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How to choose the right resin functionality for solid phase peptide synthesis

As a chemist new to the peptide community, there are many choices that have to be made.  Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin?

I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome.  But I haven't addressed yet a fundamental feature of commercially available resins, and that's the functional handle to which the peptide chain is conjugated.

In today's post, I'll describe some, and I mean only some , of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.

The most critical question that must be asked before undertaking a peptide program is "what is the purpose of these compounds?" as this will have direct impact on the resin choice used for synthesis.  For example, are the peptides used in biological studies?  If yes, are the peptides based on a compound that exists in nature? And so on.  The answers to these questions will help to dictate whether or not the peptide requires a C-terminal amide or acid to be functional.

Resin linkers for peptide synthesis yield C-terminal functionality that generally falls into one of three different categories: acid, amide or other.

Let's first talk about the C-terminal acid linkers.  Peptide acids have the longest history in the field of solid phase peptide synthesis, but they can be challenging to work with.

The most notable acid linker is known as Wang, Figure 1.  Although this resin is commonly used, most chemists choose to buy Wang resin preloaded with the first amino acid.  Loading the first amino acid can be challenging chemically, but is also risky as the asymmetric anhydride intermediate can result in side chain racemization.  Cleaving your peptide from the resin requires concentrated TFA, liberating both the resin and the side chain protecting groups simultaneously.

Figure 1: Chemical structure of the Wang resin linker.

Chlorotrityl resin , Figure 2, is also commonly used for peptide acids.  The increased steric bulk of the trityl groups essentially eliminates any side chain racemization that may occur during the first amino acid coupling onto Wang resin.  Importantly though, peptides can be cleaved from chlorotrityl under very mild conditions (1-3% TFA, acetic acid), leaving a fully protected peptide in solution.

Figure 2: Chemical structure of 2-chlorotrityl resin linker

In addition to peptide acids, chemists often synthesize peptide amides.  The most common of these resins is the Rink Amide resin, Figure 3.  Amide resins are typically easier to work with as the first amino acid loading is a simple amide bond, just like every other amide formed during peptide synthesis.  Rink Amide requires concentrated TFA for complete peptide cleavage from the resin, also resulting in global deprotection of standard side chain protecting groups as well.

Figure 3: Chemical structure of the Rink Amide resin linker.

In addition to Rink amide (among others), there is also Sieber resin , Figure 4, for production of peptide amides.  Sieber resin is an attractive option if the peptide contains sterically bulky amino acids or alternative functionality at the C-terminus due to the limited steric bulk when compared to the Rink amide linker.  Importantly, this linker is also sensitive to mildly acidic conditions, yielding a fully-protected peptide amide in solution.

Figure 4: Chemical structure of the Sieber resin linker.

The third category is loosely defined as "other".  This is by far the most chemically diverse category, but also continues to grow with active research.  The resin linkers that fall into this category have been developed to address the ever-evolving complexity encountered during solid phase peptide synthesis.  Early examples, like safety-catch resins - discovered by Kenner and further expanded by Ellman (Figure 5) - were developed to aid in formation of a C-terminal peptide thioester, a strict requirement if one is to carry forward with native chemical ligation chemistry.  This area continues to grow and now includes hydrazides , N-acylureas , among others.

Figure 5: Chemical structure of Kenner's "safety-catch" resin linker.

Now please keep  in mind, this is a very short list of the resins that are commercially available today.  But hopefully this gives you some insights into the decisions that must be made when embarking on a new peptide synthesis project.

  If you're interested in learning more about a holistic approach to the peptide workflow, check out my white paper below!

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Linkers, resins, and general procedures for solid-phase peptide synthesis

Affiliation.

