Download Synthesis of Phosphopeptides Containing O

CHAPTER9
Synthesis of Phosphopeptides
Containing
O-Phosphoserine
and 0-Phosphothreonine
Anatol
Arendt
and Paul
A Hargrave
1. Introduction
Phosphorylation and dephosphorylation of proteins represents one of
the most widespread and important reactions in the regulation of cellular
processes. Specific serine, threonine, or tyrosine residues in substrate
proteins become phosphorylated by the action of protein kinases that
catalyze the transfer of phosphate from high-energy nucleoside triphosphate. Major proteins in bones, teeth, eggs, and milk are highly phosphorylated. Preparation of phosphopeptides related to sequences of
phosphoproteins is important for the study of their properties. Enzymatic
methods for the synthesis of phosphopeptides can be useful, but are limited to very specific peptide sequences.
Chemical phosphorylation was reviewed by A. W. Frank in 1984 (I),
but at that time, only phospho-amino acids and phosphodipeptides were
prepared. In the same year, Alewood et al. (2) introduced the use of the
dibenzyl-blocking group to protect phosphoserine during peptide synthesis. Unfortunately, the dibenzyl group proved to be insufficiently
stable during typical conditions of solid-phase peptide synthesis (3).
Arendt and Hargrave (4) used the more stable diphenyl protection of
phosphoserine and phosphothreonine that made possible synthesis of
phosphopeptides by solid phase. Diphenyl triesters were deprotected by
E&ted
From Methods
by M. W Pennmgton
In Molecular
Bology,
Vol 35 PeptIde Synthesis
Protocols
and B M Dunn Copyright
01994
Humana Press Inc , Totowa,
187
NJ
188
Arendt
and Hargrave
tetrabutylammonium fluoride (TBAF), and then the peptide was cleaved
from the resin using the standard HF method (see Chapter 4). Penta and
nonapeptides containing both phosphoserine and phosphothreonine were
prepared using this method. However, this procedure is sometimes difficult to control, and some nonphosphorylated peptides are obtained when
contact with TBAF is prolonged. Catalytical hydrogenolysis of diphenyl
phosphopeptides yielded good results and eliminated this inconvenience
(5). This also made it possible to deprotect the diphenyl phosphopeptides
following HF cleavage of the peptide from the resin. This modification
was used to synthesize two heptaphosphopeptides, PSer5 and PThr5
kemptide (6). Their identity was verified by enzymatic synthesis and
mass spectrometry (see Chapter 7, PAP). Four undecaphosphopeptides
(two with PSer and two with PThr) were synthesized by the same procedure and used as substratesfor casein kinase II (7). Seven different monophosphorylated peptides from the sequence of bovine rhodopsin have
been made and tested as substrates for rhodopsin kinase (8). Although
this method has been very useful, we have also found that it has some
limitations. When we synthesized the myelin peptide AcAlaSerAlaGln
LysArgPro(P)SerGlnArgSerLysTyrNHz and then removed the diphenylblocking group by catalytic hydrogenolysis, we observed that the tyrosine
phenyl group was also catalytically reduced (Arendt, A. unpublished
results). Also, the hydrogenolysis of longer (>15) and hydrophobic peptides is more difficult, slower and synthetic yields are much smaller than
with shorter ones. This chapter describes the synthetic method we have
developed for preparation of N-(t-butoxycarbonyl)-O-(diphenylphosphone)-L-serine and N-(t-butoxycarbonyl)-O-(diphenylphosphono)-Lthreonine, and the application of these substrates in solid-phase synthesis
of peptides. Additional methods for synthesis of phosphopeptides are
described elsewhere (6,9).
2. Materials
t-Butoxycarbonyl (Boc) serine and threonine may be obtained from
Bachem Bioscience Inc. (Philadelphia, PA); trifluoroacetic acid (TFA),
pyridine, and triethylamine (NEt,) from Fisher Scientific (Pittsburgh,
PA); hydrogen fluoride (HF) from Matheson (Secaucus, NJ); and benzyl
chloride, diphenyl chlorophosphate, dicyclohexylamine (DCHA), 10%
palladium on charcoal (PdK), and platinum oxide from Aldrich (Milwaukee, WI).
Synthesis
189
of Phosphopeptides
CICH,C,H,
Boc
NH- YH
COOH
CH(RDH
*
Boc
NH-
YH-COOCH,&H5
CH(R)OH
NEt,
CIPO(C
pyridine
H2
Boc
NH-
c?i-COOH
CH(R)OW(OCGH&
Boc
N
Pd/C
NH- YH-COOCH2C6H5
CH(R)OPO(OC&H&
R = H (Serine) or R = -CH, (Threomne)
Scheme 1.
3. Methods
3.1. Preparation
of the Protected Phosphoserine
and Phosphothreonine
Derivatives
This is a three-step synthesis involving:
1. Temporary protection of the reactive carboxyl group of BocSer or BocThr;
2. Phosphorylation of the free hydroxyl group; and
3. Selective deprotection of the carboxyl group (Scheme 1).
1.
