A new protecting group for tryptophan in solid-phase peptide synthesis which protects against acid-catalyzed side reactions and facilitates purification by HPLC

The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis (SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group, which improves the solubility of the peptide during HPLC purification. On treatment of the purified peptide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully deprotected peptide and N-methylpyrrolidone.     

N-methyl-phenacyloxycarbamidomethyl (Pocam) group: a novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions

In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.     

Unexpected chain-terminating side reaction caused by histidine and acetic anhydride in solid-phase peptide synthesis

Capping is a useful technique to facilitate purification of a crude deprotected peptide in solid-phase peptide synthesis. However, we observed a serious side reaction caused by the Ac2O capping procedure, when it was applied to a synthesis of a peptide containing His. The mechanism of the side reaction was studied.     

Application of an automated synthesis suite to parallel solution-phase peptide synthesis

An in-house developed automated synthesis suite was used to prepare a library of 72 tetrapeptide derivatives, the starting materials for pharmaceutically attractive pentapeptides, employing a convergent strategy. An initial set of 18 dipeptides were synthesized on a large-scale (100-1000 g) using automated synthesis workstations, and then 72 tetrapeptides were synthesized on a medium scale (5-10 g) using an automated system. Each di- or tetrapeptide was prepared in a single operating cycle using a modified methanesulfonic acid method, then a sub-library of 56 pentapeptides were synthesized in parallel, on a small-scale (100 mg-1 g) using a robotic workstation.     

Incorporation of unprotected heterocyclic side chains into peptoid oligomers via solid-phase submonomer synthesis

Peptoids (N-substituted glycines) are an important class of biomimetic oligomers that have made a significant impact in the areas of combinatorial drug discovery, gene therapy, drug delivery, and biopolymer folding in recent years. Sequence-specific peptoid oligomers are easily assembled from primary amines by the solid-phase submonomer method. However, most amines that contain heterocyclic nitrogens in the side chain do not incorporate efficiently. We present here a straightforward revision of the submonomer method that allows efficient incorporation of unprotected imidazoles, pyridines, pyrazines, indoles, and quinolines into oligomers as long as 15 monomers in length. This improved method uses chloroacetic acid instead of bromoacetic acid in the acylation step of the monomer addition cycle, and allows for the incorporation of new side chains that should enable the synthesis of peptoids with entirely new properties.     

Enantioselective solution- and solid-phase synthesis of glutamic acid derivatives via Michael addition reactions

The enantioselective conjugate addition of Schiff base ester derivatives to Michael acceptors either in solution (56–89% e.e.) or on solid-phase (34–82% e.e.) gave optically active unnatural α-amino acid derivatives. The reaction was conducted in the presence of chiral, non-racemic quaternary salts derived from the cinchona alkaloids using neutral, non-ionic phosphazene bases.     

Phenolic resins for solid-phase peptide synthesis: Copolymerization of styrene and p-acetoxystyrene

Details are given of the synthesis and purification of p-acetoxystyrene and its solution and suspension copolymerization with styrene. Reactivity ratios, evaluated by the Tidwell-Mortimer method, were r₁ (p-acetoxystyrene) = 1.18, and r₂ (styrene) = 0.88 for (bulk) solution copolymerization. Corresponding values of the reactivity ratios for suspension copolymerization were, within experimental error, indistinguishable from unity. Thus the copolymer composition is governed simply by the monomer feed composition. Use of a specially designed reactor vessel permits convenient suspension copolymerization of styrene, p-acetoxystyrene, and divinylbenzene to give crosslinked resins having comparatively narrow particle size distributions. Acetoxy groups in the crosslinked resin are cleaved by hydrazine hydrate under very mild conditions to give crosslinked polystyrenes having phenolic groups which, in turn, provide a useful alternative to the more usual chloromethylated polystyrene resins for solid-phase peptide synthesis.     

Microwave irradiation and COMU: a potent combination for solid-phase peptide synthesis

Here we demonstrate the compatibility of Oxyma-based uronium-type coupling reagent COMU with microwave-assisted peptide synthesizers. Consistent with previous reports, COMU displayed higher efficiency than benzotriazole classical immonium salts HATU and HBTU in the demanding synthesis of the Aib derivative of Leu-Enkephalin pentapeptide and did not yield Oxyma-based byproducts. Thus, the combination of microwave irradiation and COMU resulted in a similar performance in considerably shorter time to that achieved by manual synthesis.     

Simple and efficient solid-phase synthesis of unprotected peptide aldehyde for peptide segment ligation

We describe an efficient solid-phase synthesis of C-terminal peptide aldehyde. Making use of the stability of the PAM linker towards both acid and base conditions, a pentapeptide was synthesized starting from a PAM resin according to Fmoc/tBu chemistry. The side-chains were deprotected by TFA. The peptide was cleaved by aminolysis with aminoacetaldehyde-dimethylacetal leading to a C-terminal masked aldehyde. The unprotected peptide aldehyde was then coupled to amino-oxy derivatives by chemoselective ligation in aqueous solution.     

The development of a fully automated solid-phase radioimmunoassay for digoxin

A radioimmunoassay for using antibody coated tubes and a unique radiotracer is described. The assay was designed specifically for use with the fully automated instrument Micromedic Systems «Concept 4 Automatic Radioassay». Antisera to digoxigenin-3-succinyl-BSA were raised in rabbits, purified by ammonium sulfate precipitation, and coated onto polypropylene tubes. An iodinated histamine derivative of digoxigenin which is insensitive to serum variability is utilized. The standard curve is linear for digoxin concentrations between 0.4 and 6.4 ng/ml and is parallel for two different sample sizes. Recovery of added digoxin was quantitative. Sample values obtained by this method correlate well with values obtained by other methods. The assay can either be performed manually or fully automated on Concept 4. The advantages provided by total automation are discussed.     

Traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles via Cu-catalyzed azide-alkyne cycloaddition reactions

A broadly useful acid-labile traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles is presented. A variety of alkynes were efficiently immobilized on a range of polymeric supports by Cu(I)-mediated azide-alkyne cycloadditions. Supported triazoles showed excellent compatibility with subsequent peptide chemistry. Release of pure material (typically >95%) from the solid support was readily achieved by treatment with aqueous TFA.     

Core-shell-type resins for solid-phase peptide synthesis: comparison with gel-type resins in solid-phase photolytic cleavage reaction

[reaction: see text] Novel core-shell-type resins with a rigid core and amino-functionalized flexible shell were prepared with 2,4,6-trichloro-1,3,5-triazine (CNC) and Jeffamine ED-600 starting from 1% cross-linked aminomethyl (AM) polystyrene resins. All of the amino groups were located outside the resin beads, and the loading capacity was 0.2-0.4 mmol/g. The amount of CNC treated was a determining factor in the properties of the final resins. The core-shell-type resins showed superior performances in terms of the initial loading of amino acid and the photocleavage reaction compared to the gel-type resins.     

Fmoc solid-phase synthesis of peptide thioesters using an intramolecularn,S-acyl shift

[reaction: see text] We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.     

General methodology for solid-phase synthesis of N-alkyl hydroxamic acids

Polymer-supported N-benzyloxy-2-nitrobenzenesulfonamides 1 were N-alkylated using three different routes: via Fukuyama reaction with alcohols, by N-alkylation with alkylbromides, and by Michael addition reaction with α,β-unsaturated carbonyl compounds. The N-alkylated products prepared on the linker 1b were obtained in excellent purity and yield. The 2-nitrobenzenesulfonyl (Nos) group was cleaved under mild conditions to yield polymer-supported N-alkylated benzyloxyamines. Acylation by carboxylic acids and cleavage with TFA yielded N-alkyl hydroxamic acids.     

Pd-catalyzed sequential reactions via allene intermediate for the synthesis of polycyclic frameworks containing 2,3-dihydrofuran units

A stepwise process involving Sonogashira coupling, propargyl allenyl isomerization, and consecutive [4 + 2] cyclization has been realized, leading to an efficient synthesis of polycyclic compounds containing a 2,3-dihydrofuran unit. Most attractive for synthetic interest is the finding that up to four stereogenic centers could be generated in one step with high stereoselectivity.     

Bicyclic homodetic peptide libraries: comparison of synthetic strategies for their solid-phase synthesis

Preliminary studies and synthesis development for the preparation of a bicyclic homodetic peptide library have been carried out using orthogonal protection schemes. The best results have been obtained using two Fmoc/tBu-based strategies, in which the first cycle is carried out in the solid phase through side chain functional groups previously protected with Aloc/Al groups. The second cycle is performed either in the solid phase, which requires side chain anchoring of a trifunctional amino acid and Dmb protection for the C-terminus carboxyl group, or in solution, which requires the use of highly labile resins, such as the 2-chlorotrityl (Barlos) resin. Only when the cycles are formed in a ziplike manner, that is, first the small cycle and then the larger ring, is the desired final product obtained.     

Aza-amino acid scanning of secondary structure suited for solid-phase peptide synthesis with fmoc chemistry and aza-amino acids with heteroatomic side chains

Aza-peptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit a propensity for adopting beta-turn conformations. A general Fmoc-protection protocol for the stepwise solid-phase synthesis of aza-peptides has now been developed based on the activation of N'-alkyl fluoren-9-ylmethyl carbazates with phosgene for coupling the aza-amino acid residues. This method has proven effective for introducing aza-amino acid residues with aliphatic (Ala, Leu, Val, and Gly) and aromatic (Phe, Tyr, and Trp) side chains. Acid promoted loss of aromatic side chains was noted with aza-Trp and aza-Tyr residues during peptide cleavage and suppressed by temperature control in the case of the latter. In addition, aza-peptides with heteroatomic side chain residues (Lys, Orn, Arg, and Asp) were conveniently synthesized using this protocol. Partial aza-amino acid scans were performed on three biologically active peptides: the potent tetrapeptide melanocortin receptor agonist, Ac-His-d-Phe-Arg-Trp-NH2; the growth hormone secretagogue hexapeptide, GHRP-6, His-d-Trp-Ala-Trp-d-Phe-Lys-NH2; and the human calcitonin gene-related peptide (hCGRP) antagonist, FVPTDVGPFAF-NH2. This practical procedure for aza-amino acid scanning using Fmoc-based solid-phase synthesis should find general utility for probing the existence and importance of beta-turn conformations in bioactive peptides.     

Amino acid bromides: their utilization for difficult couplings in solid-phase peptide synthesis

Use of N-protected-α-amino acid bromides for facile solid-phase synthesis of peptides (SPPS) containing extremely sterically hindered non-proteinogenic amino acids is presented. Amino acid bromides (Aaa-Br), generated in situ, were used for the synthesis of long chain homopeptides containing up to eight successive α-MeVal or Aib residues. SPPS of a heteropeptide containing a very bulky amino acid building block is also described. The choice of suitable N-protections is discussed.