Pepstatin CAS 26305-03-3
Pepstatin Basic informationBackground
| Product Name: | Pepstatin |
| Synonyms: | ahpatininc;pepsininhibitors735a;procidins735a;PEPSTATIN;PEPSTATIN A;X-VAL-VAL-STATYL-ALA-STATIN;(3S,4S)-4-AMINO-3-HYDROXY-6-METHYL-HEPTANOIC ACID;AHMHA ISOVALERYL-L-VAL-L-VAL-AHMHA-L-ALA-STA |
| CAS: | 26305-03-3 |
| MF: | C34H63N5O9 |
| MW: | 685.89 |
| EINECS: | 247-600-0 |
| Product Categories: | Antibiotics;Inhibitor;peptides;Pepetides;ProteaseInhibitors |
| Mol File: | 26305-03-3.mol |
Pepstatin Chemical Properties
| Melting point | 233 °C (dec.)(lit.) |
| alpha | D27 -90.3° (c = 0.288 in methanol) |
| Boiling point | 695.91°C (rough estimate) |
| density | 1.1340 (rough estimate) |
| refractive index | 1.7500 (estimate) |
| storage temp. | 2-8°C |
| solubility | 10% acetic acid in methanol: 1 mg/mL |
| pka | 4.17±0.10(Predicted) |
| form | White solid |
| color | Colorless needles |
| Optical Rotation | Optical rotation: -90.0 ± 5° (c = 0.5, MeOH, 20°C). |
| biological source | synthetic |
| Water Solubility | It is soluble in 10% (v/v) acetic acid in methanol (9:1 methanol:acetic acid) (1 mg/ml), ethanol (1-2 mg/ml with heat up to 60°C), DMSO (5 mg/ml), methanol (1 mg/ml), and acetic acid. Insoluble in benzene, chloroform, water, 1 M NaOH, and ether. |
| Specific Activity | ≥100,000U/mg |
| BRN | 2201362 |
| Sequence | IsoValeryl-Val-Val-Sta-Ala-Sta-OH |
| Stability: | Stable. Incompatible with strong bases, strong acids. |
| InChIKey | JKGWASGTXVCDML-LXTPJMTPSA-N |
| SMILES | CC(C)C[C@H](NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CC(C)C)C(C)C)C(C)C)[C@@H](O)CC(O)=O |
| CAS DataBase Reference | 26305-03-3(CAS DataBase Reference) |
Safety Information
| Safety Statements | 22-24/25 |
| WGK Germany | 2 |
| RTECS | SC6155000 |
| F | 10 |
| HS Code | 29241990 |
| Storage Class | 11 - Combustible Solids |
| Toxicity | LD50 in mice, rats, rabbits, dogs (mg/kg): 1090, 875, 820, 450 i.p.; all >2000 orally (Umezawa, 1970) |
| Description | Pepstatin A is a bacterial- |
| Chemical Properties | solid |
| Uses | Antiviral;Aspartic proteases irreversible inhibitor |
| Uses | As an aspartic proteases irreversible inhibitor, Pepstain A can be used in conjunction with E64-d and Leupeptin A to inhibit the degradation of autophagic cargo inside autophagosomes. |
| Uses | Pepstatin A are natural, statine-containing peptides that acts as inhibitor of aspartic protease enzymes. |
| Definition | ChEBI: Pepstatin A is a pentapeptide isolated from Streptomyces testaceus. It is a potent inhibitor of aspartyl proteases. It has a role as a bacterial metabolite and an EC 3.4.23.* (aspartic endopeptidase) inhibitor. It is a pentapeptide and a secondary carboxamide. It is a conjugate acid of a pepstatin A(1-). |
| General Description | Pepstatin A is a naturally occurring chemotactic peptide and inhibitor of aspartic proteases that was initially isolated from culture filtrates of various Actinomycetes species, with the initial name simply of “pepstatin”. This name was later modified to “pepstatin A” to distinguish the original pepstatin from later derivatives. Pepstatin A notably contains the unusual amino acid 4-amino-hydroxy-6-methylheptanoic acid (AHMHA), which is known also as statine. The amino acid sequence of pepstatin A is N-Isovaleryl-L-Valyl-L-Valyl-AHMHA-L-Alanyl-AHMHA. |
| Biochem/physiol Actions | Primary Targetaspartic proteases |
| Safety Profile | Moderately toxic by intraperitoneal route. When heated to decomposition it emits toxic fumes of NOx. |
