Figure 5.

Tandem MS spectrum of nucleoside compound #6 in negative ion mode, comparing the measured m/z values of its fragmentation pattern to those of the Ψ, m¹Ψ and s²Ψ standards. The fragment containing the uracil C2 and N3 atoms (red) has a theoretical m/z of 41.999 in negative ion mode when containing a C2-carbonyl group (the values measured here were m/z 41.997 for Ψ, and m/z 41.999 for m¹Ψ and compound #6). This fragment shifts to m/z 57.976 when C2 is thiolated (as for s²Ψ, and not for compound #6). The fragment (in green) spanning from the C4-carbonyl and the N1 atom through the glycosidic bond to the 2′-hydroxyl is seen at m/z 110.023 when unmodified (e.g. for Ψ and s²Ψ); this fragment increases to m/z 124.040 when there is an N1-methyl group (as in m¹Ψ); and with a further increase to m/z 140.017 when the C4-position is thiolated (as in compound #6). With the instrumentation used here, the m/z measurement of 140.017 is of sufficient accuracy to distinguish the composition of this fragment as C₆H₇NOS, as opposed to the m/z 140.034 and 142.050 fragments, which fit the compositions C₆H₇NO₃ and C₆H₉NO₃, respectively. The data are fully consistent with compound #6 being m¹s⁴Ψ.