2-, 6-, And 8-alkylated (methyl, ethyl, and vinyl) adenosine analogues were synthesized by a palladium-catalyzed cross-coupling of a tetraalkyltin with the halogenated purine nucleosides. The synthesis of the 8-substituted analogues was accomplished using a transient protection procedure. The 6-alkylated-9-beta-D-ribofuranosylpurines as well as 2-ethyladenosine were cytotoxic at relatively low concentrations (0.8-10 micrograms/mL). 8-Methyladenosine was a potent and selective inhibitor of vaccinia virus, whereas 8-ethyl- and 8-vinyladenosine were specifically inhibitory to respiratory syncytial virus. 8-Vinyladenosine displayed particular activity against herpes simplex virus (type 1).
N α‐9‐Fluorenylmethoxycarbonyl‐Nε‐4‐methyltrityl‐lysine, [Fmoc‐Lys(Mtt)‐OH], was prepared in two steps from lysine, in 42% overall yield. The Nε‐Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert‐butyl type and peptide ester bonds to TFA‐labile resins, such as the 2‐chlorodiphenylmethyl‐ and the Wang‐resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid‐phase synthesis of one, two or three epitopes containing antigenic peptides or template‐assembled synthetic proteins have been synthesized on Merrifield, Wang and 2‐chlorodiphenylmethyl resin. © Munksgaard 1995.
Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [ 14 C]putrescine or [14 C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (K t ϭ 24.58Ϯ 2.24 µM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (K t ϭ 3.63Ϯ 0.43 µM, K′ t ϭ 0.61Ϯ 0.10 µM). From the K i values, it is inferred that N 1 -5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N 1 -acetylspermine, and then N 1 ,N 12-diacetylspermine. N 1 -acetylspermidine and N 8 -acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows:also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N 4 ,N 9 -diacetylspermine, nor N 1 ,N 4 -bis(β-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.Keywords : acylated polyamine ; polyamine uptake; polyamine pool ; cell growth. Polyamines participate in a wide variety of growth pro-tion, two transport systems with different affinities for putrescine have been suggested in E. coli K12 cells grown in low osmolarcesses. Thus, considerable interest has been generated in elucidating the mechanism of polyamine homeostasis in vivo. It is ity medium [16]. Cross-species transfection of DNA from transport-positive cells into transport-negative cells has been used inferred from several lines of evidence that intracellular polyamine concentrations are regulated by the collective effects of a successfully by Igarashi and co-workers ([13] and references catabolic pathway and an efflux mechanism on the one hand, therein), for the isolation of genes encoding polyamine carriers and a biosynthetic pathway and an uptake mechanism on the in E. coli. The proteins encoded by pPT104 and pPT79 operons other [1]. In Escherichia coli cells, this regulation is, to a large constitute the spermidine/spermine-preferential and the putresdegree, achieved by changes in polyamine biosynthesis and up-cine-specific uptake system, respectively [13]. Another putrestake [2, 3].cine-transport system encoded by pPT71 seems to ha...
Chloramphenicol (CAM) inhibits peptide bond formation by binding to the 50S subunit of prokaryotic ribosomes and interfering competitively with the binding of the aminoacyl-tRNA 3'-terminus to ribosomal A-site. Further studies have demonstrated that CAM (I) reacts rapidly with a model initiator ribosomal complex [poly(U)-programmed ribosomes from Escherichia coli, bearing AcPhe-tRNA at the P-site], complex C, to form an encounter complex CI which is then isomerized slowly to a tighter complex, C I. Herein, we show by time-resolved footprinting analysis that CAM produces a footprint in CI complex, comprising nucleotides A2451, G2505, and U2506, all exhibiting reduced reactivity against base-specific modifying agents. When C I complex is footprinted, the reactivities of G2505 and U2506 are almost restored, while protection is observed at A2062 and altered reactivity at A2058 and A2059. Our results suggest that CAM initially binds to a hydrophobic crevice composed of nucleotides located adjacently to the A-site (CI complex). Soon after, CAM shifts slowly to a final position, in which the interaction between the p-nitrobenzyl group of CAM and the base of A2451 is conserved, while the dichloroacetyl group reorientates toward A2062. Analogous behavior is observed, if CAM is modified by replacement of dichloroacetyl group with-alanyl. However, insertion at this position of a bulkier group, such as phenylalanyl-phenylalanyl group, sterically prevents CAM accommodation to its initial binding site and favors its direct fitting into the final binding pocket. Our data correlate well with recent crystallographic results regarding CAM binding on Thermus thermophilus and E. coli ribosomes.
In a cell-free system derived from Escherichia coli,various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg 2ϩ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N 1 -acetylspermine Ͼ N 1 ,N 12 -diacetylspermine Х N 1 ,N 12 -dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg 2ϩ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect.In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.Keywords : acylated polyamine; protein synthesis; puromycin reaction; peptidyltransferase.Polyamines are organic polycations that are protonated at physiological pH and can potentially interact with a variety of cellular macromolecules including nucleic acids and proteins [1]. Substantial evidence exists suggesting a significant role of polyamines in regulating the translation process at several levels [1]. Due to its four positive charges, spermine is the most effective of the naturally occurring polyamines both in regulating the ribosomal functions and in decreasing the Mg 2ϩ requirements for protein synthesis. Tabor and Tabor [2] have reported that endogenous spermine does not exist in Escherichia coli. However, small amounts of spermine, about two orders of magnitude lower than those of spermidine, can be detected in E. coli by more sensitive methods [3]. Extensive biochemical studies have focused on the mechanism of spermine biological action in prokaryotic-cell-free systems, since this polyamine consists the parent compound of a large number of synthetic polyamine analogues with antiparasitic and antitumor activity [4Ϫ6].Evidently, spermine markedly stimulates the activity of several in vitro protein-synthesizing systems characterized by low Mg 2ϩ concentrations near those observed in vivo [7Ϫ10]. In a recent report [11], we have demonstrated that, at 6 mM Mg 2ϩ spermine exhibits a concentration-dependent allosteric biphasic
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