As a chemical probe, Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide does not have a biological target in the traditional sense. Instead, its “target” is the cohort of cell surface glycoproteins that can be covalently labeled via click chemistry. The picolyl azide group reacts with alkyne-modified sugars (e.g., via metabolic labeling with alkynylated monosaccharides like N-azidoacetylgalactosamine (GalNAz)) that have been incorporated into glycoproteins. The compound is then used as an enrichment tool to capture, isolate, and identify these glycoproteins using streptavidin beads. The acid-cleavable linker allows for gentle elution, preserving the native structure of the glycoproteins for downstream analysis. The biotin moiety ensures specific binding to streptavidin, which can be immobilized on magnetic beads or agarose resins. This probe is particularly useful for identifying changes in glycosylation patterns associated with disease, such as cancer or inflammation. It also allows for the study of glycoprotein dynamics on the cell surface. Thus, the probe serves as a tool for mapping the glycoproteome.

In vitro studies with this compound focus on its ability to label and enrich cell surface glycoproteins. A typical experiment involves first metabolically labeling cells with an alkynylated sugar, such as N-azidoacetylglucosamine (GlcNAz) or N-azidoacetylgalactosamine (GalNAz), which is incorporated into the glycan chains of glycoproteins. After washing, the cells are incubated with Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide in the presence of a copper catalyst (e.g., CuSO4 and THPTA) and a reducing agent (sodium ascorbate) to perform a click chemistry reaction. The picolyl azide reacts specifically with the alkyne groups on the glycoproteins, forming a stable triazole linkage. The cells are then lysed, and the biotin-labeled glycoproteins are captured using streptavidin-coated magnetic beads. After washing away unbound proteins, the captured glycoproteins are eluted by acidic cleavage of the silane linker (e.g., with 1-5% formic acid or 0.1 M sodium citrate, pH 3.0). The eluted proteins are then digested with trypsin and analyzed by mass spectrometry for identification and quantification. The probe's efficiency can be assessed by comparing the number of identified glycoproteins to controls (e.g., no sugar labeling, no probe, or non-cleavable probe). The acid-cleavable feature is critical for releasing the glycoproteins without the need for harsh denaturation, which could degrade the sample. The PEG4 linker enhances the solubility of the probe and reduces non-specific binding. In vitro, the compound is typically used at concentrations of 10-100 uM.

In vivo studies with this compound are not typically performed, as the probe is primarily used in ex vivo or in vitro settings for glycoprotein labeling and enrichment. However, the probe could theoretically be used for in vivo imaging or labeling if the azide group is conjugated to a fluorophore or a radionuclide and if the click chemistry reaction is performed in vivo (e.g., using bioorthogonal chemistry). The probe has been designed as a chemical biology tool for cell surface labeling, and its in vivo applications are not the primary focus. Nevertheless, the acid-cleavable silane linker might allow for in vivo release of a payload, but this is not the intended use. The compound's utility lies in the proteomic characterization of the cell surface glycoproteome, which can be performed on tissue samples after in vivo metabolic labeling with alkynylated sugars. For example, a mouse can be injected with GalNAz, the tissues are harvested, and the probe is used to enrich glycoproteins from tissue lysates. This ex vivo approach combines in vivo labeling with in vitro enrichment. Detailed in vivo studies have not been published for this specific compound.

The protocol for using this probe in vitro involves a copper-catalyzed click chemistry reaction (CuAAC) between the picolyl azide and an alkyne-modified glycoprotein. First, cells are metabolically labeled with an azido sugar (e.g., GalNAz) at 50-200 uM for 24-72 hours. The cells are washed and incubated with the Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide probe (50-100 uM) in PBS containing CuSO4 (1 mM), THPTA (1 mM), and sodium ascorbate (5 mM) for 1-2 hours at room temperature. The click reaction is quenched by washing with PBS containing 1 mM EDTA. The cells are then lysed in RIPA buffer (or a lysis buffer without reducing agents) with protease inhibitors. The lysate is incubated with streptavidin magnetic beads (100 uL of beads per 1 mg of protein) for 2 hours at 4degC. After washing 3-5 times with 0.1% SDS in PBS, the beads are transferred to a fresh tube and the labeled glycoproteins are eluted by incubating with 100 uL of 1-5% formic acid for 10-30 minutes. The eluate is neutralized with 1 M Tris-HCl pH 8.0, and proteins are digested with trypsin for LC-MS/MS analysis. For quality control, an aliquot can be run on an SDS-PAGE gel and stained with Coomassie Blue or silver stain to check for the presence of protein bands. The absence of bands in control samples (without azide sugar or without probe) confirms specificity.

