John W. Perich

Physicochemical Characterization of Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

A new, convenient and efficient general procedure for the conversion of alcohols into their dibenzyl phosphorotriesters using N,N-diethyl dibenzyl phosphoramidite

Abstract A general procedure is described for the near-quantitative preparation of alkyl dibenzyl phosphates by treating primary, secondary or tertiary alcohols with N,N-diethyl dibenzyl phosphoramidite/1H,5-methyltetrazole followed by mild oxidation of the resultant alkyl dibenzyl phosphites with 3-chloroperoxybenzoic acid.

Di-t-butyl N, N-diethylphosphoramidite and dibenzyl N, N-diethylphosphoramidite. Highly reactive reagents for the ‘phosphite-triester’ phosphorylation of serine-containing peptides

Abstract Di- t -butyl N , N -diethylphosphoramidite and dibenzyl N , N -diethylphosphoramidite were found to be highly reactive reagents for the efficient ‘phosphite-triester’ phosphorylation of protected serine derivatives (Boc-Ser-ONBzl), protected serine-containing peptides and resin-bound protected serine-containing peptides.

A synthetic β‐casein phosphopeptide and analogues as model substrates for casein kinase‐1, a ubiquitous, phosphate directed protein kinase

The phosphopeptide Ser (P)‐Ser(P)‐Ser‐(P)‐Glu‐Glu‐Ser11‐Ilc‐Thr, reproducing the 17‐24 segment of β‐casein Λ1 including the seryl residue (Ser‐22) which is targeted by casein kinase‐1 was synthesized and used as model substrate for this enzyme. Its phosphorylation efficiency is actually higher than that of intact β‐casein (similar Vmax and 14 μM vs 50 μM Km ). Conversely the fully dephosphorylated peptide SSSEESIT is not affected by CK‐1 to any detectable extent and its glutamyl derivative EEEEESIT displays a more than 50‐fold higher Km and a 5‐fold lower Vmax as compared to the parent phosphopeptide. The relevance of the individual phosphoseryl residues has been assessed by comparing the phosphorylation efficiencies of the phosphopeptides EESpEESIT, ESpEEESIT and SpEEEESIT; while the first is a substrate almost as good as the tris Ser (P)‐peptide (Km =62 μM), and the third one is almost as poor as EEEEESIT (Km =1.55mM), ESpEEESIT displays a intermediate efficiency (Km =277 μM). These data in conjunction with the finding that the phosphopentapeptide Ser(P)‐Ser(P)‐Ser‐(P)‐Ser‐Ser(P), but neither Ser(P)‐Ser(P)‐Ser‐Ser(P) nor Ser‐Ser(P)‐Ser(P)‐Glu‐Glu and Ser‐Ala‐Ala‐Ser(P)‐Ser(P), is readily phosphorylated by CK‐1, support the concept that CK‐1 is a phosphate directed protein kinase recognizing the Ser(P)‐X‐X‐Ser‐X and, less efficiently, the Ser(P)‐X‐X‐X‐Ser‐X motifs.

Fmoc-polyamide solid phase synthesis of an O-phosphotyrosine-containing tridecapeptide

The incorporation of O-phosphotyrosine into synthetic peptides using Fmoc-Tyr(PO3Me2)-OH in the Fmoc-polyamide procedure is described with the preparation of the model PTyr-tridecapeptide sequence H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-PTyr-Thr-Ala-Arg-Gln-Gly-OH.

Hydroxyapatite mineralization and demineralization in the presence of synthetic phosphorylated pentapeptides

A constant composition (CC) method was used to compare the influence of statherin-like N-terminal 5-residue fragments having different amino acids in the terminal position on hydroxyapatite (HAP) growth and dissolution. The CC experiments were done in solutions containing 4.00 x 10(-4) mol/l calcium and 2.40 x 10(-4) mol/l phosphate. The solutions used in the crystallization studies were supersaturated only with respect to HAP (pH = 7.40, sigma HAP = 3.60). The CC dissolution studies were done in solutions undersaturated with respect to HAP (pH = 6.00; sigma HAP = -0.39). The HAP mineralization and demineralization processes were markedly inhibited by the negatively charged pentapeptides. Those containing a phosphorylated terminal residue inhibited dissolution to a greater extent than the native statherin fragment having aspartate as the N-terminal residue. Strong dependencies of the degree of inhibition of growth and dissolution reaction rates on the extents of reaction were noted. As the reactions proceeded, the rate inhibition decreased in the case of crystal growth and increased for dissolution.

The facile synthesis of ala-glu-tyr(P)-ser-ala by ‘Global’ di-t-butylN,N-diethylphosphoramidite ‘Phosphite-triester’ phosphorylation of a resin-bound peptide

The O-phosphotyrosyl-containing peptide, Ala-Glu-Tyr(P)-Ser-Ala, was prepared by a methodology which featured ‘phosphite-triester’ phosphorylation of a resin-bound Tyr-containing peptide. The peptide-resin was assembled by Fmoc/solid phase peptide synthesis using HBTU as the coupling reagent and the tyrosyl residue incorporated as Fmoc-Tyr-OH. The peptide-resin was phosphorylated using di-t-butylN,N-diethylphosphonamidite/ 1H-tetrazole followed by m-chloroperoxybenzoic acid oxidation. Simultaneous peptide-resin cleavage and peptide deprotection was effected by 5% anisole/TFA to give the crude Tyr(P)-peptide in high yield and in >95% purity.

The efficient synthesis of a o-phosphotyrosine-containing peptide using modern deprotection methods

Abstract The O-phosphotyrosine-containing peptide Pro- PT yr-Val was prepared in high yield by the incorporation of Boc-Tyr(PO 3 Me 2 )-OH in peptide synthesis and the use of TFMSA/TFA/DMS/ m -cresol or 1M TMSBr/TFA/ m -cresol, for the final peptide deprotection.

Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase

The motif Ser‐Ser‐Ser‐Glu‐Glu is readily phosphorylated by casein kinase‐2 (CK‐2), a growth‐related protein kinase whose consensus sequence is Ser(Thr)‐Xaa‐Xaa‐Glu(Asp) [(1990) Biochim. Biophys. Acta. 1054, 267–283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK‐2 phosphorylation, the phosphotyrosyl peptide Ser‐Ser‐Ser‐TyrP‐TyrP actually being a substrate more efficient than Ser‐Ser‐Ser‐Glu‐Glu itself both in terms of K m (0.69 vs 2.43 mM) and V???. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK‐2. While Ser‐Ser‐Ser‐TyrP‐TyrP is better than Ser‐Ser‐Ser‐SerP‐SerP, which in turn is better than Ser‐Ser‐Ser‐Glu‐Glu, Ser‐Ser‐Ser‐ThrP‐ThrP is a less efficient substrate than Ser‐Ser‐Ser‐Glu‐Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK‐2 appears to be TyrP>SerP>ThrP.

Synthesis of phosphopeptides by the Multipin method: Evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH

The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiesterprotected derivatives, Fmoc-Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu-Glu-Asp-Pro-Ala-NH2 (Xxx=Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9)DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx (PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the α-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr-His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO3Bzl,H)-OH.