Regiospecificity of the peptidyl trna ester within the ribosomal P site

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1 S1 SURTING INFRMATIN Regiospecificity of the peptidyl trna ester within the ribosomal site Kevin S. Huang, Joshua S. Weinger, Ethan B. Butler, and Scott A. Strobel* Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT Materials All solvents and reagents for solution phase organic synthesis were purchased from Aldrich unless otherwise stated. reparatory plates (20 X 20 cm, silica gel 60 F 254, 1 mm) were purchased from Merck. olystyrene solid support (rimer support HL, amino derivatized, loading 161 mmol/g) was from Amersham Biosciences. xidizing solution (0.1 M iodine in THF/pyridine/water), activator (sublimed 1H-tetrazole in anhydrous acetonitirle), deblocking mix (3% trichloroacetic acid in CH 2 Cl 2 ), capping solution were purchased from Glen Research. HLC was carried out on a C18 reversed-phase Zorbax column (Hewlett ackard) with 50 mm triethylammonium acetate ph 7.0/acetonitrile gradient as eluent. T4 polynucleotide kinase and NK buffer were from New England Biolabs. Reverse transcriptase AMV and protector RNase inhibitor were from Roche. Synthesis of inhibitors a R NH H Si 1 R = H 2 R = DMT b DMT NH Si 3 H c DMT Si NH 4 H N Scheme 1. Coupling onto the solid support. (a) DMT-Cl, pyr. (b) succinic anhydride, pyr (c) rimer Support, EDIC, pyr, TEA, DMA. 3'N-[[(2S)-2-tert-Butyldimethylsilyloxy-3-phenyl-1-oxopropyl]amino]-3'-deoxy- N 6,N 6 -dimethyladenosine (1). 1 An oven-dried 35 ml round bottom flask equipped with a magnetic stir bar was charged with puromycin aminonucleoside (100 mg, mmol), co-evaporated with dry pyridine (3 X 1 ml), dried under vacuum for 1 hr, and finally suspended in dry pyridine (2 ml) under an atmosphere of argon. At 0 o C, 1

2 S2 chlorotrimethylsilane was added drop wise to this solution and the mixture was slowly warmed to rt (2 hrs). A solution containing 2-(tert-butyldimethylsilyloxy)-3- phenylpropanoyl chloride 1 (433 mg, 1.45 mmol, 4 equiv in 5 ml dry CH 2 Cl 2 ) was added drop wise to the mixture at 0 o C, and slowly warmed to rt (2hrs) and quenched with citric acid H 2 (2.43 mmol in 3 ml MeH), stirred for 10 min, and concentrated by rotatory evaporation. To remove the 5' ester formed during the reaction, the crude solid was suspended in 20 ml of concentrated K 2 C 3, stirred for 1 hr, concentrated and purified by preparatory plate (eluent 5% MeH in CH 2 Cl 2, R f = 0.35) to afford the title compound as a white solid [158 mg, mmol, 82%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.25 (s, 1H), 8.22 (s, 1H), 7.49 (d, J = 6.4 Hz, 1H), 7.32 (m, 5H), 6.03 (s, 1H), 4.66 (m, 1H), 4.55 (m, 1H), 4.40 (m, 1H), 4.25 (m, 2H), 4.00 (m, 3H), 3.70 (br, 6H), 3.21 (m, 1H), 2.93 (m, 1H), 0.95 (s, 9H), 0.00 (s, 3H), (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , 91.60, 85.05, 75.22, 74.41, 62.02, 50.37, 42.14, 26.06, 18.33, -5.16, -5.29]. 3'N-[[(2S)-2-tert-Butyldimethylsilyloxy-3-phenyl-1-oxopropyl]amino]-3'-deoxy-5'- dimethoxytrityl-n 6,N 6 -dimethyladenosine (2). 1 An oven dried 25 ml round bottom flask equipped with a magnetic stir bar was charged with 1 (158 mg, mmol), coevaporate with dry pyridine (2 X 1 ml), dried under the vacuum for 1 hr, suspended in dry pyridine (1.70 ml), and chilled to 0 o C. To this was added solid 4,4'-dimethoxytrityl chloride (150 mg, mmol, 1.58 equiv). Under an atmosphere of argon, the mixture was slowly warmed to rt. After 17 hrs, the mixture was quenched with 6 ml MeH, stirred for 10 min, concentrated, dried and purified by preparatory plate (eluent 5% MeH in CH 2 Cl 2, R f = 0.30) to afford the title compound as a white solid [159 mg, mmol, 66%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.54 (s, 1H), 8.29 (s, 1H), 7.53 (m, 15H), 7.02 (m, 4H), 6.51 (br, 1H), 6.27 (s, 1H), 4.82 (m, 1H), 4.76 (m, 1H), 4.59 (m, 1H), 4.44 (m, 1H), 4.00 (s, 6H), 3.80 (m, 7H), 3.58 (m, 1H), 3.35 (m, 1H), 3.06 (m, 1H), 1.03 (s, 9H), 0.00 (s, 3H), (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ , 158.3, , , , 140.5, , , 136.5, 131.2, , 129.8, 128.9, , , 126.4, , 116.1, 91.60, 88.2, 85.05, 75.22, 74.41, 62.02, 56.1, 50.37, 42.14, 26.06, 18.33, -5.16, -5.29]. 2

