In Vivo profiling and visualization of cellular protein-lipid interactions using bifunctional Fatty acids.
Haberkant, P., Raijmakers, R., Wildwater, M., Sachsenheimer, T., Brugger, B., Maeda, K., Houweling, M., Gavin, A.C., Schultz, C., van Meer, G., Heck, A.J. & Holthuis, J.C.
Angew Chem Int Ed Engl. 2013 Apr 2;52(14):4033-8. doi: 10.1002/anie.201210178.Epub 2013 Feb 28.
Bifunctional lipid technology: Cells convert externally added photoactivatable and "clickable" fatty acids into a variety of bifunctional phospholipids that can be covalently linked to their protein-binding partners by irradiation with UV light. Derivatization of the clickable group with a reporter molecule makes it possible to identify and image the lipid-bound proteins in situ.
The power of fluorogenic probes.
Nadler, A. & Schultz, C.
Angew Chem Int Ed Engl. 2013 Feb 25;52(9):2408-10. doi: 10.1002/anie.201209733.Epub 2013 Jan 21.
A definite turn-on: Turning on fluorescence only where successful labeling is happening sounds as desirable as delivering a drug only where the drug target resides. New fluorogenic xanthene derivatives from the Bertozzi research group are getting us closer to "magic bullet" dyes.
A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.
Lukinavicius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Correa IR, J.r, Luo, Z.G., Schultz, C., Lemke, E.A., Heppenstall, P., Eggeling, C., Manley, S. & Johnsson, K.
Nat Chem. 2013 Feb;5(2):132-9. doi: 10.1038/nchem.1546. Epub 2013 Jan 6.
The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.
HyPer-3: A Genetically Encoded H2O2 Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging.
Bilan, D.S., Pase, L., Joosen, L., Gorokhovatsky, A.Y., Ermakova, Y.G., Gadella, T.W., Grabher, C., Schultz, C., Lukyanov, S. & Belousov, V.V.
ACS Chem Biol. 2013 Mar 15;8(3):535-42. doi: 10.1021/cb300625g. Epub 2013 Jan 7.
High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.
The fatty acid composition of diacylglycerols determines local signaling patterns.
Nadler, A., Reither, G., Feng, S., Stein, F., Reither, S., Mueller, R. & Schultz, C.
Angew. Chem. Int. Ed. 52, in press (2013). DOI: 10.1002/anie.201301716
Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation.
Borrmann, A., Milles, S., Plass, T., Dommerholt, J., Verkade, J.M., Wiessler, M., Schultz, C., van Hest, J.C., van Delft, F.L. & Lemke, E.A.
Chembiochem. 2012 Sep 24;13(14):2094-9. doi: 10.1002/cbic.201200407. Epub 2012Sep 3.
Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.
The chemical biology of phosphoinositide 3-kinases.
Wymann, M.P. & Schultz, C.
Chembiochem. 2012 Sep 24;13(14):2022-35. doi: 10.1002/cbic.201200089. Epub 2012Sep 10.
Since its discovery in the late 1980s, phosphoinositide 3-kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug-discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms.
Protein tango: the toolbox to capture interacting partners.
Rutkowska, A. & Schultz, C.
Angew Chem Int Ed Engl. 2012 Aug 13;51(33):8166-76. doi: 10.1002/anie.201201717.Epub 2012 Jun 11.
The evaluation of protein function in the context of the whole cell is crucial for understanding of living systems. In this context, the identification and modulation of protein-protein interactions in and outside cells is of ample importance. Several methods have been developed in the past years to detect and/or actively induce protein-protein interactions in living cells. As a result, tools are now available to manipulate intracellular events by reversible or irreversible cross-linking of proteins in a specific manner. These techniques open many new doors and enable the dissection of complicated protein networks. Herein we describe which cross-linkers and inducers of dimerization are out there and how to make use of this great toolbox.
Target-activated prodrugs for the auto-regulated inhibition of MMP12.
Cobos-Correa, A., Stein, F., Schultz, C.
ACS Med. Chem. Lett., 2012, 3 (8), pp 653-657, doi: 10.1021/ml3001193.