For in vitro cell experiments, the protocol is as described above. Specifically, adherent cells (e.g., HEK293, HeLa, or cancer cell lines) are grown to 80-90% confluence in 10 cm dishes. The culture medium is replaced with medium containing 50-200 uM of a peracetylated azido sugar (e.g., Ac4GalNAz) for 48 hours to allow metabolic incorporation into glycoproteins. The cells are then washed three times with PBS to remove unincorporated sugar. The click chemistry reaction mixture is prepared fresh: Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide (100 uM), CuSO4 (1 mM), THPTA (1 mM), and sodium ascorbate (5 mM) in PBS. The mixture is added to the cells (2 mL per 10 cm dish) and incubated for 1 hour at room temperature with gentle shaking. The cells are then washed three times with PBS + 1 mM EDTA to remove copper and unreacted probe. The cells are lysed with 500 uL of RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail). The lysate is cleared by centrifugation at 13,000 rpm for 10 minutes at 4degC. The protein concentration is determined by BCA assay. An equal amount of protein (1-2 mg) is mixed with 50 uL of pre-washed streptavidin magnetic beads and incubated for 2 hours at 4degC with rotation. The beads are collected with a magnet, and the supernatant is removed. The beads are washed 3 times with 1 mL of 0.1% SDS in PBS, then 3 times with PBS. For elution, 50 uL of 1-5% formic acid is added to the beads and incubated for 15 minutes. The supernatant (eluate) is collected and neutralized with 2.5 uL of 1 M Tris-HCl pH 8.0. The eluate can be analyzed by Western blotting using streptavidin-HRP or by silver staining. For mass spectrometry, the eluate is digested with trypsin and processed for LC-MS/MS. Cell viability after the click reaction is not typically assessed, but the copper catalyst can be toxic to cells; a copper-free click chemistry (SPAAC) using a bicyclononyne (BCN)-modified sugar may be used if cell viability is a concern.

In vivo animal experiments are not commonly performed with this specific probe because the copper catalyst required for CuAAC click chemistry is toxic to animals. However, if one wishes to perform in vivo labeling, a copper-free click chemistry approach would be required, using a strained alkyne (e.g., DBCO or BCN) instead of a terminal alkyne, and the probe would need a complementary azide group. Alternatively, ex vivo labeling of tissue samples from animals that had been metabolically labeled with an azido sugar could be performed. For example, C57BL/6 mice can be injected intraperitoneally with 100 uL of 100 mM Ac4GalNAz (in DMSO) daily for 3 days. Tissues (liver, kidney, brain, tumor) are harvested, homogenized, and the lysates are subjected to the click reaction and enrichment as described in the in vitro protocol. This approach allows for the identification of tissue-specific glycoproteins. The probe's acid-cleavable linker also permits analysis of glycoproteins that may be otherwise difficult to elute from streptavidin beads. However, published in vivo studies using this exact probe are not available. For research purposes, the probe is typically used in cultured cells or tissue lysates. The compound is considered non-toxic at the concentrations used for labeling. It is not intended for in vivo therapeutic use.

Pharmacokinetic (PK) data are not available for Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide, as it is a chemical biology tool not intended for systemic administration. The compound has a molecular weight of 978.24 g/mol, which is above the typical threshold for oral absorption (>500 daltons). The PEG4 linker improves water solubility, but the overall compound is likely to have poor oral bioavailability and is not designed for in vivo absorption. The silane linker may be unstable in plasma (acidic conditions). The compound's half-life in biological fluids has not been studied. For in vitro experiments, the compound is typically prepared as a 10-100 mM stock in DMSO and stored at -80degC. Working solutions are diluted in PBS or cell culture medium. The compound's stability in solution at room temperature is limited to a few hours due to potential oxidation or hydrolysis of the silane linker. The compound should be stored as a solid powder at -20degC, protected from light and moisture. For labeling experiments, it is recommended to prepare fresh reaction mixtures each time. There is no need for PK analysis as the compound is not used as a therapeutic agent.

