Amino Acid Sidechain Deprotection HEADING_TITLE

Deprotection of Arg(Mtr) in Fmoc Peptide Synthesis[1] 

  1. If necessary, remove the Fmoc group and cleave the peptide from the resin by standard protocols.
  2. Dissolve the peptide in 5% (w/w) phenol/TFA (approximately 10 mmol/mL).
  3. Monitor the cleavage of the Mtr group by HPLC.
  4. After the cleavage is complete (approximately 7.5 hours) evaporate the solution to dryness.
  5. Partition the residue between water and dichloromethane.
  6. Wash the aqueous layer with dichloromethane (4 times).
  7. Lyophilize the aqueous layer to obtain the crude peptide.

Deprotection of Arg(Pmc) and Arg(Pbf)

This procedure will also remove tert-butyl based protecting groups and trityl based groups.

  1. Dissolve the peptide in 95% TFA/water (v/v, approximately 5 to 10 mL per gram of protected peptide) containing scavengers appropriate for the amino acid composition of the peptide.
  2. Stir the mixture at room temperature for 20 to 30 minutes.  If the peptide contains multiple Arg residues, deprotection may take longer.
  3. Slowly add ice-cold ether, methyl tert-butyl ether, or diisopropyl ether (approximately ten times the volume of TFA).
  4. Filter the precipitate and wash it with a little cold ether to obtain the crude product.

Deprotection of Cys(Acm)

The S-acetamidomethyl group can be removed using mercury (II) acetate or silver (I) tetrafluoroborate.  This group can also be removed with simultaneous oxidation to disulfides using iodine.

Removal with Mercury(II) Acetate[2] 

S-t-Butyl and S-trityl protecting groups may be removed under these conditions.

  1. Dissolve the protected peptide in water or 10% (v/v) aqueous acetic acid (100 mL/mg peptide) and carefully adjust the pH to 4.0 with glacial acetic acid.
  2. Add, with stirring, 1.0 equivalent of mercury (II) acetate per S-acetamidomethyl group in the peptide. 
  3. Readjust the pH of the solution to 4.0 with acetic acid or aqueous ammonia.  Stir the mixture at room temperature for 1 hour under an inert atmosphere.
  4. Add beta-mercaptoethanol (0.5 mL per 100 mmol of peptide) and let the mixture stand at room temperature for 5 hours.
  5. Centrifuge the mixture to remove the precipitate.  Desalt the supernatant containing the crude peptide under an inert atmosphere then lyophilize.

Removal with Ag(I) Salts[3]

S-Trityl and S-p-methoxybenzyl groups may be partially removed by this procedure.

  1. Dissolve the protected peptide in cold (4 ºC) trifluoroacetic acid (200 mL/ mmole peptide).  Add anisole (4 mL/mmol peptide) to the solution.
  2. Add 20 equivalents of silver salt (AgOTf or AgBF4) per S-acetamidomethyl group.
  3. Stir the mixture at 4 ºC for 1.5 hours, then add cold ether to precipitate the peptide silver salt.  
  4. Centrifuge to isolate the precipitated peptide silver salt.  Suspend the precipitate in 1 M aqueous acetic acid. Add dithiothreitol (40 equivalents per acetamidomethyl group) and mix at room temperature for 3 to 4 hours.
  5. Centrifuge to remove solids.  Desalt the supernatant under an inert atmosphere.  Lyophilize the desalted supernatant to obtain the crude peptide.

Iodine Oxidation[4]

S-Trityl protecting groups may also be removed under these conditions.

  1. Dissolve the protected peptide in methanol (1.25 mL/ mmol).
  2. Add 0.4 M methanolic iodine solution (2.5 equivalents per acetamidomethyl group).  Stir vigorously for 30 minutes.
  3. Add 1 M aqueous ascorbic acid or sodium thiosulfate solution (100 mL/mmol peptide).  Evaporate under reduced pressure to approximately one third of the original volume.
  4. Desalt and lyophilize the crude peptide.

Deprotection of Cys(tBu)

The S-t-butyl group is stable to trifluoroacetic acid, so it can be used with either Boc or Fmoc chemistries.  It can be removed with trifluoromethanesulfonic acid (TFMSA) or mercury (II) acetate.  The TFMSA method is usually used with Boc chemistry to simultaneously cleave the peptide from the resin and remove the S-t-butyl group from cysteine.