We describe a prodrug concept in which the target enzyme MMP12 produces its own inhibitor in a two-step activation procedure. By using an MMP12-specific peptide sequence and a known sulfonamide drug integrated in the backbone, the active inhibitor is released upon enzyme cleavage. In in vitro experiments, we present proof of concept that the activation proceeds with useful kinetics. The approach is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in a highly specific manner only at such sites where enzyme activity resides.
Can We See PIP(3) and Hydrogen Peroxide with a Single Probe?
Mishina, N.M., Bogeski, I., Bolotin, D.A., Hoth, M., Niemeyer, B.A., Schultz, C., Zagaynova, E.V., Lukyanov, S. & Belousov, V.V.
Antioxid Redox Signal. 2012 Aug 1;17(3):505-12. Epub 2012 Apr 10.
Abstract A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H(2)O(2)) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H(2)O(2). The dynamics of PIP(3) and H(2)O(2) generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts. Antioxid. Redox Signal. 17, 505-512.
Protein translocation as a tool: The current rapamycin story.
Putyrski, M. & Schultz, C.
FEBS Lett. 2012 Jul 16;586(15):2097-105. doi: 10.1016/j.febslet.2012.04.061. Epub2012 May 11.
In cell biology and pharmacology, small chemicals are mostly used as agonists and antagonists against receptors and enzymes. The immunosuppressant rapamycin can serve an entirely different purpose: if employed sensibly, it might function as an inducer of dimerization that is able to rapidly activate enzyme activity inside the intact cell. A number of very recent developments such as photoactivatable derivatives make rapamycin an even more attractive tool for basic science.
Conformational Analysis of a Genetically Encoded FRET Biosensor by SAXS.
Mertens, H.D., Piljic, A., Schultz, C. & Svergun, D.I.
Biophys J. 2012 Jun 20;102(12):2866-75. Epub 2012 Jun 19.
Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.
Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters.
Gehrig, S., Mall, M.A. & Schultz, C.
Angew Chem Int Ed Engl. 2012 Jun 18;51(25):6258-61. doi: 10.1002/anie.201109226.Epub 2012 May 3.
Finding NEmo: In a mouse model for lung inflammation, the lipidated peptide-based ratiometric fluorescent reporter NEmo-2 indicates that neutrophil elastase (NE) activity is mainly associated with the surface of neutrophils, whereas a soluble reporter variant showed no activity in the lung fluid, likely because of the abundance of antiproteases. Targeting the localization of NE activity has great potential for the development of improved antiinflammatory drugs.
Amino acids for diels-alder reactions in living cells.
Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2012 Apr 23;51(17):4166-70. doi: 10.1002/anie.201108231.Epub 2012 Mar 30.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site-specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans-cyclooctene units that react with tetrazine derivatives in an inverse-electron-demand Diels-Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide-cyclooctyne click reaction (right).
CFTR Regulates Early Pathogenesis of Chronic Obstructive Lung Disease in betaENaC-Overexpressing Mice.
Johannesson, B., Hirtz, S., Schatterny, J., Schultz, C. & Mall, M.A.
PLoS One. 2012;7(8):e44059. Epub 2012 Aug 24.
BACKGROUND: Factors determining the onset and severity of chronic obstructive pulmonary disease remain poorly understood. Previous studies demonstrated that airway surface dehydration in betaENaC-overexpressing (betaENaC-Tg) mice on a mixed genetic background caused either neonatal mortality or chronic obstructive lung disease suggesting that the onset of lung disease was modulated by the genetic background. METHODS: To test this hypothesis, we backcrossed betaENaC-Tg mice onto two inbred strains (C57BL/6 and BALB/c) and studied effects of the genetic background on neonatal mortality, airway ion transport and airway morphology. Further, we crossed betaENaC-Tg mice with CFTR-deficient mice to validate the role of CFTR in early lung disease. RESULTS: We demonstrate that the C57BL/6 background conferred increased CFTR-mediated Cl(-) secretion, which was associated with decreased mucus plugging and mortality in neonatal betaENaC-Tg C57BL/6 compared to betaENaC-Tg BALB/c mice. Conversely, genetic deletion of CFTR increased early mucus obstruction and mortality in betaENaC-Tg mice. CONCLUSIONS: We conclude that a decrease or absence of CFTR function in airway epithelia aggravates the severity of early airway mucus obstruction and related mortality in betaENaC-Tg mice. These results suggest that genetic or environmental factors that reduce CFTR activity may contribute to the onset and severity of chronic obstructive pulmonary disease and that CFTR may serve as a novel therapeutic target.
