Biomedical subjects
Publications and source records attributed to Jef Rozenski.
A strategy was developed for the synthesis of 3'-O-beta-D-ribofuranosyl 2'-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5'-O- and 5''-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.
Cyclohexene nucleic acids (CeNA) with a D-like configuration form very stable self-complementary duplexes and stable duplexes with RNA. An increased duplex stability with Delta T(m)/mod of +1.2 degrees C is observed. The duplex with DNA is less stable. Excellent mismatch discrimination has been observed as well for the duplex with DNA as for the duplex with RNA. The results obtained with mixed CeNA sequences warrant antisense studies with CeNA. The CeNAs of opposite chirality constitute a self-pairing system on their own, resembling L-RNA sequences.
Current standard therapy for the treatment of chronic infections with hepatitis C virus consists of combination therapy with (pegylated) interferon-alpha and ribavirin. 1,5-Anhydrohexitol nucleoside analogues are constrained congeners known to mimic the ribonucleoside conformation. Within this series some analogues are endowed with strong antiviral properties, particularly against herpesviruses. The six-membered anhydrohexitol ring was, therefore, combined with the triazolyl carboxamide moiety of ribavirin, thus providing a new series of ribavirin analogues. None of the newly synthesized compounds elicited any substantial antiviral activity, neither against herpes simplex virus type 1 and 2, nor against bovine viral diarrhoea virus (a surrogate for hepatitis C virus) or the yellow fever virus.
A series of new cyclohexenyl nucleosides is synthesized by coupling the heterocyclic bases with a protected cyclohexenyl precursor under Mitsunobu conditions. The compounds were evaluated for their antiviral and cytostatic activity. Pronounced activity against herpes simplex virus type 1 and type 2 was observed for the 2,6-diaminopurine analogue.
On the basis of the original lead neocryptolepine or 5-methyl-5H-indolo[2,3-b]quinoline, an alkaloid from Cryptolepis sanguinolenta, derivatives were prepared using a biradical cyclization methodology. Starting from easily accessible educts, this approach allowed the synthesis of hitherto unknown compounds with a varied substitution pattern. As a result of steric hindrance, preferential formation of the 3-substituted isomers over the 1-substituted isomers was observed when cyclizing N-(3-substituted-phenyl)-N'-[2-(2-trimethylsilylethynyl)phenyl]carbodiimides. All compounds were evaluated for their activity against chloroquine-sensitive as well as chloroquine-resistant Plasmodium falciparum strains, for their activity against Trypanosoma brucei and T. cruzi, and for their cytotoxicity on human MRC-5 cells. Mechanisms of action were investigated by testing heme complexation using ESI-MS, inhibition of beta-hematin formation, DNA interactions (DNA-methyl green assay and linear dichroism), and inhibition of human topoisomerase II. Neocryptolepine derivatives with a higher antiplasmodial activity and a lower cytotoxicity than the original lead have been obtained. This selective antiplasmodial activity was associated with inhibition of beta-hematin formation. 2-Bromoneocryptolepine was the most selective compound with an IC(50) value against chloroquine-resistant P. falciparum of 4.0 microM in the absence of cytotoxicity (IC(50) > 32 microM). Although cryptolepine, a known lead for antimalarials also originally isolated from Cryptolepis sanguinolenta, was more active (IC(50) = 2.0 microM), 2-bromoneocryptolepine showed a low affinity for DNA and no inhibition of human topoisomerase II, in contrast to cryptolepine. Although some neocryptolepine derivatives showed a higher antiplasmodial activity than 2-bromocryptolepine, these compounds also showed a higher affinity for DNA and/or a more pronounced cytotoxicity. Therefore, 2-bromoneocryptolepine is considered as the most promising lead from the present work for new antimalarial agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-9-cyanoneocryptolepine exhibited antitrypanosomal activity in the micromolar range in the absence of obvious cytotoxicity.
Hexitol nucleic acids (HNA) as well as their 1,5-anhydrohexitol triphosphate building blocks were evaluated for their ability to be recognized by several DNA metabolizing enzymes. It was found that RNA polymerases can recognize the triphosphate of the adenine analogue. However, only the incorporation of a maximum of three consecutive building block analogues was possible under the applied experimental conditions. Terminal transferase was more successful succeeding in the elongation of a DNA primer with a maximum of 15 1,5-anhydrohexitol purine nucleotides. Furthermore, it was observed that the 1,5-anhydroaltritol triphosphate analogue of adenosine was a poor substrate for terminal transferase and that HNA could not act as a primer for this enzyme. Likewise, HNA did not function as a template for restriction enzymes, ligases or methylases.
