Synthesis and solid-phase purification of anthranilic sulfonamides as CCK-2 ligands

A novel strategy for the synthesis of cholecystokinin-2 receptor ligands was developed. The route employs a solution-phase synthesis of a series of anthranilic sulfonamides followed by a resin capture purification strategy to produce multi-milligram quantities of compounds for bioassay. The synthesis was used to produce >100 compounds containing various functional groups, highlighting the general applicability of this strategy and to address specific metabolism issues in our CCK-2 program.     

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.     

Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-disubstituted ureas

A series of lysine sulfonamide analogues bearing a Nepsilon-benzylic ureas was synthesized using both solution-phase and solid-phase approaches. A novel synthetic route of Nalpha-(alkyl)-Nalpha-(sulfonamides)lysinol using alpha-amino-caprolactam was developed. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type HIV virus.     

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.     

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.     

'Ethylene-bis[phosphonate] nucleic acids': novel monomeric synthons for the solid-phase synthesis of (P-CH2-CH2-P)-bridged oligonucleotides

An efficient synthesis of modified deoxyribonucleosidic building units, containing the 'ethylene-bisphosphonate nucleic acids' (EBPNAs) 1 or 2, has been developed. By means of solution-phase incorporation of 11, the oxidized form of 2, dinucleotide analogs of type 13 were prepared. The same strategy was used to incorporate 2 into the 5'-terminal position of the 12-mer homopolynucleotide methylphosphonate 14. A preliminary approach for solid-phase incorporation of the nucleosidic building block 1 into a preselected position of an oligonucleotide sequence to give 19 is presented, a reaction performed by means of a coupling strategy involving catalytic oxazaphospholidine-ring cleavage.     

Solution-phase library generation: methods and applications in drug discovery

Solution-phase high throughput synthesis has emerged as a powerful method for the rapid generation of chemical libraries. The success of this approach is largely due to the development of novel synthetic methodologies that expedite the preparation of compounds. Several isolation/purification techniques have also been developed to eliminate the time-consuming purification procedures often associated with solution-phase chemistry. These methods are amenable to parallel synthesis and combinatorial strategies and can be fully automated. In addition, the compound libraries generated using solution-phase high throughput synthesis have been used to accelerate both lead identification and lead optimization programs at various companies.     

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.     

Solution- and solid-phase oligosaccharide synthesis using glucosyl iodides: a comparative study

Glycosyl iodide donors have been used in both solid- and solution-phase syntheses yielding alpha-(1 --> 6)-linked glucosyl oligomers in highly efficient protocols. While the solid-phase strategy offers advantages in terms of ease of purification, it requires a total of 7.5 equiv of donor and approximately 12 h to complete the incorporation of one monosaccharide unit. In contrast, solution-phase methods require only 2.5 equiv of donor and 2-3 h reaction time per glycosylation. Moreover, since the reactions are virtually quantitative (> 90%) column chromatography of the material is facile. The overall advantages of solution-phase oligosaccharide synthesis were further illustrated in the convergent synthesis of a hexamer (methoxycarbonylmethyl 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6)-tetrakis-(2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6))-2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside) that was constructed from dimer donor iodides in a two-plus-two and a two-plus-four fashion.     

Application of solid-phase chemistry for the synthesis of 3'-fluoro-3'-deoxythymidine

Reported solution-phase methods for the synthesis of 3'-fluoro-3'-deoxythymidine (FLT) are cumbersome, require purification of intermediates, and include several protecting/deprotecting steps. To circumvent these problems, a solid-phase strategy was designed for the synthesis of FLT. Thymidine was immobilized on trityl resin via the 5'-hydroxyl group. The subsequent mesylation of the free 3'-hydroxyl group in the presence of methanesulfonyl chloride afforded the polymer-bound 3'-O-mesylthymidine. Nucleophilic substitution of the mesyl moiety by hydroxyl group in the presence of sodium hydroxide produced the polymer-bound threothymidine. Fluorination with diethylaminosulfur trifluoride followed by acidic cleavage of the polymer-bound FLT in the presence of trifluoroacetic acid afforded FLT.     

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.     

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.     

A novel immobilization strategy using oligonucleotide as linker for small molecule microarrays construction

A novel immobilization method based on oligonucleotide as linker has been developed for small molecule microarrays (SMMs) construction. The oligonucleotide tail was employed as a linker in solid-phase synthesis. Small molecules could be easily conjugated at the 5′ end of the oligonucleotide, previously modified with a functional group. Being a reactive species, the oligonucleotide was activated by UV irradiation, for the attachment of the conjugate to the slide surface. The method was successfully applied to structurally distinct small molecules, including biotin, antibiotic and drug. This immobilization strategy showed high efficiency, 1.1 fmol of small molecules in the spotting solution per spot gave a detectable signal (mean S/N = 10.9). The results suggest that it is very promising for exploring interaction between small molecules and proteins, and high throughput detecting the chemical compounds.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.     

Solid-phase synthesis of FKBP12 inhibitors: N-sulfonyl and N-carbamoylprolyl/pipecolyl amides

In parallel with our work on solution-phase parallel synthesis of ligands for the rotamase enzyme FKBP12, we herein report a methodology for the solid-phase synthesis of two classes of inhibitor, N-sulfonyl and N-carbamoylprolyl and pipecolyl amides along with their in vitro/in vivo biological results.     

Tyrphostin-like compounds with ubiquitin modulatory activity as possible therapeutic agents for multiple myeloma

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,β-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(??) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(β) carbon were utilized in developing a model to explain the compound activity.