A FlAsH-Based Cross-Linker to Study Protein Interactions in Living Cells.
Rutkowska, A., Häring, C.H. & Schultz, C.
Angew Chem Int Ed Engl. 2011 Dec 23;50(52):12655-8. doi:10.1002/anie.201106404. Epub 2011 Nov 16.
As you like it: xCrAsH, a dimeric derivative of the arsenical compound FlAsH, enables the highly specific, covalent cross-linking of two proteins containing a 12 amino acid peptide tag. This inducible and (by addition of dithiols) reversible system can be used to detect and manipulate protein-protein interactions both in vitro and in living cells.
Switching heterotrimeric G protein subunits with a chemical dimerizer.
Putyrski, M. & Schultz, C.
Chem Biol. 2011 Sep 23;18(9):1126-33.
The selective manipulation of single intracellular-signaling events remains one of the key tasks when studying signaling networks. Here, we demonstrate for the first time the stimulation of FKBP fusions of various subunits of heterotrimeric G proteins by the simple addition of the chemical dimerizer rapamycin. Activation of constitutively active Galpha(q), but not its GDP-bound form, leads to sustained oscillations of intracellular calcium and myo-inositol 1,4,5-trisphosphate (InsP(3)) levels in HEK cells, independent of the activation of endogenous Galpha(q), in full agreement with the InsP(3)-Ca(2+) cross-coupling model of calcium oscillations. Rapamycin-induced translocation of wild-type Galpha(s) to the plasma membrane results in elevated cAMP levels. Activation of rapamycin-inducible Galpha(s) or Gbeta(1)gamma(2) evokes extensive modulation of ATP-induced calcium transients. The results demonstrate that inducible heterotrimeric G protein subunits will provide ways for dissecting G protein-coupled receptor signaling.
Principles for designing fluorescent sensors and reporters.
Lemke, E.A. & Schultz, C.
Nat Chem Biol. 2011 Jul 18;7(8):480-3. doi: 10.1038/nchembio.620. PubMed
Rapid development of genetically encoded FRET reporters.
Piljic, A., de Diego, I., Wilmanns, M. & Schultz, C.
ACS Chem Biol. 2011 Jul 15;6(7):685-91. Epub 2011 Apr 28.
To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II alpha (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.
The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease.
Zhou, Z., Duerr, J., Johannesson, B., Schubert, S.C., Treis, D., Harm, M., Graeber, S.Y., Dalpke, A., Schultz, C. & Mall, M.A.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S172-82.
Chronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl(-) secretion and increased Na(+) absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na(+) channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of betaENaC-transgenic mice demonstrated that increased airway Na(+) absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction an
Genetically encoded copper-free click chemistry.
Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2011 Apr 18;50(17):3878-81. doi:10.1002/anie.201008178. Epub 2011 Mar 23. PubMed
Photoactivatable and cell-membrane-permeable phosphatidylinositol 3,4,5-trisphosphate.
Mentel, M., Laketa, V., Subramanian, D., Gillandt, H. & Schultz, C.
Angew Chem Int Ed Engl. 2011 Apr 11;50(16):3811-4. doi:10.1002/anie.201007796. Epub 2011 Mar 14. PubMed
Schultz, C. & Aime, S.
Bioorg Med Chem. 2011 Feb 1;19(3):1022. PubMed
Does cellular hydrogen peroxide diffuse or act locally?
Mishina, N.M., Tyurin-Kuzmin, P.A., Markvicheva, K.N., Vorotnikov, A.V., Tkachuk, V.A., Laketa, V., Schultz, C., Lukyanov, S. & Belousov, V.V.
Antioxid Redox Signal. 2011 Jan 1;14(1):1-7. Epub 2010 Oct 12.