Standard Trifluoromethanesulfonic Acid Procedure

  1. If the peptide contains His(Dnp), remove the Dnp group.  If the peptide contains Trp(CHO), remove the N-terminal BOC group then remove the formyl group.
  2. Check that the peptide-resin has been washed and thoroughly dried.
  3. Transfer the resin into a round bottom flask equipped with a stirring bar.  For every 100 mg of peptide-resin add 200 mL of thioanisole and 100 mL of ethandithiol.   Cool the flask in an ice bath and add 2 mL of TFA for every 100 mg of resin.  Stir for 5 to 10 minutes.
  4. For every 100 mg of resin slowly add 200 mL of TMSFA dropwise. Stir vigorously during addition of the TFMSA to dissipate the heat generated.
  5. Let the mixture stir at room temperature for 30 to 60 minutes.
  6. Filter the resin with a fine sintered funnel.  Wash the resin with a small amount of TFA. Combine the filtrates and add 8-10 times the volume of cold ether.  If necessary, keep the mixture at 4°C overnight to precipitate the peptide.  Filter the peptide using a fine sintered glass funnel.  Wash the crude peptide with cold ether to remove cleavage scavengers.
  7. Desalt the peptide by ion exchange column. 

Removal with Mercury(II) Acetate[5]

S-t-Butyl and S-trityl protecting groups may be removed under these conditions.

  1. Dissolve the protected peptide in water or 10% (v/v) aqueous acetic acid (100 mL/mg peptide) and carefully adjust the pH to 4.0 with glacial acetic acid.
  2. Add, with stirring, 1.0 equivalent of mercury (II) acetate per S-acetamidomethyl group in the peptide. 
  3. Readjust the pH of the solution to 4.0 with acetic acid or aqueous ammonia.  Stir the mixture at room temperature for 1 hour under an inert atmosphere.
  4. Add beta-mercaptoethanol (0.5 mL per 100 mmol of peptide) and let the mixture stand at room temperature for 5 hours.
  5. Centrifuge the mixture to remove the precipitate.  Desalt the supernatant containing the crude peptide under an inert atmosphere.

Deprotection of Cys(Trt) with S-S Bond Formation[6]

  1. Dissolve the protected peptide in dichloromethane (DCM) (1 mL/mmol peptide).
  2. Add a 0.1 M solution of iodine in DCM (22 mL/ mmol peptide).  Stir 5 minutes at room temperature.
  3. Add 0.2 M citrate buffer containing ascorbic acid (5 mg/ mL) (Add 100 mL of buffer per mmol peptide).
  4. Isolate the peptide by chromatography on a Sephadex column.

Deprotection of His(Dnp)[7]

  1. Suspend the peptide resin in DMF (10 mL/ g of resin).
  2. Add thiophenol and triethylamine (2 mL of each/ g of resin).
  3. Shake the mixture with a mechanical shaker at room temperature for approximately 90 minutes.
  4. Filter the resin and wash it twice with DMF, twice with DCM, and twice with methanol.
  5. Dry the resin in vacuo to a constant weight.

Removal of Allyl Based Protecting Groups[8]

  1. Swell the substrate-resin in chloroform (CHCl3).
  2. Suspend the swollen resin in CHCl3 (approximately 35 mL per gram of resin).
  3. Add acetic acid (0.5 mL per gram of resin), N-methylmorpholine (2 mL per gram of resin), and Pd(PPh3)4 (3 equivalents based on resin substitution).
  4. Shake the mixture at room temperature for 4 hours.
  5. Filter the resin and resuspend it in CHCl3 (approximately 35 mL per gram of resin).
  6. Add acetic acid (0.5 mL per gram of resin), N-methylmorpholine (2 mL per gram of resin), and Pd(PPh3)4 (3 equivalents based on resin substitution).
  7. Shake the mixture at room temperature for 12 hours.  Filter and wash the deprotected resin product with dichloromethane (DCM).

Removal of Dde and ivDde Groups (Hydrazine Method)[9]

  1. If necessary, replace the N-terminal Fmoc group with Boc.
  2. Prepare a 2% (w/v) solution of hydrazine monohydrate in DMF (25 ml/g of peptide-resin).
  3. Add the hydrazine solution to the flask containing the peptide-resin.
  4. Stopper the flask and allow the mixure to stand t room temperature for 3 minutes.
  5. Filter the resin and repeat the hydrazine treatment twice.
  6. Wash the resin with DMF.

The peptide-resin is ready for further elaboration.

Removal of Dde Groups (Hydroxylamine Hydrochloride/Imidazole Method)[10]

  1. Dissolve hydroxylamine hydrochloride (1 equiv. based on Dde content of the peptide-resin) and imidazole (0.75 equiv. based on Dde content of the peptide-resin) in N-methylpyrrolidone (NMP) (approximately 10 mL per gram of peptide resin).
  2. Add the solution to the resin.
  3. Gently shake the mixture at room temperature for 30 min. to 1 hour.
  4. Filter the resin and wash three times with DMF.

The peptide-resin is ready for further elaboration.

