TECHNIQUE AND TYPICAL PROCEDURE

TMSBr procedure:

1. Add 1.0 gram of peptide resin (note 1) in a 50 mL 1 neck round bottom flask having a magnetic stir bar.

2. Dry the flask containing the peptide resin over phosphorous pentoxide in a vacuum desiccator for at least 6 hours.

3. Remove the flask containing the peptide resin from the desiccator and assemble the apparatus with an oven dried pressure equalized addition funnel having a gas inlet tube as shown to allow a nitrogen pressure on the apparatus during the cleavage.

4. Place a magnetic stirrer and cooling bath under the apparatus.

5. Add 3.5 mL of thioanisole (note 2) via the addition funnel followed by 25 mL of trifluoroacetic acid (TFA) into the stirred mixture. Caution. Wear gloves.

6. Cool the reaction flask in an ice- water bath (note 3) and start a slow flow of nitrogen gas into the inlet tube at the top of the addition funnel (note 4).

7. Add 4.0 mL of bromotrimethylsilane (TMSBr) to the dropping funnel.

8. Add the TMSBr to the reaction mixture over 1-2 minutes at the ice-water bath temperature (note 5) with rapid stirring. Stir for 10 minutes in the ice bath. For larger scale processing, increase the addition time for the TMSBr to 10-15 minutes.

9. Raise the bath temperature to 25-30 ° C and stir for 60-90 minutes. See added note.

10. Remove the addition funnel and place the flask (note 6) on a rotary evaporator with water bath set at 30-35 ° C.

11. Evacuate and evaporate to remove volatile from the reaction mixture. The thioanisole can not be removed by evaporation because it's boiling point is too high (bp= 188 ° C). The evaporation step is complete when no bubbling is noted in the evacuated flask. A strong vacuum is needed to keep the evaporation time to 10-15 minutes or less. During the evaporation, copious amounts of HBr are evacuated. For this reason, a Teflon diaphragm vacuum pump is a necessity as well as a system (CaO trap) for removing the HBr from the vacuum pump effluent to avoid environmental contamination.

12. The vacuum is released, the flask removed from the evaporator and 35 mL of ethyl ether is added immediately. Stir the mixture vigorously for 30 minutes and add 0.5 g of Celite filter aid (Celatom FW-60, Aldrich).

!3. Stir the mixture vigorously for 30-60 minutes and then collect the solids on a sintered glass filter funnel. Wash the solids several times with ethyl ether.

14. The peptide is extracted from the Celite and spent resin beads with a suitable solvent that depends on the specific peptide used, i.e., aqueous acetic acid, aqueous ammonium acetate or TFA (note 7). The peptide can purified directly (by Chromatography) or freeze dried prior to purification.


Notes

1. The amount of peptide resin and reactants can be varied in any multiples of the proportions given. For large peptides (for > 40 residues), increased amounts of TMSBr and thioanisole should be used. For larger scale processing (> 50 g of peptide resin) the optimum proportions of reagents to peptide resin should be developed to optimize the process. Go Back

2. Dried over Linde molecular sieves # 4A. Go Back

3. For quantities of peptide resin > 5 g, the temperature during the addition should be -10 to -5 ° C for best results. Go Back

4. The nitrogen flow is adjusted to keep a positive pressure on the system to keep out air. There is some belief that the nitrogen flow should not be too rapid as to displace all of the HBr from the reaction as the HBr may be a factor in the process. This remains to be studied. See added note below. Go Back

5. It is important to maintain this temperature range. Some type of a temperature monitor should be used for large scale processing. Go Back

Added note: During the initial work on this process, nitrogen sparging was not used. However the recent publication of Sparrow and Monera, (Ref. 4) called for nitrogen sparging. Work by J. Hughes following Sparrow and Monera's paper calling for sparging resulted in lower yields of cleaved peptide leading us to conclude that an excess of sparging with nitrogen removes a critical amount of HBr from the reaction media.

6. The magnetic stir bar is allowed to remain in the reaction flask during the evaporation and precipitation with ethyl ether. Go Back

7. For large scale operations (>5-10 g of peptide resin), the collected solids are removed from the filter funnel and extracted further in a beaker. The solids are slurried with extracting solvent and collected on a sintered glass funnel for more extraction if necessary. HPLC can be used to monitor the efficiency of the extraction. Go Back


Typical Procedure

The TMSBr cleavage procedure was performed by placing 0.5 g of Leu-Ala-Gly-Val- (oxymethyl)- PAM resin in a 100 mL round bottom flask containing a magnetic stir bar (all glassware was oven dried), 6 mL of TFA (Halocarbon) and 1.6 mL of thioanisole (Aldrich, dried over molecular sieves type 4A). The flask was equipped with a nitrogen inlet tube and was cooled in a dry ice-ethylene glycol bath at -10 ° C. The agitation was started and to the flask was added 1.2 mL of TMSBr (Aldrich). The mixture was stirred at -10 ° C for 10 minutes and then at 25 ° C for 90 minutes. The reaction flask was transferred to a rotary evaporator and volatile reaction components removed under a vacuum (10 to 20 millitorr) at a bath temperature of 30- 35 ° C in 5 minutes. To the flask was added 75 mL of anhydrous ethyl ether (used as obtained from the manufacturer, EM Science) to precipitate the cleaved peptide and resin beads. The mixture was stirred for 30 to 60 minutes and Celite was added followed by filtration and washing the collected solids with ether to remove residual thioanisole. The peptide was extracted from the resin beads and Celite with 10 % acetic acid (2). The solution was freeze-dried to obtain the product as a powder. HPLC analysis was per- formed on this material. The results are shown in Figure 1.

TMSCl procedure:

The TMSCl cleavage was performed by an analogous method except that in this instance 0.5 g of dry lithium bromide (Aldrich, dried in a vacuum over phosphorous pentoxide) was added to the reaction mixture prior to the addition of 1.2 mL of TMSCl (Aldrich) which was added in place of the TMSBr. In addition, the TMSCl was added to the flask while the flask was immersed in a water bath at 25 ° C. The stirred mixture was then heated in the water bath at 50 ° C for 90 min (note: TMSCl boils at 57 ° C). The reaction and work up procedures were identical to those described above. In this case, the ether washing removed the residual thioanisole and the lithium salts. The results are shown in Figure 1.

Chromatogram A .......................................................Chromatogram B

Figure 1. HPLC chromatograms of crude peptide Leu-Ala-Gly-Val cleaved from PAM resin with TMSBr (A) and using TMSCl (B). Chromatograms (A) and (B): isocratic 28 % B, detection at 214 nm. Solvent A: 0.035 M potassium phosphate, pH 5.7. Solvent B: 40 % acetonitrile in 0.035 M potassium phosphate. Column: YMC-Pack ODS-AMQ, 5 mm, 200 Å, 250 x 4.6 mm I. D.

Additional work using TMS bromide for peptide resin deprotection and cleavage has been published recently by Sparrow and Monera. See reference 4.

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Last modified 6/28/2000