Understanding of redox signaling requires data on the spatiotemporal distribution of hydrogen peroxide (H(2)O(2)) within the cell. The fluorescent reporter HyPer is a powerful instrument for H(2)O(2) imaging. However, rapid diffusion of HyPer throughout the nucleocytoplasmic compartment does not allow visualization of H(2)O(2) gradients on the micrometer scale. Here we dramatically improved the spatial resolution of H(2)O(2) imaging by applying subcytoplasmic targeting of HyPer. The membrane-attached reporters identified "microdomains" of elevated H(2)O(2) levels within the cytoplasm of the cells exposed to growth factors. We demonstrate that diffusion of H(2)O(2) across the cytoplasm was strongly limited, providing evidence that H(2)O(2) acts locally inside cells.
Covalent Labeling of Biomolecules in Living Cells.
Plass, T. and Schultz, C.
Advanced Fluorescence Reporters in Chemistry and Biology III: Applications to Sensing and Imaging. A. P. Demchenko, (Ed.); Springer Series on Fluorescence, vol. 10. (O. S. Wolfbeis, Series Ed.); ISBN: 978-3-642-18034-7.
Inositol pentakisphosphate isomers bind PH domains with varying specificity and inhibit phosphoinositide interactions.
Jackson, S.G., Al-Saigh, S., Schultz, C. & Junop, M.S.
BMC Struct Biol. 2011 Feb 10;11:11.
BACKGROUND: PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. RESULTS: In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. CONCLUSIONS: These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathways.
Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice.
Mall, M.A., Button, B., Johannesson, B., Zhou, Z., Livraghi, A., Caldwell, R.A., Schubert, S.C., Schultz, C., O'Neal, W.K., Pradervand, S., Hummler, E., Rossier, B.C., Grubb, B.R. & Boucher, R.C.
J Biol Chem. 2010 Aug 27;285(35):26945-55. Epub 2010 Jun 21.
Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.
Challenges in studying phospholipid signaling.
Nat Chem Biol. 2010 Jul;6(7):473-5. PubMed
Activation of membrane-permeant caged PtdIns(3)P induces endosomal fusion in cells.
Subramanian, D.*, Laketa, V.*, Muller, R., Tischer, C., Zarbakhsh, S., Pepperkok, R. & Schultz, C. * - equally contributing first authors.
Nat Chem Biol. 2010 May;6(5):324-6. Epub 2010 Apr 4.
Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phospholipid residing on early endosomes, where it is proposed to be involved in endosomal fusion. We synthesized membrane-permeant derivatives of PtdIns(3)P, including a caged version that is to our knowledge the first photoactivatable phosphoinositide derivative developed so far. In living cells, photoactivation of caged PtdIns(3)P induced rapid endosomal fusion in an EEA1-dependent fashion, thus providing in vivo evidence that PtdIns(3)P is a sufficient signal for driving this process.
The ßENaC-overexpressing mouse as a model of cystic fibrosis lung disease.
Zhou, Z., Duerr, J., Johannesson, B., Schubert, S. C., Treis, D., Harm, M., Gräber, S., Dalpke, A., Schultz, C., Mall, M. A.
J. Cystic Fibr. (2010). In press
Imaging lipids in living cells.
Schultz, C., Neef, A., Goedhart, J. & Gadella, T.W.J.
In 'Live Cell Imaging', Spector, D.L., Svedlow, J. & Jaron, B.F (eds), 2010, Cold Spring Harbor Press
Membrane-permeant phosphoinositide derivatives as modulators of growth factor signaling and neurite outgrowth.
Laketa, V., Zarbakhsh, S., Morbier, E., Subramanian, D., Dinkel, C., Brumbaugh, J., Zimmermann, P., Pepperkok, R. & Schultz, C.
Chem Biol. 2009 Nov 25;16(11):1190-6.
Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.
Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation.
Cobos-Correa, A., Trojanek, J.B., Diemer, S., Mall, M.A. & Schultz, C.
Nat Chem Biol. 2009 Sep;5(9):628-30. Epub 2009 Aug 2.