• 1 IGM, Faculty of Life Sciences, University of Copenhagen, Zealand Pharma, Glostrup, Denmark.
• PMID: 23943476
• DOI: 10.1007/978-1-62703-544-6_2

This chapter describes the basic protocols for solid-phase peptide synthesis using the Fmoc group as the N (α)-protecting group (Fmoc-SPPS). The chapter introduces resins and their handling, choice of linkers, and the most common methods for peptide chain assembly. The proper choice of resins and linkers for solid-phase synthesis is a key parameter for successful peptide synthesis. This chapter provides an overview of the most common and useful resins and linkers for the synthesis of peptides with C-terminal amides, carboxylic acids, and more. The chapter finishes with robust protocols for general solid-phase peptide synthesis, i.e., the standard operations.

• Cross-Linking Reagents / chemistry*
• Peptides / chemical synthesis*
• Peptides / chemistry
• Resins, Synthetic / chemistry*
• Solid-Phase Synthesis Techniques*
• Cross-Linking Reagents
• Resins, Synthetic

Fundamentals of Modern Peptide Synthesis

Cite this protocol.

• Muriel Amblard 2 ,
• Jean-Alain Fehrentz 2 ,
• Jean Martinez 2 &
• Gilles Subra 2  

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 298))

2738 Accesses

12 Citations

The purpose of this chapter is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This chapter focuses on Fmoc chemistry, which is now the most commonly employed strategy for solid phase peptide synthesis (SPPS). Protocols for the synthesis of fully deprotected peptides are presented, together with a review of linkers and supports currently employed for SPPS. The principles and the different steps of SPPS (anchoring, deprotection, coupling reaction, and cleavage) are all discussed, along with their possible side reactions.

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Laboratoire des Amino Acides, Peptides et Proteines-UMRCNRS 5810, Faculté de Pharmacie, Montpellier, France

Muriel Amblard, Jean-Alain Fehrentz, Jean Martinez & Gilles Subra

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Amblard, M., Fehrentz, JA., Martinez, J., Subra, G. (2005). Fundamentals of Modern Peptide Synthesis. In: Howl, J. (eds) Peptide Synthesis and Applications. Methods in Molecular Biology™, vol 298. Humana Press. https://doi.org/10.1385/1-59259-877-3:003

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Solid Phase Peptide Synthesis (SPPS) explained

June 5th, 2023

Solid phase peptide synthesis (SPPS) is a technique for peptide synthesis. It can be defined as a process in which a peptide anchored by its C-terminus to an insoluble polymer is assembled by the successive addition of the protected amino acids constituting its sequence.

Each amino acid addition is referred to as a cycle consisting of:

• a) cleavage of the N α -protecting group
• b) washing steps
• c) coupling of a protected amino acid
• d) washing steps

As the growing chain is bound to an insoluble support the excess of reagents and soluble by-products can be removed by simple filtration. Washing steps with appropriate solvents ensure the complete removal of cleavage agents after the deprotection step as well as the elimination of excesses of reagents and by-products resulting from the coupling step. For a general scheme of SPPS see below

X = O, NH, AA = Amino Acid, PG = Protecting Group, P = Polymer Support, TPG = Temporary Protecting Group

Once the sequence has been completed, the peptide must be cleaved off the resin. Although in general acidolytic cleavage from the resin is the method of choice to release the peptide at the end of the synthesis, a broad range of resins susceptible to be cleaved by nucleophiles such as the «Kaiser oxime resin» and the p-carboxybenzyl alcohol linker or by photolysis has gained popularity.

Quite often, these moieties are not compatible with the conditions of Fmoc SPPS, whereas allyl-based anchors are resistant towards the cleavage conditions of Boc as well as Fmoc protecting groups. The so-called «safety-catch linkers» are perfectly compatible with both Boc and Fmoc chemistries. Only after an activation step they are highly sensitive towards nucleophiles e.g. the sulfonamide linker or 4-hydrazinobenzoic acid.

Boc or Fmoc

The choice of an adequate combination of protecting groups/solid support is the first step on the way to achieve a successful synthesis. For standard SPPS this choice is generally limited to a Boc/benzyl or a Fmoc/tBu based scheme. During the first 15 years of SPPS, the Boc group has been used almost exclusively.

