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The hallmark of a molecular glue is its ability to induce cooperative protein–protein interactions, leading to the formation of a ternary complex, despite weaker binding towards one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, a second class of inducers of protein–protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence and absence of a presenter protein, using the “presenter ratio”, the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain (BRD)9 and the VHL–elongin C–elongin B (VCB) complex. Our most cooperative hit compound,13-7, exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the discovery of molecular glues for pre-selected proteins and thus facilitate the transition to a new paradigm of molecular therapeutics.

Molecular glues and bifunctional compounds that induce protein–protein associations provide a powerful and general mechanism to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two pre-selected proteins, using the first bromodomain of BRD4 and the VHL–elongin C–elongin B (VCB) complex as a test system. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ∼one million DNA-encoded compounds that possess a VHL-targeting fragment, a variety of connectors, and a diversity element generated by split- and-pool combinatorial chemistry. By screening our DEL against BRD4BD1in the presence and absence of VCB, we could identify VHL-bound molecules that simultaneously bind BRD4. For highly barcode-enriched library members, ternary complex formation leading to BRD4 degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for the most enriched library member. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.Abstract Figure

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.

Growing malignant tumors must evade destruction by the immune system, a hurdle some malignancies overcome by attracting immune-suppressive regulatory T-cells (Tregs)1. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Tregs, and IKZF2 deficiency enhances immune responses to tumors in mice2, suggesting IKZF2 may be an attractive target for cancer immunotherapy. Here we describe the discovery and characterization of DKY709, the first molecular glue degrader of IKZF2/4 which spares IKZF1/3. DKY709 was identified through a recruitment-guided medicinal chemistry campaign that redirected the degradation selectivity of CRBN binders towards IKZF2. The IKZF transcription factor selectivity of DKY709 was rationalized by the X-ray structure of the CRBN-DKY709-IKZF2(ZF2) ternary complex. Upon exposure to DKY709, human Tregs showed reduced suppressive activity and exhausted T-effector cells recovered IFNγ production. In vivo, oral treatment with DKY709 drove a rapid and sustained degradation of IKZF2 including in humans and led to delayed tumor growth in mice with humanized immune systems and enhanced immunization responses in monkeys. DKY709 is a first-in-class, potent and selective oral IKZF2/4 degrader currently being investigated in a phase 1 clinical trial as an immune-enhancing agent for cancer immunotherapy.

DNA-encoded libraries of small molecules are being explored extensively for the identification of binders in early drug-discovery efforts. Combinatorial syntheses of such libraries require water- and DNA-compatible reactions, and the paucity of these reactions currently limit the chemical features of resulting barcoded products. The present work introduces strain-promoted cycloadditions of cyclic allenes under mild conditions to DNA-encoded library synthesis. Owing to distinct cycloaddition modes of these reactive intermediates with activated olefins, 1,3-dipoles, and dienes, the process generates diverse molecular architectures from a single precursor. The resulting DNA-barcoded compounds exhibit unprecedented ring and topographic features, related to elements found to be powerful in phenotypic screening.

Cyclopropane-fused N-heterocycles are featured in various biologically active compounds and represent attractive scaffolds in medicinal chemistry. However, synthesis routes to access structurally and functionally diverse cyclopropane-fused N-heterocycles remain underexplored. Leveraging novel α-diazo acylating agents, we report a general approach for the direct and modular synthesis of cyclopropane-fused lactams from unsaturated amines. The operationally simple transformation, which proceeds through successive acylation, (3+2) cycloaddition and fragmentation, tolerates a broad range of functional groups and yields a wide spectrum of complex molecular scaffolds, including fused, bridged and spiro ring systems. We demonstrate the utility of this transformation in the concise syntheses of therapeutic agents milnaciprane and amitifadine.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

DNA-encoded libraries of small molecules are being explored extensively for the identification of binders in early drug-discovery efforts. Combinatorial syntheses of such libraries require water- and DNA-compatible reactions, and the paucity of these reactions currently limit the chemical features of resulting barcoded products. The present work introduces strain-promoted cycloadditions of cyclic allenes under mild conditions to DNA-encoded library synthesis. Owing to distinct cycloaddition modes of these reactive intermediates with activated olefins, 1,3-dipoles and dienes, the process generates diverse molecular architectures from a single precursor. The resulting DNA-barcoded compounds exhibit unprecedented ring and topographic features—related to elements found to be powerful in phenotypic screening.

