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A chemical synthesis of glycylglycyl[14C]phenylalanyl-tRNA and glycylglycylglycyl[14C]phenylalanyl-tRNA
PRELIMINARY NOTES
433
BBA 91206
A chemical synthesis of glycylglycyl[+~C]phenylalanyl-tRNA and glycylglycylglycyl[t4C]phenylalanyl-t RNA Peptidyl-tRNA's are generally considered intermediates in protein biosynthesis 1. Recently we have described a general chemical procedure for the preparation of dipeptidyl-tRNA ~,3. The tRNA was enzymatically charged with an amino acid (E14Clphenylalanine, E14Clvaline, or E14C]methionine). The aminoacyl-tRNA was condensed with N-hydroxysuccinimide ester of N-monomethoxytrityl amino acid and, after removing the N-protecting group with 5 % dichloroacetic acid, a dipeptidyltRNA was obtained. It was shown that the N-hydroxysuccinimide ester reacts specifically with the amino group of the amino acid attached to the tRNA and that the monomethoxytrityl which served as a blocking group can be removed without damaging the dipeptidyl-tRNA. In the present communication we describe the synthesis of glycylglycylE~4Cl phenylalanyl-tRNA and glycylglycylglycylE14C~phenylalanyl-tRNA. N-Monomethoxytritylglycylglycine(IIU was prepared by adding monome thoxytritylchloride (7.2 g, 23 mmoles) in portions, with continuous mixing, to a solution of glycylglycine (2.64 g, 20 mmoles) in water (20 ml), diethylamine (4.2 ml, 40 mmoles) and isopropanol (32 ml). The addition was accomplished within 3 h. After another hour, water (50 ml) was added slowly. The reaction mixture was cooled in an ice bath and acidified with a cold solution of acetic acid (1.2 ml, 20 mmoles) in water (IO ml). The monomethoxytritylglycylglycine was filtered and dried in a vacuum desiccator over PzO~ and NaOH. For purification, the product was dissolved in ethyl acetate (50 ml), the non-soluble material was removed by filtration and the product was precipitated by adding the clear solution dropwise and with continuous mixing to ether or light petroleum (IOOOml) (b.p. 40-60°). The solvents were removed by decantation and the white precipitate was dried in a vacuum desiccator. N-Hydroxysuccinimide ester of N-monomethoxytritylglycylglycine (V) was prepared by adding a solution of dicyclohexylcarbodiimide (515 mg, 2.5 mmoles) in dry ethyl acetate (5 ml) to a solution containing N-hydroxysuccinimide (287 rag, 2.5 mmoles) and N-monomethoxytritylglycylglycine (i g, 2.5 mmoles) in dry ethyl acetate (20 ml). The reaction mixture was left overnight at room temperature and the dicyclohexylurea formed was removed by filtration. The filtrate was concentrated i n vacuo to yield an oil which crystallized after drying in a desiccator. The product was purified by dissolving it in ether (or ethyl acetate) and adding the solution dropwise to light petroleum. A similar procedure was used for the synthesis of the Nhydroxysuccinimide ester of monomethoxytritylglycylglycylglycine. Glycylglycyl~14Clphenylalanyl-tRNA was prepared in the following way: E14Clphenylalanyl-tRNA (0.8 mg, 2oo/~/~moles phenylalanine; spec. activity, 504 mC/mmole) dissolved in o.i M acetate buffer (pH 5.0) (0. 4 ml), was added to a solution of N-hydroxysuccinimide ester of monomethoxytritylglycylglycine (IOO rag, 2oo/~moles) in dimethylsulfoxide (2.0 ml). The reaction mixture was shaken in a Vortex test tube mixer at room temperature for 16 h. The t-RNA was isolated by adding absolute ethanol (5.0 ml), cooling to --20 ° for I h and centrifugation (12 ooo rev./min, 15 rain, 4°). The precipitate was washed with dimethylformamide (5.0 ml) and with ethanol Biochim. Biophys. Acta, 157 (1968) 433-435
