Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.

Mangelnde Verfügbarkeit erschwert die Analyse posttranslational modifizierter Proteindomänen, denn rekombinante Methoden vermitteln nur selten die gewünschte Ortsspezifität. Die Totalsynthese ermöglicht zwar eine nahezu vollkommene Kontrolle über posttranslationale und andere Modifikationen, jedoch bleiben chemische Methoden auf kürzere Peptide beschränkt. Zur Lösung dieses Problems präsentieren wir hier eine Methode, die a) Immobilisierung N-terminaler Thio-Peptidhydrazide über Hydrazonverknüpfung, b) native chemische Ligation auf der Oberfläche mit selbstgereinigten Peptidthioestern c) radikalisch induzierte Entschwefelung und d) einen oberflächenbasierten Fluoreszenzbindungs-Assay zur funktionellen Charakterisierung kombiniert. Die Methode wurde zur schnellen Untersuchung von 20 SH3-Domänen verwendet. Die Analyse lässt schließen, dass Tyrosinphosphorylierung der SH3-Domänen in Abl-Kinasen als Schalter fungiert, der sowohl einen Verlust als auch einen Gewinn an Affinität für prolinreiche Liganden induzieren kann.

Fluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the development of high affinity fluorescent ER ligands. Cyanine dyes of the MiDye series were directly attached to the SERMs 4-hydroxytamoxifen (OHT) and raloxifene (Ral); linkers were deliberately omitted. This approach yielded conjugates with ERα binding affinities superior to the natural ligand estradiol. The OHT- and Ral-MiDye conjugates emitted in the 600–800 nm range. First round staining experiments showed that the conjugates, but not the dyes alone, accumulate in cells expressing estrogen-binding receptors.

Um den Anwendungsbereich der nativen chemischen Ligation über N-terminale Cysteinreste hinaus zu erweitern, wurden bislang thiolfunktionalisierte Auxiliare verwendet. Allerdings sind die Reaktionsgeschwindigkeiten mit den bisher eingesetzten Auxiliaren vom N-Benzyl-Typ eher gering. Um die Bindungsspaltung durch eine radikalische Fragmentierungsreaktion zu initiieren, wurde nun ein neues Auxiliar entwickelt, das in Gegenwart von TCEP und Morpholin unter mild basischen Bedingungen (pH 8.5) abgespalten wird. Das 2-Mercapto-2-phenethyl-Auxiliar ist anders als früher beschriebene Auxiliare nicht auf glycinhaltige Schnittstellen beschränkt, sondern ermöglicht auch sterisch anspruchsvolle Peptidligationen. Das Auxiliar wird in hoher Ausbeute durch reduktive Aminierung mit kommerziell erhältlichen Aminosäurebausteinen an der Festphase eingeführt. Der präparative Nutzen der Methode wird durch die Synthese der antimikrobiellen Proteine DCD-1L und Opistoporin-2 demonstriert.

Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol-containing auxiliary groups have been used to extend the reaction scope beyond N-terminal cysteine residues. However, the N-benzyl-type auxiliaries used so far result in rather low reaction rates. Herein, a new N^α-auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2-mercapto-2-phenethyl auxiliary is not limited to Gly-containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on-resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD-1L and opistoporin-2.

Described here is a method for the conjugation of phosphorothioate oligonucleotides (PSOs) with peptides. PSOs are key to antisense technology. Peptide–PSO conjugates may improve target specificity, tissue distribution, and cellular uptake of PSOs. However, the highly nucleophilic phosphorothioate structure poses a challenge to conjugation chemistry. Herein, we introduce a new method which involves a sequence of oxime ligation and strain-promoted [2+3] cycloaddition. The usefulness of the method was demonstrated in the synthesis of peptide–PSO conjugates that targeted two suppressors of both the intrinsic and the extrinsic pathway of apoptosis. It is shown that the activity of a PSO sequence targeted against mRNA from c-Flip can be enhanced by conjugation with a peptide mimetic designed to inhibit the X-linked inhibitor of apoptosis protein (XIAP).

Described here is a method for the conjugation of phosphorothioate oligonucleotides (PSOs) with peptides. PSOs are key to antisense technology. Peptide–PSO conjugates may improve target specificity, tissue distribution, and cellular uptake of PSOs. However, the highly nucleophilic phosphorothioate structure poses a challenge to conjugation chemistry. Herein, we introduce a new method which involves a sequence of oxime ligation and strain-promoted [2+3] cycloaddition. The usefulness of the method was demonstrated in the synthesis of peptide–PSO conjugates that targeted two suppressors of both the intrinsic and the extrinsic pathway of apoptosis. It is shown that the activity of a PSO sequence targeted against mRNA from c-Flip can be enhanced by conjugation with a peptide mimetic designed to inhibit the X-linked inhibitor of apoptosis protein (XIAP).

