The synthesis of the complete set of seven N-desmethyl derivatives of RA-VII (8) are described. Thus, the synthesis of the four 14-membered cycloisodityrosine derivatives 21-24 and their coupling with the two tetrapeptides 32 and 33 followed by formation of the 18-membered ring with macrocyclization provided the full set of seven desmethyl derivatives 14-20 of RA-VII (8). The solution phase conformational properties of 8 and 14-20 were examined by 1D and 2D H-1 NMR to reveal the role of N-methylation on the key conformational aspects of the natural agents. In contrast to each of the simple cycloisodityrosine derivatives 21-24 which adopt a single, rigid solution conformation possessing a secondary or tertiary trans amide central to the 14-membered ring, the natural agents including 8 adopt a single predominant solution conformation (83-88%) that corresponds closely to the X-ray structure conformation which possesses an inherently disfavored cis C-30-N-29 tertiary amide central to the 14-membered cycloisodityrosine subunit. Moreover, this cis amide is the predominant conformation (85-95%) observed with N-29-desmethyl RA-VII (14) indicating that even a secondary C-30-N-29 amide adopts this inherently disfavored cis amide stereochemistry. The minor conformation of 8 observed in solution (12-17%) is shown to be derived from a minor cis C-8-N-9 tertiary amide which was not observed with its conversion to a secondary amide. adopt exclusively a single solution conformation that corresponds to the major solution conformations of 8 and 14.