Reaction Rate and Stereoselectivity Enhancement in Glycosidations with O-Glycosyl Trihaloacetimidate Donors due to Catalysis by a Lewis Acid-Nitrile Cooperative Effect.

Activation of O-glycosyl trihaloacetimidate glycosyl donors with AuCl3 as a catalyst and pivalonitrile (tBuCN) as a ligand led to excellent glycosidation results in terms of yield and anomeric selectivity. In this way, various ß-d-gluco- and ß-d-galactopyranosides were obtained conveniently and efficiently. Experimental studies and density functional theory (DFT) calculations, in order to elucidate the reaction course, support formation of the tBuCN-AuCl2-OR(H)+ AuCl4- complex as a decisive intermediate in the glycosidation event. Proton transfer from this acceptor complex to the imidate nitrogen leads to donor activation. In this way, guided by the C-2 configuration of the glycosyl donor, the alignment of the acceptor complex enforces the stereoselective ß-glycoside formation in an intramolecular fashion, thus promoting also a fast reaction course. The high stereocontrol of this novel 'Lewis acid-nitrile cooperative effect' is independent of the glycosyl donor anomeric configuration and without the support of neighboring group or remote group participation. The power of the methodology is shown by a successful glycoalkaloid solamargine synthesis.

Synthesis of Core M3 Matriglycan Constituents via an Additive-Controlled 1,2-cis-Xylopyranosylation with O-Xylosyl Imidates as Donors.

A highly stereoselective strategy for 1,2-cis-xylopyranoside bond formation was established via a preactivation-based, additive-modulated trichloroacetimidate glycosidation strategy. The current protocol is mild, practical, and successful with various xylopyranosyl donors and glycosyl acceptors, including acceptors that are reported to be less reactive due to steric hindrance. The utility of this method was demonstrated with the facile assembly of matriglycan constituent tetra- and hexasaccharides.

Preparation of Tea Aroma Precursor Glycosides: An Efficient and Sustainable Approach via Chemical Glycosidation.

Tea aroma precursor glycosides are plant-derived natural products with great economic value. However, the preparation of these glycosides remains largely overlooked in the past decades. Herein, we report a mild, efficient, and sustainable chemocatalytic procedure for the production of tea aroma precursor glycosides. During the study of the glycosidation, the catalysts were found to be decisive in the product formation favoring different reaction pathways; in addition, the influence of molecular sieves was elucidated. With regard to these findings, the serious problem of the competing orthoester formation side reaction was successfully overcome with low catalyst loading (1 mol %) and the use of 5 Å molecular sieves, leading to the preparation of a variety of tea aroma precursor ß-d-glucopyranosides and ß-primeverosides on a gram scale in high yields in an economical way. Taken together, the current approach features catalytic glycosidation with non-toxic and low-cost catalysts, demonstrates highly favorable greenness and sustainability, and promises industrial production of tea aroma precursor glycosides.

Dual-Participation Protecting Group Solves the Anomeric Stereocontrol Problems in Glycosylation Reactions.

The 2,2-dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) group permits, via robust neighboring group participation (NGP) or long distance participation (LDP) effects, the stereocontrolled 1,2-trans, 1,2-cis, as well as ß-2,6-dideoxy glycosidic bond generation, while suppressing the undesired orthoester byproduct formation. The robust stereocontrol capability of the DMNPA is due to the dual-participation effect from both the ester functionality and the nitro group, verified by control reactions and DFT calculations and further corroborated by X-ray spectroscopy.

The 2,2-Dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) Group: A Novel Protecting Group in Carbohydrate Chemistry.

The 2,2-dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) group was introduced to synthetic carbohydrate chemistry as a protecting group (PG) for the first time. Benefiting from a unique chemical structure and novel deprotection conditions, the DMNPA group can be cleaved rapidly and mutually orthogonal to other PGs. Orchestrated application of the DMNPA group with other PGs led to the highly efficient synthesis of the glycan of thornasterside A.

1-Picolinyl-5-azido Thiosialosides: Versatile Donors for the Stereoselective Construction of Sialyl Linkages.

With the picolinyl (Pic) group as a C-1 located directing group and N3 as versatile precursor for C5-NH2 , a novel 1-Pic-5-N3 thiosialyl donor was designed and synthesized, based on which a new sialylation protocol was established. In comparison to conventional sialylation methods, the new protocol exhibited obvious advantages, including excellent α-stereoselectivity in the absence of a solvent effect, broad substrate scope encompassing the challenging sialyl 8- and 9-hydroxy groups of sialic acid acceptors, flexibility in sialoside derivative synthesis, high temperature tolerance and easy scalability. In particular, the applicability to the synthesis of complex and bioactive N-glycan antennae when combined with the MPEP glycosylation protocol via the "latent-active" strategy has been shown. Mechanistically, the excellent α-stereoselectivity of the novel sialylation protocol could be attributed to the dramatic electron-withdrawing effect of the protonated Pic groups, which was supported by control reactions and DFT calculations.

