Optically Active Cyclophane Receptors for Mono‐ and Disaccharides: The Role of Bidentate Ionic Hydrogen Bonding in Carbohydrate Recognition

A new family of optically active cyclophane receptors for the complexation of mono‐ and disaccharides in competitive protic solvent mixtures is described. Macrocycles (−)‐(R,R,R,R)‐ 1 – 4 feature preorganized binding cavities formed by four 1,1′‐binaphthalene‐2,2′‐diyl phosphate moieties bridged in the 3,3′‐positions by acetylenic or phenylacetylenic spacers. The four phosphodiester groups converge towards the binding cavity and provide efficient bidentate ionic H‐bond acceptor sites (Fig. 2). Benzyloxy groups in the 7,7′‐positions of the 1,1′‐binaphthalene moieties ensure solubility of the nanometer‐sized receptors and prevent undesirable aggregation. The construction of the macrocyclic framework of the four cyclophanes takes advantage of Pd⁰‐catalyzed aryl—acetylene cross‐coupling by the Sonogashira protocol, and oxidative acetylenic homo‐coupling methodology (Schemes 2 and 8 – 10). Several cleft‐type receptors featuring one 1,1′‐binaphthalene‐2,2′‐diyl phosphate moiety were also prepared (Schemes 1, 6, and 7). An undesired side reaction encountered during the synthesis of the target compounds was the formation of naptho[b]furan rings from 3‐ethynylnaphthalene‐2‐ol derivatives, proceeding via 5‐endo‐dig cyclization (Schemes 3 – 5). Computer‐assisted molecular modeling indicated that the macrocycles prefer nonplanar puckered, cyclobutane‐type conformations (Figs. 7 and 8). According to these calculations, receptor (−)‐(R,R,R,R)‐ 1 has, on average, a square binding site, which is complementary in size to one monosaccharide. The three other cyclophanes (−)‐(R,R,R,R)‐ 2 – 4 feature, on average, wider rectangular cavities, providing a good fit to one disaccharide, while being too large for the complexation of one monosaccharide. This substrate selectivity was fully confirmed in ¹H‐NMR binding titrations. The chiroptical properties of the cyclophanes and their nonmacrocyclic precursors were investigated by circular dichroism (CD) spectroscopy. The CD spectra of the acyclic precursors showed a large dependence from the number of 1,1′‐binaphthalene moieties (Fig. 9), and those of the cyclophanes were remarkably influenced by the nature of the functional groups lining the macrocyclic cavity (Fig. 11). Profound differences were also observed between the CD spectra of linear and macrocyclic tetrakis(1,1′‐binaphthalene) scaffolds, which feature very different molecular shapes (Fig. 10). In ¹H‐NMR binding titrations with mono‐ and disaccharides (Fig. 13), concentration ranges were chosen to favor 1 : 1 host−guest binding. This stoichiometry was experimentally established by the curve‐fitting analysis of the titration data and by Job plots. The titration data demonstrate conclusively that the strength of carbohydrate recognition is enhanced with an increasing number of bidentate ionic host−guest H‐bonds (Table 1) in the complex formed. As a result of the formation of these highly stable H‐bonds, carbohydrate complexation in competitive protic solvent mixtures becomes more favorable. Thus, cleft‐type receptors (−)‐(R)‐ 7 and (−)‐(R)‐ 38 with one phosphodiester moiety form weak 1 : 1 complexes only in CD₃CN. In contrast, macrocycle (−)‐(R,R,R,R)‐ 1 with four phosphodiester groups undergoes stable inclusion complexation with monosaccharides in CD₃CN containing 2% CD₃OD. With their larger number of H‐bonding sites, disaccharide substrates bind even more strongly to the four phosphodiester groups lining the cavity of (−)‐(R,R,R,R)‐ 2 and complexation becomes efficient in CD₃CN containing 12% CD₃OD. Finally, the introduction of two additional methyl ester residues further enhances the receptor capacity of (−)‐(R,R,R,R)‐ 3 , and efficient disaccharide complexation occurs already in CD₃CN containing 20% CD₃OD.     

