Biomedical subjects
Publications and source records attributed to H Rosemeyer.
The synthesis of base modified L-nucleosides is described with pyrrolo[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, benzimidazoles, and imidazo[1,2-a]-s-triazines as nucleobases. The conformation of the nucleosides is studied and the antiviral activity is evaluated.
Cross-talking between nucleic acids is a prerequisite for information transfer. The absence of observed base pairing interactions between pyranose and furanose nucleic acids has excluded considering the former type as a (potential) direct precursor of contemporary RNA and DNA. We observed that alpha-pyranose oligonucleotides (alpha-homo-DNA) are able to hybridize with RNA and that both nucleic acid strands are parallel oriented. Hybrids between alpha-homo-DNA and DNA are less stable. During the synthesis of alpha-homo-DNA we observed extensive conversion of N6-benzoyl-5-methylcytosine into thymine under the usual deprotection conditions of oligonucleotide synthesis. Alpha-homo-DNA:RNA represents the first hybridization system between pyranose and furanose nucleic acids. The duplex formed between alpha-homo-DNA and RNA was investigated using CD, NMR spectroscopy, and molecular modeling. The general rule that orthogonal orientation of base pairs prevents hybridization is infringed. NMR experiments demonstrate that the base moieties of alpha-homo-DNA in its complex with RNA, are equatorially oriented and that the base moieties of the parallel RNA strand are pseudoaxially oriented. Modeling experiments demonstrate that the duplex formed is different from the classical A- or B-type double stranded DNA. We observed 15 base pairs in a full helical turn. The average interphosphate distance in the RNA strand is 6.2 A and in the alpha-homo-DNA strand is 6.9 A. The interstrand P-P distance is much larger than found in the typical A- and B-DNA. Most helical parameters are different from those of natural duplexes.
7-Deaza-2'-deoxyadenosine and -guanosine phosphoramidite building blocks as well as corresponding 5'-triphosphate derivatives are described carrying in position 7 substituents such as iodo, hexyn-1-yl or 5-aminopentyn-1-yl residues. The phosphoramidites were used to synthesize a series of modified oligodeoxynucleotides. A systematic study of the thermal stabilities of these oligonucleotide duplexes demonstrated that the 7-substituents are well accommodated in the major groove of B-DNA. The 7-(aminoalkyn-1-yl)-7-deazapurine 2'-deoxynucleoside triphosphates were labeled with bulky fluorophores such as Rhodamine Green(R) or tetramethylrhodamine.
2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.
Measurements of differential scanning calorimetry, ultraviolet absorption and circular dichroism have been performed on two synthetic oligo(2'-deoxyxylonucleotides): (A) d[xA)3-(xT)3-(xA)3-(xT)3-T] and (B) d[(xA-xT)6-T], and on the oligo(2'-deoxyribonucleotide) (C) d[(A)3-(T)3-(A)3-(T)3]. Oligonucleotides having 2'-deoxyxylose instead of 2'-deoxyribose exhibit unusual thermodynamic, optical and structural features. At identical concentrations the transition temperatures of the oligo(2'-deoxyxylooligomers) are higher than those of the oligo(2'-deoxyribooligomers) indicating higher stability. The calorimetric transition enthalpy of (C) is 270 +/- 15 kJ . mol-1, the corresponding van't Hoff value is 280 +/- 15 kJ . (mol of cooperative unit)-1. The ratio of delta HvH/delta Hcal = 1.04 suggests all-or-none behaviour for the transition of the 2'-deoxyribose oligonucleotide. The analogous parameters of (A) are: delta Hcal = 310 +/- 30 kJ . mol-1, delta HvH = 220 +/- 30 kJ.(mol of cooperative unit)-1. The ratio of 0.71 indicates multistate melting for this compound. The sequence dependence of the thermodynamic quantities becomes apparent when the parameters of the alternating oligo(2'-deoxyxylonucleotide) d[(xA-xT)6-T] are compared to those of d[(xA)3-(xT)3-(xA)3-(xT)3-T). The values are delta Hcal = 330 +/- 30 kJ.mol-1; delta HvH = 180 +/- 15 kJ.(mol of cooperative unit)-1 delta HvH/ delta Hcal = 0.55. The transition enthalpy of the alternating oligo(2'-deoxyxylonucleotide) (B) is the highest but the cooperativity of transition is the lowest of the oligonucleotides studied. The circular dichroic spectra of the two oligo(2'-deoxyxylonucleotides) show unusual features in that d[(xA)3-(xT)3-(XA)3-(xT)3-T] exhibits a spectrum that is suggestive of a left-handed double helix, while the spectrum of the alternating oligo(2'-deoxyxylonucleotide) (B) resembles neither that of (C) nor that of (A).