434
PRELIMINARY NOTES
(2 × 5 ml). In order to remove the monomethoxytrityl group the tRNA was suspended in 5 % solution of dichloroacetic acid (I.O ml) at 4 ° for 20 min. Then ethanol (2.0 ml) was added and the tRNA was isolated by centrifugation (12 ooo rev./min, 15 min, 4°). The precipitate was washed with ethanol (5 ml) and then dissolved in o.I M acetate buffer (pH 5.0). An aliquot was treated with 0.5 M NaOH for 30 rain at 37 °. The hydrolysate was analyzed by use of paper electrophoresis (the solvent used was i M acetic acid (pH 2.5); 16.3 V/cm). 92 % of the radioactivity moved as glycylglycylphenylalanine and none as phenylalanine (8 % of the radioactivity moved slowly and they might be a side-product of the alkali treatment). A sample of peptidyltRNA which had not been treated with alkali was analyzed on paper chromatography and all the radioactivity remained at the origin (tRNA does not move in the solvent used). The same procedure was used for the preparation of glycylglycylglycyl[X4C]phenylalanyl-tRNA. An aliquot was treated with 0.5 M NaOH for 30 min and the hydrolyzate was analyzed by paper chromatography using butanol-acetic acidwater (4:1:5, by vol.) as solvent. All the radioactivity moved as glycylglycylglycylphenylalanine (RF o.25) and none as phenylalanine (R F 0.62). The reaction can be summarized in the following scheme: R-C1 + H,N-CHzCO [NHCH2CO]nOH I
• R-NHCH2CO [NHCH,CO]nOH III
II
CO-CH 2
III
J
CO-CH2
+ HO-~
DCC I CO-CH,
•
/ R-NHCH*CO [NHCH2CO~nON "~
IV
I CO-CH 2
V R'
]:{r
V + tRNA-OCOCHNH 2 VI
•
L
tRNA-OCOCHNH[COCH,NH]nCOCH,NHR VII R p
5% VII
I)CA
(4 °) 1 • t R N A - O C O C H N H [COCH2NH]nCOCH2NH 2 Vlll
n =
I or 2; DCC = N,N-dicyclohexylcarbodiimide; DCA = dichloroacetic acid;
R=CI-I,O ~
/--. c
~~
/ ; and
=
!
0
The effect of Mg2+ concentration on the binding of glycylglycyl[l~C]phenylalanyl-tRNA to EschericMa coli ribosomes as compared with the binding of [14C]phenylalanyl-tRNA and acetyl[14C]phenylalanyl-tRNA is given in Fig. I. The curve obtained for the binding of glycylglycyl[14Clphenylalanyl-tRNA to ribosomes is very similar to that of [14C]phenylalanyl-tRNA. Acetyl[14C]phenylalanyl-tRNA, on the other hand, binds at low Mg~+ concentration (below o.o25 M) much less than [14(;]phenylalanyl-tRNA, but at higher Mg ~+ concentration, the relative difference between the binding of the two compounds decreases. Biochim. Biophys. Acta, 157 (I968) 433-435
435
PRELIMINARY NOTES
z
lO00
m
z.g ~m
,-~
>-0
~500 ,--g I
I
I
0.01
0102
0.0~
I
0.01+
[M,2+l HOLARITY Fig. 1. Binding of glycylglycyl[14C]phenylalanyl-tRNA to E. ¢oli ribosomes as a function of Mg 8+ concentration. Twice-washed ribosomes were prepared from E. coli according to the procedure of ZNIRENBERG4. Binding assays were performed by the method developed by ZNIRENBERGAND LEDER5. Reaction mixtures in a final volume of o.i ml contained: Tris acetate, 5/,moles (pH 7.3); KC1, 5/,moles; poly U, 20/zg; ribosomes, 2.1 A260 nat* units. Glycylglycyl[ltC]phenylalanyl-tRNA, [a4C]phenylalanyl-tRNA and acetyl[liC]phenylalanyl-tRNA, 2o00 counts/rain. Incubation at 3°0 for IO min. Acetyl[l*C]phenylalanyl-tRNA was prepared according to the method of LAPIDOT, DE GROOT AND FRY-SHAFRIR6. 0 - - 0 , [14C]phenylalanyl-tRNA+poly U; (D-O, [laC]phenylalanylt R N A - - p o l y U; & - A , glycylglycyl[X4C]phenylalanyl-tRNA+poly U; A - A , glycylglycyl[l*C]phenylalanyl-tRNA--poly U; B-B, acetyl[~*C]phenylalanyl-tRNA+poly U; D-V], acetyl[~4C]phenylalanyl-tRNA-- poly U.