Selective serotonin reuptake inhibitors (SSRIs) are the only effective pharmacological treatments for obsessive–compulsive disorder (OCD). Nonetheless, their generally limited efficacy, side-effects, and delayed onset of action require improved medications for this highly prevalent disorder. Preclinical and clinical findings have suggested serotonin2C (5-HT2C) receptors as a potential drug target. Data in rats and mice are presented here on the effects of a novel 5-HT2C receptor agonist ((3S)-3-Methyl-1-[4-(trifluoromethyl)-7-benzofuranyl]-piperazine) (CPD 1) with high potency and full efficacy at 5-HT2C receptors and less potency and partial agonism at 5-HT2A and 5-HT2B receptors. Effects of CPD 1 on consummatory (schedule-induced polydipsia in rats) and non-consummatory behaviors (marble-burying and nestlet-shredding in mice) that are repetitive and non-habituating were studied. We also evaluated the effects of CPD 1 in rats with isoproterenol- and deprivation-induced drinking in rats to compare with the polydipsia studies. The SSRIs, fluoxetine, and chlomipramine decreased the high rates of drinking in rats engendered by a schedule of intermittent food delivery (schedule-induced polydipsia). The effects of fluoxetine, but not of d-amphetamine, were prevented by the selective 5-HT2C receptor antagonist SB242084. The 5-HT2C receptor agonists Ro 60-0175 and CPD 1 also decreased drinking, but unlike the SSRIs and Ro 60-0175, CPD 1 dose-dependently decreased excessive drinking without affecting lever press responses that produced food. The effects of CPD 1 were prevented by SB242084. CPD 1 also suppressed drinking induced by isoproterenol and by water deprivation without affecting normative drinking behavior. CPD 1, like fluoxetine, also suppressed marble-burying and nestlet-shredding in mice at doses that did not affect rotarod performance or locomotor activity. The behavioral specificity of effects of CPD 1 against repetitive and excessive behaviors suggests a potential therapeutic application in OCD.

A series of benzylic piperazines (e.g., 4 and 5) attached to an 'address element', the dipeptide H-D-Tic-D-p-Cl-Phe-OH, 3 has been identified as ligands for the melanocortin subtype-4 receptor (MC4R). We describe herein the structure-activity relationship (SAR) studies on the N-terminal residue of the 'address element'. Several novel dipeptides and reduced dipeptides with high MC4R binding affinities and selectivity emerged from this SAR study.

Replacement of the aryl piperazine moiety in compound 1 with a variety of substituted benzylic piperazines (6) yields compounds that afford melanocortin receptor 4 (MCR4) activity. Analogs with ortho substitution on the aromatic ring afforded the highest affinity. Resolution of the stereocenter of the benzylic piperazine based privileged structure revealed that the R-enantiomer was more active.

Substitution of the aryl sulfonamide moiety contained in MC4 agonist 1 with bicyclic heterocycles and aminotetralines produced compounds with MC4 activity. The heterocycles represent alternative privileged structures to that contained in 1. Compounds in which the polar group of the privileged structure was displayed in an endocyclic fashion were not as active as the parent agonist 1, while those with an exocyclic polar group afforded activity competitive with 1.

