SPOT Synthesis Research Articles

Segmented Solid Supports: My Personal Addiction to Merrifield’s Solid Phase Synthesis

The Merrifield solid phase synthesis turned out to be the key method to put into practice many of the concepts I conceived during my scientific work concerned with the systematic empirical search for biologically active synthetic compounds. This is true for the first combinatorial “filter discs” method invented for the simultaneous synthesis of oligonucleotides, as well as for all following methods, then dedicated to peptide and later to small molecule synthesis, such as the “cut and combine” method, the “SPOT synthesis” method and the recent “SC2” method for the generation of compound repertoires in the micro-array format.

Novel peptides with tyrosinase inhibitory activity

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as β-casein, α-lactalbumin, β-lactoglobulin, ovalbumin, gliadin, glycinin, and β-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.

Cellulose-bound Peptide Arrays: Preparation and Applications

The way of the development of the cellulose-bound peptide arrays leads via the synthesis of oligonucleotides on cellulose pieces (Frank et al., 1983) and multiple peptide synthesis on cellulose paper discs in columns (Frank and Doring, 1988). Probably inspired by the addressability of the multipin method (Geysen et al., 1984), Ronald Frank and co-workers developed SPOT synthesis and first presented the method in 1990 at the 21st European Peptide Symposium (Frank et al., 1991). Today,

Synthesis of cleavable peptides with authentic C-termini: an application for fully automated SPOT synthesis

A simple method for the synthesis of carboxyl-free peptides on cellulose membranes was improved and adapted for fully automated SPOT synthesis. Using 1,1′-carbonyl-di-imidazole (CDI) or 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) as an activator within a defined period of time, we were able to reduce the formation of di- or oligomerization of the C-terminal amino acid. The soluble peptides are obtained in a purity range of 60–95% and could be used directly for different biological assays (e.g., CD8 T-cell epitope) that require authentic C-termini.

Optimization and antiviral analysis of peptide ligands for the HIV-1 packaging signal PSI

Methods Phage displayed peptide libraries were screened with PSIRNA structures. The Trp-rich peptide motifs were optimized for specific binding on spot synthesis peptide membranes. The best binding peptide was expressed intracellularly in fusion with RFP or linked to a protein transduction domain (PTD) for intracellular delivery. The effects on virion production were analyzed using pseudotyped lentiviral particles.

Sorting and pooling strategy: A novel tool to map a virus proteome for CD8 T‐cell epitopes

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.

An efficient Mitsunobu protocol for the one-pot synthesis of S-glycosyl amino-acid building blocks and their use in combinatorial spot synthesis of glycopeptide libraries

Complex glycosylation patterns on cell surfaces are involved in many fundamental biological processes like specific cell-cell interactions and signal transduction. Furthermore, the glycon part of glycopeptides and glycosylated proteins play a crucial role in numerous ligand-receptor interactions of biological significance. However, the distinct function of complex carbohydrate structures associated with cell surfaces and proteins is still only poorly understood at a molecular level with regard to specific carbohydrate-protein interaction. Here, we present an efficient Mitsunobu protocol for the convenient chemical one-pot preparation of S-glycosyl amino-acid building blocks suitable for automated combinatorial syntheses of highly glycosylated beta-peptides, which, in turn, can serve as potential mimics for complex oligosaccharides or for studying carbohydrate-protein interactions. The protocol also describes the use of the S-glycosyl amino-acid building blocks for combinatorial spot syntheses of glycopeptide libraries and can be used for the construction of other combinatorial peptide libraries as well. This is a procedure that can be completed in approximately 7 days.

