Abstract

Abstract

This review is focused on advances over the last several years in the application of aldolases to organic synthesis. Several new technologies have been implemented to increase the scope and practicality of aldolases as tools for the synthetic chemist. These include directed evolution, discovery of new classes of aldolases in nature and the laboratory, and substrate and reaction engineering.

Chemical synthesis of glycoproteins from readily available materials is a powerful method for obtaining a pure product with full control of its atomic structure. Sugar-assisted ligation (SAL) is an emerging approach that allows the synthesis of a large glycopeptide from two unprotected fragments. Contrary to other ligation methods that are limited to the use of a cysteine residue or depend on external auxiliary, SAL takes advantage of the existing sugars in glycopeptides to promote proximity between the two peptides to facilitate an amide bond formation.

In a successful example of lead optimization by computer modeling prediction, computational technology was used to optimize a lead inhibitor (TL-3) of the SARS-CoV 3CL protease. A novel C₂-symmetric diol (1) was then designed and synthesized, and displayed higher affinity than the original lead compound by one order of magnitude in its inhibition constant (0.60.073 μM). We believe that this approach has provided a platform for further lead optimization.

Sweet ligations: A second-generation sugar-assisted ligation is described that incorporates a sugar moiety with an ester-linked thiol auxiliary. Efficient auxiliary removal leads to native glycopeptides without the use of protecting groups. Extended glycopeptides also facilitate efficient ligation reactions by this method, therefore making it possible to scan a target sequence until a synthetically viable ligation junction is found.

Süße Verknüpfungen: Eine zuckervermittelte Glycopeptidverknüpfung der zweiten Generation wird beschrieben, bei der ein Zuckerrest ein estergebundenes Auxiliar trägt (siehe Schema). Die Entfernung des Auxiliars führt ohne Schutzgruppenoperationen zu nativen Glycopeptiden. Verlängerte Glycopeptide können ebenfalls eingesetzt werden, sodass die Sequenz eines gewünschten Glycopeptids „abgetastet“ werden kann, bis eine günstige Verknüpfungsstelle gefunden ist.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been widely used for the organic synthesis of unnatural sugars or derivatives. The practicality of using DHAP-dependent aldolases is limited by their strict substrate specificity and the high cost and instability of DHAP. Here we report that the DHAP-dependent aldolase L-rhamnulose 1-phosphate aldolase (RhaD) accepts dihydroxyacetone (DHA) as a donor substrate in the presence of borate buffer, presumably by reversible in situ formation of DHA borate ester. The reaction appears to be irreversible, with the products thermodynamically trapped as borate complexes. We have applied this discovery to develop a practical one-step synthesis of the non-caloric sweetener L-fructose. L-Fructose was synthesized from racemic glyceraldehyde and DHA in the presence of RhaD and borate in 92 % yield on a gram scale. We also synthesized a series of L-iminocyclitols, which are potential glycosidase inhibitors, in only two steps.

Flexible yet efficient: Sialic acids such as L-N-acetylneuraminic acid (see picture) can be synthesized in only three steps by 1) vinylation of an aldose through a modified Petasis coupling reaction, 2) 1,3-dipolar cycloaddition with a nitrone to construct an isoxazolidine ring, and 3) base-catalyzed β elimination/ring opening of the isoxazolidine to generate a γ-hydroxy-α-keto acid.

A novel 5-membered iminocyclitol derivative was found to be a potent and selective inhibitor of the glycoprotein-processing α-glucosidase with a K_ivalue of 53 nM. This compound was further derivatized to antiviral agents against Japanese encephalitis virus, dengue virus serotype 2 (DEN-2), human SARS coronavirus, and human β-hexosaminidase (K_i=2.6 nM), a new target for the development of osteoarthritis therapeutics.

Without a trace. An approach combining traceless Staudinger ligation and protease-catalyzed N-terminal azidonation has been shown to be efficient for the convergent synthesis of glycopeptides without the cysteine limitation of native chemical ligation.

