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2-chlorotrityl resins and the TFA labile Wang resins. The success of this approach was shown by the co-synthesis of ACTH (4-10) with ACTH (4-11) and Neuropeptide Y, a C-terminal amide peptide with its corresponding C-terminal free acid analog.

*Hong A., Le T., and Phan T. Techniques in Protein Chemistry VI, 531-562 (1995). Cleavage Protocol to Produce Fully Protected Peptide Starting Resin: Chlorotrityl resins Reagents for 1 g Peptide-Resin: 1 mL acetic acid (AcOH) 2 mL trifluoroethanol (TFE) 7 mL dichloromethane (DCM)

1. Prepare above mixture.

2. Add peptide-resin to the mixture and let it stir at room temperature for 1 h.

3. Filter and wash resin with 10 mL TFE:DCM (2:8) (2x) to ensure that all of the product is

recovered.

4. Evaporate the solvent until there is less than 5 mL of liquid.

5. Add ether to a test tube containing about 100 ?L of the above solution. Check solubility of the

fully protected peptide in ether. If the product precipitates, proceed to step 6. If no precipitate is observed, proceed to step 7.

6. Add cold ether to the residual liquid in step 4 to precipitate the fully protected peptide. Filter

through a fine sintered funnel to obtain the product.

7. Some fully protected peptides are soluble in ether. In this case, add water to precipitate them

out. Filter through a fine sintered funnel to obtain the product.

Procedure for FITC Labeling of Peptides Reagents:

FITC [1 g, AnaSpec Catalog # 20151]

Fmoc-?-Ahx-OH [1 g, AnaSpec Catalog # 20957; 5 g, AnaSpec Catalog # 20958]

1. Couple Fmoc-?-Ahx-OH to the amino terminal of the peptide-resin using standard coupling

conditions.

2. \ 3. Wash resin with DMF (3-4x). 4. Swell resin with DCM and drain.

5. Prepare solution of 1.1 equivalent of FITC in pyridine/DMF/DCM (12:7:5). Use just enough

solution to form a slurry with the resin. Do not use too much solution since the rate of the reaction is proportionate to the concentration of the solution. 6. Add the solution prepared in step 2 to the resin. 7. Let mix overnight.

8. Check the completion of the reaction using ninhydrin test.

9. If the coupling of FITC to the amino group is not complete, ninhydrin test will give a blue color.

Repeat the coupling with FITC (steps 5-7) if necessary. 10. Wash resin with DMF (2x), isopropanol (2x), and DCM (2x).

Page 25 of 43

Procedure for Removing Mtt group from Fmoc-Lys(Mtt) on Solid Phase Reagent:

Fmoc-Lys(Mtt)-OH [1 g, AnaSpec Catalog # 20093; 5 g, AnaSpec Catalog #20094]

1. Swell resin in DCM.

2. Wash resin with 3% TFA/DCM (2x) (since the resin is swollen in DCM, this step of washing the

resin quickly with 3% TFA/DCM ensures that the actual concentration of TFA is 3%). 3. Shake the resin in 3% TFA for 10 min. 4. Repeat step 3.

5. Wash resin with DCM (3x), DMF (3x), isopropanol (3x), and DCM (3x). 6. Let the resin dry in air.

Procedure for Fluorescein Labeling of Peptides Reagent:

5-carboxyfluorescein (5-FAM) [0.1 g, AnaSpec Catalog # 24623; 0.5 g, AnaSpec Catalog # 24624) Use standard coupling method to couple 5-carboxyfluorescein to the amino group of the peptide. For cost saving purposes, use 2x excess compared to the mmol of resin, instead of the standard 4x excess used for Fmoc-amino acids. For 0.1 mmol synthesis, use 75 mg 5-carboxyfluorescein, 76 mg HBTU, and 70 mL DIEA.

Page 26 of 43

六、常用试剂

（一）缩合剂 1．

Name: EDC.HCl Category: Peptide Coupling Reagent

2．6-氯-1-羟基-苯并-三氮唑

Name: Cl-HOBt