Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

Summary We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the - or -piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.     

Preparation of phosphopeptide thioesters by Fmoc- and Fmoc(2-F)-solid phase synthesis

Efficient methods for the preparation of phosphopeptidethioesters were examined, using Fmoc-based solid-phase method.Phosphopeptide thioesters were obtained in good yields by theuse of 1-methylpyrrolidine, hexamethyleneimine and 1-hydroxybenzotriazole in a DMSO-DMF (1:1, v/v) solution fordeblocking the Fmoc groups. Epimerization, which is oftenobserved at the C-terminal amino acid, was effectivelysuppressed by shortening the time of deblocking process viathe use of highly base sensitive Fmoc(2-F) groups for -aminoprotection.     

Molecular and crystal structure of Nα-(9-fluorenyl)- methoxycarbonyl-L-ornithine hydrochloride diethyl ether solvate

The solid-state conformation of the first N-protected ornithine derivative has been established by X-ray analysis. The hydrochloride of N-(9-fluorenyl)methoxycarbonyl-L-ornithine crystallises as diethyl ether solvate. The backbone (₀, , , ¹) torsion angles are (174.9°, –84.0°, 145.9°, –171.0°). The conformation of the urethane amide bond is trans. The ornithine aliphatic side chain adopts preferred fully extended conformation which is stabilised by the hydrogen bonding of the –NH₃ ⁺ group to the diethyl ether molecule, carboxyl group and Cl^- anions.     

Synthesis and physicochemical characterization of the lanthionine analog of somatostatin[1–14]

Summary The synthesis of the lanthionine analog of somatostatin[1–14] on a Kaiser-oxime resin is described. The 12-residue peptide segment [3–14] was assembled and cyclized on the resin by using the method of peptide cyclization on an oxime resin (PCOR); the product was obtained with good yield (41%) and purity (94%). The Fmoc protecting group on the N-terminus was cleaved with DBU, followed by a 2+12 segment condensation in solution. The chromatographic (HPLC, CZE) and spectral (UV, NMR) properties of the lanthionine and the natural somatostatins have been studied and compared. Preliminary biological tests show that the lanthionine and the natural somatostatins exhibit similar binding affinities to somatostatin receptor SSTR2.Abbreviations Ala_L one end of a lanthionine unit - Boc tert-butyloxycarbonyl - BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - Cbz benzyloxycarbonyl - DQF-COSY double-quantum-filtered correlated NMR spectroscopy - CZE capillary zone electrophoresis - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DMSO-d₆ hexadeuterated dimethylsulfoxide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - Fmoc 9-florenylmethoxycarbonyl - For formyl - HMPA hexamethylphosphoramide - HOBt N-hydroxybenzotriazole - HOHAHA homonuclear Hartmann-Hahn experiment - HPLC high-performance liquid chromatography - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - TFA trifluoroacetic acid - PCOR peptide cyclization on an oxime resin - Tmac₂O trimethylacetic or pivalic anhydride - Tos p-toluenesulfonyl     

Design and synthesis of chiral peptidic nucleic acids

Summary Due to the increasing interest in the use of oligonucleotide analogues as antisense and antigene drugs, we designed a chiral analogue constituted of a peptidic frame bearing nucleobases in suitable positions (C-PNA). We recently reported the synthesis of four nonnatural α-amino acids with the DNA bases in the lateral chain. In this paper we present an improved synthesis of the Fmoc monomers and their polymerisation to polypeptidic oligonucleotide analogues using a modification of the standard protocol for solid phase peptide synthesis.     

Synthesis and Characterisation of a Multiphosphorylated Phosphophoryn Repeat Motif; H-[Asp-(Ser(<Emphasis Type="Italic">P</Emphasis>))<Subscript>2</Subscript>]<Subscript>3</Subscript>-Asp-OH

Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence H-[Asp-(Ser(P))₂]₃-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn using Fmoc-Ser(PO₃Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups. The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with good sensitivity (20 fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6 mol of calcium per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of a wide variety of multiphosphorylated peptides.     