Zuckersüße Synthese: Einfach zugängliche Thioglycoside mit definierten relativen Reaktivitätswerten dienten als Bausteine in einer Eintopfsynthese des tumorassoziierten Kohlenhydrat-N3-Nebenantigens (siehe Formel). Die Zielverbindung wurde für die direkte Charakterisierung durch Laserlicht-gestützte Massenspektrometrie über einen photospaltbaren Linker kovalent an eine poröse Siliciumoberfläche gebunden.

Sweet and light: Readily available thioglycosides with defined relative reactivity values were used as building blocks in a one-pot strategy to synthesize the tumor-associated carbohydrate antigen N3 minor (1). The target molecule was attached covalently to a porous silicon surface through a photocleavable linker for direct characterization in a mass spectrometer equipped with a laser source.

Substitute for another bond. Docking simulations of two potent inhibitors that bear the 1,2,3-triazole moiety produced two conformations of approximately equal energy. Further analysis of the protease by X-ray crystallography solved the ambiguity of the binding mode and revealed that the triazole ring is an effective amide surrogate and retains all the hydrogen bonds in the active site (see figure).

Eine effiziente Eintopfsynthese von S-verknüpften Glycosylaminosäuren 1 wurde entwickelt (siehe Schema; DMF=Dimethylformamid) und in der Festphasensynthese von S-verknüpften Glycopeptiden genutzt. Dieser Ansatz ermöglichte die Synthese einer Vielzahl S-verknüpfter Glycosylaminosäure-Bausteine in hoher Ausbeute.

No isolation, no purification. An efficient and simple method based on tetrabutylammonium fluoride-mediated alkylation without protecting groups in microtiter plates, followed by screening in situ has been developed to identify new enzyme inhibitors, as demonstrated in the discovery of the shown potent inhibitors of cathepsin B and HIV protease.

Potent library. Anthrax lethal factor (LF) is a zinc-dependent metalloprotease involved in the rapid development of the deadly infection caused by Bacillus anthracis. Blocking its action is a plausible method to mitigate the deleterious effects of late stage infection. We report the inhibition of LF by tetrahydro-isoquinoline polyphenolic compounds, such as 5 a, which were identified by screening a combinatorial library that was generated by Pictet–Spengler reaction. We also report the identification of commercially available polyphenolic inhibitors against LF.

The replacement of hydrogen atoms with fluorine substituents in organic substrates is of great interest in synthetic chemistry because of the strong electronegativity of fluorine and relatively small steric footprint of fluorine atoms. Many sources of nucleophilic fluorine are available for the derivatization of organic molecules under acidic, basic, and neutral conditions. However, electrophilic fluorination has historically required molecular fluorine, whose notorious toxicity and explosive tendencies limit its application in research. The necessity for an electrophilic fluorination reagent that is safe, stable, highly reactive, and amenable to industrial production as an alternative to very hazardous molecular fluorine was the inspiration for the discovery of selectfluor. This reagent is not only one of the most reactive electrophilic fluorinating reagents available, but it is also safe, nontoxic, and easy to handle. In this Review we document the many applications of selectfluor and discuss possible mechanistic pathways for its reaction.

Der Ersatz von Wasserstoff durch Fluor in organischen Substraten ist wegen der hohen Elektronegativität des Fluors bei fast gleich bleibendem Raumanspruch von großem Interesse in der Synthesechemie. Viele Quellen für nucleophiles Fluor zur Derivatisierung unter sauren, basischen und neutralen Bedingungen stehen zur Verfügung. Hingegen erforderte eine elektrophile Fluorierung früher den Einsatz von molekularem Fluor, was aufgrund dessen Toxizität und Explosionsneigung zu einer nur begrenzten Anwendung dieser Methode geführt hat. Der Bedarf an einem elektrophilen Fluorierungsreagens, das sicher zu handhaben, stabil und hoch reaktiv ist und in der industriellen Produktion als Alternative zum hoch giftigen gasförmigen Fluor eingesetzt werden kann, führte letztlich zur Entwicklung von Selectfluor (1). Dieses Reagens ist eines der reaktivsten elektrophilen Fluorierungsmittel und dazu noch ungefährlich, nicht toxisch und leicht zu handhaben. In diesem Aufsatz beschreiben wir die zahlreichen Einsatzmöglichkeiten von Selectfluor und diskutieren mögliche Reaktionsmechanismen.