No formal toxicity studies have been conducted for Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide, as it is a research reagent and not intended for human use. In cell culture, the compound (at 100 uM for 1-2 hours in the presence of copper catalyst) does not cause immediate cytotoxicity, as measured by trypan blue exclusion or the MTT assay after 24 hours. However, the copper catalyst (CuSO4) itself is toxic at concentrations above 1 mM in cells. To minimize toxicity, the copper concentration can be reduced to 100 uM if the labeling efficiency is sufficient, or a copper-free click chemistry method can be used. The probe alone (without copper) is likely non-toxic, as the biotin and PEG4 groups are generally biocompatible, and the picolyl azide group is stable. However, silane groups can hydrolyze to form silanol, which may be reactive, but at neutral pH, this hydrolysis is slow. The acid-cleavable property means that the linker is stable at pH 7.4 but cleaves at pH <5. Therefore, in cellular compartments with low pH (e.g., lysosomes), the probe may be cleaved, but this is not a toxic effect. No data are available on the compound's potential to cause genotoxicity, reproductive toxicity, or carcinogenicity. As with all chemical probes, standard laboratory safety practices should be followed: wear gloves, a lab coat, and safety glasses. Avoid inhalation of powders and skin contact. Dispose of waste according to local regulations. The compound should be considered an irritant. For any spills, clean up with absorbent material and wash the area with water. The compound is not known to be flammable.

[1]. Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells. Mol Cell. 2020 Jun 4;78(5):824-834.e15.

Other information: Biotin-PEG4-dialkoxydiphenylsilane-picolyl azide is a research-grade chemical probe for laboratory use only. It is not approved for clinical use. The compound is used in the fields of chemical biology, glycobiology, and proteomics. It is part of a family of clickable enrichment handles that allow for the selective isolation of cell surface glycoproteins. The presence of the acid-cleavable linker distinguishes it from other biotin-based enrichment probes, as it enables gentle elution of captured proteins, avoiding the need for boiling in SDS-containing buffer. The compound's design is based on the DADPS (dialkoxydiphenylsilane) linker, which is known to be stable under neutral and basic conditions but cleaved under mildly acidic conditions (pH 3-5). This feature is especially useful for mass spectrometry analysis because acid-cleavage does not introduce cross-links and leaves minimal residual mass on the glycoprotein. The molecular formula is C4₇H₆₇N₉O10SSi, and the exact mass is 977.45 g/mol. The compound is available with a purity of >95% (often >98%). Storage: as a solid at -20degC, desiccated. For use, a stock solution is prepared in DMSO (10-50 mM). The compound should be protected from light and moisture. For cell labeling, the working concentration is 10-100 uM. The compound can be used for labeling both live and fixed cells, although live cell labeling requires the copper catalyst to be non-toxic, which is achieved using copper-chelating ligands such as THPTA or BTTAA to reduce toxicity. The compound is not intended for in vivo use, but researchers have reported using similar probes for in vivo imaging with bioorthogonal chemistry after the development of copper-free click chemistry. The CAS number is 2599839-59-3. The compound is also known as “Biotin-PEG4-DADPS-picolyl azide.” It is not a drug and has no clinical applications. Researchers should be aware that the azide group can be reduced to an amine if exposed to reducing agents (e.g., DTT, TCEP), so avoid adding such agents before the click reaction. For best results, the click reaction should be carried out under inert atmosphere (N2 or Ar) and in the dark. The probe is compatible with cell lysis buffers containing detergents. For Western blot detection, the biotinylated proteins can be detected directly using streptavidin-HRP without the need for primary antibodies. The probe is available from multiple chemical suppliers. Do not use the compound after its expiration date. Always refer to the manufacturer's instructions for specific protocols. The compound is for research only, not for diagnostic or therapeutic use.

C47H67N9O10SSI

978.24

977.45

2599839-59-3

163322293

Light yellow to yellow solid powder

5

14

34

68

1540

3

[Si](C1C=CC=CC=1)(C1C=CC=CC=1)(OCCCNC(C1=CN=C(CN=[N+]=[N-])C=C1)=O)OC(C)(C)CNC(=O)CCOCCOCCOCCOCCNC(=O)CCCC[C@@H]1SC[C@]2([H])NC(=O)N[C@]12[H]

SGCWLLPKOCPPEB-UEMJTUMQSA-N

InChI=1S/C47H67N9O10SSi/c1-47(2,66-68(38-12-5-3-6-13-38,39-14-7-4-8-15-39)65-23-11-21-50-45(59)36-18-19-37(51-32-36)33-53-56-48)35-52-43(58)20-24-61-26-28-63-30-31-64-29-27-62-25-22-49-42(57)17-10-9-16-41-44-40(34-67-41)54-46(60)55-44/h3-8,12-15,18-19,32,40-41,44H,9-11,16-17,20-31,33-35H2,1-2H3,(H,49,57)(H,50,59)(H,52,58)(H2,54,55,60)/t40-,41-,44-/m0/s1

N-[3-[[1-[3-[2-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-2-methylpropan-2-yl]oxy-diphenylsilyl]oxypropyl]-6-(azidomethyl)pyridine-3-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~102.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)