Publications by authors named "Rishi K Jain"

18 Publications

  • Page 1 of 1

Systematic Chemogenetic Library Assembly.

Cell Chem Biol 2020 09 23;27(9):1124-1129. Epub 2020 Jul 23.

Novartis Institute for BioMedical Research, 181 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.
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http://dx.doi.org/10.1016/j.chembiol.2020.07.004DOI Listing
September 2020

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

Nat Chem Biol 2019 02 14;15(2):179-188. Epub 2019 Jan 14.

Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
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http://dx.doi.org/10.1038/s41589-018-0200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251565PMC
February 2019

Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action.

ACS Chem Biol 2019 01 18;14(1):20-26. Epub 2018 Dec 18.

Novartis Institutes for BioMedical Research , 250 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.
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http://dx.doi.org/10.1021/acschembio.8b00656DOI Listing
January 2019

A Photoaffinity Labeling-Based Chemoproteomics Strategy for Unbiased Target Deconvolution of Small Molecule Drug Candidates.

Methods Mol Biol 2017 ;1647:1-18

Novartis Institutes for Biomedical Research, 181 Massachusetts Avenue, Cambridge, MA, 02139, USA.

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.
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http://dx.doi.org/10.1007/978-1-4939-7201-2_1DOI Listing
April 2018

Examining the influence of specificity ligands and ATP-competitive ligands on the overall effectiveness of bivalent kinase inhibitors.

Proteome Sci 2016 17;15:17. Epub 2017 Jul 17.

Novartis Institutes for BioMedical Research, Cambridge, MA 02139 USA.

Background: Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1.

Methods: Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix.

Results: The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity.

Conclusion: Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone.
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http://dx.doi.org/10.1186/s12953-017-0125-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513037PMC
July 2017

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Elife 2015 Dec 10;4. Epub 2015 Dec 10.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.
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http://dx.doi.org/10.7554/eLife.10222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786417PMC
December 2015

Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity.

ACS Chem Biol 2016 Jan 6;11(1):121-31. Epub 2015 Nov 6.

Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.

Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2.
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http://dx.doi.org/10.1021/acschembio.5b00847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943861PMC
January 2016

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

PLoS One 2015 22;10(6):e0127498. Epub 2015 Jun 22.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127498PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476799PMC
May 2016

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Nat Chem Biol 2014 May 16;10(5):343-9. Epub 2014 Mar 16.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
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http://dx.doi.org/10.1038/nchembio.1481DOI Listing
May 2014

Accelerating the discovery of new drug targets with chemical proteomics.

IDrugs 2010 Dec;13(12):862-8

Novartis Institutes for BioMedical Research, Global Discovery Chemistry-Chemogenetics and Proteomics, 250 Massachusetts Aveune, Cambridge, MA 02139, USA.

The application of chemical proteomics to new target discovery can lead to a rapid understanding of disease mechanism and new therapeutic methods. Successful application includes a thorough understanding of SAR and the validation of target relevance using multiple genetic and biochemical methods. This feature review highlights several successful applications of chemical proteomics and outlines the strategy and approaches that lead to the discovery of novel therapeutic targets.
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December 2010

Combined solid/solution phase synthesis of large surface area scaffolds derived from aminomethyl-benzoates.

Tetrahedron Lett 2009 Jun;50(23):2787-2789

Department of Chemistry, Yale University, New Haven, CT 06520, U.S.A.

A set of macrocycles was generated by solid phase synthesis of linear trimers of 5-aminoacyl-3-aminomethyl-benzoates followed by resin cleavage and solution phase macrocyclization. These scaffolds can serve as useful building blocks for molecular recognition studies, especially where differentially functionalized molecular platforms spanning large surface areas are required.
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http://dx.doi.org/10.1016/j.tetlet.2009.03.153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843919PMC
June 2009

1-amino-4-benzylphthalazines as orally bioavailable smoothened antagonists with antitumor activity.

J Med Chem 2009 Jul;52(13):3954-68

Department of Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 01239, USA.

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.
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http://dx.doi.org/10.1021/jm900309jDOI Listing
July 2009

Identification and structure-activity relationships of ortho-biphenyl carboxamides as potent Smoothened antagonists inhibiting the Hedgehog signaling pathway.

Bioorg Med Chem Lett 2009 Jan 3;19(2):328-31. Epub 2008 Dec 3.

Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge, MA 02139, USA.

Ortho-biphenyl carboxamides, originally prepared as inhibitors of microsomal triglyceride transfer protein (MTP) have been identified as novel inhibitors of the Hedgehog signaling pathway. Structure-activity relationship studies for this class of compounds reduced MTP inhibitory activity and led to low nanomolar Hedgehog inhibitors. Binding assays revealed that the compounds act as antagonists of Smoothened and show cross-reactivity for both the human and mouse receptor.
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http://dx.doi.org/10.1016/j.bmcl.2008.11.096DOI Listing
January 2009

Substrate activity screening: a fragment-based method for the rapid identification of nonpeptidic protease inhibitors.

J Am Chem Soc 2005 Nov;127(44):15521-7

Department of Chemistry, University of California-Berkeley, Berkeley, CA 94720, USA.