Diversity-Oriented Synthesis of Steviol Glycosides.

With cheap and easily available mixtures of steviol glycosides as starting materials, a practical method for steviol acquisition has been developed, on the basis of which a facile, diversity-oriented, and economic protocol for the synthesis of structurally defined steviol glycosides was established. The novel approach is featured by the highly efficient glycosylation of sterically hindered and acid-sensitive steviol via orchestrated application of Yu glycosylation, Schmidt glycosylation, and PTC glycosylation. Hence, these high-intensity sweeteners and potential lead compounds for drug development are now readily accessible.

Regioselective One-Pot Benzoylation of Triol and Tetraol Arrays in Carbohydrates.

Protection of 2,3,4- O-unprotected α-galacto- and α-fucopyranosides with BzCN and DMAP/DIPEA as the base afforded directly and regioselectively the 3- O-unprotected derivatives. The rationale for these studies was to take advantage of the eventual cooperativity of the "cyanide effect" and "the alkoxy group mediated diol effect". This way, even the totally unprotected α-galactopyranosides could be regioselectively transformed into the corresponding 2,4,6- O-protected derivatives. The great utility of these building blocks was demonstrated in efficient trisaccharide syntheses.

Acid-Base Catalysis in Glycosidations: A Nature Derived Alternative to the Generally Employed Methodology.

Inverting glycosyltransferases enforce in the active site an intramolecular, acid-base catalyzed glycosidation that, due to proximity of the donor anomeric carbon and the acceptor hydroxyl group, follows an SN2-type reaction. Spacers, tethering donor and acceptor via nonreacting functional groups, led in intramolecular glycosidations to excellent yields and, independent of the donor anomeric configuration, to either the α- or the ß-anomer. The requirement of a demanding protecting group pattern confines the application of this efficient method. Only the method where the 2-hydroxyl group of a mannopyranosyl donor is tethered via an acetal spacer to the reacting acceptor functional group is used for ß-mannopyranoside synthesis. The most elegant method, tethering donor and acceptor covalently to the spacer via the leaving group and the reacting functional group, was so far not as efficient as hoped. This method is very efficient when donor and acceptor are temporarily assembled through a hydrogen-bond facilitating a stretched hexagon-like transition state. This follows from the stereoselective O-glucopyranosyl trichloroacetimidate transformation into O-glucopyranosyl phosphate with dibenzyl phosphoric acid as acceptor that can be regarded as A═B-C-H acceptor type. Generalizing this concept to the use of alcohols as acceptors requires reversible generation of an A-B-C-H adduct where A-H represents the acceptor (RO-H) and B═C a catalyst that has to fulfill several criteria. Among these criteria are low affinity to nitrogen, avoiding glycosyl donor activation in the absence of acceptor, and high affinity to oxygen in order to generate the A-B-C-H adduct with increased proton acidity. Thus, hydrogen-bond mediated self-assembly of donor and acceptor and concomitant donor activation via a transition state is available, which enforces an acid-base catalyzed SN2-type reaction. It could be shown that PhBF2, Ph2BF, and PhSiF3 are such catalysts that fulfill the desired four functions: reversible adduct formation with the acceptor, hydrogen-bond mediated tethering of this adduct with the donor, and acid- and base-catalysis of the glycosidation. Also Lewis acidic metal salts, particularly the dimeric gold(III) chloride, turned out to exhibit excellent B═C type catalyst properties. Worth mentioning in this context is the ability of gold(III) chloride to regioselectively activate diols. As thioureas have high affinity to anions and also to neutral compounds through strong hydrogen bonds, their binding to alcohols and concomitant activation of O-glycosyl trichloroacetimidates was of interest. Yet, even the acidic N,N'-bis[3,5-bis(trifluoromethyl)phenyl]-thiourea was unable to catalyze glycosidations. However, as a cocatalyst to acids, thiourea exerts a strong effect that, based on NMR studies, leads first to a hydrogen-bond mediated catalyst-cocatalyst-acceptor complex. This complex activates the donor in an intramolecular, acid-base catalyzed reaction that is again closely related to the action of inverting glycosyltransferases. Thus, from O-(α-glycosyl) trichloroacetimidates, good yields of the inversion products, that is, the ß-glycosides, are obtained. This novel conceptual approach to glycosidation revealed that for retention of configuration in addition a catalytic nucleophile is required that enables formation of the α-glucoside from the α-trichloroacetimidate. Preliminary studies with a catalyst possessing this 5-fold function, that is, adduct formation with the acceptor, hydrogen-bonding between the reactants, acid and base catalysis, and a catalytic nucleophile as part of a chiral framework supporting facial selection, exhibited good chances for final success in this endeavor.

