Tips for Peptide Synthesis

Reconstitution and Storage of Peptides
Peptides are usually supplied as a fluffy, freeze-dried material in serum vials. Store peptides in a freezer after they have been received. In order to reconstitute the peptide, distilled water or a buffer solution should be utilized. Some peptides have low solubility in water and must be dissolved in other solvents such as 10% acetic acid for a positively charged peptide or 10% ammonium bicarbonate solution for a negatively charged peptide. Other solvents that can be used for dissolving peptides are acetonitrile, DMSO, DMF, or isopropanol. Use the minimal amount of these non-aqueous solvents and add water or buffer to make up the desired volume. After peptides are reconstituted, they should be used as soon as possible to avoid degradation in solution. Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20°C. Repeated thawing and refreezing should be avoided.
Methods to Dissolve Peptides
The way to dissolve a peptide is to use water. For peptides that are not soluble in water, use the following procedure:

1. For acidic peptides, use a small amount of base such as 10% ammonium bicarbonate to dissolve the peptide, dilute with water to the desired concentration. Do not use base for cysteine-containing peptides.
2. For basic peptides, use a small amount of 30% acetic acid, dilute with water to the desired concentration.
3. For a very hydrophobic peptide, try dissolving the peptide in a very small amount of DMSO, dilute with water to the desired concentration.
4. For peptides that tend to aggregate (usually peptides containing cysteines), add 6 M urea, 6 M urea with 20% acetic acid, or 6 M guanidine•HCl to the peptide, then proceed with the necessary dilutions.

Preparation of HBTU/HOBt Solution for the Peptide Synthesizer

1. Preparation of 0.5 M HOBt in DMF:
• Weigh 13.5 g anhydrous HOBt (0.1 mol, MW 135.1) into a 250 mL graduated cylinder.
• Add DMF until the 200 mL level is reached.
2. Preparation of 0.45 M HBTU/HOBt solution:
• Add the solution prepared in step 1 to 37.9 g HBTU (0.1 mol, MW 379.3) contained in a beaker or an Erlenmayer flask.
3. Stir for about 15 min with a magnetic stirring bar until HBTU is dissolved.
4. Filter the solution through a fine pore size sintered glass funnel.
5. Pour the filtered solution into an appropriate bottle for attachment to a peptide synthesizer.

* This solution is stable at room temperature for at least six weeks.
Biotinylation of Amino Group

1. Wash 0.1 mmol resin with DMF.
2. Dissolve 0.244 g (+)-biotin (1 mmol, MW 244.3) in 5 mL DMF-DMSO (1:1) solution. A little warming is necessary.
3. Add 2.1 mL 0.45 M HBTU/HOBt solution and 0.3 mL DIEA to the solution prepared in step 2.
4. Add the activated biotin solution to the resin and let stir overnight.
5. Check resin to make sure coupling is complete as evidenced by negative ninhydrin test (colorless).
6. Wash resin with DMF-DMSO (1:1) (2x) to remove excess (+)-biotin.
7. Wash resin with DMF (2x) and DCM (2x).
8. Let the resin dry before proceeding to cleavage.

Procedure for Loading Fmoc-Amino Acid to 2-Chlorotrityl Chloride Resin

1. Weigh 10 g 2-chlorotrityl chloride resin (15 mmol) in a reaction vessel, wash with DMF (2x), swell the resin in 50 mL DMF for 10 min, drain vessel.
2. Weigh 10 mmol Fmoc-amino acid in a test tube, dissolve Fmoc-amino acid in 40 mL DMF, transfer the solution into the reaction vessel above, add 8.7 mL DIEA (50 mmol), swirl mixture for 30 min at room temperature.
3. Add 5 mL methanol into the reaction vessel and swirl for 5 min.
4. Drain and wash with DMF (5x).
5. Check substitution.
6. Add 50 mL 20% piperidine to remove the Fmoc group. Swirl mixture for 30 min.
7. Wash with DMF (5x), DCM (2x), put resin on tissue paper over a foam pad and let dry at room temperature overnight under the hood. Cover the resin with another piece of tissue paper, press lightly to break aggregates.
8. Weigh loaded resin.
9. Pack in appropriate container.