MMP12 is a metalloproteinase implicated in inflammation. To monitor its activity, we synthesized a membrane-targeted reporter (LaRee1) based on Foerster resonance energy transfer (FRET). Unlike existing sensors, LaRee1 detects MMP12 activity by loss of FRET plus internalization of the lipidated fragment. In bronchoalveolar lavages from a mouse model of pulmonary inflammation, LaRee1 detected MMP12 activity at the surface of activated macrophages. LaRee1 may become a powerful tool for monitoring lung disease.
Chem Biol. 2009 Feb 27;16(2):107-11.
A fluorescent protein from jellyfish changed the way life science research is performed today. Its discovery, the first expression in an animal, the determination of its structure, the details of the mechanism behind the fluorescence, and diversification of the fluorescent properties has made green fluorescent protein a unique tool in the biological sciences, and the scientists that made key contributions to these developments were awarded the 2008 Nobel Prize in Chemistry.
Chemical biologists gather in Heidelberg.
Köhn, M. & Schultz, C.
Nat Chem Biol. 2009 Feb;5(2):66-9.
Chemical biology is well defined at its core--chemistry helping to answer biological questions--yet the boundaries are rather fuzzy. What are the differences between chemical biology and pharmacology? Is intracellular imaging a branch of chemical biology, and what about screening libraries? At Chemical Biology 2008, held in Heidelberg in October, participants heard presentations covering all these topics and more.
Selective fluorescence labeling of lipids in living cells.
Neef, A.B. & Schultz, C.
Angew Chem Int Ed Engl. 2009;48(8):1498-500.
Click chemistry in vivo: Three phosphatidic acid derivatives with alkyne groups in their fatty acid chains were synthesized and incorporated into mammalian cell membranes. Copper(I)-catalyzed and strain-promoted azide-alkyne cycloaddition reactions were used for their visualization (see schematic representation and fluorescence microscopic image).
Analysis of protein complex hierarchy in living cells.
Piljic, A. & Schultz, C.
ACS Chem Biol. 2008 Dec 19;3(12):749-55. PubMed
Contribution of fluorophores to protein kinase C FRET probe performance.
Jost, C.A., Reither, G., Hoffmann, C. & Schultz, C.
Chembiochem. 2008 Jun 16;9(9):1379-84. PubMed
Simultaneous recording of multiple cellular events by FRET.
Piljic, A. & Schultz, C.
ACS Chem Biol. 2008 Mar 20;3(3):156-60.
The function of many sensors for measuring intracellular events is based on Forster resonance energy transfer (FRET). Here we demonstrate for the first time the use of multiple ratiometric FRET sensors in parallel through spatial and spectral resolution. We monitored three calcium-dependent signaling events by a cytosolic sensor for calcium/calmodulin-dependent protein kinase IIalpha, a membrane-bound sensor for protein kinase C, and a translocating FRET probe based on annexin A4. This multiparameter imaging approach gives insight into the precise timing of cellular events within one single cell, thereby providing a major advantage over single-parameter protocols. This type of imaging will likely be important for high content cell analysis and screening efforts in the future.
Heterogeneity and timing of translocation and membrane-mediated assembly of different annexins.
Skrahina, T., Piljic, A. & Schultz, C.
Exp Cell Res. 2008 Mar 10;314(5):1039-47. Epub 2007 Nov 29.
Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.
Fluorescence and bioluminescence procedures for functional proteomics.
Prinz, A., Reither, G., Diskar, M. & Schultz, C.
Proteomics. 2008 Mar;8(6):1179-96.
This review aims to provide an overview of current optical procedures used in functional proteomics, investigating protein localization, protein-protein interaction, intracellular signaling events, and second messenger generation in living cells. Reporter assays using proteins tagged with fluorescent or bioluminescent moieties are discussed. Recently, intracellular biosensor assays, flow cytometry-based techniques (fluorescent cell barcoding), as well as transfected cell microarray assays involving RNA interference coupled with automated imaging were introduced and have been adopted as screening platforms for annotating small molecules, investigating signaling events, or in phenotype analysis. These novel methodological advances include improved image acquisition and processing techniques and help linking in vitro observations to in vivo processes. In addition, the acquired data are increasingly quantitative in nature and will therefore pave the way for modeling of signaling cascades and other complex cellular events, an important step toward systems biology.
