Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe-TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe-TFA and H-Glu-Ser(P)-Ser(^P)-Glu-Glu-NHMe-TFA were prepared by the use of Boc-Ser(PO₃Ph₂)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO₃Ph₂)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH₂C1₂ used for removal of the Boc group from intermediate Boc-protected peptides.     

Further studies into the Boc/solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides

The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Are-Ala-Ser(P)-Thr-Ile-Glu-NH₂, was prepared by the use of Boc-Ser(PO₃Ph₂)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO₃Ph₂)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO₃Ph₂) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO₃Ph₂) residue. © Munksgaard 1994.     

Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection

The synthesis is of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO₃Me₂)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO₃ Me₂)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc-Tyr(PO₃^tBu₂)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P).     

Efficient Fmoc/solid-phase synthesis of Abu(P)-containing peptides using Fmoc-Abu(PO3Me2)-OH

The synthesis of the two 4-phosphono-2-aminobutanoyl-containing peptides, Leu-Arg-Arg-Val-Abu(P)-Leu-Gly-OH.CF₃CO₂H and Ile-Val-Pro-Asn-Abu(P)-Val-Glu-Glu-OH.CF₃CO₂H was accomplished by the use of Fmoc-Abu(PO₃Me₂)-OH in Fmoc solid-phase peptide synthesis. The protected phosphoamino acid, Fmoc-Abu(PO₃Me₂)-OH, was prepared from Boc-Asp-O'Bu in seven steps, the formation of the C—P linkage being effected by the treatment of Boc-Asa-O'Bu with dimethyl trimethylsilyl phosphite. Peptide synthesis was performed using Wang Resin as the polymer support with both peptides assembled by the use of PyBOP® for the coupling of Fmoc amino acids and 20%, piperidine for cleavage of the Fmoc group from the Fmoc-peptide after each coupling cycle. Cleavage of the peptide from the resin and peptide deprotection was accomplished by the treatment of the peptide-resin with 5%, thioanisole/TFA followed by cleavage of the methyl phosphonate group by 1 M bromotrimethylsilane/l M thioanisole in TFA.     

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates

A general synthetic method for the efficient preparation of Tyr(P) -containing peptides is described by the use of Fmoc-Tyr(PO₃¹Bu₂) -OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P) -Tyr(P) -OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8–13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using ³²P-radiolabeled [Tyr(P)]⁴-angiotensin II, both un-labeled synthetic [Tyr(P)]⁴-angiotensin II and Ser-Ser-Ser-Tyr(P) -Tyr(P) reduced the release of ³²P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]⁴-neurotensin 8–13 was ineffective as a competitive substrate and indicated that this particular Tyr(P) -containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P) -Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P) -Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.     

Efficient solid phase synthesis of mixed Thr(P)-, Ser(P)- and Tyr(P)-containing phosphopeptides by “global”“phosphite-triester” phosphorylation*

The synthesis of the mixed Thr(P), Tyr(P)-containing peptide, Ala-Thr(P)-Tyr(P)-Ser-Ala, was accomplished by “phosphite-triester” phosphorylation of the resin-bound Thr Tyr-containing peptide using di-t-butyl N,N-diethylphosphoramidite as the phosphitylation reagent. The pentapeptide-resin was assembled by Fmoc/ solid-phase peptide synthesis with the use of PyBOP® as coupling reagent and the hydroxy-amino acids incorporated as side-chain free Fmoc-Tyr-OH and Fmoc-Thr-OH. “Global” bis-phosphorylation of the peptide-resin was accomplished by treatment with di-t-butyl N,N-diethylphosphoramidite/1H-tetrazole followed by m-chloroperoxybenzoic acid oxidation of the intermediate di-t-butylphosphite triester. Simultaneous peptide-resin cleavage and peptide deprotection was effected by treatment of the peptide-resin with 5% anisole/TFA and gave the Thr(P) Tyr(P)-containing phosphopeptide in high yield and purity. In addition, the tyrosyl residue was found to be phosphitylated in preference to the threonyl residue since the phosphitylation of the pentapeptide-resin using only 1.1 equiv. of di-t-butyl N,N-diethylphosphoramidite gave Ala-Thr-Tyr(P)-Ser-Ala as the major product and both Ala-Thr(P)-Tyr(P)-Ser-Ala and Ala-Thr-Tyr-Scr-Ala as minor products.     

Synthesis of phosphotyrosine-containing peptides by the solid-phase method

A practical and convenient procedure for making phosphotyrosine-containing peptides by the solid-phase method was developed. Phosphotyrosine was incorporated via Boc-Tyr(PO₃Bzl₂)-OH. The completed peptide was cleaved from the solid support by treatment with 1 M TMSBr-thioanisole-TFA. By gel-phase ³¹P-NMR spectroscopy we found that one of the benzyl protecting groups on phosphate was completely removed by two consecutive runs of Boc deprotection with 50% TFA-DCM. However, the other benzyl group remained intact throughout the synthesis (35 cycles).     