A new fragment-based method for the rapid development of novel and distinct classes of nonpeptidic protease inhibitors, Substrate Activity Screening (SAS), is described. This method consists of three steps: (1) a library of N-acyl aminocoumarins with diverse, low molecular weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay, (2) the identified N-acyl aminocoumarin substrates are optimized by rapid analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. The SAS method was successfully applied to the cysteine protease cathepsin S, which is implicated in autoimmune diseases. Multiple distinct classes of nonpeptidic substrates were identified upon screening an N-acyl aminocoumarin library. Two of the nonpeptidic substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency for each class. Select nonpeptidic substrates were then directly converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity to cathepsin S. This study demonstrates the unique characteristics and merits of this first substrate-based method for the rapid identification and optimization of weak fragments and provides the framework for the development of completely nonpeptidic inhibitors to many different proteases.
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http://dx.doi.org/10.1021/ja0547230DOI Listing
November 2005

Inhibiting angiogenesis and tumorigenesis by a synthetic molecule that blocks binding of both VEGF and PDGF to their receptors.

Oncogene 2005 Jul;24(29):4701-9

Drug Discovery Program, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC-DRDIS, Tampa, FL 33612, USA.

Angiogenesis depends on vascular endothelial growth factor (VEGF) for initiation and platelet-derived growth factor (PDGF) for maintenance of blood vessels. We have designed a targeted library of compounds from which we identified a novel molecule, GFB-204, that binds PDGF and VEGF, blocks binding of PDGF and VEGF to their receptors (200-500 nM) and subsequently inhibits PDGFR and Flk-1 tyrosine phosphorylation and stimulation of the protein kinases Erk1, Erk2 and Akt and the signal transducer and activator of transcription STAT3. GFB-204 is selective for PDGF and VEGF and does not inhibit EGF, IGF-1 and FGF stimulation of Erk1/2, Akt and STAT3. GFB-204 inhibits endothelial cell migration and capillary network formation in vitro. Finally, treatment of mice with GFB-204 suppresses human tumor growth and angiogenesis. Thus, inhibition of VEGF and PDGF receptor binding with a synthetic molecule results in potent inhibition of angiogenesis and tumorigenesis.
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http://dx.doi.org/10.1038/sj.onc.1208391DOI Listing
July 2005

Blocking angiogenesis and tumorigenesis with GFA-116, a synthetic molecule that inhibits binding of vascular endothelial growth factor to its receptor.

Cancer Res 2004 May;64(10):3586-92

Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA.

A small synthetic library of cyclohexapeptidomimetic calixarenes was prepared to identify disrupters of vascular endothelial growth factor (VEGF) binding to its receptor that inhibits angiogenesis. From this library, we discovered GFA-116, which potently inhibits (125)I-VEGF binding to Flk-1 in Flk-1-overexpressing NIH 3T3 cells and human prostate tumor cells with an IC(50) of 750 nM. This inhibition is highly selective for VEGF in that (125)I- platelet-derived growth factor binding to its receptor is not affected. GFA-116 inhibits VEGF-stimulated Flk-1 tyrosine phosphorylation and subsequent activation of Erk1/2 mitogen-activated protein kinases. Furthermore, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor-dependent stimulation of Erk1/2 phosphorylation are not affected at concentrations as high as 10 microM. In vitro, GFA-116 inhibits angiogenesis as measured by inhibition of migration and formation of capillary-like structures by human endothelial cells as well as suppression of microvessel outgrowth in rat aortic rings and rat cornea angiogenesis. In vivo, GFA-116 (50 mpk/day) inhibits tumor growth and angiogenesis as measured by CD31 staining of A-549 human lung tumors in nude mice. Furthermore, GFA-116 is also effective at inhibiting tumor growth and metastasis to the lung of B16-F10 melanoma cells injected into immunocompetent mice. Taken together, these results demonstrate that a synthetic molecule capable of disrupting the binding of VEGF to its receptor selectively inhibits VEGF-dependent signaling and suppresses angiogenesis and tumorigenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-03-2673DOI Listing
May 2004

D-Ala-D-lac binding is not required for the high activity of vancomycin dimers against vancomycin resistant enterococci.

J Am Chem Soc 2003 Jul;125(29):8740-1

Department of Chemistry, University of California, Berkeley, California 94720, USA.

Covalent dimerization and oligomerization of vancomycin is an important and extensively used strategy to develop analogues active against vancomycin resistant enteroccoci (VRE). Here, we have carried out investigations to probe the role of peptide binding (Lys-d-Ala-d-Lac) in the high anti-VRE activities of covalently linked vancomycin dimers. Covalent dimers of damaged vancomycin (desleucyl) were prepared, and their anti-VRE activities and binding affinities toward various model peptides were measured. Despite the dramatic loss in affinity toward several model peptides in comparison to the corresponding intact vancomycin dimers, these damaged dimers maintained good activity against VRE. These results strongly suggest that the high anti-VRE activities of covalent vancomycin dimers are conferred from mechanisms other than Lys-d-Ala-d-Lac binding.
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http://dx.doi.org/10.1021/ja0359761DOI Listing
July 2003

Directed denaturation: room temperature and stoichiometric unfolding of cytochrome C by a metalloporphyrin dimer.

J Am Chem Soc 2003 Apr;125(15):4420-1

Department of Chemistry, P.O. Box 208107, 225 Prospect Street, Yale University, New Haven, CT 06520-8107, USA.

Using circular dichroism, UV-vis, and trypsin proteolysis, we have shown how a metalloporphyrin dimer induces the unfolding of a protein, cytochrome c, under physiologically relevant conditions and accelerates its rate of proteolytic degradation.
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http://dx.doi.org/10.1021/ja028574mDOI Listing
April 2003