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The simple model substrate AC-L-Abu(PO3H2)- NHMe was prepared by the use of the protected 4( diethylphosphono ) butanoic acid derivative Boc -Abu(PO3Et2)-OH in the Boc mode of solution phase peptide synthesis. The protected peptide model Ac-Abu(P03Et2)- NHMe was prepared by initial reaction of the isobutoxycarbonyl mixed anhydride of Boc-Abu(PO3Et2)-OH with N-methylamine followed by cleavage of the Boc group from Boc -Abu(PO3Et2)- NHMe with 4 M HCl/dioxan and N- acetylation of H-Abu(PO3Et2)-NHMe.HCl with the isobutoxycarbonyl mixed anhydride of acetic acid. Cleavage of the phosphonate ethyl groups was effected with 33% hydrogen bromidelacetic acid or 10% bromotrimethylsilane/acetonitrile to give AC-L-Abu(PO3H2)-NHMe in near-quantitative yield.

A solid phase method is described for the synthesis of O- phosphoseryl-containing peptides by the use of polystyrene resin (Merrifield) as the peptide support and protected Boc-Ser(PO3R2)-OH derivatives for the incorporation of the phosphorylated seryl residue. The viability of this solid phase approach was demonstrated by the synthesis of HBr.H-Glu-Ser (PO3Et2)-Leu-OH in high yield by the use of Bo -Ser(PO3Et2)-OH in peptide synthesis and subsequent use of HBr/CF3CO2H for cleavage of the Ser(PO3Et2)-containing tripeptide from the resin support. Similarly, the dipeptide, CF3CO2H.H-Ser(P)- Leu -OH, was prepared in high yield by using Boc -Ser(PO3But2)-OH in peptide synthesis followed by the one-step deprotection of the Ser(PO3But2)- dipeptide resin by treatment with HBr/CF3CO2H (90 min). Alternatively, the O-phosphoseryl tripeptide , CF3CO2H.H-Glu-Ser(P)- Leu -OH was prepared by using either Ppoc -Ser(PO3Bzl2)-OH or Boc-Ser(PO3Ph2)-OH in peptide synthesis. The one-step deprotection of the Ser(PO3Bzl2)-containing tripeptide and cleavage of the peptide from the resin support was effected by high-pressure hydrogenolysis (palladium acetate). In the case of phenyl phosphate protection, the Ser(PO3Ph2)-containing peptide was cleaved from the resin support by high-pressure hydrogenolysis (palladium acetate) followed by cleavage of the phenyl phosphate groups by platinum-mediated hydrogenolysis (1.0 equiv. PtO2/phenyl group) in 50% CF3CO2H/AcOH.

Benzyl phosphate groups were found to be sensitive to acid conditions, and a stability study with dibenzyl isobutyl phosphate under various acid conditions is described. While extensive acidolytic debenzylation of the dibenzyl phosphorotriester occurred on treatment with either 4 M hydrogen chloride/ dioxan or 50% trifluoroacetic acid/dichloromethane, only minor benzyl loss occurred with the use of formic acid or 1 M hydrogen chloride/acetic acid. Minimization of benzyl phosphate loss during the synthesis of a Ser(PO3Bzl2)-containing tripeptide was effected by the use of 98% formic acid (or 1 M hydrogen chloride/acetic acid) for the cleavage of the Boc group from Boc -Ser(PO3Bzl2)- Leu-OBzl . In an alternative procedure, the protected 2-phenylisopropyloxycarbonyl derivative, Ppoc -Ser(PO3Bzl2)-OH was prepared by an efficient four-step procedure and was used in a solution-phase peptide synthesis for the high yielding preparation of Boc-Glu ( OBzl )-Ser(PO3Bzl2)- Leu-OBzl . The protected Ser(PO3Bzl2) tripeptide was deprotected by palladium- catalysed hydrogenolysis in formic acid and gave Glu -Ser(P)-Leu in near-quantitative yield.

.The synthesis of five Z-Ser(PO3R2)-OH (R = phenyl, ethyl, methyl, benzyl, t-butyl) derivatives is described by a simple three-step synthetic procedure which features the use of either (A) diphenyl or dialkyl phosphorochloridate /pyridine or (B) dialkyl N,N-diethylphosphoramidite/1H-tetrazole-m-chloroperoxybenzoic acid for the phosphorylation of the serine hydroxy group. The benzyl phosphate derivative, Z-Ser(PO3Bzl2)-OH, was used in the benzyloxycarbonyl mode of peptide synthesis for the preparation of Z-Ser(PO3Bzl2)-Leu-OBzl and Z-Ser(PO3Bzl2)-Ser(PO3Bzl2)- OBzl which were deprotected by palladium- catalysed hydrogenolysis to give Ser(P)-Leu and Ser(P)-Ser(P) in high yields and high purity.

