Reaktion #1016053

ord-7dc54a020d76451896b20307fb5581fb

Reaktionsgleichung

CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O
GalNAc
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ATP
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UTP
Cl.NC(CO)(CO)CO
Tris-HCl
[Cl-].[Cl-].[Mg+2]
MgCl2
→
CC(=O)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
desired product
CC(=O)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-GalNAc

Edukte

CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O
GalNAc
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ATP
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UTP
—
uridine 5′-diphospho-2-acetamido-2-deoxy-α-D-glacopyranoside
Cl.NC(CO)(CO)CO
Tris-HCl
[Cl-].[Cl-].[Mg+2]
MgCl2
—
p-anisaldehyde sugar

Reagenzien

Keine

Lösungsmittel

CO
MeOH
O
water
O
water
CCOC(C)=O
EtOAc
O
H2O

Reaktionsbedingungen

Detaillierte Bedingungen
See reaction.notes.procedure_details.

Aufarbeitung

  1. 1
    workup.ADDITIONwas added
  2. 2
    Sonstigeat 37° C.
  3. 3
    Sonstigefor 24 h
  4. 4
    workup.ADDITIONby adding the same volume of ice-cold ethanol
  5. 5
    EinengenThe mixture was concentrated
  6. 6
    Filtrationpassed through a BioGel P-2 gel filtration column

Vorschrift

One-Pot Three-Enzyme Synthesis of uridine 5′-diphospho-2-acetamido-2-deoxy-α-D-glacopyranoside (UDP-GalNAc). GalNAc (100 mg, 1.0 eq), ATP (1.2 eq.), and UTP (1.2 eq.) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (10 mM). After the addition of NanK_ATCC55813 (3.5 mg), PmGlmU (5 mg), and PmPpA (2.5 mg), water was added to bring the volume of the reaction mixture to 20 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37° C. for 24 h with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H2O=3:2:1 by volume) with p-anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4° C. for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. Silica gel column purification (EtOAc:MeOH:H2O=4:2:1) was applied for further purification to give pure target compound. Yield, 83% (228 mg); white foam. 1H NMR (600 MHz, D2O) δ 7.93 (d, J=8.4 Hz, 1H), 5.94-5.96 (m, 2H), 5.55 (dd, J=6.6, 3.0 Hz, 1H), 4.27-4.36 (m, 2H), 4.22-4.27 (m, 3H), 4.16-4.18 (m, 2H), 4.02 (d, J=3.0 Hz, 1H), 3.95 (dd, J=10.8, 3.0 Hz, 1H), 3.71-3.78 (m, 2H), 2.06 (s, 3H). 13C NMR (150 MHz, D2O) δ 175.05, 166.31, 151.85, 141.89, 102.82, 94.75, 88.70, 83.03 (d, J=8.6 Hz), 73.97, 72.32, 69.79, 68.50, 67.64, 65.14, 61.17, 49.95 (d, J=7.8 Hz), 22.24. HRMS (ESI) m/z calcd for C17H28N3O17P2 (M+H) 608.0894, found 608.0906.

Quelle

DOI: 10.6084/m9.figshare.5104873.v1Patent: US09290530B2uspto-grants-2016_03