Regioselective Acylation of Diols and Triols: The Cyanide Effect.

Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation.

An Alternative Reaction Course in O-Glycosidation with O-Glycosyl Trichloroacetimidates as Glycosyl Donors and Lewis Acidic Metal Salts as Catalyst: Acid-Base Catalysis with Gold Chloride-Glycosyl Acceptor Adducts.

Gold(III) chloride as catalyst for O-glycosyl trichloroacetimidate activation revealed low affinity to the glycosyl donor but high affinity to the hydroxy group of the acceptor alcohol moiety, thus leading to catalyst-acceptor adduct formation. Charge separation in this adduct, increasing the proton acidity and the oxygen nucleophilicity, permits donor activation and concomitant acceptor transfer in a hydrogen-bond mediated S(N)2-type transition state. Hence, the sequential binding between acceptor and catalyst and then with the glycosyl donor enables self-organization of an ordered transition-state. This way, with various acceptors, even at temperatures below -60 °C, fast and high yielding glycosidations in high anomeric selectivities were recorded, showing the power of this gold(III) chloride acid-base catalysis. Alternative reaction courses via hydrogen chloride or HAuCl4 activation or intermediate generation of glycosyl chloride as the real donor could be excluded. With partially O-protected acceptors, prone to bidentate ligation to gold(III) chloride, particularly high reactivities and anomeric selectivities were observed. Gold(I) chloride follows the same catalyst-acceptor adduct driven acid-base catalysis reaction course.

2-Nitro-thioglycosides: α- and ß-selective generation and their potential as ß-selective glycosyl donors.

Michael-type addition of thiolates to 2-nitro-D-glucal or to 2-nitro-D-galactal derivatives readily provides 2-deoxy-2-nitro-1-thioglycosides. Kinetic and thermodynamic reaction control permitted formation of either the α- or preferentially the ß-anomers, respectively. Addition of achiral and chiral thiourea derivatives to the reaction mixture increased the reaction rate; the outcome is substrate-controlled. The 2-deoxy-2-nitro-1-thioglycosides are excellent glycosyl donors under arylsulfenyl chloride/silver triflate (ArSCl/AgOTf) activation, and they provide, anchimerically assisted by the nitro group, mostly ß-glycosides.

Human L-ficolin recognizes phosphocholine moieties of pneumococcal teichoic acid.

Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.

Cooperative catalysis in glycosidation reactions with O-glycosyl trichloroacetimidates as glycosyl donors.

Thiourea mediates cooperative glycosidation through hydrogen bonding. N,N'-Diarylthiourea as cocatalyst enforces an SN2-type acid-catalyzed glycosidation even at room temperature (see scheme; Bn=benzyl). From O-(α-glycosyl) trichloroacetimidates as glycosyl donors and various acceptors, ß-glycosides are preferentially or exclusively obtained.

C-(α-d-Glucopyranosyl)-phenyldiazomethanes-irreversible inhibitors of α-glucosidase.

Several C-(α-d-glucopyranosyl)-phenyldiazomethanes, with different substituent groups at the para-position of the phenyl ring, were prepared. The stabilities of these diazo compounds were investigated through NMR and UV monitoring. The para-cyano substituted diazo compound was found to be stable in neutral media (pH 7.0 buffer) and could be isolated. Inhibitory activity investigations indicated that this compound is an irreversible inhibitor against α-glucosidase from Saccharomyces cerevisiae.

Equivalence of students' scores on timed and untimed anatomy practical examinations.