A new approach to the synthesis of Ser(PO3R2)-containing peptides by using five protected Boc-Ser(PO3R2)-OH (R=phenyl, ethyl, methyl, benzyl, t-butyl) derivatives is described. The five Boc-protected derivatives are prepared by a simple three-step synthetic procedure which features the use of either (A) diphenyl or dialkyl phosphorochloridate/pyridine, or (B) dialkyl N,N-diethylphosphoramidite/1H-tetrazole-m-chloroperoxybenzoic acid for the phosphorylation of the serine hydroxy group. The five derivatives were utilized in the Boc mode of peptide synthesis with the use of the mixed-anhydride coupling procedure, and led to the synthesis of the protected tripeptides, Boc-Glu(OBzl)-Ser(PO3R2)-Leu-OBzl, in high yield and purity. In peptide deprotection studies, the phenyl and benzyl phosphate protecting groups were readily removed from the protected Ser(PO3Ph2) and Ser(PO3Bzl2) tripeptides by hydrogenolysis and gave Glu-Ser(P)-Leu in near quantitative yield. The t-butyl phosphate groups were cleaved from the Ser(PO3But2) dipeptide by mild acidolysis and gave Ser(P)-Leu in quantitative yield. However, attempts to cleave the ethyl or methyl phosphate groups by acidolytic or silylitic treatment of the Ser(PO3Et2) and Ser(PO3Me2) tripeptides resulted in decomposition of the O-phosphoseryl residue.

The octapeptide, Ac-Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu-NHMe (I), was synthesized by the solution-phase method by using the mixed anhydride coupling procedure for the fragment condensation of the Nα-acetyl tripeptide, Ac-Glu(OBut)-Ser(But)-Leu-OH, with the pentapeptide N-methylamide hydrochloride, Cl.H2-Ser(But)-Ser(But)-Ser(But)-Glu(OBzl)-Glu(OBzl)-NHMe, followed by palladium-catalysed hydrogenolysis of Ac-Glu(OBut)-Ser(But)-Leu-Ser(But)-Ser(But)- Ser(But) Glu(OBzl)Glu(OBzl NHMe in trifluoroacetic acid. The synthesis of the two peptide fragments was accomplished in high yields and purity by using the repetitive excess mixed anhydride procedure and the isobutoxycarbonyl mixed anhydride of acetic acid for the rapid and high yielding N-acetylation of the tripeptide fragment. 13C n.m.r. spectroscopy was routinely used to monitor the efficiency of the coupling steps and to confirm the structure of octapeptide (1), signal assignments being possible for both the protected tri- and penta-peptides.

The chemotactic dipeptide for the slime-mould Polysphondylium violaceum, glorin (N- propionyl-y-L-glutamyl-L-ornithine-δ-lac ethyl ester) (8), was synthesized convergently in five steps with 41% overall yield, starting from L- ornithine hydrochloride (1) and L- glutamic acid γ-benzyl ester (4). -1H n.m.r. spectral analysis led to the conclusion that the propionamide and the γ-L- glutamyl amide bonds both undergo slow cis-trans isomerism in solution at room temperature. From a Karplus-type analysis of the values of the Cα,Cβ coupling constants at the lactam chiral carbon, it was concluded that the six-membered ring can adopt either a half-chair or half-boat conformation, but it is not clear which. Similar calculations for the glutamyl Ca,Cβ coupling constants were performed and, from these, significantly different relative populations for the three staggered conformations about the Glu-Ca,Cβ bond were suggested. Receptor binding studies showed that glorin did not bind significantly to any of the six receptors studied, including opioid, dopamine, central benzodiazepine, peripheral benzodiazepine, α1-adrenoceptor and α2-adrenoceptor sites.

The PTyr (O-phosphotyrosine) pentapeptide H-Asn-Glu-Tyr(PO3H2)-Thr-Ala-OH.HO2CCF3, which is a naturally occurring sequence from the autophosphorylated Rous sarcoma virus pp60V-SrC, was prepared in high yield by the use of Boc-Tyr(PO3Me2)-OH in the Boc mode of solution-phase peptide synthesis. The protected pentapeptide, Z-Asn-Glu(OBzl)-Tyr(PO3Me2)-Thr(Bzl)-Ala-OBzl, was deprotected by a two-stage procedure which involved initial palladium-catalysed hydrogenolysis followed by the removal of the phosphate methyl groups by the use of one of the following treatments: (A) 10% bromotrimethylsilane/acetonitrile, (B) 1 M bromotrimethylsilane/thioanisole in trifluoroacetic acid, or (C) trifluoromethanesulfonic acid/trifluoroacetic acid/dimethyl sulfide/m-cresol.

The protected phosphoserine synthon Nα-(t-butoxycarbonyl)-O-(dibenzylphosphono)-L-serine was prepared in three steps. Stability studies on the phosphorylating agent dibenzyl phosphorochloridate were performed by 31P n.m.r. spectroscopy.

The model phosphopeptide N-acetyl-O-phosphonoserine methylamide was synthesized in five steps.