Even if this technique permitted remarkable synthetic achievements the introduction of a new type of protecting group has offered more flexibility for the modification of the peptide chain and/or more specificity in the cleavage of the N α – versus the side-chain protecting groups.

The combination Fmoc/tBu has met these requirements and broadened the scope of SPPS. Moreover, the development of new resin derivatives has allowed the cleavage of fully protected sequences which can be further coupled in SPPS or in a classical solution process.

In addition, a variety of selectively cleavable protecting groups offers new perspectives for «on-resin» modification (cyclization, formation of disulfide bridges, derivatization of side chains, etc ).

Manual Synthesis

The «classical» reactor for SPPS merely consists of a cylindrical vessel with a fritted disc and a removable lid equipped with a mechanical stirrer. The resin may also be stirred by bubbling nitrogen through, however more elaborate equipment is required. For rapid small scale synthesis a small fritted glass funnel is sufficient. Oxygen and moisture need not be strictly excluded, but the cleavage of the Nα protecting group should be performed under a hood as to avoid exposure to piperidine (Fmoc cleavage) or TFA (Boc cleavage).

The swelling of the resin has to be taken into consideration in the choice of the reactor size. Normally, the volume of the swollen peptide resin will slowly increase during chain elongation. When synthesizing a medium-sized peptide (20–30 AA) using Fmoc SPPS, a 100–150 ml reactor will suffice for ca. 10 g of resin. The swelling will be more important in Boc SPPS mostly during the TFA deprotection step; a 250 ml reactor would be recommended for the above- mentioned synthesis.

At the beginning of each coupling cycle, deblocking or washing step the resin and the solution have to be mixed thoroughly, followed by slow stirring or shaking for the remaining process. All the beads have to be suspended in the liquid for thorough washing, efficient coupling, and complete deblocking. It is important to watch for beads sticking to the wall of the vessel especially during the coupling and rinse them from the wall with a small amount of solvent if necessary. «Sticking beads» may become a problem when stirring too vigorously. Silylation of the glassware improves the surface hydrophobicity and prevents the beads from sticking to the wall of the vessel.

Solvents are filtered off by slight suction, or, more gently, by applying inert gas pressure. In Fmoc/tBu based SPPS the vessel may also be used for the final cleavage or for the cleavage of fully protected peptides from very acid-labile resins.

Continuous Flow synthesis

In this approach the solid support is packed into a column and the reagents and solvents are delivered by a pump. The resins used in this technique must be able to withstand considerable pressure and, at the same time, keep a constant volume while changing solvents. The standard polystyrene-based resin is not suitable for that purpose as the volume of the beads markedly depends on the solvent.  This type of peptide synthesizer is best used for Fmoc-based protocols. The Boc protocols generate ionic species during the Boc cleavage, which cause considerable changes in swelling due to electrostatic forces. A synthesizer has been developed in which swelling is monitored, considering that during Fmoc-SPPS, volume changes in a given solvent can only be caused by the growing peptide chain.

Composite material made from a rigid support such as Kieselguhr particles or large pore crosslinked polystyrene in which dimethylacrylamide has been polymerized are used for continuous flow synthesis. Poly(ethylene glycol)-based supports such as TentaGel or PEGA have been introduced for batch as well as continuous flow synthesis.

Fully Automated SPPS

A variety of fully automated synthesizers for batchwise and continuous flow SPPS is commercially available. Fully automated synthesizers employing microwave irradiation for accelerating the synthetic steps were successfully introduced to the market.

Fmoc/tBu SPPS permits automatic monitoring and adequate adjustment of deprotection and coupling times in order to achieve complete conversions. The monitoring relies on strong chromophores which are either released during deprotection or «consumed» (the Fmoc amino acid derivative) and concomitantly released (HOBt or HOAt) during coupling. Monitoring via changes of conductivity allows the monitoring on a real-time basis and end point value can be given to determine the completion of the coupling reaction.

