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[18] S y n t h e s i s of O - P h o s p h o s e r i n e - a n d O - P h o s p h o t h r e o n i n e Containing Peptides B y JOHN W . PERICH

The increased recognition of protein phosphorylation in many physiological processes has resulted in the need for efficient chemical methods for the synthesis of phosphorylated serine- and threonine-containing peptides for use as model substrates. Prior to 1980, phosphoserine [Ser(P)]containing peptides were generally prepared by a "global phosphorylation" approach 1'2 that involved (1) the phosphorylation of a protected Ser-containing peptide with dibenzyl or diphenyl phosphorochloridate in pyridine followed by (2) hydrogenolytic deprotection. Although this approach has been successfully used for the preparation of many simple Ser(P)-containing peptides, ~this approach is limited since the phosphorylation or deprotection steps are often incomplete and lead to major synthetic problems. Since 1980, however, these problems were overcome by the development of an alternative approach that involved the use of protected Boc-Ser(PO3R2)-OH derivatives in the tert-butyloxycarbonyl (Boc) mode of peptide synthesis. 3 This chapter describes the synthetic methods used for the preparation of Ser(P)- and phosphothreonine [Thr(P)]-containing peptides by the use of (1) Boc-Ser(PO3Ph2)-OH and Boc-Thr(PO3Phz)-OH in the Boc mode of peptide synthesis for the synthesis of protected Ser(PO3 Ph2)- o r Thr(PO 3Ph 2)-containing peptides followed by (2) their deprotection using modified hydrogenation conditions.

Equipment and Reagents The synthesis of Set(P)- and Thr(P)-peptides requires a laboratory equipped with a rotary evaporator, a high vacuum line, a fume hood, a hydrogenation apparatus, and a high-performance liquid chromatography (HPLC) instrument. In addition, the availability of nuclear magnetic resonance (NMR) spectroscopy (~H, 13C, and 3~p) and fast atom bombardment (FAB) mass spectrometry facilities is considered necessary for the proper analysis of protected amino acids and peptides.

I G. Folsch, Sven. Kem. Tidskr. 79, 38 (1967). 2 A. W. Frank, CRC Crit. Rev. Biochem. 16, 51 (1984). 3 j. W. Perich, Ph.D. Thesis, University of Melbourne, Melbourne, Australia (1986).

METHODS IN ENZYMOLOGY, VOL. 201

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

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ANALYSISOF PROTEINPHOSPHORYLATION

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Synthesis of Ser(P)- and Thr(P)-Containing Peptides The most suitable procedure for the synthesis of Ser(P)- and Thr(P)containing peptides is accomplished by (1) the use of Boc-Ser(PO3Ph2)OH or Boc-Thr(PO3Ph2)-OH in the Boc mode of peptide synthesis for the synthesis of protected Ser(PO3Ph2)- or Thr(PO3Ph2)-peptides followed by (2) the hydrogenolytic cleavage (platinum) of the phenyl phosphate groups. 4'5 This approach permits a phosphorylated serine residue to be incorporated at any specific site in a peptide during the peptide synthesis procedure, and is suitable for the synthesis of multiple Ser(P)-containing peptides and mixed Ser/Ser(P)-containing peptides. The procedure for the synthesis of Ser(P)- and Thr(P)-containing peptides is outlined as follows: (1) preparation of Boc-Ser(PO3Ph2)-OH and Boc-Thr(PO3Ph2)-OH, (2) synthesis of protected Ser(PO3Ph2)- or Thr(PO3Ph2)-containing peptides, (3) peptide deprotection (including cleavage of phosphate-protecting groups), and (4) characterization of Ser(P)- and Thr(P)-containing peptides. Preparation o f Boc-Ser(PO3Ph2)-OH and Boc-Thr(PO3Ph2)-OH

The synthesis of Boc-Ser(POaPh2)-OH and Boc-Thr(PO3PhE)-OH is accomplished by a three-step procedure which involves (1) initial protection of the carboxyl group of Boc-Ser-OH as its 4-nitrobenzyl ester, (2) phosphorylation of the hydroxyl group of Boc-Ser-ONBzl or Boc-ThrONBzl by the use of diphenyl phosphorochloridate in pyridine, and (3) hydrogenolytic cleavage of the 4-nitrobenzyl group from the carboxyl terminus (see Fig. 1). 6 Protection of the carboxy terminus prior to the phosphorylation step is necessary, since a carboxylic acid group readily reacts with diphenyl phosphorochloridate and leads to by-products. In this case, the 4-nitrobenzyl group is a suitable protecting group since it can be readily introduced to Boc-Ser-OH or Boc-Thr-OH without side-chain protection and can be cleaved in high yield by palladium-catalyzed hydrogenolysis or sodium dithionite reduction. The phosphorylation of BocSer-ONBzl and Boc-Thr-ONBzl is straightforward and is accomplished by the use of diphenyl phosphorochloridate in pyridine. The cleavage of the 4-nitrobenzyl group from Boc-Ser(PO3PhE)-ONBzl or BocThr(POaPhE)-ONBzl is best performed by palladium-catalyzed hydrogenolysis using a hydrogen column apparatus operated at 1 atm of pressure.