Selective Deprotection of Lys(Mtt)

Cleavage With TFA/TIS/DCM[11]

  1. Suspend the resin in TFA/TIS/DCM (1:2:97 v:v:v) (approximately 10 mL per gram of resin).
  2. Gently shake at room temperature for 30 minutes.  Remove a few beads and add 1-2 drops of TFA.  If the beads turn orange immediately, continue shaking for another 30 minutes and retest.
  3. Filter the resin and wash twice with DCM.
  4. Wash the resin twice with methanol (MeOH).
  5. Wash the resin twice with DCM.
  6. Wash the resin twice with 1% diisopropylamine (DIEA) in N,N-dimethylformamide (DMF).
  7. Wash the resin twice with DMF.

The resin is ready for further elaboration.

Cleavage With TES/HFIP/TFA/DCM

  1. Suspend the resin in TES/HFIP/TFE/DCM (2:1:0.5:6.5 v/v/v/v) (approximately 10 mL per gram of resin).
  2. Gently hake at room temperature for 1 hour. Remove a few beads and add 1-2 drops of TFA.  If the beads turn orange immediately, continue shaking for another hour and retest.
  3. Filter the resin and wash twice with DCM.
  4. Wash the resin twice with DMF.
  5. Wash the resin twice with 10% diisopropylamine (DIEA) in N,N-dimethylformamide (DMF).
  6. Wash the resin twice with DMF.

The resin is ready for further elaboration.

Piperidine Deprotection of Trp(CHO)[12]

  1. Mix piperidine and DMF (1:10 v/v).  Prepare 10 mL of the solution per gram of the peptide-resin.  Cool the mixture to 0 °C.
  2. Add the peptide resin and stir at 0 °C for 2 hours.
  3. Filter the resin and wash it three times with DMF.  Wash the resin 3 times with DCM, then 3 times with methanol.  Dry the resin in vacuo until a constant weight is achieved.

Post-Cleavage Reduction of Met(O)[13]

Methionine can easily be oxidized to the corresponding sulfoxide and this may occur during peptide synthesis and cleavage.  Fortunately, this oxidation can be readily reversed.  In some cases, methionine sulfoxide derivatives are used as side-chain protected methionine in peptide synthesis and then are reduced back to methionine residues following synthesis and cleavage.

Method 1

  1. Dissolve the peptide in water (approximately 100 mL/ mg of peptide).  Adjust the pH of the peptide solution to 8.0 with triethylamine.
  2. Cool the mixture in an ice bath.  Add mercaptoethanol (4 mL/ mg of peptide) and 1 M ammonium fluoride (4 mL/ mg of peptide).
  3. Stir the mixture in an ice bath for 30 minutes.
  4. Lyophilize the solution to obtain the crude product.

Method 2

  1. Dissolve the peptide in 10% v/v aqueous acetic acid (approximately 200 mL to 1000 mL / mg of peptide)
  2. Add 2-10 mg of N-(methyl)mercaptoacetamide.
  3. Warm the solution at 37 °C under inert atmosphere for 24 to 36 hours.  The reaction can be monitored by HPLC.
  4. Lyophilize the mixture to obtain the crude peptide.

 


[1] Atherton, E.; Sheppard, R. C.; Ward, P. J. Chem. Soc., Perkin Trans.1 1985, 2065-2073.

[2] Marbach, P.; Rudinger, J. Helv.Chim.Acta 1974, 57, 403-414.

[3] Fujii, N; Otaka, A.; Watanabe, T.; Okamachi, A.; Tamamura, H.; Yajima, H; Inagaki, Y.; Nomizu, M.; Asano, K. J. Chem. Soc., Chem. Commun. 1989, 283.

[4] Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber, P.; Rittel, W. Helv. Chim. Acta 1980, 63, 899-914.

[5] Marbach, P.; Rudinger, J. Helv.Chim.Acta 1974, 57, 403-414.

[6] Sieber, P.; Kamber, B.; Riniker, B.; Rittel, W. Helv. Chim. Acta 1980, 63, 2358-2363.

[7] Based of the procedure in Uhmann, R.; Bayer, E. Liebigs Ann. Chem. 1974, 1955-1964.

[8] Lee, J.; Griffin, J. H.; Nicas, T. I. J. Org. Chem. 1996, 61, 3983-3986.

[9] Based on the procedures in Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Hone, N. D. J. Chem. Soc., Chem. Commun. 1993, 778-779 and Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C.; Tetrahedron Lett. 1998, 39, 1603-1606.

[10] Based on Diaz-Mochon, J. J.; Bialy, L.; Bradley, M. Org. Lett., 2004, 6, 1127-1129.

[11] Based on Li, D; Elbert, D. L. J. Pept. Res., 2002, 60, 300-3.

[12] Based on Chowdhury, S. K.: Chait, B. T. Anal. Biochem. 1989, 180, 387-395.

[13] Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama, E.; Otaka, A. Tetrahedron 1988, 44, 805-819.