Selective DNA Cleavage Research Articles

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.

Truncated Tetrahedral CdTe QDs Act as Endonucleases through Local ROS Induction

Recently in Nature Chemistry, Kuang and co-workers described truncated tetrahedral chiral cadmium telluride quantum dots (CdTe QDs) as novel endonucleases that cause the selective cleavage of DNA upon circular polarized light excitation by locally inducing the generation of reactive oxygen species. As a result, these CdTe QDs show promise in bioapplications.

Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage

This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules.

Selective cleavage of DNA at guanosine bases which locate in DNA non-duplex portions within duplexes by ruthenium(II) complexes

Ruthenium complexes are well known for their high affinity and reactivity with DNA. There are so many reports about the interactions of ruthenium complexes and duplex DNA. However, their effects on other DNA structures are poorly studied. In this paper, the photo-activated DNA cleavage by two ruthenium(II) complexes was researched. It was found that these complexes can selectively act on the guanosine bases located in DNA non-duplex portions which were contained in the well matched duplex structures. The mechanism was also studied and considered to be a singlet oxygen oxidation. Through the molecular modeling experiment, we proposed that the strong interaction of complexes with non-duplex DNA structure cause the structural selectivity of DNA cleavage. The research show great future prospects of these ruthenium complexes to be used in anticancer drug development and nucleic acid structure research.

Investigation and improvement of DNA cleavage models of polyamide + Cu(II) nuclease + OOH- ligands bound to DNA

BackgroundCopper nucleases as a famous class of artificial metallonucleases have attracted considerable interest in relation to their diverse potentials not only as therapeutic agents but also in genomic researches. Copper nucleases present high efficient oxidative cleavage of DNA, in which DNA strand scission occurs generally after hydrogen atom abstracted from a sugar moiety. In order to achieve the selective cleavage of DNA sequences by copper nucleases, the DNA specific recognition agents of the Dervan-type hairpin and cyclic polyamides can be considered as proper carriers of copper nucleases. Investigation of the DNA cleavage selectivity of copper nucleases assisted by the hairpin and cyclic polyamides at the molecular level has not yet been elucidated.ResultsWe carried out a series of molecular dynamics simulations for the nuclease [Cu(BPA)]2+ or [Cu(IDB)]2+ bound to the hairpin/cyclic polyamide and associated with DNA to investigate the selective DNA cleavage properties of Cu(II)-based artificial nucleases. The simulated results demonstrate that the DNA cleavage selectivity of the two nucleases assisted by the hairpin polyamide is improved efficiently. The [Cu(BPA)]2+ or [Cu(IDB)]2+ nuclease with a substrate OOH- bound to the hairpin polyamide can be stably located at the minor groove of DNA, and possibly abstracts H atom from the sugar of DNA. However, the DNA cleavage properties of the two nucleases assisted by the cyclic polyamide are significantly poor due to the rigidity of linking region between the cyclic polyamide and nuclease. With introduction of the flexible linker -CH2CH2CH2NH2, the modified cyclic polyamide can assist the two copper nucleases to improve the selective DNA cleavage properties efficiently.ConclusionA flexible linker and a proper binding site of the polyamide-type recognition agents play an important role in improving the DNA cleavage selectivity of copper nucleases. Current investigations provide an insight into the DNA cleavage specificities of chemical nucleases assisted by an appropriate nucleic acid recognition agent.

Molecular Dynamics Simulations on Binding Models of Dervan-Type Polyamide + Cu(II) Nuclease Ligands to DNA

Molecular dynamics simulations for the ligand [Cu(BPA)](2+) (BPA = bis(2-pyridylmethyl)amine) nuclease bound to either the single-chain polyamide, ImPyImImPybetaDp, or the antiparallel double-polyamide, (ImImImbetaDp)(2), and associated with DNA were performed to predict the improvement of selective DNA cleavage ability of copper-based chemical nucleases. The results from the simulations indicate that either polyamide-bound [Cu(BPA)](2+) + OOH(-) (which is a necessary substrate in the redox mechanism of DNA cleavage) can locate in the minor groove of DNA in the parallel orientation similar to the X-ray structure. As a consequence of the polyamide + [Cu(BPA)](2+) + OOH(-) ligand binding to DNA, the active end oxygen atom of the OOH(-) substrate is held in close proximity to the known target C1'H or C4'H protons of the DNA. Upon examinations of six different ligands binding to DNA, the binding interaction of the entire ligand with DNA for each polyamide increases with the presence of [Cu(BPA)](2+) + OOH(-) ligand. The N-H groups of the linking regions from polyamide play an important role for the interaction by functioning as H-bond donors to N or O atoms of the nucleobase located on the floor of the minor groove of DNA. The investigation provides the feasible protocol to improve the selective DNA cleavage activity of chemical nucleases assisted by DNA recognition agents.

