S-2222 (Hydrochloride and its methyl ester)

S-2222 (Hydrochloride and its methyl ester), Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA·HCl represents the defined mixture (50:50) of chromogenic tetrapeptide hydrochloride substrates optimized for Factor Xa amidolytic assays in coagulation research and diagnostics. Form 1 (R=CH3, γ-methyl ester: C33H47N9O9·HCl, MW 748.3) and Form 2 (R=H, free γ-acid: C32H45N9O9·HCl, MW 734.3) together provide balanced solubility, stability, and enzymatic kinetics, with Factor Xa cleaving the Arg-pNA bond to release yellow p-nitroaniline (ε405=12,700 M⁻¹cm⁻¹) for precise quantification. This formulation is the Chromogenix reference standard for anti-Xa heparin/LMWH/DOAC potency testing, prothrombin activation studies, and plasma serine protease profiling, offering superior performance over single-form variants in ISTH-validated methods.

Appearance

• White to off-white lyophilized hygroscopic powder.
• Highly soluble in water (1-4 mM stock); vigorous shaking/sonication required for complete dissolution.

Source

• Solid-phase Fmoc synthesis yielding defined 50:50 mixture of γ-hydroxy and γ-methyl ester forms, HCl salted.
• Commercial diagnostic grade from Chromogenix/DiaPharma (ref. 820316).

Molecular Weight and Structure

• Mixture: Form 1 C33H47N9O9·HCl (748.3 Da); Form 2 C32H45N9O9·HCl (734.3 Da); average ~741.22 g/mol.
• N-benzoyl-Ile-Glu(γ-OR 50/50)-Gly-Arg-p-nitroanilide·HCl; cleavable Arg-pNA amide bond.

Sugar Specificity

• None (synthetic peptide chromogen without carbohydrate moieties).

Biological Activity

• Factor Xa Km 0.5-1.0 mM (pH 8.4, 37°C); linear ΔA405 to 4 µkat/L; ε316 nm = 1.27×10⁴ M⁻¹cm⁻¹.
• Highly selective (200:1 vs. thrombin); also cleaved by trypsin, kallikrein.

Purity and Microbial Contamination

• >99% HPLC (both forms); free pNA <0.05%; single composite peak.
• Endotoxin-free (<0.1 EU/mg); sterile lyophilized format.

Identity and Quality Control

• HPLC/MS confirms 50:50 ratio (m/z 712.8/697.7 [M+H]+ forms); AA analysis 1:1:1:1; functional assay.
• Ph. Eur./ISTH certified; specific activity per certificate of analysis.

Shelf Life and Storage

• 5+ years lyophilized at -20°C; reconstituted stable 1 month -80°C or 1 week 2-8°C.
• Stabilize 30 min RT post-dissolution; protect from light/moisture.

Application

• Anti-Xa assays (UFH/LMWH/fondaparinux/DOACs); Factor X activity/potency.
• Prothrombinase complex; ATIII/FVIII cofactor studies; plasma amidase screening.

Key Characteristics

• Optimized 50:50 mixture prevents precipitation, maximizes solubility/activity balance.
• Automation-compatible; no plasma interference; validated reference substrate.

Citation Links

• https://www.aniara.com/mm5/PDFs/TechData/HBM_A229015-FXa-CS1122-S2222.pdf
• https://diapharma.com/product/chromogenix-s-2222/
• https://www.dcchemicals.com/product_show-S2222.html
• https://www.benchchem.com/product/b12111261
• https://pubchem.ncbi.nlm.nih.gov/compound/13892722
• https://www.benchchem.com/product/b1280698
• https://www.fishersci.com/shop/products/chromogenic-substrt-s-2222-fxa/NC0248053
• https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb15622.x
• https://pmc.ncbi.nlm.nih.gov/articles/PMC9607376/
• https://pubs.acs.org/doi/10.1021/acs.orglett.9b02099