Triethylamine → Biotin

HRID 1517052Notes

Reaction equation

HRID 1517052 reaction equation
[CH2:1]1[S:5][C@@H:4]([CH2:6][CH2:7][CH2:8][CH2:9][C:10](NCCCCCN)=[O:11])[C@H:3]2[NH:19][C:20]([NH:22][C@@H:2]12)=[O:21].CC1(C)C(/C=C/C=C/C=C2\C(C)(C)C3C(N\2CCCCCC(ON2C(=O)CCC2=O)=[O:53])=CC=CC=3)=[N+](C)C2C1=CC=CC=2.C(N(CC)CC)C>CS(C)=O>[OH:53][C:10]([CH2:9][CH2:8][CH2:7][CH2:6][C@H:4]1[C@@H:3]2[C@@H:2]([NH:22][C:20]([NH:19]2)=[O:21])[CH2:1][S:5]1)=[O:11]

Participants

5

Reactant 3

HCID 8471TriethylamineHCID 839065-(Biotinamido)pentylamineHCID 71581510HCID 71581510, compound

Product 1

HCID 171548Biotin

Reagents, catalysts & solvents 1

HCID 679Dimethyl SulfoxideSolvent

Procedure

Biotin Cadaverine (Sigma Aldrich) was conjugated to Cy5 NHS ester (Amersham Biosciences) in DMSO by adding triethylamine for one hour. The conjugate was purified on a Sephadex LH20 column (Amersham Biosciences) by collecting the first ˜500 μl of the coloured fraction. The concentration of the conjugate was determined by preparing a standard curve of Cy5 NHS Ester dye by diluting it in steps of five (resulting in 205, 41.1, 8.21, 1.64, 0.329 and 0 nM). An aliquot of the conjugate was diluted by a factor of 100. Based on the standard curve, we evaluated the concentration to be 11 mM. For biotin assay experiments the biotin conjugate was diluted to 2.2, 1.1, 0.22, 0.044 nM. In an assay experiment, the scanner was operated in line-scanning mode (˜2 sec/line) across the streptavidin line, resulting in data collection also from areas with no biotin. After a few seconds of scanning, 40 μl of biotin solution was dispensed on the chip and data was recorded for about 3.5 minutes. A new chip was used for each experiment.

Workup

  1. customThe conjugate was purified on a Sephadex LH20 column (Amersham Biosciences)
  2. customby collecting the first ˜500 μl of the coloured fraction
  3. customThe concentration of the conjugate was determined by preparing a standard curve of Cy5 NHS Ester
  4. additionby diluting it
  5. customin steps of five (resulting in 205, 41.1, 8.21, 1.64, 0.329 and 0 nM)
  6. concentrationthe concentration
  7. additionFor biotin assay experiments the biotin conjugate was diluted to 2.2, 1.1, 0.22, 0.044 nM
  8. customwas operated in line-scanning mode (˜2 sec/line) across the streptavidin line

Sources & evidence

ORD record
ord-79a3def11ebe467ab80791dab20fab79
Source dataset
uspto-grants-2012_07
Patent number
US08228602B2

Reaction notes

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