Solid-phase synthesis of (tyrosyl-alanyl-glutamyl)n,by segment condensation

(Tyr-Ala-Glu)_n, n= 1–9, were synthesized by segment condensation using the Fmoc/tert-butyl protection strategy and solid-phase techniques. The C-terminal residue was coupled to the resin and the peptides were built out by adding Fmoc-Glu(O-r-Bu)-Tyr(t-Bu)-Ala-OH units. When the desired lengths were reached the peptides were capped with Fmoc-Tyr(t-Bu)-Ala-OH units. Fmoc-Tyr(t-Bu)-Ala-OH and Fmoc-Glu(O-t-Bu)-Tyr(t-Bu)-Ala-OH were synthesized in aqueous solution by the successive addition of N-hydroxysuccinimide esters of Fmoc-Tyr(t-Bu) and Fnioc-Glu(0-t-Bu) to the growing chain. Neither sequential amino acid addition or segment condensation techniques were successful on polystyrene supports. However, the segment condensations were highly successful on kieselguhr-supported polydimethylacrylamide based resins. (Tyr-Ala-Glu)_n, n= 1–9, were tested as inhibitors of the protein [yosine kinase, pp60C^c-src. Inhibition, as measured by IC₅₀ values, increased with increasing size of the peptide.     

N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles for efficient solid-phase peptide synthesis by segment condensation

N-Fmoc-protected(alpha-aminoacyl)benzotriazoles 1a-d readily afford chiral N-Fmoc-protected-alpha-dipeptides 2a-f (77-89%). Compounds 2a-f are further converted into N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles 3a-f (71% average yield). Under mild microwave irradiation, 3a-f are used in solid-phase peptide segment condensation syntheses to give tri-, tetra-, penta-, hexa-, and heptapeptides (20-68%).     

Synthesis of the protected tridecapeptide (56–68) of the VH domain of mouse myeloma immunoglobulin M603 and its reattachment to resin supports

A protected tridecapeptide, representing a new peptide corresponding to residues 56–68 of the V_H domain in the mouse M603 myeloma protein, has been prepared by solid phase peptide synthesis. The protected tridecapeptide was prepared using the photolabile 4-bromomethyl-(3-nitro)-benzamidomethyl-resin and the multidetachable 2-[4-bromomethyl)phenylacetoxy] propionyl-resin as solid supports. The synthetic protocol and protecting groups were the same for both syntheses. The protected tridecapeptide was removed photolytically from both supports and the sequence integrity was determined by preview analysis using the solid phase Edman degradation procedure. The protected tridecapeptide-OMPA was purified to homogeneity by DMF/H₂O precipitation and LH-60 chromatography. The purity of the protected peptide was further demonstrated by high pressure liquid chromatography on the free peptide after HF deprotection. The protected tridecapeptide was reattached to 4-bromomethyl-(3-nitro)-benzamidomethyl-resin to give the photolabile Boc-(protected) peptidyl-4 - oxymethyl - (3 - nitro) benzamidomethyl - resin in 25% yield. The protected tridecapeptide-oxymethylphenylacetic acid derivative was reattached to aminomethyl-resin to give Boc -(protected)peptidyl-2-[(4-oxymethyl)phenyl]acet-amidomethyl-resin in 45% yield and to 2-bromopropionyl-resin generating the multidetachable Boc - (protected)peptidyl - 2 - [(4 - oxymethyl) phenylacetoxy] propionyl-resin in 80% yield. The reactivity of these reattached peptides was demonstrated by the quantitative coupling of Boc-leucine to the protected peptide-resin. The advantages and disadvantages of the different resins with respect to solid phase fragment synthesis are discussed.     

Synthesis of tryptophyllin-7 and analogues

Syntheses by solution methods of tryptophyllin-7 (H-Val-Pro-Pro-Leu-Gly-Trp-Met-OH), a heptapeptide isolated from skin extracts of Phyllomedusa rhodei, and its methyl ester and amide are reported. Preliminary biological results in rats seem to indicate a growth promoting activity for both the acid and the ester derivative.     