Probing lipid- and drug-binding domains with fluorescent dyes.
Black, S.L., Stanley, W.A., Filipp, F.V., Bhairo, M., Verma, A., Wichmann, O., Sattler, M., Wilmanns, M. & Schultz, C.
Bioorg Med Chem. 2008 Feb 1;16(3):1162-73. Epub 2007 Nov 19.
A series of 2- and 3-OH Nile red dyes was prepared in order to generate water-soluble probes that could be used to probe lipid binding to proteins. Various substitutions in positions 2-/3-, 6-, and 7-shifted wavelengths while maintaining the environmental sensitivity of Nile red. In order to increase the solubility of the dyes in aqueous solutions, we attached butyric acid groups to the 2- or 3-OH position. In addition, phenothiazine dyes, which exhibited particularly long excitation properties, were synthesized and tested for the first time. All dyes showed Stoke's shifts of 70-100 nm and changes in excitation and emission of over 100 nm, depending on the hydrophobicity of the environment. Binding studies with bovine serum albumin and the non-specific lipid transfer protein SCP2 revealed emission changes of more than 30 nm upon binding to the protein and a five-fold increase in emission intensity. Titration of the dye-loaded proteins with various lipids or drugs replaced the dye and thereby reversed the shift in wavelength intensity. This allowed us to estimate the lipid binding affinity of the investigated proteins. For SCP2, isothermal calorimetry (ITC) data verified the titration experiments. NMR titration experiments of SCP2 with Nile red 2-O-butyric acid (1a) revealed that the dye is bound within the lipid binding pocket and competes with lipid ligands for this binding site. These results give valuable insight into lipid and drug transport by proteins outside and inside cells.
Simultaneous protein tagging in two colors.
Schultz, C. & Köhn, M.
Chem Biol. 2008 Feb;15(2):91-2.
The fluorescent tagging of proteins in the natural environment of the cell is an emerging technique in cell biology. In this issue of Chemistry & Biology, Gautier et al. introduce a fluorescent labeling procedure orthogonal to existing ones, enabling tagging of two different proteins in living cells.
Small molecule-based FRET probes.
Cobos Correa, A. & Schultz, C.
In "FLIM and FRET", Gadella, T. (ed.), Elsevier, 2008, pp 225-288
Molecular tools for cell and systems biology
Human Frontiers Sci. Progr. J. 2007/11 1(4) 230-248
The sequencing of the genomes of key organisms and the subsequent identification of genes merely leads us to the next real challenge in modern biology-revealing the precise functions of these genes. Further, detailed knowledge of how the products of these genes behave in space and time is required, including their interactions with other molecules. In order to tackle these considerable tasks, a large and continuously expanding toolbox is required to probe the functions of proteins on a cellular level. Here, the currently available tools are described and future developments are projected. There is no doubt that only the close interplay between the life science disciplines in addition to advances in engineering will be able to meet the challenge.
Probing phospholipase a(2) with fluorescent phospholipid substrates.
Wichmann, O., Gelb, M.H. & Schultz, C.
Chembiochem. 2007 Sep 3;8(13):1555-69.
The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.
Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay.
Stanley, W.A., Versluis, K., Schultz, C., Heck, A.J. & Wilmanns, M.
Arch Biochem Biophys. 2007 May 1;461(1):50-8. Epub 2007 Mar 15.
Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.
Structural analysis of the binding of myo-inositol pentakisphosphates by the C-terminal PH domain of pleckstrin.
Jackson, SG; Zhang, Y; Zhang, K; Bao, XK; Schultz, C.; Haslam, R; Junop, M
FASEB Journal April 2007 21(5)
Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B.
Yudushkin, I.A., Schleifenbaum, A., Kinkhabwala, A., Neel, B.G., Schultz, C. & Bastiaens, P.I.
Science. 2007 Jan 5;315(5808):115-9.
Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on Forster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells. We observed the establishment of a steady-state ES gradient across the cell. This gradient exhibited robustness to cell-to-cell variability, growth factor activation, and RTK localization, which demonstrated spatial regulation of PTP1B activity. Such regulation may be important for generating distinct cellular environments that permit RTK signal transduction and that mediate its eventual termination.
