Solid supports for the synthesis of peptides

Transcription

1 Fernando Albericio Fayna Garcia Martin Solid supports for the synthesis of peptides From the first resin used to the most sophisticated in the market ABSTRACT The most popular way to synthesize peptides is via the solidphase approach, mostly on a research scale, although progress is being made in large-scale production. The most evident example is Fuzeon, a commercial anti-hiv peptide, which is produced in multi-kilograms using a solid support for the synthesis of the fragments. Success in solid-phase peptide synthesis is heavily determined by the solid support. In this review we focus on the evolution of the solid support from the totally polystyrene-based resin used by Merrifield to the most sophisticated ones currently available on the market. These new resins offer access to previously inaccessible compounds as well as the possibility to be used in diverse applications but without losing stability. Moreover, these new supports are easy to handle. The final chapter of the review highlights the complex sequences that are difficult to achieve and the reasons for this. It then concludes by explaining the approaches that have been followed to synthesize such difficult peptides. INTRODUCTION From the first dipeptide synthesis in 1901 until now, the field of peptide synthesis has experienced considerable growth as a result of several new developments: i) The solid-support has been one of the main contributors to chemical peptide synthesis. Merrifield developed this ingenious and neat approach, which consists of using polystyrene (PS) resin as a solid matrix where the peptide chain is grown by being covalently attached at one end to the functionalized support. The solid support allows for simple stepwise syntheses, thereby avoiding a large number of individual steps. In addition, it allows a shortening of the time required and also the synthesis of previously inaccessible peptides. ii) Protecting groups, which allow a cleaner synthesis, the construction of selective disulfide bridges, and orthogonal groups, which permit new synthetic strategies and even the disruption of highly aggregated sequences. iii) The handle/linker (1), which allows cleavage of the peptide from the resin in diverse conditions and even modification of the carbonyl terminus at one s convenience. iv) New coupling reagents and additives for more efficient amide bonds, thereby permitting shorter times, better yields and non-racemization, among others. well as coupling of the consecutive amino acid. This repetitive process allows the stepwise attachment of the side-chainprotected sequence directly on the support, thereby avoiding several steps simply by ready filtration. Once the total peptide has been achieved, it is cleaved and permanent protecting groups are removed. This is followed by precipitation, characterization, purification and folding, when required (3). The success of solid-phase synthesis is strongly related to the support and its performance. The requirements to use polymeric support in this kind of synthesis include: i) uniform beads; ii) stability in variation of temperature; iii) mobile, well-solvated and reagent-accessible sites; iv) good swelling in a broad range of solvents, if applied; v) functionalized beads permitting covalent coupling of the first compound and vi) acceptable loadings (4). Finally, the resin should be chemically, mechanically and physically stable to allow ready filtration and high chemical conditions. Currently there is no general rule to decide on the most convenient solid support. However, it is important to consider the type of chemistry to be carried out during the synthesis, resin reagent compatibility, swelling solvent ratio, and the length and sequence of the desired product. When synthesizing a peptide it is also relevant to take into account several questions related to the nature of the resin, such as its uniform bead size, cross-linking, and loading. In the case of the synthesis of The solid support is made of diverse materials with improved intrinsic properties that allow more reagent diffusion, swelling in a wider range of solvents, uniform beads and an amphiphilic character. The solid support is precisely the focus of this review. MOST USEFUL RESINS FOR PEPTIDE SYNTHESIS (2) A solid-phase approach is the most popular way to synthesize peptides on small and large scales for research purposes. This is partly because this approach facilitates the production of medium-short and simple sequences with a minimum of work. Figure 1 shows how a peptide is usually constructed: it consists mostly of introducing a first amino acid conveniently protected, usually by C-terminus, to the solid matrix. The deprotection of the temporal group is then carried out, usually at the N-terminal, as difficult Figure 1. Solid-phase peptide sequences, peptide synthesis it is crucial (SPPS) to scheme. select an appropriate 29

