CARB 1 |
| Protein synthesis is a fundamental biological process. To understand the mechanism of this reaction we have developed a modified fragment assay which uses minimal A-site and P-site substrates, CC-Puromycin (CCPmn) and the trinucleotide conjugate cytidylyl-(3'5')-cytidylyl-(3'5')-3'(2')-O-(N-(6-D-(+)-biotinoylaminohexanoyl)-L-phenylalanyl)adenosine (CCA-Phe-cap-biotin), respectively. The reaction catalyzed by the 50S ribosomal subunit reacts with multiple turnover kinetics and has chemistry as at least partially rate limiting. This reaction was used successfully to measure the kinetic isotope effect (KIE) of the nucleophilic amine within the A-site substrate. We next intend to measure KIEs for heavy atom substitutions within the P-site substrate. Here, we report the synthesis of the P-site substrate isotopomers (3-15N, 4-15N-cytidylyl)-(3'5')- (3-15N, 4-15N-cytidylyl)-(3'5')-3'(2')-O-(N-(6-D-(+)-biotinoylaminohexanoyl)-(1-13C-L-phenylalanyl))adenosine [(15N4-CC)A-(1-13C-Phe)-cap-biotin], (15N4-CC)A-(2-2H-Phe)-cap-biotin and (15N4-CC)A-Phe-cap-biotin in 32, 35 and 32 steps, respectively. The syntheses were accomplished by coupling (3-15N, 4-15N-cytidylyl)-(3'5')-3-15N, 4-15N-cytidine (15N4-CC) phosphoramidite, as the common synthetic intermediate, with isotopically substituted A-Phe-cap-biotin by activation with imidazolium ion. |
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Nucleosides, Nucleotides and Oligonucleotides
9:00 AM-11:40 AM, Sunday, 10 September 2006 Hilton San Francisco -- Yosemite C, Oral
Division of Carbohydrate Chemistry |