Fast and quantitative high-performance liquid chromatography method for the determination of 9-fluorenylmethoxycarbonyl release from solid-phase synthesis resins.

Base catalyzed cleavage of 9-fluorenylmethoxycarbonyl (FMOC) group and subsequent analysis by UV spectrophotometry is a commonly used technique for measuring the loading of functional groups on solid supports. Recent works suggest that using 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) instead of piperidine makes it possible to use gas chromatography for quantitation, but due to the long deprotection time used, the method is not high-throughput. We observed that the dibenzofulvene released after DBU deprotection could be measured by reversed-phase (RP) HPLC. We have optimized the concentration of DBU as well as the time of the deprotection and coupled with a fast RP-HPLC separation results in a highly reproducible, high-throughput method. The measured loading correspond well with the manufacturer's data on several commercial resins. Using this method we have quantitated the amine loading on several polystyrene resins and we have found that the total amount of functional group can be more than twice the amount of the available ones. We concluded that the differences were the function of the resin loading as well as the level of crosslinking.

Covalent protein-oligonucleotide conjugates for efficient delivery of antisense molecules.

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.

Targeted antisense modulation of inflammatory cytokine receptors.

Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations ( approximately 100 microg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application.

Oligonucleotides tethering Hoechst 33258 derivatives: effect of the conjugation site on duplex stabilization and fluorescence properties.

A series of DNA conjugates have been prepared in which two different derivatives of Hoechst 33258 have been tethered to a sequence containing a 5'-GAATTC-3' target site. The two derivatives differ only in the length of the tether between the DNA and the Hoechst fluorophore. By using a DNA backbone labeling protocol, one in which the Hoechst dye is tethered to an internucleotide phosphoramidate residue, it was possible to easily vary the site of attachment with respect to the A-T rich binding site. When tethered outside the GAATTC sequence, little if any helix stabilization results upon hybridization of the conjugate to its complementary sequence. As the site of conjugation is moved to one end of the target sequence and finally within the AATT sequence, more effective helix stabilization results. When tethered between the two A residues, or between the A and T residue, a delta Tm of at least +20 degrees C is observed. Upon hybridization and formation of the B-form DNA, binding by the tethered Hoechst dye results, and the bound dye becomes brightly fluorescent. Upon a simple titration of the single-stranded conjugate with the complementary target sequence the quantum yield enhancement for hybridization only appears to be 5-7-fold at best. These fluorescence effects, generally less dramatic than those observed with other sequences, result from an increase in quantum yield for the single-stranded conjugate relative to the free Hoechst 33258. Heating the single-stranded conjugate reduces the inherent fluorescence of the single-stranded conjugate to a level comparable with that of the free Hoechst dye. In experiments monitoring absorbance vs temperature, a cooperative transition is observed for the single-stranded conjugate. Both the high quantum yield observed for the single-stranded conjugate and the observed thermally induced transition suggest that the single-stranded conjugate can dimerize (at the GAATTC site), mediated by the groove-binding fluorophore.

Synthesis, analgesic and anti-inflammatory activities of some 1-acyl/aracyl-5-aminopyrazole derivatives.

A series of 1-acyl/aracyl-3, 4-disubstituted-5-aminopyrazoles (IV) were synthesized by the reaction of 1-acyl/aracylhydrazines (I) with an appropriately substituted ketenedithioacetal (II/III). Compounds IV were tested for their analgesic activity by rat caudal immersion test and anti-inflammatory activity by carrageenin induced edema in rat paw test. 1-Benzoyl-3-mercapto-4-carboxamido-5-aminopyrazole (IVk) proved to be the most active compound of the series in both tests.

Synthesis and properties of a Hoechst-like minor-groove binding agent tethered to an oligodeoxynucleotide.

A small Hoechst-like DNA groove-binding fluorophore carrying a terminal bromoacetimido linker has been synthesized. Individual diastereomeric oligodeoxynucleotide dodecamers containing a thiol-based linker attached to an internucleotide phosphoramidate within a sequence of dA-dT residues were each covalently labeled with the new fluorophore. One isomer of the DNA-fluorophore conjugate (Isomer B) hybridizes to the complementary single-stranded target DNA and the presence of the tethered fluorophore results in both increased duplex stability and an enhanced fluorescent signal, presumably due to the fluorophore binding in the dA-dT rich minor groove. Duplex stability is increased by 2-3 degrees C and the fluorescence quantum yield for the fluorophore increases four-fold. In contrast, the other diastereomer (Isomer A) exhibits reduced helix stability (approximately 2 degrees C) and only slight changes in fluorescence intensity upon hybridization.

Activity of the hammerhead ribozyme upon inversion of the stereocenters for the guanosine 2'-hydroxyls.