4 j. W. Perich, P. F. Alewood, and R. B. Johns, Tetrahedron Left. 27, 1373(1986). 5j. W. Perich and R. B. Johns, J. Org. Chem. 53, 4103 (1989). 6j. W. Perich, P. F. Alewood, and R. B. Johns, Aust. J. Chem. 44, 233 (1991).

[18]

Ser(P)- AND Thr(P)-CONTAININGPEPTIDES OH

OH

I

CH3I

I

CH-R,

(1)

CH3-C-O-C-NH-CH-C-OH

t

ii

CH 3 0

227

~H3 ~

?-R

.

~ NO2

CH3- .C-O-C-NH-CH-C-O-CH 2

il

i

0

CH 3 0

.

.

0

(il)

0 II OP(OPh) 2 CH3

0 II OP(OPh) 2

I

CH-R

I I CH3-C-O-C-NH-CH-C-OH i II II CH 3 0 0

( 11 i ) tc

CHa

I

CH-R

...-.-.

I I /r'x\ CH3-C-O-C-NH-CH-C-O-CH2- ~ ( ) )-NO 2 I II II CB 3 0 0

FIc. 1. Synthesis of Boc-Ser(PO3Ph2)-OH and Boc-Thr(PO3Ph2)-OH. (i) 4-Nitrobenzyl bromide, triethylamine, ethyl acetate (80°, 6 hr); (ii) (PhO)2P(O)C1/pyridine [20°, 4 hr (R = H) or 16 hr (R = CH3)]; (iii) H E, 10% (w/v) palladium on charcoal, 5% (v/v) acetic acid/ethyl acetate,

Carboxyl Esterification Procedure 7 1. 4-Nitrobenzyl bromide (125 mmol) is added to a solution of BocSer-OH or Boc-Thr-OH (100 mmol) and triethylamine (125 mmol) in ethyl acetate (150 ml). 2. The solution is refluxed for 6 hr. 3. After cooling, water (60 ml) is added and the solution transferred to a separating funnel. 4. The aqueous phase is discarded and the organic phase successively washed with 1 M HC1 (two times, 50 ml each) and 5% (w/v) NaHCO3 (two times, 50 ml each) dried, (NazSO4), and then filtered. 5. The solvent is then evaporated on a rotary evaporator under reduced pressure. While Boc-Thr-ONBzl is obtained as a thick oil, Boc-Ser-ONBzl is obtained as a white solid and can be further purified by recrystallization from ethyl acetate/ligroine 60-80 °. In the esterification procedure, particular care should be used in the handling of 4-nitrobenzyl bromide since this reagent is extremely corrosive and causes severe skin and eye irritation. 7 p. F. Alewood, J. W. Perich, and R. B. Johns, Synth. Commun. 12, 821 (1982).

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ANALYSIS OF PROTEIN PHOSPHORYLATION

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Phosphorylation Procedure 6

1. A solution of diphenyl phosphorochloridate (12.0 mmol) in tetrahydrofuran (5 ml) is added to a solution of Boc-Ser-ONBzl or Boc-ThrONBzl (I0.0 mmol) in pyridine (10 ml) at 20°. 2. After stirring for 4 hr at 20° (for Ser) or overnight at 4° (for Thr), water (2 ml) is added and the solution stirred for 15 min. 3. Ethyl acetate (60 ml) (for Ser) or diethyl ether (60 ml) (for Thr) is added and the organic phase washed successively with 1 M HCI (three times, 30 ml each), 5% NaHCO 3 (two times, 30 ml each), 1 M HC1 (once with 30 ml), dried (Na2SO4), and filtered. 4. The solvent is then evaporated on a rotary evaporator under reduced pressure. 5. The crude Boc-Ser(PO3Ph2)-ONBzl is recrystallized from ethyl acetate/lingroine 60-80 °. While complete phosphorylation of the primary hydroxyl group of Boc-Ser-ONBzl is effected after 2 hr, the slower phosphorylation of the secondary hydroxy group of Boc-Thr-ONBzl requires the phosphorylation solution to be stirred at 4° overnight for complete hydroxyl phosphorylation. In the case of Boc-Thr(PO3Phz)-ONBzl, diethyl ether is the solvent of choice for product isolation since this low-polarity solvent permits the complete removal of diphenyl hydrogen phosphate (formed by hydrolysis of excess diphenyl phosphorochloridate) from the organic phase by sodium bicarbonate extraction. The use of ethyl acetate, dichloromethane, or chloroform is not recommended since sodium diphenyl phosphate cannot be completely extracted from these organic phases by base extraction. As Boc-Ser(PO3Ph2)-ONBzl is insoluble in diethyl ether, the isolation of this product is best performed by the use of ethyl acetate for product isolation followed by recrystallization from ethyl acetate/diethyl ether to removal diphenyl hydrogen phosphate from the crude isolated solid. Hydrogenation Procedure 6

