

Flavin 1,N6-ethenoadenine dinucleotide (eFAD) was prepared by the action of chloroacetaldehyde on flavin adenine dinucleotide. This compound, which has two potential fluorophores, e-adenine and isoalloxazine, shows extremely efficient energy transfer from the former to the latter. The fluorescences of both moieties are greatly diminished in the intact molecule. Determination of the fluorescence yields and lifetimes leads to the conclusion that at 200 in neutral aqueous solution eFAD exists mainly (90%) as an internally complexed or stacked form. In the shortened forms of the names, the abbreviation "e" now generally in use stands for the etheno bridge. * To whom to address reprint requests.t Harvey and Damle (2) announced the preparation of EFAD, but our findings on pure material are in strong contrast to those reported, especially with respect to energy transfer. We suggest that considerable error could have been introduced if the eFAD was used in the fluorescence studies without chromatographic purification, since significant amounts of hydrolysis products are present in the crude compound. The large emission with a maximum at 410 nm attributed by Harvey and Damle to the eAde moiety in EFAD is undoubtedly due to some contaminating unquenched eAde derivative, such as a mononucleotide. 941 developed with 1.0 M LiCi or isobutyric acid: NH4OH:H20 75:1:24, v/v. eFAD was extensively purified by column chromatography on DEAE-Sephadex, eluting with either 100 mM phosphate buffer (pH 6.8) or a gradient of ammonium formate (pH 4.0), 0.025-1.0 M, following the excellent method of purification of FAD supplied by Massey and Swoboda (4). Homogeneity of the fluorescence lifetimes, as determined by phase and modulation by use of the crosscorrelation fluorometer (5), was found to be the most sensitive criterion of purity and was therefore extremely useful for following the purification procedure. All purification steps were performed at 50 and in the dark.Hydrolysis of eFAD by Phosphodiesterase I from Crotalus adamanteus venom (EC 3.1.4.1) (Sigma). The assay was performed at 23°in 3-ml reaction mixtures containing 100 mM phosphate buffer (pH 7.0) and eFAD (0.2 OD at 450 nm) by following the increase in fluorescence intensity of the e-adenine moiety (410 nm) when phosphodiesterase I was added in a quantity sufficient to produce complete hydrolysis in 30-40 min. WAVELENGTH (nm) FIG. 1. Fluorescence emission spectrum of eFAD in 0.1 M aqueous phosphate buffer at pH 7.0.

1. Fluorescence lifetimes and emission spectra of 1 : N6-ethenoadenosine 5'-monophosphate (&) in aqueous solution over the pH range 1.5 to 12.0 indicate the presence of only one emitting fluorophore.2. The loss a t low pH of fluorescence emission a t 415 nm from the neutral 1 : N6-ethenoadenine fluorophore is due to the conversion of the fluorescent unprotonated form to the non-fluorescent protonated form by protonation a t N-9. This conclusion is based on the pH dependence of the fluorescence lifetimes and quantum efficiences of CAMP over the range 1.5 to 12.0.3. The observation of a fluorescence quantum efficiency of 86O/, that of & in aqueous solution (pH 6.8) for 1 : N6-etheno-9-propyladenine (E-PrAde) in dry dioxane where it cannot acquire a proton in the excited state is direct evidence that the unprotonated form of the Eadenine fluorophore is responsible for the fluorescence emission. the interpretation of the fluorescence data obtained necessitate an understanding of the excited state of the fluorophore, since quantum efficiencies, lifetimes and spectra reflect environmental conditions surrounding the fluorophore. It is our purpose here to define the excited state of 1 : N6-ethenoadenosine, especially since Penzer [14] has proposed that in aqueous solutions fluorescence is emitted from the 9-protonated form of I a .I n the initial description of the fluorescence properties of 1 : N6-ethenoadenosine [l], evidence was presented that the neutral form of &Ado is responsible for the fluorescence. Emission spectra taken a t intervals between pH 7.0 and 2.2 showed that the emission maximum a t 415 nm remained constant over this pH range while the fluorescence intensity decreased progressively beginning about pH 5.5 and continuing until, at pH 2.2, the intensity was less than loo/, of the intensity a t pH 7.0. The fluorescence excitation spectrum indicated that the long-wavelength band centered at 300 nm was responsible for fluorescence. The disappearance of the long-wavelength band in the absorption spectrum with the loss of fluorescence intensity a t 415 nm as I : N6-ethenoadenosine (pKa = 3.9) becomes protonated, suggested that only one structure, the neutral form, is the fluorophore [l].Nevertheless, Penzer has proposed that although the pKa of the ground state protonation is known to be 3.9 [l], the excited state pKa is five or more units more basic and that upon excitation, a proton is donated by water: &Ado* + H,O z &Ado*-H+ + OH-.(1) Eur. J. Biochem. 45 (1974)

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The ribosylation of several silylated pyrrolo[2,3‐d]pyrimidines by the Wittenberg procedure has produced 1,3 and 7‐ribosylpyrrolo[2,3‐d]pyrimidine derivatives in high yield. Structure assignments have been made on the basis of the ultraviolet spectra of model compounds and further confirmed by chemical conversion to derivatives of established structure. A convenient ribosylation procedure utilizing silver oxide, a halosugar, and a silylated pyrrolo[2,3‐d]pyrimidine derivative in acetonitrile has been described.