Molecular Interactions - Bringing Chemistry to Life
May 15th - 19th, 2006, Bozen, Italy
Protein-protein Interactions in Cell Regulation and Signalling: Targets for Drug Discovery
Tom L. Blundell
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
Multiprotein complexes mediate signalling processes and ensure high fidelity in signalling. Signals with useful information may not be provided by strong and enduring interactions between pairs of proteins. Rather synergistic interactions among many components can provide specificity and a sensitive regulation of cellular processes. We have addressed this question by defining by X-ray analysis the 3-D structures of multiprotein complexes involved in extracellular [2], cytoplasmic, and nuclear signals [3].
I will compare the interacting protein surfaces involved in these signalling complexes with those in other protein complexes and discuss their relevance to accurate transduction of signals. I will also discuss approaches to discovery [1,4] of new therapeutic agents that might interrupt these extensive protein-protein interactions. We suggest that proteins forming interactions with a ligand that comprises a continuous region of flexible peptide may be more druggable targets than where complexes are formed from preformed globular protein structures.
[1] Blundell, T.L., Jhoti, H. and Abell, C. (2002). Nature Reviews Drug Discovery. 1, 45-54.
[2] Pellegrini L., Burke D.F., von Delft F., Mulloy B., Blundell TL (2000) Nature 407, 1029-1034;
[3] Pellegrini L, Yu DS, Lo T, Anand S, Lee M, Blundell TL, Venkitaraman AR (2002) Nature 420, 287-293:
[4] Congreve M, Murray CW and Blundell TL (2005) Drug Discovery Today 10, 895-907
Bringing Chemistry to Life: What Does it Mean to be Alive?
Athel Cornish-Bowden and María Luz Cárdenas
CNRS-Bioénergétique et Ingénierie des Protéines, Marseille, France
Biology is commonly regarded as the study of life, but in modern practice biologists barely concern themselves with studying life, and this is especially true of molecular biologists and biological chemists — what they do is to study finer and finer details of living organisms, rarely asking what it means to be alive. As Henri Atlan put it, “Today, a molecular biologist has no need, so far as his work is concerned, for the word ‘life’.” As long as living organisms are regarded as nothing more than rather complicated machines this lack of interest in life perhaps has no importance. However, as soon as one recognizes that living organisms possess a property that no machine has — the capacity to make themselves and to repair themselves without outside intervention — it becomes evident that we shall need a much more fundamental understanding of what life is than we have now if biotechnology is ever to advance beyond the level of tinkering. Metabolism is an open system in the thermodynamic sense, and that is how living organisms can remain metabolically active for very long periods while always maintaining themselves far from thermodynamic equilibrium; however, it is a closed system in terms of its organism, as all its catalysts and nearly all of its chemical components are produced in a hugely complicated cyclic process. Understanding metabolic circularity is thus an essential part of understanding life.
The Principle of Complementarity: Chemical versus Biological Space
Stephen J. Haggarty
Broad Institute of Harvard University & MIT, Cambridge, MA, United States of America
Chemical genomics aims to systematically explore the interactions between small molecules and biological systems. These efforts intend to annotate genomes using the language of chemistry, and to provide information-rich profiles of chemical and biological systems. In addition to examining therapeutically useful drugs and known bioactive molecules, the efficient synthesis and screening of novel collections of small molecules having rich skeletal and stereochemical diversity is needed in order to fully explore chemical space. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the function of proteins in intact signalling networks is necessary. Here I describe recent conceptual and experimental advances toward the goal of mapping multidimensional chemical and biological descriptor spaces. In doing so, I will focus on the complementary nature of these efforts, the importance of recognizing the distinction between computed versus observed descriptors, and highlight on-going research projects in my laboratory aiming to explore the central nervous system and neuropsychiatric disease mechanisms.