2.
3.
4.
5.
6.
3.1.1. Synthesis of N-(t-Butoxycarbonyl)-O(Diphenylphosphono)-L-Serine
Benzyl Ester
Dissolve 100 g (01339 mol) of Boc-r.-SerOBzl in 750 mL of pyridine.
Cool the mixture in an ice bath, and add 100 g (0.373 mol) of diphenylphosphochloridate slowly with stirring.
Remove the cooling bath, and stir mixture overnight at room temperature.
Transfer the mixture to round-bottom flask, and evaporate to solid on rotatory evaporator.
Dtssolve the restdue in 800 mL of chloroform, wash 2x water, 2x 1M HCI,
2x water (2 L each), and evaporate organic layer on rotatory evaporator
(without drying).
Dissolve crystalline mass in 400 mL of warm ethyl acetate, and add equivalent amount of petroleum ether. Let crystallize.
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Arendt
and Hargrave
7. Remove crystals by filtration, wash them with a small amount of petroleum ether, and dry. You can obtain 166-170 g of product containing only
a small amount (~1%) of substrate.
8. For a better quaky product, recrystallize from rsopropanol(74 g/L). After
this, 159-163 g (89-91%) pure product can be obtained.
3.1.2. Synthesis of N-(t-Butoxycarbonyl)-O(DiphenylphosphonoJL-Serine
1. Suspend 15 g (28.4 mmol) of N-(t-butoxycarbonyl)-O-(diphenylphosphono)+serine benzyl ester and 850 mg 10% Pd/C tn the mtxture of
50 mL ethyl acetate and 50 mL of methanol m pressurrzed bottle.
2. Pressurize the bottle with hydrogen gas at 4.05 bar (4 atm), and mix the
residue magnetically When the mixture becomes homogeneous, the
hydrogenolysis is complete.
3. Filter the solution after -3 h of reaction.
4. Evaporate solvent in vucuo by rotatory evaporator.
5. Dissolve the resulting oil in 40 mL of warm isopropanol. Add water unttl
the solutron becomes slightly cloudy. Keep mixture m refrrgerator (at about
5°C) overnight.
6. Filter crystals and wash them with water. You can obtain 11.5-12 g (9296%) of crystalline product.
Hydrogenolysis can be performed with good results also under atmosphenc pressure, but more solvent must be used and the reaction is slower.
For details, see ref. 6. For longer storage, protected phosphoserine can be
converted to the stable dicyclohexylammonium
(DCHA) salt (6). BocSer[PO(OPh)J-OHSDCHA
is commercially available from AminoTech
Inc. (Canada).
3.1.3. Synthesis of N-(t-Butoxycarbonylj-L-Threonine
Benzyl Ester
1. Using conditions similar to that for the serine analog, from 38.9 g (0.177
mol) of BocThr, 43.4 mL (0.311 mol) triethylamine and 35.1 mL (0.305
mol) of benzyl chloride in 273 mL of ethyl acetate 47.7-50 g (87-91%) of
product can be obtained.
2. The syrup of BocThrOBzl crystallizes very slowly and can be used for the
next step without further purification. Ethyl ether/hexane can be used for
crystallization of product.
3.1.4. Synthesis of N-(t-Butoxycarbonyl)-O(DiphenylphosphonoJL-Threonine
Benzyl Ester
From 46.4 g (0.15 mol) BocThrOBzl and 44.3 g (0.165 mol) diphenylphosphochloridate in 332 mL of pyridine, using the procedure for the
Synthesis
of Phosphopeptides
191
serine analog, 75-77 g (92-95%) of noncrystalline, but cbromatographitally homogeneous product can be obtained.
3.1.5.
Synthesis
of DCHA Salt of N-(t-Butoxycarbonyl)-O(DiphenylphosphonoJL-Threonine
1. 15.4 g (28.4 mmol) of noncrystalline protected phosphothreonine compound from the previous synthesis can be hydrogenolyzed under similar
conditions to that for the serine analog. After evaporation of the solution,
the resulting heavy oil can be converted to a crystalline dicyclohexylammonium salt.
2. Dissolve product in 350 mL ethyl ether/hexane (1: 1 v/v), and add 5.6 mL
(28 mmol) of dicyclohexylamine.
3. Let the solution crystallize at 0°C (-12 h).
4. Product can be recrystalhzed from a mixture of isopropanol/ethyl ether.
Yield is 15-16 g (85-90%).
3.1.6. Conversion
of DCHA Salt of N-(t-ButoxycarbonyZ)-O(Diphenylphosphono)-L-Serine
or N-(t-Butoxycarbonyl-O(DiphenylphosphonoJL-Threonine
to Free Acid
Before peptide synthesis, the necessary amount of Boc-Ser[PO(OPh),]OH or Boc-Thr[PO(OPh)J-OH
is first liberated from the salt:
1. Suspend the calculated amount of DCHA salt of phosphoserme or
phosphothreonine derivative in ethyl acetate in a separatory funnel.
