MBHA Resin
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Fmoc Amino Acid

MBHA Resin

MBHA resin is the most widely used resin for preparing peptide C-terminal amides using Boc chemistry. This resin is more HF labile than BHA resin which is important for peptides containing C-terminal Phe or Leu, since these peptides are difficult to cleave from BHA resin. The resin is sold in the HCl salt form to inhibit air oxidation of the amine function attached to the resin. After treating the resin with diisopropylethylamine/dichloromethane (DIPEA/DCM) to remove the HCl, the first amino acid can be attached to the resin using standard Boc coupling protocols.

High quality MBHA resin in a range of substitution levels is available from aapptec.

  Catalog #   Quantity Price
  RBZ001 MBHA Resin HCl, 100-200 mesh, 1% DVB 5g $25.00
      25g $95.00
      100g $280.00
For more information about MBHA resin and other aapptec resins click here.
 

Attaching the First Amino Acid

1. Suspend the resin in 10% (v/v) DIPEA in DCM. Use approximately 10 mL per gram of resin. Mix the suspended resin with a mechanical shaker for 10 to 15 minutes. Filter the resin and wash it with DCM.
2. Return the resin to the flask and add 10 mL of DMF per gram of resin.
3. In a separate flask dissolve 1.5 to 2.5 equivalents (relative to the resin substitution) of the first Boc-amino acid and an equal molar amount of HOBt in a minimum amount of DMF. Add this solution to the to the resin.
4. Cool the mixture in an ice bath. Add an amount of DIC equal molar to the Boc-amino acid. Swirl the flask in the ice bath to mix the contents, then remove the ice bath and allow the mixture to come to room temperature.
5. Mix the reaction mixture at room temperature with a mechanical shaker for 4 hours.
6. Remove a small sample of the resin, wash it with DCM, and perform a Kaiser test. If the test shows there are free amine groups present, continue shaking the mixture for 1 hour. Remove another sample of the resin, wash it with DCM, and perform a Kaiser test.
7. If the Kaiser test shows free amine groups are still present 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 the resin. Remove a small sample of the resin, wash it with DCM, and perform a Kaiser test. If the test is positive for free amine groups then repeat this step until the Kaiser test is negative.
8. Filter the resin in a fine sintered funnel. Wash the resin three times with DMF, 3 times with DCM, and 3 times with methanol. Each time use enough solvent to slurry the resin.
9. Dry the resin in vacuo to a constant weight. The substitution of the resin can be estimated from the weight gain of the resin. For a more accurate determination of the resin substitution perform a picric acid test.

 

Cleavage of the Peptide from the Resin

There are many protocols for cleaving peptides from MBHA resin. Four common protocols are standard HF, low-high HF, standard TFMSA, and low-high TFMSA. In the standard HF protocol the N-terminal Boc group is removed then the peptide-resin and a mixture of scavenger are mixed with a high concentration of HF inside a special HF apparatus. With the low-high HF procedure a low concentration of HF in DMS is used to remove most of side chain protection groups followed by a standard HF cleavage. TFMSA standard and low-high procedures are alternatives to the corresponding HF procedures. The main advantage of these procedures is that they do not require special HF resistant apparatus.

In choosing a cleavage protocol, the side chain protecting groups as well as the amino acid composition of the peptide must be considered. The following chart will assist in selecting the appropriate protocol.

If peptide contains:

 
His(Dnp) The Dnp group must be removed before cleaving the peptide from the resin or removing the N-terminal Boc group of the finished peptide.
Trp(CHO) In the standard HF and TFMSA cleavage protocols the peptide resin must be treated with piperidine in DMF following the removal of the N-terminal Boc group to remove the formyl group. The formyl group can also be removed thiolytically in a "low-high" HF procedure or "low-high" TFSMA procedure where p-cresol is replaced with p-thiocresol or thiophenol. The TMSOTf procedure can remove the formyl group if ethanedithiol (EDT) is added to the cleavage mixture.
Trp Anisole should be used in the cleavage mixture to prevent alkylation of Trp by benzyl or t-butyl cations. Avoid using thioanisole in HF cleavages.
Arg(Tos) TFMSA does not deprotect Arg(Tos). Arg(Tos) is deprotected during standard HF cleavage conditions, but may require longer reaction times. The low-high HF cleavage is recommended.
Arg(NO2) TFMSA and TMSOTf will not deprotect this group. During "low-high" HF cleavage, it is cleaved under the "high" conditions.
Asp(OBzl), Glu(OBzl) The cleavage should be performed at 5°C or lower to minimize side reactions of these amino acids.
Asp(OcHx) Cleavage should be performed at 5°C or lower to minimize aspartimide formation. TFMSA does not remove the OcHx group efficiently.
Glu(OcHx) Cleavage should be performed at 5°C or lower to reduce anisylation of Glu.
Cys(ACM) This group is not deprotected by TMSOTf.
Cys(Bzl) This group is not deprotected by TMSOTf.
Cys(MeBzl) TFMSA does not remove this group efficiently. Cleavage at temperatures below 5°C may be very slow.
Met(O) This group is reduced to Met during low-high HF and low-high TFMSA cleavages. TSMOTf will not quantitatively reduce Met(O), so post-cleavage reduction is necessary.
Cys, Met DMS, p-thiocresol, and anisole should be added to the cleavage mixture to prevent alkylation of these amino acids.
 
