Solid-phase, single nucleotide primer extension of DNA/RNA hybrids by reverse transcriptases.
A method for RNA analysis based on primer extension by reverse transcriptase is described.
Base Pair Mismatch↗
Biomedical subjects
M C Pirrung
Publications and source records attributed to M C Pirrung.
Photochemically removable silyl protecting groups.
Several o-phenol-containing alkoxyvinylsilanes were prepared and their photochemistry was investigated. These materials were prepared via hydrosilylation of the corresponding o-acetoxy arylacetylenes. Two major classes of photochemical processes were identified in these reactants: trans-->cis isomerization, leading to an intramolecular nucleophilic substitution process at silicon, and 1,5-silyl shift, leading to an unsymmetrical dialkoxysilane. The major outcome of this work is a novel class of photochemically removable protecting groups. Two alkyl substitutions on silicon, the dimethyl and diisopropyl, were examined. The latter is more stable and is preferred for protecting groups that must tolerate multiple steps or reagents. Protection of alcohols is generally performed starting with the arylethynyl acetate, which can be subjected to hydrosilylation, alcohol substitution, and acetate deprotection without isolation of intermediates. Two groups were studied in detail, the phenol and 2-naphthol vinyl silane derivatives. A variety of primary and secondary alcohols were protected with these reagents. These groups can be deprotected cleanly and in high yield by irradiation from 250 to 350 nm.
Acetylene↗
3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5' --> 3' photochemical DNA synthesis.
[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synthesized probes. We have developed new pyrimidine building blocks for 5' --> 3' DNA synthesis with high cycle yields using the NPPOC (3'-nitrophenylpropyloxycarbonyl) protecting group. These phosphoramidites were proved in automated photochemical DNA synthesis on a modified synthesizer.
Automation↗
A universal, photocleavable DNA base: nitropiperonyl 2'-deoxyriboside.
A universal, photochemically cleavable DNA base analogue would add desirable versatility to a number of methods in molecular biology. A novel C-nucleoside, nitropiperonyl deoxyriboside (NPdR, P), has been investigated for this purpose. NPdR can be converted to its 5'-DMTr-3'-CE-phosphoramidite and was incorporated into pentacosanucleotides by conventional synthesis techniques. The destabilizing effect on hybrid formation with a complementary strand when this P base opposes A, T, and G was found to be 3-5 kcal/mol, but 9 kcal/mol when it opposes C. Brief irradiation (lambda > 360 nm, 20 min) of DNA containing the P base and piperidine treatment causes strand cleavage giving the 3'- and 5'-phosphates. Two significant recent interests, universal/non-hydrogen-bonding base analogues and photochemical backbone cleavage, have thus been combined in a single molecule that serves as a light-based DNA scissors.
Base Sequence↗
Synthesis of 3-indolyl-2,5-dihydroxybenzoquinones.
[figure: see text] 3-Indolylquinones can be efficiently prepared by the acid-catalyzed condensation of Indoles with 2,5-dichiorobenzoquinone, followed by DDQ oxidation. The resulting dichloroquinones are hydrolyzed to the 3-indolyidihydroxybenzoquinones. The 3-indolylquinone substructure is of interest because of its presence in natural products that modulate biological processes through protein-protein interactions, including the asterriquinones.
Benzoquinones↗
APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.
Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.
DNA Mutational Analysis↗
Thiophosphorylation of histidine.
The preparation of a novel phosphorus species, thiophosphoramidate, has enabled the specific thiophosphorylation of histidine at its 3-position. The rates of phosphorylation and thiophosphorylation of histidine are reported, as well as the spectroscopic properties of both thiophosphoramidate and 3-thiophosphohistidine. Structural assignment of the latter was made by analogy to the NMR properties of the known 3-phosphohistidine. The alkylation of 3-thiophosphohistidine by phenacyl bromide serves as a model for the introduction of labeling or probe reagents into histidine phosphorothioate-containing proteins.
Chromatography, High Pressure Liquid↗
Discovery of selective metal-binding peptoids using 19F encoded combinatorial libraries.
