234

ANALYSIS OF PROTEIN PHOSPHORYLATION

[19]

[19] S y n t h e s i s of O - P h o s p h o t y r o s i n e - C o n t a i n i n g P e p t i d e s By JOHN W. PERICH

It is only recently that tyrosine kinases have been identified and that protein phosphorylation of tyrosine residues has been recognized in numerous cellular processes. L2As past syntheses of synthetic phosphotyrosine [Tyr(P)]-containing peptides were inadequate, 3 the investigation of more efficient chemical methods was undertaken in 1980 so as to provide suitable model Tyr(P)-containing peptide substrates for biochemical study. As a result of such studies, it is now possible to prepare complex Tyr(P)containing peptides by the use of protected tyrosine derivatives in butyloxycarbonyl (Boc)- or 9-fluorenylmethyloxycarbonyl(Fmoc)-peptide synthesis. Both Boc-Tyr(PO3Bzl2)-OH and Boc-Tyr(PO3Me2)-OH have been used in peptide synthesis for the preparation of Tyr(P)-containing peptides. In the case of benzyl phosphate protection, Perich and Johns 4 prepared Tyr(P)-Leu-Gly-OH by the use of Boc-Tyr(PO3BzI2)-OH for the solutionphase synthesis of Boc-Tyr(PO3Bzlz)-Leu-Gly-OBzl followed by quantitative deprotection by palladium-catalyzed hydrogenolysis in 40% (v/v) CF3COEH/CH3COEH. However, due to the sensitivity of benzyl phosphate groups to the acid conditions used for the removal of the Boc group (i.e., 4 M HCl/dioxane and 40% CF3COzH/CH2CI24'5), this derivative is therefore limited to the synthesis of N-terminal Tyr(POaBZlE)-containing peptides. In a solid-phase approach (Merrifield, see Ref. 14), Gibson et al. 6 prepared Arg-Tyr(P)-Val-Phe by the use of Boc-Tyr(PO3Bzl2)-OH with peptide-resin cleavage and peptide deprotection effected by a final HF/10% (v/v) anisole deprotection step. However, the yields of Tyr(P)-peptides are low with this approach since extensive dephosphorylation of the Tyr(P) residue occurs in liquid hydrogen fluoride. The methyl phosphate-protected derivative, Boc-Tyr(POaMe2)-OH, has proved to be better suited to peptide synthesis and has been used for I p. j. Blackshear, A. C. Nairn, and J. F. Kuo, FASEB J. 2, 2957 (1988). 2 G. Carpenter, Annu. Rev. Biochem. 56, 881 (1987). 3 A. W. Frank, CRC Crit. Rev. Biochem. 16, 51 (1984). 4 j. W. Perich and R. B. Johns, J. Org. Chem. 54, 1750 (1989). 5 R. M. Valerio, P. F. Alewood, R. B. Johns, and B. E. K e m p , Int. J. Pept. Protein Res. 33, 428 (1989). 6 B. W. Gibson, A. M. Falick, A. L. Burlingame, L. Nadasdi, A. C. N g u y e n , and G. L. K e n y o n , J. Am. Chem. Soc. 109, 5343 (1987).

METHODS IN ENZYMOLOGY, VOL. 201

Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.

[19]

SYNTHESISOF Tyr(P)-CONTAININGPEPTIDES

235

the solution-phase synthesis of Asn-Glu-Tyr(P)-Thr-Ala and Pro-Tyr(P)Val. v,8 Depending on the sequence of the peptide, the cleavage of methyl phosphate groups is possible by the use of either (1) 33% HBr/acetic acid, (2) bromotrimethylsilane/CH3CN, (3) 1 M bromotrimethylsilane-thioanisole/CF3COzH/m-cresol, (4) trifluoromethanesulfonic acid/CF3COzH/thioanisole/m-cresol (1 : 5 : 3 : 1), or (5) 1 M trimethylsilyl triflate-thioanisole/ CF3COzH/m-cresol. The preparation of Tyr(P)-containing peptides can be accomplished by (1) the use of Boc-Tyr(PO3Mez)-OH in Boc solution or solid-phase peptide synthesis or (2) the use of Fmoc-Tyr(PO3Mez)-OH in Fmoc/polyamide solid-phase peptide synthesis. Preparation of Tyr(P)-Containing Peptides by Boc-Peptide Synthesis The procedure for the synthesis of Tyr(P)-containing peptides by Bocpeptide synthesis is outlined as follows: (1) preparation of Boc-Tyr (PO3Me2)-OH, (2) synthesis of protected Tyr(PO3Me2)-containing peptides (solution and solid phase), and (3) peptide deprotection (including cleavage of methyl phosphate groups). The synthesis of the protected derivative, Boc-Tyr(PO3Me2)-OH, is accomplished from Boc-Tyr-OH by a simple three-step procedure which involves (1) initial esterification of Boc-Tyr-OH with 4-nitrobenzyl bromide or 2-(bromomethyl)anthraquinone, (2) phosphoro triester or "phosphite triester" phosphorylation of the tyrosyl hydroxyl group, followed by (3) hydrogenolytic or reductive cleavage of the 4-nitrobenzyl or 2methyleneanthraquinonyl (Maq) ester group. Preparation o f Boc-Tyr(PO3Me2)-OH (Fig. 1) 1. Carboxyl Esterification Procedure 8'9

