SFox imidates as versatile glycosyl donors for chemical glycosylation.

Described herein is a continuation of our studies dedicated to the development of novel classes of leaving groups based on O- and S-imidates. The main focus of the study presented herein is the synthesis of novel 3,3-difluoro-3H-indol-2-ylthio (SFox) imidates and their application as glycosyl donors in chemical glycosylation. Being thioimidates, these compounds are more stable than O-imidates albeit much more reactive than conventional alkyl/arylthio glycosides. This study demonstrates that SFox imidates can be activated either with soft thiophilic reagents (N-iodosuccinimide or transition metal salts), typical for the activation of thioglycosides or thioimidates, or hard electrophilic reagents (protic or Lewis acids) common for the activation of O-imidates. Expectedly, complete ß-selectivity was obtained from SFox donors equipped with 2-O-benzoyl group. Surprisingly, complete α-selectivity was obtained from 2-O-benzylated SFox imidates in all investigated cases.

The 4K reaction.

The classical Koenigs-Knorr glycosidation of bromides or chlorides promoted with Ag2O or Ag2CO3 works only with reactive substrates (ideally both donor and acceptor). This reaction was found to be practically ineffective with unreactive donors such as per-O-benzoylated mannosyl bromide. Recently, it was discovered that the addition of catalytic (Lewis) acids to a silver salt-promoted reaction has a dramatic effect on the reaction rate and yield. A tentative mechanism for this cooperatively-catalyzed glycosylation reaction has been proposed, and the improved understanding of the reaction led to more efficient protocols and broader applications to a variety of glycosidic linkages. Since Ag2O-mediated activation was introduced by German chemists Koenigs and Knorr, and "cooperatively catalyzed" is Kooperativ Katalysiert in German, we refer to this new reaction as "the 4K reaction."

Expedient Synthesis of Superarmed Glycosyl Donors via Oxidative Thioglycosidation of Glycals.

Superarmed glycosyl donors have higher reactivity compared to their perbenzylated armed counterparts. Generally, the 2-O- benzoyl-3,4,6-tri-O-benzyl protecting group pattern gives rise to increased reactivity due to an O-2/O-5 cooperative effect. Despite having a high reactivity profile and applicability in many expeditious strategies for glycan synthesis, regioselective introduction of the superarming protecting group pattern is tedious for most sugar series. Reported herein is a streamlined synthetic route to yield superarmed glycosyl donors of the d-gluco and d-galacto series equipped with an ethylthio, phenylthio, p-tolylthio, benzoxazol-2-ylthio, O-allyl, or O-pentenyl anomeric leaving group. This streamlined approach was made possible due to the refinement of the oxidative thioglycosylation reaction of the respective glucal and galactal precursors. The applicability of this approach to the direct formation of disaccharides is also showcased.

Direct Synthesis of Glycosyl Chlorides from Thioglycosides.

Reported herein is a new method for the direct synthesis of glycosyl chlorides from thioglycosides using sulfuryl chloride at rt. A variety of thioglycosides and thioimidates could be used as substrates. Both acid- and base-sensitive protecting groups were found compatible with these reaction conditions. Preliminary investigation of the reaction mechanism indicates chlorination of the leaving group at the anomeric sulfur as the key step of the reaction.

HPLC-Based Automated Synthesis and Purification of Carbohydrates.

Reported herein is a new HPLC-based automated synthesizer (HPLC-A) capable of a temperature-controlled synthesis and purification of carbohydrates. The developed platform allows to perform various protecting group manipulations as well as the synthesis of O- and N-glycosides. A fully automated synthesis and purification was showcased in application to different carbohydrate derivatives including glycosides, oligosaccharides, glycopeptides, glycolipids, and nucleosides.

Overcoming Challenges in Chemical Glycosylation to Achieve Innovative Vaccine Adjuvants Possessing Enhanced TLR4 Activity.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity.

Chemical Synthesis of Human Milk Oligosaccharides: para-Lacto-N-hexaose and para-Lacto-N-neohexaose.

Human milk oligosaccharides (HMO) have emerged as a very active area of research in glycoscience and nutrition. HMO are involved in the early development of infants and may help to prevent certain diseases. The development of chemical methods for obtaining individual HMO aids the global effort dedicated to understanding the roles of these biomolecules. Reported herein is the chemical synthesis of two common core hexasaccharides found in human milk, i. e. para-lacto-N-hexaose (pLNH) and para-lacto-N-neohexaose (pLNnH). After screening multiple leaving groups and temporary protecting group combinations, a 3+3 convergent coupling strategy was found to work best for obtaining these linear glycans.