Herbivore-induced Volatiles in Plant Defence: Early and Late Events in Enemy-recognition and Response
Wilhelm Boland
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, Germany
Herbivore feeding elicits defence responses in the infested plants, typically the emission of a blend of volatile organic compounds (VOCs) that mediates interactions with the parasites of enemies of the herbivore. To study the impact of individual factors which may contribute to the stimulation of volatile biosynthesis or control the composition of a blend, a mechanical caterpillar (MccWorm) has been engineered, which very closely resembles the herbivore-caused tissue damage in terms of a similar physical appearance and a long lasting wounding period on defined leaf areas. In many plants the mechanical treatment was sufficient to induce a blend of VOCs as known from real herbivore feeding. The defence patterns could be modified by addition of salivary secretions from the feeding insect to the wounded leaf, demonstrating that the salivary secretions also have a strong impact on the composition of the blend. This was further established by microarrays comprising the whole genome of A. thaliana. In total about 5000 genes were either up- or down regulated after simple mechanical damage. By Principal Component analysis the different treatments of the leaves of A. thaliana, such as mechanical damage, feeding by a specialized insect (Diamond Back Moth) and a generalist herbivore (Beet Army Worm), could be clearly distinguished by a typical set of differently affected genes. Interestingly, the salivary secretions of the feeding insects seem to silence locally the gene expression in the damaged leaf, compared to the effect of mechanical wounding, but in distant leaves a significant reprogramming occurs that is not observed after the MccWorm treatment. The complexity of interactions will be presented at the molecular level; consequences and the impact on plant-insect interactions will be discussed.
Modifying Enzyme Specificity by Combinatorial Active Site Mutations
Joelle N. Pelletier
Départment de chimie, Université de Montréal, Montrèal, Quebec, Canada
Recent developments in molecular biology offer new approaches for improving our understanding of enzyme-ligand interactions. The complexity of enzyme catalysis, consisting of ligand recognition and exquisite discrimination followed by rapid catalytic turnover, is now being tapped by modifying ligand specificity. By conducting combinatorial mutagenesis specifically directed toward the active-site area of enzymes involved in drug resistances, we are gaining insights into the nature of the enzyme-ligand interactions underlying these resistance mechanisms. We screen libraries of mutated enzymes for resistance toward their target drug(s), and pull out a variety of modified active site environments that we characterize for binding. The correlation between specific active site mutations and new selectivity is further explored using a variety of methodologies including NMR protein backbone dynamics and molecular modelling. These insights into the nature of enzyme-ligand interactions will provide new information for design of more advanced drug generations.
Using the same methodology, we are also modifying enzyme specificity for synthetic applications. The design of high-throughput screening methodologies will be presented. Our work contributes to demonstrate the generality of the combinatorial active-site mutation strategy for modifying enzyme specificity.
DNA-directed Ligation – Chemistry for Genetic Analyses
Oliver Seitz
Institute of Chemistry, Humboldt University Berlin, Berlin, Germany
It is one of the chief aims of DNA diagnostics to analyse Single Nucleotide Polymorphisms (SNP) which are causally related to many diseases. The enzyme-mediated ligation of two oligonucleotides on DNA templates is one of the most powerful methods for the detection of specific DNA sequences. Ligases can discriminate a particular DNA from its single base mutant by more than 103-fold differences in ligation rate. Such high discriminative powers are required in early cancer detection where the challenge arises to detect acquired single base mutations over the background of predominant wild-type sequences. The exceptional properties of enzymatic ligation systems stimulated chemists to explore chemical means of DNA-controlled ligation. However, in comparison to enzymatic ligation there have been several drawbacks of chemical techniques, which refer to limitations as far as a) speed, b) sequence selectivity, c) applicability to genuine double-stranded DNA and d) signal amplification is concerned. In this presentation, it is reported that substantial improvements can be made by changes of the ligation chemistry and the ligation architecture [1-4].
a) To address the issue of reaction speed, a very powerful reaction from peptide chemistry, native chemical ligation, was employed. It will be shown that the use of this chemistry allows DNA-controlled ligation to proceed as rapid as enzymatic oligonucleotide ligations [3,4].
b) Enhancements of the sequence selectivity can be achieved by changing the ligation architecture. Previous ligation systems have been designed in analogy to enzymatic nick ligation.