Peptide-based probes that fluoresce upon proteolytic cleavage are invaluable tools for monitoring protease activity. The read-out of protease activity through pyrene excimer signaling would be a valuable asset because the large Stokes shift and the long lifetime of the excimer emission facilitate measurements in autofluorescent media such as blood serum. However, proteolytic cleavage abolishes rather than installs the proximity relationships required for excimer signaling. Herein, we introduce a new probe architecture to enable the switching on of pyrene excimer emission upon proteolytic scission. The method relies on hairpin-structured peptide nucleic acid (PNA)/peptide hybrids with pyrene units and anthraquinone-based quencher residues positioned in a zipper-like arrangement within the PNA stem. The excimer hairpin peptide beacons afforded up to a 50-fold enhancement of the pyrene excimer emission. Time-resolved measurements allowed the detection of matrix metalloprotease 7 in human blood serum.

Peptidbasierte Sonden, die nach einer proteolytischen Spaltung fluoreszieren, sind unschätzbare Hilfsmittel für Untersuchungen von Proteaseaktivitäten. Eine Signalerzeugung durch Pyrenexcimere ist vorteilhaft, da die große Stokes-Verschiebung und die lange Lebensdauer der Excimeremission Messungen in autofluoreszierenden Medien wie Blutserum erleichtern. Die proteolytische Spaltung stört jedoch die Proximitätsbeziehungen, die für eine excimerbasierte Signalgebung notwendig sind. Wir stellen eine neue Sondenarchitektur vor, bei der die Pyrenexcimeremission durch Spaltung eingeschaltet wird. Die Methode beruht auf haarnadelförmigen Peptidnukleinsäure(PNA)-Peptid-Hybriden. Im PNA-Stamm werden Pyreneinheiten und Anthrachinonlöscher reißverschlussartig positioniert. Die Excimer Hairpin Peptide Beacons erbringen eine bis zu 50-fache Verstärkung der Pyrenexcimeremission. Zeitaufgelöste Messungen ermöglichen die Detektion der Matrixmetalloprotease 7 im humanen Blutserum.

Bei der Analyse der RNA-Dynamik in lebenden Zellen werden üblicherweise transgene Methoden eingesetzt. Diese erfordern modifizierte RNAs und Zellen. Hingegen ermöglichen Hybridisierungs-Fluoreszenzsonden Untersuchungen an Wildtyp-Zellen. Wir haben Nuklease-resistente FIT(“DNA-forced intercalation”)-Sonden entwickelt, in denen hohe Anstiege der Fluoreszenz durch Hybridisierung mit einer großen Helligkeit einhergehen, sodass einzelne Ribonukleotidpartikel (RNP) verfolgt werden können. Hierbei dient ein einzelner Thiazolorange(TO)-Interkalationsfarbstoff als Nukleobasensurrogat während eine benachbarte LNA(“locked nucleic acid”)-Modifikation die Umgebung konformativ fixiert. Dies schließt Fluoreszenzabklingkanäle und erhöht so die Helligkeit von TO in Sonden-Ziel-Komplexen. Zwei FIT-Sonden reichen aus, um oskar-RNPs in lebenden Wildtyp-Oozyten von Drosophila melanogaster zu verfolgen.

Eine neue Methode zur chemischen Proteinmarkierung läuft mit hoher Zielspezifität innerhalb weniger Minuten ab und ermöglicht eine freie Wahl des Reportermoleküls. Sie beruht auf Peptidtemplaten, die einen Thioester und ein N-terminales Cystein so ausrichten, dass eine Acyltransferreaktion bei nanomolarer Konzentration möglich wird. Das Zielprotein wird N-terminal mit einem 22 Aminosäure langen Cys-E3-Peptid (Akzeptor) ausgestattet, das mit einem Reporter-tragenden K3-Peptid (Donor) ein Coiled-Coil bilden kann. Dadurch wird die Übertragung des Reportermoleküls auf den Akzeptor am Zielprotein ausgelöst. Weil die Ligierung der wechselwirkenden Peptide vermieden wird, ist der Massenzuwachs am Zielprotein minimal. Zur Veranschaulichung der Methode werden die rasche Fluoreszenzmarkierung und die fluoreszenzmikroskopische Untersuchung des humanen Y2-Rezeptors an lebenden Zellen gezeigt.