1. 4-Nitrobenzyl bromide or 2-(bromomethyl)anthraquinone (12.5 mmol) is added to a solution of Boc-Tyr-OH (10 mmol) and triethylamine (12.5 mmol) in ethyl acetate (30 ml). 2. The solution is refluxed for 6 hr. 3. After cooling, water is added and the organic phase successively washed with 1 M HC1 and 5% NaHCO 3, dried (Na2SO4), filtered, and the solvent then evaporated under reduced pressure. 7 R. M. Valerio, J. W. Perich, E. A. Kitas, P. F. Alewood,and R. B. Johns, Aust. J. Chem. 42, 1519(1989). 8 E. A. Kitas, J. W. Perich, R. B. Johns, and G. W. Tregear, J. Org. Chem. 55, 4181(1990). 9 j. Jentsch and E. Wunsch, Justus Liebigs Ann. Chem. 97, 2490(1964).

236

ANALYSIS OF PROTEIN PHOSPHORYLATION OH

CH3

OH

(l)

?H2

I I

II

H

?.3

?H2

CH3-C-O-C-NH-CH-C-OR I II II CH3 0 0

CH3-C-O-~-NH-CH-C-OH CH3 0

0

(ii)

0

~ CH3 0

or (iii)

tJ

OP(OMe) 2

Me) 2

CIt3-C-O-C-I~-CtI-C-OH

II

R = NBzl o r Maq

0

II

I

[19]

II

0

~ or

CH3-C-O-C-I~-CH-C-OR (v)

[

It

CH3 0

II

0

R = NBzl o r Maq

FIG. 1. Preparation of Boc-Tyr(PO3Me2)-OH.(i) 4-Nitrobenzylbromideor 2-(bromomethyl)anthraquinone, triethylamine, ethyl acetate (80°, 6 hr); (ii) Nail (30 rain), then (MeO)z_ P(O)CI (30 min, 20°); (iii) (MeO)zPNEh/1H-tetrazole(20 min, 20°), then MCPBA (0°, 10 min); (iv) H2, 10% palladium on charcoal, 5% acetic acid/ethyl acetate; and (v) Na2SzO4/Na2CO3 (1 hr, 50°).

The phosphorylation of Boc-Tyr-ONBzl or Boc-Tyr-OMaq is achieved by its initial treatment with 1 Eq of sodium hydride at - 6 0 ° followed by in situ treatment of the resultant sodium phenoxide with dimethyl phosphorochloridate. In this phosphorylation, the generation of the phenoxide intermediate gives an orange solution which, after addition of dimethyl phosphorochloridate, becomes colorless due to rapid phosphorylation of the sodium phenoxide intermediate. As this phosphorylation step is water sensitive, it is necessary that dry tetrahydrofuran or dioxane is used. An advantage in the use of the 2methylanthraquinone group is that this protecting group generally leads to solid products; for example, Boc-Tyr(PO3Mez)-ONBzl is isolated as a yellow oil while Boc-Tyr(PO3Me2)-OMaq is obtained as a yellow solid and can be purified by crystallization.