The synthesis and structure-activity relationship of N(6)-cyclopentyl-3'-substituted-xylofuranosyladenosine analogues with respect to various adenosine receptors were explored in order to identify selective and potent antagonists and inverse agonists for the adenosine A(1) receptor. In particular, the effects of removal of the 5'-OH group and introduction of selected substituents at the 3'-NH(2) position of 9-(3-amino-3-deoxy-beta-D-xylofuranosyl)-N(6)-cyclopentyladenine were probed. A solid phase-assisted synthetic approach was used to optimize the 3'-amide functionality. In view of the general concern of the presence of a 5'-OH moiety with regard to cellular toxicity, the present study describes 5'-deoxy compounds with reasonable affinity for the human adenosine A(1) receptor. Interestingly, this study shows that optimization of the 3'-"up" amide substituent can substantially compensate for the drop in affinity for the adenosine A(1) receptor, which is generally observed upon removal of the 5'-OH group. The fact that for several 3'-amido-substituted (5'-deoxy)-N(6)-cyclopentyladenosine derivatives, guanosine 5'-triphosphate-induced shifts in K(i) values were significantly lower than 1 implies that these analogues behave as inverse agonists. This is further supported by their 1,3-dipropyl-8-cyclopentylxanthine-like capacity to increase forskolin-induced adenosine cyclic 3',5'-phosphate production.
The base moiety of 1-N-methyladenosine can be protected with a chloroacetyl group for incorporation of this modified nucleoside into DNA and RNA. Carefully controlled anhydrous conditions are needed for deprotection of the oligonucleotides.
The synthesis of a new series of D-erythro-homoceramide analogues is described. Several synthetic approaches were investigated. Homoceramides can be successfully synthesized from L-homoserine as chiral building block and a protected Weinreb-amide as a key intermediate. The synthesis of short-chain analogues with a heptyl side chain, as well as with a phenyl residue in the sphingoid part (instead of the naturally occurring tridecyl side chain), was effected. The homoceramides 15-17 and 24 were investigated for their potential to reverse the inhibitory effect of fumonisin B(1) on axonal growth. Unfortunately, none of the tested compounds showed any biological activity due to their lack of metabolism to glucosylhomoceramide.
Electrospray ionization linked to quadrupole/orthogonal-acceleration time-of-flight (Q/oaTOF) and ion trap equipment was used to study the fragmentation behavior of the linear side-chain cyclized peptides of the polymyxin B and E antibiotics. This study exemplifies both the benefits and the drawbacks of mass spectrometric techniques for the determination of the sequence of such complex linear side-chain cyclized peptides. Q/oaTOF accurate mass measurements did not help sufficiently to assign the product ions observed in the product ion spectra. An ion trap mass spectrometer providing MS(n) capability was used to eliminate ambiguities encountered with a single MS/MS approach. The complex fragmentation behavior of these compounds of well-established structure is described which could be useful for structural characterization of unknown substances related to polymyxin B and E in the future.
The content of 24 12-membered heptapeptide libraries was investigated using capillary liquid chromatography coupled with an electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometer. Adjustment of the chromatographic parameters led to the separation of most of the components. Extraction of the [M + 2H]2+ ions allowed us to demonstrate the presence of all expected species in the library and to evaluate their relative abundance in the mixture. Rapid sequence confirmation was achieved by subtraction of product ion spectra, a way to eliminate common ions and to simplify the spectra for interpretation. This technique can also easily be applied to other libraries consisting of components with a common core.
