Substrates for Ribosomal Peptidyl Transferase: Synthesis of 3′-N-Aminoacyl and 5′-O-Nucleotidyl Analogues of Puromycin

Abstract

The chemical synthesis, on a 10–30 μmol scale, of two series of puromycin analogues is described: the first is the type 3′-N-aminoacyl-puromycin aminonucleoside (e.g. 3′-N-glycyl-PANS or PANS-Gly) in which the O-methyl-L-tyrosyl residue of puromycin is replaced by various aminoacyl residues, and the second is the type NpPANS-Gly in which the 5′-hydroxyl of PANS-Gly is substituted with phosphate, 3′-AMP, 3′-CMP, 3′-GMP, or 3′-UMP. The 3′-N-aminoacyl-PANS derivatives were synthesized either by coupling N-protected amino acids via their N-hydroxysuccinimide esters to PANS in 70% aqueous pyridine or by direct coupling in ethanol or methanol of N-protected amino acids to PANS using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as condensing agent After removal of the protecting groups, reaction mixtures were purified by preparative thin-layer chromatography on silicic acid, paper chromatography, and paper electrophoresis to give the 3′-N-aminoacyl-PANS derivatives in yields of 40–80%.The 5′-O-nucleotidyl derivatives of PANS-Gly were synthesized by three methods: (i) by the coupling of fully acetylated 3′-mononucleotides to 3′-N-(Boc-glycyl)-PANS, (ii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranylcytidine or N2,O2′,O5′-tritetrahydropyranylguanosine to 5′-O-phosphoryl-(Boc-glycyl)-PANS, and (iii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranyl-3′-CMP or N2,O2′,O5′-tritetrahydropyranyl-3′-GMP to 3′-N-Boc-PANS or 3′-N-trifluoroacetyl-PANS followed by aminoacylation of the CpPANS or GpPANS produced with Boc-glycine and EEDQ. Coupling reactions were carried out using either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulfonyl chloride as condensing agents in anhydrous pyridine. After removal of protecting groups, reaction mixtures were purified by paper chromatography and paper electrophoresis to give the NpPANS-Gly derivatives in 3–30% yield.

Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site

“…were purchased from New England Nuclear. A-Phenylalanylpuromycin aminonucleoside (PhePANS) was prepared as described (Harris et al, 1972). [3H]Puromycin and [3H]-PhePANS (1.5 Ci/mol) labeled in the H-8 position, [8-3H]puromycin and [8-3H]PhePANS, were prepared by exchange with tritiated water, as described (Shelton and Clark, 1967).…”

Antibiotic effects on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin

“…Tetracycline, lincomycin, blasticidin S, and sparsomycin were gifts from Dr. J. G. Flaks (University of Pennsylvania). A-Phenylalanylpuromycin aminonucleoside (PhePANS) was prepared as described (Harris et al, 1972). Cytidylyl-(3/-5,)-3'-0-phenylalanyladenosine (CAPhe) was a gift of Dr. S. Chladek (Michigan Cancer Foundation).…”