Lability of N-alkylated peptides towards TFA cleavage

Trifluoroacetic acid (TFA) is a common reagent in both solid-phase and solution peptide synthesis. It is used for the deprotection and/or cleavage of the synthesized peptide from the resin. The use of TFA under these standardized conditions is thought to be sufficiently mild, thereby preventing degradation of the desired product. However, peptides of the general structure R¹-(N-alkyl X₁)-X₂-R² are hydrolyzed by standard TFA solid-phase peptide synthesis (SPPS) cleavage/deprotection conditions providing fragments R¹-(N-alkyl X₁)-OH and H-X₂-R². The fragmentation is observed during a TFA cleavage both from the resin and in solution. The hydrolysis is proposed to proceed via an oxazolone-like intermediate in which equilibration of the chiral center of the N-alkylated residue occurs. This mechanism is supported by H/D exchange as observed by MS and NMR in conjunction with HPLC. © Munksgaard 1996.     

Synthesis of O-phosphonotyrosyl peptides

A general method for the synthesis of O-phosphonotyrosyl peptides using solid phase methodology is described. Protected O-phosphonotyrosine derivatives with the general structure Boc-Tyr(R₂PO₃)-OH (R = methyl, ethyl or benzyl) were prepared as potential synthons for the introduction of O-phosphonotyrosine residues into peptide sequences. Using ³¹P n.m.r. spectroscopy, the alkyl phosphate protecting groups (R = methyl or ethyl) were shown to be stable to the coupling, deprotection and neutralization cycles of the Merrifield method of solid phase peptide synthesis. Facile removal of the methyl phosphate protecting groups from the O-phosphonotyrosyl peptide analogue Ac-Tyr(Me₂PO₃)-NHMe was demonstrated using 45% HBr/acetic acid. The O-phosphonotyrosyl heptapeptide H-Leu-Arg-Arg-Ala-PTyr-Leu-Gly-OH was subsequently prepared using solid phase methodology via incorporation of N²-tert-butyloxycarbonyl-O-dimethylphosphonotyrosine.     

Solid-phase synthesis of cionin,a protochordate-derived octapeptide related to the gastrin/cholecystokinin family of peptides,and its mono-tyrosine-sulfate-containing derivatives

Cionin, a protochordate-derived octapeptide amide related to the gastrin/cholecystokinin family of peptides, contains two consecutive tyrosine sulfate residues. In order to gain insight into the role of the respective tyrosine sulfate residue in biological activity, cionin and its derivatives in which one of the two tyrosine sulfate residues was replaced by tyrosine, were prepared by two Fmoc-based solid-phase approaches. In approach ( 1 ) Fmoc-Tyr(SO₃Na)-OH was employed as a building block to assemble the Tyr(SO₃Na)-containing peptide-resin, and a global deprotection cleavage was conducted with 90% aqueous TFA in the presence of m-cresol and 2-methylindole at 4°C. In approach ( 2 ) the Tyr(Msib) [Msib =p-(methylsulfinyl)benzyl] derivative was used for the peptide-chain assembly to achieve sulfation on the selective Tyr residue. Partially protected peptide with the Msib Msz protecting groups [Msz =p-(methylsulfinyl)benzyloxycarbonyl], obtained after peptide-resin cleavage, was treated with DMF-SO₃ complex in the presence of ethanedithiol to achieve the sulfation of free Tyr residue and the reduction of the Msib/Msz groups to TFA-labile Mtb/Mtz groups [Mtb =p-(methyithio)benzyl, Mtz =p-(methylthio)benzyloxycarbonyl]. Final deprotection of the Mtb/ Mtz groups with 90° aqueous TFA in the presence of m-cresol and 2-methylindole gave the desired cionin derivative, which contains the tyrosine sulfate residue at the selective position. Yields obtained with approach ( 2 ) were considerably higher than those obtained with approach ( 1 ). Cionin and mono-Tyr(SO³H)-containing derivatives were assayed on exocrine pancreas in dogs.     

Synthesis of oligophosphoseryl sequences occurring in casein

The protected oligophosphoseryl peptides from bovine caseins, Z-Xxx-{Ser[PO(OPh)₂]}₃-Glu(OBzl)-OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc-Ser[PO(OPh)₂]-OH as acylating carboxyl component and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO₂) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)₂]₃-Glu(OBzl)-OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2-Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc-Ser[PO(OTc)₂]-OBzl, Z-Ser[PO(OTc)₂]-OBzl and Boc-Glu(OBzl)-Ser[PO(OTc)₂]-OBzl were synthesized in high yields using bis-(2,2,2-trichloroethyl) phosphochloridate.     

