Anomeric O-Functionalization of Carbohydrates for Chemical Conjugation to Vaccine Constructs.

Carbohydrates mediate a wide range of biological interactions, and understanding these processes benefits the development of new therapeutics. Isolating sufficient quantities of glycoconjugates from biological samples remains a significant challenge. With advances in chemical and enzymatic carbohydrate synthesis, the availability of complex carbohydrates is increasing and developing methods for stereoselective conjugation these polar head groups to proteins and lipids is critically important for pharmaceutical applications. The aim of this review is to provide an overview of commonly employed strategies for installing a functionalized linker at the anomeric position as well as examples of further transformations that have successfully led to glycoconjugation to vaccine constructs for biological evaluation as carbohydrate-based therapeutics.

Taming the Reactivity of Glycosyl Iodides To Achieve Stereoselective Glycosidation.

Although glycosyl iodides have been known for more than 100 years, it was not until the 21st century that their full potential began to be harnessed for complex glycoconjugate synthesis. Mechanistic studies in the late 1990s probed glycosyl iodide formation by NMR spectroscopy and revealed important reactivity features embedded in protecting-group stereoelectronics. Differentially protected sugars having an anomeric acetate were reacted with trimethylsilyl iodide (TMSI) to generate the glycosyl iodides. In the absence of C-2 participation, generation of the glycosyl iodide proceeded by inversion of the starting anomeric acetate stereochemistry. Once formed, the glycosyl iodide readily underwent in situ anomerization, and in the presence of excess iodide, equilibrium concentrations of α- and ß-iodides were established. Reactivity profiles depended upon the identity of the sugar and the protecting groups adorning it. Consistent with the modern idea of disarmed versus armed sugars, ester protecting groups diminished the reactivity of glycosyl iodides and ether protecting groups enhanced the reactivity. Thus, acetylated sugars were slower to form the iodide and anomerize than their benzylated analogues, and these disarmed glycosyl iodides could be isolated and purified, whereas armed ether-protected iodides could only be generated and reacted in situ. All other things being equal, the ß-iodide was orders of magnitude more reactive than the thermodynamically more stable α-iodide, consistent with the idea of in situ anomerization introduced by Lemieux in the mid-20th century. Glycosyl iodides are far more reactive than the corresponding bromides, and with the increased reactivity comes increased stereocontrol, particularly when forming α-linked linear and branched oligosaccharides. Reactions with per-O-silylated glycosyl iodides are especially useful for the synthesis of α-linked glycoconjugates. Silyl ether protecting groups make the glycosyl iodide so reactive that even highly functionalized aglycon acceptors add. Following the coupling event, the TMS ethers are readily removed by methanolysis, and since all of the byproducts are volatile, multiple reactions can be performed in a single reaction vessel without isolation of intermediates. In this fashion, per-O-TMS monosaccharides can be converted to biologically relevant α-linked glycolipids in one pot. The stereochemical outcome of these reactions can also be switched to ß-glycoside formation by addition of silver to chelate the iodide, thus favoring SN2 displacement of the α-iodide. While iodides derived from benzyl and silyl ether-protected oligosaccharides are susceptible to interglycosidic bond cleavage when treated with TMSI, the introduction of a single acetate protecting group prevents this unwanted side reaction. Partial acetylation of armed glycosyl iodides also attenuates HI elimination side reactions. Conversely, fully acetylated glycosyl iodides are deactivated and require metal catalysis in order for glycosidation to occur. Recent findings indicate that I2 activation of per-O-acetylated mono-, di-, and trisaccharides promotes glycosidation of cyclic ethers to give ß-linked iodoalkyl glycoconjugates in one step. Products of these reactions have been converted into multivalent carbohydrate displays. With these synthetic pathways elucidated, chemical reactivity can be exquisitely controlled by the judicious selection of protecting groups to achieve high stereocontrol in step-economical processes.

Synthesis of cholesteryl-α-D-lactoside via generation and trapping of a stable ß-lactosyl iodide.

The generation of ß-lactosyl iodide was carried out under non-in situ-anomerization, metal free conditions by reacting commercially available ß-per-O-acetylated lactose with trimethylsilyl iodide (TMSI). The ß-iodide was surprisingly stable as evidenced by NMR spectroscopy. Introduction of octanol or cholesterol under microwave conditions gave high yields of α-linked glycoconjugates. Careful analysis of the reaction products and mechanistic considerations suggest an acid catalyzed rearrangement that provides α-linked glycosylation products with a free C2-hydroxyl. Accessibility to these compounds may further advance glycolipidomic profiling of immune modulating bacterial derived-glycans.