Controlling protein function by caged compounds.
Giordano, A., Zarbahksh, S. & Schultz, C.
In "Chemical Biology" S. Schreiber, T. Kapoor, G. Wess (eds.), Wiley/VCH, 2007, pp140-173
Nat Chem Biol. 2006 Aug;2(8):396-8. PubMed
Annexin a4 self-association modulates general membrane protein mobility in living cells.
Piljic, A. & Schultz, C.
Mol Biol Cell. 2006 Jul;17(7):3318-28. Epub 2006 May 10.
Annexins are Ca(2+)-regulated phospholipid-binding proteins whose function is only partially understood. Annexin A4 is a member of this family that is believed to be involved in exocytosis and regulation of epithelial Cl(-) secretion. In this work, fluorescent protein fusions of annexin A4 were used to investigate Ca(2+)-induced annexin A4 translocation and self-association on membrane surfaces in living cells. We designed a novel, genetically encoded, FRET sensor (CYNEX4) that allowed for easy quantification of translocation and self-association. Mobility of annexin A4 on membrane surfaces was investigated by FRAP. The experiments revealed the immobile nature of annexin A4 aggregates on membrane surfaces, which in turn strongly reduced the mobility of transmembrane and plasma membrane associated proteins. Our work provides mechanistic insight into how annexin A4 may regulate plasma membrane protein function.
Cellular uptake of PNA-terpyridine conjugates and its enhancement by Zn(2+) Ions.
Füssl, A., Schleifenbaum, A., Goritz, M., Riddell, A., Schultz, C. & Kramer, R.
J Am Chem Soc. 2006 May 10;128(18):5986-7.
Conjugation of usually impermeable peptide nucleic acids (PNA) to the chelator 2,2':6',2' '-terpyridine strongly promotes cellular and nuclear uptake by cultured HeLa cells. Cellular accumulation is further enhanced in the presence of extracellular Zn(2+).
Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells.
Tabary, O., Boncoeur, E., de Martin, R., Pepperkok, R., Clement, A., Schultz, C. & Jacquot, J.
Cell Signal. 2006 May;18(5):652-60. Epub 2005 Aug 9.
Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.
A small-molecule FRET probe to monitor phospholipase A2 activity in cells and organisms.
Wichmann, O., Wittbrodt, J. & Schultz, C.
Angew Chem Int Ed Engl. 2006 Jan 9;45(3):508-12. PubMed
A dual parameter FRET probe for measuring PKC and PKA activity in living cells.
Brumbaugh, J., Schleifenbaum, A., Gasch, A., Sattler, M. & Schultz, C.
J Am Chem Soc. 2006 Jan 11;128(1):24-5.
Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.
An inositol polyphosphate derivative inhibits Na+ flux and improves fluid dynamics in cystic fibrosis airway epithelia.
Moody, M., Pennington, C., Schultz, C., Caldwell, R., Dinkel, C., Rossi, M.W., McNamara, S., Widdicombe, J., Gabriel, S. & Traynor-Kaplan, A.E.
Am J Physiol Cell Physiol 2005 Sep;289(3):C512-20. Epub 2005 Apr 27.
Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.
Multiparameter imaging for the analysis of intracellular signaling.
Schultz, C., Schleifenbaum, A., Goedhart, J. & Gadella TW, J.r
Chembiochem 2005 Aug;6(8):1323-30.
In biological experimentation and especially in drug discovery there is a trend towards more complex test systems. Cell-based assays are replacing conventional binding or enzyme assays more and more. This development is strongly driven by novel fluorescent probes that give insight into cellular processes. Target proteins are studied in their natural environment; this gives much more realistic test results, especially with respect to enzyme location and kinetics. However, in the complex environment of cells, many parameters contribute to the performance of the protein of interest. Therefore, it would be desirable to monitor simultaneously as many of the relevant cellular processes as possible. Here, we discuss the possibilities and limitations provided by multiparameter monitoring of cellular events with fluorescent probes. Some novel examples of the use of fluorescent probes and multiparameter imaging are shown.
Closing the gap between chemistry and biology.
Scharer, O. & Schultz, C.