Synthesis of phosphotyrosine-containing peptides using bis-(2, 2, 2-trichloro)ethyl groups for phosphate protection

Suitability of bis-(2, 2, 2-trichloro)ethyl (Tc) groups for protection of phosphate moiety in Boc-mode synthesis of phosphotyrosine peptides is demonstrated Boc-Tyr(PO³ Tc²)-OH and Fmoc-Tyr(PO³Tc²)-OH were prepared by acylating H-Tyr(PO³Tc²)-OH with (Boc)²O and Fmoc-ONSu, respectively. Phosphorus introduction was achieved by phosphorylating Boc-Tyr-OBzl with Tc phosphochloride. The Tc-phosphorus protector was found to be incompatible with the Fmoc group because the conditions of Fmoc removal (piperidine treatment) caused dephosphorylation. Complete NMR spectral assignments in the described compounds is presented. (Contribution No. 2398 from the Centre for Food and Animal Research).     

A facile method for preparation of t-butyloxycarbonylamino acid p-nitroanilides

A series of p-nitroanilides of t-butyloxycarbonylamino acids, including Boc-Trp, Boc-Asn, Boc-Gln, Boc-Ser(Bu′), Boc-Thr(Bu′), Boc-Asp(OBu′), Boc-Lys(TFA), and Boc-His(Boc), were prepared conveniently by the mixed anhydride method using 2,2-dimethylpropanoic chloride (pivaloyl chloride). The products were obtained in 40-60% yields after purification by column chromatography on Sephadex LH-20. p-Nitroanilide of histidine was purified after deprotection of Boc-His(Boc)-pNA and obtained as H-His-pNA-2HCl.     

Use of 10% sulfuric acid/dioxane for removal of N-α-tertiary-butyloxycarbonyl group during solid phase peptide synthesis

The hazards and high costs associated with the use of trifluoroacetic acid (TFA) in the removal of the N-α-tertiary-butyloxycarbonyl (Boc) group during solid phase peptide synthesis prompted an examination of alternative acidolytic reagents for α-amino group deprotection. N-α-Boc-glycine and N-α-Boc-isoleucine resins as well as an N-α-Boc-peptide resin were used to test the lability to various deprotection mixtures of both the N-α-Boc resin group as well as the amino acid or peptide-O benzyl ester resin linkage. Of the combinations tried, several were found, including 10% H₂SO₄/dioxane, which gave results roughly comparable to 50% TFA/CH₂Cl₂. Several peptides, 5–10 amino acid residues in length, have been successfully synthesized using the 10% H₂SO₄/dioxane mixture and were found to be comparable in purity to the same peptides prepared using the standard TFA/CH₂Cl₂ method of N-α-Boc removal. Thus, for the peptides examined, 10% H₂SO₄dioxane was found to be an inexpensive, safe, and practical alternative reagent to the more costly and hazardous 50% TFA/CH₂Cl₂ commonly used in the deprotection step of solid phase peptide synthesis.     

Pegylated peptides II

General procedures are presented for the site-specific pegylation of peptides at the NH₂-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH₂, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH₂-Terminal pegylation was carried out by coupling PEG-CH₂CO-Nle-OH to the free NH₂-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH₂CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH₂CH₂-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH₂CO-Nle-OH or H-Nle-NH-(CH₂)₂-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH₂CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, ¹H-NMR spectroscopy and laser desorption mass spectrometry.     

Further studies on the use of 2,2,2-trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis

Boc-Ser(PO₃Tc₂)-OH, Z-Ser(PO₃Tc₂)-OH and Fmoc-Ser(PO₃Tc₂)-OH, derivatives useful for peptide synthesis, have been obtained in high yields by acylation of H-Ser(PO₃Tc₂)-OHCF₃COOH. The latter was obtained from Boc- or Z-Ser(PO₃Tc₂)-OBzl by simultaneous removal of the amino- and carboxy-protecting groups by Pd-catalyzed hydrogenolysis in acetic acid-trifiuoroacetic acid solution. Removal of the Tc-protecting group was efficiently achieved by hydrogenolysis in aqueous ethanol.     

