Attaching the First Amino Acid to a Resin HEADING_TITLE

The following are general procedures.  Although these procedures generally produce good results, they may not be applicable to all resins or substrates.

Storing and Using Amino Acid Solutions

Dissolved amino acids have a finite life span and will need to be replaced if they are not used soon after preparation. 

Amino acid solutions are typically made up 0.5 M in an HOBt solution that is itself 0.5 M. This preparation can typically sit open to air at room temperature throughout the course of a synthesis. Unused solution can be refrigerated for weeks awaiting later use. When the solution becomes discolored it should be discarded.

Certain amino acids are particularly prone to oxidation and should not be kept longer than a week in solution. These include cysteine, methionine, tryptophan, and histidine.

Merrifield Resin

Cesium Salt Method (Gisin Method)[1]

  1. Dissolve the carboxylic acid in methanol (5 mL/mmol) and add water (0.5 mL/mmol).  Titrate the solution to pH 7.0 with a 20% aqueous solution of cesium carbonate.  Evaporate the mixture to dryness.  Add DMF (2.5 mL/mmol) and evaporate to dryness (45°C).  Add a second portion of DMF (2.5 mL/mmol) and evaporate to dryness (45°C).
  2. Set up a flask with a heating mantle and thermometer on an orbital shaker.
  3. Swell the resin in DMF (6-8 mL per gram of resin).  Add the dry carboxylic acid cesium salt (1.0 equivalent based on the chlorine substitution of the resin).  The cesium salt must be completely dry to obtain satisfactory results.
  4. Shake the mixture at 50°C for 24 hrs.
  5. Filter the resin.  Wash the resin thoroughly with DMF, then 50% (v/v) aqueous DMF, then 50% (v/v) aqueous methanol, and finally methanol.  Dry the resin in vacuo to a constant weight.

 

Potassium Fluoride Method[2]

  1. Set up a flask with a heating mantle and thermometer on an orbital shaker.
  2. Dissolve the Boc-amino acid (1.5 equivalents based on the chlorine substitution of the Merrifield resin) in DMF (6 mL/g resin) and add it to the flask.
  3. Add the Merrifield resin (1 equivalent) and anhydrous potassium fluoride (3 equivalents based on the chlorine substitution of the Merrifield resin).
  4. Shake the mixture at 50°C for 24 hrs.
  5. Filter the resin.  Wash the resin thoroughly with DMF, then 50% (v/v) aqueous DMF, then 50% (v/v) aqueous methanol, and finally methanol.  Dry the resin in vacuo to a constant weight.

 

2-Chlorotrityl Chloride Resin

Attachment of Amino Acids to Trityl Chloride Resins[3]

  1. Use 1.0 equivalent of the protected amino acid.  If a lower substitution resin is required, reduce the amount of acid.  Dissolve the amino acid in DCM (approximately 10 mL per gram of resin).  If the acid does not dissolve in completely, add a small amount of DMF.
  2. Add the amino acid solution to the resin.  Add 1.0 equivalent (respective to the acid) of DIPEA.  Agitate the mixture with a shaker for 5 minutes, then add 1.5 equivalents (respective to the acid) of DIPEA.  Agitate the mixture vigorously for 30 to 60 minutes. 
  3. To endcap any remaining reactive trityl groups, add HPLC grade methanol, 0.8 mL per gram of resin, and mix for 15 minutes.  Filter the resin and wash it three times with DCM, using approximately 10 mL per gram of resin.  Wash the resin twice with DMF, twice with DCM, and three times with methanol.  Dry the resin in vacuo.  The substitution of the resin can be estimated from the weight gain.

 

Attachment of Alcohols and Phenols to Trityl Chloride Resins[4]

  1. Dissolve 2 equivalents (relative to the resin substitution) of the alcohol or phenol in pyridine (8 mL per gram of resin).
  2. Add the resin to this solution and mix at room temperature.  Reaction may take up to two days.
  3. Filter the resin and wash it 4 times DCM.  Wash the resin 4 times with dry ether.
  4. Dry in vacuo to a constant weight.  The substitution of the resulting resin can be estimated from the weight gain of the resin.

