Standard Coupling Procedures HEADING_TITLE

Converting CHA and DCHA Salts to Free Acids

For stability considereations and easier handling, some N-protected amino acids are available as cyclohexylamine (CHA) or dicyclohexylamine (DCHA) salts.  Before they can be utilized in carbodiimide coupling, they must be converted from the salt form to the free acid.

  1. Dissolve the DCHA or CHA salt in dichloromethane (DCM).
  2. Extract the DCM solution with ice-cold aqueous KHSO4 solution 3 times.
  3. Dry the organic layer over MgSO4 and filter.
  4. Evaporate the solvent under reduced pressure to obtain the free acid. 

Standard DIC/HOBt Coupling

  1. Remove the N-terminal protecting group by standard deprotection protocols.Suspend the resin in dichloromethane (DCM, 10 mL per gram resin)
  2. Dissolve 5 equivalents (based on resin substitution) in DMF (approximately 1 mL per gram) of amino acid derivative.
  3. Dissolve 5.5 equivalents (based on resin substitution) of HOBt in DMF (minimum volume necessary for complete solution).
  4. Add the amino acid solution and the HOBt solution to the resin suspension.
  5. Add 5.5 equivalents (based on resin substitution) of DIC.
  6. Shake the mixture at room temperature under inert gas. Monitor the reaction using the ninhydrin test.  When the ninhydrin test is negative, filter and wash the resin three times with DMF, three times with DIC, then three times with either methanol or DCM.  If the ninhydrin test is not negative within four hours, repeat the coupling procedure.

Coupling with EDC

  1. Dissolve the N-protected amino acid and the amino acid ester to be coupled in dichloromethane (DCM).
  2. Cool the mixture in an ice bath.
  3. Add 1.2 equivalents of EDC and stir the mixture. 
  4. When the reaction is complete, wash the mixture with water to remove excess EDC and urea by-product.
  5. Dry the organic phase over sodium sulfate, filter, and evaporate to obtain the crude product.

Coupling with BOP Reagent[1]

  1. Remove the N-protecting group using standard deprotection protocols.
  2. Dissolve 2.0 equivalents (based on resin substitution) of the protected amino acid in DMF (5 mL/g of resin) and add to the resin.
  3. Add 2.0 equivalents (based on resin substitution) of 1.0 M BOP solution and 4.0 equivalents (based on resin substitution) of diisopropylethylamine (DIPEA).  2.0 equivalents (based on resin substitution) of 0.5 M HOBt solution in DMF can be added to suppress racemization.
  4. Mix for 10-60 minutes until the Kaiser test is negative.

Coupling with PyBOP[2]

  1. Remove the N-protecting group using standard deprotection protocols.
  2. Dissolve 1.1 equivalents (based on resin substitution) of the protected amino acid in DMF (5 mL/g of resin) and add to the resin.
  3. Add 1.1 equivalents (based on resin substitution) of 1.0 M PyBOP solution and 2.2 equivalents (based on resin substitution) of diisoproplyethylamine (DIPEA).  1.1 equivalents (based on resin substitution) of 0.5 M HOBt solution in DMF can be added to suppress racemization.
  4. Mix for 10-60 minutes until the Kaiser test is negative.

Coupling N-Methyl Amino Acids with Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate[3]

  1. Remove the N-protecting group from the resin peptide using standard procedures.
  2. Suspend the resin in DCM (10 mL/gram resin).
  3. Dissolve 2 equivalents (based on resin substitution) of the protected amino acid in DCM or DMF.  Add the solution to the resin.
  4. Add 2 equivalents (based on resin substitution) of PyBroP®.  Cool the mixture to 0 °C.
  5. Add 6 equivalents of diisopropylethylamine (DIPEA).  Mix 1 minute cold and 1 hour at room temperature.
  6. Filter the resin and wash with DCM.

Coupling with HATU, HBTU, HCTU or TBTU

  1. Remove the N-protecting group using standard deprotection protocols.
  2. Dissolve 2.0 equivalents (based on resin substitution) of the protected amino acid in DMF (5 mL/g of resin) and add to the resin.
  3. Add 2.0 equivalents (based on resin substitution) of 1.0 M HBTU solution and 4.0 equivalents (based on resin substitution) of diisoproplyethylamine (DIPEA).  2.0 equivalents (based on resin substitution) of 0.5 M HOBt solution in DMF can be added to suppress racemization.
  4. Mix for 10-60 minutes until the Kaiser test is negative.
  5. Filter and wash the resin with DMF.