Acylation and sulfonylation of the N,N′-unsubstituted glucosylidenespirodiaziridines 1A/1B 95 : 5 with Ac2O, BzCl, FmocCl, TsCl, (naphthalen-2-yl)sulfonyl, and (2,4,6-triisopropylphenyl)sulfonyl chloride, and concomitant rearrangement gave the acylated and sulfonylated gluconolactone hydrazones 2B–2G in 40–83% yield (Scheme 2). Similarly, the galacto and manno analogues 3A/3B 95 : 5 and 5A/5B 55 : 45 and the mannofuransoylidene-diaziridine 30 were acetylated and tosylated to give 4A, 4B, 6, 31A, and 31B (55–73% yield; Schemes 2 and 5). 15N-Labelling of 11A/11B and 14A/14B showed that the pseudoequatorial NH of the gluco diaziridines 1 and the pseudoaxial NH of the galacto diaziridines 3 were preferentially acetylated and tosylated (Scheme 3). Sulfonylation of the N-methylated diaziridines 19A/19B 72 : 28, 22A/22B 85 : 15, 25A/25B 85 : 15, 28A/28B 80 : 20, and 33A/33B/33C/33D 76 : 4 : 12 : 8 yielded the N-methyl-N-tosylglyconolactone hydrazones 20, 23, 26, 29, and 34 (44–66%; Schemes 4 and 5). The methylated N-atom of the diaziridines proved more reactive, irrespective of the configuration at C(2) and C(4). The products were readily hydrolysed to glyconolactones.

The diastereoselectivity of the addition of NH3 and MeNH2 to glyconolactone oxime sulfonates and the structures of the resulting N-unsubstituted and N-methylated glycosylidene diaziridines were The 15N-labelled glucono- and galactono-1,5-lactone oxime mesylates 1* and 9* add NH3 mostly axially (>3 : 1; Scheme 4), while the 15N-labelled mannono-1,5-lactone oxime sulfonate 19* adds NH3 mostly equatorially (9 : 1; Scheme 7). The 15N-labelled mannono-1,4-lactone oxime sulfonate 30* adds NH3 mostly from the exo side (>4 : 1; Scheme 9). The configuration of the N-methylated pyranosylidene diaziridines 17, 18, 28, and 29 suggests that MeNH2 adds to 1, 9, 19, and 23 mostly to exclusively from the equatorial direction (>7 : 3; Schemes 5 and 8). The mannono-1,4-lactone oxime sulfonate 30 adds MeNH2 mostly from the exo side (85 : 15; Scheme 10), while the ribo analogue 37 adds MeNH2 mostly from the endo side (4 : 1; Scheme 10). Analysis of the preferred and of the reactive conformers of the tetrahedral intermediates suggests that the addition of the amine to lactone oxime sulfonates is kinetically controlled. The diastereoselectivity of the diaziridine formation is rationalized as the result of the competing influences of intramolecular H-bonding during addition of the amines, steric interactions (addition of MeNH2), and the kinetic anomeric effect. The diaziridines obtained from 2,3,5-tri-O-benzyl-D-ribono- and -D-arabinono-1,4-lactone oxime methanesulfonate (42 and 48; Scheme 11) decomposed readily to mixtures of 1,4-dihydro-1,2,4,5-tetrazines, pentono-1,4-lactones, and pentonamides. The N-unsubstituted gluco- and galactopyranosylidene diaziridines 2, 4, 6, 8, and 10 are mixtures of two trans-substituted isomers (S/Rca. 19 : 1, Scheme 2). The main, (S,S)-configured isomers S are stabilised by a weak intramolecular H-bond from the pseudoaxial NH to ROC(2). The diaziridines 12, derived from GlcNAc, cannot form such a H-bond; the (R,R)-isomer dominates (R/S 85 : 15; Scheme 3). The 2,3-di-O-benzyl-D-mannopyranosylidene diaziridines 20 and 22 adopt a 4C1 conformation, which does not allow an intramolecular H-bond; they are nearly 1 : 1 mixtures of R and S diastereoisomers, whereas the OH5 conformation of the 2,3:5,6-di-O-isopropylidene-D-mannopyranosylidene diaziridines 24 is compatible with a weak H-bond from the equatorial NH to OC(2); the (R,R)-isomer is favoured (R/S ≥7 : 3; Scheme 6). The mannofuranosylidene diaziridine 31 completely prefers the (R,R)-configuration (Scheme 9).

