Solid-phase synthesis of functionalized bis-peptides

We demonstrate the first solid-phase synthesis of highly functionalized bis-peptides. Bis-peptides are ladder oligomers composed of stereochemically pure, cyclic bis-amino acids joined by substituted diketopiperazine linkages. They have a shape-programmable backbone that is controlled by controlling the stereochemistry and sequence of the monomers within each oligomer. Functionalized bis-peptides are assembled using a new amide bond forming reaction (acyl-transfer coupling) that we have previously developed and a novel activation strategy that allows the sequential formation of penta- and hexa-substituted diketopiperazines from extremely hindered N-alkyl-alpha,alpha-disubstituted amino acids. We present mechanistic evidence that acyl-transfer coupling is competitive with direct acylation in the formation of hindered amide bonds. We also detail the synthesis of four functionalized bis-peptides, and that by combining bis-peptides with amino acids through diketopiperazine linkages, bis-peptides can mimic the display of residues i, i+4, i+7 of an alpha-helical peptide.     

Enhancing solid phase synthesis by a noncovalent protection strategy-efficient coupling of rhodamine to resin-bound peptide nucleic acids.

Resins for solid-phase synthesis can affect coupling efficiencies by interacting with reactants. We have observed that polyethylene glycol-polystyrene (PEG-PS) solid support absorbs added activated fluorophores, preventing efficient labeling of peptide nucleic acids (PNAs). We now report that addition of an inexpensive unactivated fluorophore blocks the resin and allows efficient labeling. This protection strategy may have general benefits for peptide and combinatorial synthesis.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

Novel heterobifunctionalized polystyrene-polyethylene glycol resin for simultaneous preparation of free and immobilized peptides and biological activity detected by confocal microscopy

Summary Fast and convenient binding assays using synthetic peptides are of utmost and increasing importance, especially in the search for lead structures or in the field of diagnostics. A polymeric support suitable for solid-phase peptide synthesis was functionalized with two different anchor groups. The interior part of the aminomethylated polystyrene-1%-divinylbenzene resin beads, comprising about 98% of the total loading capacity, was modified by the acid-labile ADPV anchor whereas the 2% outer surface of the polymer was covalently coated with a PEG 10 000 derivative which renders the resin surface hydrophilic and biocompatible. The novel resin was characterized by introducing marker amino acids and by infrared spectroscopy. Employing this bifunctionalized resin for peptide synthesis, free as well as polymer-bound peptides were obtained which were tested for recognition by antibody. The resin-bound peptides proved to be suitable for ELISA and fluorescence assays, as shown by confocal laser microscopic investigations. Peptides from the interior part were obtained in high yield and purity as analyzed by HPLC, electrospray mass spectrometry and Edman degradation.     

Combined solid-phase and solution approach for the synthesis of large peptides or proteins.

In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid-phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free alpha-carboxyl group from the solid support, base-labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N-[9-(hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2-bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base-catalysed conditions, the base-resistant 3-pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side-chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid-phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors.     

Solid-phase peptide synthesis without side-chain hydroxyl protection of threonine.

A peptide containing four threonine residues was synthesised by the solid-phase method using fluorenyl-methoxycarbonylamino acid reactive esters or coupling by preactivation with 1-hydroxybenzotriazole and Castro's reagent. In two separate experiments the synthesis was carried out with or without protection of the side-chain hydroxyl group of threonine as the tert.-butyl ether. Comparison of the crude peptides after deprotection and detachment from the synthesis resin suggests that side-chain protection of threonine is unnecessary under the synthetic conditions employed.     

Developments in peptide and amide synthesis

The solid-phase methodology is key for an effective synthesis of peptides, from a milligram scale for research to a multi-kilo scale for drug production. Indeed, small peptides containing up to 20-30 amino acids are most readily synthesized by a solid-phase strategy. Larger peptides (up to 60 amino acids) should be synthesized by a convergent approach (i.e. synthesis of protected constituent peptides in solid-phase and combination of these units in solution). Larger peptides and proteins are prepared by chemical ligation, where unprotected segments have been prepared in solid-phase.     