Two-step functionalization of oligosaccharides using glycosyl iodide and trimethylene oxide and its applications to multivalent glycoconjugates.

Oligosaccharide conjugates, such as glycoproteins and glycolipids, are potential chemotherapeutics and also serve as useful tools for understanding the biological roles of carbohydrates. With many modern isolation and synthetic technologies providing access to a wide variety of free sugars, there is increasing need for general methodologies for carbohydrate functionalization. Herein, we report a two-step methodology for the conjugation of per-O-acetylated oligosaccharides to functionalized linkers that can be used for various displays. Oligosaccharides obtained from both synthetic and commercial sources were converted to glycosyl iodides and activated with I2 to form reactive donors that were subsequently trapped with trimethylene oxide to form iodopropyl conjugates in a single step. The terminal iodide served as a chemical handle for further modification. Conversion into the corresponding azide followed by copper-catalyzed azide-alkyne cycloaddition afforded multivalent glycoconjugates of Gb3 for further investigation as anti-cancer therapeutics.

Tandem glycosyl iodide glycosylation and regioselective enzymatic acylation affords 6-O-tetradecanoyl-α-d-cholesterylglycosides.

A generalized synthesis of α-d-cholesterylglycosides has been achieved using one-pot per-O-trimethylsilyl glycosyl iodide glycosidation. Both cholesteryl α-d-glucopyranoside (αCG) and cholesteryl α-d-galactopyranoside were prepared in high yield. These compounds were further esterified using regioselective enzymatic acylation with tetradecanoyl vinyl ester to afford 6-O-tetradecanoyl-α-d-cholesteryl glucopyranoside (αCAG) of Helicobacter pylori and the corresponding galactose analogue in 66-78% overall yields from free sugars. The tandem step-economy sequence provides novel analogues to facilitate glycolipidomic profiling.

Integrating ReSET with glycosyl iodide glycosylation in step-economy syntheses of tumor-associated carbohydrate antigens and immunogenic glycolipids.

Carbohydrates mediate a wide range of biological processes, and understanding these events and how they might be influenced is a complex undertaking that requires access to pure glycoconjugates. The isolation of sufficient quantities of carbohydrates and glycolipids from biological samples remains a significant challenge that has redirected efforts toward chemical synthesis. However, progress toward complex glycoconjugate total synthesis has been slowed by the need for multiple protection and deprotection steps owing to the large number of similarly reactive hydroxyls in carbohydrates. Two methodologies, regioselective silyl exchange technology (ReSET) and glycosyl iodide glycosylation have now been integrated to streamline the synthesis of the globo series trisaccharides (globotriaose and isoglobotriaose) and α-lactosylceramide (α-LacCer). These glycoconjugates include tumor-associated carbohydrate antigens (TACAs) and immunostimulatory glycolipids that hold promise as immunotherapeutics. Beyond the utility of the step-economy syntheses afforded by this synthetic platform, the studies also reveal a unique electronic interplay between acetate and silyl ether protecting groups. Incorporation of acetates proximal to silyl ethers attenuates their reactivity while reducing undesirable side reactions. This phenomenon can be used to fine-tune the reactivity of silylated/acetylated sugar building blocks.

Synthesis and structural characterization of three unique Helicobacter pylori α-cholesteryl phosphatidyl glucosides.

Steryl glycosides produced by bacteria play important biological roles in the evasion and modulation of host immunity. Step-economical syntheses of three cholesteryl-6-O-phosphatidyl-α-D-glucopyranosides (αCPG) unique to Helicobacter pylori have been achieved. The approach relies upon regioselective deprotection of per-O-trimethylsilyl-α-D-cholesterylglucoside at C6 followed by phosphoramidite coupling. Global TMS ether deprotection in the presence of oxygen and subsequent deprotection of the cyano ethyl phosphoester afforded the target compounds in 16-21 % overall yield starting from D-glucose. The structures of these natural products were determined using a combination of 2D NMR methods and mass spectrometry. These robust synthesis and characterization protocols provide analogues to facilitate glycolipidomic profiling and biological studies.

Sterol-lipids enable large-scale, liquid-liquid phase separation in membranes of only 2 components.