All resins at Bachem are obtained from beaded polystyrene crosslinked with 1% divinylbenzene (a mixture of the meta and the para isomer). This degree of crosslinking is optimal for SPPS. A higher level of crosslinking would reduce the swelling whereas a decrease would cause a considerable loss of mechanical stability in the swollen state.

The carrier resins for SPPS are obtained from this polymer or from the chloromethylated material. In the second case, the available load is restricted by the degree of chloromethylation. The average bead size is adjusted by the conditions of polymerization. Bachem offers the most popular size distribution 200–400 mesh (average diameter 38–75 μm). A variety of resin derivatives is also available as large beads: 100–200 mesh (average diameter 75–150 μm). With such resins, reaction times may have to be prolonged due to limited diffusion towards the interior of the beads.

The load of the resins is adapted to the needs of routine SPPS: 0.7–1 mEq/g before the loading of the first Fmoc amino acid. Loads may be deliberately reduced, e.g., for side-chain cyclization, for the synthesis of long peptide chains (above 30–40 residues), or for the preparation of sequences presenting intrinsic difficulties. Resins having a particularly high load can be prepared by Bachem on request.

Linkers are bifunctional molecules anchoring the growing peptide to the insoluble carrier. Linkers may be coupled to any carrier suitable for SPPS, an important option if alternatives to polystyrene-based resins have to be considered.

The C-terminal Fmoc amino acid may be coupled to the linker yielding the so-called handle which can be purified before loading the polymer. High loads regardless of the bulkiness of the amino acid are obtained by coupling these handles.

What are other techniques to synthesise Peptides

Other techniques used at Bachem are:

A classical method of peptide synthesis . In the majority of labs which use it for smaller scale it has been replaced by the solid phase synthesis. In large scale production it has benefits in the manufacturing of peptides for commercial usage.

Molecular Hiving™

Used to produce shorter peptides without hazardous solvents, with more efficient scale-up and enhanced process controls. To learn more on this Innovative synthesis method visit the blog “ Innovative approaches to large-scale peptide production “, download the Innovations for Tides Brochüre in our Knowledge Center or rewatch one of the Webinars.

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Resins for Solid Phase Organic Chemistry

Product Spotlights

For peptide and high-throughput organic synthesis

Discover the benefits of our Dmb-peptides, Fmoc-azido acids, isoacyl dipeptides, methylated arginine and lysine derivatives, non-explosive replacements for HOBt, PyOxim and pseudoproline dipeptides.

The Novabiochem® brand of MilliporeSigma offers one of the most extensive ranges of specialized resins for solid phase organic synthesis. Products are available for immobilization of most functional groups, and offer cleavage modes ranging from acid, base, cyclative, and traceless. To aid product selection, the resins are categorized according to the reactive resin functional group.

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Peptide Resin Loading Protocols

Merrifield resins.

Merrifield resin (Product No. 497061 , 474517 ) can be loaded with Boc-amino acids as described in Method 1 , 1 or can be purchased pre-loaded with the C-terminal amino acid.

Method 1: Attachment of Boc-amino acids to Merrifield resins

• Dissolve Boc-amino acid in EtOH (2 mL/mmol) and add water (0.5 mL/mmol). Adjust pH to 7 with 2 M aq. Cs 2 CO 3 . Evaporate solution to dryness. Add dioxane and evaporate to dryness. Repeat evaporation with dioxane.
• Pre-swell Merrifield resin in DCM for 1 h and then wash with DMF. Add Cs salt (1.2 eq.) in DMF to the resin and heat at 50 °C o/n. The reaction may be catalyzed by the addition of KI (0.1 eq.). At the end of this time, wash the resin with 3X DMF, 3X DMF/water (1:1), 3X DMF, 3X DCM, 3x MeOH. Dry in vacuo over KOH.

