Stepwise Solid-phase Synthesis Research Articles

Stepwise Solid-Phase Synthesis of Peptide-Oligonucleotide Conjugates on New Solid Supports

Several peptide-oligonucleotide and peptide-(oligonucleotide phosphorothioate) conjugates were synthesized on new solid supports. These supports are designed to link the 3′-terminus of an oligonucleotide to the C-end of a peptide via a phosphodiester or phosphorothioate bond in the process of stepwise solid-phase assembly.

Fmoc-Based Synthesis of Peptide-αThioesters: Application to the Total Chemical Synthesis of a Glycoprotein by Native Chemical Ligation

The technique of native chemical ligation has enabled the total chemical synthesis of proteins with molecular weights far in excess of those achievable by conventional stepwise solid-phase peptide synthesis. The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal αthioester and the other an N-terminal cysteine residue, to afford a protein with a native amide linkage at the site of ligation. Here we report an extension of the native chemical ligation method to the total synthesis of a glycosylated protein, the antimicrobial O-linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-residue glycopeptide-αthioester segment, which was complicated by the incompatibility of glycosidic linkages with Boc chemistry and by the incompatibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide “safety-catch” linker circumvented this problem and permitted the solid-phase synthesis of the glycopeptide-αthioester using standa...

Stepwise Solid-Phase Synthesis of Polyamides as Linkers

Solid-phase peptide synthesis (SPPS) yields well-defined polyamides of repeat unit NH-Z-CO but requires protected derivatives and deprotection steps. Polyamides of repeat unit NH-Y-NH-CO-X-CO (e.g., Nylon 66) are made by polymerizing diacids with diamines, but the absence of protecting groups leads to heterogeneity of chain length. We have produced a new class of polymers by automated solid-phase synthesis without protecting groups: polyamide chains NH-Y-NH-CO-X-CO containing a precise number of monomer units where X and Y can be varied independently at each step. Dimers and multimeric molecules which display phage-derived binding peptides in a synthetic construction were easily assembled with these precision length polyamide chains, which can usefully replace polypeptides and poly(ethylene glycol) as linkers.

The Stepwise Solid-Phase Synthesis Methodology is Suitable for the Preparation of a Great Variety of Nucleopeptides

The preparation of nucleopeptides containing tryptophan and basic residues (lysine, arginine) is described. Different solid supports and the necessity of primary carboxamide protection have also been evaluated.

Investigation of crudes of synthesis of carbetocin by liquid chromatography coupled to electrospray ionization mass spectrometry

Synthetic peptides are becoming increasingly important as drugs in the pharmaceutical industry. Stepwise solid-phase peptide synthesis, commonly results in unwanted side products associated with incomplete peptide chains. Here, LC–electrospray MS has been used to examine a crude of synthesis of carbetocin, a peptide with recognized therapeutical application for stimulation of uterine contractions to facilitate parturition. The molecular masses of various side products obtained within the crude of synthesis have been determined and these side products have been identified on the basis of their molecular masses. The knowledge of the impurities present in the crude peptide permits the optimization of the synthetic procedure.

Solid phase synthesis of oligomeric guanidiniums

Oligomers containing guanidinium linkages prepared via solid phase organic synthesis are of interest as possible therapeutic agents and in the assembly of supramolecular architectures. Efficient routes to these oligomers must be developed before their potential may be fully realized. Herein, four routes for their stepwise solid phase synthesis are described. In the first, a resin-bound thiourea was converted to a guanidinium using 2-chloro-1-methylpyridinium iodide. The second method utilized aza-Wittig couplings to prepare guanidiniums from resin-bound carbodiimides. Next, highly activated monomers prepared from bis- tert-butyloxycarbonythioureas and 2,4-dinitrofluorobenzene formed guanidiniums upon reaction with terminal amines. The optimum route, however, relied upon the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride promoted coupling of a protected thiourea monomer with a resin-bound amine to produce the guanidinium linkage. The thiourea monomers for this method are easily prepared from mono-protected diamines and benzoyl- or Fmoc-isothiocyanate. The procedure is straightforward, proceeds cleanly in a relatively short period of time, and is compatible with several functional groups.