| Synthesis | 503-74-2 68858-20-8 35661-39-3 158257-40-0 26305-03-3 General steps:4.2 General Procedure A: Resin LoadingSolid phase peptide synthesis was performed manually in a sintered polypropylene syringe. The 2-chlorotrityl chloride (CTC) resin was pre-immersed in dichloromethane (DCM) for 15 minutes and drained. The first amino acid in a DCM solution of 0.4 M N,N-diisopropylethylamine (DIPEA) was added and the mixture was stirred for 3 hours. After draining the solvent, the resin was treated with a 17:2:1 DCM/methanol (MeOH)/DIPEA (3 × 3 mL × 5 min) solution, and then any free 2-CTC resin joints were blocked with an 8:1:1 N,N-dimethylformamide (DMF)/DIPEA/acetic anhydride (2 × 3 mL × 10 min) solution. The resin was finally washed with DCM (2 × 3 mL × 1 min), DMF (2 × 3 mL × 1 min), DCM (2 × 3 mL × 1 min), and DMF (2 × 3 mL × 1 min).4.3 General method B: Fmoc deprotectionThe resin was stirred with a 10% piperidine solution of DMF (2 x 3mL x 3 min) and subsequently washed with DMF (3 x 3mL x 1 min), DCM (3 x 3mL x 1 min), and DMF (5 x 3mL x 1 min). The deprotected solutions were combined and diluted appropriately (100-fold, 0.05 mmol resin loading). Resin loading was estimated by measuring the absorbance of piperidine-fulvene adduct with 10% piperidine in DMF as reference (λ = 301 nm; ε = 7800 M-1cm-1).4.4 Generalized method C: peptide coupling with HBTUsSolutions of amino acids protected by appropriate Fmoc (3 eq. relative to resin loading) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (2.9 eq. relative to resin loading) were prepared in minimal amounts of DMF. DIPEA (6 equivalents relative to resin loading) was added and the resin was stirred for 1.5 hours. The resin was then drained and washed with DMF (3 x 3 mL x 1 min), DCM (3 x 3 mL x 1 min) and DMF (5 x 3 mL x 1 min).4.5 Generalized Method D: Peptide Coupling with HATUsSolutions of amino acids protected by appropriate Fmoc (3 equivalents relative to resin loading) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (2.9 equivalents relative to resin loading) were prepared in minimal DMF. DIPEA (6 equiv. vs. resin loading) was added to the solution and the mixture was immediately added to the resin and stirred. The reaction time was varied based on the coupled residues: Phe (NMe) and Ala (2 × 2 h); Thr and Sta (1 × 2 h); Asn, Leu, D-Val, and L-Val (2 × 2 h); and DMVal (3 × 3 h). Once the reaction was complete, the resin was drained and washed with DMF (3 × 3 mL × 1 min), DCM (3 × 3 mL × 1 min), and DMF (3 × 3 mL × 1 min).4.6 Generalized Method E: Peptide Coupling to DICsA solution of amino acids protected by appropriate Fmoc (1.5 equivalents relative to resin loading), 1-hydroxybenzotriazole (HOBt) (1.5 equivalents relative to resin loading), and N,N'-diisopropylcarbodiimide (DIC) (1.5 equivalents relative to resin loading) in minimal DMF was prepared. The solution was stirred for 20 minutes, then added to the resin and stirred overnight. The resin was drained and washed with DMF (3 × 3 mL × 1 min), DCM (3 × 3 mL × 1 min), and DMF (5 × 3 mL × 1 min). Application of coupled fluorinated amino acids followed by double coupling of the next amino acid.4.7 Generalized Procedure F: Resin CleavageAfter final Fmoc deprotection, the resin was washed with DMF (3 x 3mL x 1 min) and DCM (3 x 3mL x 1 min) and then vacuum dried. The resin was stirred with 95:2.5:2.5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (3 mL) solution for 2 hours. The resin was drained and washed with the same TFA mixture described above (2 × 3 mL × 1 min). The combined lysis solutions were concentrated under a stream of nitrogen. Ether was added and the supernatant was decanted off (3×). The residue was then dried under vacuum to give a crude linear peptide. |