[19]

SYNTHESIS OF Tyr(P)-CONTAINING PEPTIDES

237

2a. Phosphoro Triester Phosphorylation Procedure 5'8 1. Sodium hydride is washed with pentane (three times, 5 ml each) and dried under a stream of dry nitrogen. 2. Sodium hydride (5.0 mmol) is suspended in dry tetrahydrofuran (5 ml) (or dioxane) and cooled to 10°. 3. A solution of Boc-Tyr-ONBzl or Boc-Tyr-OMaq (4.0 retool) in dry tetrahydrofuran (5 ml) (or dioxane) is added to the hydride suspension with vigorous stirring. 4. After stirring for 30 min at 20°, the solution is cooled to 10° and dimethyl phosphorochloridate (6.0 mmol) is added in one portion to the bright orange solution (the solution becomes colorless). 5. After stirring for 30 min at 20°, water (1 ml) is added and the solvents are evaporated under reduced pressure. 6. The residue is dissolved in ethyl acetate (30 ml) and the organic phase washed successively with 1 M HC1 (10 ml) and 5% NaHCO3 (10 ml). The organic phase is dried (Na2SO4), filtered, and the solvent then evaporated under reduced pressure. 7. In the case of Boc-Tyr(PO3Me2)-OMaq, the solid is triturated with diethyl ether and then recrystallized from ethyl acetate/ligroine 60-80 ° (mp 149-150°). This derivative can also be prepared by the "phosphite triester" phosphorylation of Boc-Tyr-ONBzl or Boc-Tyr-OMaq with (1) (Meo)zPNEt2/ 1H-tetrazole followed by (2) m-chloroperoxybenzoic acid (MCPBA) oxidation. It should be noted that dimethyl N,N-diethylphosphoramidite is particularly pungent and that its synthesis uses dangerous reagents and therefore should only be performed by experienced personnel. Also, as phosphite triester phosphorylation reagents are very sensitive to water, all reagents and tetrahydrofuran should be thoroughly dried. If possible, tetrahydrofuran should be freshly distilled from a potassium/benzophenone still.

2b. Phosphite Triester Phosphorylation Procedure 8 1. Boc-Tyr-ONBzl or Boc-Tyr-OMaq (5.0 mmol) and dimethyl N,Ndiethylphosphoramidite (5.5 mmol) are dissolved in dry tetrahydrofuran (5 ml) at 20°. 2. 1H-Tetrazole (16.5 mmol) is added in one portion to the stirred solution at 20°. 3. After stirring for 20 min, the solution is cooled to - 4 0 ° and a solution of 85% m-chloroperoxybenzoic acid (6.0 mmol) in CHzCI 2 (12 ml) or 14% tert-butyl hydroperoxide (3.8 ml, 6.0 mmol) is added so that the temperature of the reaction solution is kept below 0 °.

238

ANALYSIS OF PROTEIN PHOSPHORYLATION

[19]

4. After stirring for 10 min, a solution of 10% Na2S205 (5 ml) is added to the solution at 0 °. 5. The solution is transferred to a separating funnel using ethyl acetate (30 ml) and the organic phase washed successively with 5% NaHCO3 (15 ml) and 1 M HC1 (15 ml), The organic phase is then dried (Na2SO4), filtered, and the solvent then evaporated under reduced pressure. 6. In the case of Boc-Tyr(PO3Me2)-OMaq, the solid is triturated with diethyl ether and then recrystallized from ethyl acetate/ligroine 60-80 ° (mp 149-152°). 3a. Hydrogenation Procedure 5

1. Boc-Tyr(PO3Mez)-ONBzl or Boc-Tyr(PO3Me2)-OMaq (10.0 mmol) is dissolved in 5% acetic acid/methanol (50 ml) and 10% palladium on charcoal (0.5 g) added. 2. The hydrogenation column is charged with hydrogen and the solution vigorously stirred until hydrogen uptake ceases. 3. The catalyst is removed by gravity filtration through filter paper [Whatman (Clifton, NJ) No. 1] and the solvent evaporated under reduced pressure. 4. The residue is dissolved in diethyl ether (60 ml) and the organic phase washed with 1 M HC1 (30 ml). 5. The organic phase is extracted with 5% NaHCO3 (three times, 15 ml each) and the aqueous phase then washed with diethyl ether (15 ml). 6. The aqueous phase is then acidified to pH 1 with 2 M HCI and the aqueous solution then extracted with dichloromethane (three times, 30 ml each). 7. The solvent is then evaporated under reduced pressure. In the case where laboratories lack a hydrogenation apparatus, the synthesis of Boc-Tyr(PO3Me2)-OH can be accomplished by the sodium dithionite reduction of the 2-methylanthraquinone-protecting group. The methylanthraquinone group is preferred over the use of the 4-nitrobenzyl group in this route since, unlike dithionite reduction of the 4-nitrobenzyl group, dithionite reduction of the methylanthraquinone group does not proceed with the formation of polymeric by-products. 3b. S o d i u m Dithionite Reduction Procedure 8

1. Boc-Tyr(PO3Me2)-OMaq (8.5 mmol) is dissolved in acetonitrile (50 ml) and a solution of sodium dithionite (34.0 mmol) and sodium carbonate (34.0 mmol) in hot water (25 ml) added. 2. The solution is vigorously stirred at 50 ° for 1 hr, cooled at 20°, and then acidified to pH I with 1 M HCI.