The possibility of peptidase activity on glycylglycylEliClphenylalanyl-tRNA during the binding reaction was investigated. A binding reaction mixture with glycylglycyl[14C]phenylalanyl-tRNA in the presence of poly U (for conditions see legend of Fig. I) was poured on a Millipore filter, washed, and the filter was soaked in o.05 ml of 0.5 M NaOH for I h at room temperature. The filter was then washed with 0. 5 ml of water, the solution was concentrated in vacuo, and the residue was run on paper electrophoresis, pH 2.5 (the solvent used was I M acetic acid). All the radioactivity moved as glycylglycylphenylalanine. This experiment shows that there was no peptidase activity on glycylglycylE14C]phenylalanyl-tRNA during the binding reaction.
Department o[ Biological Chemistry, The Hebrew University o~ Jerusalem, Jerusalem (Israel)
Y. LAPIDOT N. I)E GROOT S. RAPPOPORT A. PANET
I D. NATHANS AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 4972 Y. LAPIDOT, N. DE CrROOT, IV[.WEISS, R. PELED AND Y. WOLMAN, Biochim. Biophys. Acta, 138
(1967) 241. 3 Y. LAPIDOT, N. DE GROOT, A. D. HAMBURGER AND S. RAPPOPORT, Biochim. Biophys. Acta, 149 (1967) 532 • 4 W. M. NIRENBERG, in S. P. CoLowIcK AND N. O. KAPLAN, Methods in Enzymology, Vol. VI, Academic Press, New York, 1963, p. 17. 5 M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. 6 Y. LAPIDOT, N. DE GROOT AND I. FRY-SHAFRIR, Biochim. Biophys. Acta, 145 (1967) 292.
Received February 2nd, 1968 Biochim. Biophys. Acta, 157 (1968) 433-435
433
BBA 91206
A chemical synthesis of glycylglycyl[+~C]phenylalanyl-tRNA and glycylglycylglycyl[t4C]phenylalanyl-t RNA Peptidyl-tRNA's are generally considered intermediates in protein biosynthesis 1. Recently we have described a general chemical procedure for the preparation of dipeptidyl-tRNA ~,3. The tRNA was enzymatically charged with an amino acid (E14Clphenylalanine, E14Clvaline, or E14C]methionine). The aminoacyl-tRNA was condensed with N-hydroxysuccinimide ester of N-monomethoxytrityl amino acid and, after removing the N-protecting group with 5 % dichloroacetic acid, a dipeptidyltRNA was obtained. It was shown that the N-hydroxysuccinimide ester reacts specifically with the amino group of the amino acid attached to the tRNA and that the monomethoxytrityl which served as a blocking group can be removed without damaging the dipeptidyl-tRNA. In the present communication we describe the synthesis of glycylglycylE~4Cl phenylalanyl-tRNA and glycylglycylglycylE14C~phenylalanyl-tRNA. N-Monomethoxytritylglycylglycine(IIU was prepared by adding monome thoxytritylchloride (7.2 g, 23 mmoles) in portions, with continuous mixing, to a solution of glycylglycine (2.64 g, 20 mmoles) in water (20 ml), diethylamine (4.2 ml, 40 mmoles) and isopropanol (32 ml). The addition was accomplished within 3 h. After another hour, water (50 ml) was added slowly. The reaction mixture was cooled in an ice bath and acidified with a cold solution of acetic acid (1.2 ml, 20 mmoles) in water (IO ml). The monomethoxytritylglycylglycine was filtered and dried in a vacuum desiccator over PzO~ and NaOH. For purification, the product was dissolved in ethyl acetate (50 ml), the non-soluble material was removed by filtration and the product was precipitated by adding the clear solution dropwise and with continuous mixing to ether or light petroleum (IOOOml) (b.p. 40-60°). The