Exogenous glycosaminoglycans (GAG) differentially modulate GAG synthesis by anchorage-independent cultures of the outer cells from neonatal rat calvaria in the absence and presence of TGF-β

In anchorage-dependent (AD) cultures of the outer cell population (OCP) from neonatal rat calvaria, transforming growth factor-₁ (TGF-) specifically upregulated the synthesis of chondroitin sulfate (CS) proteoglycan (PG) and uncoupled the inhibitory effect of increasing cell density on CS PG synthesis (reference #30). Utilizing the same cell population, we have further examined the possibility that glycosaminoglycans (GAG) known to be synthesized and secreted by bone cells might exert feedback effects on GAG synthesis and/or its stimulation by TGF-. Although addition of TGF- alone stimulated net synthesis of HA and CS in both AD and anchorage-independent (AI) cultures, significant alterations of basal and TGF--stimulated GAG synthesis by exogenous GAGs were observed only in AI cultures. In AI cultures exogenously added hyaluronic acid (HA) markedly enhanced the basal synthesis of HA and CS while heparin (H) suppressed the basal synthesis of HA, CS as well as dermatan sulfate (DS). Also, the addition of HA markedly potentiated the stimulation by TGF- of HA and CS synthesis as did heparan sulfate (HS) for CS and DS synthesis. H suppressed the stimulation of the synthesis of HA, CS and DS by TGF-. Overall, our results indicate specific effects of individual GAGs on basal and TGF--stimulated GAG synthesis in OCP cultures. We suggest that some of the GAGs in the OCP microenvironment (which with the exception of HA are covalently linked to protein cores of secreted PGs), acting in concert with TGF-, may serve as an amplification system for upregulating GAG synthesis in the rapidly growing neonatal calvarium.     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

Untersuchungen zur Kompartimentierung der freien Aminosäure Alanin in den Farnvorkeimen von Dryopteris filix-mas (L.) Schott im Rotlicht und im Blaulicht

Summary In fern gametophytes (= sporelings) there is a strong correlation between the degree of blue light mediated photomorphogenesis and the protein content of the organism (cf. Mohr, 1963). In a previous paper (Payer et al., 1969) we have shown that blue light specifically increases the rate of protein synthesis in the fern sporelings over the rate which is maintained under red light. — In the present paper blue light mediated protein synthesis has been dealt with further using one representative amino acid, alanine, which was labelled with ¹⁴C from ¹⁴CO₂ under steady state conditions of photosynthetic ¹⁴C incorporation under blue or red light.Synthesis of free alanine is proportional to the rate of photosynthesis (Table 1). For a number of reasons we conclude that alanine is derived directly from primary photosynthetic products. Since the pool size of the thoroughly ¹⁴C-labelled pool of free alanine is much less than the actual, pool size of this amino acid, (Table 1), and since the specific activity of the isolated ¹⁴C-alanine is much below the value we can expect on the basis of the specific activity of the ¹⁴CO₂ applied we conclude that there are separate pools of free alanine; active (with respect to protein synthesis) and inactive pools which do not mingle. Taking into account this possibility of compartmentation of pools of free amino acids we have calculated in the case of ¹⁴C-alanine the rate of protein synthesis for two extreme instances (Table 2). A comparison of the theoretical values with the actual data indicates that indeed protein synthesis is fed from active pools of amino acids while the inactive pools are possibly located in the vacuoles. The total pool of alanine is much larger in red grown than in blue grown sporelings while the active pools seem to have the same size under both conditions. The cells of the red grown sporelings have much larger vacuoles than the cells of the blue grown sporelings.The rate of protein synthesis is under our conditions 1.8 times higher in blue light than in red light. The rate of turnover of the total protein is 0.29% per hour in the blue and 0.23% in the red light. The absolute turnover of protein is 1.5 times higher in blue light than in red light. It is concluded that the blue light mediated increase of protein synthesis is very real. Blue light must act specifically at the level of polypeptide synthesis.     

