Peptide Synthesis Resins HEADING_TITLE

Linkers and Synthesis Resins

The core resins, by themselves, have limited utility as peptide synthesis resins.  Peptides can be cleaved from Merrifield resin and MBHA resin in good yield only with strong acid and are sledom used with Fmoc-amino acids.  Peptides attached to aminomethyl resin can not be removed  without destroying or seriously damaging the peptide.
The cleavage properties of the resins can be modified by permanently attaching suitable linkers.  By manipulating the structure of the linker, resins ranging from extremely acid labile to base labile can be prepared.  Using linkers additionally allows preparation of resins with special applications such as DHP resin utilized as a solid phase support for alcohols or Weinreb resin used for preparing aldehydes and ketones.
PAM ResinLinkers and Synthesis Resins


The core resins, by themselves, have limited utility as peptide synthesis resins.  Peptides can be cleaved from Merrifield resin and MBHA resin in good yield only with strong acid and are seldom used with Fmoc-amino acids.  Peptides attached to aminomethyl resin can not be removed  without destroying or seriously damaging the peptide.


The cleavage properties of the resins can be modified by permanently attaching suitable linkers.  By manipulating the structure of the linker, resins ranging from extremely acid labile to base labile can be prepared.  Using linkers additionally allows preparation of resins with special applications such as DHP resin utilized as a solid phase support for alcohols or Weinreb resin utilized for preparing aldehydes and ketones.

PAM Resin

 

PAM Resin

PAM resin is widely used for solid phase synthesis of peptides utilizing the Boc strategy.  The numerous Boc deprotection reactions with trifluoroacetic acid (TFA) required in the synthesis of large peptides leads to significant losses of peptide from Merrifield resin.1  PAM resin provides better stability to TFA,2 but the finished products are harder to cleave.  Since typical cleavage conditions require a strong acid such a HF, this resin has found limited use in solid phase organic chemistry.3  

Wang Resin

Wang Resin

 

Wang resin is the most widely used solid phase support for acid substrates.  The linker attached to the polystyrene core is a 4-hydroxybenzyl alcohol moiety.4  The linker is bound to the resin through a phenyl ether bond and the substrate is generally attached to the linker by a benzylic ester or ether bond.  This linkage has good stability to a variety of reaction conditions, but can be readily cleaved by moderate treatment with an acid, generally trifluoroacetic acid.  Impurities can form if a portion of the linker is attached to the resin through the benzylic position leaving a reactive phenolic site.  This can occur during attachment of the linker if exact reaction conditions are not maintained.5  

Wang resin is the most widely used solid phase support for acid substrates.  The linker attached to the polystyrene core is a 4-hydroxybenzyl alcohol moiety.6  The linker is bound to the resin through a phenyl ether bond and the substrate is generally attached to the linker by a benzylic ester or ether bond.  This linkage has good stability to a variety of reaction conditions, but can be readily cleaved by moderate treatment with an acid, generally trifluoroacetic acid.  Impurities can form if a portion of the linker is attached to the resin through the benzylic position leaving a reactive phenolic site.  This can occur during attachment of the linker if exact reaction conditions are not maintained.7

Amide/Amine Forming Resins for Fmoc/tBu

Rink Amide Resin PAL Resin Sieber Amide Resin
Rink Amide Resin PAL Resin Sieber Amide Resin

 

The most popular solid phase supports for the formation of amide products include Rink and PAL resins.  All of these resins were originally developed for peptide amide synthesis using the Fmoc strategy.  These resins are favored due to their higher acid lability; cleavage can be performed under conditions as mild as 1% TFA.8  In solid phase organic chemistry, these resins have been used to produce amines by reductive alkylation.9  To provide stability on storage, each of these resins is supplied with the amine protected by a Fmoc group, therefore pretreatment with piperidine is required to render the free amine.  Acids can be coupled using standard amide forming conditions such as DIC/HOBt, HBTU or BOP.

Rink and PAL resins exhibit similar characteristics with respect to cleavage conditions and the type of products formed.  Rink resin, however, has been more widely utilized.  PAL is somewhat more acid labile.10  PAL resin has been found to give cleaner products with long peptide sequences.11  

Sieber Amide Resin is useful for preparing amides and amines and fully protected peptide amide fragments. Products can be cleaved under mild conditions,12 using 1%TFA in DCM.  This resin is less sterically hindered than Rink resin and thus allows for higher loading in sterically demanding applications than Rink resins.  This is illustrated in a recent synthesis of secondary amides.13

MBHA Resin

MBHA Resin

MBHA (methylbenzhydryl amine) is an amide-forming resin structurally similar to Rink. Although it is not Fmoc protected it must be activated by treatment with base (e.g. diisopropylethylamine).

