反応 #420096

ord-f5b5b9418ce840ee8119eee3d7664960

反応方程式

Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
O=S(=O)(O)CCN1CCOCC1
MES
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
ClCCCl
EDC
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O.[K]
ADP monopotassium
O=S(=O)(O)CCCN1CCN(CCO)CC1
EPPS
OC[C@H]1O[C@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1O
trehalose
ClCCCl
EDC
→
ClCCCl.Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP EDC

反応物

—
inorganic phosphate
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
O=S(=O)(O)CCN1CCOCC1
MES
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
ClCCCl
EDC
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O.[K]
ADP monopotassium
O=S(=O)(O)CCCN1CCN(CCO)CC1
EPPS
OC[C@H]1O[C@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1O
trehalose
ClCCCl
EDC

試薬

[Au]
Gold

溶媒

O
water
O
water
O
water

反応条件

詳細条件
See reaction.notes.procedure_details.

後処理

  1. 1
    その他dried
  2. 2
    その他prepared
  3. 3
    その他was removed
  4. 4
    workup.ADDITIONThe fractions containing protein
  5. 5
    その他subjected to acid hydrolysis
  6. 6
    その他conjugates prepared

実験手順

500 ul of 20 mM ADP monopotassium salt (Fluka product code 01899) was mixed with 200 ul of 0.5M EPPS pH 8.5, 100 ul of trehalose (1 g in 2 ml water), 200 ul of water and 5 μl of either 2M EDC (final concentration 10 mM) or water. Aliquots of 100 ul were freeze dried using the drying program given in Example 6. The two types of dry powders (i.e. with and without EDC) were reconstituted with 100 ul of cationised BSA prepared as described in Example 15 (but desalted into water instead of EPPS buffer) and 100 ul of 0.5M MES pH 6.2. After reaction for 18 hours at 25° C. free ADP was removed by desalting on a NAP-5 column (GE Healthcare) equilibrated with 0.15M NaCl. The fractions containing protein were pooled (0.3 ml) and subjected to acid hydrolysis by heating at 100° C. for 5 min with 75 ul of PiColorlock Gold colorimetric detection reagent for inorganic phosphate (Innova Biosciences product code 303-0030). Blank subtracted absorbance values (A650) for 200 ul samples of hydrolysed BSA-ADP conjugates prepared either with or without EDC were 1.22 and 0.08, respectively.

出典

DOI: 10.6084/m9.figshare.5104873.v1特許: US08877892B2uspto-grants-2014_11