Molecular Recognition of Pyranosides by a Family of Trimeric, 1,1′-Binaphthalene-Derived Cyclophane Receptors

The synthesis and carbohydrate-recognition properties of a new family of optically active cyclophane receptors, 1 – 3 , in which three 1,1′-binaphthalene-2,2′-diol spacers are interconnected by three buta-1,3-diynediyl linkers, are described. The macrocycles all contain highly preorganized cavities lined with six convergent OH groups for H-bonding and complementary in size and shape to monosaccharides. Compounds 1 – 3 differ by the functionality attached to the major groove of the 1,1′-binaphthalene-2,2′-diol spacers. The major grooves of the spacers in 2 are unsubstituted, whereas those in 1 bear benzyloxy (BnO) groups in the 7,7′-positions and those in 3 2-phenylethyl groups in the 6,6′-positions. The preparation of the more planar, D₃-symmetrical receptors (R,R,R)- 1 (Schemes 1 and 2), (S,S,S)- 1 (Scheme 4), (S,S,S)- 2 (Scheme 5), and (S,S,S)- 3 (Scheme 8) involved as key step the Glaser-Hay cyclotrimerization of the corresponding OH-protected 3,3′-diethynyl-1,1′-binaphthalene-2,2′-diol precursors, which yielded tetrameric and pentameric macrocycles in addition to the desired trimeric compounds. The synthesis of the less planar, C₂-symmetrical receptors (R,R,S)- 2 (Scheme 6) and (S,S,R)- 3 (Scheme 9) proceeded via two Glaser-Hay coupling steps. First, two monomeric precursors of identical configuration were oxidatively coupled to give a dimeric intermediate which was then subjected to macrocyclization with a third monomeric 1,1′-binaphthalene precursor of opposite configuration. The 3,3′-dialkynylation of the OH-protected 1,1′-binaphthalene-2,2′-diol precursors for the macrocyclizations was either performed by Stille (Scheme 1) or by Sonogashira (Schemes 4, 5, and 8) cross-coupling reactions. The flat D₃-symmetrical receptors (R,R,R)- 1 and (S,S,S)- 1 formed 1 : 1 cavity inclusion complexes with octyl 1-O-pyranosides in CDCl₃ (300 K) with moderate stability (ΔG⁰ ca. −3 kcal mol⁻¹) as well as moderate diastereo- (Δ(ΔG⁰) up to 0.7 kcal mol⁻¹) and enantioselectivity (Δ(ΔG⁰)=0.4 kcal mol⁻¹) (Table 1). Stoichiometric 1 : 1 complexation by (S,S,S)- 2 and (S,S,S)- 3 could not be investigated by ¹H-NMR binding titrations, due to very strong signal broadening. This broadening of the ¹H-NMR resonances is presumably indicative of higher-order associations, in which the planar macrocycles sandwich the carbohydrate guests. The less planar C₂-symmetrical receptor (S,S,R)- 3 formed stable 1 : 1 complexes with binding free enthalpies of up to ΔG⁰=−5.0 kcal mol⁻¹ (Table 2). With diastereoselectivities up to Δ(ΔG⁰)=1.3 kcal mol⁻¹ and enantioselectivities of Δ(ΔG⁰)=0.9 kcal mol⁻¹, (S,S,R)- 3 is among the most selective artificial carbohydrate receptors known.     