Modification of nuclear gene expression by inhibition of mitochondrial translation during sporulation in MAT alpha/MATa diploids of Saccharomyces cerevisiae

Summary Sporulation of S. cerevisiae MAT/MATa was accompanied by a novel pattern of protein synthesis as shown by the disappearance of some mitotic polypeptides and by the appearance of a new set of meiotic polypeptides. Inhibition of mitochondrial protein synthesis by erythromycin within the 1st h caused the disappearance of several meiotic polypeptides. These meiotic polypeptides were also sensitive to cycloheximide and were localized in the cytosol, demonstrating that they were not mitochondrial translational products. Since erythromycin affected neither protein synthesis nor sporulation in a mitochondrially inherited er y mutant, we conclude that mitochondrial protein synthesis is needed for the expression of some nuclear genes during sporulation.     

The Necessary Site for Initiation of RNA Synthesis in the 3′-Noncoding Region of Classical Swine Fever Virus Genome

Classical swine fever virus (CSFV) is the causative agent of swine fever, which represents an economically important disease in hogs. We previously made a prediction about the recognition sites of replication initiation of CSFV by using the information content method, and it was predicted that the 21 nucleotides located at the 3 end of the CSFV genome 3UTR were essential to CSFV replication. In this paper, we experimentally studied these 21 nucleotides by site-directed mutagenesis. It was found that the 3UTRs with the 21 nucleotides could initiate RNA synthesis, while the 3UTRs without the 21 nucleotides could not. The 21 nucleotides alone, without the rest of 3UTR, were able to initiate RNA synthesis, though with a slump. Most probably the 21 nucleotides were the necessary site for the CSFV genome replication initiation, and the elements required for sufficient RNA synthesis were in the other part of 3UTR. It was assumed that the CSFV replicase bound to the site and initiated the replication of the CSFV genome. In the 21 nucleotides, it was found that the mutation of position 216 and destruction of the 3 terminus in the 3UTR precluded initiation of RNA synthesis, where the mutation of position 212 did not affect the capacity for initiation of RNA synthesis but attenuated the synthesis of RNA. Among the four mutants of 3UTR at position 219, three proved inactive and one partly active in initiating RNA synthesis. Therefore, it could be concluded that T216 was the most important while T212 was the least important, and that G219 and C228 were also important for RNA synthesis.     

人脑利钠肽的Fmoc固相合成

探讨生物活性肽人脑利钠肽(BNP)的固相合成工艺,并为工业化合成提供理论依据.本文以二氯三甲基树脂(以下简称为二氯树脂)为载体,采用9-芴甲氧羰基(Fmoc)保护的氨基酸,以1-氧-3-双二甲胺羰基苯骈三氮唑四氟化硼盐(TBTU)/1-羟基苯并三氮唑(HOBT)/二异丙基乙胺(DIEA)缩合,以碘作为环化试剂,用切割试剂将BNP粗品从树脂上切割下来.通过MALDI-MS质谱仪检测,所合成环肽的分子量与理论分子量一致,使用RP-HPLC液相色谱仪对合成的环肽进行纯化,得到的BNP纯度达到97%以上.本合成工艺具有快捷、简便、高效的特点,适合于大批量的生产目的肽.     

Zygote Formation in Blakeslea trispora: Morphological Peculiarities and Relationship with Carotenoid Synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking -carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene producion results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on -carotene synthesis.     

β-Adrenergic receptors of frog erythrocytes

Following persistent stimulation of -adrenergic receptors of frog erythrocytes with (–)-isoproterenol, the cyclic adenosine 3,5-monophosphate-dependent protein kinase (cAMP-dependent protein kinase) (EC 2.7.1.37) was activated for several hours. This activation outlasted the duration of the increase of cAMP content. Following a persistant stimulation of -adrenergic receptors with isoproterenol, the phosphorylation of selective membrane proteins was increased. This increase in phosphorylation lasted longer than 4 hr but less than 12 hr. Between 2 and 4 hr after receptor stimulation the loss of -adrenergic receptor form plasma membrane was maximal, and the phosphorylation of two membrane proteins characterized by molecular weights of 60,000 and 38,000 daltons was selectively enhanced. In addition we found that isolated erythrocytes are capable of synthesizing RNA and polypeptides and that incubation with (–)-isoproterenol induces a longterm delayed increase of the synthesis of erythrocyte proteins. This increase in the synthesis of proteins appears to require new RNA synthesis. Thus the possibility can be entertained that this delayed increase in protein synthesis participates in the new synthesis of receptor and is operative in the termination of -adrenergic receptor subsensitivity elicited by a persistent stimulation with (–)-isoproterenol.     