2 resin composition (PS, polyamide, PS containing poly(ethylene glycol) (PEG), totally PEG-based, among others) (5, 6). The polymeric network of the resin can be considered in two models: the rigid support model and the co-solvent model. The former considers the resin as a compact sphere while the latter accepts the effect of the resin during reactions. A number of direct and indirect studies have been carried out to establish which of the two is the most appropriate to describe the solid support. Addressing the microenvironment within the resin, Regen studied two kinds of supports, cross-linked totally PS-based and PEG-PS polymers. After performing several chemical studies he concluded that both the PS polymer backbone and PEG groups had significant mobility; therefore the resin tends to fit the co-solvent model. This observation was corroborated by other researchers, such as Czarnik, who considered the resin not as a ball, but as a cosolvent, or Meldal who introduced PEG chains into the structure following the principle like dissolves like (7). Success in solid-phase peptide synthesis is highly dependent on the accessibility of the free amino termini of the peptidyl-resin. Association of the reactants with the solid support can influence the concerted reaction pathway and therefore the rate of the reaction. It has also been shown that peptide-resin solvation and swelling affect the efficiency of the coupling reaction in solidphase peptide synthesis. Solvation of the peptide-resin depends on the composition of the solid support, its cross-linking, the weight of the peptide chain, the sequence, the swelling solvent and the Fmoc/Boc chemistry chosen. Here we provide an overview of the most used resins and their nature. Polystyrene (PS) resin Used by Merrifield (8) for solid-phase peptide synthesis, these spherical beads are based on a chloromethylated polymer of styrene and divinylbenzene (DVB) (Figure 2). The resin (2 percent DVB, mesh beads) allows the diffusion of reagents when the polymer swells in the presence of appropriate solvents. Although Merrifield found that the reaction rates were slower in solid phase than in solution, the final result showed improvements in time and yields since several purification steps could be avoided. Taking advantage of these convenient properties, some years later Merrifield and Stewart developed and set up an instrument for the automated synthesis of peptides (Figure 3). The amount of cross-linking alters the solvation and swelling properties of the polymeric matrix. Standard crosslinking is about 1-2 percent DVB and therefore it is a high-hydrophobic resin that may affect peptide assembly. Although forty years later we can find more sophisticated supports in the market, those most commonly used are still very similar to that employed by Merrifield. Resins incorporating Poly(ethylene glycol) moieties Although PS-DVB resin has been widely used for solid-phase organic synthesis, it has been proven that the rate of incorporation of particular amino acids decreases, especially with increased peptide length. In an attempt to solve these intrinsic problems, Figure 2. Merrifield resin polymer net. Figure 3. Peptide synthesizer designed by R.B. Merrifield and J. M. Stewart (F. Garcia-Martin). poly(ethylene glycol) moiety was incorporated into the PS structure. The crucial role of the resin composition during the amide formation reaction has been extensively studied by numerous researchers, such as Czarnik and collaborators, who compared PS resin and PS cross-linked resin with distinct amounts of PEG chains (with a 40, 70 and percent PEG respectively) (9). Under identical conditions, faster kinetics was observed on PEG-PS resin: the greater the PEG content in the resin, the faster the reaction. A possible explanation for this observation is that PEG chains interfere with solvation, dielectric properties and hydrogen bonding. In this second generation of resins, many combinations of PEG chains into the resin (proportions and mode of structure) have been conceived to achieve a more convenient solid support. Herein we give a summary of the most used PEG-containing resins. Grafted PEG-PS In general terms, a PEG implant is introduced on a PS matrix while maintaining a PS core with a more hydrophilic exterior. The main handicap of this support is its lower loading and chemical stability. Figure 4 illustrates the main PS resin with PEG grafts. POE-PS (TentaGel ) POE-PS was developed by Bayer et al. and prepared by grafting ethylene oxide onto PS resin. Chemical architecture is formed by a PS web cross-linked by PEG chains and functionalized at the end of the chain (Figure 4a). These copolymers contain percent of PEG, and are mono-sized and useful for library applications (10). Tentagel shows good and uniform swelling in a wide range of solvents, from toluene to water. However, the first generation of PEG-PS grafted