A method for encoding solid-phase split/mix combinatorial libraries using the chemical shift of synthetic fluoroarenes ('F-codes') has been developed. They have wide chemical shift dispersion and are detectable at the sub-micromol level. 19F NMR is used for decoding. Nine fluoroarenes bearing linkers for attachment to solid-phase synthesis supports through a photocleavable group were prepared. A library of 90 N-alkylglycines bearing substituted succinamides was prepared on solid phase from nine amines, in which the amine is encoded by the fluorinated tag, and 10 anhydrides. Metal binding studies followed by decoding identified unique, specific binders of copper(II) and iron(III) with microM K(D)s.
Amides↗
Synthesis of (+/-)-homohistidine.
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Enzyme Inhibitors↗
Dipolar cycloaddition of rhodium-generated carbonyl ylides with p-quinones.
[reaction: see text] The dipolar cycloaddition of carbonyl ylides generated by the rhodium-catalyzed decomposition of delta- and epsilon-carbonyl-alpha-diazoketones with p-quinones leads to both C=O and C=C addition products. The product ratio is solvent- and catalyst-dependent and has been optimized to favor formation of either product. The C=C addition products of naphthoquinones are used in the assembly of structures hybridizing the illudin and anthraquinone anticancer agents.
Antibiotics, Antineoplastic↗
Available pathways database (APD): an essential resource for combinatorial biology.
A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for combinatorial biology experiments. It provides the same type of resource for combinatorial biology as the Available Chemicals Directory (ACD) does for combinatorial chemical library generation. Its cataloging ability can also provide insight into novel aspects of biosynthetic routes. In particular, no 10-unit Type I polyketides were found in the compilation of this edition of the APD (Version I).
Bacteria↗
Histidine kinases and two-component signal transduction systems.
The phosphorylation of histidine is the first step in many signal transduction cascades in bacteria, yeast and higher plants. The transfer of a very reactive phosphoryl group from phosphorylated histidine kinase to an acceptor is an essential step in many cellular signaling responses.
Bacteria↗
Ethylene biosynthesis: processing of a substrate analog supports a radical mechanism for the ethylene-forming enzyme.
BACKGROUND: The chemical mechanism of the final step of ethylene biosynthesis (the conversion of 1-aminocyclopropanecarboxylic acid, ACC, to ethylene by ACC oxidase, the ethylene-forming enzyme, EFE) is poorly understood. Two possibilities have been suggested: a radical mechanism and an N-hydroxylation mechanism. We investigated reaction pathways available to radical intermediates in this reaction using an ACC analog, 1-aminocyclobutanecarboxylic acid (ACBC) as a substrate. RESULTS: ACBC was converted to dehydroproline (delta 1-pyrroline-2-carboxylic acid) by the EFE via a ring expansion process. The possibility that an N-hydroxy-aminoacid (produced during two-electron oxidation) acts as an intermediate in this process was eliminated by control experiments. Chemical model reactions involving two-electron oxidants, such as a positive halogen (X+), which presumably generate N-halo derivatives, produce only decarboxylation products. Radical-based oxidants, in contrast, generate dehydroproline. Model reactions involving sequential single-electron transfer mechanisms also produce dehydroproline; thus our results support the proposal that the EFE-catalyzed step of ethylene biosynthesis proceeds using a radical-based mechanism. CONCLUSIONS: Our results provide support for a radical mechanism in the final step of ethylene biosynthesis and refute an alternative N-hydroxylation mechanism. This work extends the idea that the intrinsic chemical reactivity of a high energy iron-oxo intermediate can account for the observed products in ethylene biosynthesis.
Amino Acids↗
Discovery of a novel tetrahydroacridine acetylcholinesterase inhibitor through an indexed combinatorial library.