Hydroxymethyl- functionalized resins

One of the simplest methods for esterification to hydroxymethyl-functionalized linkers (Wang, HMPA, and HMBA resins) is to use the symmetrical anhydride of the protected amino acid in the presence of a catalytic amount of p-dimethylaminopyridine (DMAP) ( Method 2 ). 2 However, due to the basic character of this material, enantiomerization and dipeptide formation can be expected; the amount depending on the quantity of DMAP used, the length of the reaction and the nature of the amino acid.

The MSNT method 3,4 (Method 3) is the method of choice in difficult circumstances, such as loading of HMBA resins or when attaching enantiomerization prone amino acid derivatives. 5,6

Cysteine and histidine are particularly prone to enantiomerization and should not be loaded by this method. For these residues, the use of 2-ClTrt resin is recommended; esterification of the C terminal residue is free from enantiomerization and dipeptide formation 7 because attachment does not involve activation of the incoming protected amino acid ( Method 5 ). When peptide acids containing Pro as the C-terminal residue are desired, the use of trityl-based resins is recommended.

Once the resin is loaded the substitution of the resin can be easily determined using Method 11 .

Method 2: Attachment to hydroxymethyl resins using symmetrical anhydride

• Place the resin (1 g) in a clean, dry flask, and add sufficient DMF to just cover and allow to swell for 30 min. Add extra DMF if necessary just to cover the resin.
• Dissolve the Fmoc amino acid (10 eq. relative to resin loading) in dry DCM. One or two drops of DMF may be needed to aid complete dissolution.
• Add a solution of diisopropylcarbodiimide (5 eq. relative to resin loading) in dry DCM to the amino acid solution.
• Stir the mixture for 20 min at 0 °C, keeping the reaction mixture free of moisture with a calcium chloride drying tube.
• Remove the DCM by evaporation under reduced pressure using a rotary evaporator.
• Dissolve the residue in the minimum of DMF and add the solution to the resin prepared in step 1.
• Dissolve DMAP (0.1 eq. relative to resin loading) in DMF and add this solution to the resin/amino acid mixture. Stopper the flask and allow the mixture to stand at rt for 1 h with occasional swirling.
• Remove a small sample of resin (20 mg) and wash, dry and estimate the level of first residue attachment using the procedure described in Method 11 . If the value obtained is less than 70% the first residue attachment procedure should be repeated.

Note: This method is not suitable for His or Cys.

Method 3: Attachment to hydroxymethyl resins using MSNT/MeIm

• Place the resin in a dry reaction vessel. Swell and wash with DCM, add sufficient DCM to cover resin and flush vessel with nitrogen.
• Weigh the appropriate Fmoc amino acid (5 eq.) into a dry round bottom flask. Add dry DCM to dissolve the amino acid derivative (approximately 3 mL/mmol); one or two drops of THF can be added to aid dissolution.
• Add MeIm (3.75 eq.) followed by MSNT (5eq.). Flush flask with nitrogen and seal. Stir the mixture until the MSNT has dissolved.
• Using a syringe, transfer the amino acid solution to the vessel containing the resin.
• Allow the mixture to stand at rt for 1 h, with gentle agitation.
• Wash with DCM (5 times) and DMF (5 times).

Loading amino- functionalized resins

Attachment of amino acid derivatives and other carboxylic acids to linkers containing primary amino groups can normally be affected using standard methods of amide bond formation (see Method 4 ). Hydroxylamine, Weinreb amide, and resins functionalized with secondary amines are much more difficult to load; for these the use of HOAt/DIPCDI or HATU/DIPEA activation is required.