The H-phosphonates and phosphoramidites of 2'-deoxyisoguanosine, 2'-deoxyisoinosine, 5-aza-7-deaza-2'-deoxyguanosine, and N1-methyl-2'-deoxyformycin A were prepared. The diphenylcarbamoyl group was chosen for the 2-O-protection of 2'-deoxyisoinosine and 2'-deoxyisoguanosine, and dimethylaminoalkylidene groups were used to block the amino function of the various monomers. The synthesis of isoguanine oligonucleotides was found to be much more efficient using the 2-O-protected building blocks compared to those without oxygen protection. Oligodeoxynucleotides containing 2'-deoxyisoguanosine and 2'-deoxycytidine form parallel duplex structures. The self-complementary duplex containing 5-aza-7-deaza-2'-deoxyguanosine and 2'-deoxycytidine forms a stable duplex in acidic solution (pH = 5.0) while it is destabilized under neutral conditions.
1-(2'-Deoxy-beta-D-threo-pentofuranosyl)thymine (xTd) and -adenine (xAd) were converted into their appropriately protected 3'-phosphonates 1a, 2a as well as their 2-cyanoethyl phosphoramidites 1b, 2b. These compounds were used for solid-phase syntheses of the oligo(2'-deoxy-beta-D-xylonucleotides) 5-8. Structural properties and behavior against nucleases is described. Apart from oligo(2'-deoxyxylonucleotides) the PCR-amplification of a pUC18 DNA fragment with Taq polymerase was studied in the presence of the 7-deazapurine derivatives of dGTP, dATP, and dITP. The incorporation efficiency of the modified compounds was compared with those of the parent nucleotides. 7-Deaza-2'-deoxyguanosine protected the DNA-fragment from hydrolysis by the restriction endodeoxyribonuclease Eco RI, Pst I, Bam HI, and Sma I if the nucleoside was located within the recognition site.
Decanucleotide duplexes of the parent sequence d(GGCA6C).d(CCGT6G) containing various numbers of 2'-deoxytubercidin (c7Ad) in place of 2'-deoxyadenosine have been synthesized. Phosphoramidites of protected c7Ad (3a,b) were used in automated solid-phase synthesis together with those of regular nucleosides. Upon enzymic 5'-phosphorylation and ligation, multimers of 5 and 7-11 were analyzed by polyacrylamide gel electrophoresis and compared with regard to intrinsic, sequence-directed bending. Replacement of dA by c7Ad within the oligomers decreased bending, but the extent depends strongly on the position of incorporation: strong bending was still observed if the 3'- and 5'-terminal dA residues of the dA tract were replaced while the interruption of the d(A)6 tract by c7Ad reduced bending strongly.
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The 2'-deoxyguanosine isostere 1 as well as the pyrrolo [3,2-c] pyridine 2'-deoxyribofuranosides 2a,b and 3a-d have been synthesized in high yield by solid-liquid phase-transfer glycosylation. It was shown that only the strongly nucleophilic pyrrolo [3,2-c] pyridines formed exclusively beta-2'-deoxyribofuranosides, whereas weakly nucleophilic imidazo [1,2-a]-s-triazines gave mixtures of anomers.
The N-methyl isomers of 2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (2a) have been synthesized regiospecifically and their structures assigned. The 3-methyl compound 3 was obtained by alkylation of the parent chromophore 2a with dimethyl sulfate, and the 1-methyl isomer 5b was obtained by condensation of ethyl 2-cyano-4,4-diethoxybutyrate with N-methylguanidine and subsequent cyclization. Methylation of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (7b), however, with methyl iodide in the presence of 50% NaOH, by phase-transfer techniques, followed by the replacement of halide by hydroxyl, yielded the 7-methyl compound 2b. The N-methyl isomers of 2a were all found to be inhibitors of xanthine oxidase from cow's milk. While the 3-methyl isomer 3 exhibits a Ki of 40 microM, the 7- and 1-isomers show Ki values of 4.5 and 3 microM, respectively.
7-Deazahypoxanthine was found to be oxidised by cow's milk xanthine oxidase exclusively at carbon 2. The resulting 7-deazaxanthine is a strong inhibitor of the enzymatic reaction. This offers a possibility for determining the structural requirements of ligand binding separately for the first step. All the monomethyl isomers of 7-deazahypoxanthine were tested as probes by measuring their Km, Ki and V values. While the N-3-methyl and C-7-methyl isomers are still processed, the N-9-methyl and 6-O-methyl isomers are bound as inhibitors to the active site. The N-1-methyl compound is neither an inhibitor nor a substrate. This demonstrates that HN(1) and O = C(6) are essential for the binding. Replacement of O = C(6) by S = C(6) changes the substrate into a strong inhibitor (Ki = 9 microM), implying that the electron transfer to the enzyme is hindered. Methylation of the thioxo group (S =) reduces the inhibition significantly. In contrast to 7-deazahypoxanthine, 2-thioxo-7-deazaxanthine is an activator at concentrations below 87 microM and a partial competitive inhibitor above this concentration, which implies the presence of a second binding site.