BACKGROUND: Methods for the rapid and efficient preparation of drug candidates through combinatorial chemistry are of increasing interest. We have previously reported an indexed combinatorial library method that allows both the preparation and testing of compounds in solution. We set out to apply this method to develop more effective analogs of the known, marketed drug tacrine, an acetylcholinesterase inhibitor. RESULTS: A one-step condensation of cyclohexanones with cyanoanilines to generate tetrahydroacridine pools was developed. The resulting library of (formally) 72 tetrahydroacridines was screened against acetylcholinesterase, and a compound 10-fold more potent than tacrine, 7-nitrotacrine, was discovered. Its increased potency could be readily explained by examining the known structure of the complex of acetylcholinesterase with tetrahydroacridine. CONCLUSIONS: In this work, we have provided a relatively rare example of carbon-carbon bond formation in a pool synthesis and have discovered a potentially useful acetylcholinesterase inhibitor.
Acridines↗
Purification and properties of the apple fruit ethylene-forming enzyme.
The enzyme that oxidatively converts 1-aminocyclopropanecarboxylic acid (ACC) to ethylene, a key plant growth hormone, has been classified, on the basis of a comparison of homologous protein sequences (derived from the cDNA sequences), as a member of a family of non-heme iron proteins that includes plant and bacterial oxidative enzymes. This knowledge has facilitated the purification of the relatively abundant ethylene-forming enzyme to homogeneity from apple tissue. The properties of the enzyme are consistent with two other recent reports that describes its purification by different protocols, lending credence to the assertion that the key protein has been isolated. New characterizations of the protein have been conducted. Electrospray mass spectrometry shows that its molecular weight (35 331.8 +/- 5 amu) is approximately 50 amu higher than that predicted from the cDNA sequence, identifying the blocking group at the N-terminus as acetyl. The enzyme is activated by bicarbonate at low concentration but is inhibited at high concentration, with the maximum activation occurring at 5 mM. The iron concentration leading to half-maximal activity is 1 microM. The enzyme self-inactivates during turnover. The availability of the purified enzyme will permit definitive studies of the mechanism by which ethylene is produced and provide opportunities to discover molecules that inhibit the process.
Amino Acid Sequence↗
Light-directed, spatially addressable parallel chemical synthesis.
Solid-phase chemistry, photolabile protecting groups, and photolithography have been combined to achieve light-directed, spatially addressable parallel chemical synthesis to yield a highly diverse set of chemical products. Binary masking, one of many possible combinatorial synthesis strategies, yields 2n compounds in n chemical steps. An array of 1024 peptides was synthesized in ten steps, and its interaction with a monoclonal antibody was assayed by epifluorescence microscopy. High-density arrays formed by light-directed synthesis are potentially rich sources of chemical diversity for discovering new ligands that bind to biological receptors and for elucidating principles governing molecular interactions. The generality of this approach is illustrated by the light-directed synthesis of a dinucleotide. Spatially directed synthesis of complex compounds could also be used for microfabrication of devices.
Amino Acid Sequence↗
De novo synthesis of carbohydrates by stereoselective aldol reaction: L-cladinose.
Aldol reactions of methyl 2-methoxypropanoate (4), the corresponding ester of 2-methoxypropanoic acid with 4-methyl-2,6-di-(tert-butyl)phenol (13), and silylketene acetals 14 and 15 with (S)-2-(phenyl-methoxy)propanal (17) have been investigated. The lithium enolate of 4 reacts with 17 to give primarily beta-hydroxy ester 18a. If the reaction is carried out with the bis-silylketene acetal 14 under the influence of stannic chloride, beta-hydroxy acid 20c is produced. Compound 20c is cleanly inverted, via the beta-lactone 26, to provide beta-hydroxy acid 19c. Compound 18a has been converted into L-cladinose by the sequence of steps: 18a----35----39----40----41----42----1.
Chemical Phenomena↗
Caged chemotactic peptides.
This work has as its ultimate goal the creation of a concentration spike of a chemoattractant peptide in a time-resolved and spatially defined way using a light pulse. This strategy requires "caging" the peptide with a photochemically removable group. Model studies used alanine ethyl ester in reductive amination with nitrobenzaldehydes to form two different N-nitrobenzyl derivatives. An fMLF peptide bearing these two N-terminal nitrobenzyl groups was also prepared. The yield and kinetics of their deprotection to return the fMLF peptide were determined. It was established that the caged peptides have vastly reduced biological activity as chemoattractants, as designed.
Animals↗