Ergolines were recently identified as a novel class of H3 receptor (H3R) inverse agonists. Although their optimization led to drug candidates with encouraging properties for the treatment of narcolepsy, brain penetration remained low. To overcome this issue, ergoline 1 ((6aR,9R,10aR)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9,10,10a-hexahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide)) was transformed into a series of indole derivatives with high H3R affinity. These new molecules were profiled by simultaneous determination of their brain receptor occupancy (RO) levels and pharmacodynamic (PD) effects in mice. These efforts culminated in the discovery of 15 m ((R)-1-isopropyl-5-(1-(2-(2-methylpyrrolidin-1-yl)ethyl)-1H-indol-4-yl)pyridin-2(1H)-one), which has an ideal profile showing a strong correlation of PD effects with RO, and no measurable safety liabilities. Its desirably short duration of action was confirmed by electroencephalography (EEG) measurements in rats.

Two intramolecular routes were experimented to reach the hexahydrobenzofuro(2,3-c)pyridine platform: a Heck and a radical reaction. The radical route was applicable to all substrates, whereas the Heck route was of limited use. The key adducts were obtained via a Mitsunobu condensation between halogenated phenols and an allylic alcohol, the 3-hydroxy-tetra- hydropyridine. © 2001 Elsevier Science Ltd. All rights reserved.

Abstract: The challenges for the design of new antidepressant drugs are threefold: rapid onset of antidepressant response, broader efficacy, and fewer adverse effects. While progress has been made to reduce side-effects, the currently available drugs show no convincing evidence for a shorter delay of onset nor for improved efficacy. This review summarizes some of the hypotheses that try to explain the delayed onset and that are currently used for antidepressant drug design. These approaches include combinations of SSRI activity with either NA reuptake inhibition or 5-HT1 A-, 5-HT2-, or a.2-adrenergic receptor specificity, as well as targeting interactions with specific receptor subtypes (5-HT1A, 5-HTJA/5-HT2, 5-HTJB/D, a.2/5-HT2, D2/D3). Structure activity relationship studies to obtain molecular entities with the desired pharmacological profile are discussed. Many structural modifications have exploited successfully the acquired knowledge on structural features that are necessary for monoamine reuptake inhibition as well as for affinities at various receptors. Most of the current drug design efforts are focused on these approaches, which are based on the monoamine theory of depression; however, novel biological concepts of depression involving other receptor systems or intracellular targets are briefly mentioned. Although their impact on the onset of antidepressant response is only a matter of speculation at this moment, they may lead to the identification of targets for truly novel antidepressants.

The syntheses of glycosides from the diazirine 1 and a range of alcohols under thermal and/or photolytic conditions are described. Yields and diastereoselectivities depend upon the pKHA values of the alcohols, the solvent, and the reaction temperature. The glycosidation of weakly acidic alcohols (MeOH, EtOH, i-PrOH, and t-BuOH, 1 equiv. each) in CH2Cl2 at room temperature leads to the glycosides 2–5 in yields between 60 and 34% (Scheme 1 and Table 1). At −70 to −60°, yields are markedly higher. In CH2Cl2, diastereoselectivities are very low. In THF, at −70 to −60°, however, glycosidation of i-PrOH leads to α-D-/β-D-4 in a ratio of 8:92. More strongly acidic alcohols, such as CF3CH2OH, (CF3)2 CHOH, and (CF3)2C(Me)OH, and the highly fluorinated long-chain alcohols CF3(CF2)5(CH2)2OH (11) and CHF2(CF2)9CH2OH (13) react (CH2Cl2, r.t.) in yields between 73 and 85% and lead mainly to the β-D-glucosides β-D-6 to β-D-8, β-D-12, and β-D-14 (d.e. 14–68%). Yields and diastereoselectivities are markedly improved, when toluene, dioxane, 1,2-dimetoxyethane, or THF are used, as examined for the glycosidation of (CF3)2C(Me)OH, yielding (1,2-dimethoxyethane, 25°) 80% of α-D-/ β-D-8 in a ratio of 2:98 (d.e. 96%; Table 4). In EtCN, (CF3)2C(Me)OH yields up to 55% of the imidate 10. Glycosidation of di-O-isopropylideneglucose 15 leads to 16 (CH2Cl2, r.t.; 65%, α-D/ β-D = 33:67). That glycosidation occurs by initial protonation of the intermediate glycosylidene carbene is evidenced, for strongly acidic alcohols, by the formation of 10, derived from the attack of (CF3)2MeCO− on an intermediate nitrilium ion (Scheme 4), and for weakly acidic alcohols, by the formation of α-D-9 and β-D-9, derived by attack of i-PrO− on intermediate tetrahydrofuranylium ions. A working hypothesis is presented (Scheme 3). The diastereoselectivities are rationalized on the basis of a protonation in the σ plane of the intermediate carbene, the stabilization of the thereby generated ion pair by interaction with the BnOC(2) group, with the solvent, and/or with the alcohol, and the final nucleophilic attack by RO− in the π plane of the (solvated) oxonium ion.