Activation of thioglycosides under mild alkylation conditions.

Reported herein is the development of a novel method for the activation of thioglycosides and thioimidates using benzyl trichloroacetimidate in the presence of catalytic triflic acid. Excellent yields have been achieved with reactive substrates, whereas efficiency of reactions with unreactive glycosyl donors and/or acceptors was modest.

Cooperatively Catalyzed Activation of Thioglycosides That Bypasses Intermediacy of Glycosyl Halides.

Reported herein is the development of a novel method for activating thioglycosides without a glycosyl halide intermediate. This has been achieved through the use of a silver salt coupled with an acid additive and molecular iodine. The enhanced stereocontrol was achieved via the H-bond mediated aglycone delivery (HAD) method, and the extended trisaccharide synthesis was achieved via iteration of deprotection and glycosylation steps.

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold.

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, drug loading and release, and the development of catalysts have unquestionably accelerated the usage of nanoporous gold (NPG)-based nanomaterials in research and development. This article describes the process of the generation of hierarchical bimodal nanoporous gold (hb-NPG) by employing a step-wise procedure involving electrochemical alloying, chemical dealloying techniques, and annealing to create both macro- and mesopores. This is done to improve the utility of NPG by creating a bicontinuous solid/void morphology. The area available for surface modification is enhanced by smaller pores, while molecular transport benefits from the network of larger pores. The bimodal architecture, which is the result of a series of fabrication steps, is visualized using scanning electron microscopy (SEM) as a network of pores that are less than 100 nm in size and connected by ligaments to larger pores that are several hundred nanometers in size. The electrochemically active surface area of the hb-NPG is assessed using cyclic voltammetry (CV), with a focus on the critical roles that both dealloying and annealing play in creating the necessary structure. The adsorption of different proteins is measured by solution depletion technique, revealing the better performance of hb-NPG in terms of protein loading. By changing the surface area to volume ratio, the created hb-NPG electrode offers tremendous potential for biosensor development. The manuscript discusses a scalable method to create hb-NPG surface structures, as they offer a large surface area for the immobilization of small molecules and improved transport pathways for faster reactions.

N-Alkylated Analogues of Indolylthio Glycosides as Glycosyl Donors with Enhanced Activation Profile.

While studying indolylthio glycosides, previously we determined their activation profile that required large excess of activators. This drawback was partially addressed in the present study of N-alkylated SInR derivatives. The activation process was studied by NMR and the increased understanding of the mechanism led to a discovery of different activation pathways taking place with SIn versus SInR derivatives. Also investigated was orthogonality of the SInR leaving groups versus thioglycosides and selective activation of thioimidates over SInR glycosides.

Facile fabrication of hierarchically nanostructured gold electrode for bio-electrochemical applications.

Nanoporous gold (NPG) is one of the most extensively investigated nanomaterials owing to its tunable pore size, ease of surface modification, and range of applications from catalysis, actuation, and molecular release to the development of electrochemical sensors. In an effort to improve the usefulness of NPG, a simple and robust method for the fabrication of hierarchical and bimodal nanoporous gold electrodes (hb-NPG) containing both macro-and mesopores is reported using electrochemical alloying and dealloying processes to engineer a bicontinuous solid/void morphology. Scanning electron microscopy (color SEM) images depict the hierarchical pore structure created after the multistep synthesis with an ensemble of tiny pores below 100 nm in size located in ligaments spanning larger pores of several hundred nanometers. Smaller-sized pores are exploited for surface modification, and the network of larger pores aids in molecular transport. Cyclic voltammetry (CV) was used to compare the electrochemically active surface area of the hierarchical bimodal structure with that of the regular unimodal NPG with an emphasis on the critical role of both dealloying and annealing in creating the desired structure. The adsorption of different proteins was followed using UV-vis absorbance measurements of solution depletion revealing the high loading capacity of hb-NPG. The surface coverage of lipoic acid on the hb-NPG was analyzed using thermogravimetric analysis (TGA) and reductive desorption. The roughness factor determinations suggest that the fabricated hb-NPG electrode has tremendous potential for biosensor development by changing the scaling relations between volume and surface area which may lead to improved analytical performance. We have chosen to take advantage of the surface architectures of hb-NPG due to the presence of a large specific surface area for functionalization and rapid transport pathways for faster response. It is shown that the hb-NPG electrode has a higher sensitivity for the amperometric detection of glucose than does an NPG electrode of the same geometric surface area.