3 S3 3'N-[[(2S)-2-tert-Butyldimethylsilyloxy-3-phenyl-1-oxopropyl]amino]-3'-deoxy-5'- dimethoxytrityl-2'-succinyl-n 6,N 6 -dimethyladenosine (3). 1 An oven dried 25 ml round bottom flask equipped with a magnetic stir bar was charged with 2 (159 mmol, mmol), co-evaporated with dry pyridine (2 X 1 ml), dried under the vacuum for 1 hr, and suspended in dry pyridine (1.60 ml). To this solution was added DMA (37 mg, mmol, 1.63 equiv), succinic anhydride (65 mg, mmol, 3.51 equiv), and the mixture was stirred at rt under an atmosphere of argon (24 hrs). The mixture was concentrated, dried and purified by preparatory plate (eluent 5% MeH in CH 2 Cl 2, R f = 0.33) to afford the title compound as a white solid [91 mg, 0.10 mmol, 50%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.67 (br, 1H), 8.61 (s, 1H), 8.32 (s, 1H), 7.48 (m, 14H), 7.00 (m, 4H), 6.87 (d, J = 9 Hz, 1H), 6.45 (s, 1H), 5.55 (m, 2H), 4.51 (m, 1H), 4.34 (m, 1H), 4.02 (s, 6H), 3.76 (br, 6H), 3.68 (m, 1H), 3.53 (m, 1H), 3.24 (m, 1H), 3.15 (m, 1H), 2.91 (m, 4H), 1.08 (s, 9H), 0.04 (s, 3H), 0.00 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , , , , , , 99.22, 86.84, 82.17, 62.25, 55.65, 52.33, 48.50, 41.68, 32.00, 29.26, 26.07, 23.07, 18.30, 14.56, -5.03]. Derivatization of puromycin analogue 3 onto the polymer support. An oven dried 25 ml flask was charged with 3 (91 mg, 0.10 mmol), anhydrous pyridine (10 ml), triethylamine (80 µl, 0.57 mmol), DMA (12 mg, 0.1 mmol), 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (0.38 g, 2 mmol), and amino derivatized rimer Support (1 g, 161 µmol). This mixture was slowly agitated on a Red Rotor (Hoefer) at rt until the desired loading of 30 µmol/g was obtained. Loading was determined by measuring the UV absorbance at 504 nm of a solution containing 3 mg of puromycin immobilized polystyrene 4 and diluted to 25 ml of 3% trichloroacetic acid (DMT extinction coefficient is 70 ml/µmol). The resin was filtered, washed with 20 ml pyridine, 20 ml MeH, 20 ml CH 2 Cl 2, and air dried. To cap unreacted amino sites, the resin was transferred to another 25 ml round bottom flask and charged with 10 ml of acetic anhydride solution (10% in acetonitirle), and 10 ml of N-methylimidazole (10% in acetic anhydride), and the mixture agitated for 3 hrs. The resin was then filtered, washed with 20 ml MeH, 20 ml CH 2 Cl 2, and air dried. Final UV absorbance of 4 at 504 nm gave a loading of 30 µmol/g. 3

4 S4 R 4 R = tbdms 5 R = H DMT DMT CE A(Bn) 6 tbdms DMT CpCp H A H H 3' Inhibitor pcpc DMT C(Ac) tbdms CE A(Bn) CE tbdms DMT C(Ac) tbdms CE Scheme 2. Solid phase synthesis of 3' and 2' inhibitors. 7 Solid phase organic synthesis of 3' and 2' inhibitors was essentially as described 1 except using the following phosphoramidites: 5'-DMTr-N-Ac-cytidine phosphoramidite (Glen research), 5'-DMTr-N-AC-adenosine phosphoramidite(glen research), and 5'- DMTr-N-bz-3'-tBDMS-Adenosine phosphoramidite (ChemGenes Corp). After cleaving from the solid support, the crude inhibitors were dried and suspended in a 250 µl of a TEA 3HF/NM solution (750 µl TEA, 1.5 ml N-methylpyrrolidinone, and 1 ml of TEA 3HF). 2 This mixture was incubated at 65 o C for 1hr, cooled to rt and transferred to a 2 ml Eppendorf tube. Fluoride scavenger (isopropoxytrimethylsilane, 500 µl) was added and slowly mixed for 10 min. 3 Each inhibitor was precipitated by the addition of ethyl ether (1 ml) and spun at 14,000 rpm for 30 min. The inhibitors were purified by HLC on a Microsorb C18 column (Varian) using 50 mm triethylamine acetate (ph 7.0)/acetonitirle gradient (0-30% in 30 min; retention time for 3' inhibitor was min; retention time for 2' inhibitor was min). Mass spectra: calculated C 67 H 86 N Da; found Da (3' inhibitor); found Da (2' inhibitor). 4

