Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

2'-OH-protection by the p-nitrophenylethylsulfonyl group in oligocytidylate synthesis

A new approach to oligoribonucleotide synthesis has been developed by use of the p-nitrophenylethylsulfonyl group as a new versatile sugar protecting group. The synthesis of 3'-5'- and 2'-5'-cydidylyl-cytidine is performed in high yield.     

Effect of RNA synthesis inhibitors on insulin-induced protein synthesis by 3T3 cells

1. The increased protein synthesis of quiescent 3T3 cells in response to insulin was separated into three distinct phases based on their response to various inhibitors of RNA synthesis. 2. The first increase in protein synthesis was insensitive to the inhibitors used, and probably resulted from activation of existing protein synthesizing mechanism. 3. The second phase was sensitive to a varying extent to alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, implying the need for new mRNA synthesis as well as the production of new ribosomes indicated by its further sensitivity to low concentration (10 ng/ml) of Actinomycin D. 4. The final phase was insensitive to inhibitors of new ribosome formation, but still depended on new mRNA. alpha-difluoromethylornithine, an inhibitor of de novo polyamine synthesis, partly inhibited the insulin induced stimulation of protein synthesis.     

Light-dependent regulation of the synthesis of soluble and intracytoplasmic membrane proteins of Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides grown under saturating light conditions (30 W/m2) and then shifted to low light intensity (3 W/m2) required 2.5 h to adapt to the new lower light conditions. After the shift, cell growth, whole cell protein accumulation, and bacteriochlorophyll accumulation ceased immediately. Approximately midway into the adaptation period, bacteriochlorophyll synthesis commenced at a new, higher rate, which continued through the beginning of the low-light growth period until new steady-state levels were reached. Immediately after the downshift, the rate of cellular protein synthesis declined to 22% of its preshift rate. Pulse-labeling of protein throughout the adaptation period and comparison with a steady-state prelabel culture revealed that synthesis of two of the three light-harvesting proteins, as well as two additional high-molecular-weight photosynthetic membrane proteins, was derepressed three- to fivefold compared with bulk cellular protein. Finally, the synthesis of at least three soluble proteins showed light-dependent regulation after the light downshift. These results are discussed in terms of the light-dependent regulation of synthesis of the photosynthetic membrane macromolecular components and the division of protein synthesis between the photosynthetic membranes and the soluble cell phase.     

RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry

The chemical synthesis of RNA oligonucleotides is a valuable resource for biological research. A new approach for RNA synthesis that is now as reliable and efficient as DNA synthesis methods is described in this report. A 5'-O-silyl ether is used in conjunction with acid-labile orthoester protecting groups on the 2'-hydroxyls. RNA synthesis proceeds efficiently on commercial synthesizers in high yields. Analysis by anion-exchange HPLC shows that the quality and yields of RNA synthesized with this chemistry are unprecedented. Furthermore, this chemistry enables analysis and purification of stable 2'-O-protected RNA. This property serves to minimize possibilities for degradation of the RNA. In addition, it now possible to analyze troublesome sequences, which, when fully 2'-O-deprotected, do not easily resolve into one major conformation due to strong secondary structure. When ready for use, the RNA is easily 2'-O-deprotected in mild-acidic aqueous buffers in 30 min. This new RNA chemistry has enabled the routine high-quality synthesis of RNA oligonucleotides up to 50 bases in length regardless of sequence or secondary structure.     

Solid phase synthesis of nucleobase and ribose modified inosine nucleoside analogues

The synthesis and the use of new N-1-dinitrophenyl-inosine based solid support is reported. The support, which binds the nucleoside by a 5'-O-monomethoxytrityl function, reacting with N-nucleophiles allowed the synthesis of a small library of N-1 alkylated inosine and AICAR derivatives. Moreover, the obtained supports, after the cleavage of the 2' -3' ribose bond, furnished a set of new N-1 alkylated-2' -3' -secoinosine derivatives in high yields.     

Application of lipase-catalyzed transformations for the synthesis of insect pheromones and related compounds

This review describes efficient means of preparing optically pure insect pheromones and related compounds via lipase-catalyzed enantioselective reaction on a large scale. (1) A new synthesis of the Japanese beetle pheromone, (R,Z)-(−)-5-(1-decenyl)oxacyclopentan-2-one, established by a combination of two lipase-catalyzed transformation was demonstrated. (2) A chemico-enzymatic procedure for the syntheses of both enantiomers of cupreous chafer beetle pheromone, (R,Z)- and (S,Z)-5-(1-octenyl)oxacyclopentan-2-one, was described. (3) An optical resolution of (±)-2,3-epoxy-8-methyl-1-nonanol, the key intermediate of the synthesis of gypsy moth pheromone, was demonstrated. (4) A practical chemico-enzymatic synthesis of (+)-disparlure in large scale was demonstrated. (5) A facile synthesis of carboxyalkyl acrylate, which is special monomers in the synthesis of the new polymers, by two lipase-catalyzed regioselective reactions was described.     

