Synthesis and intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]phenylalanyl-tRNAPhe (yeast), n = 0, 5, 11 and 15.
Rigid, variable-length oligoproline crosslinking reagents, which we call molecular rulers, are a potentially powerful tool for probing the solution structures of tRNA and other biological macromolecules. We wish to demonstrate the feasibility of molecular rulers on a well-studied model system, yeast phenylalanine tRNA, before applying them to less well understood structures. We have found chlorambucil (4-(4-(bis(2-chloroethyl)amino)phenyl)-butanoic acid) to be suitable for use as an alkylating function attached to the imino end of oligo-L-proline spacers which are peptide bonded at their carboxyl ends to the alpha-amine of [3H]Phe-tRNAPhe (yeast). Chlorambucil and chlorambucilyl oligoprolines may be readily and sensitively assayed by their alkylation kinetics with aqueous pyridine as measured by optical absorbance of the product. The pyridine reaction seemed to be the first order in chlorambucil, k1 = (5.4 +/- 1.0) X 10(-3) min-1, zero order in pyridine, and was strongly inhibited by Me2SO. Filter assays of tRNA alkylation by chlorambucilyl [3H]prolyl proline suggested that this reaction is also first order in alkylation reagent, but somewhat dependent on tRNA concentration, and also strongly inhibited by Me2SO. Full alkylation activity was regained upon removal of Me2SO. Modification of [3H]Phe-tRNAPhe (yeast) with the N-hydroxysuccinimide esters of chlorambucilyl (prolyl)n was accomplished with yields of 100% for n = 0, 92% for n = 5, 94% for n = 11 and 44% for n = 15, in 80% Me2SO/CHCl3 at pH 9, 37 degrees C, conditions under which chlorambucil alkylation of tRNA is strongly inhibited. The rates of intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]Phe-tRNAPhe (yeast) were measured assuming a first-order process, giving K1 = (5.3 +/- 0.2) X 10(-3) min-1 for n = 0, (3.2 +/- 0.4) X 10(-4) min-1 for n = 5, (6.8 +/- 0.8) X 10(-5) min-1 for n = 11 and (1.6 +/- 0.4) X 10(-4) min-1 for n = 15. Yields of intramolecularly crosslinked tRNA were 80% for n = 0 after 4 h in 10 mM NH4OAc (pH 6)/1 mM Mg(OAc)2 at 37 degrees C, 7% for n = 5, 3% for n = 11, and 5% for n = 15.
D055598:Chemical Phenomena; D002621:Chemistry; D002699:Chlorambucil / Q000031:analogs & derivatives / Q000138:chemical synthesis; D002855:Chromatography, Thin Layer; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004121:Dimethyl Sulfoxide; D009682:Magnetic Resonance Spectroscopy; D008722:Methods; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis*; D012441:Saccharomyces cerevisiae
D003432:Cross-Linking Reagents; D012346:RNA, Transfer, Amino Acyl; D002699:Chlorambucil; D004121:Dimethyl Sulfoxide