 

Activation of Trityl Alcohol Resins

 2-Chlorotrityl chloride resin can react with atmospheric moisture and convert to inactive 2-chlorotrityl alcohol resin.  This will occur is the resin is stored for a long period of time or is not stored in a tightly sealed container.  The resin can be reactived using this proceedure. 

  1. Suspend the resin in DCM (approximately 10 mL per gram of resin).
  2. Cool the suspension in an ice bath.
  3. Add 1.2 equivalents of thionyl chloride (SO2Cl2) and 2.4 equivalents of pyridine.  Caution: Thionyl chloride is corrosive and reacts with moisture to release HCl.  Wear safety goggles and chemical resistant gloves.  Perform this reaction in an efficient fume hood.
  4. Heat at reflux for 2 to 4 hours.
  5. Filter the resin.  Wash it 6 times with DCM.  Dry the resin in vacuo over KOH.

 

Wang Resin, PAM Resin, HMBA Resin, Hydroxymethyl Polystyrene

Attachment of Carboxylic Acids to Hydroxy-Substituted Resins (Wang, PAM, Hydroxymethyl polystyrene, HMBA)

  1. In a round bottom flask suspend the resin in 9:1 v/v CH2Cl2/DMF (approximately 15 mL per gram of resin). 
  2. In a separate flask dissolve 1.5 to 2.5 equivalents (relative to the resin) of the carboxylic acid in a minimum amount of DMF.  Add the same equivalency of HOBt.  Stir the mixture until the HOBt dissolves.  If the HOBt doesn’t dissolve completely, add DMF to bring it into solution.  Add this solution to the resin.
  3. In a separate flask dissolve 0.1 equivalent (relative to the resin) of DMAP in a minimum amount of DMF.
  4. Add 1.0 equivalent (relative to the carboxylic acid) of DIC to the resin mixture then add the DMAP solution.  Equip the flask with a drying tube.
  5. Agitate the mixture with a mechanical shaker for 2 to 3 hours at room temperature.  Add 2 equivalents (relative to the resin) of acetic anhydride and pyridine to the reaction flask and mix an additional 30 minutes at room temperature to end cap any unreacted hydroxyl groups on the resin.   
  6. Filter the resin in a fine porosity sintered glass funnel and wash it 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol.  In each wash use enough solvent to slurry the resin.  After the final methanol wash, dry the resin in vacuo to a constant weight.

 

Mitsunobu Coupling to Hydroxy-Substituted Resins[5] 

  1. Dissolve the acid or phenol (5 equivalents based on resin substitution) in THF (50 mL/g resin).  Add the resin and flush the flask with argon.
  2. Add triphenylphosphine (10 equivalents based on resin substitution).
  3. Add 10 equivalents (based on resin substitution) of a 2M solution of diethyldiazodicarboxylate in THF dropwise at 5 ºC.  
  4. Shake the mixture with a mechanical shaker for 4 hours at room temperature.  Filter the resin and wash it with THF, MeOH, and Et2O.  Dry the resin under vacuum to a constant weight.  The coupling can be repeated to improve coupling yield.

 

Attachment of Fmoc-Amino Acids to Rink Amide or Sieber Amide Resins

  1. In a round bottom flask suspend the resin in 20% v/v piperidine/DMF (approximately 15 mL per gram of resin).
  2. In a separate flask dissolve 1.5 to 2.5 equivalents (relative to the resin) of the Fmoc-amino acid in a minimum amount of DMF.  Add the same equivalency of HOBt.  Stir the mixture until the HOBt dissolves.  If the HOBt doesn’t dissolve completely, add DMF to bring it into solution. 
  3. Add 1.0 equivalent (relative to the amino acid) of DIC to the Fmoc-amino acid/HOBt mixture.  Equip the flask with a drying tube.  Let the mixture stand at room temperature for 10 minutes.
  4. Add the activated amino acid solution to the resin and equip the flask with a drying tube.  Agitate the mixture with a mechanical shaker for 2 to 3 hours at room temperature.
  5. Remove a small sample of the resin and wash it with DCM.  Test for free amino groups using the Kaiser test.  If there are free amino groups, add 1 equivalent of acetic anhydride and pyridine to the reaction flask and mix for 30 minutes. 
  6. Filter the resin in a fine porosity sintered glass funnel and wash it 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol.  In each wash use enough solvent to slurry the resin.  After the final methanol wash, dry the resin in vacuo to a constant weight.  The substitution of the resin can be estimated from the weight gain of the resin.