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH₂Tyr¹,D-Ala²,Ala¹⁵]-GRF(1-29)-NH₂ ( 4 ), from the precursor, [Ala^15,29]-GRF(4-29)-OH ( 1 ). C-Terminal amidation of 1 to form [Ala¹⁵]-GRF(4-29)-NH₂ ( 2 ) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala²⁹-OH for Arg-NH₂. The target analog 4 was then obtained by acylation of segment 2 with desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) (R = CH₃CH₂ or 4-NO₂C₆H 4 CH₂) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3 . GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 °C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp³-Ala⁴ and Asp²⁵-Ile²⁶. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH₂Tyr-D-Ala-Asp(OH)-OR ( 3 ) [R = CH₃CH_2- ( 3a ), CH_3- ( 3b ), ClCH₂CH_2- ( 3c ), C₆H₅CH_2- ( 3d ), 4-No₂C₆H₄CH_2- ( 3e )] revealed that the rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4 , increase with increasing specificity (V_max/K_m) of GSE for the tripeptide ester. The specificity varies in the order 3e>3d>3c>3b>3a and appears to depend on an increase in the maximum turnover rate (V_max) with increasing basicity of R. This work demonstrates the coupling of a small peptide segment containing unnatural amino acids to the N-terminus of an intermediate-length polypeptide, without side-chain protection, by GSE, a potentially less costly and more stable alternative to the V8 protease.     

Synthesis of phosphopeptides containing O-phosphoserine or O-phosphothreonine

Peptides containing phosphoserine or phosphothreonine were synthesized by solid phase methods. Phosphoserine and phosphothreonine were incorporated into peptides using Boc-diphenylphosphono esters of serine and threonine and standard DCC/HOBt coupling. The phenylphosphoesters were not removed when the peptides were cleaved from the resin by HF or by trifluoromethane sulfonic acid, but were subsequently removed by catalytic hydrogenation. Phosphopeptides were purified by HPLC and by Fe⁺³-Chelex chromatography and their identity verified by mass spectrometry. Two peptides, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly and Leu-Arg-Arg-Ala-Thr(P)-Leu-Gly, were prepared by both enzymatic and chemical methods and had identical properties.     

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.     

Synthesis of partial sequences of ribosomal proteins and their derivatization with acryloyl residues

Peptide sequences B-X-B (B = Arg and/or Lys; X = Glu or Asp) are of considerable interest because of their possible interactions with ribosomal RNA. The syntheses of various protected peptides with the sequence Ala-B-X-B-Ala (X = Glu or Ala) and [Arg]_n-Pro (n = 1–3) are described. They are carried out in solution according to the conventional peptide synthesis method. The carbobenzoxy group is used for Nα-protection and the methyl group for the protection of the terminal carboxyl group. The side chains of Lys and Glu are respectively blocked with the tert.-butyloxycarbonyl and the tertiary butyl group and the guanidinic function of arginine with the NO₂-group. The intermediate peptides are purified either by extraction or by size exclusion chromatography. A specially adapted strategy of peptide synthesis allows removal of the amino terminal Cbo-group at the end of the synthesis and introduction of an acryloyl group. By radical copolymerization with cross-linking agents these acryloyl derivatives can be transferred into insoluble peptide gels suitable for affinity chromatography and for investigating peptide-oligonucleotide interactions. The isolation of the unprotected peptides Arg-Arg-Arg-Pro, Ala-Arg-Glu-Arg-Ala, Ala-Arg-Glu-Lys-Ala, Ala-Arg-Ala-Lys-Ala, Ala-Lys-Glu-Lys-Ala and their characterization using amino acid analysis, electrophoresis, and FAB-mass spectrometry is also reported.     