Activation of Thioglycosides with Copper(II) Bromide.

Reported herein is a new protocol for glycosidation of alkyl and aryl thioglycosides in the presence of copper(II) bromide. While the activation with CuBr2 alone was proven suitable for reactive glycosyl donors, the activation of less reactive donors was more efficient in the presence of triflic acid as an additive. A variety of thioglycoside donors in reactions with different glycosyl acceptors were investigated to determine the initial scope of this reaction.

Synthesis and glycosidation of building blocks of D-altrosamine.

Presented herein is a streamlined synthesis of building blocks of a rare sugar D-altrosamine. Also investigated was the glycosylation of different glycosyl acceptors with differentially protected altrosamine donors. High facial stereoselectivity was achieved with 3-O-picoloyl donors and reactive glycosyl acceptors via the H-bond-mediated aglycone delivery (HAD) pathway. In contrast, glycosidations of the altrosamine donor equipped with the 3-O-benzoyl group were poorly stereoselective.

Expanding the scope of stereoselective α-galactosylation using glycosyl chlorides.

Recently, we reported that silver(I) oxide mediated Koenigs-Knorr glycosylation reaction can be dramatically accelerated in the presence of catalytic acid additives. We have also investigated how well this reaction works in application to differentially protected galactosyl bromides. Reported herein is the stereoselective synthesis of α-galactosides with galactosyl chlorides as glycosyl donors. Chlorides are easily accessible, stable, and can be efficiently activated for glycosylation. In this application, the most favorable reactions conditions comprised cooperative Ag2SO4 and Bi(OTf)3 promoter system.

Broadening the Scope of the Reverse Orthogonal Strategy for Oligosaccharide Synthesis.

The reverse orthogonal strategy was invented in 2011 in an attempt to address drawbacks of other strategies for glycan assembly. Different from the classical orthogonal approach that relies on the orthogonality of leaving groups, the reverse strategy is based on orthogonal protecting groups that could be removed during the glycosylation step. This strategy remained largely unexplored due to only one combination of orthogonal protecting groups that would fit into this concept. Reported herein are new orthogonal combinations of leaving and protecting groups that help to streamline the glycan assembly. Also reported is further refinement of the previously reported reaction conditions.

Synthesis and Glycosidation of Anomeric Halides: Evolution from Early Studies to Modern Methods of the 21st Century.

Advances in synthetic carbohydrate chemistry have dramatically improved access to common glycans. However, many novel methods still fail to adequately address challenges associated with chemical glycosylation and glycan synthesis. Since a challenge of glycosylation has remained, scientists have been frequently returning to the traditional glycosyl donors. This review is dedicated to glycosyl halides that have played crucial roles in shaping the field of glycosciences and continue to pave the way toward our understanding of chemical glycosylation.

Development of a Simple and Effective Lipid-A Antagonist Based on Computational Prediction.

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

HPLC-Based Automated Synthesis of Glycans in Solution.

As the 21st century unfolds with rapid changes, new challenges in research and development emerge. These new challenges prompted us to repurpose our HPLC-A platform that was previously used in solid phase glycan synthesis to a solution phase batch synthesis described herein. The modular character of HPLC allows for implementing new attachments. To enable sequential synthesis of multiple oligosaccharides with the single press of a button, we supplemented our system with a four-way split valve and an automated fraction collector. This enabled the operator to load all reagents and all reactants in the autosampler, press the button to start the repetitive automation sequence, leave the lab, and upon return find products of multiple reactions ready for purification, analysis, and subsequent application.

Streamlined access to carbohydrate building blocks: Methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside.

Presented herein is an improved synthesis of a common 3-OH glycosyl acceptor. This compound is a building block that is routinely synthesized by many research groups to be used in glycosylation refinement studies. The only known direct synthesis by Koto lacks regioselectivity and relies on chromatography separation using hazardous solvents. Our improved synthetic approach relies on Koto's selective benzylation protocol, but it is followed by acylation-purification-deacylation sequence. Although this approach involves additional manipulations, it provides consistent results and is superior to other indirect strategies. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.