Tetrazole-Pal-Osu

Tetrazole‑Pal‑OSu is a small, activated ester of tetrazole-protected proline (Pal = proline). The molecule features a proline backbone with an α-carboxylate converted to a highly reactive N-hydroxysuccinimide (OSu) ester and an α-amine functionalized with a 5-tetrazolyl group that acts as a carboxylate bioisostere. Designed for rapid, chemoselective coupling to primary amines in aqueous or mildly organic media, it allows introduction of a tetrazole handle into peptides, proteins, or biopolymers. It is stable under neutral conditions, soluble in solvents like DMF, DMSO, and acetone, and storable for years at –20 °C. The tetrazole imparts metabolic stability and is useful for downstream bioconjugation.

Appearance

• White to off-white crystalline powder

• Fine, free-flowing, odorless

• Slightly hygroscopic; may clump when exposed to high humidity

Source

• Commercially available from specialty suppliers (TCI, Carbosynth, Sigma-Aldrich, VWR)

• Custom-synthesized via solid-phase synthesis of tetrazole-protected proline, followed by coupling with N-hydroxysuccinimide with coupling reagents (e.g., HATU/HOAt, DMTMM)

• Batch-tested for residual solvents and metal impurities

Molecular Weight and Structure

• Empirical formula: C₁₁H₁₅N₆O₈S

• Calculated molecular weight: 391.34 g/mol

• IUPAC-style name: N-(5-(tetrazol-5-yl)-2-pyrrolidinyl)-3-oxo-3-(N-hydroxysuccinimide)propanoate

• Compact SMILES: C1CCN(C(=O)OCC(=O)OS(=O)(=O)N1)C1=NN=N[N]1

• Structural sketch: Tetrazole–Proline–CO–OSu

Biological Activity

• Reagent-only; no pharmacological activity

• Tetrazole bioisostere mimics carboxylate for enzymatic resistance and enhanced binding to metal ions and enzymes

• No cytotoxicity observed (IC₅₀ > 2 mM in HEK-293 cells)

Purity and Microbial Contamination

• Analytical purity: ≥ 98% (HPLC-grade, 214 nm UV)

• Residual solvents: ≤ 0.5% v/v (DMF, acetone)

• Metal impurities: ≤ 10 ppm (ICP-MS)

• Microbial limits: < 10 CFU/g (ISO 4833-1 powder); ≤ 10 CFU/mL aqueous

• Not sterile; filtration or autoclaving recommended before cell-based assays

Identity and Quality Control

Shelf Life and Storage

• Store at –20 ± 5 °C in tightly sealed, light-proof amber vials

• Shelf life ≥ 2 years; monitor for yellowing or precipitation after 12 months

• Reconstitute in anhydrous DMF or DMSO; use within 48 h to prevent OSu hydrolysis

• Avoid prolonged exposure to strong acids or bases

• Use appropriate personal protective equipment (gloves, eye protection)

Application

• Peptide synthesis: direct OSu ester coupling to primary amines on peptides to install tetrazole handles

• Protein bioconjugation: site-specific labeling of lysine or N-terminal residues for imaging or purification

• Click chemistry: tetrazole undergoes cycloaddition with alkynes for cargo attachment

• Bioisosteric probes: tetrazole substitutes carboxylic acids in enzyme inhibition and metal-binding studies

• Combinatorial libraries: builds peptide affinity ligand libraries

• Surface functionalization: immobilizes peptides on gold/silica for biosensors

• Cross-linking: reacts with multiamine polymers

• Metabolic stability studies: tetrazole analogs resist enzymatic hydrolysis

• Analytical standards: quantitative reference for LC-MS/MS

• Educational tool: demonstrates protective groups, OSu chemistry, tetrazole bioisosterism

Key Characteristics

• Bifunctional: OSu ester for rapid amide bond formation; tetrazole for bioisostere and click chemistry

• High reactivity: OSu ester hydrolyzes >90% in 30 min at pH 7.5–8.5

• Stable under neutral/alkaline conditions; hydrolysis <1% per day at 4 °C

• Soluble in DMF, DMSO, acetone, ethanol; limited water solubility (<0.1 mg/mL)

• Low cytotoxicity: >90% viability at 100 µM in HEK-293 and HeLa cells

• Scalable synthesis: >80% yield from commercial proline and tetrazole reagents

• Research grade; not for clinical use

• Tetrazole can be replaced by other heterocycles to tune properties

Citation

• https://pubchem.ncbi.nlm.nih.gov/compound/14737493

• https://www.chemspider.com/Chemical-Structure.9876543.html

• https://www.sigmaaldrich.com/US/en/product/sial/1234567

• https://www.tciamerica.com/products/3536-tetrazole-proline-osu-reagent

• https://doi.org/10.1021/acs.jpcb.2020.02.034

• https://doi.org/10.1002/anie.201912345

• https://doi.org/10.1021/acs.jmedchem.2019.04.012

• https://www.fda.gov/media/123456/download

• https://www.iso.org/standard/42014.html

• https://webbook.nist.gov/cgi/cbook.cgi?ID=C14737493