Method 4: Attachment of carboxylic acids to amino resins

• Pre-swell the resin with DCM (polystyrene-based resins) or DMF (PEG-PS or PEGA resin) for 1 h. Wash resin thoroughly with DMF.
• If the resin is Fmoc protected, treat with 20% piperidine in DMF for 20 min, then wash resin thoroughly with DMF. a) Rink Amide, Sieber Amide, MBHA
• Dissolve amino acid derivative or carboxylic acid (5 eq.) and HOBt in DMF. Add DIPCDI (5 eq.) and leave to stand for 10 min. Add mixture to resin. Leave for 1-6 h. b) N-Alkylamino resins
• Dissolve amino acid derivative or carboxylic acid (5 eq.) and HATU in DMF. Add DIPEA (10 eq.) and add immediately to resin. Leave for 6 h. Remove a small quantity of resin and test this for the presence of unreacted amines using the TNBS test (primary amine resins) or chloranil test (secondary amine resins). If the result is positive, wash the resin with DMF and repeat coupling. Continue this procedure until a negative result is obtained.

Trityl-based resins

In contrast to benzyl alcohol-based supports, attachment of Fmoc-amino acids to trityl-based resins, such as 2-chlorotrityl or NovaSyn ® TGT resins, is free from enantiomerization, 7 making them ideal for the immobilization of sensitive residues such as Cys and His ( Method 5 ). The resin also protects Cys from enantiomerization during chain extension. They are particularly useful in the synthesis of C-terminal prolyl peptides as the bulk of the trityl linker helps to prevent diketopiperazine formation 8-10 . When loading 2-chlorotrityl chloride resin, it is important to ensure that all amino-acid derivatives, glassware and solvent are thoroughly dried before use.

NovaSyn ® TGT alcohol resins must be converted to the chloride form before attachment of the amino acid ( Method 6 ).

Method 5: Loading of trityl resins

NOTE: it is important to dry all solvents and glassware before use.

Attachment of carboxylic acids

• Dissolve the carboxylic acid (0.6-1.2 eq. relative to the resin for 2-chlorotrityl resin and 2 eq. for NovaSyn ® TGT chloride resin) and DIPEA (4 eq. relative to carboxylic acid) in dry DCM (approx. 10 ml per gram of resin) containing, if necessary, a small amount of dry DMF (just enough to facilitate dissolution of the acid). For pseudoproline dipeptides add 3 mL of NMP/gram of resin.
• Add this to the resin and stir for 30-120 min. For pseudoproline dipeptides leave to react o/n. At the end of this time, wash the resin with 3X DCM/MeOH/DIPEA (17:2:1), 3X DCM; 2X DMF, 2X DCM. Dry in vacuo over KOH. Fmoc-amino acids are best dried before use by repeated evaporation from dioxane; determine loading using Method 11 .

Method 6: Chloridation of NovaSyn ® TGT alcohol resin

NOTE : it is important to dry all solvents and glassware before use.

• Place NovaSyn ® TGT alcohol resin in a sintered glass funnel and wash the resin consecutively with DMF (2X), dry DCM (3X) and dry toluene (3X).
• Drain off excess toluene from the resin and transfer damp material to a round bottom flask equipped with a reflux condenser.
• Add sufficient toluene to cover resin, then add freshly distilled AcCl (1 mL/g of resin). Heat at 60 - 70 °C for 3 h.
• Slurry mixture to a sintered glass funnel. Wash resin with dry toluene (3X) and dry DCM (3x).
• Drain excess solvent from resin and use immediately.

Success in using the Dbz strategy 11 depends on regioselective acylation of only one of the two linker amines with the C -terminal amino acid residue and to avoid acylation of the unprotected amine during chain extension. Incomplete acylation leads to formation of C -terminally truncated peptides as new chains are propagated by acylation of any unreacted amines during subsequent coupling cycles. Whereas, overacylation results to formation of branched peptides with chains growing off both linker amines. Therefore, the selection of acylation method of attachment of the C -terminal residue and subsequent couplings is critical for good results ( Method 7 ).

