Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potentmammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongationfactors. Kinetic isotope effect studies have established that RTA operates via a D
N*A
N mechanism involvingan oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L.(2000)
J. Am. Chem. Soc.
122, 1609-1617]. On the basis of this information, stem-loop RNA moleculeswere chemically synthesized, incorporating structural features of the oxacarbenium ion-like transitionstate. A 10-base RNA stem-loop incorporating (1
S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-
D-ribitol at the depurination site binds four times better (0.57
M) than the 10-base RNA stem-loop withadenosine at the depurination site (2.2
M). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2
R,3
S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a
Kd of 3.2
M andtightens to 0.75
M in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-
D-ribitol at the depurination site binds with a
Kd of 1.3
M and improves to 0.65
M with9-deazaadenine added. When (3
S,4
R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at thedepurination site of a 14-base RNA stem-loop, the
Kd was 0.48
M. Addition of 9-deazaadenine tightensthe binding to 0.10
M whereas added adenine increases the affinity to 12 nM. The results of this studyare consistent with the unusual dissociative D
N*A
N mechanism determined for RTA. Knowledge of thisintermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalyticsite of RTA.