We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(؊)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.

N-Acyl homoserine lactones (AHLs) are molecules that are synthesized and detected by many gram-negative bacteria to monitor the population density, a phenomenon known as quorum sensing. Salmonella enterica serovar Typhimurium is an exceptional species since it does not synthesize its own AHLs, while it does encode a LuxR homologue, SdiA, which enables this bacterium to detect AHLs that are produced by other species. To obtain more information about the specificity of the ligand binding by SdiA, we synthesized and screened a limited library of AHL analogues. We identified two classes of analogues that are strong activators of SdiA: the N-(3-oxo-acyl)-homocysteine thiolactones (3O-AHTLs) and the N-(3-oxo-acyl)-trans-2-aminocyclohexanols. To our knowledge, this is the first report of compounds (the 3O-AHTLs) that are able to activate a LuxR homologue at concentrations that are lower than the concentrations of the most active AHLs. SdiA responds with greatest sensitivity to AHTLs that have a keto modification at the third carbon atom and an acyl chain that is seven or eight carbon atoms long. The N-(3-oxo-acyl)-trans-2-aminocyclohexanols were found to be less sensitive to deactivation by lactonase and alkaline pH than the 3O-AHTLs and the AHLs are. We also examined the activity of our library with LuxR of Vibrio fischeri and identified three new inhibitors of LuxR. Finally, we performed preliminary binding experiments which suggested that SdiA binds its activators reversibly. These results increase our understanding of the specificity of the SdiA-ligand interaction, which could have uses in the development of anti-quorum-sensing-based antimicrobials.

The synthesis of different cycloSal-phosphotriesters of the acyclic nucleoside analogues acyclovir (ACV), penciclovir (PCV) and T-penciclovir (T-PCV) as potential new lipophilic, membrane-soluble pronucleotides is described. The introduction of the cycloSal moiety was achieved by using reactive cyclic chlorophosphane reagents. In addition to the cycloSal-PCV monophosphate (MP) phosphotriesters, a second derivative bearing an acetyl group at the second primary alcohol function was prepared. In hydrolysis studies the cycloSal-ACVMPs showed the expected range of hydrolytic stability dependent on the substituent in the masking group (8-17 h). In contrast, the cycloSal-PCVMP derivatives exhibited a 11- to 15-fold increase in hydrolytic lability as compared to the corresponding cycloSal-ACVMP derivatives. We demonstrated that the free primary alcohol group is responsible for this rate acceleration because cycloSal-OAc-PCVMP, in which the hydroxyl group was blocked by acetylation, did not show the aforementioned acceleration. Unexpectedly, the hydrolysis product was not PCVMP but according to NMR and mass spectrometry it was cycloPCVMP (cPCVMP). The title compounds were evaluated in vitro for their ability to inhibit herpes simplex virus type 1 (HSV-1) and thymidine kinase-negative (TK-) HSV-1 replication in Vero cells. The cycloSal-ACVMP compounds exhibited high antiviral activity in HSV-1-infected cells. More importantly, one derivative retained all activity from the wild-type virus strain in HSV-1/TK(-)-infected Vero cells. The PCV derivatives were markedly less active. The reason for the failure of the cycloSal-PCVMPs seems to be due to the formation of cPCVMP instead of the desired PCVMP.