How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes

Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60–62 residues and four disulfide bonds) and long-chain toxins (66–74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine α7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.

Racemization studies of Fmoc-Ser(tBu)-OH during stepwise continuous-flow solid-phase peptide synthesis

We observed unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH 2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serine with less than 1 % racemization. We describe unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH 2 , in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serin with less than 1% racemization.

Solid-phase synthesis of single-chain monellin, a sweet protein.

Single-chain monellin, a sweet protein, was synthesized by stepwise solid-phase synthesis. After deprotection, and purification with HPLC, single-chain monellin was obtained in a yield of 0.3% based on the starting resin.

Total chemical synthesis of enzymatically active human type II secretory phospholipase A2.

Human group II secretory phospholipase A2 (sPLA2) is an enzyme found in the alpha granules of platelets and at inflammatory sites. Although its physiological function is unclear, sPLA2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA2 activities, we developed a total chemical synthesis of sPLA2 by chemical ligation of large unprotected peptides. The synthetic segments PLA2-(1-58)-alphaCOSCH2COOH and PLA2-(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly58 and Cys59. The 124-residue polypeptide product (mass: 13,920 +/- 2 Da) was folded to yield one major product (mass: 13,905 +/- 1 Da), the loss of 15 +/- 3 Da reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA2 showed alpha-helix, beta-structure, and random coil contents consistent with those found in the crystal structure of sPLA2. Synthetic sPLA2 had kcat and Km values identical to those of recombinant sPLA2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA2, like recombinant sPLA2, inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca2+, and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca2+. Thus, synthetic sPLA2 is a phospholipid-independent anticoagulant like recombinant or natural sPLA2. This study demonstrates that chemical synthesis of sPLA2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA2 analogs for structure-activity studies of anticoagulant, lipolytic, or inflammatory activities.

Solid-Phase Synthesis of Glycopeptides: Immunological Studies with T Cell Stimulating Glycopeptides

Stepwise solid-phase synthesis using glycosylated amino acids as puilding blocks has been shown to be the most general and efficient approach for preparation of glycopeptides. The a-amino group of the glycosylated amino acids should be protected with the fluoren-9-ylmethoxy­ carbonyl (Fmoc) group whereas acetyl groups, or acid labile protective groups, are suitable for the carbohydrate hydroxyl groups. Due to the recent rapid development of Fmoc solid-phase glycopeptide synthesis glycopeptides are increasingly being made available for investigations in biological sciences, such as studies of the cellular immune response to glycopeptides. These studies have established that glycopeptides are bound well by both class I and . II MHC molecules and elicit a carbohydrate specific T cell response after immunization, provided that the position for the glycan is chosen carefully in the centre of the peptide. Such a location most likely allows contacts between carbohydrate and the CDR3s of the T cell receptor. Investigations using viral glycoproteins suggest that the large N-linked glycans influence the T cell response to glycoproteins, but that this does not involve specific interactions between the glycan and the T cell. Furthermore, viruses seem to exploit mutations which introduce or delete N-linked glycans to escape from attack by T cells. In contrast, studies have revealed that the smaller 0-linked carbohydrates found on collagen or mucins are able to elicit a carbohydrate specific T cell response. Altogether, these immunological studies have suggested novel applications in medicine, including generation of a cellular immune response to carbohydrate antigens found on pathogens or tumour cells, and induction of tolerance in autoimmune rheumatoid arthritis.

An Approach to the Solid Phase Synthesis of Oligonucleotides Containing N-Acylphosphoramidate Internucleosidic Linkages

(2-Cyanoethoxy)(N,N-diisopropylamino)(1-(1-thyminyl)-1,2-dideoxy-3-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranuronamido)phosphine 7 was used as a building unit in a stepwise solid phase synthesis of an immobilized decamer employing 5-mercapto-1-methyltetrazole as activation reagent. Decyanoethylation and release of the immobilized decamer from the support with methanolic potassium carbonate furnished modified oligonucleotide 16.