Trapping of peroxidic intermediates with sulfur and phosphorus centered electrophiles

The trapping ability of a new peroxidic trapping agent relative to several well-established trapping agents was measured. Two different methods for this measurement were utilized. It was shown that adamantylidene adamantane reacts under the trapping conditions to give adamantylidene adamantane epoxide via a more complicated process than previously recognized and is consequently an inappropriate system to make this measurement. In contrast, the use of diethyl sulfide is straightforward and gives reliable values. © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:51–56, 1998     

A resolution measure for three-dimensional microscopy

A three-dimensional (3D) resolution measure for the conventional optical microscope is introduced which overcomes the drawbacks of the classical 3D (axial) resolution limit. Formulated within the context of a parameter estimation problem and based on the Cramer-Rao lower bound, this 3D resolution measure indicates the accuracy with which a given distance between two objects in 3D space can be determined from the acquired image. It predicts that, given enough photons from the objects of interest, arbitrarily small distances of separation can be estimated with prespecified accuracy. Using simulated images of point source pairs, we show that the maximum likelihood estimator is capable of attaining the accuracy predicted by the resolution measure. We also demonstrate how different factors, such as extraneous noise sources and the spatial orientation of the imaged object pair, can affect the accuracy with which a given distance of separation can be determined.     

Electrophysiological Responses of Bactrocera kraussi (Hardy) (Tephritidae) to Rectal Gland Secretions and Headspace Volatiles Emitted by Conspecific Males and Females

Pheromones are biologically important in fruit fly mating systems, and also have potential applications as attractants or mating disrupters for pest management. Bactrocera kraussi (Hardy) (Diptera: Tephritidae) is a polyphagous pest fruit fly for which the chemical profile of rectal glands is available for males but not for females. There have been no studies of the volatile emissions of either sex or of electrophysiological responses to these compounds. The present study (i) establishes the chemical profiles of rectal gland contents and volatiles emitted by both sexes of B. kraussi by gas chromatography–mass spectrometry (GC–MS) and (ii) evaluates the detection of the identified compounds by gas chromatography–electroantennogram detection (GC–EAD) and –electropalpogram detection (GC–EPD). Sixteen compounds are identified in the rectal glands of male B. kraussi and 29 compounds are identified in the rectal glands of females. Of these compounds, 5 were detected in the headspace of males and 13 were detected in the headspace of females. GC–EPD assays recorded strong signals in both sexes against (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-ethyl-7-mehtyl-1,6-dioxaspiro[4.5]decane isomer 2, (E,Z)/(Z,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, and (Z,Z)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. Male antennae responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-methyl-6-pentyl-3,4-dihydro-2H-pyran, 6-hexyl-2-methyl-3,4-dihydro-2H-pyran, 6-oxononan-1-ol, ethyl dodecanoate, ethyl tetradecanoate and ethyl (Z)-hexadec-9-enoate, whereas female antennae responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and 2-methyl-6-pentyl-3,4-dihydro-2H-pyran only. These compounds are candidates as pheromones mediating sexual interactions in B. kraussi.     

THM PyGC–MS of wood fragment and vegetable fibre forensic samples

Wood fragments and vegetable fibres were investigated using thermally assisted hydrolysis and methylation with pyrolysis gas chromatography–mass spectrometry (THM PyGC–MS). Multiple ion chromatography was used to decrease the interference from cellulosic peaks, and to obtain greater resolution between the lignin peaks. Forty-four wood samples were analysed using THM PyGC–MS. The wood fragments were able to be differentiated into angiosperms (hardwoods) and gymnosperms (softwoods) using principle component analysis (PCA), hierarchical cluster analysis (HCA), and the ratio of syringyl to guaiacyl lignin fragments (S/G ratio). PCA and HCA also differentiated several Monterey pine samples from the rest of the gymnosperms, primarily by the presence of β-pinene, an extractive compound. Other gymnosperm species and the individual angiosperm species were unable to be differentiated. A pilot study investigating the use of THM PyGC–MS for the analysis of vegetable fibres in forensic science found that the fibre types tended to group into two clusters, with one containing cotton, hemp and linen; and the other consisting of hessian, sisal, jute and coir. The seagrass sample was able to be differentiated from both groups. These groups were well separated using PCA, HCA and by the ratio of cinnamyl phenolic derivatives to guaiacyl lignin derivatives (C/G ratio). Some grouping of each fibre type was evident within each cluster, however the separation between the clusters was insufficient to differentiate them using these statistical techniques. THM PyGC–MS of vegetable fibres showed some potential for future use in forensic science.     

