Polystyrene-bound peptide

Peptides can be chemically transformed on insoluble supports to yield other types of oligomer. These transformations include N-alkylation and reduction. N-Alkylation of polystyrene-bound peptides enables the preparation of peptide mimetics with improved enzymatic stability [270], Polyamines have been prepared by exhaustive reduction of peptides with borane [271] (see Section 10.1.6). N-Alkylated polyamines can be prepared on solid phase by reduction of the corresponding N-alkylated peptides [272]. 

In the stepwise synthesis of peptides using cross-linked polystyrene supports, the rate of incorporation of a particular amino acid residue has been found to decrease with increasing chain length in a number of instances 41 -43). These occurrences have been attributed to steric hindrance at the various functional sites on the heterogeneous network. Sheppard investigated the origin of this steric hindrance and its sudden and unpredictable onset on the reactivity and physicochemical characteristics in the case of the cross-linked polystyrene-bound peptides 44). These studies suggested that the physicochemical incompatibility of the polystyrene matrix with the attached peptides is the factor responsible for the undesired influences of the solid support on the synthetic manipulations 44 45>. 

Thus, in the non-polar medium, due to the collapse of the otherwise partially extended peptide chain within itself, one can rationalize the steric hindrance in the polar medium, due to the collapse of the otherwise extended polystyrene backbone, some of the peptide chains can be visualized as buried and hence hindered to the approach of reagents and solvents. Thus, based on these models of polystyrene-bound peptides in different environments (Fig. 1), an ideal situation would be that where both the polymer and the peptide chains are extended. This is likely to be attained most easily if the polymer and peptide are of comparable polarities and are placed in a good solvating medium. This is in contrast to the polystyrene case where the macromolecular support is a pure hydrocarbon physicochemically quite dissimilar to the peptide chain being synthesized 44). 

Fig. 1. Hypothetical representation of polystyrene-bound peptides in (A) non-polar solvent and (B) polar solvent...

Scheme 4. Chain-doubling in Polystyrene-Bound Peptides.

Andreatta and Rink U9) synthesized model-peptides on a linear polystyrene of molecular weight 20,000, employing the gel filtration technique for the separation of the polystyrene-bound peptides from excess reagents. These investigators observed that the maximum leading to maintain satisfactory solubility properties of the polymer-peptide was 0.5 mmol peptide per gram of polymer. 

For example, Yu et al. showed that polystyrene-bound peptides could be hydrolyzed in 7 min in a domestic MW oven, a process normally taking 24 h. Furthermore, traditional soHd-phase peptide couplings were achieved in 4 min in 99-100% conversion with no detected racemization. A broad range of solid-phase reactions was found to undergo substantial rate acceleration, including Claisen and Knoeve-nagel condensations, nucleophilic substitutions, sucdnimide and hydantoin formation, and Suzuki coupHngs. 

A series of polystyrene-bound substituted benzophenone oximes 71a-d have been synthesized and tested by DeGrado and Kaiser as potential supports for the solid-phase preparation of protected peptide fragments (Scheme 38). ... 

IR spectroscopy is not a very sensitive analytical tool and is, therefore, not well suited to the detection of small amounts of material. If, however, intermediates have intense and well-resolved IR absorptions, the progress of their chemical transformation can be followed by IR spectroscopy [83,88,91-93], Near-infrared spectroscopy, in combination with an acousto-optic tunable filter, can be sufficiently sensitive to enable the on-bead identification of polystyrene-bound di- and tripeptides, even if the peptides have very similar structures (e.g., Leu-Ala-Gly-PS and Val-Ala-Gly-PS) or differ only in their amino acid sequence (e.g., Leu-Val-Gly-PS and Val-Leu-Gly-PS) [94]. Special resins displaying an IR and Raman barcode have been developed, which may facilitate the deconvolution of combinatorial compound libraries prepared by the mix-and-split method [48]. 

The preparation of cyclic peptides by intramolecular nucleophilic cleavage of polystyrene-bound 2-nitrophenyl esters has also been reported [287],... 