Fmoc 策略固相合成磷酰化肽研究进展

蛋白质的磷酰化和去磷酰化对多种生命活动的调节起着关键的作用,磷酰化肽是研究这些生命调节过程中一类非常重要的物质。自 20 世纪 40 年代人类首次成功地合成出磷酰化肽以来,磷酰化肽的研究就引起了化学家和生物学家的广泛关注。由于Fmoc 固相合成策略在多肽的合成中被普遍应用,因此,Fmoc 固相合成策略也已经成为目前磷酰化肽最主要的合成手段。该文对近年来采用 Fmoc 固相合成策略进行磷酰化肽合成的方法（包括整体磷酰化法和磷酰化单体合成法）进行了总结,并对各种合成方法的优缺点进行了讨论。     

Synthesis and conformation of the trimeric coiled-coil segment of laminin*

A disulfide linked 95-mer parallel hetero-trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32-mer), B1 (30-mer) and B2 (33-mer), were prepared by Boc-based solid-phase peptide synthesis involving a two-step trimethylsilyl bromide-thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air-oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero-dimer, B1-B2, one of the homo-dimers, B1-B1, and the trinier are 30 to 40% in the α-helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61°) than the hetero-dimer, B1-B2 (Tm = 36°).     

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).     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

Synthesis of homologous peptides using fragment condensation: analogs of an HIV proteinase substrate

Two protected peptides Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)OH and Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)-ProOH were synthesized on a resin substituted by 9-(hydroxymethyl)-2-fluoreneacetic acid. After cleavage with piperidine/DMF, desalting, and activation, these peptides were used for the synthesis of 11 analogs of an HIV proteinase nonapeptide substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH₂ using fragment condensation in solid phase. The fragment condensation was made in an ultrasonic bath. Using only 2 equivalents of the activated peptide in a DMF solution, this reaction was complete in 2h. All nonapeptides were assayed as substrates for HIV-1 and HIV-2 proteinases.     

Synthesis of TPH-13, a new tridecapeptide from Phyllomedusa rohdei

The synthesis of TPH-13 (Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH), a tridecapeptide isolated from the skin of the South American frog Phyllomedusa rohdei, is described and alternative approaches are discussed.     

Chemical synthesis of α-inhibin-92 by the thiocarboxyl segment coupling method

The 92 amino acid residue peptide, α-inhibin-92 (α-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS³⁴]-α-IB-92-(l-34) (I), CF₃CO-[GlyS⁶⁵]-α-IB-92-(35–65) (II), and Msc-α-IB-92-(66–92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural α-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural α-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.     

Synthesis and pharmacological activity of dermorphin and its N-terminal sequences

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂ (demorphin), an opiate-like peptide, and tri-, tetra-, penta- and hexapeptide-amide analogs, were synthesized by conventional methods in solution, to determine the minimum peptide chain-length, required for analgesic activity.     

Synthesis of the protected repeat sequence of glutelin-2 of maize,Boc-Val-His-Leu-Pro-Pro-Pro-OH,via nucleophilic cleavage from an Nbb-resin

The protected hexapeptide Boc-Val-His-Leu-Pro-Pro-Pro-OH, the N-terminal protected repeat sequence of glutelin-2 of maize, has been synthesized on a bromomethyl-Nbb-resin. After cleavage of the peptide-resin bond by two 3-min treatments with dioxane/methanol/4n NaOH (30:9:1), gel filtration followed by anion-exchange chromatography allowed the isolation of the target peptide (37% yield) and its methyl ester derivative (52% yield). Cleavage with dioxane/2-dimethylaminoethanol/4n NaOH (30:9:1) has been found to be a simple and effective alternative to the photolysis of the peptide-Nbb-resin bond. After two 15-min treatments with this reagent the protected hexapeptide was isolated by chromatography without apparent epimerization.     

Racemization free coupling of peptide segments

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂ has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10-20% of the d -epimer.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

Synthesis of tetrapeptide p-nitroanilides catalyzed by pepsin

Swine pepsin at pH 5 efficiently catalyzes a condensation between Z-Ala-Ala-Phe-OH and p-nitroanilides of Leu, Phe, Val, Ala and Arg that leads to formation of corresponding benzyloxycarbonyl-tetrapeptide p-nitroanilides with yields of 70–90%. These reactions are complicated by co-precipitation of pepsin and the reaction products that necessitates the use of a relatively high concentration of pepsin.