solvents were removed by decantation and the white precipitate was dried in a vacuum desiccator. N-Hydroxysuccinimide ester of N-monomethoxytritylglycylglycine (V) was prepared by adding a solution of dicyclohexylcarbodiimide (515 mg, 2.5 mmoles) in dry ethyl acetate (5 ml) to a solution containing N-hydroxysuccinimide (287 rag, 2.5 mmoles) and N-monomethoxytritylglycylglycine (i g, 2.5 mmoles) in dry ethyl acetate (20 ml). The reaction mixture was left overnight at room temperature and the dicyclohexylurea formed was removed by filtration. The filtrate was concentrated i n vacuo to yield an oil which crystallized after drying in a desiccator. The product was purified by dissolving it in ether (or ethyl acetate) and adding the solution dropwise to light petroleum. A similar procedure was used for the synthesis of the Nhydroxysuccinimide ester of monomethoxytritylglycylglycylglycine. Glycylglycyl~14Clphenylalanyl-tRNA was prepared in the following way: E14Clphenylalanyl-tRNA (0.8 mg, 2oo/~/~moles phenylalanine; spec. activity, 504 mC/mmole) dissolved in o.i M acetate buffer (pH 5.0) (0. 4 ml), was added to a solution of N-hydroxysuccinimide ester of monomethoxytritylglycylglycine (IOO rag, 2oo/~moles) in dimethylsulfoxide (2.0 ml). The reaction mixture was shaken in a Vortex test tube mixer at room temperature for 16 h. The t-RNA was isolated by adding absolute ethanol (5.0 ml), cooling to --20 ° for I h and centrifugation (12 ooo rev./min, 15 rain, 4°). The precipitate was washed with dimethylformamide (5.0 ml) and with ethanol Biochim. Biophys. Acta, 157 (1968) 433-435
434
PRELIMINARY NOTES
(2 × 5 ml). In order to remove the monomethoxytrityl group the tRNA was suspended in 5 % solution of dichloroacetic acid (I.O ml) at 4 ° for 20 min. Then ethanol (2.0 ml) was added and the tRNA was isolated by centrifugation (12 ooo rev./min, 15 min, 4°). The precipitate was washed with ethanol (5 ml) and then dissolved in o.I M acetate buffer (pH 5.0). An aliquot was treated with 0.5 M NaOH for 30 rain at 37 °. The hydrolysate was analyzed by use of paper electrophoresis (the solvent used was i M acetic acid (pH 2.5); 16.3 V/cm). 92 % of the radioactivity moved as glycylglycylphenylalanine and none as phenylalanine (8 % of the radioactivity moved slowly and they might be a side-product of the alkali treatment). A sample of peptidyltRNA which had not been treated with alkali was analyzed on paper chromatography and all the radioactivity remained at the origin (tRNA does not move in the solvent used). The same procedure was used for the preparation of glycylglycylglycyl[X4C]phenylalanyl-tRNA. An aliquot was treated with 0.5 M NaOH for 30 min and the hydrolyzate was analyzed by paper chromatography using butanol-acetic acidwater (4:1:5, by vol.) as solvent. All the radioactivity moved as glycylglycylglycylphenylalanine (RF o.25) and none as phenylalanine (R F 0.62). The reaction can be summarized in the following scheme: R-C1 + H,N-CHzCO [NHCH2CO]nOH I
• R-NHCH2CO [NHCH,CO]nOH III
II
CO-CH 2
III
J
CO-CH2
+ HO-~
DCC I CO-CH,
•
/ R-NHCH*CO [NHCH2CO~nON "~
IV
I CO-CH 2
V R'
]:{r
V + tRNA-OCOCHNH 2 VI
•
L
tRNA-OCOCHNH[COCH,NH]nCOCH,NHR VII R p
5% VII
I)CA
(4 °) 1 • t R N A - O C O C H N H [COCH2NH]nCOCH2NH 2 Vlll
n =
I or 2; DCC = N,N-dicyclohexylcarbodiimide; DCA = dichloroacetic acid;
R=CI-I,O ~
/--. c
~~
/ ; and
=
!