resins did not result in such a convenient resin because of its low loading and loss of PEG chains during strong acidic treatments, such as the TFA applied in the cleavage step. A new generation of PEG-PS resins has been developed to overcome these drawbacks. It is believed that these problems are due to the direct linkage of the benzyl from the PS to the PEG chain. Thus, greater stability and loading has been achieved by using a longer linkage and in some cases by branching it. PEG-PS Albericio and Barany developed a solid support (11) in which PEG chains of defined molecular weight were coupled by amide linkage onto a suitable amino functionalized gel-type PS resin (1 percent DVB). As shown in Figure 4b, the amine of the PS core is functionalized by a linker (usually a Nle or an Orn in the second PEG-PS generation) and the PEG chain is incorporated on it. Assembly of the peptide is performed at the end of the PEG chain, where a linker has been previously attached. A potential advantage of this grafting procedure is that the final proportion PEG:PS can be controlled. At first, PEG-PS resins had a moderate loading of mmol/g and were suitable for either Fmoc or Boc protocols. Later on, superior loadings ( mmol/g) have been obtained by introducing a branch point. 30

3 PEG/PS (ArgoGel ) This resin was produced by Argonaut Technologies and conceived as a PEG-PS resin which has all the advantages of this resin while showing improved loading. The graft component of ArgoGel is a bifunctional PEG chain attached to the PS core at its centre (Figure 4c). Branching allows the preparation of a new PEG-PS graft copolymer that doubles the loading (approximately 0.45 mmol/g) of conventional PEG-PS graft copolymers containing monofunctional PEG chains. Moreover, the use of chemically inert aliphatic ethers to join PEG chains to the polymer backbone, in contrast to a benzylic or amide-based connection, makes the resin inert to the presence of strong acids and nucleophilic reagents (12). Figure 4. PEG-PS grafted resins architecture. PEG-PS (Champion I and II) This resin was developed by Adams et al. (13), and is based on the incorporation of PEG chains onto the PS core but attaching a spacer between them to improve stability and loading. The resin is basically formed by PS grafted with 40 percent PEG (Champion I) and 60 percent PEG (Champion II), but with higher loading (0.7 mmol/g). This superior loading, which is almost twice that of a normal resin, is achieved through a urethane linkage between the PEG and the original resin (See Figure 4 d). This support has various formulations on the basis of its moiety, named Champion I and Champion II. Copolymers The main difference with the previous resin group is the introduction of PEG chains into the structure, not simply as a graft. This introduction modifies the cross-linking changes and the physicochemical properties of the solid support, but at the same time maintains the notable properties of the classical support. The main copolymerized resins are summarized and illustrated in Figure 5. PEGA This resin was developed by Meldal et al. (14) as an improvement of Sheppard s polyamide resin (15). Sheppard s polymer was developed as an alternative to hydrophobic PS resin and its main characteristic is that it mimics the peptide chain. PEGA resin is based upon the same principle but in this case poly(ethylene glycol) is its major constituent. PEG chains work as cross-linkers and as the starting point of the synthesis (Figure 5a). Furthermore, these chains also helps to give more flexibility and stability. It has high swelling properties and is suitable for enzymatic reactions despite its initially low loading ( mmol/g). In addition, the swelling of its polar solvent and its biocompatibility allow direct screening on it. Thus, this architecture clearly facilitates the penetration of large biomolecules into the macromolecular matrix in aqueous milieu. Several applications have been explored using PEGA resin, such as chemical ligation on the resin, direct screening of phosphopeptide-binding proteins as a useful technique for proteomics, direct monitoring on the resin by gel phase NMR, and biotransformation on solid support. TTEGDA-PS The features of this resin are illustrated in Figure 5b. It is a PS resin with tetra(ethylene glycol) diacrylate (TTEGDA) as crosslinker (16). It resembles a gel more than a solid support. The introduction of tetra(ethylene glycol) groups into the PS structure by copolymerization renders more uniformly functionalized supports and has the advantage of not having contaminants remaining from incomplete polymerization. Furthermore, studies comparing several cross-linked densities concluded that a 4 percent TTEGDA was the optimum balance between good swelling and good mechanical properties, similar to PS. This resin is mechanically stable, exhibits good-swelling, and presents an optimum hydrophilic-hydrophobic