Resins for Solid Phase Peptide Synthesis (Part 2)

In our previous newsletter, we reported on the different parameters describing resins, e.g. bead size, swelling, cross-linkage, loading.

Herein, we provide a summary of the different resins available at Iris Biotech.

Merrifield

Substrates are attached to the Merrifield resin by nucleophilic displacement of its chlorine. The cleavage of substrates from this type of resin requires strongly acidic conditions. Aside from the standard HF procedure, low-high HF methods, as well as trifluoromethanesulfonic acid (TFMSA) and low-high TFMSA protocols can be employed. Besides, methods such as saponification, transesterification and cyclization-release have proven effective.

Hydroxymethyl

The hydroxymethyl resin analog was invented to avoid unwanted side reactions, which can occur for unreacted chloromethyl groups in the case of incomplete Merrifield resin loading. In order to avoid such side reactions, remaining free hydroxyl groups can be capped with acetic anhydride. Cleavage conditions are the same as for the conventional Merrifield resin.

Amino Core Resins, e.g. methylbenzyhydryl amine (MBHA), benzhydylamine (BHA)

Amino core resins were originally developed for preparing C-terminal peptide amides via the Boc SPPS strategy.

HMBA Resin

HMBA linkers attached to amine base resins are stable to acidic cleaving conditions. Cleavage from the resin is achieved by a range of nucleophiles, thus various C-terminal modifications such as alcohols, methyl esters, hydrazides or amides can be synthesized.

TentaGel®

These resins consist of functionalized polyethyleneglycols grafted onto polystyrene beads and are thus especially developed for the use in polar solvents which are mostly incompatible with the traditional polystyrene resins. For large peptides, unfavorable folding of the growing peptide on the resin can cause coupling difficulties. The more hydrophilic environment of the TentaGel® resins as well as their typically high swelling promotes better solvation, decreases repulsion, reduces aggregation, and thus allows for better coupling and improved yields. A variety of functional groups is available ranging from electrophilic bromine leaving groups to nucleophilic groups like hydroxy, amino, and carboxy functions.

Hypogel®

HypoGel® is a hydrophilic gel type resin which combines high capacities with good solvent compatibility. Glycol spacers with only n = 5 or 10 units separate the reactive sites from the polystyrene matrix.

Trityl, 2-chlorotrityl chloride, 4-methyl/methoxy-trityl chloride, TCP

Resins functionalized with trityl linkers are highly acid sensitive, as the three phenyl rings stabilize the benzylic carbocation that is generated during cleavage. Cleavage can be performed with only 1% of TFA or acetic acid. Besides, protected peptides can be cleaved using 20% hexafluoroisopropanol keeping all sidechain protecting groups intact; even trityl groups on sulfhydryl function of homocysteine. Various differently substituted trityl-based linkers exist allowing to fine-tune the cleavage conditions.

Chlorotrityl resins are reported to have better stability during peptide synthesis than trityl resins. However, chlorotrityl resins are extremely moisture sensitive. In case the resin was deactivated during storage, treatment with thionyl chloride immediately before usage restores activity.

A further variation of the basic trityl resin are the TCP resins. In addition to the advantages of regular trityl resins mentioned above, the p-carboxamide group of the TCP-resin exhibits a deactivating effect on the trityl ring system, so that TCP resins are stable under standard storage conditions.

Wang

The Wang resin is the most widely used solid phase support for acid substrates. Finished products can readily be cleaved by moderate treatment with an acid, generally 50% (v/v) TFA in DCM.

Phenylacetamidomethyl (PAM)

The 4-hydroxymethyl-phenylacetamidomethyl functionalized PAM resin is widely used in Boc-chemistry, as it is more stable towards acids and allows the synthesis of medium-sized to large peptides.

Benzhydryl

Due to their steric demand, these resins are less prone to side reactions than the phenoxy-substituted Wang-resins. Cleavage conditions are similar – at least 25% TFA in DCM with scavengers allow to liberate the peptide from the resin. In case a more acid sensitive resin is required, for example for the production of protected peptide fragments, typically 2-chlorotrityl resin is being used. However, if the sterical hindrance becomes too high, 4-methoxybenzhydryl resin can be used as an alternative.