[19]

SYNTHESIS OF Tyr(P)-CONTAINING PEPTIDES

239

3. The acetonitrile is then evaporated under reduced pressure. 4. Diethyl ether (60 ml) is then added and the aqueous phase discarded. 5. The organic phase is washed with 1 M HCI; (30 ml) and the organic phase extracted with 5% NaHCO3 (three times, 15 ml each). The combined base extracts are combined and the aqueous phase then washed with diethyl ether (15 ml). 6. The aqueous phase is then acidified to pH 1 with 2 M HC1 and the aqueous solution then extracted with dichloromethane (three times, 30 ml each). 7. The solvent is then evaporated under reduced pressure and the thick oil dried under high vacuum. In addition, Boc-Tyr(PO3Me2)-OH can also be prepared by a onepot procedure which uses in situ tert-butyldimethylsilyl protection of the carboxyl terminus of Boc-Tyr-OH followed by phosphite triester phosphorylation of the tyrosyl hydroxyl group using dimethyl N,N-diethylphosphoramidite. 10

One-Pot Procedure 1. Boc-Tyr-OH (3.0 mmol) is dissolved in tetrahydrofuran (9 ml) and solutions of N-methylmorpholine (3.0 mmol) in tetrahydrofuran (1 ml) and tert-butyldimethylchlorosilane (3.0 mmol) in tetrahydrofuran (1 ml) are added, respectively. 2. After 2 min at 20°, a solution of dimethyl N,N-diethylphosphoramidite (4.0 mmol) in tetrahydrofuran (THF) (1 ml) is added followed by the addition of 1H-tetrazole (9.0 mmol) in one portion. 3. After 20 min at 20°, the reaction solution is cooled to - 4 0 ° and a solution of iodine (4.0 retool) in tetrahydrofuran/water (3 : 1, 4 ml) added. 4. After 10 min at 20°, a solution of 10% N a ] S 2 0 5 (10 ml) is added and the solution stirred for a further 10 min. 5. The solution is transferred to a separating funnel using diethyl ether (40 ml) and the aqueous phase discarded. 6. The organic phase is washed with 10% Na2S205 (30 ml) and then extracted with 5% NaHCO3 (three times, 15 ml each). 7. The combined base extracts are washed with diethyl ether (30 ml) and then acidified to pH 1 with 30% HCI. 8. The organic extracts are combined, dried (NazSO4), filtered, and evaporated under reduced pressure to give Boc-Tyr(PO3Me2)-OH as a thick oil. l0 j. W. Perich and R. B. Johns, Synthesis p. 701 (1989).

240

ANALYSIS OF PROTEIN PHOSPHORYLATION

[19]

Solution-Phase Synthesis of Tyr(P)-Containing Peptides The isobutoxycarbonyl mixed anhydride coupling procedure ~1 is the method of choice for the addition of protected amino acids and BocTyr(PO3Mez)-OH to peptides. This coupling method is favored since it is simple, rapid, gives high product yields, and uses readily available reagents. In addition to the mixed anhydride coupling procedure, the dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) and active ester coupling procedures can also be used.

Procedure 1. Dissolve Boc-Tyr(PO3Me2)-OH (1.4 Eq) in tetrahydrofuran and cool the solution to - 2 0 °, using dry ice/acetone cooling. 2. Add solution of N-methylmorpholine (1.4 Eq) in THF. 3. Add solution of isobutyl chloroformate (1.3 Eq) in THF so that the temperature of the coupling solution is maintained at - 2 0 °. 4. After an activation period of 3 min, a solution of the peptide trifluoroacetate (1.0 Eq) and N-methylmorpholine (1.0 Eq) in either tetrahydrofuran (or dichloromethane or N-methylpyrrolidone, depending on the solubility of the peptide) is added at - 20 °. 5. After a coupling period of 1.5 to 2 hr, 5% NaHCO 3 is added and the solution stirred for a further 30 min. 6. The solution is transferred to a separating funnel, using a suitable solvent (the selection of diethyl ether, ethyl acetate, dichloromethane, or chloroform being determined by the solubility of the peptide in the solvent). (Note: In some cases, the peptide is isolated by aqueous precipitation.) 7. The organic phase is washed with 5% NaHCO3 (two times, 30 ml each), 1 M HC1 (two times, 30 ml each), the organic phase evaporated under reduced pressure, and then dried under high vacuum.

