Solid-phase Synthesis Strategy Research Articles

A convenient solid-phase synthesis methodology for preparing peptide-derived molecular imaging agents — Synthesis, characterization, and in vitro screening of Tc(I) – chemotactic peptide conjugates

A versatile solid-phase synthesis strategy for preparing peptide–chelate conjugates was developed. The methodology was optimized using a series of ligands, designed to bind Tc(I)/Re(I), and a chemotactic peptide fMFL, which was exploited as a model targeting vector. The peptide derivatives were prepared in parallel using a conventional automated peptide synthesizer in multi-milligram quantities, which provided sufficient material to perform complete characterization, radiolabelling, and in vitro screening studies. Because of the robust nature of the metal–chelate complexes, the Re complex of a chelate–peptide conjugate was prepared on the resin using the same methodology employed to prepare the free ligand conjugates. As such, the reported methodology is amenable to the preparation of libraries of novel Tc radiopharmaceutical ligands and their corresponding Re reference standards in which several factors, including peptide sequence, site of derivatization, and both the type and length of the spacer, can be easily varied.Key words: radiopharmaceuticals, technetium, rhenium, peptides, solid-phase synthesis.

Reversible and Chemically Programmable Micelle Assembly with DNA Block-Copolymer Amphiphiles

We report a general solid-phase synthesis strategy for a novel class of DNA block copolymer amphiphiles. The assembled spherical micelle structures exhibit recognition properties defined by their DNA sequences and can be used to build higher-ordered structures through hybridization with materials that possess complementary DNA. This work is an important step toward creating DNA modified structures that can act as phase transfer agents and carriers of nanovesicles.

An efficient approach for monosulfide bridge formation in solid-phase peptide synthesis

An efficient approach for the synthesis of cyclic peptides containing unnatural thioether side-chain bridges, based on the use of (2S)-9-fluorenylmethyl-2-[(tert-butoxycarbonyl)amino]-4-iodobutanoate and its homologue 5-iodopentanoate, derived from Boc-l-Asp-OFm and Boc-l-Glu-OFm, respectively, is reported. The synthesis was performed by a tandem combination of solid-phase peptide synthesis and microwave-assisted cyclization strategy.

Methionine anchoring applied to the solid-phase synthesis of lysine-containing ‘head-to-tail’ cyclic peptides

Lysine-containing ‘head-to-tail’ cyclic peptides can be prepared via a side-chain anchoring solid-phase synthesis strategy. A handle is prepared using a methionine residue, theCα-carboxyl of which forms an, amide with theNe-amine of lysine. Subsequently, the linear peptide sequence is assembled, appropriate deblocking steps are carried out, and on-resin head-to-tail cyclization follows. Optionally, acid-labile protecting groups may be removed while the peptide remains resin-bound. The final cleavage step uses CNBr, and releases the free or protected cyclic peptide into solution.

A new strategy for solid phase synthesis of a secondary amide library using sulfonamide linker via radical traceless cleavage.

A new strategy for solid phase synthesis of a secondary amide library using sulfonamide linker via radical traceless cleavage is reported. Polystyrylsulfonyl chloride (1) reacted with primary amines to afford polystyryl-supported N-alkyl sulfonamides (2), which were acylated with acid chlorides and followed by radical cleavage with TiCl4/Zn to afford secondary amides. It was interestingly found that the products released from acyl alkanesulfonamide resins are closely dependent on the substituents of benzene rings of alkyl or acyl groups on the resins. When the substituent on benzene ring of N-benzyl group of sulfonamides is an electron rich MeO-group, the products released from sulfonamide resins are dependent on the substitution position on benzene ring: para-MeO- to yield 1,2-bis (p-methoxylphenyl)ethane and N--p-methoxylbenzyl benzamide (30:1); ortho-MeO- to give 1,2-bis (o-methoxylphenyl)ethane and N--o-methoxylbenzyl benzamide (1:15); and meta-MeO- only to release N--m-methoxylbenzyl benzamide. Neither N-benzoyl sulfonamide resins on benzene ring with electron-drawing para-O2N-, nor the one with electron-donating para-H2N- could release any amide product, while the N-benzoyl sulfonamide resins on benzene ring with para-acetamido group released para-acetamidobenzamides. The conjugation effect to stabilize the radical groups in the radical cleaving process was observed.

Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides.

Phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides were prepared via a solid-phase synthesis strategy. Under Reformatsky reaction conditions, novel esterified acetic acid phosphinodiamidites were synthesized and condensed with appropriately protected 5'-O-(4, 4'-dimethoxytrityl)-2'-deoxynucleosides to yield 3'-O-phosphinoamidite reactive monomers. These synthons when activated with tetrazole were used with an automated DNA synthesizer to prepare phosphonoacetic acid modified internucleotide linkages on controlled pore glass. The phosphinoacetate coupling products were quantitatively oxidized at each step with (1S)-(+)-(10-camphorsulfonyl)oxaziridine or 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce mixed sequence phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides with an average per cycle coupling efficiency of greater than 97%. Completely deprotected, modified oligodeoxynucleotides were purified by reverse-phase HPLC and characterized by ion exchange HPLC, (31)P NMR, and MALDI/TOF mass spectroscopy. Both analogues were stable toward hydrolysis with snake venom phosphodiesterase and stimulated RNase H1 activity.

Side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine.

Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C-terminus. Furthermore, installation of C-terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of N(alpha)-Fmoc, S-protected C-terminal cysteine as an ester onto the support for solid-phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C-terminal 3-(1-piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a). conversion of the eventual C-terminal cysteine residue, with Fmoc for N(alpha)-amino protection and tert-butyl for C(alpha)-carboxyl protection, to a corresponding S-xanthenyl ((2)XAL(4)) preformed handle derivative; and (b). attachment of the resultant preformed handle to amino-containing supports. This approach uses key intermediates that are similar to previously reported Fmoc-XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3-(1-piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80-90%) of TFA plus appropriate thiols or silanes as scavengers. High-yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1-5%) of TFA. In peptides with two cysteine residues, the corresponding intramolecular disulfide-bridged peptide is obtained by either (a). oxidation, in solution, of the dithiol product released by acid; (b). simultaneous acidolytic cleavage and disulfide formation, achieved by addition of the mild oxidant DMSO to the cleavage cocktail; or (c). concomitant cleavage/cooxidation (involving a downstream S-Xan protected cysteine), using reagents such as iodine or thallium tris(trifluoroacetate) in acetic acid.

Imaging polymer supported organic molecules by mass spectrometry Application in combinatorial chemistry

ABSTRACTIn combinatorial chemistry, most libraries are prepared according to solid-phase synthesis strategies using resins or pins. Although synthesis and purification steps are facilitated, reaction monitoring presents difficulties. Since the polymeric support is not soluble, an analytical method able to cope with a solid sample is thus required to perform direct identification of the anchored molecules without any chemical treatment. Static-Secondary Ion Mass Spectrometry (S-SIMS) was investigated in that purpose. Positive and negative ion mass spectra were acquired to identify single beads whereas mixtures (Mix and Split libraries or pooled beads issued from different batches) were profiled through imaging experiments.

Preparation of core 2 type tetrasaccharide carrying decapeptide by benzyl protection-based solid-phase synthesis strategy

β- d-Gal-(1→4)-β- d-GlcNAc-[β- d-Gal-(1→3)]-α- d-GalNAc-(1→3)- l-Ser/Thr building blocks for solid-phase synthesis of glycopeptide were stereoselectively synthesized in a benzyl-protected form. The key glycosylation reaction to form β- d-GlcNAc linkage was established by the use of protected N-trichloroacetyl- d-lactosaminyl fluoride. Usefulness of the building block was demonstrated by solid-phase synthesis of a segment of human leukosialin. Benzyl protecting group was efficiently removed by ‘low acidity TfOH’ conditions.

Solid-phase synthesis of deglycobleomycins: a C-terminal tetraamine linker that permits direct evaluation of resin-bound bleomycins.

Deglycobleomycin analogues having different length polyamine side chains at the C-terminus were synthesized using a novel solid-phase synthesis strategy that produces fully deprotected deglycobleomycin congeners attached to the resin. Detailed studies of DNA cleavage by these compounds and their resin-bound counterparts using supercoiled plasmid DNAs and DNA restriction fragments as substrates revealed that (i) the length of the polyamine side chain of free deglycoBLM had limited effect on its DNA cleavage potency or sequence selectivity, and (ii) the nature of the linker moiety between the resin and attached deglycobleomycin had a more substantial effect on the potency of DNA cleavage, but no effect on sequence selectivity of resin-bound deglycoBLMs. Resin-bound 4 exhibited efficient DNA cleavage, indicating that its tetraamine linker moiety could be used for the elaboration and direct evaluation of bleomycin congeners attached to resins.

Solid-phase synthesis of C-terminal peptide amides from N-tetrachlorophthaloyl protected amino acids

A new strategy for solid-phase synthesis of C-terminal peptide amides based on the use of N-tetrachlorophthaloyl protected amino acids with acid-labile side-chain protection is described.

Solid-phase synthesis of peptide aldehydes directly on acetal resin

A practical strategy for solid-phase synthesis of peptide aldehydes has been developed employing a 10,11-dihydroxyundecanoic acid (DHUA) functionalized resin. High loading levels are obtained for amino aldehydes as their respective acetals. After classical Fmoc SPPS, treatment of the derivatized resin with 95% TFA/H 2O affords peptide aldehydes in high yield and purity.

