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Potent and selective EP3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24-95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.
The synthesis of several omega-chloroalkyl tetrazoles and their subsequent attachment to a solid support is described. Using an in situ Finkelstein reaction, a variety of nucleophiles were alkylated and then cleaved from the resin to give pure alkyl tetrazole derivatives. A sample library of 5 x 6 demonstrates the general utility of this sequence.
The cleavable diamines cystamine, 5, and 1,6-diamino-3,4-dihydroxyhexane, 1, were bonded to solid supports and, with a simple, newly developed dinitrofluorobenzene-based assay, were used to define (a) titers of ligands and (b) chemistry distal to the support. Compound 1, which is cleavable with periodate, becomes a linking molecule which is stable to almost all conditions encountered in biochemistry and enjoys considerable hydrophilic character. Compound 5, which is cleavable with dithiothreitol, can be usefully applied to those systems which do not require reducing agents. These nucleophilic linking moieties were converted to cleavable electrophilic linkers by succinylation and p-nitrophenyl ester activation. The first preparation of a polysaccharide-linked support is described. The method also allows the chemical definition of ligands containing amino groups which are prepared by deblocking of protecting groups while on the support. The methodology should promote greater understanding of affinity chromatography materials and processes.
Hydrophobically assisted switching phase (HASP) synthesis is a concept that allows the choice between the advantages of solid-supported chemistry and those of solution-phase synthesis. Starting from the examination of adsorption and desorption properties of hydrophobic molecules to and from reversed-phase silica, we designed a dilipid as a quantitative and fully reversible HASP anchor, permitting final product release. The utility of this new tool in synthetic organic chemistry was demonstrated on oligosaccharide preparation. The synthesis of a pentarhamnoside was accomplished by repetitive glycosylation reactions. Glycosylations were conducted preferably in solution, whereas all protecting group manipulations were performed on solid support. Without the need for chromatographic purification of intermediates, the HASP system furnished the final product after 12 linear steps with average yields of 94% per step at a scale of 0.1 mmol, thus overcoming several of the limitations encountered in the solid-phase synthesis of complex carbohydrates.
With increasing interest in automated synthesis and screening protocols, solid supported chemistry and biochemistry are attractive technologies. Studies with surface-immobilised substrates have been carried out to analyse enzyme accessibility, kinetics and thermodynamics. Several interesting new methods have been developed to monitor enzyme action on substrates attached to a solid phase such as polymer beads glass or gold surfaces. These include fluorescence measurements, MALDI-TOF mass spectrometry, and the use of quartz crystal microbalances to measure weight changes of immobilised molecules directly on the surface. Approaches that allow spatial resolution in single beads have also been reported. The ability of enzymes to reach the inside of beads is becoming better characterised and new supports have been developed that allow improved accessibility. The equilibrium position of reactions on the solid surface can be substantially shifted compared with reactions in solution, and this can be usefully exploited using hydrolases in reverse. Research is also starting to tackle the way in which kinetics are modified when the substrates are surface immobilised.
Resin-bound 4H-1,3-oxazines are synthesized by the stepwise condensation of an amide resin, an aldehyde, and an alkyne. Upon DDQ activation, oxazines are converted into oxazinium salts. When treated with hydrazines, these resin-bound beta-diketone equivalents yield pyrazoles through a functionalizing release process. This multicomponent capture strategy, tedious to handle in classical synthesis in solution, is well-suited to solid-supported chemistry. It facilitates the handling of sensitive and unstable intermediates, such as N-alpha-methoxyalkylamides and 1,3-oxazinium salts.
REsin Activation/Capture APproach or REACAP Technology, a novel approach to the synthesis of compound libraries, capitalizes on the formation and retention of a resin-bound reactive intermediate, which can be subsequently transformed into a stable, covalently attached molecule. Any unreacted "reactive intermediate" is quenched and removed from the resin upon work-up, leaving only the desired product on the solid support. In contrast to more traditional solid-supported chemistry that must address issues such as resin-loading, capping of unreactive functionalized moieties, and reaction yields, REACAP offers an attractive alternative with the focus more on the purity of the released products and less on yield. In an endeavor to generate truly non-peptide leads, we describe herein the synthesis of N-acyl-2-substituted-dihydro-4-pyridones, dihydro-4-pyridones, 4-ketopiperidines, tetrahydropyridines, and 2-acyl-3,7,8-substituted-5-oxo-2-azabicyclo[2.2.2]octane and triaza analogs using REACAP Technology.