| Enzyme inhibitor | This naturally occurring statine-containing peptidomimetic (FWfree-acid = 685.90 g/mol; CAS 26305-03-3), also called pepstatin A and isovalerylpepstatin and named systematically as N-(isovaleryl)-L-valyl-L-valyl-statylL-alanyl-statine (where the statyl residue is a (3S,4S)-4-amino-3-hydroxy-6- methylheptanoyl residue and the C-terminal statine is the corresponding acid), is a presumptive transition-state analogue for prototypical aspartate proteinase pepsin (Ki = 46 pM). Pepstatin will also inhibit other carboxy proteinases. The inhibitory effectiveness of statine-containing peptides has been widely exploited by incorporating the statine residue into peptides that otherwise match sequence preferences of the target enzyme’s sub-sites. Because pepstatin has a low solubility in water, it is often dissolved indimethyl sulfoxide, methanol, or ethanol. Note that many derivatives of pepstatin are available, each displaying a different spectrum of inhibitory effects. Pepstatin B and C are the N-(n-caproyl)- and N-(iso-caproyl)- derivatives, respectively. Note: Above a critical concentration of 0.1 mM in low ionic-strength and neutral buffers, pepstatin often polymerizes into filaments that be several micrometers in length and have characteristic diameters ranging between 6 and 12 nm |
| storage | +4°C |
| Background | Pepstatin A is a low molecular weight inhibitor of aspartyl proteases, including pepsin, renin, cathepsin D and cathepsin E. Cathepsin D is a ubiquitously expressed lysosomal aspartyl protease involved in the normal degradation of proteins; dysregulation of cathepsin D plays a role in neuronal degradation and malignant transformation. Cathespin D is highly expressed in triple-negative breast cancer cells. Pepstatin A treatment induced apoptosis and autophagy while reducing the proliferation, invasion, and migration of TNBC cells. Increased expression and leakage of lysosomal cathepsin D is associated with cardiomyocyte death related to hyperglycemia. Treatment with Pepstatin A markedly diminished high glucose-induced cardiomyocyte death. Pepstatin A treatment of osteoclast cells suppressed receptor activator of NF-κB ligand-induced differentiation to mononuclear osteoclast cells by blocking NFATc1 expression and inhibiting ERK signaling. Pepstatin A also inhibits human immunodeficiency virus protease. Pepstatin A treatment of HIV-infected H9 cells decreased the level of HIV core antigen in the culture medium. |
| References | [1] Merck’s Index, Nachtrag 1930. Verlag von E. Merck, Darmstadt. 14 Seiten[J]. Archiv der Pharmazie, 1930, 268 6: 444-445. DOI:10.1002/ardp.19302680608 [2] J MARCINISZYN J T J A Hartsuck. Mode of inhibition of acid proteases by pepstatin.[J]. The Journal of Biological Chemistry, 1976, 251 22: 7088-7094. [3] ODA K. New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases.[J]. Journal of biochemistry, 2012, 151 1: 13-25. DOI:10.1093/jb/mvr129 [4] HAJIME YOSHIDA. Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation.[J]. Journal of biochemistry, 2006, 139 3: 583-590. DOI:10.1093/jb/mvj066 |
Pepstatin Preparation Products And Raw materials
| Raw materials | FMOC-Ala-OH-->FMOC-STA-OH-->FMOC-L-Valine-->Isovaleric acid-->Piperidine-->N,N-Dimethylformamide-->N,N-Diisopropylethylamine-->Dichloromethane |