The method of attaching the first amino acid is the same as Rink; ordinary amide bond forming conditions. Coupling the first amino acid is no different from coupling the rest.

In contrast, conditions for cleaving peptide products from MBHA are much harsher than from Rink. MBHA requires treatment with hydrofluoric acid or trifluoromethanesulfonic acid (TFMSA).

Trityl and 2-Chlorotrityl Resins

2-Cl-Trt Chloride Resin

Trityl resins have been widely used in both solid phase organic and peptide chemistry.  These resins are very acid labile and can be cleaved with acetic acid.14  Protected peptides can be cleaved with 1:4 v/v hexafluoroisopropyl alcohol/dichloromethane15 with all sidechain protecting groups intact, even trityl groups on sulfhydryl function of homocysteine.16  These resins are particularly useful when less acid labile protecting groups are required on the substrate following cleavage, or in cases where the substrate can cyclize on the anchoring linkage causing premature cleavage.  The bulky triphenylmethyl group prevents such attack through steric hindrance.  In addition to being used to immobilize acids and alcohols, trityl resins can also be used to immobilize amines or thiols.  

Barlos has reported that the 2-chlorotrityl resin has better stability during peptide synthesis than the trityl resin.14  The 2-chlorotrityl resins are available in the chloride form.  The chloride form is exceedingly moisture sensitive and must be handled and stored under inert conditions.  Should the resin become deactivated, treatment with thionyl chloride immediately before use restores the activity.

Base Labile Resins

Oxime Resin   HMBA Resin
Oxime Resin   HMBA Resin

 

Both oxime resin and hydroxymethyl benzoic acid linked resin (HMBA resin) can be cleaved with a variety of nucleophilic agents [ammonia or primary amines (amides),17 hydrazine (hydrazides),18 methanol/triethylamine (methyl esters),19 sodium borohydride (alcohols), sodium hydroxide (acids)] to produce the wide range of products indicated from the same precursor resin.  A special application of oxime resin is the formation of cyclic peptides by cyclization cleavage.20

Although oxime resin is compatible with Boc chemistry, the oxime ester linkage is susceptible to TFA.  Therefore the Boc group is removed with 25% TFA in DCM during synthesis and end-capping should be performed after each coupling to block any active sites on the resin that may have been exposed.  For best results, the peptide should not exceed 10 residues.

DHP Resin

DHP Resin

DHP resin was developed as a solid phase support for alcohols.  The DHP linker is less sterically hindered than the trityl linkers are, hence it is preferred for sterically bulky substrates such a s secondary alcohols.  Alcohols are attached to the resin under anhydrous catalytic acid conditions, forming an acetal.  The bound alcohols can be cleaved from the resin under acidic conditions.21  DHP resin has been utilized as a sidechain anchor for serine and threonine,22 as well as hydroxyproline.23 

Weinreb Aminomethyl Resin

Weinreb Resin

 

This resin was developed by Fehrentz and coworkers to prepare aldehydes by solid phase methodology.24  An amino acid or carboxylic acid is attached to the resin by standard coupling procedures.  Reduction with LiAlH4 releases the product aldehyde.  Reacting the resin-substrate with Grignard reagents produces ketones.25

Polyethylene Glycol-Polystyrene Grafted Resins

Aggregation resulting from inter and intra chain hydrogen bonding can leave the N-terminal of the growing peptide unavailable for reaction.  Grafting polyethylene glycol (PEG) onto the surface of polystyrene beads provides a more polar environment and helps the growing peptide to remain highly solvated.  Several brands of PEG-grafted resins are commercially available.  These types of resins are often utilized in the synthesis of very long peptides.  Since the PEG grafted on the surface of these resins allow greater solvation by water and other protic solvents, these resins are also utilized when protic solvents are required.

Amino Acid Loaded Resins

Attaching a substrate unit onto a resin is a step crucial to the success of solid phase syntheses, especially library syntheses.  One problem often encountered in this step incomplete reaction of the reactive sites on the resin.  Unless they are blocked, these unreacted sites can react with other reagents in later steps of the synthesis and generate impurities that may be difficult to remove.  Inefficient attachment of the amino acid also lowers the useful substitution, reducing the overall yield and efficiency of the solid phase synthesis.  Partial racemization of protected amino acids is another problem that can occur during the loading reaction.