Untimed examinations are popular with students because there is a perception that first impressions may be incorrect, and that difficult questions require more time for reflection. In this report, we tested the hypothesis that timed anatomy practical examinations are inherently more difficult than untimed examinations. Students in the Doctor of Physical Therapy program at Thomas Jefferson University were assessed on their understanding of anatomic relationships using multiple-choice questions. For the class of 2012 (n = 46), students were allowed to circulate freely among 40 testing stations during the 40-minute testing session. For the class of 2013 (n = 46), students were required to move sequentially through the 40 testing stations (one minute per item). Students in both years were given three practical examinations covering the back/upper limb, lower limb, and trunk. An identical set of questions was used for both groups of students (untimed and timed examinations). Our results indicate that there is no significant difference between student performance on untimed and timed examinations (final percent scores of 87.3 and 88.9, respectively). This result also held true for students in the top and bottom 20th percentiles of the class. Moreover, time limits did not lead to errors on even the most difficult, higher-order questions (i.e., items with P-values < 0.70). Thus, limiting time at testing stations during an anatomy practical examination does not adversely affect student performance.

Synthesis of glycosylthiols and reactivity studies.

The acid-catalyzed reaction of 1,2-anhydro-3,4,6-tri-O-benzyl-α-d-glucopyranose (7) as glycosyl donor with bis-trimethylsilyl sulfide as acceptor affords the α-thiol. Hence, this sterically hindered S-nucleophile as acceptor should provide with O-glycosyl trichloroacetimidates as glycosyl donors that have nonparticipating groups at C-2, glycosylthiols with the thiol group in axial position. This was confirmed for various donors (4, 16-19) with the exception of the corresponding mannosyl donor (20). However, powerful participating groups at C-2 of the donor (23-28) governed the anomeric selectivity.

Glycoside bond formation via acid-base catalysis.

Acid-base catalyzed glycosyl donor and then glycosyl acceptor activation with phenylboron difluoride or diphenylboron fluoride permits hydrogen bond mediated intramolecular S(N)2-type glycosidation in generally high anomeric selectivity.

Chemical synthesis of bacterial lipoteichoic acids: an insight on its biological significance.

During infections caused by Gram-negative bacteria, lipopolysaccharide (LPS, endotoxin) has a dominant role leading to fulminant pro-inflammatory reactions in the host. As there is no LPS in Gram-positive bacteria, other microbial cell wall components have been identified to be the causative agent for the pro-inflammatory activity since also Gram-positive bacterial infections lead to comparable clinical symptoms and reactions. On search for the "Gram-positive endotoxin" a widely accepted hypothesis has been raised in that the lipoteichoic acids (LTAs) serve as pathogen-associated molecular patterns (PAMPs) during Gram-positive sepsis, although the amount necessary for a pro-inflammatory in vitro response is several orders of magnitude higher than that for LPS. Therefore, LTA cannot be considered to be "the (endo)toxin of Gram-positive bacteria". Although LPS and LTA show structural relatedness (amphiphilic, negatively charged glycophospholipids), they are structurally quite different from each other and one might expect that they are also recognized by different receptors of the innate immune system, the so called toll-like receptors 4 and 2 (TLR4 and TLR2), respectively. Based on their chemical structure, the LTAs were classified into four types (type I-IV) of which we have carefully investigated the LTA of Staphylococcus aureus (type I), Lactococcus garvieae (type II) and Streptococcus pneumoniae (type IV). Hence, these LTAs have been synthesized in our group and biologically evaluated with respect to their potency to activate cytokines in transiently TLR2/CD14-transfected human endothelial kidney cells (HEK 293) or human macrophages and whole blood cells. Although LTA of type I and IV are structurally quite different, especially in their hydrophilic moiety, they originally were believed to interact with the same receptor (TLR2). Hence, the chemical syntheses leading to structurally defined, non-contaminated stimuli have a major impact on the outcome and interpretation of these biological studies of the innate immune system. With this material, it became evident that synthetic LTA from S. aureus and S. pneumoniae are not recognized by TLR2. Instead, another receptor of the innate immune system, the lectin pathway of the complement, known since many years to interact with LTA in quite a specific way, has gained increasing attractivity. With the help of synthetic LTA we obtained first evidences that this receptor is indeed the pathogen recognition receptor (PRR) for LTA.

Synthesis of the core structure of the lipoteichoic acid of Streptococcus pneumoniae.

Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2-acetamino-4-amino-2,4,6-trideoxygalactose (AATDgal) residues that are each differently linked, one ribitol and one diacylated glycerol (DAG) residue. Suitable building blocks for the glucose and the AATDgal residues were designed and their synthesis is described in this paper. These building blocks permitted the successful synthesis of the core structure Glcß(1-3)AATDgalß(1-3)Glcα(1-O)DAG in a suitably protected form for further chain extension (1 b, 1 c) and as unprotected glycolipid (1 a) that was employed in biological studies. These studies revealed that 1 a as well as 1 lead to interleukin-8 release, however not via TLR2 or TLR4 as receptor.