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Udp-Galactose → alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-Glc

HRID 1456914
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HRID 1456914 반응식
[OH:1][CH:2]1[O:21][C@H:20]([CH2:22][OH:23])[C@@H:7]([O:8][C@@H:9]2[O:17][C@H:16]([CH2:18][OH:19])[C@H:14]([OH:15])[C@H:12]([OH:13])[C@H:10]2[OH:11])[C@H:5]([OH:6])[C@H:3]1[OH:4].C1C(=O)NC(=O)N([C@@H]2O[C@H](COP(OP(O[C@H:47]3[O:52][C@H:51]([CH2:53][OH:54])[C@H:50]([OH:55])[C@H:49]([OH:56])[C@H:48]3[OH:57])(O)=O)(O)=O)[C@@H](O)[C@H]2O)C=1.CCC(COC(C(N(CC[NH+](C)C)C)=O)(C1C=CC=CC=1)C1C=CC=CC=1)CC.[Cl-]>[As](C)(C)(=O)[O-].[Na+]>[O:13]([C@H:12]1[C@@H:14]([OH:15])[C@@H:16]([CH2:18][OH:19])[O:17][C@@H:9]([O:8][C@@H:7]2[C@@H:20]([CH2:22][OH:23])[O:21][CH:2]([OH:1])[C@H:3]([OH:4])[C@H:5]2[OH:6])[C@@H:10]1[OH:11])[C@H:47]1[O:52][C@H:51]([CH2:53][OH:54])[C@H:50]([OH:55])[C@H:49]([OH:56])[C@H:48]1[OH:57]
참가 물질실험 절차후처리출처 및 근거반응 노트

참가 물질

5

반응물 3

HCID 18068Udp-Galactose3회HCID 19153X 100HCID 440995(+)-Lactose

생성물 1

HCID 11249135alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-Glc수율 58.9%

시약·촉매·용매 1

HCID 2724247Sodium Cacodylate용매

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').

후처리

  1. customwas incubated at 37° C
  2. waitwas incubated for an additional 72 hours
  3. customto give about 95% conversion to product
  4. filtrationThe reaction mixture was filtered through a 0.2 μm Nalgene nylon
  5. filtrationfilter
  6. washthe column was eluted with water
  7. dissolutionThe dry residue was dissolved in 50 mM potassium phosphate buffer
  8. additionpH 7.5, β-galactosidase (150 mU) was added to the mixture
  9. customto destroy unreacted lactose
  10. waitthe sample left at ambient temperature (24° C.) for 18 hours
  11. waitThe mixture was then boiled for 2 minutes
  12. filtrationfiltered through a 0.2 μm
  13. filtrationfilter
  14. washThe columns were eluted with water (200 mL)
  15. concentrationthe aqueous eluents were concentrated to dryness under reduced pressure
  16. dissolutionThe residue was dissolved in water (5mL)

출처 및 근거

ORD 기록
ord-0cc97167ef5640679f47af8e5cb7db37
출처 데이터셋
uspto-grants-1998_12
DOI
10.6084/m9.figshare.5104873.v1
특허 번호
US05846943

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