Despite longstanding excitement and progress toward understanding liquid-liquid phase separation in natural and artificial membranes, fundamental questions have persisted about which molecules are required for this phenomenon. Except in extraordinary circumstances, the smallest number of components that has produced large-scale, liquid-liquid phase separation in bilayers has stubbornly remained at three: a sterol, a phospholipid with ordered chains, and a phospholipid with disordered chains. This requirement of three components is puzzling for two reasons: (1) the Gibbs Phase Rule states that only two components are necessary, and (2) only two components are required for liquid-liquid phase separation in lipid monolayers, which resemble half of a bilayer. Inspired by reports that sterols interact closely with lipids with ordered chains, we tested whether phase separation would occur in bilayers in which a sterol and lipid were replaced by a single, joined sterol-lipid. By evaluating a panel of sterol-lipids, we discovered a minimal bilayer of only two components (PChemsPC and diPhyPC) that demixes into micron-scale, liquid phases. In this system, the sterol-lipid behaves as a 3:1 ratio of cholesterol to phospholipid. Our system gives the computation and theory community a two-component membrane that maps directly onto simplified theories and that can be used to validate simulation force fields. It suggests a new role for sterol-lipids in nature, and it gives experimental communities a membrane in which tie-lines (and, therefore, the lipid composition of each phase) are easily determined and will be consistent across multiple laboratories. Significance Statement: A wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined "sterol-lipid" molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

The effect of roast profiles on the dynamics of titratable acidity during coffee roasting.

Coffee professionals have long known that the "roast profile," i.e., the temperature versus time inside the roaster, strongly affects the flavor and quality of the coffee. A particularly important attribute of brewed coffee is the perceived sourness, which is known to be strongly correlated to the total titratable acidity (TA). Most prior work has focused on laboratory-scale roasters with little control over the roast profile, so the relationship between roast profile in a commercial-scale roaster and the corresponding development of TA to date remains unclear. Here we investigate roast profiles of the same total duration but very different dynamics inside a 5-kg commercial drum roaster, and we show that the TA invariably peaks during first crack and then decays to its original value by second crack. Although the dynamics of the TA development varied with roast profile, the peak TA surprisingly exhibited almost no statistically significant differences among roast profiles. Our results provide insight on how to manipulate and achieve desired sourness during roasting.

A Thr/Ser dual residue motif in the cytoplasmic tail of human CD1d is important for the down-regulation of antigen presentation following a herpes simplex virus 1 infection.

CD1d-restricted T (natural killer T; NKT) cells are important for controlling herpesvirus infections. Interestingly, herpes simplex virus (HSV) can down-regulate CD1d-mediated activation of NKT cells. We have previously shown that the Thr322 residue in the cytoplasmic tail of human CD1d is important for its intracellular trafficking and functional expression. We proposed that the phosphorylation of T322 is a signal for CD1d lysosomal targeting and subsequent degradation. In the current study, we generated dual mutants by substituting the T322 and S323 residues of wild-type (WT) CD1d with Ala (non-phosphorylatable) or Asp (mimicking phosphorylation) and ectopically expressed them in human embryonic kidney 293 cells. We found that the surface expression levels of the CD1d mutants was in this order: T322AS323A > WT > T322A > S323A > S323D > T322D > T322DS323D. Our results therefore suggest that mimicking the phosphorylation of both T322 and S323 has a cumulative negative effect on the functional expression of CD1d. As previously reported, we also found that upon an HSV infection, antigen presentation by WT CD1d is reduced and the CD1d molecule is degraded. Interestingly, the T322A/S323A double mutation inhibited CD1d degradation and rescued CD1d-mediated antigen presentation following an HSV-1 infection. This suggests that the T322/S323 dyad may be phosphorylated, which then targets CD1d for lysosomal degradation post-infection as a means of immune evasion, explaining (at least in part) the reduced antigen presentation observed. Hence, our findings strongly suggest that T322 and S323 form a dual residue motif that can regulate the functional expression of CD1d during a viral infection.

Solution phase conformation and proteolytic stability of amide-linked neuraminic acid analogues.

