Merrifield solid-phase synthesis steps

Write out all of the steps in a Merrifield solid-phase synthesis of Leu—Pro. 

Then N-Boc-O-benzylserine is coupled to the free amino group with DCC. This concludes one cycle (N° -deprotection, neutralization, coupling) in solid-phase synthesis. All three steps can be driven to very high total yields (< 99.5%) since excesses of Boc-amino acids and DCC (about fourfold) in CHjClj can be used and since side-reactions which lead to soluble products do not lower the yield of condensation product. One side-reaction in DCC-promoted condensations leads to N-acylated ureas. These products will remain in solution and not reaa with the polymer-bound amine. At the end of the reaction time, the polymer is filtered off and washed. The times consumed for 99% completion of condensation vary from 5 min for small amino acids to several hours for a bulky amino acid, e.g. Boc-Ile, with other bulky amino acids on a resin. A new cycle can begin without any workup problems (R.B. Merrifield, 1969 B.W. Erickson, 1976 M. Bodanszky, 1976). 

The polymeric resin used for Merrifield solid-phase peptide synthesis (Section 26.8) is prepared by treating polystyrene with iV-(hydroxymethyl) phthalimide and trifluoromethanesulfonic acid, followed by reaction with hydrazine. Propose a mechanism for both steps. 

A variety of cleavage conditions have been reported for the release of amines from a solid support. Triazene linker 52 prepared from Merrifield resin in three steps was used for the solid-phase synthesis of aliphatic amines (Scheme 22) [61]. The triazenes were stable to basic conditions and the amino products were released in high yields upon treatment with mild acids. Alternatively, base labile linker 53 synthesized from a-bromo-p-toluic acid in two steps was used to anchor amino functions (Scheme 23) [62]. Cleavage was accomplished by oxidation of the thioether to the sulfone with m-chloroperbenzoic acid followed by 13-elimination with a 10% solution of NH4OH in 2,2,2-trifluoroethanol. A linker based on l-(4,4 -dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) primary amine protecting group was developed for attaching amino functions (Scheme 24) [65]. Linker 54 was stable to both acidic and basic conditions and the final products were cleaved from the resin by treatment with hydrazine or transamination with ra-propylamine. 

The solid-phase synthesis of dendritic polyamides was explored by Frechet et al. [49]. Inspired by the technique used by Merrifield for peptide synthesis, the same strategy was used to build hyperbranched polyamides onto a polymeric support. The idea was to ensure the preservation of the focal point and to ease the purification between successive steps. The resulting polymers were cleaved from the solid support, allowing ordinary polymer characterization. The reaction was found to be extremely sluggish beyond the fourth generation. 

The polymer-bound p-nitrobenzophenone oxime (71d) has been found to be a suitable support for stepwise peptide synthesis. Protected peptides can be assembled on 70d by coupling and deprotection steps similar to those employed in the usual Merrifield solid-phase procedures (Scheme 39). Cleavage of peptides from 71d can be accomplished with hydrazine and amino acid esters under mild conditions, which do not affect benzyl ester side-chain protecting groups. 

In a more recent study, Westman and Lundin described the solid-phase synthesis of aminopropenones and aminopropenoates, respectively30 as intermediates for heterocyclic synthesis. Two different three-step methods for the preparation of heterocycles have been developed. The first method involved formation of a polymer-bound ester from a IV-protected glycine derivative and Merrifield resin (Scheme 7.10a), while the second method employed an interesting approach utilising simple aqueous methy-lamine solution for functionalisation of the solid support (Scheme 7.10b). In this latter approach, a variety of hetero cycles were readily synthesised from the generated polymer-bound benzylamine using a two-step protocol (see Section 5.3.3). 

The major disadvantage of solid-phase peptide synthesis is the fact that all the by-products attached to the resin can only be removed at the final stages of synthesis. Another problem is the relatively low local concentration of peptide which can be obtained on the polymer, and this limits the turnover of all other educts. Preparation of large quantities (> 1 g) is therefore difficult. Thirdly, the racemization-safe methods for acid activation, e.g. with azides, are too mild (= slow) for solid-phase synthesis. For these reasons the convenient Merrifield procedures are quite generally used for syntheses of small peptides, whereas for larger polypeptides many research groups adhere to classic solution methods and purification after each condensation step (F.M. Finn, 1976). 