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA₃). GA₃ greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA₃. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA₃ caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA₃ was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA₃ on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA₃ reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA₃ and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.     

Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine     

Regulation of uteroglobin/Clara cell protein expression after acute lung exposure to an organophosphoreted insecticide

Uteroglobin (UG) or Clara cell protein (CC16), the main secretory product of bronchiolar Clara cells, plays an important protective role in the respiratory tract against inflammatory processes. In the lung, protein secretion is regulated by glucocorticoids, but also proinflammatory cytokines, such as interferon-gamma (IFN-) and TNF-, have been found to modulate the expression of this peptide. We have previously demonstrated that the acute exposure to an organophosphoreted insecticide induces an enhanced production of UG/CC16 by Clara cells. In the present report, we worked with intact and adrenalectomised (ADX) animals to study the mechanism involved in the UG/CC16 increase caused by the insecticide and the role played by a glucocorticoid (dexamethasone; DEX). In intact rats we found that DEX treatment could not reproduce such an increase of UG/CC16 synthesis with pharmacological doses. In ADX rats, even though glucocorticoid deprivation provoked a strong inhibition of UG/CC16 synthesis, the exposure to the organophosphoreted insecticide stimulated the synthesis of the protein, shown by the great accumulation of secretory granules in the cytoplasm of Clara cells and the increase of UG/CC16 detected by immunocytochemistry and western blot. These results imply that glucocorticoids are not essential to trigger the increase of UG/CC16 in response to an injury, and they also suggest an involvement of other molecules associated with inflammation. In coincidence with these observations, we have found that IFN-, a proinflammatory cytokine, increased after insecticide exposition in both groups, intact and ADX, mainly in ADX rats. The stimulation of UG/CC16 synthesis occurring during inflammatory processes of the respiratory tract caused by acute inhalation of a toxicant appears to be functional without the intervention of glucocorticoids and mediated by IFN- as a mechanism for local control of the inflammatory response.     

Disturbed Cooperation of Hepatocytes in the Rhythm of Protein Synthesis by Chelating Agent of Cytoplasmic Calcium BAPTA-AM

Previously we demonstrated synchronized oscillations of protein synthesis rate in hepatocyte cultures upon accumulation of monosialoganglioside GM1 in the medium or after introduction of exogenous GM1 to the medium. The synchronized oscillations of the protein synthesis rate in dense hepatocyte cultures were blocked 30 min after their treatment with 10–20 M BAPTA-AM, a chelating agent of cytoplasmic calcium. Enzyme immunoassay for GM1 demonstrated similar amounts of GM1 in the medium conditioned for 3 h by dense hepatocyte cultures pretreated with 20 M BAPTA-AM for 1 h and in the medium of normal dense cultures: 0.0060 ± 0.0005 and 0.0055 ± 0.0005 pmol/1000 cells, respectively. The content of GM1 was also similar in the normal and BAPTA-AM-pretreated hepatocytes: 0.158 ± 0.013 and 0.183 ± 0.014 pmol/1000 cells, respectively. The synchronized rhythm of protein synthesis has been confirmed in the diluted cultures in the medium conditioned by the normal dense cultures. However, the medium conditioned by the dense cultures pretreated with BAPTA-AM induced no synchronization of the diluted cultures. Since GM1 concentration was normal in this medium, we propose the effect of a physicochemical form of the gangliosides accumulated in the medium on their ability to synchronize the rhythm of protein synthesis.