Two intramolecular routes were experimented to reach the hexahydrobenzofuro(2,3-c)pyridine platform: a Heck and a radical reaction. The radical route was applicable to all substrates, whereas the Heck route was of limited use. The key adducts were obtained via a Mitsunobu condensation between halogenated phenols and an allylic alcohol, the 3-hydroxy-tetra- hydropyridine. © 2001 Elsevier Science Ltd. All rights reserved.

The syntheses of glycosides from the diazirine 1 and a range of alcohols under thermal and/or photolytic conditions are described. Yields and diastereoselectivities depend upon the pKHA values of the alcohols, the solvent, and the reaction temperature. The glycosidation of weakly acidic alcohols (MeOH, EtOH, i-PrOH, and t-BuOH, 1 equiv. each) in CH2Cl2 at room temperature leads to the glycosides 2–5 in yields between 60 and 34% (Scheme 1 and Table 1). At −70 to −60°, yields are markedly higher. In CH2Cl2, diastereoselectivities are very low. In THF, at −70 to −60°, however, glycosidation of i-PrOH leads to α-D-/β-D-4 in a ratio of 8:92. More strongly acidic alcohols, such as CF3CH2OH, (CF3)2 CHOH, and (CF3)2C(Me)OH, and the highly fluorinated long-chain alcohols CF3(CF2)5(CH2)2OH (11) and CHF2(CF2)9CH2OH (13) react (CH2Cl2, r.t.) in yields between 73 and 85% and lead mainly to the β-D-glucosides β-D-6 to β-D-8, β-D-12, and β-D-14 (d.e. 14–68%). Yields and diastereoselectivities are markedly improved, when toluene, dioxane, 1,2-dimetoxyethane, or THF are used, as examined for the glycosidation of (CF3)2C(Me)OH, yielding (1,2-dimethoxyethane, 25°) 80% of α-D-/ β-D-8 in a ratio of 2:98 (d.e. 96%; Table 4). In EtCN, (CF3)2C(Me)OH yields up to 55% of the imidate 10. Glycosidation of di-O-isopropylideneglucose 15 leads to 16 (CH2Cl2, r.t.; 65%, α-D/ β-D = 33:67). That glycosidation occurs by initial protonation of the intermediate glycosylidene carbene is evidenced, for strongly acidic alcohols, by the formation of 10, derived from the attack of (CF3)2MeCO− on an intermediate nitrilium ion (Scheme 4), and for weakly acidic alcohols, by the formation of α-D-9 and β-D-9, derived by attack of i-PrO− on intermediate tetrahydrofuranylium ions. A working hypothesis is presented (Scheme 3). The diastereoselectivities are rationalized on the basis of a protonation in the σ plane of the intermediate carbene, the stabilization of the thereby generated ion pair by interaction with the BnOC(2) group, with the solvent, and/or with the alcohol, and the final nucleophilic attack by RO− in the π plane of the (solvated) oxonium ion.

Laser flash photolysis of a glycosylidene-derived diazirine produces the corresponding carbene. The carbene can be intercepted with pyridine to form an ylide. The absolute rate constants for the reaction of the glycosylidene carbene with alcohols can be obtained by monitoring the absolute rate of formation of the pyridinium ylide. The kinetic data favors a mechanism involving a proton transfer from the alcohol to the carbene

[formula: see text] The reactions of glycals with ceric(IV) ammonium nitrate and sodium iodide in the presence of acetic acid provides 2-deoxy-2-iodo-alpha-mannopyranosyl acetates with good stereoselectivity. In the majority of the cases examined, the selectivity was considerably better than that from reactions using N-iodosuccinimide and HOAc.