Peptide Deprotection (Including Cleavage of Methyl Phosphate Groups) The deprotection of protected Tyr(PO3Me2)-containing peptides is performed in accordance with standard peptide deprotection procedures. J2 The cleavage of the methyl phosphate groups is accomplished by acidolytic II j. Meienhofer, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds), Vol. 1, Chapter 6. Academic Press, New York, 1983. i2 H. Yajima and N. Fujii, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds), Vol. 5, Chapter 2. Academic Press, New York, 1983.

[19]

SYNTHESIS OF Tyr(P)-CONTAINING PEPTIDES

241

or silylitic treatment and, if possible, can be monitored by 3~p nuclear magnetic resonance (NMR) spectroscopy.

Procedure M e t h o d 15: The peptide (0.1 mmol) is dissolved in 33% HBr/acetic acid (5 ml) and left at 20° for 24 hr (or as determined by 31p NMR spectroscopy to be complete). The solvent is evaporated under reduced pressure, the residue triturated with diethyl ether, and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) or anion-exchange chromatography. Method27: The peptide (0.1 retool) is dissolved in 10% bromotrimethylsilane/CH3CN (20 ml) and left at 20° for 5 hr (or as determined by 3)p NMR spectroscopy to be complete). The solvent is evaporated under reduced pressure, the residue triturated with diethyl ether, and then purified by RP-HPLC or anion-exchange chromatography. Method 3 7'8'13" The peptide (0.1 mmol) is dissolved in 1 M bromotrimethylsilane and thioanisole in CF3COEH (3 ml) containing m-cresol (10 mEq/peptide) at 0° and left at 20° for 12 hr (or as methyl cleavage was determined by 31p NMR spectroscopy to be complete). The solvent is evaporated under reduced pressure and cold diethyl ether is added. The precipitated residue is washed with diethyl ether and then purified by RPHPLC or anion-exchange chromatography. Method 4 7'8'13" The peptide (0.1 mmol) is dissolved in trifluoromethanesulfonic acid/CF3COzH/thioanisole (or dimethyl sulfide)/m-cresol ( 1 : 5 : 3 : 1 ) (3 ml) and left at 20° for 1 hr (or as methyl cleavage was determined by 31p NMR spectroscopy to be complete). The solvent is evaporated under reduced pressure and cold diethyl ether is added. The precipitated residue is washed with diethyl ether and then purified by RPHPLC or anion-exchange chromatography. M e t h o d 58: The peptide (0.1 mmol) is dissolved in 1 M trimethylsilyl triflate and thioanisole in CF3COEH (3 ml) containing m-cresol (I0 mEq/ peptide) and left at 20° for 24 hr (or as methyl cleavage was determined by 31p NMR spectroscopy to be complete). Methanol is added to the solution and solvent is evaporated under reduced pressure. Cold diethyl ether is added and the precipitated residue is washed with diethyl ether and then purified by RP-HPLC or anion-exchange chromatography.

13E. A. Kitas, J. W. Perich, R. B. Johns, and G. W. Tregear, Tetrahedron Lett. 29, 3591 (1988).

242

ANALYSIS OF PROTEIN PHOSPHORYLATION

[19]

Boc/Solid-Phase Synthesis of Tyr(P)-Containing Peptides The synthesis of Tyr(P)-containing peptides is possible by the use of Boc-Tyr(PO3Me2)-OH in the Merrifield solid-phase methodology. 14