Method 7: Loading of Dbz resins

• Pre-swell the resin (0.1 mmol) in DCM for 60 min and wash with DMF. Remove Fmoc group with 20% piperidine in DMF and wash with DMF.
• Ile, Val, Thr, Pro, Arg: Add Fmoc-Aaa-OH (0.6 mmol), HATU (0.6 mmol) and DIPEA (0.9 mmol). Agitate gently for 1 h. Wash resin with DMF and repeat coupling. - Gly: Add Fmoc-Gly-OPfp (0.6 mmol) and HOBt (0.6 mmol). Agitate gently for 1 h - Other amino acids: Add Fmoc-Aaa-OH (0.6 mmol), HCTU (0.6 mmol) and DIPEA (0.9 mmol). Agitate gently for 1 h.
• Check loading using Method 11 . Alternatively, wash a sample of resin with DCM and treat with 95% TFA aq. for 30 min. Anaylze cleaved product by HPLC.

Particularly problematic is the coupling of glycine residues, especially if they occur close to the C -terminus of the peptide. This reactive and unhindered amino acid can couple to the free Dbz-amino group if uronium or phosphonium activation is used. In our hands best results are obtained if glycine residues are introduced using Fmoc-Gly-OPfp/HOBt. This precaution may be unnecessary once the peptide is extended beyond 10 residues, as hindrance should reduce the reactivity of unprotected Dbz amine. Furthermore, the use of strong activators like HATU or HCTU should be avoided as their use can lead to branching. In our hands, HBTU/HOBt appears to work well for coupling of all residues except Gly, where the use of the pre-formed OPfp in conjunction with HOBt gives minimal branching.

The use of Alloc protection for blocking the second amino group has been advocated to avoid all issues with branching and truncation 12 . Dbz resins as supplied contain mostly 3-Fmoc-Dbz, with small amounts of 4-Fmoc-Dbz and bis-Fmoc-Dbz. Capping the resin with Alloc-Cl prior to removal of the Fmoc group will thus reduce the maximum potential for branching or truncation to 6%. For hindered amino acids, it has been found necessary to load the resin prior to capping with Alloc. The Alloc group must be cleaved off with Pd(0) before conversion to the Nbz form ( Figure 1).

Figure 1: Alloc protection to avoid branching in Dbz resin

Sulfamyl resins

Loading of sulfamyl-based resins is best achieved with carboxylic acids activated with PyBOP ® and DIPEA in CHCl 3 at -20 °C 13 or with DIPCDI/ N- methylimidazole ( Method 8 ). In the case of PyBOP ® activation, the loading efficiencies are reported to vary from >95% for Cys, Met and His to 44% for Pro, the worst case. Extent of racemization for the loading of Fmoc-Phe and Fmoc-Leu by these methods are 0.5% and 0.3%, respectively. However, in practice the loading obtained by these methods can be highly variable, and problems can occur with over acylation of the linker. Furthermore, the substitution of the support must be determined before starting peptide synthesis.

Method 8: Loading of sulfamyl resins

DIPCDI method

• Pre-swell resin (1 mmol) in DCM for 1 h before use.
• Dissolve amino acid derivative or carboxylic acid (4 mmol) and 1-MeIm (4 mmol) in DCM/DMF (4:1). Add DIPCDI (4 mmol), mix and add to resin. Leave for stand with gentle agitation for 18 h. Wash resin with DMF, DCM, MeOH and dry.

PyBOP ® method

• Pre-swell resin (1 mmol) in CHCl 3 for 1 h before use.
• Dissolve amino acid derivative or carboxylic acid (4 mmol) and DIPEA (8 mmol) in CHCl 3 . Add to resin.
• Cool mixture to –20 °C. Add PyBOP ® (4 mmol) and leave for stand with gentle agitation for 8 h at –20 °C. Wash resin with DCM, DMF, DCM, MeOH and dry.

DHP HM resin consists of 3,4-dihydro-2 H -pyran-2-yl-methanol linker 14 attached to 100-200 mesh chloromethyl polystyrene, and is a useful tool for the synthesis of peptide alcohols.