Procedure for Checking Substitution of Fmoc-Amino Acid Loaded Resins

1. Weigh duplicate samples of 5 to 10 mg loaded resin in an eppendorf tube, add 1.00 mL 20% piperidine/DMF, shake for 20 min, centrifuge down the resin.
2. Transfer 100 µL of the above solution into a tube containing 10 mL DMF, mix well.
3. Pipette 2 mL DMF into each of the two cells (reference cell and sample cell), set spectrophotometer to zero. Empty the sample cell, transfer 2 mL of the solution from step 2 into the sample cell, check absorbance.
4. Subs = 101(A)/7.8(w)
   A = absorbance
   w = mg of resin
5. Check absorbance three times at 301 nm, calculate average substitution.

Manual Fmoc Synthesis (0.25 mmol)

1. Wash resin with DMF (4x) and then drain completely.
2. Add approximately 10 mL 20% piperidine/DMF to resin. Shake for one min and drain.
3. Add another 10 mL 20% piperidine/DMF. Shake for 30 min.
4. Drain reaction vessel and wash resin with DMF (4x). Make sure there is no piperidine remaining. Check beads using ninhydrin test, beads should be blue.
5. Coupling Step - Prepare the following solution:
   1 mmol Fmoc-amino acid
   2.1 mL 0.45 M HBTU/HOBT (1mmol)
   348 µL DIEA (2 mmol)
   Add above solution to the resin and shake for a less of 30 min. This coupling step can be longer if desired.
6. Drain reaction vessel and wash resin with DMF (4x).
7. Perform Ninhydrin test:
• If negative (colorless), proceed to step 2 and continue synthesis.
• If positive (blue), return to step 5 and re-couple the same Fmoc-amino acid. Increase the coupling time if necessary.

Synthesis of Phosphotyrosine-Containing Peptides Using Fmoc-Phosphotyrosine
Reagent: N-a-Fmoc-O-phosphotyrosine

1. For 0.1 mmol or 0.25 mmol synthesis, use 0.483 g Fmoc-Tyr(PO3H2)-OH (1 mmol, MW 483.4) . For ABI synthesizers, pack Fmoc-Tyr(PO3H2)-OH in a cartridge.
2. The cycle program for coupling Fmoc-Tyr (PO3H2)-OH is the same as for other Fmoc-amino acids except for the coupling time (see step 3). (Note: ABI synthesizers use HBTU/HOBT as the activating reagent.)
3. The coupling time for Fmoc-Tyr(PO3H2)-OH needs to be increased. For ABI model 430A peptide synthesizer, insert several steps (i.e., vortex on, wait 990 sec, vortex off, to increase the coupling time). For ABI model 431A peptide synthesizer, add additional "I"s. Overnight coupling may be necessary for some sequences.
4. After the coupling step for Fmoc-Tyr(PO3H2)-OH, perform ninhydrin test to ensure complete coupling. Negative (colorless) ninhydrin test indicates complete coupling, while a positive (blue) ninhydrin test indicates incomplete coupling.
5. Increase the coupling time of the amino acid residues after the phosphotyrosine or perform double coupling. (Note: The coupling of amino acids after the phosphotyrosine can be difficult.)
6. There is a limit on the number of amino acid residues that can be coupled after the hosphotyrosine. Since the phospho group is unprotected, side reactions are likely to ccur. (Note: Peptides have been successfully coupled with sequences containing up o ten additional amino acids following the phosphotyrosine residue.)

Simultaneous Synthesis of Peptides Which Differ in the C-Termini Using 2-Chlorotrityl Resin and Wang Resin*
Peptides which differ in the C-termini can be simultaneously synthesized in one reaction vessel by employing resins that possess different cleavage properties. The resins used were the weak acid labile 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 mL 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 Removing Mtt group from Fmoc-Lys(Mtt) on Solid Phase
Reagent:
Fmoc-Lys(Mtt)-OH

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 FITC Labeling of Peptides
Reagents:
FITC
Fmoc-e-Ahx-OH

1. Couple Fmoc-e-Ahx-OH to the amino terminal of the peptide-resin using standard coupling conditions.
2. "De-Fmoc" with piperidine using the standard 20% piperidine procedure.
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).

Procedure for Fluorescein Labeling of Peptides
Reagent:
5-carboxyfluorescein (5-FAM)
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.