Protection of asparagine and glutamine during N alpha-Bpoc-based solid-phase peptide synthesis.

In this paper we describe the synthesis and properties of Bpoc-Asn(Trt)-OH, Bpoc-Asn(Trt)-OPfp, Bpoc-Gln(Trt)-OH and Bpoc-Gln(Trt)-OPfp. These derivatives are highly soluble in CH2Cl2 and can be coupled efficiently in solid-phase peptide synthesis. The peptides, acetyl-Ala-Phe-Asn(Trt)-Gly-Leu-Ala-O-Dbf-SH and Boc-Cys(Acm)-Ala-Phe-Gln(Trt)-Gly-Leu-Ala-O-Dbf-SH (where O-Dbf-SH is the peptide ester of 4-mercapto-6-hydroxydibenzofuran) were synthesized by stepwise solid-phase peptide synthesis using N alpha-Bpoc amino acids. We have observed that less than 0.1% of the trityl group is removed from the carboxamide of Gln and Asn during a standard 15 min N alpha-Bpoc deprotection in 0.5% TFA in CH2Cl2.

Synthesis and characterization of indolicidin, a tryptophan-rich antimicrobial peptide from bovine neutrophils.

Indolicidin, a novel tryptophan-rich microbicidal tridecapeptide amide isolated originally from granules of bovine neutrophils, has been prepared by optimized manual and automated protocols of stepwise solid-phase synthesis with N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) amino acid derivatives. Both standard polystyrene (PS) and polyethylene glycol-polystyrene (PEG-PS) graft supports were used in combination with handles that provide C-terminal peptide amides: 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) or 5-(9-Fmoc-aminoxanthen-2-oxy)valeric acid (XAL). Final deprotection/cleavage was carried out with reagent K, trifluoroacetic acid-phenol-water-thioanisole-1,2-ethanedithiol (82.5:5:5:5:2.5), or reagent B, trifluoroacetic acid-phenol-water-tri(isopropyl)silane (88:5:5:2), and related cocktails. Initial purities as high as 93% were obtained immediately following cleavage. In the largest-scale synthesis carried out, 0.8 g of HPLC-purified indolicidin (> 99% pure) was obtained, representing a 39% overall yield based on C-terminal Arg(Pmc) anchored to PAL-PS-resin. The main synthetic product, and some by-products, were characterized by analytical high-performance liquid chromatography (HPLC), sequencing, and fast atom bombardment mass spectrometry (FABMS). The antimicrobial potencies of natural and synthetic indolicidin, as determined by in vitro antibacterial and antifungal assays, were identical. Further, the reactivities of natural and synthetic peptides with anti-indolicidin antibody were indistinguishable.

Purification of synthetic peptides with the aid of reversible chromatographic probes

The application of reversible Chromatographic probes for the purification of synthetic peptides by reversed-phase (RP) and affinity Chromatography is described. In previous work the concept of a one-step purification procedure based on the acid-stable fluorenylmethoxycarbonyl (Fmoc) group was considered. Similar probes were used to purify peptides from 17 to 104 residues in length, using ion-exchange and RP media. The RP probes described here represent simplified versions of those reported previously. Further, the application of a new probe containing biotin, for affinity Chromatography, is demonstrated. Stepwise solid-phase peptide synthesis was performed using both Boc and Fmoc chemistries and N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping reagent. The Chromatographic probes were incorporated on to the “target” peptide as active succinimidyl carbonate derivatives. After cleavage using either hydrogen fluoride or trifluoroacetic acid, purification was performed on either RP media or immobilized avidin. Treatment with organic base yielded the free purified product. The efficiency of the new probe molecules and the capping procedure was demonstrated by the purification of two polypeptides, 46 and 101 residues in length.

