Glycine 4-nitroanilide
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Glycine 4-nitroanilide

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A substrate for glycine aminopeptidase.

Category
Other Unnatural Amino Acids
Catalog number
BAT-004263
CAS number
1205-88-5
Molecular Formula
C8H9N3O3
Molecular Weight
195.18
Glycine 4-nitroanilide
IUPAC Name
2-amino-N-(4-nitrophenyl)acetamide
Synonyms
Gly-pNAN'-p-Nitrophenylglycinamide; 2-amino-N-(4-nitrophenyl)acetamide; Glycine p-nitroanilide
Appearance
Yellow crystalline to light yellow powder
Purity
≥ 99% (Assay by titration, HPLC, TLC)
Density
1.413 g/cm3
Melting Point
167-172 °C
Boiling Point
451.7 °C at 760 mmHg
Storage
Store at 2-8 °C
InChI
InChI=1S/C8H9N3O3/c9-5-8(12)10-6-1-3-7(4-2-6)11(13)14/h1-4H,5,9H2,(H,10,12)
InChI Key
VZZMNSJSZXVCCW-UHFFFAOYSA-N
Canonical SMILES
C1=CC(=CC=C1NC(=O)CN)[N+](=O)[O-]
1. The complement component C1s catalysed hydrolysis of peptide 4-nitroanilide substrates
S J Keogh, D R Harding, M J Hardman Biochim Biophys Acta. 1987 May 27;913(1):39-44. doi: 10.1016/0167-4838(87)90229-9.
The kinetic parameter kcat/Km has been determined for the hydrolysis of peptide 4-nitroanilides, catalysed by complement component C1s. Substrates based on the C-terminal sequence of human C4a (Leu-Gln-Arg) were synthesised. Replacement of the glutamine residue by glycine or serine increased kcat/Km. Substitution of valine for the leucine residue increased kcat/Km, while substitution of glycine or lysine for the leucine residue decreased kcat/Km slightly. D-Val-Ser-Arg 4-nitroanilide is the most reactive 4-nitroanilide substrate towards C1s, so far. These results are discussed in relation to the amino acid sequences near the bonds cleaved by C1s in C4, C2 and C1 inhibitor.
2. Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein
J C Powers, B J McRae, T Tanaka, K Cho, R R Cook Biochem J. 1984 Jun 1;220(2):569-73. doi: 10.1042/bj2200569.
A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.
3. Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters
Ilka Knütter, et al. FEBS J. 2007 Nov;274(22):5905-14. doi: 10.1111/j.1742-4658.2007.06113.x. Epub 2007 Oct 18.
In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.
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