تفاعل #969867

ord-3619111ce1b54746beae6e022093081f

معادلة التفاعل

O=C(CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@@H]21)NCCCCC(C(=O)[O-])(N1C(=O)CCC1=O)S(=O)(=O)O
sulfosuccinimidyl-6-(biotinamido)hexanoate
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Glc
N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalN
→
N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O.O=C(O)CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@@H]21.O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP GalN biotin

المتفاعلات

O=C(CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@@H]21)NCCCCC(C(=O)[O-])(N1C(=O)CCC1=O)S(=O)(=O)O
sulfosuccinimidyl-6-(biotinamido)hexanoate
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Glc
N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalN
—
nucleotide sugar

الكواشف

لا شيء

المذيبات

—
NH4HCO3

ظروف التفاعل

الظروف التفصيلية
See reaction.notes.procedure_details.

المعالجة

  1. 1
    أخرىat room temperature
  2. 2
    أخرىfor 2.5 hours
  3. 3
    ترشيحThe product, UDP-GalN-biotin, uridine 5′-diphospho-N-(6-biotinamidohexanoyl)galactosamine, is purified by gel filtration

الإجراء التجريبي

UDP-galactosamine (UDP-GalN) is formed from UDP-Glc and galactosamine-1-phosphate by the action of galactose-1-phosphate uridyltransferase (E.C. 2.7.7.12; Sigma, USA). A typical synthesis protocol is described below. The reaction mixture contains 10 mM galactosamine-1-phosphate, 20 mM UDP-Glc, 5 U/ml of galactose-1-phosphate uridyltransferase, 100 mM Na-HEPES pH 8.0, 5 mM MgCl2, and 5 mM β-mercaptoethanol. The reaction vessel is incubated at room temperature under nitrogen atmosphere for 3 days, after which nucleotide sugars are isolated from the reaction mixture with a graphitised carbon column essentially as in (Mäki et al., 2002). The nucleotide sugar mixture, containing UDP-Glc and UDP-GalN, is incubated with a molar excess of sulfosuccinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin; Pierce, USA) in 50 mM NH4HCO3 at room temperature for 2.5 hours. The product, UDP-GalN-biotin, uridine 5′-diphospho-N-(6-biotinamidohexanoyl)galactosamine, is purified by gel filtration and reversed phase HPLC.

المصدر

DOI: 10.6084/m9.figshare.5104873.v1براءة الاختراع: US08236487B2uspto-grants-2012_08