2. Wash this with 1M sulfuric acid solution and separate layers.
3. Wash the organic phase 2x with water, dry with MgS04, and evaporate
in vacua.
4. Dissolve in calculated volume of a suitable solvent and use for synthesis of
phosphopeptide.
3.2. Synthesis
of Phosphopeptides
Synthesis of phosphopeptides on solid phase can be performed manually, on an automated synthesizer, or on a multiple-peptide synthesizer,
using any procedure suitable for Boc amino acids. Incorporation of BocSer[OP(OPh)J and Boc-Thr[OP(OPh)z] into the peptide chain proceeds
in the same manner as nonphosphorylated amino acids.
3.3. Cleavage
of Phosphopeptides
from Resin
Peptides can be cleaved from the resin using the standard water-free
HF (see Chapter 4) or TFMSA (see Chapter 5) procedure. Phenyl protection of phosphoserine or phosphotbreonine is usually stable under these
conditions. In some cases, lO-20% of nonphosphorylated peptide and
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Arendt and Hargrave
monophenyl phosphopeptide is present (calculated from HPLC chromatogram; see Chapter 3, PAP). Product can be easily separated from
the substrate at this step because of the great difference in polarity of
these compounds.
3.4, Cleavage
of Phenyl
Group from Phosphopeptides
1, Dissolve peptide with phenyl-protected phospho group in a mixture of 40%
trifluoroacetic
acid in acetic acid (optimal concentration lo-40 mL/mrnol).
2. Add 482 mg of amorphous Pt02/phospho-group/mmol
(1 Eq).
3. Perform hydrogenolysis for 24 h at room temperature under 4.05 bar (4 atm)
of hydrogen pressure. Under atmospheric pressure, the process is too slow.
4. Evaporate the mixture to dryness under vacuum, suspend in water, and
remove the catalyst by filtration.
3.5. Phosphopeptide
Purification
Phosphopeptide can be purified by preparative HPLC on a reversephase or ion-exchange column using a mixture of volatile solvents. A
good method for separation of phosphopeptides from nonphosphorylated
impurities is affinity chromatography on Fe2+Chelex gel (iminodiacetic
acid epoxy activated Sepharose 6B, Sigma) (6).
Free phosphopeptide cannot be stored dry, even in the freezer, for a long
time. However, storing the DCHA salt of the phosphopeptide resulted in
no observable dephosphorylation for at least 1 yr of storage at -20°C.
Acknowledgments
This work was supported in part by research grants EY06225 and
EY06226 from the National Eye Institute of the National Institutes of
Health, an unrestricted departmental award from Research to Prevent
Blindness, Inc., and an International Human Frontier Science Program
award. P. A. H. is Francis N. Bullard Professor of Ophthalmology.
References
1. Frank, A. W. (1984) Synthesis and properties of N-, 0-, and S-phospho derivatives
of amino acids, peptides, and protems CRC Cnt. Rev. Biochem. 16,51-101.
2. Alewood, P. F., Pench, J. W., and Johns, R. B. (1984) Preparation of N-(tButoxycarbonyl)-0-(dibenzylphosphono)~L-serine.
Aust. J. Chem. 37,429433.
3. Alewood, P F , Pench, J. W., and Johns, R. B. (1984) A novel approach to
phosphopeptide synthesis-preparation
of Glu-PSer-Leu Tetrahedron Lett. 25,
987-990.
4. Arendt, A. and Hargrave, P. A. (1985) Solid-phase synthesis of phosphopeptides*
synthesis of phosphopeptides from the carboxyl-terminus of rhodopsin, in Pep-
Synthesis of Phosphopeptides
193
tides: Structure and Function (Deber, C. M., Hruby, V. I., and Kopple, K. P., eds.),
Pierce Chemical Co., Rockford, IL, pp. 237-240.
5. Perich, J. W., Valerio, R. M , and Johns, R. B. (1986) Solution-phase synthesis of
an 0-phosphoseryl-contaming peptide using phenyl phosphorotriester protection.
Tetrahedron Lett 27, 1373-1376
6 Arendt, A., Palczewski, K., Moore, W. T , Caprioli, R M., McDowell, J. H., and
Hargrave P. A. (1989) Synthesis of phosphopeptides containing 0-phosphoserine
or 0-phosphothreonine Int J. Peptide Protein Res. 33,468-476
7. Litchfield, D. W., Arendt, A., Lozeman, F. J., Krebs, E. G., Hargrave, P A , and
Palczewski K. (1990) Synthetic phosphopeptides are substrates for casein kinase
II. FEBS Lett. 261, 117-120.
8. Adamus, G , Arendt, A , Hargrave, P A., Heyduk, T , and Palczewski, K. (1993)
The kinetics of multi-phosphorylation of rhodopsin. Arch. Biochem. Biophys. 304,
443-447.
9. Perich, J. W. (1991) Synthesis of 0-Phosphoserine- and O-Phosphothreonme-conmining peptides. Methods in Enzymology 201,225-233.