Solid-phase synthesis and biochemical evaluation of conformationally constrained analogues of deglycobleomycin A5

Deglycobleomycin binds to and degrades the self-complementary oligonucleotide d(CGCTAGCG)2 in a sequence selective fashion. A previous modeling study [J. Am. Chem. Soc. 120, (1998), 7450] had shown that, during binding to double stranded DNA, the conformation of the methylvalerate domain of deglycoBLM approximated that of S-proline. In the belief that an analogue of deglycoBLM structurally constrained to mimic the DNA-bound conformation might exhibit facilitated DNA binding and cleavage, an analogue of deglycoBLM was prepared in which the methylvalerate moiety was replaced by S-proline. This deglycoBLM analogue, as well as the related analogue containing R-proline, was synthesized on a TentaGel resin. Both of the analogues were found to be capable of binding Fe2+ and activating O2 for transfer to styrene. However, both deglycoBLM analogues exhibited diminished abilities to effect the relaxation of supercoiled plasmid DNA, and neither mediated sequence selective DNA cleavage.

Synthesis, DNA cleavage, and cytotoxicity of a series of bis(propargylic) sulfone crown ethers

Compounds that couple molecular recognition of specific alkali metal ions with DNA damage may display selective cleavage of DNA under conditions of elevated alkali metal ion levels reported to exist in certain cancer cells. We have prepared a homologous series of compounds in which a DNA reactive moiety, a bis(propargylic) sulfone, is incorporated into an alkali metal ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ethers to bind Li+, Na+, and K+ ions was determined. For the series of crown ethers, the association constants for Li+ ions are generally low (<2×104M−1). Only two of the bis(propargylic) sulfone crown ethers associate with Na+ or K+ ions (Ka 4–8×104M−1), with little discrimination between Na+ or K+ ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of Li+, Na+, and K+ ions was determined. The two crown ethers that bind Na+ and K+ display a modest increase in DNA cleavage efficiency in the presence of Na+ or K+ ions as compared to Li+ ions. These two bis(propargylic) sulfone crown ethers are also more cytotoxic against a panel of human cancer cell lines when compared to a non-crown ether macrocyclic bis(propargylic) sulfone.

Triplet state mechanism for electron transfer oxidation of DNA

The interaction of anthraquinone-2-sulfonate with nucleotides and DNA in acetonitrile and acetonitrile water solvent mixture have been studied using KrF laser photolysis aimed at elucidation of the reaction mechanism. Laser spectroscopy directly demonstrates that the initial species from interaction of anthraquinone-2-sulfonate with nucleotides are radical cations of nucleotides and radical anion of anthraquinone-2-sulfonate. In addition, formation of ion pair from interaction of any of mucleotides with anthraquinone-2-sulfonate is synchronous with decay of triplet anthraquinone-2-sulfonate, which has provided dynamic evidence for initiation of electron transfer from DNA bases to triplet anthraquinone-2-sulfonate. Moreover, direct observation of stabilized DNA guanyl radical cation from interaction of anthraquinone-2-sulfonate with DNA has provided initial evidence for selective cleavage of DNA at guanine moiety. The solvent-separated ion pairs in acetonitrile have evidently dissociated into free ions, thereby enabling independent study of the behavior of guanyl radical cations and radical anion of anthraquinone-2-sulfonate.

Strand Selective Cleavage of DNA by Diastereomers of Hairpin Polyamide-seco-CBI Conjugates

Pyrrole-imidazole polyamides are synthetic ligands that bind predetermined DNA sequences with subnanomolar affinity. We report the synthesis and characterization of an eight-ring hairpin polyamide conjugated at the turn to both enantiomers of 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI), an alkylating moiety related to CC-1065. Alkylation yields and specificity were determined on a restriction fragment containing six base pair match and mismatch sites. Alkylation was observed at a single adenine flanking the polyamide binding site, and strand selective cleavage could be achieved based on the enantiomer of seco-CBI chosen. At 1 nM concentrations of polyamide-seco-CBI conjugate, near quantitative cleavage was observed after 12 h. These bifunctional molecules could be useful for targeting coding regions of genes and inhibition of transcription.