A Novel Silicon-Based Linkage and Cleavage Strategy for Solid-Phase Synthesis: Formation of Resin-Linked Zwitterionic Pentacoordinate Silicates as the Key Step and Release of the Target Molecules in a Traceless Fashion

A novel silicon-based linkage and cleavage strategy for solid-phase synthesis of aromatic organic compounds has been developed. The method is based on the following steps: (i) attachment of silanes of the formula type (R1 = aryl; R2 = aryl, alkyl; R3, R4 = alkyl; n = 1, 3) to a Merrifield resin via R4, (ii) reactions with the resin-linked silanes (chemical transformations of the aryl group R1; R1 → → → R1*), and (iii) treatment of the resin with 1,2-dihydroxybenzene in acetonitrile at 50 °C to give the cleavage products R1*H (release of the target molecules in a traceless fashion), R2H, and R3OH, along with the resin-linked zwitterionic pentacoordinate silicate of the formula type

An original traceless linker strategy for solid-phase synthesis of N,N',N''-substituted guanidines.

An original sequence for solution- and solid-phase synthesis of N,N',N"-trisubstituted guanidines is described. The sequence involves as key intermediate a bis-electrophilic chlorothioformamidine that is stable, easy to prepare and also easy to handle. Supported chlorothioformamidine, prepared in two steps from Merrifield resin, undergoes smooth nucleophilic addition of a primary amine to afford the corresponding supported isothiourea. The guanidine is obtained in satisfactory yield and good purity through a functionalizing-release process by heating the supported isothiourea in the presence of a primary amine in toluene at 100 degrees C. Compatibility of this sequence with several functional groups is demonstrated.

A modified backbone amide linker (BAL) solid-phase peptide synthesis strategy accommodating prolyl, N-alkylamino acyl, or histidyl derivatives at the C-terminus

A new variation of the Backbone Amide Linker (BAL) approach has been developed for N α-Fmoc solid-phase synthesis (SPS) of C-terminal modified peptides; this provides a convenient route to peptides containing prolyl, N-alkylamino acyl, or histidyl derivatives at the C-terminus. To illustrate the principles, several model peptides were prepared in high yields and excellent purities; diketopiperazines and racemized byproducts which otherwise form when alternative procedures are used were absent from the syntheses reported herein.

Oligomerization and Toxicity of β-Amyloid-42 Implicated in Alzheimer's Disease

β-Amyloid protein (Aβ) is the major component of senile plaques found in the brains of Alzheimer's patients. A novel ELISA has been developed which probes the early stages of oligomerization of Aβ. Incubation of Aβ solutions at 37°C and pH 7.4 produces soluble oligomers in a concentration-dependent manner. Fresh Aβ42 solutions rapidly form soluble oligomers, whereas Aβ40 solutions require prolonged incubation to produce oligomers. Fresh Aβ42 solutions are more toxic to human neuroblastoma SH-SY5Y cells than Aβ40 solutions, possibly mediated by soluble oligomers. The differences between Aβ42 and Aβ40 could explain the association of the longer form with familial early-onset Alzheimer's disease. We also report a new strategy for solid-phase synthesis of Aβ peptides which gives high yield and purity of the initial crude preparation.

Reductive cleavage of N-O bonds using Samarium(II) iodide in a traceless release strategy for solid-phase synthesis

A strategy for making amides and ureas using a polymer-supported hydroxylamine resin as a traceless linker is described. The cleavage of the linker by samarium(II) iodide is reported for the first time.

A solid-phase approach towards the development of 3-aza-6,8-dioxabicyclo[3.2.1]octane scaffolds.

The development of new strategies for solid-phase synthesis of 3-aza-6,8-dioxabicyclo[3.2.1]octane scaffolds, named BTKa, is described. The preparation was made possible by the combination of three components: amines, alpha-halo-acetophenones, and sugar or tartaric acid derivatives. By anchoring each of the three components it was possible to synthesize BTKa compounds either as amino alcohols or amido esters. The compatibility of the protocols with different classes of amines and substituted alpha-halo-acetophenones was demonstrated.

Studies Towards the Synthesis of Peptide-Oligonucleotide Conjugates

We describe a peptide fragment, solid-phase coupling strategy for synthesis of peptide-oligonucleotide conjugates. Model conjugates contained a hydrophobic tetrapeptide, a hydrophobic influenza virus fusion nonapeptide, or a basic octapeptide of the HIV-1 Tat protein coupled to either dT12 or a 16-mer anti-Tat oligodeoxyribonucleotide. Conjugation yields were improved by removal of internucleotide 2-cyanoethyl groups prior to peptide coupling and by use of a C12 spacer between peptide and oligonucleotide.

Contulakin-G, an O-Glycosylated Invertebrate Neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.