We provide evidence that the fidelity of a ligation system can be improved by more than one order of magnitude when performing the ligation opposite to unpaired nucleobases [4].
c) The practicability of chemical ligation on double-stranded DNA templates was demonstrated. Here, the favourable properties of peptide nucleic acids (PNA) allowed rapid single base mutation analyses even on double-stranded PCR DNA templates [3].
d) Usually, template-directed reactions, particularly ligation reactions, suffer from product inhibition since the product usually binds the template with higher affinity than the reactants before ligation. A potentially general approach to obtain signal amplification in template-controlled ligation reactions is presented. It is shown that high turnover numbers can be achieved by means of a ligation-rearrangement sequence in which the rearrangement is designed to reduce the template affinity of the initially formed ligation product.
[1] A. Mattes, O. Seitz, Chem. Commun. 2001, 2050-2051.
[2] A. Mattes, O. Seitz, Angew. Chem. Int. Ed. 2001, 40, 3178-3181.
[3] S. Ficht, A. Mattes and O. Ficht J. Am. Chem. Soc. 2004, 126, 9970-9981.
[4] S. Ficht, C. Dose, O. Seitz, ChemBioChem. 2005, 6, 2098 – 2103.
Reprogrammed Protein Translation and Expanded Genetic Code
Nediljko Budisa
Department of Molecular Biotechnology, Max Planck Institute for Biochemistry, Martinsried, Germany
The chemistry of the life is based on defined number of the generic monomeric building blocks. For example, twenty canonical alpha-amino acids are encoded for basic protein syntheses in all organisms. The central issue of the research presented here is the development of the experimental strategies and techniques to expand the number of the amino acids for protein biosyntheses. This requires the reprogramming of protein translation machinery by changing the coding capacities of standard genetic code. Bacterial host cells should serve as a platform for large-scale expression of custom-made proteins by coupling metabolically engineered biosynthetic pathways with reprogrammed protein translation machinery. Such genetically encoded protein modifications achieved by introducing non-canonical amino acids have greatly expanded the repertoire of accessible proteins for basic research and biotechnological application.
Controlling Biomolecular Recognition with Designed Peptides
Marcey L. Waters
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, United States of America
Research in the Waters group focuses on the connection between molecular structure and biological function, specifically with regard to protein folding and biomolecular recognition. To this end, we utilize structured peptides to investigate the role of perturbations in molecular structure, including posttranslational modifications such as glycosylation, acylation, and methylation, on folding. We apply these findings to the design and study structured peptides as miniproteins to define the minimal requirements for biomolecular recognition.
Molecular Simulations of Enzyme Catalysis
Martin J. Field
Laboratoire de Dynamique Moléculaire, Institut de Biologie Structurale – Jean Pierre Ebel, Grenoble, France
An important goal of computational and theoretical biochemistry is helping elucidate how enzymes achieve their catalytic efficiency. The differing length and time scales of processes that contribute to catalysis, however, makes this a challenging task for molecular simulation techniques. An approach that has proved particularly powerful for the investigation of the chemical steps in enzymatic and other condensed phase reaction processes is the use of hybrid quantum mechanical (QM) and molecular mechanical (MM) potentials. This talk will introduce the concept of a hybrid potential and will discuss their application to a number of enzyme systems.
Biological Communication via Molecular Surfaces
Tim Clark
Computer Chemistry Center, University of Erlangen-Nürnberg, Erlangen, Germany
Switching events are central to biology but the selectivity (or lack of it) and mechanism of biological signalling via intermolecular interactions are poorly understood. We have recently introduced modelling techniques that are not based on the conventional atomistic concepts, but rather on local properties projected onto the molecular surface (expressed as an isodensity surface). These techniques have now been extended by the concept of molecular surface information content, which is based on the Shannon entropy (also a local property) at the molecular surface. The relationship between the information content of a ligand and its receptor will be discussed and ideas as to what determines the selectivity of ligand-receptor binding in specific cases will be presented.
Remote Control of Stereochemistry – Communicating Information via Conformation
Jonathan Clayden
School of Chemistry, University of Manchester, Manchester, United Kingdom
Remote stereocontrol (transmission of information over more than about 4 bond lengths) can be viewed as telecommunication of information (on a molecular scale). Using conformation to transmit information is a relatively undeveloped idea in chemistry, but is highly developed in biology, where conformational relay of information is a feature of many enzymes and receptors. The lecture will present recent work aimed towards the control of conformation in a range of semi-rigid systems, some of them atropisomeric. The importance of dipole interactions for conformational control will be emphasised – dipole interactions between “auxiliary” groups and conformationally flexible functional groups have permitted the synthesis of atropisomeric molecules. Dipole interactions also allow remote stereocontrol, and hence the conformational transmission of information, in molecules such as 1, in which a series of amide groups to relay information stepwise across a linear, acyclic series of rigid, but flexibly joined, xanthene units [1]. Each amide passes information to its neighbour through repulsive interactions between the amide dipoles, the first amide in the sequence being orientated [2] by proximity to an adjacent “auxiliary” A, and the “read-out” from the last amide taking the form of a diastereoselective nucleophilic addition at B [3].