Molecular mechanisms of DNA recognition and function by bioactive compounds

Molecular mechanisms of DNA recognition and functional expression by bioactive compounds such as bleomycin, enediyne antibiotics, and zinc finger proteins are an important research subject in the pharmaceutical scientific field. Cleavage of cellular DNA by bleomycin substantially contributes to the antitumor activity of this drug. Some physicochemical data have clearly demonstrated that the bleomycin-iron (II) complex forms a dioxygen adduct species. Of special interest is the fact that the bleomycin-iron complex and cytochrome P450 have a similar dioxygen activation cycle. Probably, the site-specific iron-peroxide species of bleomycin accounts for the action mechanism of selective DNA cleavage by bleomycin. The enediyne antibiotics possess an unprecedented chemical structure, potent anticancer activity, and a fascinating mode of biological action. A new compound, C-1027, consists of a carrier apoprotein and an enediyne chromophore. The high-resolution NMR experiments clarified that novel chromophore interacts with DNA through its benzoxazolinate and aminosugar moiety, and also with apoprotein through the benzoxazolinate and macrocyclic moiety. The results provide a molecular basis for the host-recognition mode, the reaction mechanism, and the drug delivery system of chromoprotein C-1027. In addition, we found unique reactivity of C-1027 chromophore toward a tRNA(Phe). RNA as well as DNA might be also a potent biological target of the enediyne antibiotics. On the basis of characteristic DNA binding mode of Cys2His2-type zinc finger motif, artificial zinc finger proteins have been created, and new functions such as DNA cleavage, long sequence binding, and DNA bending are produced. The first artificial His4-type zinc finger protein is also made from Cys-->His mutations of the Cys2His2-type transcription factor Sp1. Such novel zinc finger proteins may be useful as a gene therapeutic agent and a tool for genetic engineering.

N-methyl threonine analogues of deglycobleomycin A 2: Synthesis and evaluation

The synthesis of 5 and its D- allo-threonine epimer 6 and the comparison of their DNA cleavage efficiency and selectivity with that of deglycobleomycin A 2 ( 3) are detailed. The studies illustrate that N-methylation of the l-threonine subunit within deglycobleomycin A 2 dramatically reduces the DNA cleavage efficiency (10–15-fold), weakens and nearly abolishes the inherent DNA cleavage selectivity, but has little effect on the inherent oxidation capabilities of the activated Fe(III) complexes. The results are consistent with a previously unrecognized prominent role for the threonine NH and the potential importance of a hydrogen bond to the Fe(III) hydroperoxide complex of bleomycin or a subsequent activated complex implicated in recent structural models.

Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity.

Non-conservative changes, consisting of Y76E, Y76L, Y76Q and Y76W, have been made to tyrosine 76, one of the key DNA binding residues in DNase I. Normally Y76 inserts into the minor groove of DNA and makes an unusual, hydrophobic, stacking interaction with one of the sugars. All four mutants bind to DNA more tightly than the wild type, but cut it more slowly as assessed by Kunitz assays. This gives a rather small decrease in the specificity constants (Vmax/K(m)) for the hydrolysis of DNA, which is roughly paralleled by the loss of activity towards the non-DNA small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)phosphate. These non-conservative mutants, therefore, show different behaviour to Y76A and Y76G, studied previously [Doherty A.J., Worrall A.F. and Connolly B.A. (1995) J: Mol. Biol., 251, 366-377]. These two mutants both bind to and cut DNA poorly, resulting in large decreases in Vmax/K(m) values. However, they showed little reduction in rates with the chromophoric substrate. It is likely that the altered side chains in the non-conservative mutants are still able to interact productively with the DNA and contribute to the observed DNA distortion that is essential for efficient catalysis. However, these mutations disrupt the active site, most probably by interference with the hydrogen bonded Y76-E78-H134 triad. H134 is a critical hydrolytic residue of DNase I that is essential for catalysis. The DNA cleavage selectivity of the Y76E, Y76L, Y76Q and Y76W mutants were little altered as compared with the wild-type enzyme as measured using the cutting patterns of a 160 base-pair Escherichia coli Tyr T promoter DNA fragment. This confirms earlier observations, with Y76F, Y76A and Y76G, that showed that this tyrosine has little role in DNA cleavage specificity.

