Combination Of Solid-phase Synthesis Research Articles

Functional DNA-Polymer Conjugates.

DNA nanotechnology has seen large developments over the last 30 years through the combination of solid phase synthesis and the discovery of DNA nanostructures. Solid phase synthesis has facilitated the availability of short DNA sequences and the expansion of the DNA toolbox to increase the chemical functionalities afforded on DNA, which in turn enabled the conception and synthesis of sophisticated and complex 2D and 3D nanostructures. In parallel, polymer science has developed several polymerization approaches to build di- and triblock copolymers bearing hydrophilic, hydrophobic, and amphiphilic properties. By bringing together these two emerging technologies, complementary properties of both materials have been explored; for example, the synthesis of amphiphilic DNA–polymer conjugates has enabled the production of several nanostructures, such as spherical and rod-like micelles. Through both the DNA and polymer parts, stimuli-responsiveness can be instilled. Nanostructures have consequently been developed with responsive structural changes to physical properties, such as pH and temperature, as well as short DNA through competitive complementary binding. These responsive changes have enabled the application of DNA–polymer conjugates in biomedical applications including drug delivery. This review discusses the progress of DNA–polymer conjugates, exploring the synthetic routes and state-of-the-art applications afforded through the combination of nucleic acids and synthetic polymers.

Synthesis of octapeptin C4 and biological profiling against NDM-1 and polymyxin-resistant bacteria

The first synthesis of octapeptin C4 was achieved using a combination of solid phase synthesis and off-resin cyclisation. Octapeptin C4 displayed antibiotic activity against multi-drug resistant, NDM-1 and polymyxin-resistant Gram-negative bacteria, with moderate activity against Staphylococcus aureus. The linear analogue of octapeptin C4 was also prepared, which showed reduced activity.

Sequence-Controlled Glycopolymers via Step-Growth Polymerization of Precision Glycomacromolecules for Lectin Receptor Clustering.

A versatile approach for the synthesis of sequence-controlled multiblock copolymers, using a combination of solid phase synthesis and step-growth polymerization by photoinduced thiol-ene coupling (TEC) is presented. Following this strategy, a series of sequence-controlled glycopolymers is derived from the polymerization of a hydrophilic spacer macromonomer and different glycomacromonomers bearing between one to five α-d-Mannose (Man) ligands. Through the solid phase assembly of the macromonomers, the number and positioning of spacer and sugar moieties is controlled and translates into the sequence-control of the final polymer. A maximum M̅n of 16 kDa, corresponding to a X̅n of 10, for the applied macromonomers is accessible with optimized polymerization conditions. The binding behavior of the resulting multiblock glycopolymers toward the model lectin Concanavalin A (ConA) is studied via turbidity assays and surface plasmon resonance (SPR) measurements, comparing the ability of precision glycomacromolecules and glycopolymers to bind to and cross-link ConA in dependence of the number of sugar moieties and overall molecular weight. The results show that there is a clear correlation between number of Man ligands and Con A binding and clustering, whereas the length of the glycooligomer- or polymer backbone seems to have no effect.

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.

Deoxyribozyme-mediated ligation for incorporating EPR spin labels and reporter groups into RNA.

Preparation of site-specifically labeled RNA for spectroscopic studies is a multistep process and requires handling of delicate samples. This chapter is focused on the synthesis of spin-labeled RNA using convertible nucleosides and the application of the deoxyribozyme 9DB1* for the ligation of RNA fragments. The convertible nucleoside approach enables the attachment of nitroxyls as paramagnetic reporters at the exocyclic amino groups of cytidine, adenosine, and guanosine nucleobases in synthetic RNA. The deoxyribozyme 9DB1* is a synthetic single-stranded DNA with RNA ligase activity that can be used as an alternative to protein enzymes (T4 RNA/DNA ligases) for covalently joining RNA fragments via native 3'-5' phosphodiester bonds. The combination of solid-phase synthesis and DNA-catalyzed RNA ligation provides reliable access to site-specifically labeled functional RNAs for spectroscopic studies. A particular advantage of using deoxyribozymes for RNA ligation lies in the mild reaction conditions that prevent chemical damage to sensitive labels. As an example, we describe a detailed protocol for the synthesis of TEMPO-labeled SAM-I riboswitch RNA.

Back Cover: Synthesis of PAF, an Antifungal Protein fromP. chrysogenum, by Native Chemical Ligation: Native Disulfide Pattern and Fold Obtained upon Oxidative Refolding (Chem. Eur. J. 38/2013)

PAF, an antifungal protein found in P. chrysogenum, is stabilized with three disulfide bonds that are essential for bioactivity. G. Váradi, G. K. Tóth and co-workers in their Full Paper on page 12684 ff. show that the protein with its native fold and disulfide pattern can be prepared in good yield by using a combination of solid-phase synthesis and native chemical ligation followed by oxidative refolding.

New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes

The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (trans-ligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cis-ligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of site-specific modifications and is applicable to the synthesis of many biologically important RNA molecules.

Synthesis and NMR studies of cyclopeptides containing a sugar amino Acid.