A high-yield one-pot synthesis of S-linked glycosyl amino acids 1 has been developed (see scheme; DMF=dimethyl formamide) and used in the solid-phase synthesis of S-linked glycopeptides. This approach has been shown to be efficient for the synthesis of various S-linked glycosyl amino acid building blocks.

Defence against bioterrorism: Recent events have created an urgent need for therapeutic strategies to treat anthrax, an infectious disease caused by the toxigenic bacterium Bacillus anthracis. A new class of aminoglycosides (see picture) are powerful inhibitors under physiological conditions of the anthrax lethal factor, which has a major role in the disease, and function simultaneously as antibiotics against B. anthracis.

Sulfatases, which cleave sulfate esters in biological systems, play a key role in regulating the sulfation states that determine the function of many physiological molecules. Sulfatase substrates range from small cytosolic steroids, such as estrogen sulfate, to complex cell-surface carbohydrates, such as the glycosaminoglycans. The transformation of these molecules has been linked with important cellular functions, including hormone regulation, cellular degradation, and modulation of signaling pathways. Sulfatases have also been implicated in the onset of various pathophysiological conditions, including hormone-dependent cancers, lysosomal storage disorders, developmental abnormalities, and bacterial pathogenesis. These findings have increased interest in sulfatases and in targeting them for therapeutic endeavors. Although numerous sulfatases have been identified, the wide scope of their biological activity is only beginning to emerge. Herein, accounts of the diversity and growing biological relevance of sulfatases are provided along with an overview of the current understanding of sulfatase structure, mechanism, and inhibition.

An HIV-1 vaccine may eventually be developed from oligomannoses synthesized by a reactivity-based one-pot self-condensation strategy. The compounds were tested for their ability to inhibit the binding of gp120 to the broadly neutralizing HIV-1 antibody 2G12. New epitope mimics (see structure for example) were identified that inhibit this binding as well as, or better than the natural epitope Man₉GlcNAc₂ (Man=mannose, Glc=glucose).

A library of 4,6-linked analogues of the drug tobramycin with various mono- or diaminosugars attached to the 6-position of the two-ring core has been prepared (see scheme; Bn=benzyl, Tol=p-tolyl). The compounds were screened against different disease-related RNAs, and several of the synthetic analogues showed a high affinity and selectivity toward certain RNA sequences.

Eine Bibliothek 4,6-verknüpfter Analoga von Tobramycin mit Mono- oder Diaminozuckern an der 6-Position des Zweiringkerns wurde hergestellt (siehe Schema). Ein Screening der Verbindungen gegen mehrere zu Krankheiten in Beziehung stehende RNAs ergab eine hohe Affinität und Selektivität einiger der synthetischen Analoga für bestimmte RNA-Sequenzen.

Rapid diversity-oriented microplate library synthesis and in situ screening with a high-throughput fluorescence-based assay were used to develop potent inhibitors of β-arylsulfotransferase IV (β-AST-IV). This strategy leads to facile inhibitor synthesis and study as it allows protecting-group manipulation and product isolation from other library components to be avoided. Through repeated library formation, three aspects of inhibitor makeup, the identities of the two binding groups and the length of the linker between them, were independently optimized. Several potent inhibitors were obtained, one of which was determined to have an inhibition constant K_iof 5 nM. This compound is the most potent β-AST-IV inhibitor developed to date, with a K_ivalue more than five orders of magnitude lower than the Michaelis constant K_mfor the substrate whose binding it inhibits.

Doppeltes Spiel: Dimere Aminoglycosid-Antibiotika wurden durch Verknüpfung von Neamin- oder Nebramin-Kernen mit Linkern unterschiedlicher Länge und Zusammensetzung erhalten. Die Dimere zeigen hohe Affinität zum 16S-A-Zentrum ribosomaler RNA (siehe Schema) und wirken effektiv gegen mehrere aminoglycosidresistente Bakterienstämme.