 

Attachment of Boc-Amino Acids to MBHA Resins

  1. In a round bottom flask suspend the resin in 10% v/v DIPEA/DMF (approximately 15 mL per gram of resin).  Agitate the suspension with a mechanical shaker for 5 minutes. Filter the resin.
  2. Wash the resin with DMF (10 ml of DMF per gram of resin) and filter.  Repeat 2 more times.
  3. In a separate flask dissolve 1.5 to 2.5 equivalents (relative to the resin) of the Boc-amino acid in a minimum amount of DMF.  Add the same equivalency of HOBt.  Stir the mixture until the HOBt dissolves.  If the HOBt doesn’t dissolve completely, add DMF to bring it into solution. 
  4. Add 1.0 equivalent (relative to the amino acid) of DIC to the Boc-amino acid/HOBt mixture.  Equip the flask with a drying tube.  Let the mixture stand at room temperature for 10 minutes.
  5. Add the activated amino acid solution to the resin and equip the flask with a drying tube.  Agitate the mixture with a mechanical shaker for 2 to 3 hours at room temperature.
  6. Remove a small sample of the resin and wash it with DCM.  Test for free amino groups using the Kaiser test.  If there are free amino groups, add 1 equivalent of acetic anhydride and pyridine to the reaction flask and mix for 30 minutes. 
  7. Filter the resin in a fine porosity sintered glass funnel and wash it 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol.  In each wash use enough solvent to slurry the resin.  After the final methanol wash, dry the resin in vacuo to a constant weight.  The substitution of the resin can be estimated from the weight gain of the resin.

 

Attachment of Amines to 2-Chlorotrityl Chloride Resin

  1. Dissolve 2 to 4 equivalents (relative to the resin substitution) of the amine in dry THF (10 to 12 mL per gram of resin).
  2. Add the trityl chloride resin to the solution. Mix with a mechanical shaker for 2 hours.
  3. Filter the resin and wash it 3 times with 17:2:1 DCM/MeOH/DIPEA (v/v/v).  Wash the resin 3 times with DCM.
  4. Dry in vacuo to a constant weight.  The substitution of the resulting resin may be estimated from the weight gained by the resin.

 

Attaching Alcohols to DHP Resin[6]

  1. Dissolve the alcohol (approximately 5 to 10 equivalents based on the resin substitution) in dichloromethane (DCM, approximately 10 ml per gram of resin).
  2. Add the alcohol solution to the resin.
  3. Add catalytic pyridinium p-toluenesulfonate (approximately 0.1 to 0.5 equiv. based on resin substitution).
  4. Shake the mixture at 40-45 ˚C for 20 hours.
  5. Filter and was the resin with DCM.
  6. Dry the resin in vacuo to a constant weight. 

 

Alcohol Coupling to Carboxypolystyrene

Carbodiimide Activation

  1. In a round bottom flask suspend the resin in DMF (approximately 15 mL per gram of resin). 
  2. In a separate flask dissolve 1.5 to 2.5 equivalents (relative to the resin) of the compound to be attached in a minimum amount of DMF.  Add the same equivalency of HOBt.  Stir the mixture until the HOBt dissolves.  If the HOBt doesn’t dissolve completely, add just enough DMF to bring it into solution.  Add this solution to the resin.
  3. In a separate flask dissolve 0.1 equivalent (relative to the resin) of DMAP in a minimum amount of DMF.
  4. Add 1.0 equivalent (relative to the amino acid) of DIC to the resin mixture then add the DMAP solution.  Equip the flask with a drying tube.
  5. Agitate the mixture with a mechanical shaker for 4 to 6 hours at room temperature.   
  6. Filter the resin in a fine porosity sintered glass funnel and wash it 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol.  In each wash use enough solvent to slurry the resin.  After the final methanol wash, dry the resin in vacuo to a constant weight.  The substitution of the resin can be estimated from the weight gain of the resin.