Phenol, 4-methoxyphenol, 4-nitrophenol, methyl orsellinate (1), and 2,6-di(tert-butyl)-4-methylphenol (BHT; 2) have been glycosylated by thermal reaction (20-60°) with various glycosylidene-derived diazirines

Homologation and cyclization back to the chiral methine of compound 3 yields achiral 4,4-disubstituted piperidine privileged structures (e.g., 8a) useful in the construction of melanocortin 4 receptor (MC4R) ligands. The piperidine nitrogen was replaced with carbon, oxygen, sulfur, and sulfone with minor erosion of binding. The methyl cyclohexane substituent was the most potent while significant affinity was still seen for smaller lipophilic groups such as ethyl.

Phosphonate analogue 5 of the lipid A precursor 4 has been prepared from phosphonate 2 and nucleotide 3 with the help of lipid A synthase, isolated from the overproducing Escherichia coli mutant MC 1061 (delta 2512) or JB1104 (delta 2514). The biological properties of phosphonate 5 and phosphate 4 are quite similar to each other as compared in the limulus amoebocyte lysate assay, by the activation of the RAW264 murine macrophagelike cell line (determined by stimulation of ornithine decarboxylase), and by the pyrogenicity in rabbits. Hydrolytic removal of the 1-phosphate group of 4 is thus not a prerequisite for its biological activity.

Phenol, 4-methoxyphenol, 4-nitrophenol, methyl orsellinate (1), and 2,6-di(tert-butyl)-4-methylphenol (BHT; 2) have been glycosylated by thermal reaction (20-60°) with various glycosylidene-derived diazirines

A preparation of glycosylphosphonates (27, 28, 36, 38, and 39) from 2-azido-2-deoxy-glycoses (26,35, and 37) and the synthesis of the non-isosteric phosphonate analogue 3a of lipid X(2) are described. The 2-azido group was introduced by azidonitration. Treatment of the 1-O-acetyl-2-azido-2-deoxy-β-D-galactopyranose 22 with 1.5-3 equiv. of P(OMe)3 and 1.2-2.5 equiv. of TfOSiMe3 gave mainly recovered starting material. In P(OMe)3 as the solvent, the dimethyl phosphoramidate 24 was obtained by way of a Staudinger reaction, even in the presence of TfOSiMe3. Treatment of the benzylated α-D-galacto-trichloroacetimidate 26, however, with P(OMe)3 and TfOSiMe3 gave a 1:1 mixture of the α- and β-D-galacto-phosphonates 27 and 28, while the acetylated α-D-gluco- imidate 35 led to the α-D-gluco-configurated phosphonate 36. The stereoselectivity of the phosphonate formation is related to the relative ease of formation of oxonium-ion intermediates from 26 and 35. Starting from the phosphonate 36, deacetylation, benzylidenation, reduction of the azido group, acylation with (R)-3-(benzyloxy)tetradecanoic acid and deprotection yielded the desired compound 3a which was crystallized in the presence of 2 equiv. of (aminomethylidyne)trimethanol (Tris.). The structure of the phosphonates was deduced from their 1H-, 13C-, and 31P-NMR spectra.

A new approach towards the synthesis of glycosides based upon a (formal) insertion of glycosylidene carbenes into OH bonds is presented. The synthesis and characterization of the glycosylidene-derived diazirines 25–28, precursors of glycosylidene carbenes, are described. The diazirines were prepared by the rapid, high-yielding oxidation of the diaziridines 20 and 22–24 with I2/Et3N. The diaziridines, the first examples of C- alkoxy-diaziridines, were formed in high yields by the reaction of the [(glycosylidene)-amino]methanesulfonates 14 and 17–19 with a saturated solution of NH3 in MeOH. The diazirines are highly reactive compounds, losing N2 at room temperature or below. The reaction of the gluco-configurated diazirine 25 with i-PrOH yielding a mixture of the α- and β-D-glucosides 29 and 30 illustrates the potential of glycosylidene-derived diazirines as a new type of glycosyl donors.