To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino- and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of omega-amino- and alpha,omega-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the omega-amino- and alpha,omega-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.

The cyclization kinetics of five backbone-cyclic tetrapeptides was investigated both experimentally and computationally. The aim was to both accurately measure the cyclization rates in solution and develop a method that efficiently estimates the relative cyclization tendencies computationally. Progression of the cyclization reaction was monitored directly, yielding the kinetics of changes in the amounts of the linear precursor and the products. These measurements were used to calculate the reaction rates; the results were consistent with a first-order reaction kinetics. In order to predict the cyclization rates computationally, the conformation space of the linear precursors was mapped and used to construct an approximate partition function. We assumed that the cyclization tendency was correlated with the relative probability of being found in a cyclization-prone conformation of the backbone, this probability was estimated from the partition function. The results supported this assumption and demonstrated that, within reasonable accuracy, we are able to predict the relative cyclization tendencies of the peptides measured.

537In recent years there was considerable interest in the synthesis of conformationally restricted bioactive peptides [1 -3]. Due to the reduced flexibility of the peptide backbone these constrained structures are expected to possess improved potency, receptor subtype selectivity and enhanced resistance against proteolytic degradation. Besides other cyclization approaches backbone cyclization has been proven a promising concept for introducing conformational constraints on peptides without affecting the amino acid side chains [4]. To achieve N-backbone cyclization two amide nitrogens of the peptidic backbone have to be modified by alkylation. A variety of N-functionalized amino acids have been prepared and incorporated in peptide sequences [5 -9]. To circumvent difficult coupling steps during peptide synthesis using the solid phase methodology we have developed a method to introduce different types of N-functionalized dipeptide building units which are prepared in solution before their use in SPPS [10,11]. We were further interested in the application of the method of backbone cyclization to somatostatin analogues to probe our synthetic approach of incorporating preformed diAbstract. Somatostatin octapeptide analogues of the general sequence DPhe 5 -Phe 6 -Tyr 7 -DTrp 8 -Lys 9 -Val 10 -Phe 11 -Thr 12 -NH 2 containing two types of backbone cyclization have been synthesized by the solid phase methodology. Backbone cyclization in these peptides was achieved via N-modified phenylalanines in position 6 and 11. The N-modified amino acids were incorporated as dipeptide building units which have been prepared in solution prior to the solid phase synthesis. Two dipeptide units of structure a) Fmoc-aa 1 Ψ[CO-N((CH 2 ) n -X)]Phe-OH or b) Fmoc-aa 1 Ψ[CH 2 -N(CO(CH 2 ) n -X)]Phe-OH peptide units as well as to produce analogues with bioactivity, receptor subtype selectivity and enzymatic stability.The cyclic tetradecapeptide somatostatin (SST-14, SRIF) which was first isolated from ovine hypothalamus [12] is a potent inhibitor of endocrine and exocrine secretion of several hormones including growth hormone, glucagon, insulin and gastrin [13 -15]. It also regulates many other physiological activities and is considered an inhibitor of tumor cell growth [16] through binding to its specific cell surface receptors [17 -19]. Since SRIF and somatostatin-28, a 14 residues longer native form [13], bind to the five known receptors with low selectivity extensive structure-activity-studies have been carried out with a variety of synthetic analogues derived from small peptide structures [20 -22]. In both native peptides the Phe 7 -DTrp 8 -Lys 9 -Thr 10 -moiety has been disclosed as the pharmacophore sequence essential for biological activity of the molecule [20]. This core sequence allows further modifications and therefore a substitution of Phe 7 and Thr 10 by Tyr and Val, respectively, was found out to lead to biologically active comhave been introduced into the peptide sequence. Different resins and linkers were examined for an optimize...

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