Ethylene stimulates endoreduplication but inhibits cytokinesis in cucumber hypocotyl epidermis

The effects of ethylene on cell division are generally considered inhibitory. In this study, we demonstrate that transient ethylene exposure, while suppressing cytokinesis, stimulates DNA synthesis. We monitored DNA synthesis and cytokinesis in the epidermis of cucumber (Cucumis sativus) hypocotyls, an organ whose post-germination development involves strictly limited cell division. During exposure to ethylene, DNA synthesis, assessed by the incorporation of the thymidine homolog 5-bromo-2'-deoxyuridine, was detected in 20% of the epidermal cells, whereas DNA synthesis was nearly undetectable in normal air. Cytofluorometric analysis of nuclei in affected cells showed an up to 8-fold increase in DNA content. During this time, new cell plate formation was not detected. However, shortly after ethylene was removed, DNA content was rapidly restored to 2C (diploid) levels in all cells, and new cell plate formation dramatically increased. These results demonstrate that ethylene promotes DNA synthesis and its endoreduplication but inhibits cytokinesis, thereby maintaining some cells in G2 phase.     

Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation

We have found a striking difference between the modes of action of amdinocillin (mecillinam) and compound A22, both of which inhibit cell elongation. This was made possible by employment of a new method using an Escherichia coli peptidoglycan (PG)-recycling mutant, lacking ampD, to analyze PG degradation during cell elongation and septation. Using this method, we have found that A22, which is known to prevent MreB function, strongly inhibited PG synthesis during elongation. In contrast, treatment of elongating cells with amdinocillin, which inhibits penicillin-binding protein 2 (PBP2), allowed PG glycan synthesis to proceed at a nearly normal rate with concomitant rapid degradation of the new glycan strands. By treating cells with A22 to inhibit sidewall synthesis, the method could also be applied to study septum synthesis. To our surprise, over 30% of newly synthesized septal PG was degraded during septation. Thus, excess PG sufficient to form at least one additional pole was being synthesized and rapidly degraded during septation. We propose that during cell division, rapid removal of the excess PG serves to separate the new poles of the daughter cells. We have also employed this new method to demonstrate that PBP2 and RodA are required for the synthesis of glycan strands during elongation and that the periplasmic amidases that aid in cell separation are minor players, cleaving only one-sixth of the PG that is turned over by the lytic transglycosylases.     

Design,synthesis and biological evaluation of trans 2-(thiophen-2-yl)vinyl heteroaromatic iodides

A modeling approach based on physico-chemical and pharmacokinetic properties, called Volsurf+, was used to design new trans 2-(thiophen-2-yl)vinyl heteroaromatic iodides with antiproliferative activity. The synthesis and in vitro antitumor tests on two cell lines (MCF-7 and LNCap) confirmed Volsurf predicted activity values. An Almond model, derived to have an overall structural insight on the above compounds, supported the validity of Volsurf and provided guidelines for the synthesis of new compounds.     

Prolonging cell-free protein synthesis with a novel ATP regeneration system

A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.     

Total synthesis and properties of prostaglandins. XXI. Synthesis of a 4,4-dimethyl-4-sil analog of 11-deoxyprostaglandin E1

Total synthesis of a new prostaglandin analogue, 11-deoxy-4,4-dimethyl-4-sil-prostaglandin E1, is carried out through synthesis of a silicon-containing alpha-chain precursor and a 2-substituted cyclopentenone derivative followed by their cuprate-induced interaction.     

Oligoribonucleotide synthesis by use of the 2-(methylthio)-phenylthiomethyl (MPTM) group

The 2-(methylthio)phenylthiomethyl (MPTM) group was developed as a new type of 2'-hydroxyl protecting group in oligoribonucleotide synthesis. The building monomer units of uridine and cytidine for the phosphotriester approach were synthesized from 2'-O-(1,3-benzodithiol-2-yl)-3',5'-O- (1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)uridine and successfully utilized for the synthesis of CpUpG.     

The kinetics of the incorporation of newly synthesized ribonucleic acid and protein into the ribosomes of the uterus of the oestrogen-stimulated immature rat.

The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.     

Synthesis of new sugar amino acid derivatives of D-glucosamine

The synthesis of several new sugar amino acid derivatives of 2-acetamido-2-deoxy-D-glucose, bearing a C-glycosyl functionality as building blocks for the design and synthesis of natural glycoconjugates mimetics, is described. These compounds were prepared from the readily accessible per-benzylated amino C-allyl glucopyranosyl compounds, with TMSOTf/Ac(2)O-mediated selective acetolysis of the 6-O-benzyl group as the key step.     

Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.     

Hydrostatic pressure-induced changes in cellular protein synthesis

Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.     

Solid phase synthesis of gastrin I. Comparison of methods utilizing strong acid for deprotection and cleavage

A successful synthesis of human gastrin I in 60% overall yield based on the first residue attached to a benzhydrylamine-resin was achieved by the stepwise solidphase method. The synthesis was carried out on a 1% crosslinked polystyrene support, using conventional benzyl-based side chain protecting groups and final deprotection with different acidic protocols. Several improvements in this general approach were applied, including new scavengers, new resin attachment and especially a new technique that allows the strong acid reactions to occur by an SN2 mechanism.