5'-Atp → HCID 58423310, compound

HRID 1398027Notes

Reaction equation

HRID 1398027 reaction equation
N(F)[C@H:2]([C:7]([OH:9])=O)[CH2:3][CH2:4][CH2:5][CH3:6].P(OC[C@H:25]1O[C@@H:28]([N:30]2[C:39]3N=CN=C(N)[C:33]=3[N:32]=[CH:31]2)[C@H:27](O)[C@@H:26]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.N[C@H](C=O)CCSC.[Mg+2].[Cl-:51].[Cl-].C1N(CCO)CCN(CCS(O)(=O)=O)C1.C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O>>[NH2:32][CH:33]1[CH:26]([CH3:25])[CH2:27][CH2:28][N:30]([C:31]2[C:6]([Cl:51])=[CH:5][CH:4]=[CH:3][C:2]=2[CH:7]=[O:9])[CH2:39]1

Participants

7

Reactant 6

HCID 59575'-Atp2×HCID 6049Ethylenediaminetetraacetic AcidHCID 238314-(2-Hydroxyethyl)-1-piperazineethanesulfonic acidHCID 446793Methionin ALHCID 5360315Magnesium ChlorideHCID 54290789HCID 54290789, compound2×

Product 1

HCID 58423310HCID 58423310, compound

Procedure

One determines phosphorylation of the FL-Ahx-Bad in the presence and absence of the compound of interest. One preincubates 5u1 of Enzyme/Substrate/adenosine triphosphate (ATP) mix consisting of 2.4 nM PIM1, 3.6 uM FL-Ahx-Bad, and 240 uM ATP in 1.2× buffer with 2 ul of compound for 20 minutes at 25° C. One initiates reactions with 5 ul of Metal mix consisting of 24 mM MgCl2 in 1.2× buffer and incubated at 25° C. for 90 minutes and stops the reactions by addition of 5 ul of Stop mix consisting of 100 mM HEPES, 121 mM ethylenediamine tetraacetic acid, 0.8% Coatin Reagent 3 (Caliper, Mass.), and 0.01% Tween. One detects phosphorylated and unphosphorylated substrate by a Caliper LC3000 reader (Caliper, Mass.) in the presence of separation buffer consisting of 100 mM HEPES, 16 mM ethylenediamine tetraacetic acid, 0.1% Coatin Reagent 3 (Caliper, Mass.), 0.015% Brij-35, 5% DMSO, and 5.6 mM MgCl2. One may use the following separation conditions for a Caliper LC3000: −1.0 PSI, −2000 V upstream voltage, −400 V downstream voltage, 0.2 second sample sip, 45 second post sip, 10% laser strength.

Workup

  1. additionmix
  2. additionmix
  3. additionby addition of 5 ul of Stop
  4. additionmix
  5. customunphosphorylated substrate by a Caliper LC3000 reader (Caliper, Mass.) in the presence of separation
  6. customthe following separation conditions for a Caliper LC3000
  7. customdownstream voltage, 0.2 second sample sip, 45 second post sip, 10% laser strength

Sources & evidence

ORD record
ord-cf82d0eaf0cd418ab41dcce0a35f6863
Source dataset
uspto-grants-2014_12
Patent number
US08901307B2

Reaction notes

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