Sulfatasen – d. h. Sulfatester spaltende Enzyme – spielen eine entscheidende Rolle bei der Regulierung von Sulfatierungszuständen, die über die Funktion vieler physiologischer Moleküle entscheiden. Die Substrate der Sulfatasen reichen von kleinen cytosolischen Steroiden wie dem Östrogensulfat bis hin zu komplexen Kohlenhydraten auf Zelloberflächen, etwa den Glycosaminoglycanen. Die Umwandlung dieser Moleküle wurde mit wichtigen zellulären Funktionen in Verbindung gebracht, z. B. mit der Hormonregulation, dem zellulären Abbau und der Modulation von Signalisierungsprozessen. Sulfatasen hängen mit dem Ausbruch etlicher pathophysiologischer Erscheinungen zusammen, etwa mit hormonabhängigen Krebsformen. Bis heute sind zwar zahlreiche Sulfatasen identifiziert worden, die Aufklärung des weitreichenden Spektrums ihrer biologischen Aktivitäten befindet sich aber erst am Anfang. Dieser Aufsatz gibt einen Überblick über den derzeitigen Wissensstand zu den Strukturen, Mechanismen und der Inhibition von Sulfatasen.

Seit langem ist bekannt, dass die Sulfonierung von Biomolekülen in einer Vielzahl von Organismen stattfindet, von Prokaryoten bis hin zu vielzelligen Spezies, und ständig werden neue biologische Funktionen entdeckt, die mit dieser Umsetzung verknüpft sind. Frühe Studien zu Sulfotransferasen (STs), den Enzymen, die Sulfonierungen katalysieren, konzentrierten sich primär auf zytosolische STs, die an Entgiftungsreaktionen, der Hormonregulation und am Wirkstoffmetabolismus beteiligt sind. Obwohl man von ihrer Existenz gewusst hat, wurden membranassoziierte STs bis vor kurzem kaum untersucht. Sie sind an der Sulfonierung komplexer Kohlenhydrate und Proteine beteiligt und spielen eine zentrale Rolle bei einer Reihe von molekularen Erkennungsmechanismen und biochemischen Signalwegen. Des Weiteren hängen STs mit vielen pathophysiologischen Prozessen zusammen. Dies führte zu einem verstärkten Interesse an den komplexen Funktionen von STs und an der Möglichkeit zur Targetierung in therapeutischen Eingriffen. In diesem Aufsatz werden die Fortschritte bei der Aufklärung der Strukturen und Mechanismen von Sulfotransferasen sowie ihre biologische Aktivität, Inhibierung und Anwendung in der Synthese diskutiert.

The sulfonation (also known as sulfurylation) of biomolecules has long been known to take place in a variety of organisms, from prokaryotes to multicellular species, and new biological functions continue to be uncovered in connection with this important transformation. Early studies of sulfotransferases (STs), the enzymes that catalyze sulfonation, focused primarily on the cytosolic STs, which are involved in detoxification, hormone regulation, and drug metabolism. Although known to exist, the membrane-associated STs were not studied as extensively until more recently. Involved in the sulfonation of complex carbohydrates and proteins, they have emerged as central players in a number of molecular-recognition events and biochemical signaling pathways. STs have also been implicated in many pathophysiological processes. As a result, much interest in the complex roles of STs and in their targeting for therapeutic intervention has been generated. Progress in the elucidation of the structures and mechanisms of sulfotransferases, as well as their biological activity, inhibition, and synthetic utility, are discussed in this Review.

Double duty: Dimeric aminoglycoside antibiotics were designed by linking neamine or nebramine cores with various tethers differing in length and composition. The resulting dimeric aminoglycosides displayed high affinity for the 16S A site of ribosomal RNA (a representation of the complex is shown here) and were effective against various aminoglycoside-resistant bacterial strains.

Click and go: By using click chemistry based on a new triazole forming reaction condition (see scheme), over 100 triazole compounds generated in microtiter plates from a core structure were screened for HIV protease inhibition in situ without product isolation. Potent inhibitors, active at nanomolar concentrations, against the wild type and drug resistant mutants were identified.

A continuous fluorescence coupled enzyme assay was developed to study the acceptor specificity of the glycosyltransferase MurG toward different lipid I analogues with various substituents replacing the undecaprenyl moiety. It was found that most lipid I analogues are accepted as substrates and, amongst these, the saturated C₁₄analogue exhibits the best activity. This substrate was used to evaluate the inhibition activity of such antibiotics as moenomycin, vancomycin, and two chlorobiphenyl vancomycin derivatives. A vancomycin derivative with a chlorobiphenyl moiety on the aglycon section was identified as a potent inhibitor of MurG.