Mata et al. also described a new and robust protocol for the solid-phase synthesis of 2(3-methyl substituted penam derivatives using Merrifield resin as support [210]. The work begins with immobilization of 6,6-dibromopenicillanic acid (171) onto Merrifield resin followed by oxidation with m-chloroperbenzoic acid (MCPBA) to obtain the resin-bound sulfoxide (173). The key-step involves the thermal rearrangement of the corresponding penicillin sulfoxide (Scheme 49). 

As is apparent from this example, the synthesis of a polypeptide requires numerous steps. At each step, the product must be isolated, and if you have worked in an organic chemistry laboratory, you are certainly aware of how much time and energy are required to isolate and purify a product. This makes a polypeptide synthesis quite tedious. In addition, the mechanical losses that occur in each isolation step contribute to lower yields for the overall process. Motivated by these problems, R. B. Merrifield developed a method, called solid phase synthesis, that makes the preparation of a polypeptide much easier. Merrifield was awarded the 1984 Nobel Prize in chemistry for this work. Let s see how it works. 

The breakthrough in peptide chemistry, which opened up applications in biochemistry and molecular biology, was the development of solid phase synthesis by Merrifield in 1963. This formed the basis of automated synthetic procedures in which the nascent peptide chain was covalently linked to a solid support such as a styrene-divinylbenzene copolymer the complex isolation and purification procedures needed to separate reactants and products at the end of each reaction cycle, which characterised previous solution methods of peptide synthesis, were replaced by a simple washing step. With modern automated methods of peptide synthesis, the time for an Fmoc reaction cycle has been reduced to 20 min, so that a 50-residue peptide can be synthesised in a day (Chan and White 2000). 

Solid phase synthesis lends itself easily to automation. This faet was readily apparent to Merrifield, who built the first automated synthesizer (S,5S,5P). Merri-field s pioneering effort was soon followed by other laboratories (60-87) and several synthesizers appeared on the market. The development of the Fmoe synthetic strategy (88,89) allowed for the substantial simplification of automatic synthesizers, since handling of the very unfriendly reagent, hydrofluoric acid, was no longer necessary. Cambridge Research Biochemicals was the first to introduce a Fmoc-based synthesizer, PEPSYNthesizer, to the market. This machine was capable of only one step of the synthesis, but it applied the... 

Synthesis on solid supports was first developed by Merrifield [1] for the assembly of peptides. It has expanded to include many different applications including oligonucleotide, carbohydrate, and small-molecule assembly (see Chapters 11 and 14). The repetitive cycle of steps involved in the solid-phase synthesis of biopolymers can be performed manually using simple laboratory equipment or fully automated with sophisticated instrumentation. This chapter examines typical solid-phase reaction kinetics to identify factors that can improve the efficiency of both manual and automated synthesis. The hardware and software features of automated solid-phase instruments are also discussed. The focus of this discussion is not on particular commercial model synthesizers but on the basic principles of instrument operation. These considerations can assist in the design, purchase, or use of automated equipment for solid-phase synthesis. Most contrasting features have advantages and disadvantages and the proper choice of instrumentation depends on the synthetic needs of the user. 

Any chemist knows that the more steps there are in a chemical synthesis, the lower the final yield. For example, if each step in a 10-step synthesis furnishes a 90 percent yield of product, the yield of the final product will be only about 35 percent. That is why it is not possible to extend Du Vigneaud s masterful syntheses (see chapter 6) of the hormones oxytocin and vasopressin (9 amino acid residues each) to proteins, even small ones such as ribonuclease A (124 amino acid residues). In order to pursue this daunting challenge Robert Bruce Merrifield (1921-2006), at Rockefeller University, devised a new concept solid-phase synthesis. The idea is disarmingly simple covalently attach an amino acid to a macroscopic particle that can be exposed to the reaction, washed, and then separated by simple filtration. Each reaction step requires no chromatography and no crystallization, just washing and filtering. At the end, completed peptide chains are chemically released from the particles. 

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