Solid phase peptide library screening followed by extension of a lead recognition element for binding to a dsDNA sequence (NF binding site of IL6) using solution phase screening, delivered a new DNA binding peptide, Ac-Arg-Ual-Sar-Chi-Chi-Tal-Arg-CONH2. In the present research, the contribution of the different amino acid side chains to the binding strength of the peptide to dsDNA was investigated using an ethidium bromide displacement test. Based on these results, the lead structure was optimized by deconvolution. Eight new unnatural amino acids were evaluated at two positions of the heptapeptide replacing the Ual-Sar fragment. The strongest dsDNA binding was observed using ([(3-chlorophenyl)methyl]amino)acetic acid (Cbg) and beta-cyclohexyl-l-alanine (Cha) respectively, at those two positions. A 10-fold increase in affinity compared to the Ual-Sar sequence was obtained. Further enhancement of dsDNA binding was obtained with hybrid molecules linking the newly developed peptide fragment to an acridine derivative with a flexible spacer. This resulted in ligands with affinities in the microM range for the dsDNA target (K(d) of 2.1 x 10(-6) M). DNase I footprinting with the newly developed oligopeptide motifs showed the presence of a pronounced pyrimidine specificity, while conjugation to an intercalator seems to redirect the interaction to mixed sequences. This way, new unnatural oligopeptide motifs and hybrid molecules have been developed endowed with different sequence selectivities. The results demonstrate that the unnatural peptide library approach combined with subsequent modification of selected amino acid positions, is very suited for the discovery of novel sequence-specific dsDNA binding ligands.
Mass spectra of oligonucleotides derived from collision-induced dissociation following electrospray ionization provide an effective means of sequence determination, at the 20-mer level and below. An interactive, stand-alone computer program, Simple Oligonucleotide Sequencer (SOS) has been developed for rapid oligonucleotide sequencing from mass spectra, under user control on a residue by residue basis. Modifications can be defined in any combination for the base, sugar or backbone. Sequence ladders can be independently constructed in both the 5' --> 3' directions and 3' --> 5' directions, and graphically compared for homology and overlap. A particular advantage of this method is the ability to easily erase and rebuild alternate subsequences. The program can be used for ab initio sequencing of modified or unmodified oligonucleotides, for rapid verification of sequence, and in studies of fragmentation processes of model oligonucleotide derivatives.
Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the imported tRNA(Trp) in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNA(Trp) as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific posttranscriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA(Trp) variants presented shows a single base-pair reversal in the anticodon stem of tRNA(Trp) is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.
The cyclohexane nucleosides with a 1,4-relationship between nucleoside base and hydroxymethyl moiety were synthesized using a conjugated addition reaction of the nucleobases to ethyl 1,3-cyclohexadiene-1-carboxylate and hydroboration of the cyclohexenyl precursor. The lack of antiviral activity of the compounds was correlated with the conformation of these nucleosides as deduced from NMR and X-ray analysis.
1,5-Anhydro-2,3-dideoxy-D-ribohexitol nucleosides were synthesized starting from 4,6-di-O-benzyl-1,5-di-O-pivaloyl-3-deoxy-D-glucitol using a ring closure procedure. The target nucleoside adopts a (4)C(1) conformation as also demonstrated for the corresponding 1,5-anhydrohexitol nucleosides with beta-configuration of the base-substituted carbon atom. The cytosine congener demonstrated a moderate but selective activity against Herpes simplex virus types 1 and 2.
For the currently used (99m)Tc-labeled diphosphonates such as (99m)Tc-MDP and (99m)Tc-HDP, the required interval of 2.5 to 3 h between injection and the scintigraphic bone imaging is an inconvenience. The present study was set up in an attempt to develop a technetium-99m-labeled diphosphonate with efficient bone uptake and more rapid clearance from blood and soft tissue by renal extraction and excretion so that it would be possible to start imaging as early as 1 h after injection. A conjugate of the new renal tracer agent (99m)Tc-ethylene dicysteine ((99m)Tc-L,L-EC), covalently bound via one of its carboxylates with aminomethylenediphosphonic acid (AMDP), was synthesized in seven steps. EC-AMDP could be labeled easily and efficiently with (99m)Tc at pH > or = 12 and room temperature. Analysis using ion pair reversed phase high performance liquid chromatography showed the formation of a mixture of two main compounds with reproducible relative ratios, which were stable as a function of time. In a baboon, the scintigraphic images obtained with the new agent showed good quality bone scans, with clear visualization of the skeleton and low soft tissue activity at respectively 1 and 2 h after injection.
Ribonuclease mimics based on diimidazole derived constructs in combination with or without additional amino groups have been synthesized and conjugated to oligonucleotides. The imidazole moiety was used either unprotected, protected with a monomethoxytrityl group or a tert-butyloxy carbonyl group. Acylation reactions were carried out using the 3-acyl-1,3-thiazolidine-2-thione activation strategy. The peptides were coupled to the oligonucleotides with a mixture of PyBOP, DIEA an HOBt in DMF on solid support. The conjugates were purified by RP-HPLC and identified using negative ion mode mass spectrometry. Unfortunately, no cleavage of a linear RNA target under physiological conditions could be observed.