Preparation and applications of polyethylene glycol-polystyrene graft resin supports for solid-phase peptide synthesis

Heterobifunctional polyethylene glycol (PEG) derivatives BocNH(CH 2CH 2O) nCONHCH 2CO 2H were attached cleanly onto aminomethyl copoly(styrene-1% divinylbenzene) (PS) resins by oxidation-reduction couplings mediated by tri- n-butylphosphine plus 2,2′-dipyridyl disulfide. The amount of PEG incorporated into the resultant PEG-PS graft resins can be controlled through the degree of aminomethylation of the starting cross-linked PS and/or by use of PEG derivatives of different molecular weight ( i.e., ∼ 2000 for n = 45 ; ∼ 4000 for n = 90 ). The new PEG-PS supports, which have good swelling properties in an extended range of solvents, were evaluated as supports for stepwise solid-phase peptide synthesis (SPPS) using tert.-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) or dithiasuccinoyl (Dts) for N α-amino protection, along with compatible anchoring handles for the C-terminal residue. Several peptide syntheses were carried out in parallel in order to compare PEG-PS supports with the corresponding parent PS resins; reproducible advantages were observed with PEG-PS. In addition, PEG-PS supports were shown to be uniquely suited for SPPS using acetonitrile as the solvent for all coupling steps and washes.

Stepwise solid-phase synthesis of oligonucleotide-peptide hybrids

Oligonucleotide-peptide hybrids containing a nuclear transport signal have been synthesized stepwise on a polyethyleneglycol-polystyrene support using base labile protecting groups for the nucleobases and aminoacid side chains.

Structure-activity relationships of human epidermal growth factor(h-EGF)

The 53 amino acid regulatory peptide, human epidermal growth factor (h-EGF), is a potent mitogen that stimulates cellular proliferation and differentiation in a wide variety of cells. To identify the critical residues that elicit the biological activity of h-EGF, peptides were constructed by stepwise solid-phase synthesis using the Boc-HF strategy. These synthetic peptides were characterized by HPLC, FAB-MS, amino acid analysis and thermolytic digestion. The mitogenic activity of thes h-EGF analogues was determined by the stimulation of [ 3H]-thymidine uptake into DNA in NIH-3T3 fibroblast cell lines. Substituting Tyr with Phe at position's 37 and 13 had little effect on the mitogenic activity of h-EGF. In contrast, Ala at these positions resulted in a severe loss of activity (20 and 10 3-fold). These results indicate that the hydrophobicity of the side chain at positions 13 and 37 of h-EGF is essential for its biological activity. A semiconservative substitution of Leu with Ala at position 15 and a conservative change of Lys at position 41 also drastically reduced mitogenic activity (10 4 and 10 5-fold). Thus, the bulky hydrophobic side chain at position 15 and the guanidino group at position 41 are indispensable in determining the biological activity of h-EGF.

固相ペプチド合成における“Ｄｉｆｆｉｃｕｌｔ Ｓｅｑｕｅｎｃｅ”とペプチド鎖の溶媒和

Recently, “difficult sequences” have been often found as a key word in solid-phase peptide synthesis. “Difficult sequences” which cause the most serious potential problem during stepwise solid-phase peptide synthesis result from the intermolecular aggregation, namely, the formation of β-sheet structure by intermolecular hydrogen bonds between protected peptide chains. This β-sheet structure hinders the coupling reaction, the deprotection of the N-protecting group, and even the liberation of amino groups at each step for peptide chain elongation and results in the incomplete peptides with the properties similar to the target peptide. In liquid phase peptide synthesis, the “difficult sequence” is closely related to the solubility of protected peptides. The decreasing solubility of protected peptides in organic solvents along with increasing their chain length is due to the intermolecular β-sheet structure formation. The conversion of “difficult sequence” to easy sequence by disrupting the β-sheet structure can be achieved wherether the protected peptides are completely solvated by organic solvents or they are subject to peptide segment separation.

Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.