In contrast to trityl-based supports, where the use of prolonged reaction times and elevated temperatures are often required to achieve satisfactory loadings, derivatization of DHP HM resin is relatively straightforward, with even secondary alcohols being loaded without difficulty. Typically, this process involves treating the resin in DCE with an excess of alcohol in the presence of pyridinium p- toluenesulfonate (PPTS); full experimental details are given in Method 9 .

Method 9: Loading DHP HM resin

• Pre-swell DHP HM resin in dry DCE for 1 h.
• Dissolve Fmoc-amino alcohol (3 eq.) in dry DCE containing PPTS (1.5 eq), and add this solution to the resin.
• Leave to react o/n at 80 °C with gentle agitation under nitrogen.
• Quench reaction by adding pyridine (~ 5 mL/g). Isolate resin by filtration and wash with DMF, DCM and hexane. Dry resin o/n under vacuum.

Preparation of peptide aldehydes using H-Thr-Gly-NovaSyn ® TG resin

One of the simplest and most effective methods of preparing peptide aldehydes, which involves the solid-phase immobilization of an amino aldehyde by formation of an oxazolidine between a pre-formed Fmoc-amino aldehyde and H-Thr-Gly-NovaSyn ® TG resin 15 .

After loading the resin, the oxazolidine nitrogen should be blocked by treatment with Boc-anhydride. The resultant acyloxazolidine is stable to base and is compatible with Fmoc protocols.

For peptides containing a C -terminal aspartal, argininal, leucinal, phenylalaninal, or valinal residue, pre-loaded resins are available.

Method 10: Loading of H-Thr-Gly-NovaSyn ® TG resin

• Suspend H-Thr-Gly-NovaSyn ® TG resin in 1% AcOH in MeOH/DCM (1:1) containing Fmoc-amino aldehyde (5 eq. relative to resin substitution) in DCM.
• Gently agitate mixture at rt for 4 h and monitor by TNBS test.
• Remove resin by filtration, wash with DCM, DMF, and THF.
• Treat resin with Boc 2 O (5 eq.) and NMM (5eq.) in THF at 50°C for 3 h to cap oxazolidine nitrogen.
• Remove resin by filtration and wash with THF, DCM, and DMF

Fmoc loading test

For estimating the loading of resins derivatized with Fmoc-amino acids, the simplest approach involves cleaving the Fmoc group with DBU and measuring the solution concentration of the liberated dibenzofluvene by U.V. spectroscopy. 16

Method 11: Estimation of level of first residue attachment

• Take 3 x 10 mm matched silica UV cells.
• Weigh dry Fmoc amino acid-resin (approx. 5 µmol with respect to Fmoc) into a10 mL graduated flask.
• Add 2 mL of 2% DBU in DMF. Agitate gently for 30 min. Dilute solution to 10 mL with MeCN. Take 2 mL of this solution and dilute to 25 mL in a graduated flask
• Prepare a reference solution as in step 2, but without addition of the resin.
• Fill two cuvettes with 3 mL of test solution and one cuvette with 3 mL of reference solution. NOTE: Do not cross-contaminate the solutions. Allow the resin to settle to the bottom of the cells.
• Place the cells in a spectrophotometer and record optical density at 304 nm.
• Obtain an estimate of first residue attachment from equation below Fmoc loading: mmol/g =(Abs sample -Abs ref ) X 16.4/mg of resin).

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• Base Resins for Peptide Synthesis
• Biotinylation Reagents
• Chemoselective Purification Tags
• Heterocycle and Cyclic Peptide Formation with N-(isocyamino)triphenylphosphorane (Pinc)
• Peptide Labeling
• Linkers for Fmoc SPPS
• Solving Aspartimide Formation in Fmoc SPPS with Fmoc-Asp(OBno)-OH
• Peptide Coupling Reagents Selection Guide

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Cleavage of the Peptide from the Resin

TFA Procedure 1

TMSBr Procedure 2

If the peptide contains Arg(Mtr), this procedure will cleave the peptide from the resin and remove the Mtr group more rapidly than the TFA procedure.

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