[1] Clayden, J.; Lund, A.; Vallverdú, L.; Helliwell, M., Nature (London) 2004, 431, 966.
[2] Clayden, J.; Lai, L. W., Tetrahedron Lett. 2001, 42, 3163.
[3] Clayden, J.; McCarthy, C.; Westlund, N.; Frampton, C. S., J. Chem. Soc. Perkin Trans. 1 2000, 1363.
Designing Natural Product-derived Focused Libraries
Gisbert Schneider
Institute of Organic Chemistry and Chemical Biology, University of Frankfurt, Frankfurt/Main, Germany
Natural products provide a rich source of novel pharmacologically active substances. We have developed cheminformatics methods for the design of natural product-derived combinatorial libraries with a potential for hit and lead structure finding. Two principal design routes are followed: i) scaffold-extraction from natural products and decoration with suitable side-chains, and ii) pharmacophore modelling and "scaffold-hopping". Technical concepts of both approaches and first virtual screening applications will be presented and discussed.
Coarse-grained Modelling of Membrane Systems
Jonathan W. Essex
School of Chemistry, University of Southampton, Southampton, United Kingdom
Biological membranes play a crucial role in biology. Not only do they act to separate the various compartments of the cell, but small molecules such as drugs have to pass through membranes to reach their targets. The cell membrane itself is not composed of a single molecular species, but is rather a complicated mixture of different types of lipid, proteins and carbohydrates. Molecular dynamics computer simulations offer a powerful way of studying such systems, allowing, for example the permeation of small molecules through simple model membranes to be simulated, and permeability coefficients calculated. However, these simulations are limited by computational resources, such that membrane simulations of atom-based models may only be performed on small areas of the membrane, at most tens of nanometers in length on each side, and for typically a few hundred nanoseconds. If simulations of larger systems over longer timescales are needed, alternative representations of the lipid environment must be sought.
Coarse-graining is a process by which groups of atoms in a molecule are subsumed into a single interaction site, thereby reducing the number of explicitly simulated particles in the system, and allowing larger systems to be simulated for longer. In this presentation, the current state of coarse-grained membrane models will be reviewed. A coarse-grained membrane model developed in my laboratory, based on the popular Gay-Berne model of liquid crystals, will be presented in some detail, and the advantages and disadvantages of this particular methodology over the alternatives discussed.
Molecular Simulations of Membrane Proteins
Mark S.P. Sansom
Department of Biochemistry, University of Oxford, Oxford, United Kingdom
Membrane proteins account for ~25% of all genes. Interactions of membrane proteins with their phospholipid environment play a key role in structure, stability and function. Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. For example, simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid binding site at the interface between adjacent protein monomers [1]. High throughput simulation and coarse-grained simulations [2] enable us to perform comparative analysis of lipid-protein interactions across whole families of membrane proteins. Multi-scale simulations are being used to predict the structure of simple membrane proteins.
[1] Deol, S.S., Domene, C., Bond, P.J. and Sansom, M.S.P. (2005) Anionic phospholipids interactions with the potassium channel KcsA: simulation studies Biophys. J. (in press)
[2] Bond, P.J. and Sansom, M.S.P. (2005) Insertion and assembly of membrane proteins via simulation. J. Amer. Chem. Soc. (submitted)
Artificial Micelles and Liposomes
Andreas Hirsch
Institute of Organic Chemistry, University of Erlangen-Nürnberg, Erlangen, Germany
The synthesis of a variety of new lipophilic and amphiphilic molecular dendrimer architectures based on fullerenes and calixarenes as core units will be presented. The programmed supramolecular aggregation of these molecular building blocks leads to the formation unprecedented aggregates such and calix-micelles being the first examples of stable and robust micelles that can be characterized with molecular resolution. These systemes can be loaded with lipophilic guest molecules and are accessible to chemical functionalization allowing for bio-compatibilization and docking to antibodies. Concepts for the use of buckysomes as drug delivery systems will be introduced. Electrostatic and solvophobic hybridization of these aggregates with electroactive molecules such as porphyrines will be reported.