Dextran-linked adenosine deaminase (EC 3.5.4.4) has been prepared. The polymer-linked enzyme possesses an optimal enzymatic activity of 27 units/mg immobilized protein (non-bound enzyme: 200 units/mg protein). Support-bound adenosine deaminase (4.5 microgram protein/mg dextran) shows an enhanced heat stability, a moderately increased Km, and a decreased V value compared to those of the free enzyme. The pH dependences of V and pKm values of dextran-linked adenosine deaminase show only two inflection points compared to three for the free enzyme, which are equivalent to the pK values of the enzyme. Since the missing third inflection point (pH 9.8) can be assigned to the pK value of the epsilon-amino group of lysyl residues, it can be concluded, that immobilization of adenosine deaminase on cyanogen-bromide-activated dextran took place via these lysyl residues. The remaining pK values found from the other inflection points are moderately shifted owing to the altered secondary structure. From the temperature dependence of the enzymatic activity, a 40% decrease of the activation energy of the support-bound enzyme was found, indicating diffusion controlled deamination. The immobilization of adenosine deaminase results in a fluorescence quenching of 80%, without shifting the ultraviolet maximum of the emission spectrum. As already shown for unmodified dextran, the matrix of polymer-linked adenosine deaminase is degradable by a bacterial endodextranase (EC 3.2.1.11).
Dextran-bound adenosine, inosine, and nebularine have been prepared by carbodiimide coupling of their 2',3'-O-(4-carboxyethyl-1-methylbutylidene) cyclic acetal derivatives to 6-aminohexyldextran or 12-aminododecanyldextran. The latter polymers were prepared by cyanogen-bromide activation of dextran T80 followed by reaction with 1,6-diaminohexane or 1,12-diaminododecane. A high CNBr concentration leads to high-molecular-weight material, probably due to cross-linking, accompanied by a decrease in the digestion velocity using endo-dextranase from Penicillium species (EC 3.2.1.11). The dextran-bound nucleosides, as well as the nucleoside 2',3'-O-(4-ethoxycarbonyl-1-methylbutylidene) acetal derivatives, were tested as substrates and inhibitors for adenosine deaminase. The Km of the adenosine acetal ester is identical to that of adenosine which shows that acetalation does not hinder complex formation. Since the maximum velocity of deamination is decreased fourfold, the modified substrate does not fit as well as the nucleoside. The polymer-bound acetals show a 3-8-fold increase of Km or Ki and unchanged V compared to the corresponding acetals while dextranase digestion of the support does not alter the kinetic data. This indicates that the length of the polysaccharide chain does not interfere either with the complex formation or with the catalytic activity of the modified substrate. Since the activation energies of the deamination reactions of adenosine, its acetal ester, and dextran-linked adenosine are all similar (29.8-32.3 kJ mol-1) it is concluded that no diffusion control of the enzymatic reaction results from the binding of the nucleoside acetals to dextran T80.
Polymer-bound xanthosine (4) has been prepared. Condensation of xanthosine with ethyl 4-oxovalerate and saponification of the product gave 2',3'-O-[1-(2-carboxyethyl)ethylidene] xanthosine. The latter was coupled to 6-aminohexylagarose through its carboxylic group, to yield the polymer 4. The content of bound ligand was 8 mumol/g of moist gel, a value that agrees with the number of free amino groups determined by the trinitrobenzenesulfonic acid assay before coupling. Immobilised xanthosine was used as a biospecific resin (inhibitor resin) for guanine aminohydrolase (EC 3.5.4.3), to separate the enzyme from a mixture containing adenosine deaminase (EC 3.5.4.4).
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Condensation of 6-azauridine with ethyl levulinate, followed by saponification or phosphorylation, leads to 2',3'-O-[1-(2-carboxyethyl)ethylidene]-6-azauridine and its 5'-monophosphate. The latter was coupled to 6-aminohexylagarose via its carboxylic group. Using the same synthetic route, agarose-linked uridine 5'-monophosphate has been prepared. Both polymers show specific binding toward orotidine-5'-monophosphate decarboxylase. The immobilized inhibitor (6-azauridine 5'-monophosphat) binds the enzyme more strongly than the immobilized uridine 5'-monophosphate. Both resins have been used to separate orotidine-5'-monophosphate decarboxylase from orotidine-5'-monophosphate pyrophosphorylase.
Immobilised inosine (6a) and adenosine (6c) and their 5'-phosphates have been synthesized. Reaction of the nucleosides with ethyl levulinate, followed by saponification or phosphorylation and then saponification, gave the 2',3'-O-[1-(2-carboxyethyl)ethylidene] derivatives 3 and 4 and the corresponding 5'-phosphates 2b and 2d. 6-Aminohexylagarose (5) was severally coupled to 2b, 2d, 3, and 4 through the carboxyl groups to give the polymers 6a-d. Adenosine deaminase converts 3 into 4, and 6c into 6a. The polymers can be used as affinity resins for adenosine deaminase, which is bound more strongly to 6c than to 6a. The operational capacity of 6a for adenosine deaminase is constant at 15--25 degrees, but decreases by approximately 16% from 25 degrees to 35 degrees. The resin 6a has been used to separate adenosine deaminase from mixtures containing other enzymes, for example, guanase or alcohol dehydrogenase.