Chembiochem 2005 Jan;6(1):3-5. PubMed
A genetically encoded FRET probe for PKC activity based on pleckstrin.
Schleifenbaum, A., Stier, G., Gasch, A., Sattler, M. & Schultz, C.
J Am Chem Soc 2004 Sep 29;126(38):11786-7.
We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.
Antagonists of myo-inositol 3,4,5,6-tetrakisphosphate allow repeated epithelial chloride secretion.
Rudolf, M.T., Dinkel, C., Traynor-Kaplan, A.E. & Schultz, C.
Bioorg Med Chem 2003 Jul 31;11(15):3315-29.
Cystic fibrosis (CF) patients suffer from a defect in hydration of mucosal membranes due to mutations in the cystic fibrosis transmembrane regulator (CFTR), an apical chloride channel in mucosal epithelia. Disease expression in CF knockout mice is organ specific, varying with the level of expression of calcium activated Cl(-) channels (CLCA). Therefore, restoring transepithelial Cl(-) secretion by augmenting alternate Cl(-) channels, such as CLCA, could be beneficial. However, CLCA-mediated Cl(-) secretion is transient, due in part to the inhibitory effects of myo-inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)]. This suggests that antagonists of Ins(3,4,5,6)P(4) could be useful in treatment of CF. We have, therefore, synthesized a series of membrane-permeant Ins(3,4,5,6)P(4) derivatives, carrying alkyl substituents on the hydroxyl groups and screened them for effects on Cl(-) secretion in a human colonic epithelial cell line, T(84). While membrane-permeant Ins(3,4,5,6)P(4) derivatives had no direct effects on carbachol-stimulated Cl(-) secretion, Ins(3,4,5,6)P(4) derivatives, but not enantiomeric Ins(1,4,5,6)P(4) derivatives, reversed the inhibitory effect of Ins(3,4,5,6)P(4) on subsequent thapsigargin activation of Cl(-) secretion. The extent of the antagonistic effect of the Ins(3,4,5,6)P(4) derivatives varied with the position of the alkyl substituents. Derivatives with a cyclohexylidene ketal or a butyl-chain at the 1-position reversed the Ins(3,4,5,6)P(4)-mediated inhibition of Cl(-) secretion by up to 96 and 85%, respectively, whereas butylation of the 1- and 2-position generated a reversal effect of only 65%. Derivatives carrying the butyl chain only at the 2-position showed no antagonistic effect. These data: (1) Support the hypothesis that Ins(3,4,5,6)P(4) stereospecifically inhibits Ca(2+) activated Cl(-) secretion and that Ins(3,4,5,6)P(4) mediates most, if not all of the cholinergic-mediated inhibition of chloride secretion in T(84) cells; (2) Demonstrate Ins(3,4,5,6)P(4)-mediated inhibition can be completely reversed with rationally designed membrane-permeant Ins(3,4,5,6)P(4) antagonists; (3) Demonstrate that a SAR for membrane-permeant Ins(3,4,5,6) P(4) antagonists can be generated and screened in a physiologically relevant cell-based assay; (4) Indicate that Ins(3,4,5,6)P(4) derivatives could serve as a starting point for the development of therapeutics to treat cystic fibrosis.
Prodrugs of biologically active phosphate esters.
Bioorg Med Chem 2003 Mar 20;11(6):885-98.
Bioactivatable protecting groups represent an enormously powerful tool to increase bioavailability or to generally help deliver drugs to cells. This approach is particularly valuable in the case of biologically active phosphates because of the high intrinsic hydrophilicity and the multitude of biological functions phosphate esters exhibit inside cells. Here, the most prominent masking groups used so far are introduced. The stability and toxicology of the resulting prodrugs is discussed. Finally, this review tries to cover briefly some of the work that describes the usefulness and efficiency of the approach in various application areas.
Versatile reagents to introduce caged phosphates.
Dinkel, C., Wichmann, O., Schultz, C.
Tetrahedron Lett. 2003 (44) 1153-1155
Synthesis of caged myo-inositol 1,3,4,5-tetrakisphosphate.
Dinkel, C., Schultz, C.
Tetrahedron Lett. 2003 (44) 1157-1159
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