Two guanosine 2'-hydroxyls in the hammerhead RNA complex at positions G5 and G8 are critical for efficient cleavage by this RNA catalyst. These two functional groups are likely involved in the binding of the metal cofactor, or they are involved in specific interresidue hydrogen-bonding interactions. The importance of the stereochemical positioning of both critical 2'-hydroxyls was investigated by comparing the cleavage rates of three arabinosylguanine-substituted complexes (in which the positions of specific guanosine 2'-hydroxyls were stereochemically altered by inverting the C2' stereocenter) with that of the native complex, as well as with the rates of the dG- and dFG-substituted complexes [in which the 2'-hydroxyls are absent as the result of substitution by 2'-deoxyguanosine (dG) or 2'-deoxy-2'-fluoroguanosine (dFG)]. The G5araG and G8araG complexes exhibit dramatically different cleavage rates. The G5araG complex is essentially inactive, at least 10(5)-fold slower than the native complex. RNA cleavage by this analogue ribozyme is also 1000-fold slower than cleavage by either the G5dG or the G5dFG ribozyme, both of which lack the 2'-hydroxyl at G5. By comparison, catlytic efficiency of the G8araG complex as expressed by kcat/Km is comparable with that of the native complex and some 2 orders of magnitude more active than either the G8dG or the G8dFG complex.

Specific purine N7-nitrogens are critical for high affinity binding by the trp repressor.

We have analysed the interaction of the trp repressor with the trpEDCBA operator using a series of modified trp operator sequences incorporating two isosteric purine analogues that lack N7-nitrogens. Our results suggest that as well as the direct contact between Arg69 and G-9, three additional purine N7-nitrogens, implicated in specific, water-mediated contacts to the repressor, are critical for formation of the high-affinity repressor-operator complex. We conclude that the crystal structure obtained by Otwinowski et al. reflects high-affinity sequence-specific binding of the trp repressor to the trp operator, and that in some cases proteins can use water molecules to extend amino acid side chains in order to derive favorable binding energy in complex formation.

Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme.

Seven modified hammerhead ribozyme/substrate complexes have been prepared in which individual purine nitrogens, the guanine N7-, the guanine N2-, or the adenine N6-nitrogen, have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaguanosine (c7G), inosine (I), or nebularine (purine riboside, P) base analogue as appropriate for residues G5, G8, G12, A13, A14, or A15. Two of the base analogues, c7G5 and C7G8, occur in a 19-mer ribozyme, while the remaining three residues are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes, G5c7G, G8c7G, G12c7G, and A14P, are cleaved with relatively little change in rate when compared with the native complex. Two complexes, A13P and A15P, are cleaved some 6-8-fold slower than the native complex, while the G12I complex is reduced in rate by 50-fold. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM values, for the G5c7G, G8c7G, and G12c7G complexes are only marginally reduced relative to the native complex. The values for the A13P, A14P, and A15P complexes are reduced by 25-, 15-, and 60-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the kcat/KM value for the G12I complex is decreased 450-fold relative to the native complex and is characterized by an 8-fold increase in KM and a kcat value that is reduced nearly 60-fold. These results indicate that the N2-amino group of G12 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.

Synthesis of 11-phenyl-2,3,4,5-tetrahydro-1H-(1,4)diazepino(1,2-a)indoles and 1-(3-aminopropyl)-2-hydroxymethyl-3-phenylindoles as 5-hydroxytryptamine antagonists.

A series of novel 11-phenyl-2,3,4,5-tetrahydro-1H-(1,4)diazepino(1,2-a) indoles (5a-f) and 1-(3-aminopropyl)-2-hydroxymethyl-3-phenylindoles (6a-f) are reported. The compounds (5a-f) were prepared by the lithium aluminum hydride (LAH) reduction of corresponding 11-phenyl-1H-1-oxo-2,3,4,5-tetrahydro(1,4)diazepino(1,2-a)indoles (4a-f). The precursors (4a-f) were, in turn, prepared by the catalytic reduction of 1-(2-cyanoethyl)-3-phenylindole-2-carboxylates (2a-f) and cyclization of the resulting 1-(3-aminopropyl)-3-phenylindole-2-carboxylates (3a-f) with sodium hydride in xylene. The LAH reduction of 2a-f gave exclusively 1-(3-aminopropyl)-2-hydroxymethyl-3-phenylindoles (6a-f) and not the diazepinoindoles (5a-f) which were expected. The title compounds and the intermediates have been screened for their anti-5-hydroxytryptamine (anti-5-HT) activity. The most potent anti-5-HT compounds of this series, 6a and 6e, were found to be weak compared with cyproheptadiene, a standard anti-5-HT drug.

Synthesis and antimicrobial activity of new substituted 10-methyl-1,2-dihydro-1-oxo-1,2,4-triazino(4,5-a)indoles.

Ten new 4,8-disubstituted 10-methyl-1,2-dihydro-1-oxo-1,2,4-triazino(4,5-a)indoles (5 and 6) were prepared by refluxing various 5-substituted indole-2-carbohydrazides (4) with triethylorthoformate or triethylorthoacetate in dimethylformamide. These derivatives were evaluated for their antimicrobial activity. Some of the title compounds possess fairly potent antimicrobial activity.