Peptide arrays for the determination of humoral responses induced by active immunization with a monoclonal antibody against EpCAM

Epithelial cell adhesion molecule (EpCAM) is an attractive target for monoclonal antibody serotherapy because it is over-expressed in ∼ 70% of epithelial cancers and their metastatic lesions. IGN101, the immunogenic formulation of the murine monoclonal anti-EpCAM antibody Mab17-1A, has been shown to evoke a strong humoral immune response in both monkey studies and early clinical trials. Notably, there was a reduction in the number of circulating EpCAM-positive tumor cells in the peripheral blood of treated cancer patients. In contrast to earlier publications by other groups, we could not detect an anti-EpCAM immune response upon treatment with Mab17-1A using a conventional but optimized anti-EpCAM ELISA. Therefore, in a novel experimental setup, sera of healthy immunized monkeys, normal human donors and cancer patients immunized with IGN101 were tested for reactivity against a series of overlapping synthetic peptides encompassing the entire sequence of EpCAM prepared by SPOT synthesis on cellular supports. Using this method, sera from normal donors reacted with different peptides compared to sera from healthy monkeys. However, the peptides were clustered in the same regions of EpCAM. Cancer patients generally had a lower reactivity to EpCAM peptides and immunization with IGN101 induced reactivity against a different set of peptides. Antibodies cross-reacting with both the IgG2a framework and with the Mab17-1A idiotype were identified. In summary, our data indicate that some EpCAM peptides may be recognized in a species-specific manner. At least seven EpCAM-derived peptides could be of diagnostic interest (QCQCTSVGAQ, ERVRTYWIII, ALQKEITTRY, TYWIIIELKH, IADVAYYFEK, AYYFEKDVKG, GQTLIYYVDE), while four out of these seven peptides may also possess therapeutic relevance (TYWIIIELKH, ALQKEITTRY, IADVAYYFEK, AYYFEKDVKG).

A novel route to perovskite lead titanate from lead and titanium glycolates via the sol–gel process

AbstractPure perovskite lead titanate powder (PbTiO3) is successfully produced via the sol–gel process using lead and titanium glycolates as starting precursors and has been synthesized by the oxide one spot synthesis process. The obtained lead titanate is of the tetragonal form of the perovskite phase, with high purity and nearly zero moisture content. From high‐resolution mass spectra, the XRD technique, Raman‐FTIR and TGA‐DTA analysis, the lead–titanium glycolates undergo sol–gel transition through the formation of PbOTi bonds. From the SEM micrographs, the PbTiO3 particle shape transforms from an agglomerate sphere to a needle and fiber‐like shapes as the calcination temperature is varied above Tc. The corresponding molecular structural transformation, from the tetragonal form to the cubic form, occurs at 430 °C. The lead titanate powder calcined at 300 °C for 3 h has the highest dielectric constant and electrical conductivity values, namely 17470 and 1.83 × 10−3, respectively. Copyright © 2006 John Wiley & Sons, Ltd.

Engineering the Recruitment of Phosphotyrosine Binding Domain-containing Adaptor Proteins Reveals Distinct Roles for RET Receptor-mediated Cell Survival

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.

Identification of Lipoxygenase Inhibitory Peptides from β‐Casein by Using SPOT Synthesis

The dark and light grey boxes in the legend to Figure 3 in this article were inverted. The correct figure is given below. The editorial office apologises for this mistake. Inhibition (I) of LA oxidation, either catalyzed by LOX (▪) or AAPH (▪), by binding and nonbinding (43 and 154) peptides and two standards (β-casein and L-carnosine) at pH 7.4.

Identification of Lipoxygenase Inhibitory Peptides from β‐Casein by Using SPOT Synthesis

Identification of Lipoxygenase Inhibitory Peptides from β‐Casein by Using SPOT Synthesis

A novel approach to identifying β-secretase inhibitors: Bis-statine peptide mimetics discovered using structure and spot synthesis

β-Secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel “bis-statine” inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.

Selective Attachment of Mammalian Cells and Polystyrene Microbeads to Functional Amine Patterns Defined by Perfluoroalkyl Silane Surfaces