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N-terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y₂ receptor on living cells.

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe–target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA-programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well-defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill-defined assemblies of L-selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N-acetyllactosamine (LacNAc), sialyl-Lewis-X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L-selectin assemblies were probed. This study involved the bivalent display both of the weak-affinity sLe^X ligand and of a high-affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self-assemblies were targeted against L-selectin on gold nanoparticles. Spatial screening of L-selectin on the surfaces of leukocytes showed higher affinity enhancements (25-fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L-selectin.

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation–cyclization sequence (“cycligation”) of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2–3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25–0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real-time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance-dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false-positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open-chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12-fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false-positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.

Nucleic acid–peptide conjugates have frequently been used to manipulate gene expression in living cells or to facilitate on-array immobilization of peptides or proteins. Recently, it has been recognized that the programmed self-assembly of nucleic acids can be used as a control element to confine the spatial arrangement or the conformation of peptides. This renders the hybrid molecules useful for studies of proteins and protein function, and could allow switching of the bioactivity of the appended peptides and thus the targeted protein by means of nucleic acid hybridization. A prerequisite of such studies is a feasible access to the desired chimeric molecules. This review describes recent efforts in the synthesis of nucleic acid–peptide hybrid molecules.

Peptide thioesters play a key role in convergent protein synthesis strategies such as native chemical ligation, traceless Staudinger ligation, and Ag⁺-mediated thioester ligation. The Boc-based solid-phase synthesis provides a very reliable access to peptide thioesters. However, the acid lability of many peptide modifications and the requirements of most parallel peptide synthesizers call for the milder Fmoc-based solid-phase synthesis. The Fmoc-based synthesis of peptide thioesters is more cumbersome and typically proceeds with lower yields than the synthesis of peptide acids and peptide amides. The success of native chemical ligation and related technologies has sparked intensive research effort devoted to the development of new methods. The recent progress in this rapidly expanding field is reviewed.

Peptidthioester sind Schlüsselverbindungen in der konvergenten Proteinsynthese, wie es die native chemische Verknüpfung, die spurlose Staudinger-Verknüpfung und die Ag⁺-vermittelte Thioesterverknüpfung beispielhaft belegen. Den zuverlässigsten Zugang zu Peptidthioestern bietet die Boc-basierte Festphasensynthese. Die Säurelabilität vieler Peptidmodifikationen und die technische Ausstattung der meisten Parallelpeptidsyntheseautomaten erhöhen jedoch die Nachfrage nach der milderen Fmoc-Synthesechemie. Die Fmoc-basierte Peptidthioestersynthese ist oftmals mühevoll und verläuft in wesentlich geringeren Ausbeuten als die Synthese von Peptidsäuren und -amiden. Der Erfolg der nativen chemischen Verknüpfung und verwandter Techniken hat allerdings die Entwicklung neuer Synthesestrategien vorangetrieben. Gegenstand dieses Aufsatzes ist es, die neueren Entwicklungen auf diesem schnell expandierenden Forschungsgebiet aufzuzeigen.

Thioester-mediated peptide coupling reactions are powerful tools in protein synthesis. The fragment coupling occurs extremely fast at ligation sites that contain an N-terminal cysteine residue. This native chemical ligation involves a capture step that affixes the acyl component in the immediate vicinity of the N-terminal amino group. The subsequent intramolecular S,N-acyl transfer step proceeds via an entropically favored five-membered ring intermediate. In this study we investigated whether sequence internal cysteine residues that lead to the formation of macrocyclic intermediates are also able to accelerate the rate of thioester-based fragment couplings. It was the aim to identify distance requirements that enable internal cysteine-mediated ligation reactions to proceed at synthetically useful rates. It was found that appropriately positioned cysteine residues can induce up to 25-fold rate enhancements compared to a cysteine-lacking control peptide. Highest ligation rates and yields were obtained when the internal thiol amino acid was incorporated at the fifth or sixth position from the N-terminus of the C-terminal coupling segment. The findings reveal a correlation between the size of the macrocyclic ring intermediate and the ease of the peptide ligation. Internal cysteine ligation may provide the opportunity to shift amino acid cysteine bonds that are difficult to access through native chemical ligation (such as Pro–Cys bonds) by 4–5 amino acids to the N-terminus.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)