Amide-linked homopolymers of sialic acid offer the advantages of stable secondary structure and increased bioavailability making them useful constructs for pharmaceutical design and drug delivery. Defining the structural characteristics that give rise to secondary structure in aqueous solution is challenging in homopolymeric material due to spectral overlap in NMR spectra. Having previously developed computational tools for heteroologomers with resolved spectra, we now report that application of these methods in combination with circular dichroism, NH/ND NMR exchange rates and nOe data has enabled the structural determination of a neutral, δ-amide-linked homopolymer of a sialic acid analogue called Neu2en. The results show that the inherent planarity of the pyranose ring in Neu2en brought about by the α,δ-conjugated amide bond serves as the primary driving force of the overall conformation of the homooligomer. This peptide surrogate has an excellent bioavailability profile, with half-life of ∼12 h in human blood serum, which offers a viable peptide scaffold that is resistant to proteolytic degradation. Furthermore, a proof-of-principle study illustrates that Neu2en oligomers are functionalizable with small molecule ligands using 1,3-dipolar cycloaddition chemistry.

Regioselective silyl/acetate exchange of disaccharides yields advanced glycosyl donor and acceptor precursors.

Glycoconjugates are composed of carbohydrate building blocks linked together in a multitude of ways giving rise to diverse biological functions. Carbohydrates are especially difficult to synthetically manipulate because of the similar reactivity of their numerous and largely equivalent hydroxyl groups. Hence, methodologies for both the efficient protection and selective modification of carbohydrate alcohols are considered important synthetic tools in organic chemistry. When per-O-TMS protected mono- or disaccharides in a mixture of pyridine and acetic anhydride are treated with acetic acid, regioselective exchange of silicon for acetate protecting groups occurs. Acid concentration, thermal conditions, and microwave assistance mediate the silyl/acetate exchange reaction. Regiocontrol is achieved by limiting the equivalents of acetic acid, and microwave irradiation hastens the process. We coined the term Regioselective Silyl Exchange Technology (ReSET) to describe this process, which essentially sets the protecting groups anew. To demonstrate the scope of the reaction, the conditions were applied to lactose, melibiose, cellobiose, and trehalose. ReSET provided rapid access to a wide range of orthogonally protected disaccharides that would otherwise require multiple synthetic steps to acquire. The resulting bifunctional molecules are poised to serve as modular building blocks for more complex glycoconjugates.

A fused [3.3.0]-neoglycoside lactone derived from glucuronic acid.

The bridged next-generation aminoglycoside (neoglycoside), 1-deoxy-1-[(methoxy)methylamino)]-2,5-di-O-triethylsilyl-ß-D-glucofuranurono-γ-lactone {systematic name: (3S,3aS,5R,6R,6aS)-5-[methoxy(methyl)amino]-3,6-bis[(triethylsilyl)oxy]-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-2-one}, C20H41NO6Si2, was synthesized in a one-pot manner from commercially available D-glucuronic acid. This structure supports the properties associated with the anomeric effect for furanosides and can be employed to provide insight into the mechanisms by which alkoxyamine-appended natural products derive their enhanced biological activity. To the best of our knowledge, this is the first published crystal structure of a bicyclic neoglycoside and is the first neoglycoside to be completely and unambiguously characterized.

Synthesis of ß-C-galactosyl D- and L-alanines.

Synthesis of ß-C-D-galactosyl D- and L-alanines is carried out via a highly stereoselective Grignard reaction of glycosyl iodides, Sharpless dihydroxylation and S(N)2 displacement of the corresponding mesylate or tosylate. Alternatively, attempted triflation of the intermediate alcohols triggers a stereoselective debenzylative cyclization leading to interesting bicyclic trans-fused compounds.

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.

A threonine-based targeting signal in the human CD1d cytoplasmic tail controls its functional expression.

CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called NKT cells. The cytoplasmic tail of human CD1d possesses a tyrosine-based endosomal targeting motif (YXXZ). As such, these molecules traffic through the endocytic pathway, where it is believed that they are loaded with the antigenic lipid that stimulates NKT cells. In the current study, it was found that the T322 residue in the human CD1d tail is a major signal controlling transport to the cell surface and thus its functional expression. Mimicking the phosphorylation of this residue or removal of the entire cytoplasmic tail negates its ability to regulate CD1d trafficking, resulting in lysosomal targeting and degradation. These results demonstrate an important role of a heretofore unknown signal in the cytoplasmic tail of CD1d that may have relevance to other type I integral membrane proteins that traverse through the endocytic pathway.

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

Synthesis of a trimeric gp120 epitope mimic conjugated to a T-helper peptide to improve antigenicity.

A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.

Statins impair CD1d-mediated antigen presentation through the inhibition of prenylation.

Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.

Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma.

Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) alpha chain rearrangement (Valpha14 Jalpha18 in mice; Valpha24 Jalpha18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host's innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b(+)Gr1(+)) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT-deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b(+)Gr1(+) cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.