5 S5 CMCT modification. 50S ribosomal subunits 1 (1 µm) were activated in 200mM KCl, 20mM MgCl 2, and 50mM Tris HCl ph 8.0 at 37 o C for 10 min and then diluted to 11 nm. Various concentrations of inhibitor were incubated with 50S subunits in 100 µl volume at 37 o C for 1 hr. Solid CMCT (3-4 mg) was added to each reaction and incubated an additional 10 min. Reactions were quenched with ethanol and precipitated with 3M NaAc. Ribosomal proteins were removed by phenol extraction and the products were precipitated and stored at -20 o C. Reverse Transcription. A solution containing 32 radiolabeled primer (TACTAGGAGCAGCCCCCC), CMCT modified rrna, and 50mM Tris HCl ph 8.0, 60mM NaCl, and 10mM DTT was heated to 90 o C for 1 min, then allowed to slowly cool to 42 o C. dnts, RNAse inhibitor, AMV-reverse transcriptase, and buffer containing 80mM Tris*HCl ph 8.0, 90mM NaCl, 15mM DTT, and 45mM MgCl 2 were added to the primer:cmct modified RNA solution. This solution was incubated at 42 o C for 45 min, and the reaction products were resolved by denaturing gel electrophoresis. Fraction of inhibitor bound as a function of inhibitor concentration (log scale) was used to determine the relative dissociation constants (K d ) of 3' inhibitor using the equation: I = I sat + (I 0 -I sat )/(1+[inhibitor]/K d ), where I is band intensity of U2585, I sat is band intensity of U2585 when inhibitor is saturating, and I 0 is band intensity of U2585 in the absence of inhibitor. All band intensities were normalized relative to CMCT dependent bands for gel loading and for extent of CMCT reactivity with the 50S rrna. Finally, the U2585 band intensities were normalized relative to I 0 and subtracted by 1 to obtain the fraction of inhibitor bound. Inhibition of the modified peptidyl transfer fragment assay. Inhibition constants for the intermediate mimics were determined using the modified fragment assay as described 4 with some modifications. CCA-phenylalanyl-caproyl-biotin (CCApcb) was used as the -site substrate, and C-puromycin (Cmn) was used as the A-site substrate. These substrates react to form CCA and C-puromycin-phenylalanyl-caproylbiotin. Reaction rates were measured by monitoring the CCA formation, and correcting for the rate of background hydrolysis. Reactions contained 100nM 50S ribosomal subunits from E. coli (pre-heated for 5 minutes at 42 ºC in the presence of 20mM MgCl 2 ), 200mM KCl, 20mM MgCl 2, 50 mm MS ph 7.0, 400 nm unphosphorylated CCApcb, 5

6 S6 approximately 3000 cpm of 32-5'-end-labelled CCApcb, µm Cmn, and 0 or 300nM intermediate mimics, in a total volume of 25µl. Reactions were performed in the absence of methanol. Reactions were initiated by addition of the 50S ribosomal subunits, and reaction timepoints were collected by adding 2µl of the reaction mix to 2µl of low-ph formamide loading buffer (95% formamide, 20mM EDTA, 50mM Tris-sodium phosphate ph 6.5,.01% bromophenol blue,.01% xylene cyanol). roducts were resolved by electrophoresis on 12% denaturing polyacrylamide low-ph gels containing 7M urea and 50mM Tris-sodium phosphate, ph 6.5. Conversion of CCApcb to CCA was monitored and the fractions reacted were quantified using a Molecular Dynamics STRM phosphorimager. Reaction rates were determined by fitting the fraction reacted as a function of time to a single exponential and subtracting the background hydrolysis rate in the absence of the A-site substrate Cmn. K i values were determined by fitting the reaction rates as a function of Cmn concentration to the formula: k T = (k max *[S])/((1+[I]/K I )*K M +[S]) Where k T is the measured rate, [S] is the A-site substrate concentration, K M is the Michaelis constant of the A-site substrate, and k max is the maximum rate. [I] is the inhibitor concentration and K I is the inhibition constant. Values for k max and K M were determined by fitting the formula to the rates of the uninhibited ([I]=0) reactions. Reference: 1. Weinger, J.S., Kitchen, D., Scaringe, S.A., Strobel, S.A. & Muth, G.W. Nucleic Acids Res 32, (2004). 2. Wincott, F. et al. Nucleic Acids Res 23, (1995). 3. Song, Q. & Jones, R. Tetrahedron Letters 40, (1999). 4. arnell, M.K., Seila, A.C. & Strobel, S.A. roc Natl Acad Sci U S A, 99, (2002). 6