Boc Method 1. Prepare Boc-Tyr(PO3Me2)-OH as outlined above. 2. Assemble the peptide according to the method described by Merrifield, using a polystyrene support, 40% CF3CO2H/CHzC12 for Boc cleavage, and DCC/HOBt amino acid couplings performed with 3 Eq of the Boc-amino acid. 3. Peptide-resin cleavage: Dry hydrogen bromide is bubbled into a suspension of the peptide-resin in trifluoroacetic acid (10 ml) for 90 min (at 20°). The solvent is evaporated under reduced pressure, the residue dissolved in 10% acetic acid/water, and the solution filtered (Whatman No. 1) to remove resinous material. The solvent is then evaporated under reduced pressure, the residue triturated with diethyl ether (two times, 30 ml), and then dried under high vacuum. 4. Methyl phosphate cleavage: The above isolated solid is dissolved in 33% HBr/acetic acid (5 ml) and the solution kept at 20° for 15 hr. 5. The solvent is evaporated under reduced pressure, the residue triturated with diethyl ether, and the residue then dried under high vacuum. 6. The peptide is purified by semipreparative reversed phase HPLC or anion-exchange chromatography. In the first demonstration of this method, Valerio et al. 5'15prepared LeuArg-Arg-Ala-Tyr(P)-Leu-Gly with the use of nitro protection for the arginine side-chain groups; the nitro groups were cleaved by palladium acetatemediated hydrogenolysis in acetic acid (60 psi, 24 hr).

Fmoc/Solid-Phase Synthesis of Tyr(P)-Containing Peptides The procedure for the synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc-polyamide solid-phase synthesis j6 is outlined as follows: (1) preparation of Fmoc-Tyr(PO3Me2)-OH, (2) solidphase synthesis of protected Tyr(PO3Me2)-containing peptides, and (3) peptide deprotection (including cleavage of methyl phosphate groups). 14 G. Baramy and R. B. Merrifield, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds), Vol. 2, Chapter 1. Academic Press, New York, 1983. z5 R. M. Valerio, P. F. Alewood, R. B. Johns, and B. E. Kemp, Tetrahedron Lett. 25, 2609 (1984). 16 E. Atherton and R. C. Sheppard, "Solid Phase Peptide Synthesis--A Practical Approach." IRL Press at Oxford Univ. Press, Oxford, 1989.

[19]

SYNTHESIS OF Tyr(P)-cONTAINING PEPTIDES OH

243

Oil

c~2

~2

(1)

I

Fmoc -NB-CH-C-OI~

>,

II

Fmoc-NE-CB-C -O~aq

II

o

o

(ii)

O

O

Ii

11

OP(O~e)2

OP(OMe)2

~FI2 Fmoc-NI-I-CH-C-Oll II o

(iii) ~(

~I'I2 Fmoc-N'B-CII-C-OMaq II o

FIG. 2. Preparation of Fmoc-Tyr(PO3Me2)-OH. (i) 2-(Bromomethyl)anthraquinone, triethylamine, ethyl acetate (80 °, 6 hr); (ii) (MeO)2PNEtJ1H-tetrazole (20 rain, 20°), then MCPBA (0 °, 10 rain); and (iii) Na2S204/NazCO 3 (1 hr, 50°).

Preparation of Fmoc-Tyr(POsMe2)-OH. 17Phosphite triester phosphorylation procedure (Fig. 2) 1. Fmoc-Tyr-OMaq (5.0 retool) and dimethyl N,N-diethylphosphoramidite (5.5 mmol) are dissolved in dry tetrahydrofuran (5 ml) at 20 °. 2. 1H-Tetrazole (16.5 mmol) is added in one portion to the stirred solution at 20 ° . 3. After stirring for 30 rain at 20% the solution is cooled to - 4 0 ° and a solution of m-chloroperoxybenzoic acid (6.0 mmol) in CHzCI 2 (12 ml) or 14% tert-butyl hydroperoxide (3.8 ml) is added so that the temperature of the reaction solution is kept below 0% 4. After stirring for 10 min at 0 °, a solution of 10% Na2S205 (5 ml) is added to the reaction solution at 0 °. 5. The solution is transferred to a separating funnel using ethyl acetate 17 E. A. Kitas, J. W. Perich, J. D. Wade, R. B. Johns, and G. W. Tregear, Tetrahedron Lett. 30, 6229 (1989).

244

ANALYSIS OF PROTEIN PHOSPHORYLATION

[19]

(30 ml) and the organic phase washed successively with 5% NaHCO3 (15 ml) and I MHC1 (15 ml). The organic phase is then dried (Na2SO4), filtered, and the solvent then evaporated under reduced pressure. 6. The solid is triturated with diethyl ether and then dried under high vacuum.