The synthesis of 5′,5′‐O‐di‐(3′‐azido‐2′,3′‐dideoxythymidinyl)‐O′‐benzylphosphotriesters 1 as potential prodrugs of nucleoside monophosphates is described. The concept is applied to the antiretroviral nucleoside analog 3′‐azido‐2′,3′‐deoxythymidine (AZT) 4. All derivatives 1 were synthesized by reaction of the tetra‐n‐butylammonium salt of di‐AZT‐phosphate 2b with different benzyl bromides or ‐chlorides 9. Compound 2b was obtained by a combination of phosphoamidite/H‐phosphonate chemistry, subsequent oxidation to 2a, and cation exchange. The partition coefficients of 1 in an 1‐octanol/water mixture show that all compounds exhibit a much higher lipophilicity than the parent nucleoside AZT (4). It was also shown, that 1 decomposes spontaneously under mild aqueous basic conditions (phosphate buffer (pH 7.5) and RPMI culture medium containing heat‐deactivated fetal calf serum) releasing selectively the di‐AZT phosphate anion 2. The half‐lives of 1 could be adjusted within a wide range by changing the ring substituents of the benzyl group. Additionally, the mechanism of hydrolysis varies if the substituent is changed from a donor to an acceptor one. The described phosphotriesters 1 exhibit considerable antiviral activity in HIV‐1‐ and HIV‐2‐infected CEM/O cells, whereas no activity was detected in the HIV‐2‐infected thymidine kinase‐deficient CEM cell line. On the other hand, we could not detect any cytotoxicity of the described phosphotriesters. Consequently, compounds 1 should act as prodrugs or depot forms at least of antiviral nucleoside analogs.

No abstract

The a-hydroxybenzylphosphonates 1 a-l j of the antiviral drug 3'-azido-2',3'-dideoxythymidine 5 (AZT) as potential lipophilic prodrugs were readily accessible in 49% to 87 YO yield via a four-step synthetic pathway introducing the modifications in the aromatic ring system in the last step by making use of intermediate 6. All compounds l a -l j exhibited higher partition coefficients in 1-octanol/water than AZT (5).In hydrolysis studies at pH 7.5 we observed that precursors to bioactive compounds were delivered by simple hydrolysis of the lipophilic precursors l a -l j via two different mechanisms: the phosphonate-phosphate rearrangement leading to the benzylphosphotriesters 2 and/or the direct cleavage into the di-AZT phosphonate 6. Both compounds 2 and 6 were further degraded yielding the potentially antiviral active compounds 4 and 8 , respectively. The hydrolysis pathway could be controlled by the substitution pattern in the benzylic moiety. Identical hydrolytic behavior of 1 was detected in "biological" hydrolysis kinetics by using a RPMI culture medium containing 10 % heat-inactivated fetal calf serum (FCS). The title compounds la-1 j exhibited considerable HIV-1 and HIV-2 activity in wild-type CEM/O cells.Today, the most successful therapy against retroviral infections such as AIDS is based on the use of analogs of natural nucleosides [']. Most of these nucleoside analogs are 2',3'-dideoxynucleosides (e.g. 3'-azido-2',3'-dideoxythymidine (AZT)n). The mode of action of these nucleoside analogs is the inhibition of the replicating enzyme of HIV, reverse transcripta~e [~,~] or the incorporation into the growing DNA chain which results in chain terminati~n [~]. Hence, in order to develop their biological activity, the nucleoside analogs have to be metabolized into their 5'-triphosphate derivatives inside the infected cell. This biotransformation is effected by host cell kinases in three steps via the nucleoside monophosphate and the nucleoside diphosphateI2I. Some nucleoside analogs are known to be suitable inhibitors of the reverse transcriptase in vitro when used as the triphosphates, but are not active in vivo because no phosphorylation occurs [5]. In most cases the first kinase step leading to the monophosphate is the metabolization determining step. So by-passing this kinase by releasing monophosphorylated nucleosides from a prodrug is one attempt to improve the therapeutic potential of the drugsL6I. The advantage of a prodrug is obvious, because also non-active analogs such as 2',3'-dideoxyuridine (ddU) could be used in antiviral chemotherapy. A prodrug for that purpose has to fulfill two requirements: i) it has to be lipophilic for passive membrane diffusion and it has to be transported through the blood brain barrier; ii) furthermore, it should be able to deliver the nucleoside monophosphate spontaneously or enzymatically leaving a non-toxic masking group. Lipophilic nucleoside phosphoric acid triesters were studied with the purpose of releasing bioactive, phosphorylated compounds by hydrolytical or en...

No abstract

No abstract