Surface modification technologies have been widely utilized for the investigation of the effect of surface properties on selective attachment of cells and cell functions. Recently, hydrophobic surfaces modified with organosilanes such as dichlorodimethyl silane and perfluoroalkyl silane have been shown to form a resistant barrier for cell attachment owing to their low surface energy. Moreover, they provide a chemically inert background and prevent the cross-contamination of liquid droplets on different spots. These characteristics have the potential to be utilized in the application of spot synthesis and array-type cell-based assays. To date, there have been a few reports on the formation of amine patterns defined by hydrophobic silane layers for the spatial organization of cells. However, several problems, such as the use of the limited types of organosilane for the cell-adhesive patterns, complicated deep UV irradiation for the patterning, and possible contamination of patterns from organosilane coupling still remain to be overcome. Therefore, it is necessary to develop a new patterning technique that extends the versatile surface chemistry and eliminates the contamination problem. Here, we present a novel and simple surface modification method for preparing amine-spacer patterns defined by perfluoroalkyl silane layers and their feasibility for the directed attachment of human retinal pigment epithelial cell line (ARPE-19) and functional polystyrene microbeads. In particular, we have devoted special efforts to solving the contamination problem, which is generated from the conventional photolithography and organosilane coupling method. As illustrated in Figure 1, our strategy is based on the two-step silanization with a simple photoresist (PR) process and then subsequent diamine coupling. To simplify the PR lift-off process, we have designed a home-made rubber stamp for PR printing directly on the substrate in a short time without any further treatment. Formation of isocyanate layers using isocyanatopropyl triethoxysilane (IPTS) on the substrates makes it possible to introduce a variety of amino functional spacers on the surface via urea linkages. In this study, we employed hydrophilic diamine spacers such as 4,7,10-trioxa-1,13-tridecanediamine (TEGdiamine) and Jeffamine ED-600 (PEGdiamine) for the creation of amino-terminal groups that can serve as charged domains promoting cell adhesion, as well as the starting point for the versatile surface modification. Here, we investigated two methods for preparing the fundamental amine-spacer patterns defined by the perfluoroalkyl silane. As illustrated in Figure 1 (Method 1), the PR was patterned on the acid-treated glass substrate by stamping. Thereafter, perfluoroalkyl silane layers (1H,1H,2H,2H-perfluorodecylmethyldichlorosilane, 17F) were formed on the exposed glass surface of the patterned substrates. Subsequently, after the removal of the PR on the substrates with acetone, the substrates were reacted with IPTS to produce patterned reactive sites. However, we found that the 17F layers were contaminated by IPTS coupling and the following amination process, so that substrates were no longer resistant to cell adhesion. In general, functional silane compounds having

Evaluation of a sensitive detection method for peptide arrays prepared by SPOT synthesis

The growing range of applications for peptide arrays prepared by SPOT synthesis confirms that they are a powerful proteomics technique to study numerous aspects of molecular interaction events. The most frequent application for peptide arrays prepared by SPOT synthesis is the identification of linear epitopes that are recognized by antibodies. In the conventional format using secondary antibodies for detection unspecific binding and high background have been observed. This leads to difficulties in evaluation of developed membranes. Especially for application with combinatorial libraries false positive results are to be avoided. To circumvent this issue, we directly labeled compounds of interest with biotin and detected binding by incubation with streptavidin–horseradish peroxidase via chemiluminescence. Optimization of method conditions led to a very sensitive detection technique with no or low number of unspecific spots, which is superior to conventional detection with secondary antibodies. As one consequence, evaluation of competitive assays got more reliable.

Complete Substitutional Analysis of a Sunflower Trypsin Inhibitor with Different Serine Proteases

Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis. Inhibitor variants optimized for elastase or proteinase K inhibition by several rounds of substitutional analyses exhibit K(i) values in the micromolar range and high specificity for the corresponding protease. The results of this easy-to-perform assay can be used to design an improved peptide library using classical methods.

Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures.

Chemical Synthesis of TASP Arrays and Their Application in Protein Design

AbstractFor Abstract see ChemInform Abstract in Full Text.

Unraveling Sub-Site Specificities of Peptidic Serine Protease Inhibitors by Substitutional and Structural Analysis

The interaction of peptidic inhibitors with serine proteases is investigated using peptide spot synthesis on cellulose sheets. This method permits a highly parallel analysis of subsite specificities and an optimization of peptidic inhibitors towards higher affinity and specificity for a given target protease. Information from crystal structures of corresponding protease/inhibitor complexes may help to explain the observed binding pattern.

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.