Bringing Supramolecular Chemistry to Life
Sijbren Otto
Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
Supramolecular chemists have been trying for a long time to develop synthetic hosts that bind strongly and selectively to guests of interest. What they have achieved is impressive, but only very rarely approaches the efficiency with which proteins bind their ligands. And most proteins are not even optimised for strong binding.
A recent statistical analysis of binding affinities in biological and synthetic systems indicates that, whereas many biological systems have binding affinities in the nanomolar range, most synthetic systems have at least three orders of magnitude lower affinities (see Figure 1) [1].
So how can we bring supramolecular chemistry to life (and new life to supramolecular chemistry)? I'll show that using a dynamic combinatorial approach it is possible to develop a synthetic receptor that binds a biologically relevant molecule (spermine) with nanomolar affinity in aqueous solution. This affinity is high enough to unbind spermine from DNA and thereby indirectly influence the conformation of DNA.
I'll also argue that we may have been overlooking an important aspect while designing synthetic receptors.
In collaboration with Stefan Kubik we have studied a class of cyclic peptide receptors for anions and found that interactions between the peptide units that do not directly involve the guest are reinforcing anion binding [2]. Understanding exactly how secondary interactions can reinforce molecular recognition creates exciting new prospects for the development of improved synthetic receptors, which are specifically designed to harness these interactions. While obtaining such (probably complex) receptors will not be trivial, I expect this approach might well produce synthetic receptors with affinities on a par with or even exceeding those of biomolecules.
[1] Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Y. Angew. Chem., Int. Ed. 2003, 42, 4872.
[2] Otto, S.; Pascu, S. I.; Kubik, S.; submitted
From the Bench to the Clinic: Story and Lessons from VRX496, the First Lentivector Ever Tested in a Phase I Clinical Trial
Laurent M. Humeau
VIRxSYS Corporation, Gaithersburg, MD, United States of America
It is estimated there are more than 40 million people worldwide infected with HIV, with one million people in the United States. While effective in prolonging patients’ lives, triple cocktail drug therapy (HAART) has not proven to be a cure for AIDS. HIV resistance to drug therapy is increasing, and currently greater than 20% of newly transmitted HIV is already drug resistant. Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long-term therapy.
VIRxSYS has developed a HIV-based lentiviral vector platform for delivery of genetic therapies. HIV-1 was used as the vector’s backbone since its biology is well characterized and the associated pathology in Humans is well understood. For the first clinical application of a HIV-based lentivector, the genetic treatment of HIV/AIDS appeared promising since a) it was answering a new unmet medical need with the increasing development of HAART resistance and b) no foreign sequence would be introduced into the patient already laden with HIV.
For this purpose, we developed VRX496, a lentiviral vector expressing a 937-base long antisense against the HIV envelope gene. Along with the lentivector, a packaging vector was created based on a single plasmid approach for production via transient transfection. VIRPAC contains all the elements required for particle formation (gag, pol, rev, tat and VSV-G) and this two-plasmid approach was found to provide superior vector titers than the typical three or more plasmid for transient production, a key point for large-scale production at clinical grade level. Many safety features were incorporated into VRX496 and VIRPAC in order to avoid Recombinant Competent Lentivirus (RCL) generation. Codon degeneration was used to minimize homologous regions that could lead to recombination events between wt-HIV, VRX496 and VIRPAC. Furthermore, even in the unfortunate event a recombination should occur, a stop codon was introduced in the remaining VRX496 gag sequence while a pause signal and a cis acting ribozyme was engineered into VIRPAC to prevent gag read-through and packaging of the VSV-G RNA sequence into the lentiviral particles.