Fast Boc Deprotection and Coupling with in situ Neutralization[4] 

  1. Suspend the resin in 50% (v/v) TFA/dichloromethane (DCM), using 1 mL of TFA/DCM per gram of resin.
  2. Shake the resin at room temperature for 3 minutes.
  3. Filter the resin.
  4. Add a second portion of 50% TFA/DCM.
  5. Shake the resin at room temperature for 5 minutes.
  6. Dissolve 2.0 equivalents (based on resin substitution) of the protected amino acid in DMF (5 mL/g of resin).
  7. Add 2.0 equivalents (based on resin substitution) of 1.0 M HBTU solution and 4.0 equivalents (based on resin substitution) of diisoproplyethylamine (DIPEA).  2.0 equivalents (based on resin substitution) of 0.5 M 6-ClHOBt solution in DMF can be added to suppress racemization.
  8. Mix for 5-10 minutes until the Kaiser test is negative.
  9. Filter and wash the resin with DMF.

Coupling with TSTU in Aqueous Solvent Mixtures[5] 

  1. Dissolve the acid in a 2:2:1 mixture of DMF/dioxane/water.
  2. Add 3 equivalents of diisopropylethylamine and 1.3 equivalents of TSTU.
  3. After the formation of the -OSu ester is complete, add 1.5 equivalents of the amine.
  4. After the reaction is complete, the solvents are removed and the crude product is isolated.

Fragment Coupling with TDBTU[6]

  1. Dissolve the peptide ester in DMF (N,N-dimethylformamide) (approximately 16 ml per mmole of peptide ester).  Add 1.25 equivalents of the N-protected peptide fragment.
  2. Cool the solution to -25˚ C.
  3. Add 2.25 equivalents of DIEA (diisopropylethylamine). 
  4. Add 1.05 equivalents of TDBTU.
  5. Stir at –25˚ C for one hour.
  6. Stir overnight at room temperature
  7. Pour the mixture into water (10 times the volume of DMF) containing 8% w/w sodum carbonate.
  8. Stir for 30 minutes
  9. Collect the solid by filtration, wash with water, and dry in vacuo to obtain the crude product.

Solid Phase Coupling with DEPBT [7]

  1. Suspend the resin in dichloromethane (DCM) or N,N-dimethylformamide (DMF) (approximately 10 ml per gram of resin).  Add 1.5 equivalents (based on resin substitution) of the protected amino acid.
  2. Add 3 equivalents of DIPEA or Et3N.
  3. Add 1.5 equivalents of DEPBT and shake the mixture at room temperature for 1-2 hours.
  4. Filter the resin.
  5. Wash the resin 3 times with DMF then 3 times with DCM.

Solution Phase Coupling with DEPBT [8]

  1. Dissolve the protected amino acid and amino acid ester in THF (tetrahydrofuran) (approximately 2 ml per mmole of amino acid ester).  Add 1.1 –1.2 equivalents of DEPBT and 2.0 equivalents of triethylamine (Et3N) or diisopropylethylamine (DIPEA).  If the coupling involves amino acids highly susceptible to epimerization, cool the mixture to 0˚ C before adding the DEPBT and use sodium carbonate in place of triethylamine.
  2. Stir for 1 to 2 hours.
  3. Filter to remove any solid.  Rinse solid with a little THF. 
  4. Evaporate in vacuo to obtain the crude peptide.

Cyclization with DEPBT 

  1. Prepare a 1x10-3 M to 2x10-3 M solution of the linear peptide in DMF.
  2. Cool the solution in an ice bath and add 4 equivalents of Et3N and 2 equivalents of DEPBT.
  3. Stir 30 minutes at 0˚ C, then warm to room temperature and stir for 24 hours.
  4. Evaporate the DMF in vacuo
  5. Purify the residue by column chromatography to obtain the crude peptide.

 


[1] Rovero, P.; Quartara, L.; Fabbri, G. Tetrahedron Lett. 1991, 32, 2639-2642.

[2] Based on the procedure in Hoeg-Jensen, T.; Jakobsen, M. H.; Holm, A. Tetrahedron Lett. 1991, 32, 6387-6390.

[3] Based on procedure of Coste, J.; Frérot, E.; Jouin, P.; Castro, B. Tettrhedron Lett. 1991, 32, 1967-1970.

[4] Alewood, D.; Kent, S.B.H.; et al. Int. J. Peptide Protein Res., 1992, 40, 180-193.

[5] Bannwarth, W.; Knorr, R. Tetrahedron Lett. 1991, 32, 1157-1160.

[6] Hiebl, J; et al. J. Peptide Res., 1999, 54, 54-65.

[7] Tang, Y.-C.; Xie, H.-B.;Tian, G.-L.; Ye, Y.-H. J. Peptide Res. 2002, 60, 95-103.

[8] Boger, D.L.; Kim, S.H.; Miyazaki, S.; Strittmatter, H.; Weng, J.-H.; Mori, Y.; Rogel, O.; Castle, S.L.; McAtee, J.J. J. Am. Chem. Soc. 2000, 122, 7416-7417.