  Removal of the N-Terminal BOC Group
1. Suspend the resin in 50% (v/v) TFA/DCM. Agitate the mixture with a mechanical shaker for 5 minutes at room temperature.
2. Filter the resin in a fine sintered glass funnel.
3. Suspend the resin in fresh 50% (v/v) TFA/DCM. Agitate the mixture with a mechanical shaker for 20 to 25 minutes at room temperature.
4. Filter the resin in a fine sintered glass funnel. Wash the resin three times with DCM and twice with methanol.
5. Dry the peptide resin under high vacuum overnight with KOH or P2O5.
 
 Standard HF Proceedure1
1. If the peptide contains His(Dnp), remove the Dnp group. Remove the N-terminal BOC group. If the peptide contains Trp(CHO), remove the formyl group.
2. Place a Teflon-coated stirring bar and the peptide-resin into the reaction vessel of the HF apparatus. Add the appropriate mixture of scavengers. For most peptides, add 1 mL of anisole and 1 mL of dimethylsulfide for every 0.2 mmol of peptide resin. If the peptide resin contains Cys add 1 mL anisole, 1 mL dimethylsulfide, and 0.2 mL of p-thiocresol for every 0.2 mmol of peptide resin.
3. Secure the cap onto the reaction vessel and cool it in a dry ice/methanol bath for at least 5 minutes. For every 0.2 mmol of peptide-resin, distill 10 mL of HF into the reaction vessel. Maintain the temperature between -5°C and 0°C while collecting the HF.
4. Maintain the temperature between 0°C and 5°C for 30 to 60 minutes as the cleavage mixture is stirred. If the peptide contains Arg(Tos), the cleavage may take up to 2 hours. After the end of the reaction time, evaporate the HF under a stream of nitrogen.
5. Filter the resin, wash it 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.
 
 Low-High HF Procedure2
1. If the peptide contains His(Dnp), remove the Dnp group. Remove the N-terminal BOC group.
2. Place a Teflon-coated stirring bar and the peptide-resin into the reaction vessel of the HF apparatus. Add the appropriate mixture of scavengers. For most peptides, add 1 mL of p-cresol and 6.5 mL dimethylsulfide for every 0.2 mmol of peptide resin. If the peptide resin contains Cys add 1 mL p-cresol, 6.5 mL dimethylsulfide, and 0.2 mL of p-thiocresol for every 0.2 mmol of peptide resin.
3. Fasten the cap onto the reaction vessel and cool it in a dry ice/methanol bath for at least 5 minutes. For every 0.2 mmol of peptide-resin, distill 2.5 mL of HF into the reaction vessel. Maintain the temperature between -5°C and 0°C while collecting the HF.
4. Maintain the temperature at 0°C for 2 hours as the cleavage mixture is stirred. After the end of the reaction time, evaporate the HF and DMS in vacuo at 0 ºC.
5. Filter the resin, wash it with DCM or EtOAc to remove scavenger by products and suction dry.
6. Return the resin to the reaction vessel and add 1 mL of p-cresol for every 0.2 mmol of peptide-resin. If the peptide contains Trp(CHO), substitute thiocresol or thiophenol for p-cresol.
7. Fasten the cap onto the reaction vessel and cool it in a dry ice/methanol bath for at least 5 minutes. For every 0.2 mmol of peptide-resin, distill 10 mL of HF into the reaction vessel Maintain the temperature between -5°C and 0°C while collecting the HF.
8. Maintain the temperature between 0°C and 5°C for 30 to 60 minutes as the cleavage mixture is stirred. If the peptide contains Arg(Tos), the cleavage may take up to 2 hours. After the end of the reaction time, evaporate the HF under a stream of nitrogen.
9. Filter the resin with a fine sintered glass funnel. Wash the resin with a small amount or 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.
 
  Standard Trifluoromethanesulfonic Acid Procedure3
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 or Sephadex columns.
 
 
Low-High TFMSA Cleavage2
1. If the peptide contains His(Dnp), remove the Dnp group.
2. Transfer the resin to round bottom flask equipped with a stirring bar. For every 100 mg of resin add 100 mL of m-cresol and 300 mL dimethyl-sulfide. Cool the mixture to 0 ºC in an ice bath and 0.5 mL of TFA for every 100 mg of resin. If the peptide contains Trp(CHO), add 20 mL of EDT for each 100 mg of peptide resin.
3. Maintain the mixture at 0°C in an ice bath. For every 100 mg of peptide-resin slowly add 100 mL of TFMSA. Stir vigorously during addition of the TFMSA to dissipate the heat generated. Stir the mixture for 3 hours while maintaining the temperature between 0°C and 5°C.
4. Filter the resin in a medium sintered glass funnel. Wash the resin with several volumes of ether. Dry the resin under high vacuum over KOH or P2O5 for at least 4 hours.
5. Place the dried resin in a round bottom flask equipped with a stirring bar. For every 100 mg of resin add 100 mL of thioanisole and 30 mL of EDT. Cool the flask to between 0°C and 5°C using an ice bath. For every 100 mg of resin add 1.0 mL of TFA and mix for 5 to 10 minutes. For every 100 mg of resin slowly add 100 mL of TFMSA while stirring vigorously to dissipate the heat generated.
6. Warm the flask to room temperature and continue stirring for 90 to 120 minutes.
7. 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.
8. Desalt the peptide by ion exchange or Sephadex columns.
Footnotes
1. Based on procedures in J. M. Stewart and J. D. Young in "Solid Phase Peptide Synthesis", Pierce Chemical Company, Rockford, Illinois, 1984.
2. Based on the procedures in Tam, JP; et al. J. Am. Chem. Soc. 1983, 105, 6442.
3. Based on procedures in "Introduction to Cleavage Techniques", Applied Biosystems Inc. Foster City, California, 1990.