Optimized Solid-phase Synthesis Research Articles

An optimized Fmoc synthesis of human defensin 5

Human α-defensin 5 (DEF5), expressed by the Paneth cells of human small intestine, plays an important role in host defense against microbial infections. DEF5, a 32-residue peptide adopting a three-stranded β-sheet fold stabilized by three internal disulfide bonds, is not efficiently produced by recombinant expression techniques and is, therefore, an interesting goal for chemical synthesis. While DEF5 production by Boc-based solid-phase synthesis has been described, to date no synthetic account by the more convenient Fmoc method has been published. Herein, we report an optimized solid-phase synthesis of DEF5 using the Fmoc strategy. Starting from a rather problematic initial synthesis using standard Wang resin and coupling protocols, the sequence elongation process has been monitored by mini-cleavage and MS analysis at strategic points, to identify problematic spots and act accordingly. For expediency, some of the optimization rounds have been run on defensin 5 amide. Main modifications have included the ChemMatrix(®) resin, known to decrease chain aggregation, and the use of pseudoproline dipeptide units at selected positions. Combination of some of these improvements results in a significantly purer product, to the extent that it can undergo in situ anaerobic oxidative folding to the native form without the need of an intermediate purification step. A typical synthesis run yielded about 15 mg of >95 % pure material. This approach should facilitate production of DEF5 and of selected analogs for structure-activity studies and other applications.

Efficient access to highly pure β-amyloid peptide by optimized solid-phase synthesis

Feasible and reproducible synthesis of full-length Aβ peptides has been one of the major challenges in Alzheimer’s disease research. By using dimethyl sulfoxide as an anti-aggregation solvent and as an agent to promote double-coupling of two phenylalanine that frequently experience residual deletion, we developed a reliable manual Fmoc solid phase peptide synthesis procedure to produce biologically active Aβ in large quantities at relatively high purity. The amyloidogenic activity of the synthesized Aβ was confirmed via thioflavin T assay, transmission electron microscopic analysis and electrophoresis.

Optimized Fmoc solid‐phase synthesis of the cysteine‐rich peptide linaclotide

Linaclotide, a small 14-mer peptide highly rich in cysteines, is currently in phase III clinical trials for the treatment of gastrointestinal disorders. The challenge in the assembly of linaclotide consists of achieving the correct and clean folding of its three disulfide bridges. For this purpose, a number of regioselective, semiregioselective, and random strategies have been studied. In addition to selecting distinct protecting groups for the thiol function, their position in the sequence, the influence of the neighboring protecting groups, as well as the order in which the disulfides fold were studied. Here we describe an optimized solid-phase synthesis of linaclotide that should allow the production of this peptide in multigram amounts.

Solid-phase S-3CR generates N-substituted α-aminonitriles for the synthesis of α-phenyl-α-(1-piperazinyl) substituted amino acids

Structurally diverse amino acids were prepared as versatile synthons for combinatorial chemistry. Using an optimized solid-phase synthesis by Strecker-three-component-reaction (S-3CR), two different polymer linker constructs carrying piperazine were investigated. (a) Acrylate derived base-labile linker yielded alpha-aminonitriles with N-alkylated piperazines via Hofmann elimination after quarternisation with an alkyl halide. The crude product purities were in the range of 54-87%. (b) A urethane type linker yielded alpha-aminonitriles with the free piperazine nitrogen when cleaved with acid and the product purities were 72-93%. The alpha-aminonitriles were easily converted to novel Nepsilon) - Fmoc-protected alpha-amino acids with alpha-(1-piperazinyl) and alpha-phenyl substituents.

Activity-based probes that target diverse cysteine protease families

Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.

Design and synthesis of an optimized positional scanning library of peptoids: identification of novel multidrug resistance reversal agents

Herein is reported the optimized solid-phase synthesis of a library of 5,120 trimeric N-alkylglycines (peptoids) using the positional scanning format and the submonomer strategy. Diversity at the N-terminal position was generated from 20 commercially available primary amines, whereas 16 primary amines were employed for the middle and C-terminal positions of the trimers. Formation of undesirable side-products observed in a previous library synthesis (Humet, M. et al. J. Comb. Chem. 2003, 5, 597–605) was averted by restricting the use of primary amines functionalized with tertiary amino groups to the third amination step. Screening of the new library for the identification of chemosensitizers yielded two peptoids, compounds 1 and 2, with potent in vitro activity as multidrug resistance (MDR) reversal agents. The structures of the lead peptoids are consistent with a pharmacophore model generated from the interaction of various known inhibitors with the MDR-implicated transmembrane glycoprotein P-gp.

The use of coiled-coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.

Synthesis and triple helix-forming ability of oligonucleotides with N,N-dimethylaminoethyl phosphoramidate linkages

Two oligonucleotides, partially modified with N,N-dimethylaminoethyl phosphoramidate groups, were obtained by an optimized solid-phase synthesis cycle based on H-phosphonate chemistry. Their use as third strands in parallel triple helices was shown to produce a decrease in stability with respect to all-phosphodiester oligonucleotide complexes, most probably due to unfavourable steric effects. Phosphoramidate-modified oligonucleotides were shown to be notably stable to exonucleases.

Chemical and enzymatic synthesis of multivalent sialoglycopeptides

Linear and branched glycopeptides containing multiple sialyl- N-acetyllactosamine side chains have been synthesized using a combined chemical and enzymatic approach. Peptide backbones in which β-GlcNAc-Asn residues were incorporated were obtained in good yields by optimized solid-phase synthesis following the Boc strategy. The resulting multivalent glycopeptides were galactosylated in near-quantitative yields using bovine galactosyltransferase, UDP-galactose, and calf alkaline phosphatase that destroys the inhibiting side product UDP. Subsequent enzymatic sialylation yielded the desired glycopeptides containing asparagine-sialyl- N-acetyllactosamine side chains. The compounds were characterized by 1H NMR and FABMS. Recombinant sialyltransferase and CMP-sialate synthetase were used for the enzymatic synthesis of sialosides on a preparative scale. The synthetic glycopeptides were tested as inhibitors of influenza virus to cells, revealing that most of the multivalent sialoglycopeptides exhibit increased binding that depends on the spacing when compared to monovalent compounds. A possible mechanism for increased binding is proposed.

Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol.

The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.