Bitan:SATA conjugation to Aβ

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Procedure for SATA-Aβ40 conjugation

Procedure for SATA-Aβ40 conjugation

Small-scale prepartion

  1. Weigh out 10−12 mg of peptide-bound resin. This resin has a Wang substitution of 0.27 mmol peptide/g resin. This amount will give 2.7−3.2 μmol Aβ40.
  2. Swell the resin in 0.5 mL DMF (40 min) followed by DCM (0.5 mL, 40 min) and DMF (0.5 mL, 40 min) while shaken on the platform shaker/rocker.
  3. Perform the Kaiser test

a.    Take some resin out

b.    Add the Kaiser reagents (2 drops or 15−20 μL)

c.    Mix well by vortex.

d.    Heat at 100−120 °C for 4−6 min

e.    Assess the color change. Blue color suggests presence of free amines.

  1. If free amines are present, there is no need for deprotection.
  2. Calculate amount of SATA, HBTU and DIPEA. Need to use 10-fold excess to Aβ.

a.    Mole of SATA needed=27 μmol; SATA FW=231.23 g/mol; mass of SATA needed=27×10−6 mol×231.23 = 6.24×13−3 g =6.2 mg

b.    Mole of HBTU needed=27 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=27×10−6 mol×379.3 g/mol=10.24 mg

c.    Mole of DIPEA needed is 27 μmol. DPEA ρ=0.742 g/mL; FW=129.24 g/mol. Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7×10−3 mol/mL= 5.7 M. Volume of DIPEA needed= 27×10−6 mol÷5.7 mol/L=4.7 μL.

  1. Mix SATA, HBTU and DIPEA together and check the pH.
  2. Add to resin and incubate in the CEM Microwave Discover running the appropriate coupling method. Run the coupling method once more.
  3. Wash the resin profusely with DMF.
  4. Wash the resin profusely with DCM.
  5. Keep an aliquot for test cleavage.
  6. Store the beads at −20°C or follow on to cleave the products using the CEM discover.

 


Procedure for SATA-Aβ40 conjugation

Large-scale prepartion

  1. Weigh out 100 mg of peptide-bound resin. This resin has a Wang substitution of 0.27 mmol peptide/g resin. This amount will give ~27 μmol Aβ40.
  2. Swell the resin in 1–2 mL DMF (40 min) followed by DCM (1–2 mL, 40 min) and DMF (1–2 mL, 40 min) while shaken on the platform shaker/rocker.
  3. Perform the Kaiser test

a.    Take some resin out

b.    Add the Kaiser reagents (2 drops or 15−20 μL)

c.    Mix well by vortex.

d.    Heat at 100−120 °C for 4−6 min

e.    Assess the color change. Blue color suggests presence of free amines.

  1. If free amino group is present there is not need for deprotection.
  2. Calculate amount of SATA, HBTU and DIPEA. Need to use 5-fold excess to Aβ.

a.    Mole of SATA needed=135 μmol; SATA FW=231.23 g/mol; mass of SATA needed=135×10−6 mol×231.23 = 3.12×13−2 g =31.2 mg

b.    Mole of HBTU needed=135 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=135×10−6 mol×379.3 g/mol=51.2 mg

c.    Mole of DIPEA needed is 135 μmol. DPEA ρ=0.742 g/mL; FW=129.24 g/mol. Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7×10−3 mol/mL= 5.7 M. Volume of DIPEA needed= 135×10−6 mol÷5.7 mol/L=23.5 μL.

  1. Mix SATA, HBTU and DIPEA together and check the pH.
  2. Add to resin and incubate in the CEM Microwave Discover running the appropriate coupling method. Run the coupling method once more.
  3. Wash the resin profusely with DMF.
  4. Wash the resin profusely with DCM.
  5. Keep an aliquot for test cleavage.
  6. Store the beads at −20°C or follow on to cleave the products using the CEM discover.
  7. Cleave with cleavage solution 825 μL TFA + 50 μL H2O + 50 μL thioanisol + 25 μL EDT. For test-cleavage, use 30–60 μL of this solution. Incubate shakingly for 3–4 hours.
  8. Add 5 μL of this solution to 200 μL of 50% methanol in water and inject 150–170 μL into LC-MS.

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