Over the past year, numerous techniques have been used to study the resins commonly utilised in solid-phase synthesis to allow a greater understanding of the chemical nature and the physical properties of the supports. In addition, to overcome some of the drawbacks of existing materials, several new resins and new methods of handling solid supports have been developed. New methodologies have also been introduced to simplify the preparation of solid supports.
Chemical modifications of oligonucleotides are routinely employed to enhance their functional properties. Amino-modifiers serve as versatile chemical handles for postsynthetic (bio)conjugation, nucleic acid immobilization on solid supports, and investigations into nonenzymatic genome replication relevant to the origins of life, to name a few. Here, we report a cost-effective, disulfide-containing solid-support linkage that enables the on-column synthesis of nucleic acids with 3'-amino or 3'-phosphate modifications. The orthogonality of this solid-support linker facilitates an on-column protecting group strategy, enabling the synthesis of DNA and RNA containing 3'-amino-2',3'-dideoxyribosides from commercial unprotected mononucleosides. Additionally, we present an on-column deprotection protocol for DNA and RNA, prior to cleavage from the solid support, eliminating the precipitation step typically required in conventional RNA workflows, leading to higher recovery for certain strands. Expanding on our previous work, we introduce a versatile modular tandem oligonucleotide synthesis (mTOS) approach, allowing selective release of downstream strands from the one directly bound to the solid-support via the disulfide-containing linker. Together, these advances in solid-support design and oligonucleotide synthesis unlock new opportunities in bioconjugation, biotechnology, and the study of prebiotic replication mechanisms, broadening the utility of chemically modified nucleic acids across research disciplines.
An efficient solid-phase synthesis of 2-substituted 4-aminopyrido[2,3-d]pyrimidines 15 is reported. The procedure started by solid supporting a p-hydroxybenzaldehyde 8 to the Wang resin by using the Mitsunobu protocol. The resulting aldehyde 17 was treated with a substituted acid methyl malonate 10 to afford the corresponding alpha, beta-unsaturated ester 18, which was converted to the Michael adduct 21 by reaction with malononitrile. Cyclization of 21 with an amidine system 13 yielded the solid supported pyridopyrimidine 22, which afforded the corresponding 2-substituted 4-aminopyrido[2,3-d]pyrimidine 15 upon treatment with TFA:DCM. Compounds 15 present three diversity centers R1, R2 and R3. Having validated the chemistry on solid support, a 32-membered combinatorial library was obtained using this protocol.
An efficient solid-phase synthesis of 2-substituted 4-aminopyrido[2,3-d]pyrimidines 12 by cyclization-assisted cleavage from resin is reported. The procedure starts by solid supporting an alpha,beta-unsaturated acid 8 to the Wang resin 13 by using DCC and 4-DMAP in THF. The resulting alpha,beta-unsaturated ester 14 is converted to the Michael adduct by treatment with malononitrile in NaOMe/THF. Such Michael addition constitutes the first example of a Michael reaction with malononitrile in solid-phase. Finally, the Michael adducts 15 are treated with an amidine system in MeOH to yield the corresponding pyridopyrimidines 12. Compounds 12 present three diversity centers R1, R2 and G. Having validated the chemistry on solid support, a 40-membered combinatorial library was obtained using this protocol.
An efficient solid phase regioselective alkylation at the N4 position of a 3-oxo-1,4-benzodiazepine template exemplified by 4-H-2,3,4,5-tetrahydro-7-iodo-3-oxo-1H-1,4-benzodiazepine-2-acetate-polymer ester is described. Further chemical elaboration at position 7, utilizing a modified Heck reaction, allows the incorporation of amides from primary or secondary amines. The two diversity points at positions 4 and 7 were utilized to synthesize a 28-membered, combinatorial array on Sasrin resin in moderate yields and > 80% purity. Having validated the chemistry on solid support, a combine and split approach to prepare a bead-bound combinatorial library is achievable utilizing similar experimental practices and procedures as in the array synthesis.
