Peptide Synthesis

  1. What resin choice should I use for my peptide?

  2. What type of chemistry  (Fmoc vs Boc) should I use for my peptide?

  3. How do I use this coupling reagent?

  4. What can help me to make a difficult or hydrophobic sequence?

  5. In what direction are the peptides synthesized?

  6. What do Boc- and Fmoc- strategy mean?

  7. How much resin do I need to use?

  8. How can I prepare peptide esters?




  1. What resin choice should I use for my peptide?


    These charts will help you select the appropriate resin for your peptide synthesis.

     

    Acid forming resins

    Amide forming resins

     

     

     

     

     



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  2. What type of chemistry  (Fmoc vs Boc) should I use for my peptide?


    With average peptides, Fmoc and Boc chemistries both produce excellent peptides.  In preparing hydrophobic peptides that are suceptible to aggregation, Boc chemistry may provide some advantages.  Removal of the Boc group under acid conditions protonates the exposed amine terminal of the peptide which reduces its participation in hydrogen bonding and increases its availablity for coupling.  Neutralization in the presence of an activated amino acid (in situ neutralization) can produce higher yields and purer peptides in difficult sequences.

    Preparing protected peptide fragments to assemble into larger peptides and small proteins is often easier with Fmoc chemistry.  Special resins are available that can be cleaved under very mild conditions which leave all other protecting groups intact.  In Boc chemistry, cleavage of the peptide product from the resin typically requires very strong acid. 

     

               

    Boc

    Fmoc

    Requires special equipment

    No

    No

    Cost of reagents

    Similar

    Similar

    Solubility of protected peptides

    Higher

    Lower

    Purity of hydrophobic peptides

    High

    May be lower

    Problems with aggregation

    Less frequently

    More frequently

    Synthesis time

    10 -20 min/amino acid

    10-20 min/amino acid

    Final deprotection

    TFMSA in TFA

    TFA

    Safety

    Relatively safe

    Relatively safe

    Orthogonal

    No

    Yes



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  3. How do I use this coupling reagent?


    Standard DIC/HOBt Coupling

    1. Remove the N-terminal protecting group by standard deprotection protocols.
    2. Suspend the resin in dichloromethane (DCM, 10 mL per gram resin)
    3. Dissolve 5 equivalents (based on resin substitution) in DMF (approximately 1 mL per gram of amino acid derivative.
    4. Dissolve 5.5 equivalents (based on resin substitution) of HOBt in DMF (minimum volume necessary for complete solution).5. Add the amino acid solution and the HOBt solution to the resin suspension.
    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. 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 Benzotriazole-1-yloxy-tris-pyrrolidinophosphonium Hexafluorophosphate

    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

    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 HBTU 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.

    Coupling with TSTU in Aqueous Solvent Mixtures

    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.



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  4. What can help me to make a difficult or hydrophobic sequence?


    Incorporating Pseudoproline and Isoacyl dipeptides, Hmb & Dmb amino acid and Dmb-dipeptides may be helpful in enhancing synthetic efficency of difficult or hydrophobic sequences.  AAPPTec carries a wide variety to help our customers synthesize their peptide of interest. 

    Performing the coupling reactions at a higher temperature often reduces reaction time and improves the yield of peptide.  AAPPTec offers the Vantage and the Focus XC, two automated peptide synthesizers with the capability of performing reactions above ambient temperature.



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  5. In what direction are the peptides synthesized?


    Peptides are synthesized from the C-terminal to the N-terminal.



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  6. What do Boc- and Fmoc- strategy mean?


    Two SPPS protocols are now considered standard procedures, the Boc/Bzl protocol and the Fmoc/tBu protocol.  Fmoc/tBu- and Boc/Bzl-SPPS differ in the choice of the temporary Nα (Boc or Fmoc) and permanent (side-chain: tBu-related or Bzl-related) protecting groups. Boc/Bzl-SPPS utilizes Boc for the temporary protecting group.  The Boc group is removed with trifluoroacetic acid (TFA).  The completed peptide and the side chain protecting groups are typically removed with hydrofluoric acid (HF) or trifluoromethanesulfonic acid (TFMSA). Fmoc/tBu-SPPS utilizes the Fmoc group for protecting Nα .  The Fmoc group is removed with bases, typically piperidine.  Final release of the completed peptide and removal of the side chain protection is performed with TFA.  Fmoc-SPPS is considered the milder method.



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  7. How much resin do I need to use?


     

    You can use the chart below to determine the amount of resin, or you can divide the scale (mmoles of peptide to be synthesized) by the substitutuon of the resin(mmole/gram) you are using.

     

    Grams of Resin Required For Peptide Synthesis Scale

    Resin Subst. (mM/g)

    0.05 mM

    0.1 mM

    0.2 mM

    0.5 mM

    1 mM

    5 mM

    10 mM

    15 mM

    30 mM

    45 mM

    0.2

    0.250

    0.500

    1.000

    2.500

    5.000

    25.00

    50.00

    75.00

    150.0

    225.0

    0.3

    0.167

    0.333

    0.667

    1.667

    3.333

    16.67

    33.33

    50.00

    100.0

    150.0

    0.4

    0.125

    0.250

    0.500

    1.250

    2.500

    12.50

    25.00

    37.50

    75.00

    112.5

    0.5

    0.100

    0.200

    0.400

    1.000

    2.000

    10.00

    20.00

    30.00

    60.00

    90.00

    0.6

    0.083

    0.167

    0.333

    0.833

    1.667

    8.333

    16.67

    25.00

    50.00

    75.00

    0.7

    0,071

    0.143

    0.286

    0.714

    1.429

    7.14

    14.29

    21.43

    42.86

    64.29

    0.8

    0.063

    0.125

    0.250

    0.625

    1.250

    6.25

    12.50

    18.75

    37.50

    56.25

    0.9

    0.056

    0.111

    0.222

    0556

    1.111

    5,55

    11.11

    16.67

    33.33

    50.00



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  8. How can I prepare peptide esters?


    Peptides prepared on 2-Cl-Trt resin can be directly converted to peptide esters by treating the peptide-resin with a solution of HCl in the appropiate alcohol.  The alcoholic HCl solution is prepared by slowly adding acetyl chloride to the alcohol.

    This method was reported by R.S. Lokey and co-workers in Organic Letters.  "Direct Conversion of Resin-Bound Peptides into C-Terminal Esters" Yasuhiro Uozumi, Yoichi M. A. Yamada, Maki MinakawaR. A. Turner, R. J. Weber, R. S. Lokey, Org. Lett. 2010, 12, 1852-1855. 

    Yasuhiro Uozumi, Yoichi M. A. Yamada, Maki Minakawa
    R. A. Turner, R. J. Weber, R. S. LokeyYasuhiro Uozumi, Yoichi M. A. Yamada, Maki MinakawaR. A. Turner, R. J. Weber, R. S. Lokey

     



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