Interaction of the periplasmic dG-selective Streptomyces antibioticus nuclease with oligodeoxynucleotide substrates.

The interaction of a periplasmic nuclease, isolated from Streptomyces antibioticus, with several oligodeoxynucleotide substrates has been studied. Double-stranded oligonucleotides that contain sequences of four or more consecutive deoxyguanosine residues are preferentially hydrolyzed, with the strongest cutting site occurring at GGG decreases G. The enzyme does not hydrolyze these sequences in single-stranded DNA. However the sequence selectivity of the nuclease is far from absolute. Other sequences can also be cut, albeit more poorly, and differences in cutting rates are observed for runs of dG bases that differ in their flanking sequences. An oligonucleotide, thirty-six bases in length, that contains a central run of five dG bases has been used to evaluate the importance of the individual deoxyguanosines in recognition and cleavage. With this oligonucleotide cutting takes place at GG[symbol: see text]G decreases G[symbol: see text]G (decreases, most prominent cut; [symbol: see text], less prominent cuts). The use of dG base analogues revealed that two bases, one and two steps removed from the cleavage site in the 5' direction (*G*GG decreases), were of most importance in the determination of the nuclease DNA cleavage selectivity. Of these the inner starred dG was the most critical. The use of 5-methyldeoxycytidine also showed that the dC, base paired to this critical dG, influenced cleavage specificity. The overall pattern of results seen with the base analogues suggested that the nuclease interacted with both strands of the DNA and also contacted the nucleic acid in both the major and minor grooves. Gel retardation analysis together with footprinting experiments using hydroxyl radicals, dimethyl sulfate, and ethylnitrosourea indicated that the nuclease does not form a tight and specific complex with sequences containing dG runs, at least in the absence of the essential co-factor, Mg2+.

Synthesis of key analogs of bleomycin A 2 that permit a systematic evaluation of the linker region: Identification of an exceptionally prominent role for the l-threonine substituent

The synthesis of a full series of analogs 2b-k of deglycobleomycin A 2 ( 2a) containing systematic variations in the linker domain of bleomycin A 2 ( 1) is described. The agents 2b-k, which are not accessible through structural modification of 1 or 2a, constitute key substructure analogs incorporating deep-seated structural modifications in the linker domain capable of delineating the contribution of the individual backbone substituents to the DNA cleavage efficiency, characteristic DNA cleavage selectivity, and double strand to single strand DNA cleavage ratio. The comparative examination of the DNA cleavage properties of the Fe(II) and Fe(III) complexes of 2a-k upon activation by O 2-thiol or H 2O 2, respectively, revealed several characteristic features and trends. First, none of the substituents affect the characteristic 5′-GC, 5′-GT > 5′-GA DNA cleavage selectivity of bleomycin A 2. In contrast, an exceptionally prominent role for the l-threonine substituent and an important role for the C4-methyl substituent of the (2 S,3 S,4 R)-4-amino-3-hydroxy-2-methylpentanoic acid subunit were observed on the DNA cleavage efficiency of the agents. Similarly, the l-threonine substituent was found to substantially increase the ratio of double strand to single strand DNA cleavage events (2–3 times). In a w794 DNA cleavage assay, shortening the linker region by two carbons resulted in an exceptionally large reduction in DNA cleavage efficiency (125 times) and provided an agent that was only 1.3 times more effective than Fe(III) indicating that this deep-seated modification essentially destroys the DNA cleavage capabilities of the agent. The l-threonine substituent contributes in an exceptional manner, and its removal resulted in a 25 times reduction in DNA cleavage efficiency. A substantial contribution was observed for the C4-methyl group on the 4-aminobutanoic acid subunit and its removal resulted in a 7 times reduction in DNA cleavage efficiency. Little effect for the C3-hydroxyl and C2-methyl substituents on the 4- aminobutanoic acid subunit was observed (0–2.5 times) and even their inversion of stereochemistry had little impact on DNA cleavage efficiency or selectivity. Notably, the magnitude of the previously unappreciated l-threonine substituent contribution to the DNA cleavage efficiency and on the ratio of double to single strand DNA cleavage events is the largest effect observed to date including the well recognized disaccharide potentiation (6 times) of the DNA cleavage properties. Consequently, the past role and relative importance of the l-threonine subunit and substituent has been underestimated. Moreover, the cumulative effect of the two important linker chain substituents clearly illustrate that the functional role of this domain is much more important than its simply serving as a linker.