Four low molecular mass lysine dendrimers were synthesized by Boc chemistry in solution (155 and 169) and Fmoc chemistry on solid support (P2 and P13). The structure and fragmentation mode of the above dendrimers was investigated in gas phase by the LSI-MS and ESI-MS techniques. (1)H and (13)C NMR analysis in solution (d(6)-DMSO) allowed to confirm the correct structure. Antimicrobial activities of the dendrimers against Staphylococcus aureus, Escherichia coli and Candida albicans confirmed our hypothesis that the dendrimer structure can be used for construction of molecules interacting with biological membranes.
Spatially resolved single bead analysis in the micrometer range was employed as a tool for evaluating homogeneity, diffusion, and adsorption in solid-phase supported reactions. Fluorescence microscopy (confocal and non-confocal) as well as IR microscopy were used to detect both the distribution of products and the formation of product gradients in representative reactions. For the first time, the optical slices of whole beads obtained by confocal fluorescence microscopy were compared with the fluorescence images of microtome-sliced beads. The experiments revealed that only physical slices of polystyrene beads deliver realistic representations of the distribution of fluorophores, and confirmed-in contrast to a recent report-the homogeneity of functional site distribution in polystyrene beads. Moreover, the pattern of product formation obtained from an acylation reaction as well as from an alkylation reaction were employed as probes to study the impact of bead size, diffusion, and adsorption on the reaction progress. A simulation of the diffusion process was conducted and compared with the experimental results. Diffusional control was found neither in the case of the alkylation nor in the case of the acylation reaction under investigation. As a consequence, the reaction progress was not a function of the bead sizes as proposed in the literature. Interestingly, in the case of rhodamine acylation with substoichiometric amounts an adsorption-controlled reaction was found. This result highlights the significance of adsorptive effects in solid-phase supported chemistry.
A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein. The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E. coli tryptophan promoter. The fusion protein, termed L0-[Thr59]-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay. Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells. After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed [Thr59]-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays. It was also mitogenic in Balb/c 3T3 cells. These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.
A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.
Triple helix-forming oligonucleotides (TFOs) offer the potential to specifically modulate expression of gene in a sequence dependent manner. TFOs containing G and T residues that bind to duplex DNA, forming a series of GGC and TAT base triplets, have been well studied. It has been observed that T is relatively nonspecific in that it binds with similar affinity to AT, GC, and CG base pairs. This may significantly reduce the specificity of a given TFO, leading to undesired effects on the expression of genes unrelated to the intended target. We have now prepared 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-pyrido[2,3-d]pyrimidine-2,7(8H )- dione (P) and incorporated it into TFOs using the solid-support, phosphoramidite chemistry. It has been demonstrated that a limited substitution of P for T in a G-rich 26-mer TFO can improve binding specificity for AT base pairs in antiparallel motif under certain conditions. The specificity exhibited by P is suggestive of base pair specific interactions that influence the binding strength and consequently enhance the potential therapeutic application of TFOs. However, the effect of substitution of P for T is dependent on the binding conditions, as well as the number of position of substitutions.
An efficient and expeditious method for the synthesis of S6-(cyanoethyl)-N2-isobutyryl (or trifluoroacetyl)-2'-deoxy-6-thioguanosine (7 and 2) from 2'-deoxyguanosine (G) has been developed. Compound 7 has been incorporated into several G-rich triple-helix-forming oligonucleotides (TFOs) using solid-support, phosphoramidite chemistry. The purified oligonucleotides containing 2'-deoxy-6-thioguanosine (S6-dG) residues in the place of G have been characterized by nucleoside composition analysis. These modified TFOs have been shown to be stable in aqueous, as well as buffered, solutions normally used to assay triple-helix formation. It has also been demonstrated that partial incorporation of S6-dG is effective in inhibiting the formation of G tetrads in G-rich oligodeoxyribonucleotides, thus facilitating triple-helix formation in potassium-containing buffers.