Development of a metabolomics-based data analysis approach for identifying drug metabolites based on high-resolution mass spectrometry.

A metabolomics-based approach to data analysis is required for drug metabolites to be identified quickly. This study developed such an approach based on high-resolution mass spectrometry. Our approach is a two-stage one that combines a time-course experiment with stable isotope tracing. Pioglitazone (PIO) was used to improve glycemic management for type 2 diabetes mellitus. Consequently, PIO was taken as a model drug for identifying metabolites. During Stage I of data analysis, 704 out of 26626 ions exhibited a positive relationship between ion abundance ratio and incubation time in a time-course experiment. During Stage II, 25 isotope pairs were identified among the 704 ions. Among these 25 ions, 18 exhibited a dose-response relationship. Finally, 14 of the 18 ions were verified to be PIO structure-related metabolite ions. Otherwise, orthogonal partial least squares-discriminant analysis (OPLS-DA) was adopted to mine PIO metabolite ions, and 10 PIO structure-related metabolite ions were identified. However, only four ions were identified by both our developed approach and OPLS-DA, indicating that differences in the designs of metabolomics-based approaches to data analysis can result in differences in which metabolites are identified. A total of 20 PIO structure-related metabolites were identified by our developed approach and OPLS-DA, and six metabolites were novel. The results demonstrated that our developed two-stage data analysis approach can be used to effectively mine data on PIO metabolite ions from a relatively complex matrix.

Investigating a Boronate-Affinity-Guided Acylation Reaction for Labelling Native Antibodies.

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate-affinity-based Ab ligand featuring a 4-(dimethylamino)pyridine and an S-aryl thioester to label full-length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide-labelled Ab (N3 -Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N3 -Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.

Acceptor-mediated regioselective enzyme catalyzed sialylation: chemoenzymatic synthesis of GAA-7 ganglioside glycan.

Herein, we applied PmST1 (a sialyltransferase) to achieve acceptor-mediated regioselective sialylation (AMRS) on the nonreducing end GalNH2 or GalAz (2-azido-2-deoxy galactose). Thus, C5 and C8-modified sialic acid was efficiently assembled on GalNH2 (or GalAz) to achieve the synthesis of the GAA-7 (one of the echinodermatous gangliosides with higher neuritogenic activity) glycan moiety.

A Modular Chemoenzymatic Synthesis of Disialosyl Globopentaosylceramide (DSGb5Cer) Glycan.

The total synthesis of the oligosaccharide moiety of disialosyl globopentaosylceramide (DSGb5 Cer), a dominant ganglioside isolated from malignant renal cell carcinoma tissues, is reported. The synthetic strategy relies on a chemical α(2,6)-sialylation at the internal GalNAc unit of a Gb5 pentasaccharide backbone that furnishes a Neu5Acα(2,6)GalNAc-linked hexasaccharide, suitable for an enzymatic α(2,3)-sialylation of the terminal Gal residue to construct a heptasaccharide glycan. Convergent access to this key α(2,6)-sialylated hexasaccharide was also achieved through a [3+3] glycosylation building upon a Galß(1,3)[Neu5Acα(2,6)]GalNAc-based trisaccharide donor and a Gb3 acceptor. The synthetic DSGb5 glycan bearing a 6-azidohexyl aglycon at the reducing end could undergo further regioselective functionalization. This approach represents a viable chemoenzymatic method for accessing complex ganglioside glycans and should be useful for the synthesis and biological investigation of DSGb5 derivatives.

Fluorescence "Turn-on" Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein-Glycoprotein Interactions.

Elucidation of protein-protein interactions (PPIs) is often very challenging and yields complex and unclear results. Lectin-glycoprotein interactions are especially difficult to study due to the noncovalent nature of the interactions and inherently low binding affinities of proteins to glycan ligands on glycoproteins. Here, we report a "ligand-directed labeling probe (LLP)"-based approach to fabricate protein probes for elucidating protein-glycoprotein interactions. LLP was designed with dual photoactivatable groups for the introduction of an alkyne handle proximal to the carbohydrate-binding pocket of lectins, Ricinus communis agglutinin 120 (RCA120) and recombinant human Siglec-2-Fc. In proof-of-principle studies, alkynylated lectins were conjugated with a photoreactive diazirine cross-linker and an environment-sensitive fluorophore, respectively, by the bioorthogonal click reaction. The modified RCA120 or Siglec-2-Fc was used for detecting the interaction with the target glycoprotein in the solution or endogenously expressed glycoproteins on live HeLa cells. We anticipate that the fabrication of these protein probes will accelerate the discovery of novel PPIs.

Regioselective SN2-Type Reaction for the Oriented and Irreversible Immobilization of Antibodies to a Glass Surface Assisted by Boronate Formation.

Antibodies have exquisite specificities for molecular recognition, which have led to their incorporation into array sensors that are crucial for research, diagnostic, and therapeutic applications. Many of these platforms rely heavily on surface-bound reactive groups to covalently tether antibodies to solid substrates; however, this strategy is hindered by a lack of orientation control over antibody immobilization. Here, we report a mild electrophilic phenylsulfonate (tosylate) ester-containing boronic acid affinity ligand for attaching antibodies to glass slides. A high level of antibody coupling located near the Fc region of the boronated antibody complex could be achieved by the proximal nucleophilic amino acid driven substitution reaction at the phenylsulfonate center. This enabled the full-length antibodies to be permanently tethered onto surfaces in an oriented manner. The advantages of this strategy were demonstrated through the individual and multiplex detection of protein and serum biomarkers. This strategy not only confers stability to the immobilized antibodies but also presents a different direction for the irreversible attachment of antibodies to solid supports in an orientation-controlled way.

Chemoenzymatic Synthesis of DSGb5 and Sialylated Globo-series Glycans.

Sialic-acid-binding, immunoglobulin-type lectin-7 (Siglec-7) is present on the surface of natural killer cells. Siglec-7 shows preference for disialylated glycans, including α(2,8)-α(2,3)-disialic acids or internally branched α(2,6)-NeuAc, such as disialosylglobopentaose (DSGb5). Herein, DSGb5 was synthesized by a one-pot multiple enzyme method from Gb5 by α2,3-sialylation (with PmST1) followed by α2,6-sialylation (with Psp2,6ST) in 23 % overall yield. DSGb5 was also chemoenzymatically synthesized. The protection of the nonreducing-end galactose of Gb5 as 3,4-O-acetonide, 3,4-O-benzylidene, and 4,6-O-benzylidene derivatives provided DSGb5 in overall yields of 26 %, 12 %, and 19 %, respectively. Gb3, Gb4, and Gb5 were enzymatically sialylated to afford a range of globo-glycans. Surprisingly, DSGb5 shows a low affinity for Siglec-7 in a glycan microarray binding affinity assay. Among the synthesized globo-series glycans, α6α3DSGb4 shows the highest binding affinity for Siglec-7.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.