Rink Amide/Knorr

C-Terminal amides are the most common modification in peptides. For their synthesis via the Fmoc strategy the Rink amide linker has been developed. Different modifications of the Rink linker have been developed towards varying uses. The standard Rink amide resin is cleaved in 10% TFA in DCM, as higher concentrations of acid can cleave the Rink linker from the polystyrene resin, producing highly colored impurities. The formation of these byproducts can be minimized by adding trialkylsilanes to the cleavage mixture. The Knorr resin avoids this unwanted linker cleavage through the introduction of a more stable acetamide spacer between the Rink linker and the resin.

An additional variant of the Knorr linker is the attachment to an even more acid labile 2CT resin. Through this combination, the protected peptide can be cleaved from the 2CT resin while still carrying the Knorr linker as protecting group for the C-terminus for subsequent modifications using solution phase chemistry.

Ramage

Alternative to the commonly used Rink-resin. The three-circular structure of the Ramage linker prevents fragmentation of the linker during cleavage from the resin followed by back alkylation. A common phenomenon observed during peptide synthesis with the open structure of the Rink-amide linker. Hence, Ramage linker delivers peptides with higher purity and less impurities, than peptides produced with the Rink-amide linker. Ramage resin is particularly recommended for C-terminal Phe, Tyr and Ile.

Sieber

The Sieber amide resin is ideally suited to synthesize side-chain protected peptide amides, as cleavage occurs in 1% TFA in DCM. Since the Sieber linker is less bulky through the fixation of the two aryl rings via the phenolether bridge, it is better suited for the synthesis of C-terminal secondary amides. Sieber resin is less sterically hindered than Rink resin and thus allows for higher loading in sterically demanding applications than Rink resins.

Hydrazone

Peptide hydrazides can be easily synthesized using the novel hydrazone resin. The hydrazone linker is completely stable in the course of standard Fmoc SPPS, and tolerates treatment with 5% TFA/DCM, thus permitting selective removal of Mtt or similar acid-labile protecting groups. Subsequent application of tried and tested cleavage cocktails (TFA:water:TIS 95:2.5:2.5) permits to obtain the peptides in good yields and purity.

Oxime

Oxime resin is a synthesis resin compatible with Boc chemistry. Substrates can be cleaved from this resin under basic conditions, which leave the side chain and N-terminal protecting groups in place, making this resin very useful for preparing protected fragments that can be used in the segment condensation synthesis of larger substrates. A special application of oxime resin is the formation of cyclic peptides by cyclization cleavage.

Although this 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 is performed after each coupling to block any active sites on the resin that may have been exposed.

Weinreb Amide

This support is useful for the preparation of peptide aldehydes.

➔ For detailed protocols and further information, please download our resin guideline! 

References:

Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide; R. B. Merrfield; J. Am. Chem. Soc. 1963; 85(14): 2149-2154. https://doi.org/10.1021/ja00897a025

Preparation, structure and morphology of polymer supports; D. C. Sherrington; Chem. Commun. 1998; 2275-286. https://doi.org/10.1039/a803757d

Calculating Resin Functionalization in Solid-Phase Peptide Synthesis Using a Standardized Method based on Fmoc Determination; O. Al Musaimi, A. Basso, B. G. de la Torre, F. Albericio; ACS Comb. Sci. 2019; 21(11): 717-721. https://doi.org/10.1021/acscombsci.9b00154

Guide for Resin and Linker Selection in Solid-Phase Peptide Synthesis; J. A. Moss; Curr. Protoc. Sci. 2005; 18.7.1-18.7.19. https://doi.org/10.1002/0471140864.ps1807s40

Linkers, resins, and general procedures for solid-phase peptide synthesis; P. Tofteng Shelton, K. J. Jensen; Methods Mol Biol. 2013; 1047: 23. https://doi.org/10.1007/978-1-62703-544-6_2

Synthesis of the Merrifield resin esters of N-protected amino acids with the aid of hydrogen bonding; H. Kusuo, I. Ken’ichi, I. Ken; Chem. Lett. 1978; 7: 165-168. https://doi.org/10.1246/cl.1978.165