Oxalyl Chloride/Thionyl Chloride Activation[7]

  1. Suspend the resin in dry benzene (15 - 20 mL/g resin).
  2. Add oxalyl chloride or thionyl chloride (4.0 equivalents based on resin substitution).
  3. Heat the mixture at reflux for 12 hours.
  4. Filter the resin and wash it with dry benzene under nitrogen.  Dry the resin in vacuo to a constant weight.
  5. Suspend the resin in DCM (10 mL/g resin).  Add 1.5 equivalents (based on starting resin substitution) of the alcohol, phenol, or amine to be attached.  Add 1.5 equivalents (based on starting resin substitution) of triethylamine and 0.1 equivalent of DMAP. 
  6. Attach a drying tube to the flask and shake the mixture with a mechanical shaker at room temperature for 6 to 48 hours.  The progress of the reaction can be monitored by the disappearance of the acid chloride C=O stretching band in the IR spectrum.
  7. When the reaction is complete, filter the resin and wash it with DCM, then with 50% (v/v) DCM/methanol, and finally with methanol.  Dry the resin in vacuo to a constant weight.  The resin substitution can be approximated from the weight gain of the resin.

 

Alkylation with Primary Alkyl Halides[8]

  1. Suspend the resin in N-methylpyrrolidone (NMP, 10 mL/g resin).
  2. Add cesium carbonate (2.5 equivalents based on resin substitution), potassium iodide (0.1 equivalents based on resin substitution) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN, 0.1 equivalents based on resin substitution).
  3. Add the alkyl halide (4.0 equivalents based on resin substitution).
  4. Shake the mixture at 70 °C for 72 hours.
  5. Filter the resin, then wash it with NMP, 1:1v/v NMP/water, NMP, DCM and methanol.
  6. Dry the resin in vacuo to a constant weight.

 

Measuring Substitution of Fmoc–Amino Acid Resins[9] 

  1. Accurately weigh approximately 10 mg of resin into a 2 mL microcentrifuge tube.
  2. Add 1 mL of 20% (v/v) piperidine in DMF.
  3. Vortex briefly and agitate 1 hour at room temperature on a rotary shaker.
  4. Vortex the tube and allow the resin to settle for 2 minutes.
  5. Dilute 50 microliters of the supernate to 5 mL with DMF (dilution factor = 0.01).
  6. Prepare a blank by diluting 50 microliters of 20% (v/v) piperidine to 5 mL with DMF.
  7. Measure the absorbance of the sample versus the blank at 278 nm.
  8. Calculate the absorbance using the following formula:

S(mmol/g)=(1000·A)/(M·7800·D)

            S=substitution of the resin in mmol/g

            A=absorbance of the sample-absorbance of the blank

            M=mass of the resin used (in milligrams)

            D=dilution factor

 


[1] Geison, B. F.; Helv. Cheim. Acta, 1973, 56, 1476-1482.

[2] Based on procedures in Yajima, H.; Fujii, N.; Funokoshi, S.; Watanabe, T.; Murayama,E.; Otaka, A. Tetrahedron 1988, 44, 805-819.

[3] Based on Barlos, K.; et al. Tetrahedron Lett. 1989, 30, 3943-3946.

[4] Fyles, T. M.; Leznoff, C. C. Can. J. Chem. 1976, 54, 935-942.

[5] Fancelli, D.; Fagnola, M. C.; Severino, D.; Bedeschi, A. Tetrahedron Lett. 1997, 38, 2311-2314.

[6] Based on procedures in Thomson, L.A.; Ellman, J.A. Tetrahedron Lett. 1994, 35, 9333 and Nugel, D.A., et al, J. Org. Chem. 1997, 62, 201.

[7] Based on the procedure in Panek, J.S.; Zhu, B. Tetrahedron Lett. 1996, 37, 8151-8154.

[8] Veerman, J.J.N.; et al. Eur.J.Org.Chem. 1998, 2583-2589.

[9] Newcomb, WB; Deegan, TL; Miller, M; Porco, JA Jr. Biotech. Bioeng. (Comb. Chem.), 1998, 61, 55-60.