반응 #1544096

ord-0cc97167ef5640679f47af8e5cb7db37

반응 방정식

CCC(CC)COC(C(=O)N(C)CC[NH+](C)C)(c1ccccc1)c1ccccc1.[Cl-]
X-100
OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O
lactose
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-Gal
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-Gal
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-Gal
→
OC[C@H]1O[C@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H](O)[C@@H](O)C(O)O[C@@H]3CO)[C@@H]2O)[C@H](O)[C@@H](O)[C@H]1O
αGal(1-3)βGal(1-4)Glc
수율 58.9%

반응물

CCC(CC)COC(C(=O)N(C)CC[NH+](C)C)(c1ccccc1)c1ccccc1.[Cl-]
X-100
—
MnCl2
OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O
lactose
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-Gal
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-Gal
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-Gal

시약

없음

용매

C[As](C)(=O)[O-].[Na+]
sodium cacodylate

반응 조건

상세 조건
See reaction.notes.procedure_details.

후처리

  1. 1
    기타was incubated at 37° C
  2. 2
    workup.WAITwas incubated for an additional 72 hours
  3. 3
    기타to give about 95% conversion to product
  4. 4
    여과The reaction mixture was filtered through a 0.2 μm Nalgene nylon
  5. 5
    여과filter
  6. 6
    세척the column was eluted with water
  7. 7
    workup.DISSOLUTIONThe dry residue was dissolved in 50 mM potassium phosphate buffer
  8. 8
    workup.ADDITIONpH 7.5, β-galactosidase (150 mU) was added to the mixture
  9. 9
    기타to destroy unreacted lactose
  10. 10
    workup.WAITthe sample left at ambient temperature (24° C.) for 18 hours
  11. 11
    workup.WAITThe mixture was then boiled for 2 minutes
  12. 12
    여과filtered through a 0.2 μm
  13. 13
    여과filter
  14. 14
    세척The columns were eluted with water (200 mL)
  15. 15
    농축the aqueous eluents were concentrated to dryness under reduced pressure
  16. 16
    workup.DISSOLUTIONThe residue was dissolved in water (5mL)

실험 절차

A reaction mixture containing lactose (50 mg), UDP-Gal(20 mg), α(1-3)-galactosyltransferase (60 mU), alkaline phosphatase (20 U), 20 mM MnCl2 and 0. 1% Triton X-100 in 50 mM sodium cacodylate buffer (3 mL) at pH 6.5, was incubated at 37° C. Additional UDP-Gal was added to the mixture after 24 hours (20 mg), and 48 hours (50 mg). After 120 hours, fresh α(1-3)-galactosyltransferase (20 mU) and UDP-Gal (10 mg) were added to the mixture, which was incubated for an additional 72 hours to give about 95% conversion to product. The reaction mixture was filtered through a 0.2 μm Nalgene nylon filter, the filtrate was applied to a Bio-Rad AG 1X8 column (Cl-form 2.5×20 cm, 0.6 mL/min) and the column was eluted with water. Saccharide fractions were combined and lyophilized. The dry residue was dissolved in 50 mM potassium phosphate buffer, pH 7.5, β-galactosidase (150 mU) was added to the mixture to destroy unreacted lactose, and the sample left at ambient temperature (24° C.) for 18 hours. The mixture was then boiled for 2 minutes, filtered through a 0.2 μm filter and divided into three portions each of which was loaded onto a C-18 silica gel column (20 g). The columns were eluted with water (200 mL) and the aqueous eluents were concentrated to dryness under reduced pressure. The residue was dissolved in water (5mL) and applied to a Bio-Gel P-2 column (2.5×100 cm, H2O, 0.2 mL/min). Fractions which contained the trisaccharide were combined and lyophilized to give 10.5 mg of αGal(1-3)βGal(1-4)Glc. 1H n.m.r. data (500 MHz, D2 O): δ=5.22 (d, 0.36H, J 3.6 Hz, H-1α), 5.14 (d, 1 H, J 3.0 Hz, H-1"), 4.66 (d, 0.64 H, J 8.0 Hz, H-1β), 4.51 (d, 1 H, J 8.0 Hz, H-1').

출처

DOI: 10.6084/m9.figshare.5104873.v1특허: US05846943uspto-grants-1998_12