Numerous catalysts, activating agents, additives and reaction conditions have been tested to find the optimum conditions for the preparation of substrate-attached resins.  As may be expected, these conditions vary for the different resins.  AAPPTec substrate-attached resins are prepared by proven procedures that ensure good substitution and minimize racemization of the substrates.  Each substituted resin is end-capped to block any remaining unreacted active resin sites that could interfere later in the synthesis.

 

 

 


 

Footnotes

  1 Gutte, B.; Merrifield, R. B., J. Biol. Chem., 1971, 246, 1922-1941.

  2 Mitchell, A. R.; Erickson, B. W.; Ryabtsev, M. N.; Hodges, R. S.; Merrifield, R. B. J. Am. Chem. Soc. 1976, 98, 7357-7362; Mitchell, A. R.; Kent, S. B. H.; Engelhard, M.; Merrifield, R. B., J. Org. Chem., 1978, 43, 2845-2852.

  3 For examples of solid phase organic chemistry performed on PAM resin, see: Pulley, S. R.; Hegedus, L. S., J. Am. Chem. Soc., 1993, 115, 9037-9047; Smith, J.; Liras, J. L.; Schneider, S. E.; Anslyn, E. V., J. Org. Chem., 1996, 61, 8811-8818.

  4 Wang, S., J. Am. Chem. Soc., 1973, 95, 1328-1333.

  5 Lu, G.; Mojsov, S.; Tam, J. P.; Merrifield, R. B., J. Org. Chem., 1981, 46, 3433-3436.

  6 Wang, S., J. Am. Chem. Soc., 1973, 95, 1328-1333.

  7 Lu, G.; Mojsov, S.; Tam, J. P.; Merrifield, R. B., J. Org. Chem., 1981, 46, 3433-3436.

  8 Rink, H., Tetrahedron Lett., 1987, 28, 3787-3790.

  9 Purandare, A. V.; Poss, M. A., Tetrahedron Lett., 1998, 39, 935-938.

 10 Bernatowicz, M.S.; Daniels, S.B.; Köster, H., Tetrahedron Lett., 1989, 30, 4645-4648.

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

 12 Sieber, P., Tetrahedron Lett., 1987, 28, 2107-2110.

 13 Chan, W.C.; Mellor, S. L. J., Chem. Soc., Chem. Commun., 1995, 1475-1477.

 14 Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenqing, Y.; Schäfer, W., Tetrahedron Lett., 1989, 30, 3943-3946.

 15 Bollhagen, R.; Schirdberge, M.; Barlos, K. Grell, E. J., Chem. Soc., Chem. Commun., 1994, 2559-2560.

 16 Cyr, J. E.; Pearson, D. A.; Wilson, D. M.; Nelson, C. A.; Guaraldi, M. Azure, M. T.; Lister-James, J. Dinkelborg, L. M.; Dean, R. T., J. Med. Chem., 2007, 50, 1354-1364.

 17 Story, S. C.; Aldrich, J. V., Int. J. Peptide Protein Res., 1992, 39, 87-92; Niu, J.; Lawrence, D. S., J. Biol. Chem., 1997, 272, 1493-1499; Mohan, R.; Chou, Y.-L.; Morrissey, M.M., Tetrahedron Lett., 1996, 37 , 3963-3966.

 18 DeGrado, W. F.; Kaiser, E. T., J. Org. Chem., 1980, 45, 1295-1300.

 19 Pichette, A.; Voyer, N.; Larouche, R.; Meillon, J.-C., Tetrahedron Lett., 1997, 38, 1279-1282; Hutchins, S. M.; Chapman, K. T., Tetrahedron Lett., 1996, 37, 4869-4872.

 20 Dutton, F. E.; Lee, B. H., Tetrahedron Lett., 1998, 39, 5313-5316; Lee, B. H., Tetrahedron Lett., 1997, 38, 757-760; Paulitz, C.; Steglich, W., J. Org. Chem., 1997, 62, 8474-8478.

 21 Thompson, L.A.; Ellman, J.A., Tetrahedron Lett., 1994, 35, 9333-9336.

 22 Ramaseshan, M.; Dory, Y.L.; Deslongchamps, P., J. Comb. Chem., 2000, 2, 615-623.

 23 Anderson, M.O.; Shelat, A.A.; Guy, R.K., J. Org. Chem., 2005, 70, 4578-4584.

 24 Fehrentz, J.A.; Paris, M.; Heitz, A.; Velek, J.; Liu, C.-F.; Winternitz, J.; Martinez, J., Tetrahedron Lett., 1995, 36, 7871-7874.

 25 Dinh, T.Q.; Armstrong, R.W., Tetrahedron Lett., 1996, 37, 1161-1164.