balance. However, for long automatic syntheses (from twenty residues and over) it still presents several problems because of packing down the gel in a glass column. As a result of backpressure, the solvent is squeezed by the pressure, thus its volume is drastically reduced and the highly solvated internal structure is destroyed. This effect is most probably due to the presence of ester bonds that confer reduced chemical stability versus PS resin with DVB as cross-linker. CLEAR The cross-linked ethoxylate acrylate resin (CLEAR ) solid support was developed by Kempe and Barany (17). As shown in Figure 5c, it is structurally characterized by a high degree of cross-linking caused by a key trivalent branched linker (> 95 percent by weight of cross-linker). It presents excellent swelling conditions, and performs well in the synthesis of difficult sequences. Moreover, in on-resin native chemical ligation it performs better than its counterparts PEG-PS and PEGA. This unique architecture shows high stability, since nonleaking of material is detected under extreme SPPS conditions. Although this resin shows high mechanical and chemical stability, it is labile in the presence of strong bases. Figure 5. Principal copolymerized solid supports for SPPS. Totally PEG-based resin The next generation of solid supports is based on a totally poly(ethylene glycol)-based resin, a more hydrophilic polymer that allows diverse applications. Its amphiphilic nature helps to achieve the synthesis of particular sequences that were not accessible previously by stepwise synthesis. Among its advantages, of relevance is high swelling in a wide range of solvents, thus increasing reactivity in coupling and deprotection steps. These favourable swelling properties, 32

4 probably also present in PEG-grafted resins, may be due to the stretched helical superstructure adopted by PEG in aqueous solution. Straight PEG was used in the 70s for synthesis, but its application in solid phase has occurred only in recent years. In the following lines the most used totally PEG-based resins are summarized. Straight PEG resin for liquid phase synthesis of peptides Step-wise assembly of peptides reversibly linked to PEG chains corresponds to a hybrid between traditional solution and solidphase approaches. The main inconvenience of solid-phase peptide synthesis is the impossibility to quantitatively control the amino acids coupled during assembly. Individual couplings are non-quantitative so erroneous sequences accumulate during the synthesis of the desired peptide. Mutter et al. developed a method which combines the advantages of solution synthesis with those of the solid-phase method. This technique consists of incorporating polyethylene glycol (molecular weight from 4000 to 20000) or other soluble supports into the carboxylic group of the first amino acid; separation can be achieved via ultra filtration or precipitation (18). Synthesis follows a solid-phase protocol with the further advantage that the intermediates can be purified during the assembly, without being cleaved from the support. Unfortunately, when ethers (Et 2 O or methyl-tertbutyl ether) or alcohols (EtOH or cold isopropanol) are used to precipitate the polymer, a number of impurities in the reaction mixture can also be precipitated. Due to the simplicity of use of this support, an exhaustive analysis of the coupling rates was carried out, providing useful information about PEG chain size (19), and compared to solution peptide synthesis. This methodology has been used for synthesis of other biopolymers such as oligosaccharides and oligonucleotides, and even for the combinatorial chemistry of small molecules. SPOCC Meldal et al. were interested in obtaining a resin that could be used for peptide synthesis and also for enzymatic reactions. PEG- PS resins were not useful for this aim, and the closest resin that could be applied in enzymatic chemistry was PEGA. However, due to the presence of amide backbone in PEGA resins, this kind of support is not compatible with other chemical reactions, such as solid-phase glycosylation. The first published attempt of this family of resin was the POEPOP (polyoxyethylene/ polyoxypropylene) copolymer, which contains only ether bonds. This resin was successfully used for the synthesis of peptides. The main problem, however, was its lability under strong Lewis acidic conditions as a result of the presence of secondary ether bonds. The next generation of Meldal resin was the SPOCC (Solid-phase organic and combinatorial chemistry) (20), which has a chemical backbone based on long-chain PEG terminally substituted with oxetane units. Thus, this resin contains only primary ether bonds and alcohol functionalities to overcome the same problem encountered by the previous one. Moreover, it retains all the swelling properties of the former PEGA. SPOCC resin is much more stable under strong conditions compared with the