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 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.     

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 solution phase synthesis and use of multiple O-phosphothreonyl-containing peptides for calcium phosphate binding studies

The protected phosphothreonine derivative Boc-Thr(PO₃Ph₂)-OH was prepared in high yield from Boc-Thr-OH by a simple three-step procedure which involved 4-nitrobenzylcarboxyl protection, either phosphorotriester (diphenyl phosphorochloridate) or “phosphite-triester” (diphenyl N.N-diethylphosphoramidite) phosphorylation of the thrconine hydroxyl group of Boc-Thr-ONb followed by hydrogenolytic carboxyl deprotection. The three Thr(P)-containing peptides, H-Thr(P)-Glu-Glu-NHMe.TFA, H-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA and H-Thr(P)-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA, were prepared in high yield by the use of Boc-Thr(PO₃Ph₂)-OH in the Boc mode of peptide synthesis (mixed anhydride method) followed by platinum-mediated hydrogenolytic deprotection of the Thr(PO₃3Ph₂)-containing peptides. The use of the phosphopeptides in calcium phosphate binding studies showed that the triple Thr(P)-cluster was a basic structural requirement, since only the pentapeptide was able to bind calcium phosphate efficiently.     

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.     

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.     

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 peptides containing the non-hydrolysable analog of (O)phosphotyrosine,P(CH2PO3H2)Phe

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH₂PO₃H₂) Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the β₂ adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH₂PO₃H₂)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc- p (CH₂PO₃Et₂)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (~60% total yield) and further deprotections provided both the p(CH₂PO₃H₂)Phe and p(CH₂PO₃HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenyl-thiocarbamyl form (PTC).     

INOSITOL-1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] AND INS(1,4,5)P3 RECEPTOR CONCENTRATIONS IN HEART TISSUES

1. The isolation and culture of neonatal cardiomyocytes causes changes in the metabolism of inositol(1,4,5) trisphosphate (Ins(1,4,5)P₃) from primarily dephosphorylation in the intact tissue to a combination of phosphorylation and dephosphorylation in the cultured cells (Woodcock et al. 1992). 2. The content of Ins(1,4,5)P₃ was found to be higher in intact heart tissue than in the isolated neonatal cells (10.9 ± 1.3 and 0.5 ± 0.1 pmol/mg tissue, mean ± s.e.m., n= 4, P<0.002, respectively). 3. Despite this difference, Ins(1,4,5)P₃ receptors in intact tissue and in isolated cells were not different in terms of affinity (8.0 ± 1.7 and 10.9 ± 1.6 nmol/L, n= 3, respectively) or concentration (143.3 ± 20.5 and 91.2 ± 16.0 fmol/mg protein, n = 3, respectively). 4. Thus, while there appears to be a relationship between the tissue content of Ins(1,4,5)P₃ and its metabolism, no relationship to the properties of Ins(1,4,5)P₃ receptors could be demonstrated.     

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,conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(Bu^t)-Ala-Thr(Bu^t)-Thr(Bu^t)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO₂)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO₃H group in the sulfated-tyrosine by the non-hydrolyzable -CH₂SO₃H group was the first described modification of the sulfate ester that does not affect CCK₈ activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH₂SO₃Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH₂SO₃Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK₈ by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK₈ analogues containing the l - or the d -Phe(p-CH₂SO₃Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH₂SO₃Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure l isomer was used. Both CCK₈ analogues displayed high affinities for peripheral and central receptors (KI ～ 1 nm ) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The ?stabilized-CCK₈ peptide”, easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH₂SO₃Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Synthesis of N-tert.-butoxycarbonyl(α-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide α-carboxamides

N-tert.-butoxycarbonyl-aminomethyl(α-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide α-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-(p-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH₂Cl₂ (< 1% loss after 10h). Upon treatment for 30min with HF:anisole(9:1) at 0°, 92% cleavage of glycinamide from Glyhandle-resin was obtained. In a test synthesis of a peptide α-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminornethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.     

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).     

Acidolytic cleavage of tris(alkoxy)benzylamide (PAL) “internal reference” amino acyl (IRAA) anchoring linkages: validation of accepted procedures in solid-phase peptide synthesis (SPPS)

Under the normal conditions of acidolytic cleavage/deprotection of tris(a1koxy)benzylamide (PAL) anchoring linkages in Fmoc solid-phase peptide synthesis (SPPS), product release occurs by a straightforward single-step pathway. A recently reported cleavage of the NH-VH bond of an amino acyl residue adjacent to PAL [see Int. J. Peptide Protein Res. 38 , 146–153 (1991)] could not be confirmed in novel experiments incorporating a double “internal reference” amino acid (IRAA) design. The results of the present work revalidate the widely accepted application of IRAAs to monitor yields in SPPS, and confirm the reliability of PAL methodology for the preparation of C-terminal peptide amides.     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH₂及其类似物Phe-Pro-Arg-Phe-NH₂,并观察了此二种肽对大鼠血压和心率的影响。     

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.     

Synthesis and photolytic cleavage of bovine insulin B22–30 on a nitrobenzoylglycyl-poly (ethylene glycol) support

The synthesis of the C -terminal nonapeptide of bovine insulin B-chain is desscribed. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) (Mr = 15 000) was used as soluble support. The C -terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.     

Synthesis and properties of cyclic peptides containing the activation site of plasminogen

The activation of plasminogen results from proteolytic cleavage of the Arg₅₆₀-Val₅₆₁ bond by plasminogen activators (Sottrup-Jensen et al. PNAS (1975) 72, 2577). This region of the zymogen occurs in a small disulfide loop that must restrict the conformation around this bond. The nonapeptide sequence NH₂-Cys-Pro-Gly-Arg-Val-Val-Gly-Gly-Cys-NH₂ of plasminogen containing the activator sensitive arginyl valine bond was synthesized by carbodiimide coupling of Boc-Cys-Pro-Gly-OH(S-4-methylbenzyl) to NH₂-Arg(NO₂)-Val-Val-Gly-Gly-Cys-NH₂(S-4-methylbenz(S-4-methylbenzyl), followed by HF treatment and K₃Fe(CN)₆ oxidation to form a disulfide bond. Purified peptide was not a substrate for urokinase (UK) or plasminogen activator (PA) but possessed a slightly inhibitory activity towards PA. Addition of a lysine to the N-terminus of the nonapeptide yielded a decapeptide sequence of plasminogen that was a better substrate for UK but not for PA. The decapeptide inhibits PA slightly but not UK. These results suggest that active site geometry for PA must be more restrictive than that of UK and that other regions may be involved in the productive interactions with the activators inducing a better fit of the cyclic peptide loop.     

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO₃(Bzl)₂)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifu-gation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the α-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. © Munksgaard 1996.