1. Boc-Ser(PO3Ph2)-ONBzl or Boc-Thr(PO3Ph2)-ONBzl (10.0 mmol) is dissolved in 5% acetic acid/ethyl acetate (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 solution

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Ser(P)- AND Thr(P)-cONTAINING PEPTIDES

229

transferred to a separating funnel and washed with 1 M HC1 (once with 30 ml). 5. The organic phase is extracted with 5% NaHCO 3 (three times, 15 ml each) and the combined base extractions then washed with diethyl ether (once with 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 on a rotary evaporator under reduced pressure. While Boc-Thr(PO3Ph2)-OH is obtained as a thick oil, Boc-Ser (PO3Ph2)-OH is obtained as a clear oil which solidifies on storage and can be recrystallized from diethyl ether. Also, both products can be converted to their dicyclohexylamine (DCHA) derivatives and purified, if necessary, by recrystallization.

Synthesis of Protected Ser(PO3Ph2)- or Thr(PO3Phe)-Containing Peptides The protected Ser(PO3Ph2)-containing peptide is assembled by standard chemical procedures used in the Boc mode of solution-phase peptide synthesis. In solution-phase synthesis, the mixed anhydride condensation procedure 8 is generally the method of choice for the coupling of protected amino acids to peptides. An advantage in the use of phenyl phosphate groups is that, depending on the peptide sequence and its length, protected Ser(PO3Ph2)-containing peptides are generally soluble in common organic solvents (such as ethyl acetate and dichloromethane) and thereby peptide isolation and synthesis are facilitated. Also, the N-methylamine group is used for the peptide carboxyl terminus because peptide N-methylamides are generally obtained as solids and have good solubility properties.

Procedure I. Dissolve Boc-Ser(PO3Ph2)-OH (1.4 Eq) in tetrahydrofuran and cool the solution to - 2 0 ° using dry ice/acetone. 2. Add a solution of N-methylmorpholine (1.4 Eq) in tetrahydrofuran. 3. Add a solution of isobutyl chloroformate (1.3 Eq) in tetrahydrofuran 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 tetrahydrofuran 8 j. Meienhofer, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 1, Chapter 6. Academic Press, New York, 1983.

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ANALYSIS OF PROTEIN PHOSPHORYLATION

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(or dichloromethane or N-methylpyrrolidone, depending on the solubility of the peptide) is added at -20 °. 5. After a coupling period of 2 hr, 5% NaHCO 3 (5 ml) is added and the solution stirred for a further 30 min. 6. Transfer the solution to a separating funnel with the use of a suitable solvent (60 to 100 ml) (the selection ofdiethyl ether, ethyl acetate, dichloromethane, or chloroform being determined by the solubility of the peptide in the solvent). (Note: In some cases, a low-solubility peptide is isolated by aqueous precipitation.) 7. The organic phase is washed with 5% NaHCO3 (two times, 30 ml each), 1 M HCI (two times, 30 ml each), the organic phase evaporated under reduced pressure, and then dried under high vacuum.

Peptide Deprotection (Including Cleavage of Phosphate-Protecting Groups) The cleavage of the phenyl phosphate groups from Ser(PO3Ph:)- and Thr(PO3Ph2)-containing peptides is readily achieved by hydrogenolysis over platinum. While early phenyl reductions using catalytic quantities were often incomplete,1 complete phenyl reduction is effected by the use of (1) 50% (v/v) CF3CO2H/CH3CO2H as hydrogenation solvent and (2) molar equivalents of platinum oxide per phenyl group.4-6 Also, preliminary palladium-catalyzed cleavage of peptides containing benzylic groups (benzyl ether and ester, benzyloxycarbonyl, etc.) is unnecessary because the platinum-mediated hydrogenation cleaves the benzylic protecting groups simultaneously.