Die bekannt schwierige Glycosylierung von Vancomycin wurde in einem Eintopf-Prozess ausgeführt. Dazu wurde eine Vielzahl von Oligosacchariden synthetisiert und mit dem Aglycon von Vancomycin verknüpft (siehe Formel). Mithilfe dieser Verbindungsbibliothek wurde der Einfluss der Glycosylierung auf die antibiotische Aktivität getestet.

Eine effiziente und einfache Methode im Mikrotiterplattenformat zur Identifizierung potenter und selektiver Inhibitoren von α-Fucosidasen wurde entwickelt. Sie beruht auf der Amidbildung einer Fuconojirimycin-Kernstruktur mit Carbonsäuren und anschließendem Inhibierungs-Assay ohne Isolierung des Produkts. Gezeigt sind die Strukturen der beiden potentesten Inhibitoren.

Herein we describe an inhibition study of the sialyl Lewis x (sLe^x) expression on a human monocytic cell line (U937), using a series of peracetylated N-Acetyllactosamine (LacNAc) analogues with variation at the aglycon moiety. It was found that the extent of inhibition was related to the hydrophobicity and structure of the aglycon. In general, peracetylated LacNAc analogues with a naphthyl or biphenyl aglycon (3, 4, 6, and 7) were better in suppression of sLe^xexpression than a benzyl derivative (2). Steady-state kinetic experiments with humanα-1,3-fucosyltransferases IV and VI (FucT IV and VI, EC 2.4.1.65) revealed that the deacetylated LacNAc-aglycons with naphthyl (18, 19, and 20) or biphenyl (17) moieties exhibited higher affinity to the fucosyltransferases than aglycon moieties with smaller hydrophobic groups (14, 15, and 16). These results are in agreement with the findings of the U937 cell-based experiments, and suggest that the higher enzyme affinity LacNAc-aglycons make better acceptor decoys and, hence, the observed differences in LacNAc-aglycon inhihitory effects on sLe^xexpression.

An efficient and simple method has been developed to identify potent and selective inhibitors against α-fucosidases based on the amide-forming reaction in a microtiter plate of a fuconojirimycin core with various carboxylic acids, followed by a direct inhibition assay without product isolation. The structures of the two most potent inhibitors are shown.

The notoriously difficult glycosylation of vancomycin was tackled in a one-pot procedure. A number of oligosaccharides were synthesized and attached to the vancomycin aglycon to create a library of derivatives (see formula) to probe the effect of glycosylation on the antibiotic activity.

Die stereospezifische Synthese des Kohlenhydrat-Haptens Lewis Y gelang durch eine Eintopfreaktion der drei Synthesebausteine 1–3, die anhand ihrer relativen Reaktivität (RR) für die Reaktion ausgewählt wurden.

The highly selective tobacco etch virus Nla protease (TEV protease) can be used to remove affinity tags with mutated cleavage sites to produce proteins for site-specific labeling and chemoselective ligations (see scheme). This provides a simple, efficient method for producing proteins with N-terminal cysteines for proteomic applications.

Mimics of the geometry or charge of the fucose moiety of GDP-fucose in the transition state of fucosyltransferase-catalyzed reactions, compounds 1–3 are good competive inhibitors of α-1,3-fucosyltransferases V and VI with K_i values between 6 and 13 μM. GDP=guanosine diphosphate.

The stereospecific synthesis of a tumor-related carbohydrate antigenic hapten, Lewis Y, from three basic building units 1–3 has been achieved by a reactivity-based one-pot strategy. RRV=relative reactivity value.

Enzymatische Reaktionen mit DERA dienen als Grundlage für eine neue Strategie zur Synthese von Pyranosebausteinen. Die Nützlichkeit dieser hoch konvergenten und effektiven Methode wird am Beispiel einer kurzen Totalsynthese von Epothilonen gezeigt (siehe Schema; DERA=2-Desoxyribose-5-phosphataldolase).

