Boc-Ile-Glu-Gly-Arg-AMC (IEGR-AMC) is an activating factor X (FXa)-specific fluorescent peptide substrate for the determination of factor VIII.

Physicochemical Properties

Molecular Formula	C34H50N8O10
Molecular Weight	730.81
Exact Mass	730.365
CAS #	65147-06-0
Sequence	Boc-Ile-Glu-Gly-Arg-AMC
SequenceShortening	Boc-IEGR-AMC
Appearance	White to off-white solid powder
Density	1.36 g/cm3
Index of Refraction	1.61
LogP	4.02
Hydrogen Bond Donor Count	8
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	20
Heavy Atom Count	52
Complexity	1370
Defined Atom Stereocenter Count	4
SMILES	CC[C@@H]([C@H](NC(OC(C)(C)C)=O)C(N[C@H](C(NCC(N[C@H](C(NC1=CC2=C(C(C)=CC(O2)=O)C=C1)=O)CCCNC(N)=N)=O)=O)CCC(O)=O)=O)C
InChi Key	PWOIMWXLMWHALO-SBZQNBARSA-N
InChi Code	InChI=1S/C34H50N8O10/c1-7-18(2)28(42-33(50)52-34(4,5)6)31(49)40-23(12-13-26(44)45)29(47)38-17-25(43)41-30(48)22(9-8-14-37-32(35)36)39-20-10-11-21-19(3)15-27(46)51-24(21)16-20/h10-11,15-16,18,22-23,28,39H,7-9,12-14,17H2,1-6H3,(H,38,47)(H,40,49)(H,42,50)(H,44,45)(H4,35,36,37)(H,41,43,48)/t18-,22-,23-,28-/m0/s1
Chemical Name	(4S)-5-[[2-[[(2S)-5-(diaminomethylideneamino)-2-[(4-methyl-2-oxochromen-7-yl)amino]pentanoyl]amino]-2-oxoethyl]amino]-4-[[(2S,3S)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoyl]amino]-5-oxopentanoic acid
Synonyms	Biggam; 65147-06-0; Boc-ile-glu-gly-arg-mca; tertiary-Butyloxycarbonyl-isoleucyl-glutamyl-glycyl-arginyl-7-amino-4-methylcoumarin; L-Argininamide, N-((1,1-dimethylethoxy)carbonyl)-L-isoleucyl-L-alpha-glutamylglycyl-N-(4-methyl-2-oxo-2H-1-benzopyran-7-yl)-; (4S)-5-[[2-[[(2S)-5-(diaminomethylideneamino)-2-[(4-methyl-2-oxochromen-7-yl)amino]pentanoyl]amino]-2-oxoethyl]amino]-4-[[(2S,3S)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoyl]amino]-5-oxopentanoic acid; DTXSID90983717; 4-[(2-{[tert-Butoxy(hydroxy)methylidene]amino}-1-hydroxy-3-methylpentylidene)amino]-5-hydroxy-5-[(2-hydroxy-2-{[N~2~-(4-methyl-2-oxo-2H-1-benzopyran-7-yl)arginyl]imino}ethyl)imino]pentanoic acid
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Activated coagulation factor X (FXa) [1]
Enzyme Assay	Boc-Ile-Glu-Gly-Arg-AMC was used as a fluorogenic substrate for FXa activity measurement in a novel Factor VIII (FVIII) assay. The substrate was reconstituted in DMSO to 1.5 mM stock solution and diluted in assay buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl₂, pH 8.0) to 150 μM working concentration. Test samples (plasma or FVIII concentrates) were activated with thrombin (0.5 U/mL) for 10 min at 37°C. Activated samples were incubated with FXa reagent (containing phospholipids) for 3 min at 37°C. Reactions were initiated by adding Boc-Ile-Glu-Gly-Arg-AMC substrate and monitored kinetically for 10 min at 37°C using fluorescence spectrophotometry (excitation 380 nm, emission 460 nm). FXa cleaves the substrate to release fluorescent AMC, with fluorescence intensity proportional to FVIII activity. [1]
References	[1]. Factor VIII determination in patient's plasma and concentrates: a novel test equally suited for both matrices. Blood Coagul Fibrinolysis. 2003 Jun;14(4):347-53.
Additional Infomation	Boc-Ile-Glu-Gly-Arg-AMC is a peptide substrate specifically hydrolyzed by FXa. It enables a one-stage FVIII activity assay applicable to both patient plasma and therapeutic concentrates. The assay correlates well with conventional methods (r=0.98) and shows high precision (CV <5%). [1]

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3683 mL	6.8417 mL	13.6834 mL
	5 mM	0.2737 mL	1.3683 mL	2.7367 mL
	10 mM	0.1368 mL	0.6842 mL	1.3683 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.