Mutation in Cys662 of Escherichia coliDNA Topoisomerase I Confers Temperature Sensitivity and Change in DNA Cleavage Selectivity

Cys662 is one of the 12 cysteine residues proposed to be co-ordination sites for binding of three Zn(II) in Escherichia coliDNA topoisomerase I. Oligonucleotide-directed mutagenesis was used to convert Cys662 to either serine or histidine. The mutant enzymes were overexpressed and purified to homogeneity. Analysis of the purified enzymes demonstrated that the mutations resulted in loss of one tightly bound Zn(II). In vivocomplementation tests and in vitrorelaxation activity assays at different temperatures showed that the partial relaxation activities retained in the two mutant enzymes were temperature sensitive. Fluorescence measurements indicated that the wild-type and mutant enzymes have structural differences. When DNA cleavage specificity was examined, the mutant enzymes were found to have altered cleavage site preferences. The preferred cleavage sites of the wild-type enzyme all had a cytosine residue four nucleotides to the minus side of the break. The cleavage sites produced by the mutant enzymes did not show a preference for cytosine at that position. f2 f2 Abbreviations used: ICP, inductively coupled plasma; PAR, 4-(2-pyridylazo)resorcinol; PMPS, p-(hydroxymercuri)benzenesulfonate.

Design, synthesis and sequence selective DNA cleavage of functional models of bleomycin—II. 1,2- trans-disubstituted cyclopropane units as novel linkers

The design and synthesis of functional models for bleomycin in which AMPHIS, a simplified model of the metal-chelating subunit of bleomycin is connected to distamycin analogs with a series of linkers, are described. Kinetic studies and DNA cleavage assay show that 1,2- trans-disubstituted cyclopropane units are the best linkers within this series. Study of selective DNA cleavage on high resolution polyacrylamide sequencing gels indicates that the linker modified hybrids generally cleave selectively at the 5′ end of poly T sites at the 3′ end of poly A sites. Cleavage activity is enhanced for most of the compounds related to those with shorter linkers, previously reported, (Huang, L.; Quada, Jr J.C.; Lown, J.W. Bioconjugate Chem. 1995, 6, 21, Ref. 1) probably as a result of the linker allowing the active complex to approach the target deoxyribose more closely and efficiently. Certain of the compounds, ones containing a ( S)-methyl in the linker and the (S,S)-cyclopropyl linker, exhibit unique cleavage sites, indicating that these linkers allow the hybrids to locate novel, individual DNA binding sites.

Design, synthesis, and sequence selective DNA cleavage of functional models of bleomycin. 1. Hybrids incorporating a simple offal-complexing moiety of bleomycin and lexitropsin carriers.

The syntheses of functional models for bleomycin, which are composed of a simple analog of the metal-complexing moiety of bleomycin and oligo-N-methylpyrrole peptide DNA-binding moieties, are described. The extent and the relative rate of their cleavage of DNA in the presence of reductants were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The rate of DNA cleavage increases with the number of N-methylpyrrole units in the carrier moiety. The sequence selectivity of DNA cleavage was demonstrated by polyacrylamide sequencing gel electrophoresis of cleavage reactions on two 5'-32P labeled restriction fragments: a 158 bp GC-rich fragment from pBR322 and a 241 bp AT-rich fragment fragment from SV40. Comparison of the sequence selectivity with that of bleomycin A2 indicates that the poly-N-methylpyrrole moiety directs the hybrid compounds to AT-rich sequences of DNA. High-resolution denaturing gel electrophoresis of DNA cleaved by the model compounds reveals that 3' phosphate is produced exclusively, indicating that the chemistry of DNA cleavage differs from that of bleomycin.

A Novel Zinc Finger-Based DNA Cutter: Biosynthetic Design and Highly Selective DNA Cleavage

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA Novel Zinc Finger-Based DNA Cutter: Biosynthetic Design and Highly Selective DNA CleavageMakoto Nagaoka, Masaki Hagihara, Jun Kuwahara, and Yukio SugiuraCite this: J. Am. Chem. Soc. 1994, 116, 9, 4085–4086Publication Date (Print):May 1, 1994Publication History Published online1 May 2002Published inissue 1 May 1994https://doi.org/10.1021/ja00088a057RIGHTS & PERMISSIONSArticle Views154Altmetric-Citations49LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (885 KB) Get e-Alerts Get e-Alerts