Orthogonal protecting groups for N(alpha)-amino and C-terminal carboxyl functions in solid-phase peptide synthesis

For the controlled synthesis of even the simplest dipeptide, the N(alpha)-amino group of one of the amino acids and the C-terminal carboxyl group of the other should both be blocked with suitable protecting groups. Formation of the desired amide bond can now occur upon activation of the free carboxyl group. After coupling, peptide synthesis can be continued by removal of either of the two protecting groups and coupling with the free C-terminus or N(alpha)-amino group of another protected amino acid. When three functional amino acids are present in the sequence, the side chain of these residues also has to be protected. It is important that there is a high degree of compatibility between the different types of protecting groups such that one type may be removed selectively in the presence of the others. At the end of the synthesis, the protecting groups must be removed to give the desired peptide. Thus, it is clear that the protection scheme adopted is of the utmost importance and makes the difference between success and failure in a given synthesis. Since R. B. Merrifield introduced the solid-phase strategy for the synthesis of peptides, this prerequisite has been readily accepted. This strategy is usually carried out using two main protection schemes: the tert-butoxycarbonyl/benzyl and the 9-flourenylmethoxycarbonyl/tert-butyl methods. However, for the solid-phase preparation of complex or fragile peptides, as well as for the construction of libraries of peptides or small molecules using a combinatorial approach, a range of other protecting groups is also needed. This review summarizes other protecting groups for both the N(alpha)-amino and C-terminal carboxyl functions.     

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.     

The synthesis and study of side-chain lactam-bridged peptides

Side-chain lactam bridges linking amino acid residues that are spaced several residues apart in the linear sequence offer a convenient and flexible method for introducing conformational constraints into a peptide structure. The availability of a variety of selectively cleavable protecting groups for amines and carboxylic acids allows for several approaches to the synthesis of monocyclic, dicyclic, and bicyclic lactam-bridged peptides by solid-phase methods. Multicyclic structures are also accessible, but segment-condensation syntheses with solution-phase cyclizations are most likely to provide the best synthetic approach to these more complex constrained peptides. Lactam bridges linking (i, i + 3)-, (i, i + 4), and (i, i + 7)-spaced residue pairs have all proven useful for stabilization of alpha helices, and (i, i + 3)-linked residues have also been demonstrated to stabilize beta-turns. These structures are finding an increasing number of applications in protein biology, including studies of protein folding, protein aggregation, peptide ligand-receptor recognition, and the development of more potent peptide therapeutics. Defining the functional roles of the amphiphilic alpha-helices in medium-sized peptide hormones, and studying helix propagation from rigid, alpha-helix initiating bicyclic peptides are among the most exciting developments currently underway in this field.     

Solution-phase synthesis and evaluation of tetraproline chiral stationary phases

A protocol was developed for the solution-phase synthesis of multigram amounts of two 9-fluorenylmethoxycarbonyl (Fmoc)-protected tetraproline peptides. These tetraproline peptides were then attached to amino derivatized silica gel. The replacement of the Fmoc group with the trimethylacetyl group lead to two tetraproline chiral stationary phases (CSPs). A comparison of the chromatographic behavior of these two solution-phase-synthesized tetraproline CSPs with that prepared by stepwise solid-phase synthesis revealed that all three had similar chromatographic performance for resolving 53 model analytes. This suggests that the solution-phase synthesis of oligoprolines, which allows for the specific benefits of good batch reproducibility, selector homogeneity, and possibly low cost, is a feasible alternative to the solid-phase synthesis of oligoproline CSPs.     

Solid-phase/solution-phase combinatorial synthesis of neuroimmunophilin ligands

A novel solid-phase/solution-phase strategy for the synthesis of neuroimmunophilin ligands based on GPI 1046 was developed. The synthesis employs a solid-phase esterification strategy followed by a solution-phase pyruvic amide formation to produce multi-milligram quantities of discrete compounds for assay. The protocol was applied to a production library of 880 discrete compounds. A highlight of the strategy is an aqueous extractive purification of the final compounds using a novel liquid/ice extraction system developed for high throughput.     

