The large-scale extraction and purification to homogeneity of cyclic CMP and its unequivocal identification are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and boronate column chromatography, polyacrylamide-gel column electrophoresis and high-voltage paper electrophoresis. The purified sample co-chromatographed with authentic cyclic CMP on t.l.c. and high-pressure liquid chromatography and was positive in a cyclic CMP radio-immunoassay. The u.v., i.r. and p.m.r. spectra were each essentially identical with those of authentic cyclic CMP. Fast-atom bombardment of authentic cyclic CMP yielded a mass spectrum containing a molecular protonated ion: mass-ion-kinetic-energy scanning of this ion produced a spectrum unique to 3',5'-cyclic CMP. The extracted nucleotide produced an identical mass-ion-kinetic-energy spectrum.

The large-scale extraction and partial purification of endogenous 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and Sephadex ion-exchange chromatography and preparative electrophoresis. The samples thus obtained co-chromatographed with authentic cyclic UMP, cyclic IMP and cyclic dTMP on t.l.c. and h.p.l.c. and the u.v. spectra of the extracted samples were identical with those of the standards. Fast atom bombardment of the three cyclic nucleotide standards yielded mass spectra containing a molecular protonated ion in each case; mass-analysed ion kinetic-energy spectrometry ('m.i.k.e.s') of these ions produced a spectrum unique to the parent cyclic nucleotide. The extracted putative cyclic UMP, cyclic IMP and cyclic dTMP each produced a m.i.k.e.s. identical with that obtained with the corresponding cyclic nucleotide standard. Rat liver, heart, kidney, brain, intestine, spleen, testis and lung protein preparations were each found capable of the synthesis of cyclic UMP, cyclic IMP and cyclic dTMP from the corresponding nucleoside triphosphate, of the hydrolysis of these cyclic nucleotides and of their binding, with the exception that cyclic dTMP was not synthesized by the kidney preparation.

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Though fast atom bombardment ionization makes possible the ionization and molecular weight determination of polar or thermally labile biological compounds, the resulting mass spectra commonly give few or no fragment ions which would allow detailed structural analysis. In particular, isomeric compounds often give identical spectra. Collision-induced dissociation of ions resulting from fast atom bombardment ionization is shown to be a powerful combination which can differentiate isomeric substances. The technique is applied to isomeric bile acid salts and steroid conjugates and is capable of differentiating structural isomers which have similar fast atom bombardment mass spectra. A range of isomeric cyclic nucleotides is also shown to be amenable to the method. Sensitivity limits are examined and the unequivocal identification of two 3',5'-cyclic nucleotides isolated from living systems is demonstrated.

Molecular protonated ions of ally1 phenyl ether undergo a Claisen rearrangement both in the ion source and along the flight path. The rearranged ions undergo fragmentation, the predominant loss being ethene, and only a small contribution from loss of carbon monoxide is observed. Collision-indoced dissociation spectra are used to verify the structures of the daughter ions. These spectra, together with other evidence of an acid-induced ortho rearrangement, allow a mechanism to be proposed for the ethene loss. In contrast, molecular protonated ions of propargyl phenyl ether lose exclusively carbon monoxide.