9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

Organic Synthesis on Soluble Polymer Supports: Liquid-Phase Methodologies.

It is a privilege to be able to contribute to this volume in which Professor Zervas’ friends, students, and colleagues have joined together to honor him and his remarkable contributions to peptide chemistry. Although I did not know him until after his days in the Bergmann Laboratory at the Rockefeller Institute, I can claim the distinction of now working in those very same rooms that they occupied back in the mid-1930s. Like all peptide chemists, I am greatly indebted to Professor Zervas, having depended so heavily on the carbobenzoxy group and on the various modified urethan protecting groups which have been direct extensions of the revolutionary advance that Bergmann and Zervas made.

Solid-phase peptide synthesis.

Soluble polymers as scaffolds for recoverable catalysts and reagents.

Poly(ethylene glycol)s grated onto suitably functionalized, crosslinked polystyrenes were introduced as a new class of multidetachably immobilized hydrophilic macromolecular supports for the synthesis of peptides. This new polymeric carrier system facilitates both the homogeneous solvation of the reaction site and the insolubilization of the substrate, which are prerequisites for an efficient multistep synthesis using polymeric supports. In these support systems, a strongly solvated hydrophilic linear chain molecule, poly(ethylene glycol) (PEG)1 , is attached to an insoluble, crosslinked polystyrene through suitable anchoring linkages. At the free chain end of this supported PEG, amino acid residues were stepwise incorporated following the standard synthetic methodology for peptides. A special feature of these new carriers is that the use of an orthogonal set of anchoring linkages to connect the polystyrene, PEG and the peptide provides access to the PEG-bound solubilized peptide or to the free peptide, as desired. The applications of these new polymeric supports are illustrated by the synthesis of thioredoxin partial sequences in the free and PEG-bound solubilized forms.

Poly(ethylene glycol)s grafted onto crosslinked polystyrenes .2. multidetachably anchored polymer systems for the synthesis of solubilized peptides

Polyethyleneglycols grafted onto crosslinked polystyrenes: A new class of hydrophilic polymeric supports for peptide synthesis

The synthesis of the following oligo- and co-oligopeptides by the liquid-phase method is described: (L-Met)15 (I), [L-Glu(OBzl)]20 (II), (L-Val)8-Gly (IV), (L-Ile)8-Gly (V), (L-Ile)4-Gly-(L-Ile)4 (VI), (L-Ile)4-Pro-(L-Ile)4 (VII), (L-Met)5-L-Pro-(L-Met)5 (VIII), [L-Glu(OBzl)]7-L-Pro-[L-Glu(OBzl)]7 (IX). The oligomers are covalently bound to bifunctional polyethylene glycol (PEG) and monofunctional PEG-M of Mr 5 × 103−2 × 104. Analytical controls were carried out after each step of synthesis in order to ensure quantitative coupling yields. All products could be obtained in high purity as indicated by amino acid analysis, thin-layer chromatography and chiroptical methods. The solubility of the oligomers was strongly enhanced by the presence of the C-terminal PEG group, enabling conformational investigations in a variety of solvents. A significant relationship between conformation and physicochemical properties of the oligopeptides was observed. Oligomers with tendencies to adopt α-helical (I, II) or unordered structures (VI–IX) showed no pronounced change in solubility or coupling kinetics during chain elongation, whereas the onset of a β-structure (IV, V) was paralleled by a drastic decrease in solubility and reactivity of the terminal amino groups. Most notably, the insertion of a proline or glycine in the middle of a β-forming peptide chain (VI, VII) resulted in a considerable increase in solubility compared to the corresponding homo-oligomers. The impact of the conformational properties of a peptide chain on strategic considerations of peptide synthesis in solution is delineated.

Relationship between conformation and physicochemical properties of polypeptides. I. Synthesis of homo‐ and co‐oligopeptides by the liquid‐phase method

Primary and tertiary amine-initiated polymerizations of L-alanine-N-carboxyanhydride (L-Ala-NCA) were conducted at 20 or 100°C in a variety of solvents. The 75.5-MHz 13C-nmr CP/MAS spectra of the resulting poly(L-alanines) revealed that all samples contain both α-helix and pleated-sheet structures. Depending on the reaction conditions the α-helix content varied between ca. 1 and 99%. Reprecipitation from aprotic nonsolvents does not change the α-helix/β-sheet ratio, indicating that this ratio is thermodynamically controlled. Since relatively large amounts of oligopeptides of degree of polymerization (DP) 4–6 can be extracted by means of acetic acid, it is concluded that (a) most poly(L-alanines) possess a bimodal molecular weight distribution, (b) the oligopeptide fraction with DP ⩽ 11 is responsible for the β-sheet fraction of all samples, and (c) the two-stage crystal growth proposed by Komoto and Kawai is not correct. Solubilizing initiators such as poly(ethylene oxide) NH2 prevent the precipitation of oligoalanine and, thus, the formation of a β-sheet structure. 13C-nmr CP/MAS measurements also show that tri- and tetra-L-alanines form insoluble β-sheet structures.

Manfred Mutter

Papers

Poly(ethylene glycol)s grafted onto crosslinked polystyrenes .2. multidetachably anchored polymer systems for the synthesis of solubilized peptides

Polyethyleneglycols grafted onto crosslinked polystyrenes: A new class of hydrophilic polymeric supports for peptide synthesis

Relationship between conformation and physicochemical properties of polypeptides. I. Synthesis of homo‐ and co‐oligopeptides by the liquid‐phase method

Secondary structure of peptides. 4: 13C‐Nmr CP/MAS investigation of solid oligo‐ and poly(L‐alanines)

Applications of soluble polymeric supports