Hydrogenation Procedure5,6 1. The protected Ser(PO 3Ph2)-containing peptide (generally 0.1 mmol) is dissolved in 50% CF3CO2H/CH3CO2H (4 ml) and 83% platinum oxide (1. I mEq of PtO2/phenyl group) added. 2. The hydrogenation column is charged with hydrogen and the solution vigorously stirred until hydrogen uptake ceases. 3. The platinum is removed by gravity filtration through filter paper (Whatman No. 1) and the solvent evaporated under reduced pressure. (Note: The platinum is also washed with water and the water washings separately evaporated under reduced pressure.) 4. The residue is triturated with diethyl ether (three times, 30 ml each) and the solid residue then dried under high vacuum.

Summary A feature of this synthetic approach is that since protected peptides are obtained in high yield and the hydrogenolytic deprotection procedure

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Ser(P)- AND Thr(P)-CONTAININGPEPTIDES

231

proceeds cleanly, the isolated Ser(P)- and Thr(P)-containing peptides are often obtained in high purity (>99.5%) and, in most cases, do not require any further HPLC or anion-exchange chromatographic purification. The advantages of the above phenyl phosphate-based protection system is that this approach permits the synthesis of(l) large, complex Ser(P)and Thr(P)-containing peptides [such as Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)Ser(P)-GIu-GIu-NHMe], (2) mixed Ser/Ser(P)-containing peptides [such as H-Ser-Ser(P)-Ser(P)-NHMe • trifluoroacetic acid (TFA) and H-Ser(P)Ser-Ser(P)-NHMe • TFA], and (3) multiple Ser(P)- and Thr(P)-containing peptides [such as H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe • TFA and HThr(P)-Thr(P)-Thr(P)-GIu-GIu-NHMe • TFA].

Characterization of Ser(P)- and Thr(P)-Containing Peptides The characterization of Ser(P)- and Thr(P)-containing peptides is accomplished by the use of NMR spectroscopy (~3C and 31p) and FAB mass spectrometry. In the 13C NMR spectrum of these peptides, the C~ and the C a carbons of the Ser(P) and the Thr(P) residues are observed as phosphorus-coupled doublet signals with coupling constants varying from 3 to 9 Hz. In the case of FAB mass spectrometry, greater information is obtained in the positive mode with the use of aqueous acetic acid/glycerol as the matrix phase (thioglycerol can also be used) and either argon or xenon as the ionization gas. 9 Apart from high-intensity pseudomolecular ions being obtained for Ser(P)- and Thr(P)-containing peptides, this spectrometry technique is also useful in establishing complete phenyl cleavage after hydrogenolytic deprotection. The interpretation of the FAB mass spectra for multiple Ser(P)- and Thr(P)-containing peptides can often be complicated due to the high-molecular-weight mass spectral region containing ion peaks due to extensive sodium and potassium complexation. Also, trace amounts of platinum in the peptide can give rise to ion peaks of +96 mass units due to platinum complexation of the peptide in the matrix. However, the observation of platinum-complex ions can be overcome by HPLC purification of the peptide.

Solid-Phase Synthesis of Ser(P)- and Thr(P) Peptides The solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides is also possible by the use of Boc-Ser(PO3Ph2)-OH in the Merrifield mode ~° of solid-phase peptide synthesis. 9 R. B. Johns, P. F. Alewood,J. W. Perich, A. L. Chaffee,and J. K. MacLeod,Tetrahedron Lett. 27, 4791 (1986). 10G. Baramy and R. B. Merrifield, in "The Peptides: Analysis, Synthesis, Biology"(E. Gross and J. Meienhofer,eds), Vol. 2, Chapter 1. AcademicPress, New York, 1983.