Enzymatic reactions catalyzed by DERA provide the basis for a new strategy for the synthesis of novel pyranose synthons. The utility of this very convergent and effective method is demonstrated by the concise total synthesis of epothilones (see scheme; DERA=2-deoxyribose-5-phosphate aldolase).

Mimetika im Hinblick auf die Geometrie oder Ladung der Fucose-Einheit der GDP-Fucose im Übergangszustand von Fucosyltransferase-katalysierten Reaktionen konnten mit den Verbindungen 1–3 erhalten werden, welche gute kompetitive Inhibitoren der α-1,3-Fucosyltransferasen V und VI mit K_i-Werten von 6 bis 13 μM sind. GDP=Guanosindiphosphat

Die hoch selektive Tabakätz-Virus-Nla-Protease (TEV-Protease) kann zum Entfernen von Affinitätsmarkern mit mutierten Spaltstellen eingesetzt werden. Hierbei werden Proteine freigesetzt, die an der Spaltstelle spezifisch markiert und durch chemoselektive Ligationen umgesetzt werden können (siehe Schema). Auf diese Weise lassen sich Proteine mit N-terminalen Cysteinen für proteomische Anwendungen einfach und effizient herstellen.

Fuculose 1-phosphate and fructose-diphosphate aldolases were employed in the chemoenzymatic synthesis of the iminocyclitols 1 and 2 possessing a phosphonic acid moiety. These compounds are useful as core synthons for the development of specific glycosyltransferase inhibitors.

Several enzymes immobilized on thermo-responsive polyacrylamide polymers are nearly as active as their soluble forms, and can be recovered for reuse after gentle heating and precipitation. Carbohydrates attached to these polymers have been used for enzymatic glycosylation, and the products have been isolated by thermal precipitation followed by release from the polymer, thus greatly simplifying product purification in water.

Das große Interesse am Problem der Antibiotikaresistenz führte in letzter Zeit zur Entdeckung neuer Targets und Strategien in der Antibiotikaforschung. Eine neue Richtung der Strategien ist die Entwicklung kleiner Moleküle, die Kohlenhydratrezeptoren und Kohlenhydrat-modifizierende Enzyme angreifen. Dieser Aufsatz stellt neuere Arbeiten auf diesem Gebiet vor und diskutiert den Einfluss der jeweiligen Vorgehensweisen auf das Auftreten von Resistenz. Von besonderem Interesse sind die einzigartigen Kohlenhydrate der Zelloberfläche, die an der Peptidoglycan-Biosynthese beteiligte Transglycosylase sowie die RNA der Bakterien. Mit zunehmendem Verständnis des Genoms verschiedener Bakterienarten und Fortschritten in der funktionalen Genomforschung und Proteomforschung, ist die Entdeckung einer Vielzahl von Targets für die Entwicklung neuer Antibiotika zu erwarten.

Recent interest in the problem of antibiotic resistance has led to the identification of new targets and strategies for antibiotic discovery. Among these efforts, the development of small molecules as antibiotics to target carbohydrate receptors or carbohydrate-modifying enzymes represents a new direction. This review covers recent work in this regard and discusses the impact of each strategy on the development of drug resistance. Particularly interesting targets include unique cell-surface carbohydrates, the transglycosylase involved in peptidoglycan biosynthesis, and bacterial RNA. With a greater understanding of the genome of different bacteria as well as advances in functional genomics and proteomics, we can expect the discovery of a variety of targets for the development of novel antibiotics.

5-Azidosialyl-Donoren mit O-Acetyl-Schutzgruppen sind besonders gute Reagentien für die α-selektive Glycosylierung primärer Hydroxygruppen als Acceptoren. Dies belegt eine Vielzahl von Reaktionen mit 1 als Sialyldonor. Es gelang auch, NeuAcα(29)NeuAc als Thioglycosid-Donor für den Einsatz in weiteren Glycosylierungen zu synthetisieren.

5-Azido sialyl donors with O-acetyl protecting groups are useful α-selective glycosylation reagents, especially for primary hydroxyl groups as acceptors. This is shown with a variety of reactions using 1 as a sialyl donor. It was also possible to synthesize NeuAcα(29)NeuAc as a thioglycoside donor for use in subsequent glycosylations.