Nitro- or 2,4-dinitrobenzenesulfonamides of primary or secondary amines can be hydrolyzed under mildly basic conditions, and are increasingly being used for amine protection (see Section 10.1.10.7 [123,139,140]). /V-(2-Nitrobenzenesulfonyl)amino acids can be used as an alternative to TV-Fmoc amino acids for the solid-phase synthesis of peptides [141]. Deprotection is achieved by treatment of the polystyrene-bound sulfonamide with a solution of PhSH (0.5 mol/L) and K2C03 (2 mol/L) in DMF for 10 min at room temperature [141], conditions that do not lead to cleavage of esters (e.g. of the Wang linker) or to racemization. The condensation of polystyrene-bound sulfinamides H2N-SO-Pol with aldehydes yields /V-sulfinylimines, which add... 

Acrylic acid esterified with cross-linked hydroxymethyl polystyrene or Wang resin reacts smoothly with primary or secondary aliphatic amines at room temperature (Entries 1 and 2, Table 10.6). Only sterically demanding amines or amines of low nucleophilicity (anilines, a-amino acid esters) fail to add to polystyrene-bound acrylate. Support-bound acrylamides are less reactive than acrylic esters, and generally require heating to undergo addition with amines (Entries 4 and 5, Table 10.6). a, 3-Unsaturated esters with substituents in the 3-position (e.g. crotonates, Entry 3, Table 10.6) react significantly more slowly with nucleophiles than do acrylates. The examples in Table 10.6 also show that polystyrene-bound esters are rather stable towards aminolysis, and provide for robust attachment even in the presence of high concentrations of amines. Entry 10 in Table 10.6 is an example of the alkylation of a resin-bound amine with an electron-poor alkene to yield a fluorinated peptide mimetic. 

Polystyrene-bound amides, including peptides, can be reduced to the corresponding amines by treatment with borane in ethereal solvents. Other reagents, such as lithium aluminum hydride, are less convenient for reductions on insoluble supports, because insoluble precipitates can readily form and clog frits. Carbamates, tert-butyl ethers or thioethers, and trityl or benzhydryl amines remain unchanged upon treatment with borane, but carboxylic esters may undergo partial or complete reduction [178],... 

Phthalimide protection is stable towards acids and bases, but can be cleaved with strong nucleophiles, such as hydrazines or sulfides, or by reduction with sodium boro-hydride [230]. More sensitive towards nucleophilic attack than unsubstituted phthalimide is tetrachlorophthalimide [33]. This group has been successfully used as N(a) protection of amino acids in the solid-phase synthesis of peptides (deprotection N2H4/DMF (15 85), 40 °C, 1 h coupling DIC/HOAt/amino acid (1 1 1), 3 equiv. of each, DMF, 25 °C, 4 h [294]). Typical conditions for the removal of phthaloyl protection on cross-linked polystyrene include treatment of the resin with hydrazine hydrate [295,296], with methyl hydrazine [297], or with primary aliphatic amines [298] in DMF, EtOH, or solvent mixtures for several hours at room temperature or above [296,299,300]. Illustrative examples are sketched in Figure 10.15. It has been claimed that the hydrazinolysis of polystyrene-bound phthalimides proceeds more readily in DCM or DCE than in DMF [301]. 

Support-bound 1,2-diamines can be readily converted into imidazolidinones by treatment with carbonyl diimidazole [128,129]. The required diamines have been prepared on cross-linked polystyrene by reduction of peptides bound to MBHA resin with borane. Similarly, bicyclic imidazolines have been prepared from triamines and thiocarbonyl diimidazole (Entry 10, Table 14.3). Dehydration of polystyrene-bound monoacyl ethylene-1,2-diamines yields 4,5-dihydroimidazoles (cyclic amidines, Entry 5, Table 13.18). Several groups have reported the synthesis of 2-aminoimidazol-4-ones from resin-bound amino acid derivatives (e.g., Entry 6, Table 15.11). Most of these compounds are, however, unstable, and slowly decompose if dissolved in DMSO (Jesper Lau, private communication). 

Based on the principle of the equal and simultaneous solvation of the polymer and the bound peptide chains in different solvents, Sheppard and coworkers developed a number of polyacrylamide-type supports for solid phase peptide synthesis 50 55). In this case, the crosslinked polymeric support, in addition to possessing the good mechanical characteristics like polystyrene, is much more structurally related to the peptide than in the case of polystyrene. The polar polyacrylamide support in this case is prepared by the emulsion copolymerization of a mixture of dimethylacrylamide (7), ethylenebisacrylamide (2) and acryloylsarcosine methylester (5), initiated by ammo-nium persulphatesl). 