previous POEPOP polymer, and is suitable for general organic chemistry using parallel and combinatorial synthesis as well as for enzymatic reactions. Its main drawback is the high cost of manufacture, which implies non-attractive commercial production. ChemMatrix (CM) resin A new solid support of totally PEG-based chains is the CM, which was developed in recent years by Côté (21). As a distinctive feature, it is an amphiphilic resin that contains exclusively primary ether bonds and is highly cross-linked (See Figure 6). Thus a highly stable resin with increased loading has been obtained and has wide possibilities for application in solid-phase synthesis. The first published solid-phase peptide synthesis application using this resin was the generation of a library of short peptides (22). This resin was chosen for its biocompability, which allows screening directly on the solid support in aqueous solution. Our work using CM resin has achieved the successful synthesis of challenging peptides. This result is attributed to, among other features, the stability and amphiphilic character this resin. Evidence of Figure 6. Aminomethyl-ChemMatrix resin the superior structure. performance of this support has been certified by the success of highly structured peptide synthesis such as the HIV protease, which contains 99 amino acids, Rantes (1-68) and CCL4-L1 chemokines by combining with pseudoprolines, and in the most striking example, the synthesis of b-amyloid (1-42) (23). Moreover, the synthesis of other biomolecules, such as oligonucleotides and oligopeptides, has been achieved using CM resin. DIFFICULT PEPTIDES TO SYNTHESIZE The success of solid-phase peptide synthesis is mainly due to the high repetitive yield in the coupling reaction to the growing peptide chains. Despite these successes, difficulties in chain assembly and failure in obtaining large complex peptides have been reported (24). The main obstacles posed by difficult peptide sequences have been debated and researched. It is believed that the major features are related to i) the sequence; ii) sterically hindered amino acids; and iii) hydrogen bonding between peptidic chains and the polymeric matrix (25). Sequence-dependent factors are associated with the tendency of several residues or sequences to aggregate. Although this aggregation depends on the nature of the peptide chain, sequences containing Ala, Val, Ile, Met, Asp or Gln are prone to this effect, while secondary amino acids, such as Pro, are thought to inhibit aggregation. Sterically hindered amino acids are mainly due to the side chain or lateral protecting group. For instance, β-ramified amino acids such as Val or Thr are considered inconvenient for coupling. Moreover, sequences with sterically hindered amino acids, like alkylated α-amino, lead to very low yields of crude products. Also, several side-chain-protecting groups can affect the steric hindrance and hydrophobicity of the microenvironment. Among these factors, the contribution of hydrogen bonding is probably the most critical, and has often resulted in the impossibility of reagents to access the free α-amino group in the chain due to sudden shrinkage or insufficient swelling of the peptidyl-resin networks. The main hydrogen bonding effect in SPPS is made by peptide-polymeric backbone, and intrachain and interchain interactions in the growing peptide. Intrachain and interchain interactions are frequently sequencedependent. The former usually occur at reverse turns whereas interchain bindings arise when α-helix- or β-sheet-like structures are formed within the peptidyl-resin. Under these conditions, the peptidyl-resin affects solvation, thus limiting the diffusion of reagents into the matrix. As a result, coupling and deprotection reactions are often slow and incomplete, and the colorimetric test may give false negatives. Peptide-polymeric backbone interactions and solvation strongly contribute to the tendency to aggregate. Several studies have shown that simply changing the support allows access to previously inaccessible peptides (26). PEGcontaining resin contributes to a more hydrophilic microenvironment as well as the presence of hydrogen bonding 33

5 donor and acceptor groups within the matrix. These groups, which are in the vicinity of the growing peptide chain, could interact preferentially with the matrix, thus inhibiting the formation of ordered structures (27). Table 1 summarizes the main difficult peptides to achieve and ways to solve the problems related to interaction of the chains and peptidyl-resin. Approaches to prevent aggregation by external factors include: i) incorporating disaggregating compounds into the coupling solvent and or deprotection mixture; ii) heating during coupling; and iii) using microwave energy. In contrast, the preferred internal approaches are related to iv) incorporating moieties, as pseudoprolines or Hmb amino acids, which prevent aggregation during