Synthesis of cyclic penta- and hexapeptides: A general synthetic strategy on DAS resin

The active part or receptor-binding sequence of peptide hormones can usually be defined by a span of 4–8 amino acids. Cyclic penta- and hexapeptides are excellent model systems for performing conformational and structure-function studies on this class of bioactive molecules. A synthetic scheme has been devised comprising solid-phase Fmoc chemistry followed by resin cleavage, cyclization in solution, and, finally, side-chain deprotection. A new resin, DAS, cleaved under weak acid conditions, is an excellent solid-phase synthesis support, and HBTU or PyBOP are the activation reagents of choice, not only during synthesis, but also for the cyclization reaction. Three cyclic peptides were synthesized using this method, one requiring extensive side-chain protection, and this method has general applicability for any cyclic pentapeptide or hexapeptide, giving good yields and high purity.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group

In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Fmoc-based chemical synthesis and selective binding to supercoiled DNA of the p53 C-terminal segment and its phosphorylated and acetylated derivatives.

The C-terminal domain of p53 comprises a linker, the tetramerization domain and the regulatory domain, and contains at least seven sites of potential post-translational modification. An improved strategy was developed for the synthesis of large peptides that contain phosphorylated amino acids and p53(303-393), a 91-amino acid peptide, and three post-translationally modified derivatives were synthesized through the sequential condensation of three partially protected segments. Peptide thiolesters were prepared using the sulfonamide-based 'safety-catch' resin approach and employing Fmoc-based solid-phase peptide synthesis. At the N-terminus of the middle building block, a photolabile protecting group, 3,4-dimethoxy-6-nitrobenzyloxycarbonyl, was incorporated to differentiate the N-terminal amino group from the side-chain amino groups. Two sequential couplings were accomplished following this protection strategy. The synthetic products, p53(303-393) and its phosphorylated or acetylated derivatives, exhibited the ability to bind specifically to supercoiled DNA, which is one of the characteristics of this domain.     

Exploring Solution-Phase Cyclization and Sulfamyl Safety-Catch Resin Strategies for the Total Synthesis of the Marine Antimicrobial Cyclic Tetrapeptide Cyclo(GSPE)

Head-to-tail cyclization of 12-membered tetrapeptides has been reported to be synthetically challenging due to their highly strained ring systems. Previously, we reported the total synthesis of the marine antimicrobial cyclic tetrapeptide cyclo(GSPE) using the glutamic acid side-chain Wang resin tethering strategy with a 5 % yield under microwave irradiation (Lim et al., Int J Pept Res Ther 16:145–152, 2010). Besides the low yield, this strategy was also constrained to glutamic acid-containing peptides. In this communication, we report the outcomes of two alternate strategies to synthesize cyclo(GSPE): (A) solution-phase cyclization and (B) solid-phase synthesis using the sulfamylbutyryl safety-catch resin. Experimental results using all four possible linear precursors revealed that the former strategy failed to produce the target cyclic peptide while the latter approach afforded an improved overall yield of 8–9 % using the linear precursor H-Ser(tBu)-Pro-Glu(OtBu)-Gly-resin. Interestingly, we discovered that the placement of proline at the N- or C-termini of the linear peptide precursors failed to produce the target cyclic peptide. We also concluded that strategy B could provide the versatility needed to make a cyclic tetrapeptide library for bioactivity screening.     

New Allyl Ester Linker and Solid-phase Block Synthesis of the Serglycin Core Region

The prototype glycopeptidyl fragments of serglycin, a proteoglycan with the characteristic peptide sequence of repeating L-seryl-L-glycine, were synthesized by a convergent method involving block condensation on a solid support. In order to facilitate detachment of the protected glycopeptides from the resin, a new allyl ester type of linker, which is cleavable by Pd(0)-catalysis, was designed and used in combination with the commercial acid-labile Sieber amide resin for the solid-phase synthesis. Glycopeptide blocks consisting of [O-(2,3,4-tri-O-acetyl-D-xylosyl)-L-seryl-L-glycine]n (n=1-8) were produced in good yields. Block condensation in a solution was also successful to synthesize up to the hexadecapeptide (n=8).