For the effective coupling of amino acid residues to the polymer-bound peptide chain, the reaction should be carried out in solvents which swell the resin. Wieland et al. determined the swelling factors for a 2% cross-linked polystyrene-bound Boc-Phe resin in different solvents152>. The volumes of 1 g of this Boc-Phe resin after treatment for 50 hours in different solvents are given in Table 1. These values show that in the case of the polystyrene resin, swelling is minimum in very polar solvents (like methanol) or in very nonpolar solvents (like hexane) and is greatest in chlorinated solvents (like methylenechloride). 

The free amide peptide was synthesized on 4-methylbenzhydrylamine-substituted polystyrene resin, and the resin-bound peptide for antibody production was prepar on aminomethyl polystyrene. Cleavage/deprotection was done with hydrogen fluoride/anisole at 0 C. The free amide was not active in either bioassay. However, polyclonal antibody prepared against the resin-bound (22 24) sequence was immunologically reactive to EDNH reverse phase column fractions. We are now attempting to determine if the sequence represents an inactive fragment of authentic EDNH. 

Most synthetic methods for the generation of peptide libraries have been derived from various multiple parallel peptide synthesis techniques developed since 1984.bs-i l Consequently, the sohd supports used for hbrary synthesis are essentially the same as those used for multiple peptide synthesis. Standard divinylbenzene cross-linked polystyrene resins are typically used for hbraries that are cleaved from the resin and screened in solution.P Polyoxyethylene-grafted polystyrene resins,f l or acrylamide-polyoxyethylene copolymers, P on the other hand, are the sohd supports of choice for the synthesis of resin-bound peptide hbraries screened in sohd-phase binding assays.P Such resins are compatible with both organic solvents used for peptide synthesis, as well as aqueous buffers used in the bioassays. Various segmental supports previously employed for multiple peptide syntheses have also been utilized for the synthesis of peptide libraries, including polypropylene pins, PI cotton,t cellulose membrane,and glass shdes.P ... 

Najera, C. (2005) Polystyrene-bound 1-Hydroxybenzotriazole (PS-HOBT), in Handbook of Reagents for Organic Synthesis, Reagents for Glycoside, Nucleotide and Peptide Synthesis (ed. D. Crich), John Wiley Sons Ltd, Chichester, pp. 523-5. 

One key advantage of SPPS, which is often overlooked, is the tremendous solvation of the peptide on the solid support. As discussed before, fully protected peptides are poorly soluble in organic solvents such as dimethylformamide (DMF). However, as the polypeptide grows on a solid support (typically cross-linked polystyrene, although many new resins have been introduced in recent years) the peptide remains soluble and the peptide resin swells as much as 10-fold in volume. As a result, resin bound peptides are effectively in solution at a much higher concentration than the same peptide that is free in solution [20]. 

In general, the synthesis of relatively nonpolar sequences (cf. 18-19 in Fig. 17), proceeds efficiently on the nonpolar polymer matrix, polystyrene, but the assembly of strongly polar sequences (cf 20-21 in Fig. 17) is particularly difficult on this polymer [70a]. This arises because the hydrophilic grafts (20-21) are not compatible with the nonpolar polymer backbone. As a result, the polymer-bound peptide chains interact within themselves, and become inaccessible as a result of intra-resin H-bonding [71]. Interestingly, an opposite problem of polymer-peptide incompatibility is observed in the case of the polar polymer, dimethylacrylamide. In this case, peptide synthesis proceeds favorably for polar sequences (cf 20-21), but the synthesis of strongly hydrophobic sequences (e.g. 18-19, in Fig. 17) is not practicable because of intra-resin hydrophobic aggregation [70b]. For a recent study of peptide-peptide and peptide-polymer interactions and solvation in solid phase synthesis see Ref [72]. 

This solid phase comprises a polystyrene-bound benzylamine unit, in which removal of the prepared peptide unit is triggered by the formation of a benzylic carbocation, stabilized by the three alkoxy electron donating groups (EDG). With the tinker botmd to the Rink resin (11), the authors completed the 0-acylation with N-Fmoc-Phe to obtain 17 (Scheme 12.9). 

Akil985 Akiyama, M., Shimizu, K., Aiba, S. and Katoh, H., N-Hydroxy Amides. III. Active Esters of Polystyrene-bound 1-Hydroxy-2-pyrrolidinone and Their Use in Peptide Synthesis, Bull. Chem. Soc. Jpn, 58 (1985) 1421-1425. 

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