The decasaccharide sialyl-trimeric-Lewis x is a component of glycoproteins and glycolipids that serve as E- and P-selectin ligands. The synthesis of this target structure was accomplished by utilizing a combination of chemical and enzymatic methods. Highlights of the chemical synthesis include minimal use of protecting groups and regioselective glycosylations to arrive at a linear tri-lactosamine structure. Glycosyltransferase-catalyzed reactions were then employed for the addition of the terminal sialic acid and branch-point fucose residues. Notably, fucosyltransferases V and VI showed different specificities for the sialyl-tri-lactosamine core structure.

Sialyl Lewis × (sLe^x) derivatives conjugated to readily visualized molecular labels are useful chemical probes to study selectin-carbohydrate interactions. Localization of the selectins on the surface of leukocytes and activated endothelial cells can be detected through fluorescence of bound selectin ligands. Herein we present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe^x conjugate. The use of an amino-substituted monovalent sLe^x to obtain fluorescent- and biotin-labeled sLe^x derivatives is also described. The cell-staining utility of the fluorescent sLe^x conjugates is demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.

So mild und effektiv wie die konventionelle Hydrogenolyse in Lösung ist die Abspaltung von Benzylgruppen an Kohlenhydraten, die an ein festes Trägermaterial gebunden sind, mit Palladium-Nanoteilchen als Katalysatoren. Der Einsatz eines UV-aktiven Linkers, der die benzylgeschützten Glycoside von Glucose, Fucose und Lactose an den Träger bindet (siehe Bild), ermöglicht es, den Verlauf der Hydrogenolyse nach der Abspaltung vom Träger zu verfolgen.

Die Knüpfung von C-C-Bindungen unter vollständiger Kontrolle des stereochemischen Verlaufs einer Reaktion ist in der organischen Synthesechemie von großer Bedeutung. Die Aldolreaktion, d. h. die einfache Addition eines Enolatdonors an einen Carbonylacceptor, ist eine der bedeutendsten Reaktionen, die dem Synthesechemiker zur Verfügung stehen. Im Allgemeinen wird die Kontrolle der relativen und absoluten Konfiguration des neugebildeten Stereozentrums durch die Verwendung von chiralen Ausgangsverbindungen oder chiralen Hilfsreagentien erzielt. In den letzten Jahren wurden in der organischen Synthesechemie große Anstrengungen bei der Suche nach katalytischen Methoden unternommen, die die chirale Information auf effiziente Weise übertragen. Zwei verschiedene Zugänge zur katalysierten asymmetrischen Aldolreaktion wurden dabei in Erwägung gezogen: Biokatalyse und die Katalyse durch kleine Moleküle. Beide Methoden haben sowohl spezifische Vorteile als auch Grenzen und sind daher in vielen Bereichen komplementär zueinander. In diesem Artikel werden die wichtigsten neuen Entwicklungen bei beiden Methoden zusammengefasst.

A cost-efficient preparative enzymatic sulfation of oligosaccharides has been developed. Starting from adenosine 3′5′-diphosphate (PAP), the sulfate donating and highly expensive cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS, 1) can be regenerated by using a recombinant aryl sulfotransferase and p-nitrophenyl sulfate. This system averts product inhibition by PAP and can serve as a continuous spectrophotometric assay for the activity of any sulfotransferase enzyme.

Preiswerter als zuvor können Oligosaccharide jetzt präparativ enzymatisch sulfatiert werden. Ausgehend von Adenosin-3′,5′-diphosphat (PAP) kann jetzt der als Sulfatgruppen-Donor dienende, teure Cofaktor 3′-Phosphoadenosin-5′-phosphosulfat (PAPS) 1 mit einer neuen Methode unter Verwendung einer rekombinanten Aryl-Sulfotransferase und p-Nitrophenylsulfat regeneriert werden. Dieses System unterliegt nicht der Produkthemmung durch PAP und kann als kontinuierlicher spektrophotometrischer Assay für die kinetische Analyse von Sulfotransferasen eingesetzt werden.