VRX496 pre-clinical efficacy was demonstrated in vitro by achieving high transduction efficiencies with stable gene transfer into human primary CD4+ T lymphocytes and by showing selective resistance to CD4 down regulation with over 4 logs (99.99%) of HIV replication inhibition in challenge assays using various X4, R5 or dual tropism strains of HIV. Noteworthy, since the long anti-env antisense is driven off the native HIV LTR, the payload expression and its anti-HIV action in transduced cells is tat and rev dependent, thus occurring after transduced cells are newly infected. Toxicity and biodistribution studies in SCID mice transplanted with VRX496-modified human primary T lymphocytes supported the safety of VRX496 and its clinical grade cGMP production methods.
FDA approval in December 2002 lead to the first ever Phase I clinical trial of lentiviral vectors in humans, testing the safety and tolerability of a single infusion of autologous HIV infected CD4+ T-cells transduced with VRX496. To be eligible for the study, subjects must have failed at least two HAART regimens as a result of drug resistance or be intolerant to antiretrovirals, with a viral load of 5000 copies per mL and CD4 counts between 150 and 500. 5 patients were serially enrolled in the study, received a single infusion of ~1 x 1010, VRX496 modified cells, and were monitored for 6 months. No adverse event due to the product was observed. Integration studies using LAM-PCR have shown that VRX496 has a similar insertion pattern to wtHIV in patients’ cells. Although the purpose of the Phase I clinical trial was to establish the safety of the therapy, and the number of patients in the Phase I clinical trial is too small to make any conclusions with respect to efficacy, potential effects of VRX496 were observed in patients during the monitoring of patients’ circulating CD4 counts and HIV viral load. Three of the patients experienced declining or stable viral loads compared to baseline values, and had elevated or stable CD4 counts. Interestingly, patients showed at different degrees some signs of restored immunity against HIV itself, but also to a much common pathogenic agent, diphtheria. Currently, VRX496 is the only lentiviral vector permitted for use in human phase II clinical trial worldwide. The first part of the study is evaluating the safety and tolerability of multiple dosing. Additional enrolment of patients will help determine an optimal dosing regimen for future confirmatory trials. To date, none of the patients in this clinical trial have experienced any adverse event due to treatment demonstrating so far the safety and tolerability of repeat doses of autologous CD4 T cells modified with VRX496.
Modelling the Evolution of Influenza
Richard A. Goldstein
Division of Mathematical Biology, National Institute of Medical Research, London, United Kingdom
Influenza evolves rapidly, seeking to avoid recognition by the immune system. The rapid evolution of influenza viruses presents a challenge to those who develop vaccines to prevent infection. In addition to current biochemical, microbiological, and structural studies, one of the major sources of information about the nature of future threats from influenza is a study of its history as an evolving threat. The nature of the molecular evolution as the virus evades the immune response provides information that can give an insight into how the immune system has dealt with the virus in the past, and how the virus may change in the future to evade detection.
Chemical Glycomics: From Carbohydrate Arrays to a Malaria Vaccine
Peter H. Seeberger
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology, Zürich, Switzerland
The importance of cell surface oligosaccharides and glycosaminoglycans in signal transduction processes of biomedical significance is now well established. Described is the application of an automated solid-phase oligosaccharide synthesizer we developed recently [1] to all classes of glycoconjugates.
Based on the synthetic platform, a suite of tools for glycobiologists has been developed that includes carbohydrate arrays, fluorescently labelled oligosaccharides for imaging studies and other synthetic tools [2]. Using the specific binding of certain bacteria to particular sugars was used to develop a visual detection system to test body fluids and water for the presence of pathogens including E. coli. This system is now the basis for applications in the medical field and in food handling [3]. Described will be the development of carbohydrate based vaccines against a series of diseases on the example of an anti-toxin malaria vaccine that is currently in preclinical development [4].
[1] Plante, O.J.; Palmacci, E.R.; Seeberger, P.H.; Science 2001, 291, 1523.
[2] Ratner, D. M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H.; ChemBioChem. 2004, 5, 1375-1383.
[3] Disney, M. D.; Zheng, J.; Swager, T.; Seeberger, P. H.; J. Am. Chem. Soc. 2004, 126, 13343-13346.
[4] Schofield, L.; Hewitt, M.C.; Evans, K.; Siomos, M.A.; Seeberger, P.H.; Nature, 2002, 418, 785.