Functionalization of crosslinked polystyrene resins: 2. Preparation of nucleophilic resins containing hydroxyl or thiol functionalities; J. M. J. Fréchet, M. D. de Smet, M. J. Farrall; Polymer 1979; 20: 675-680. https://doi.org/10.1016/0032-3861(79)90241-6

Preparation of aminomethyl-polystyrene resin by direct amidomethylation; A. R. Mitchell, S. B. H. Kent, B. W. Erickson, R. B. Merrifield; Tetrahedron Lett. 1976; 17: 3795-3798. https://doi.org/10.1016/s0040-4039(00)93112-5

Preparation and use of benzhydrylamine polymers in peptide synthesis. II. Syntheses of thyrotropin releasing hormone, thyrocalcitonin 26-32, and eledoisin; P. G. Pietta, P. F. Cavallo, K. Takahashi, G. R. Marshall; J. Org. Chem. 1974; 39: 44-48. https://doi.org/10.1021/jo00915a008

Comparative use of benzhydrylamine and chloromethylated resins in solid-phase synthesis of carboxamide terminal peptides. Synthesis of oxytocin derivatives; V. J. Hruby, D. A. Upson, N. S. Agarwal; J. Org. Chem. 1977; 42: 3552-3556. https://doi.org/10.1021/jo00442a024

A p-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides; G. R. Matsueda, J. M. Stewart; Peptides 1981; 2: 45-50. https://doi.org/10.1016/s0196-9781(81)80010-1

Substituted Benzhydrylamine Resins in Solid Phase Peptide Synthesis of Peptide Amides and Peptides with C-terminal Asparagine monitored by Potentiometric Titration with Perchloric Acid. Classification of Acid Lability of Different Resins.; M. Christensen, O. Schou, V. S. Pedersen; Acta Chem. Scand. B 1981; 35B: 573-581.

2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage; K. Barlos, O. Chatzi, D. Gatos, G. Stavropoulos; Int J Pept Protein Res 1991; 37: 513-520.

p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments; S. S. Wang; J. Am. Chem. Soc. 1973; 95: 1328-1333. https://doi.org/10.1021/ja00785a602

α-(Phenylacetamido)benzylpolystyrene (pab-resin); E. Giralt, D. Andreu, M. Pons, E. Pedroso; Tetrahedron 1981; 37: 2007-2010. https://doi.org/10.1016/s0040-4020(01)97954-2

tert-Butoxycarbonylaminoacyl-4-(oxymethyl)-phenylacetamidomethyl-resin, a more acid-resistant support for solid-phase peptide synthesis; A. R. Mitchell, B. W. Erickson, M. N. Ryabtsev, R. S. Hodges, R. B. Merrifield; J. Am. Chem. Soc. 1976; 98: 7357-7362. https://doi.org/10.1021/ja00439a041

Acid-labile resin linkage agents for use in solid phase peptide synthesis; R. C. Sheppard, B. J. Williams; Int. J. Pept. Protein Res. 1982; 20: 451-454. https://doi.org/10.1111/j.1399-3011.1982.tb03067.x

Wang Linker Free of Side Reactions; V. Castro, H. Rodriguez, F. Albericio; Org. Lett. 2013; 15(2): 246-249.  https://doi.org/10.1021/ol303367s

A comparison of acid labile linkage agents for the synthesis of peptide C-terminal amides; M. S. Bernatowicz, S. B. Daniels, H. Köster; Tetrahedron Lett. 1989; 30: 4645-4648. https://doi.org/10.1016/s0040-4039(01)80764-4

A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method; P. Sieber; Tetrahedron Lett. 1987; 28: 2107-2110. https://doi.org/10.1016/s0040-4039(00)96055-6

Convenient method of peptide hydrazide synthesis using a new hydrazone resin; P. S. Chelushkin, K. V. Polyanichko, M. V. Leko, M. Y. Dorosh, T. Bruckdorfer, S. V. Burov; Tetrahedron Lett. 2015; 56: 619–622. https://doi.org/10.1016/j.tetlet.2014.12.056