Side reaction during the deprotection of (S-acetamidomethyl)cysteine in a peptide with a high serine and threonine content

The acetamidomethyl (Acm) group is a widely used protecting group for the thiol of cysteine during the SPPS process. We prepared the amino terminal loop of the snake α-neurotoxin, [Cys³, Cys²³, Ser¹⁷(1–24) amide, from the linear peptide [Cys(Acm)^3,23,Ser¹⁷](1–24) amide obtained by SPPS. Three different methods of deprotection of Cys(Acm) and disulfide bond formation were used: iodine, thallium(III) trifluoroacetate and mercuric acetate/potassium ferricyanide. The iodine method failed to yield the expected peptide, and gave instead the mono-iodinated tyrosine analog. The disulfide cyclized peptide obtained by thallium(III) or Hg(II) procedures displayed a MW value observed by mass spectrometry that was higher than the calculated value. The difference (MW_obs - MW_calc) corresponded to a multiple of the Acm moiety, which is shifted intra-and/or intermoleculary. Furthermore, we observed, in addition to the Acm shift in the disulfide cyclized decapeptide with a high Ser and Thr content (model peptide II), the dimerization phenomenon in the Tl(TFA)₃ process. Therefore we conclude that a side reaction, a S–O(Ser,Thr) Acm shift, occured during the Cys(Acm) deprotection. This shift was supported by the demonstration of Ser(O-Acm) formation in the reaction of Boc-(L)-Cys(Acm) with Tl(TFA)₃ in the presence of an equimolar amount of (L)Ser. We report here the efficiency of a trivalent alcohol, glycerol, as scavenger in the both Tl(TFA)₃ and mercuric/ferricyanide methods, in an attempt to circumvent this side-reaction during the disulfide bond formation step starting from a bis-Cys(Acm) peptide with a high Ser and Thr content, such as the N-terminal loop of neurotoxin, model peptide II or a similar peptide.     

Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5–1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr¹-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM–TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions. © Munksgaard 1996.     

Preparations of Boc-Cys(S-Pyr)-OH and Z-Cys(S-Pyr)-OH and their applications in orthogonal coupling of unprotected peptide segments

Boc-Cys(S-Pyr)-OH and Z-Cys(S-Pyr)-OH were prepared by addition of their cysteine derivatives to 3 equiv of 2,2′-dipyridyldisulfide in one portion. 2-Mercaptopyridine was removed by addition of 0.1 M CU(NO₃)₂ to the solution. Both derivatives are white solids and can be used to facilitate the formations of heterodisulfide bonds. Two methods of synthesizing peptides with N-terminal Cys(S-Pyr) were also provided. Two peptide thiocarboxylic acids H-Tyr-Ser-Ala-Glu-Leu-Val-SH and H-Tyr-Ser-Ala-Glu-Leu-Gly-SH were prepared on the thioester benzhydryl resin with the cleavage condition of 1.0 m TFMS A/TFA instead of HF. From the orthogonal couplings of these peptides with H-Cys(S-Pyr)-Tyr-Ser-Glu-Leu-Ala-NH₂, both intramolecular acyl transfers finished at pH 7 at about 15 to 20 min. The intermediate acyl disulfide peptide was collected by high-performance liquid chromatography and identified by liquid chromatography-mass spectrometry.     

Chemical synthesis of o-thiophosphotyrosyl peptides

The synthon for O-thiophosphotyrosine, Fmoc-Tyr[PS(OBzl)₂]-OH ( 1c ), was prepared in 63%;, yield from Fmoc-Tyr-OH by first transient protection as the ^tBuMe₂Si-ester and phosphinylation with (BzlO)₂PNⁱPr₂/ tetrazole followed by oxidation of P(III) to P(V) vith S₈x in CS₂. Building block 1c was incorporated in the Fmoc solid-phase synthesis of two O-thiophosphotyrosine-containing peptides H-Thr-Glu-Pro-Gln-Tyr(PS)-Gln-Pro-Gly-Glu-OH ( 2 ) and H-Thr-Arg-Asp-Ile-Tyr(PS)-Glu-Thr-Asp-Phe-Phe-Arg-Lys-OH ( 3 ), corresponding to sequences of the p60^src (523–531) protein and an insulin receptor ( IR ) (1142–1153) analogue, respectively. An alternative approach of synthesis, the global phosphorylation of a resin-bound peptide, also proved useful. Thus, the free tyrosyl side-chain containing-peptide IR (1142–1153) on support was phosphinylated with the above phosphoramidite reagent followed by oxidation with either S₈/CS₂ or tetraethylthiuram disulfide/CH₃CN solutions. Deprotection and peptide-resin cleavage was performed with a TFA/thiophenol (H₂O) mixture. Crude peptides 2 and 3 were stable to the acidolytic deprotection. Preparative RP(C₁₈)HPLC was initially performed using 0.1% TFA(aq) EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosphopeptide in solution. Stability measurements both as a function of time and pH. further confirmed this initial finding. Purifications performed at intermediate pH using a triethylammonium acetatc (pH 7.5) CH₃CN solvent system overcame this problem.