Solid-phase synthesis ofC-terminally modified peptides

Biopolymers, 2003

In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.

Tetrahedron Letters, 1989

Two general methods for labeling synthetic peptides with a 5-dimethylamino-1-napthalenesulfonyl idansyl) group at the C-terminal residue using solid phase peptide synthesis (SPPS) are described. Dansylated peptides are ideal substrates for fluorometric proteolytic enzyme assays. We have shown the utility of peptides labeled at their amino termini with a fluorescent 5-dimethylamino-l-napthalenesulfonyl (dansyl) moiety as substrates for proteolytic enzyme assays. The method involves reversephase HPLC separation of substrate and enzymically generated product which are detected and quantified fluorometrically. An example is the assay used to follow the rates of conversion of model glycine-extended peptides to C-terminal peptide amides by the peptidyl d-amidation enzyme.' It is also possible to assay enzymes which perform amino terminal peptide modifications or endoproteolytic cleavages using peptide substrates dansylated at the C-terminus. This communication outlines two strategies for the specific labeltng of peptides at the C-terminus using solid phase peptide synthesis

Journal of the American Chemical Society, 1998

Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptidessincluding cyclic peptidessby either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the R-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly-(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene-1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for N R-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.

Journal of the American Chemical Society, 1998

Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptidessincluding cyclic peptidessby either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the R-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly-(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene-1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for N R-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.

Journal of Combinatorial Chemistry, 2000

A method for solid-phase peptide synthesis in the N-to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA 1 -AA 2 -AA 3 ) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N,N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA 2 in the coupling of AA 3 was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA 1 and AA 2 . Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2000

A new method is described for the selective`in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined A-amino group of a lysine residue. The K-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially-available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective`in synthesis' labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported.

Journal of Peptide Science, 1996

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modillcations including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C-and/or N-terminals. After incubation with living cultured cells these two probes were locallzed and it is concluded that the post-synthesis modillcations can strongly modify the localization of the peptide.

Amino Acids, 2014

Here we review the strategies for the solidphase synthesis of peptides starting from the side chain of the C-terminal amino acid. Furthermore, we provide experimental data to support that C-terminal and side-chain syntheses give similar results in terms of purity. However, the stability of the two bonds that anchor the peptide to the polymer may determine the overall yield and this should be considered for the large-scale production of peptides. In addition, resins/linkers which do not subject to side reactions can be preferred for some peptides.

The Journal of Organic Chemistry, 1994

Two novel handles for peptide amide preparation under mild conditions were developed for use in highly efficient solid-phase peptide synthesis. These handles, 5-{ [(R,S)-5-[(9-fluorenylmethoxy-carbony1)aminol-l0,l l-dihydrodibenzo[u,dlcyclohepten-2-ylloxy}valeric acid (CHA) and 5-{ [(R,S)-5-[(9-fluorenylmethoxycarbonyl)aminoldibenzo[u,dlcyclohepten-2-ylloxy}vale~c acid (CHE), were attached to the solid support and were used for syntheses of peptides having a C-terminal amide by the fluorenylmethoxycarbonyl strategy. The cleavability of CHA and CHE was determined and compared with the that commercially available amide handles. CHA and CHE handles can be rapidly cleaved from the polymer support without significant side reactions using lower acid concentrations than those required for conventional handles. As CHA can be easily synthesized in large amounts, it is suitable for peptide amide preparation for pharmaceuticals. As CHE can be cleaved at very low concentrations of acid, it is especially suitable for preparing side chainprotected peptide amides. Several brain-gut peptides having a C-terminal amide were synthesized in high yield and high purity with these novel handles.

Analytical Biochemistry, 1996

of peptides is also studied to reproduce specific determi-Recent developments in allyl chemistry and pallanants of high-molecular-weight proteins. The length of dium solubilization allow automated continuous-flow the peptides used for these studies is in general limited solid-phase synthesis of cyclic or branched peptides, (up to 15 amino acids) to overcome the problems that with specific side-chain cleavage and on-line cyclizaarise with increasing length of the peptide. However, tion. In this paper, we adapted the method to the synthese peptides show a higher flexibility compared to thesis of cyclic peptides bearing an anchoring tail on the native protein. For this reason, their biological aca side chain of the cycle. Side products were obtained tivity is usually lower than in the native conformation with the standard procedure and an additional washunless they are in some way rigidified. One way to ing step had to be introduced in the synthesis protocol achieve this is to synthesize different head-to-tail cyclic to remove side products resulting from the palladium peptides with restricted conformational flexibility and allyl cleavage step. The method is illustrated by the to determine which of these analogs retains biological automated synthesis of cyclo[-DVal-Arg-Gly-Asp-Glu activity (1-5). (-eAhx-Cys-NH 2)-] which contains the Arg-Gly-Asp ad-Series of cyclic peptides have been used to study the hesion motif (RGD) recognized by cellular integrins. conformation-activity relationship of the Arg-Gly-Asp The tail of the peptide was designed with a thiol at the (RGD) 3 sequence (6-9), which is found in many procarboxylic end to allow subsequent grafting by covateins of the extracellular matrix like fibronectin, collalent attachment. Such tailed cyclic peptides can be gens, laminin, or tenascin (10) but also in dysintegrins grafted on different supports for new applications in like kistrin (11) or echistatin (12) present in venoms. biomaterial design, cell adhesion assays, affinity chro-This RGD sequence has been shown to be specifically matography, immunization, vaccine development, recognized by cell membrane receptors known as inte-ELISA kits, and the building of libraries of conformationally constrained peptides.