Solid phase synthesis and binding affinity of peptidyl transferase transition state mimics containing 2'-OH at P-site position A76

Abstract

All living cells are dependent on ribosomes to catalyze the peptidyl transfer reaction, by which amino acids are assembled into proteins. The previously studied peptidyl transferase transition state analog CC-dA-phosphate-puromycin (CCdApPmn) has important differences from the transition state, yet current models of the ribosomal active site have been heavily influenced by the properties of this molecule. One significant difference is the substitution of deoxyadenosine for riboadenosine at A76, which mimics the 3' end of a P-site tRNA. We have developed a solid phase synthetic approach to produce inhibitors that more closely match the transition state, including the critical P-site 2'-OH. Inclusion of the 2'-OH or an even bulkier OCH3 group causes significant changes in binding affinity. We also investigated the effects of changing the A-site amino acid side chain from phenylalanine to alanine. These results indicate that the absence of the 2'-OH is likely to play a significant role in the binding and conformation of CCdApPmn in the ribosomal active site by eliminating steric clash between the 2'-OH and the tetrahedral phosphate oxygen. The conformation of the actual transition state must allow for the presence of the 2'-OH, and transition state mimics that include this critical hydroxyl group must bind in a different conformation from that seen in prior analog structures. These new inhibitors will provide valuable insights into the geometry and mechanism of the ribosomal active site.

Figure 1

The peptidyl transferase reaction, including the theoretical transition state. Peptidyl transferase occurs when the α-amino group of the A-site amino acid (in this case, tyrosine) nucleophilically attacks the carboxyl carbon of the P-site nascent peptide’s C-terminal amino acid. The resulting transition state contains a tetrahedral carbon with a single oxyanion. Subsequent release of the carboxyl carbon from the P-site tRNA yields the reaction products, a deacylated P-site tRNA and an N + 1 length peptide linked to the A-site tRNA.

Figure 2

Peptidyl transferase transition state analogs. (A) CCdApPmn. The puromycin portion of the molecule mimics the terminal adenosine and amino acid of an amino-acylated A-site tRNA. The trinucleotide CCdA mimics the 3′ end of a P-site tRNA, and the tetrahedral phosphate mimics the tetrahedral carbon of the transition state. Significant differences between the actual transition state and this molecule include the absence of a 2′-OH from the P-site A and the distribution of the negative charge between two oxygen atoms. (B) Improved transition state analogs. Six new molecules were synthesized, varying in their P-site A76 2′ functional group (R₁) and their A-site amino acid R group (R₂). (C) The structure of CCdApPmn bound in the peptidyl transferase center of 50S ribosomal subunits from H.marismortui. CCdApPuro is shown in green, and nearby rRNA bases are shown in gray. rRNA base numbers are H.marismortui numbering with E.coli equivalents in parentheses. One of the non-bridging oxygens of the tetrahedral phosphate is within 2.8 Å of the 2′ carbon of the P-site A76, shown in red. The methyl tyrosine portion is stacked between A2486 and U2487.

Figure 3

Solid phase synthesis of transition state analogs. The initial reaction shown here is the coupling of TMS protected puromycin aminonucleoside with the acyl chloride of l-hydroxy acids where R₁ = CH₃ (alanine analogs) or CH₂C₆H₅ (phenylalanine analogs). Standard protection and deprotection conditions afforded compounds 5a and 5b in an overall yield of 42–48% from the starting nucleoside. Polymer bound puromycin was obtained by coupling compounds 5a and 5b to N-terminus polystyrene supports using standard techniques. The fluoride labile silyl protecting group was removed with TEMED/HF in MeCN prior to the first phosphoramidite reaction. Three separate adenosine phosphoramidites were followed by cytidine phosphoramidites to generate the desired addition of CCA to the A-site puromycin analog. Oxidation, deprotection and cleavage reactions were all conducted using established protocols.

Figure 4

Binding of transition state mimics in the peptidyl transferase center of E.coli based upon chemical modification of the 23S rRNA. Molecules bound in the active site protect U2585 from modification by CMCT, resulting in the disappearance of a CMCT-dependent reverse transcriptase stop. (A) A typical gel of reverse transcripts of 23S rRNA from ribosomes modified by CMCT in the presence of increasing concentrations of CCdApOPmn_phe and (B) CCApOPmn_phe. (C) U2585 band intensities were normalized for overall CMCT reactivity and gel loading and then relative to the intensity for no inhibitor present. Binding curves were fit to these data and dissociation constants were derived from the fits.