0
The effect of Mg2+ concentration on the binding of glycylglycyl[l~C]phenylalanyl-tRNA to EschericMa coli ribosomes as compared with the binding of [14C]phenylalanyl-tRNA and acetyl[14C]phenylalanyl-tRNA is given in Fig. I. The curve obtained for the binding of glycylglycyl[14Clphenylalanyl-tRNA to ribosomes is very similar to that of [14C]phenylalanyl-tRNA. Acetyl[14C]phenylalanyl-tRNA, on the other hand, binds at low Mg~+ concentration (below o.o25 M) much less than [14(;]phenylalanyl-tRNA, but at higher Mg ~+ concentration, the relative difference between the binding of the two compounds decreases. Biochim. Biophys. Acta, 157 (I968) 433-435
435
PRELIMINARY NOTES
z
lO00
m
z.g ~m
,-~
>-0
~500 ,--g I
I
I
0.01
0102
0.0~
I
0.01+
[M,2+l HOLARITY Fig. 1. Binding of glycylglycyl[14C]phenylalanyl-tRNA to E. ¢oli ribosomes as a function of Mg 8+ concentration. Twice-washed ribosomes were prepared from E. coli according to the procedure of ZNIRENBERG4. Binding assays were performed by the method developed by ZNIRENBERGAND LEDER5. Reaction mixtures in a final volume of o.i ml contained: Tris acetate, 5/,moles (pH 7.3); KC1, 5/,moles; poly U, 20/zg; ribosomes, 2.1 A260 nat* units. Glycylglycyl[ltC]phenylalanyl-tRNA, [a4C]phenylalanyl-tRNA and acetyl[liC]phenylalanyl-tRNA, 2o00 counts/rain. Incubation at 3°0 for IO min. Acetyl[l*C]phenylalanyl-tRNA was prepared according to the method of LAPIDOT, DE GROOT AND FRY-SHAFRIR6. 0 - - 0 , [14C]phenylalanyl-tRNA+poly U; (D-O, [laC]phenylalanylt R N A - - p o l y U; & - A , glycylglycyl[X4C]phenylalanyl-tRNA+poly U; A - A , glycylglycyl[l*C]phenylalanyl-tRNA--poly U; B-B, acetyl[~*C]phenylalanyl-tRNA+poly U; D-V], acetyl[~4C]phenylalanyl-tRNA-- poly U.
The possibility of peptidase activity on glycylglycylEliClphenylalanyl-tRNA during the binding reaction was investigated. A binding reaction mixture with glycylglycyl[14C]phenylalanyl-tRNA in the presence of poly U (for conditions see legend of Fig. I) was poured on a Millipore filter, washed, and the filter was soaked in o.05 ml of 0.5 M NaOH for I h at room temperature. The filter was then washed with 0. 5 ml of water, the solution was concentrated in vacuo, and the residue was run on paper electrophoresis, pH 2.5 (the solvent used was I M acetic acid). All the radioactivity moved as glycylglycylphenylalanine. This experiment shows that there was no peptidase activity on glycylglycylE14C]phenylalanyl-tRNA during the binding reaction.
Department o[ Biological Chemistry, The Hebrew University o~ Jerusalem, Jerusalem (Israel)
Y. LAPIDOT N. I)E GROOT S. RAPPOPORT A. PANET
I D. NATHANS AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 4972 Y. LAPIDOT, N. DE CrROOT, IV[.WEISS, R. PELED AND Y. WOLMAN, Biochim. Biophys. Acta, 138
(1967) 241. 3 Y. LAPIDOT, N. DE GROOT, A. D. HAMBURGER AND S. RAPPOPORT, Biochim. Biophys. Acta, 149 (1967) 532 • 4 W. M. NIRENBERG, in S. P. CoLowIcK AND N. O. KAPLAN, Methods in Enzymology, Vol. VI, Academic Press, New York, 1963, p. 17. 5 M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. 6 Y. LAPIDOT, N. DE GROOT AND I. FRY-SHAFRIR, Biochim. Biophys. Acta, 145 (1967) 292.
Received February 2nd, 1968 Biochim. Biophys. Acta, 157 (1968) 433-435