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.     

A general chemical synthesis platform for crosslinking multivalent single chain variable fragments

Multivalent single chain variable fragments (scFv) show increased affinity to tumor-associated antigens compared to monovalent scFv and intact monoclonal antibodies (mAb). Multivalent constructs can be derived from self-associating or covalent scFv with covalent constructs offering improved in vivo and in vitro stability. Covalent attachment of scFv can be achieved using genetically engineered expression vectors that afford scFv with site specific cysteine functionality. Expression vectors for di-scFv-C wherein the cysteine is located in the center of two scFv have also been developed for attaching chemically reactive linkers. In the example illustrated here, the di-scFv-C is derived from a mAb directed against the MUC1 epitope, which is presented on cancer cells. To achieve multivalency, a chemical crosslinking strategy utilizing various azide and multi-alkyne functionalized polyethylene glycol (PEG) linkers was implemented. Conjugation was achieved by attachment of these linkers to the scFv thiol functionality. Chemoselective ligation was employed to covalently link different protein conjugates via copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition reaction (CuAAC) chemistry. Ligations were achieved in >70% yield using a specific set of linkers as determined by SDS-PAGE and densitometry. ELISA showed increased tumor binding of a tetravalent scFv providing a versatile chemical crosslinking strategy for construction of multivalent and bi-specific immunoconjugates that retain biological activity and have potential application in pre-targeted radioimmunotherapy and imaging.     

Substantial improvements in large-scale redocking and screening using the novel HYDE scoring function

The HYDE scoring function consistently describes hydrogen bonding, the hydrophobic effect and desolvation. It relies on HYdration and DEsolvation terms which are calibrated using octanol/water partition coefficients of small molecules. We do not use affinity data for calibration, therefore HYDE is generally applicable to all protein targets. HYDE reflects the Gibbs free energy of binding while only considering the essential interactions of protein-ligand complexes. The greatest benefit of HYDE is that it yields a very intuitive atom-based score, which can be mapped onto the ligand and protein atoms. This allows the direct visualization of the score and consequently facilitates analysis of protein-ligand complexes during the lead optimization process. In this study, we validated our new scoring function by applying it in large-scale docking experiments. We could successfully predict the correct binding mode in 93% of complexes in redocking calculations on the Astex diverse set, while our performance in virtual screening experiments using the DUD dataset showed significant enrichment values with a mean AUC of 0.77 across all protein targets with little or no structural defects. As part of these studies, we also carried out a very detailed analysis of the data that revealed interesting pitfalls, which we highlight here and which should be addressed in future benchmark datasets.     

Geometric graph homomorphisms

A geometric graph is a simple graph drawn on points in the plane, in general position, with straightline edges. A geometric homomorphism from to is a vertex map that preserves adjacencies and crossings. This work proves some basic properties of geometric homomorphisms and defines the geochromatic number as the minimum n so that there is a geometric homomorphism from to a geometric n‐clique. The geochromatic number is related to both the chromatic number and to the minimum number of plane layers of . By providing an infinite family of bipartite geometric graphs, each of which is constructed of two plane layers, which take on all possible values of geochromatic number, we show that these relationships do not determine the geochromatic number. This article also gives necessary (but not sufficient) and sufficient (but not necessary) conditions for a geometric graph to have geochromatic number at most four. As a corollary, we get precise criteria for a bipartite geometric graph to have geochromatic number at most four. This article also gives criteria for a geometric graph to be homomorphic to certain geometric realizations of K_{2, 2} and K_{3, 3}. © 2011 Wiley Periodicals, Inc. J Graph Theory 69:97‐113, 2012