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ANALYSIS OF PROTEIN PHOSPHORYLATION

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Procedure

1. Prepare Boc-Ser(PO3Ph2)-OH as described above. 2. Assemble peptide according to the method described by Baramy and Merrifield, 1° using 3 Eq of the Boc-amino acid for dicyclohexycarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) couplings and 50% CF3CO2H/CH2CI 2 for Boc cleavage. 3. The Ser(PO3Ph2)-containing peptide is cleaved from the resin using method a, b, or c. a. A suspension of the peptide-resin in dry dimethylformamide (DMF) (30 ml) containing palladium acetate (0.3 g) is hydrogenated at 60 psi for 24 hr (60°). The catalyst is removed by gravity filtration (Whatman No. 1) and the solvent evaporated under reduced pressure. b. Dry hydrogen bromide is bubbled into a suspension of the peptide-resin in trifluoroacetic acid (10 ml) for 90 min (20°). The solvent is evaporated under reduced pressure and the residue triturated with diethyl ether (two times, 30 ml each) and dried under high vacuum. c. Hydrogen fluoride is distilled into a Kel-F vessel (Peninsula, CA) containing the peptide-resin and anisole (10%) and the solution allowed to stand at 0° for 45 min. The HF is evaporated under reduced pressure and the residue is triturated with diethyl ether (two times, 30 ml each) and dried under high vacuum. 4. The isolated peptide residue is hydrogenated with platinum oxide as described above. 5. The solvent is evaporated under reduced pressure and the residue is triturated with diethyl ether and then dried under high vacuum. 6. The peptide is purified by semipreparative reversed-phase HPLC or anion-exchange chromatography. This procedure was used by Perich et al. 1~ for the synthesis of GluSer(P)-Leu using palladium acetate-mediated hydrogenation (step 3,a above) or HBr/CF3COzH (step 3,b above) for the cleavage of the Ser(PO3Ph2)-containing tripeptide from the polystyrene resin. Also, Arendt et al.12 prepared Leu-Arg-Arg-Ser(P)-Leu-Glyusing liquid hydrogen fluoride (step 3,c above) for the cleavage of a Ser(PO3Ph2)-containing peptide from a polystyrene resin (or phenylacetamidomethyl resin) support. 11 j. W. Perich, R. M. Valerio, and R. B. Johns, Tetrahedron Lett. 27, 1377 (1986). 12 A. Arendt, K. Palczewski, W. T. Moore, R. M. Caprioli, J. H. McDowell, and P. A. Hargrave, Int. J. Pept. Protein Res. 33, 468 (1989).

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Synthesis of Ser(P)-Containing Peptides through Benzyl Phosphate Protection The synthesis of Ser(P)- and Thr(P)-containing peptides is also possible by the use of benzyl phosphate protection. A feature of this approach is that the benzyl phosphate groups are quantitatively cleaved by palladiumcatalyzed hydrogenolysis. Three variations of this approach are briefly described. Using Boc-Ser(PO3Bzl2)-OH. 6'13 Boc-Glu(OBzl)-Ser(PO3Bzl2)-LeuOBzl was prepared in 81% yield 6'14by the use of Boc-Ser(PO3Bz12)-OH in peptide synthesis with formic acid used for cleavage of the Boc group from the Boc-dipeptide. 6 However, the use of Boc-Ser(PO3Bzl2)-OH in peptide synthesis is limited due to the acid sensitivity of benzyl phosphate groups; approximately 50, 10, and 1% benzyl loss occurs after a 60-min treatment with 4 M HCl/dioxane, 1 M HC1/acetic acid, or formic acid, respectively. Using Ppoc-Ser(PO3Bzl2)-OH. By changing to the more acid-labile 2phenylisopropyloxycarbonyl group (Ppoc), Boc-Glu(OBzl)-Ser(PO3Bzlz)Leu-OBzl was prepared in 94% yield by the use of Ppoc-Ser(PO3Bz12)-OH in peptide synthesis with 0.5 M HC1/dioxane used for cleavage of the Ppoc group from the Ppoc-dipeptide. However, in general applications, the use of Ppoc-Ser(PO3Bzlz)-OH in peptide synthesis requires subsequent peptide extension of the Ser(PO3Bz12)-containing peptide to adopt the Ppoc or Bpoc mode of peptide synthesis. Using Boc-Ser(PO3BrBzle)-OH. The 4-bromobenzyl group can also be used for phosphate protection and takes advantage of the fourfold increase in acid stability of the 4-bromobenzyl group over the benzyl group in 1 M HCl/acetic acid or formic acid. For example, Boc-Glu(OBzl)Ser(PO3BrBz12)-Leu-OBzl was prepared in 94% yield by the use of BocSer(PO3BrBzlz)-OH in peptide synthesis and formic acid for the cleavage of the Boc group from the Boc-dipeptide.

13p. F. Alewood,J. W. Perich, and R. B. Johns, Aust. J. Chem. 37, 429 (1984). 14p. F. Alewood,J. W. Perich, ld R. B. Johns, TetrahedronLett. 25, 987 0984).

Synthesis of O-phosphoserine- and O-phosphothreonine-containing peptides.

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