elongation; v) using disaggregating presequences as linkers; and vi) using PEG-containing resins or other more hydrophilic supports. Other chemical methods to achieve the desired peptide consist of native chemical ligation, convergent synthesis or O-N intramolecular acyl migration after solid-phase synthesis. ACKNOWLEDGEMENTS The work carried out in the author s laboratory was partially supported by CICYT (CTQ /BQU), the Instituto de Salud Carlos III (CB06_01_0074), the Generalitat de Catalunya (2005SGR 00662), the Institute for Research in Biomedicine and the Barcelona Science Park. REFERENCES AND NOTES 1. Even some authors tend to confuse the terms handle /linker and solid support, the term handle /linker may be used to indicate the way in which the resin has been functionalized. 2. Detailed references are available on request from the author. 3. I. Coin, M. Beyermann, Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences, Nature Prot. 2, pp (2007). 4. W. Van den Nest, F. Albericio, The choice of the solid support, In optimization of solid-phase combinatorial synthesis (B. Yan, A. W. Czarnik, eds.) Marcel Dekker, New York (NY, USA), pp (2001). 5. A. R. Vaino, K. D. Janda, Solid-phase organic synthesis: a critical understanding of the resin, J Comb Chem. 2, pp (2000). 6. D. Hudson, Matrix assisted synthetic transformations: a mosaic of diverse contributions. I. The pattern emerges, J Comb Chem. 1, pp (1999). 7. M. Meldal, Properties of solid supports, Meth Enzymol. 289, pp (1997). 8. R. B. Merrifield, Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide, J Am Chem Soc. 85, pp (1963). 9. W. Li, X. Xiao et al., Kinetic comparison of amide formation on various cross-linked polystyrene resins, J Comb Chem. 1, pp (1999). FAYNA GARCIA MARTIN 1,2, FERNANDO ALBERICIO 1,2,3 1. Institute for Research in Biomedicine Barcelona Science Park, University of Barcelona 2. CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine Barcelona Science Park 3. Department of Organic Chemistry, University of Barcelona Table 1. Syntheses of difficult peptides to obtain and the protocols used to achieve them. 10. E. Bayer, M. Dengler et al., Peptide synthesis on the new polyoxyethylenepolystyrene graft copolymer, synthesis of insulin B[21-30], Int J Peptide Protein Res. 25, pp (1985). 11. S. Zalipsky, J.L. Chang et al., Preparation and applications of polyethylene glycol-polystyrene graft resin supports for solid-phase peptide synthesis, React Polym. 22, pp (1994). 12. O.W. Gooding, S. Baurdart et al., On the development of new poly(styreneoxyethylene) graft copolymer resin supports for solid-phase organic synthesis, J Comb Chem. 1, pp (1999). 13. J.H. Adams, R.M. Cook et al., A reinvestigation of the preparation, properties, and applications of aminomethyl and 4-methylbenzhydrylamine polystyrene resins, J Org Chem. 63, pp (1998). 14. F-I Auzanneau, M. Meldal et al., Synthesis, characterization and biocompatibility of PEGA resins, J Pept Sci. 1, pp (1995). 15. E. Atherton, D. L. Clive et al., Polyamide supports for polypeptide synthesis, J Am Chem Soc. 97, pp (1975). 16. M. Renil, R. Pillai, Synthesis, characterization and application of tetraethylene glycol diacrylate crosslinked polystyrene support for gel phase peptide synthesis, J Appl Polym Sci. 61, pp (1998). 17. M. Kempe, G. Barany, CLEAR: A novel family of highly cross-linked polymeric supports for solid-phase peptide synthesis, J Am Chem Soc. 118, pp (1996). 18. E. Bayer, M. Mutter, Liquid phase synthesis of peptides, Nature 237, pp (1972). 19. In this kinetic study they observed that during the first coupling lower PEG molecular weight gave higher coupling rates. However, in the case of the tripeptide synthesis, using the major PEG chain (~20000 molecular weight) the highest reaction rate was achieved. 20. J. Rademann, M. Grøtli et al., SPOCC: a resin for solid-phase organic chemistry and enzymatic reactions on solid phase, J Am Chem Soc. 121, pp (1999). 21. S. Côté, New polyether based monomers and highly cross-linked amphiphile resins WO S.A. Camperi, M.M. Marani et al., An efficient strategy for the preparation of one-bead-one-peptide libraries on a new biocompatible solid support, Tetrahedron Lett. 46, pp (2005). 23. F. García-Martín, M. Quintanar-Audelo et al., ChemMatrix, a poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides, J Comb Chem. 8, pp (2006). 24. S.B.H. Kent, Chemical synthesis of peptides and proteins, Ann Rev Biochem. 57, pp (1988). 25. J. Tam, Y. Lu, Coupling difficulty associated with interchain clustering and phase transition in solid phase peptide synthesis, J Am Chem Soc. 117, pp (1995). 26. M. Delgado, K. Janda, Polymeric supports for solid phase organic synthesis, Curr Org Chem. 6, pp (2002). 27. N. Zinieris, C. Zikos et al., Improved solid-phase peptide synthesis of difficult peptides by altering the microenvironment of the developing sequence, Tetrahedron Lett. 47, pp (2006). 34