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.

These electron transfer agents often have unusually low redox potentials. Eighteen are known from higher plants and bacteria, and are involved in nitrogen fixation as well as photosynthesis.

Iron-sulphur proteins.

Peptides are a very important biologically active class of naturally occurring compounds, and the number of newly discovered peptides has grown rapidly. The need for a large number of synthetic peptides and peptide analogs has grown even faster, and the most effective preparative methods are essential. This chapter describes solid-phase peptide synthesis. In the original and simplest form, solid-phase peptide synthesis involves a heterogeneous reaction mixture composed of an insoluble resin-bound peptide chain, a soluble activated amino acid derivative, and a solvent. The solid-phase principle for peptide synthesis was conceived and developed for providing a rapid, simplified, and effective way to prepare peptides and small proteins. This chapter focuses on solid supports, methods for attachment and removal of the peptide chain, protecting group schemes, and coupling methods. It also discusses difficult couplings and peptide ligation methods. The chapter also describes special synthetic applications including protein–nucleic acid compounds and the de novo design and synthesis of peptides and proteins.

3 – Solid-Phase Peptide Synthesis

Die chemische Totalsynthese von Proteinen tritt in die Phase der Realisierbarkeit ein. Damit wird Wirklichkeit, was Emil Fischer schon 1902 voraussagte11 : „Ich sehe deshalb die Zeit voraus wo die physiologische Chemie … sich auch kunstliche Fermente bereitet.” Die bisher fur Peptide aus bis zu zwanzig Aminosauren sicher etablierte Festphasensynthese an polymeren Tragern konnte durch die Entwicklung neuer Polymere und moderner Methoden zur Trennung und Charakterisierung von Polypeptiden wesentlich verbessert werden. Hydrophilie, moglichst homogene Korngrosenverteilung, gleiche Quellung in verschiedenen zur Peptidsynthese verwendeten Losungsmitteln sowie Druckstabilitat sind die wesentlichsten Eigenschaften der neuen Trager fur schnelle Peptidsynthesen im kontinuierlichen Durchflus. Mit Pfropfcopolymeren aus schwach vernetztem Polystyrol und linearem Polyethylenglycol kann im Routinebetrieb der Synthesecyclus fur die Anknupfung eines Aminosaurerestes auf 10-20 min verkurzt und zugleich eine hohere Ausbeute erzielt werden. Mit monodispersen Pfropfcopolymeren von 10 μm Durchmesser last sich die Dauer eines Synthesecyclus im Prinzip auf 1-5 min verringern. Mit dieser Hochgeschwindigkeits-Festphasensynthese konnen auch langere Peptide bis in den Bereich von Proteinen in wenigen Stunden aufgebaut werden. Mit neueren massenspektrometrischen Methoden, wie der Ionenspray-Massenspektrometrie, konnen Proteine bis zu Molmassen von 100 kDa charakterisiert werden, und mit der Kapillarzonenelektrophorese steht neben der HPLC eine weitere effiziente Trennmethode zur Verfugung.

Auf dem Weg zur chemischen Synthese von Proteinen

The stepwise synthesis and conformational studies of the N-terminal helical partial sequence of the membrane-modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N-t-Boc-L-Pro-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH and N-Ac-Aib-L-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α-helix formation of the N-protected peptide polymers beginning with the nonapeptide and of the N-protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature- and solvent-dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random-coil conformation.



For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib-L-Ala)n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right-handed α-helix with increasing peptide length. This change is observed for the N-protected peptides at a chain length of 8 residues and for the N-protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene-bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t-Boc-(Aib-L-Ala)n-OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.

H. Hagenmaier

Papers

Untersuchungen zur totalsynthese des ferredoxins—I

Untersuchungen zur optimierung der peptidsynthese an polymeren trägern—III