[structure: see text] Cyclopeptides containing Glucuronic acid methylamine (Gum) alternating with Gly, L-Ala, D-Ala, L-Phe, D-Phe, L-Lys, or D-Lys were synthesized by a combination of solid-phase synthesis and solution chemistry. A more effective pathway to synthesize the sugar amino acid Gum in higher yields and in a shorter period of time was developed. Gum is employed in the benzylated and deprotected form. The cyclopeptides were characterized by NMR and the structure of one deprotected cyclic peptide solved.

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif.

Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.

PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro.

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Design and synthesis of potent in vivo antagonists of oxytocin.

We have previously shown that the substitution of 8-ornithine and 2-O-methyltyrosine alone and in combination in [1-deaminopenicillamine] oxytocin (dPOT) brought about enhancements in antagonistic potencies to responses to oxytocin in vivo. To explore the effects of these substitutions in analogs of dPOT containing larger alkyl substitutents on the beta carbon at position 1 and on the tyrosine residue at position two, the following six analogs were synthesized: [1-(beta-mercapto-beta, beta-diethylpropionic acid), 8-ornithine] vasotocin (1, dEt2OVT); (1-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 8-ornithine] vasotocin (2, d(CH2)5OVT): [1-beta-mercapto-beta, beta-diethylpropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [3, dEt2 Tyr(Me)OVT]; [1-(beta-mercapto-beta, beta-diethylpropionic acid), 2-O-ethyltyrosine, 8-ornithine]vasotocin [4, dEt2 Tyr(Et)OVT]; [1-beta-mercapto-beta', beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [5, d(CH2)5 Tyr(me)OVT]: [1-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [6,d(CH2)5 Tyr(Et)OVT]. The required protected intermediates were synthesized by a combination of solid-phase synthesis and by individual 8 + 1 couplings in solution. All six analogs antagonize the actions of oxytocin on the rat uterus in the absence of Mg2+, in the presence of 0.5 mM Mg2+ and in situ. They also antagonize milk ejection responses to oxytocin, and the vasopressor responses to arginine vasopressin, and all have very low antidiuretic activities. With pA2 values of 7.35 +/- 0.08 and 7.37 +/- 0.17, respectively, compounds 3 and 5 are the two most potent in vivo antagonists of oxytocin reported to date.

1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),4-valine,-8-D-arginine]vasopressin, a potent and selective inhibitor of the vasopressor response to arginine-vasopressin.

As part of a program in which we are attempting the design and synthesis of an antagonist of the antidiuretic response to arginine-vasopressin (AVP) [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),4-valine,8-D-arginine]vasopressin [d(CH2)5VDAVP] was synthesized and assayed for antidiuretic, vasopressor, and oxytocic activities. The required protected intermediate was synthesized by a combination of solid-phase synthesis and an [8 + 1] coupling in solution. d(CH2)5VDAVP has an antidiuretic potency of 0.10 +/- 0.02 unit/mg, less than 1/10000 that of its parent [deamino,4-valine,8-D-arginine]vasopressin (dVDAVP). d(CH2)5VDAVP is a specific antagonist of the vasopressor responses to AVP. It has an antivasopressor pA2 value of 7.68 +/- 0.05 when tested against AVP. It is also an antagonist of the in vitro oxytocic response to oxytocin (pA2 value = 6.62 +/- 0.07). With its negligible antidiuretic activity, absence of oxytocic activity, and its potent and specific ability to antagonize the vasopressor effects of AVP, d(CH2)5VDAVP is one of the most potent and selective vasopressor antagonists reported to date. It should thus be a useful tool with which to probe the possible role(s) that AVP may play in cardiovascular regulation under normal and pathological conditions.

1-Deaminopenicillamine,4-threonine]oxytocin, a potent inhibitor of oxytocin.

[1-Deaminopenicillamine,4-threonine]oxytocin was prepared in duplicate from S-benzyl-3-mercapto-3,3-dimethylpropanoyl-Tyr(Bzl)-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) by removal of the Bzl-protecting groups with Na-NH3, followed by cyclization of the resulting disulfhydryl compound with K3Fo(CN)6. The analogue was purified by desalting on Sephadex G-15 in 50% acetic acid and gel filtration of Sephadex G-15. The protected peptide I was synthesized (a) by the solid-phase method and (b) by a combination of solid-phase synthesis and an [8 + 1] coupling in solution. The analogue has no detectable agonist activity in rat vasopressor or isolated rat uterus assays. It has an antivasopressor pA2 of 6.67 +/- 0.09. It is a potent inhibitor of the in vitro oxytocic response to oxytocin and has a pA2 value of 7.46 +/- 0.04. (Material from the repeat synthesis has a pA2 value of 7.59 +/- 0.08.) Thus the substitution of threonine for glutamine in the antagonist [1-deaminopenicilliamine]oxytocin (pA2, 7.14 +/- 0.05) has effected a twofold increase in inhibitory potency. [1-deaminopenicillamine,4-threonine]oxytocin is one of the most potent inhibitors of oxytocin known to date.