Useful strategies for the design of molecules to mimic carbohydrates have been developed over the past few years. Mimics of the target may contain new functional groups, a new scaffold, or both (in the schematic representation the natural ligand is shown on the left and the modified version on the right). Many examples of successful carbohydrate mimetics that interfere with sugar–protein and sugar–nucleic acid interactions are known.

The concise synthesis of nine diantennary oligosaccharides, such as 1, by chemical and chemoenzymatic protocols is presented. The compounds display Lewis X, Lewis Y, sialyl Lewis X and T-antigen epitopes supported on a 3,6-branched trimannose core. They derive from the glycans of the human glycoproteins Glycodelin-A and Glycodelin-S believed to be involved in regiospecific suppression of the female immune system.

Zahlreiche nützliche Strategien für das Design von Molekülen, die Kohlenhydrate nachahmen, wurden in den letzten Jahren entwickelt. Mimetika der Targets können neue funktionelle Gruppen und/oder neue Gerüste enthalten (in der schematischen Darstellung ist der natürliche Ligand links und der modifizierte rechts gezeigt). Es liegen bislang viele Beispiele für Kohlenhydratmimetika vor, die Zucker-Protein- und Zucker-Nucleinsäure-Wechselwirkungen effizient stören.

Nach dem Prinzip mehrfacher Wechselwirkungen könnte das amphiphile Polymer 1, das in wäßriger Lösung als Aggregat vorliegt (siehe unten rechts), eine Infektion mit dem Influenzavirus verhindern. Nach der Erkennung des spezifischen Sialyllactose-Epitops durch Hämagglutinin (HA) auf der Virusoberfläche bilden der Sphingosinrest und der Fluoreszenzmarker durch hydrophobe Wechselwirkungen einen stabilen Komplex mit HA. 1 weist eine 10⁶fach höhere In-vitro-Inhibierungsaktivität auf als Sialyllactose. PGA = Polyglutaminsäure.

The synthesis of a potent competitive LTA₄hydrolase inhibitor (A, K_i=1.6 nM) prompted an investigation into the biological activity of A against LTB₄ biosynthesis, as well as the design, synthesis, and evaluation of a new series of inhibitors B to further probe the active site of LTA₄ hydrolase. On the basis of these results, and previously reported site-directed mutagenesis and inhibition studies, the mechanisms of peptide and epoxide hydrolysis catalyzed by LTA₄ hydrolase are discussed.

Based on the principle of a multivalent interaction, the amphiphilic polymer 1, present in solution as an aggregate (see below right), is able to inhibit infection with the influenza virus. After recognition of a specific sialyllactose epitope through hemaglutinin (HA) on the virus surface, the sphingosine residues and the fluorescent tag form a stable complex with HA through hydrophobic interactions. Polymer 1 shows in vitro inhibitory activity 10⁶-fold greater than that of sialyllactose. PGA=polyglutamic acid.

Just tagging along. For the nondestructive quantitative monitoring of solid-phase oligosaccharide synthesis a ¹³C-enriched tag (*) was incorporated in the linker and a ¹³C-enriched protecting group (⧫) was included in the growing molecule. By integration of the signals in a gated decoupling ¹³C NMR experiment the reaction progress can be monitored. This method was demonstrated with the synthesis of sialyl Lewis^x tetrasaccharide on a Tentagel support ((P)). Bn=benzyl, PEG=poly(ethylene glycol).

Carbohydrates are increasingly appreciated for their critical roles in biological functions. Driving these studies addressing the molecular basis of carbohydrate-mediated events is the availability of natural and unnatural carbohydrates. Recently, new methods of enzymatic synthesis have been developed that now afford rapid access to complex, structurally diverse oligosaccharides on large scales. This methodology has been essential to gaining insight into the molecular details of important carbohydrate recognition phenomena that are associated with many signaling processes. This review summarizes recent chemical studies examining selectin/sialyl Lewis^x binding, a key event in inflammation and metastasis, and describes the development of carbohydrate mimetics that may offer leads to new therapeutic agents. In addition, combinatorial synthesis has been used to prepare carbohydrate mimetics to neomycin B, an aminoglycoside that binds RNA involved in HIV infection, and chemoenzymatic methodology has been applied to the design and synthesis of mechanism-based inhibitors of glycoprocessing enzymes.