Reduction procedure 1. Fmoc-Tyr(PO3Me2)-OMaq (4.25 mmol) is dissolved in acetonitrile (25 ml) and a solution of sodium dithionite (17.0 mmol) and sodium carbonate (17.0 mmol) in hot water (12.5 ml) added. 2. The solution is vigorously stirred at 50° for 1 hr, cooled to 20°, and then acidified to pH 1 with 1 M HC1. 3. The acetonitrile is then evaporated under reduced pressure. 4. Diethyl ether (60 ml) is then added and the aqueous phase discarded. 5. The organic phase is washed with I M HCI (30 ml) and the organic phase extracted with 5% NaHCO3 (three times, 15 ml each). The combined base extracts are combined and the aqueous phase then washed with diethyl ether (15 ml). 6. The aqueous phase is acidified to pH 1 with 2 M HCI and the aqueous solution then extracted with dichloromethane (three times, 30 ml each). 7. The solvent is then evaporated on a rotary evaporator under reduced pressure. 8. The isolated oil is dried under high vacuum to give a light white honeycomb solid.

Solid-Phase Synthesis of Protected Tyr(PO3Me2)-Containing Peptides.~7 The solid-phase synthesis is performed using a kieselguhr-polydimethylacrylamide resin that is functionalized with the acid-labile 4-hydroxymethylphenoxyacetic acid linkage and can include a fl-alanine internal reference. Due to variations in commercially available solid-phase peptide synthesizers, the synthesis of peptides should be performed in accordance with the manufacturer's recommendations.

Procedure 1. Prepare Fmoc-Tyr(PO3Mez)-OH as outlined above. 2. Assemble peptide according to the method described by Atherton and Sheppard, 16 using a polyamide support, the use of 3 Eq of the Fmocamino acid for BOP/HOBt or DCC/HOBt couplings, and 20% piperidine/ dimethylformamide (DMF) for Fmoc cleavage. 3. Peptide-resin cleavage and methyl cleavage: The peptide resin (0.25 mmol) is suspended in 1 M bromotrimethylsilane and thioanisole in CF3CO2H (5 ml) containing m-cresol (10 mEq) at 0° and stirred for 16 hr

[20]

STOICHIOMETRY

OF PHOSPHORYLATION

245

at 4% The solvent is evaporated under reduced pressure, the residue dissolved in 10% (v/v) acetic acid/water and the solution filtered (Whatman No. l) to remove resinous material. The solvent is then evaporated under reduced pressure, the residue triturated with diethyl ether (two times, 30 ml each), and then dried under high vacuum. 4. The peptide is purified by semipreparative reversed phase HPLC or anion-exchange chromatography. This approach has been used for the synthesis of Arg-Leu-Ile-Glu-AspAsn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly in 70% yield, using BOP/HOBt (3 Eq) as the coupling procedure. While the repeated piperidine cleavage of the Fmoc group also causes partial methyl phosphate cleavage (tl/2 = 7 min), this side reaction does not seem to effect peptide extension. Summary As a consequence of developments in peptide synthesis, it is now well established that Tyr(P)-containing peptides can be prepared in high yield by the use of Boc-Tyr(PO3MeE)-OH in Boc solution or solid-phase peptide synthesis or Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid-phase peptide synthesis. It is considered that with further developments in solid-phase synthesis techniques and the use of alternative phosphate-protecting groups, the synthesis of large and complex Tyr(P)-containing peptides will be routine.

[20] M e a s u r e m e n t o f S t o i c h i o m e t r y of P r o t e i n Phosphorylation by Biosynthetic Labeling

By BARTHOLOMEW M. SEFTON Determination of the extent of phosphorylation of proteins that can be obtained only in trace amounts or in impure form is not possible by chemical means. The stoichiometry of phosphorylation of such proteins can, however, be inferred if the protein of interest can be labeled biosynthetically. In the most general sense, this is accomplished by labeling cells to equilibrium with a radioactive amino acid and with [32P]Pi, measurement of the specific activity of total cellular protein and intracellular ATP, isolation of the protein of interest by immunoprecipitation, quantification of the protein from the recovery of amino acid label, and calculation of the number of moles of phosphate present in the protein from the recovery o f 32p. This approach has been used to quantify the phosphorylation of METHODS IN ENZYMOLOGY, VOL. 201

Copyright © 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

Synthesis of O-phosphotyrosine-containing peptides.

As a consequence of developments in peptide synthesis, it is now well established that Tyr(P)-containing peptides can be prepared in high yield by the...
588KB Sizes 0 Downloads 0 Views