Rapid Routes of Synthesis of Oligonucleotide Conjugates from Non-Protected Oligonucleotides and Ligands Possessing Different Nucleophilic or Electrophilic Functional Groups

Abstract

Optimized methods are described for post-synthetic conjugation of non-protected oligodeoxyribonucleotides to different ligands. Methods for the terminal functionalization of oligonucleotides by amino, sulfhydryl, thiophosphate or carboxyl groups using different chemical reactions and linkers in both organic and aqueous media are described and compared. Experimental conditions for subsequent coupling of ligands containing aliphatic and aromatic amines, aromatic alcohols, carboxylic, sulfiydryl, alkylating, aldehydic and other reactive nucleophilic and electrophilic groups to oligonucleotides were established, including covalent linkage to other oligonucleotides.     

A general method for the synthesis of 3''-sulfhydryl and phosphate group containing oligonucleotides.

The syntheses are described of polymer supports useful for the synthesis of 3'-partially protected sulfhydryl, free sulfhydryl or phosphate group containing oligonucleotides. The supports are compatible with established phosphoramidite chemistry of oligonucleotide synthesis giving rise to oligonucleotides with terminal 3'-partially protected sulfhydryl, free sulfhydryl or phosphate function during final deprotection. Crosslinking of the thiol group containing oligonucleotide to sulfhydryl group specific fluorescent probes was carried out with high selectivity, in high yields under mild conditions. 3-Aminopropylated Controlled Pore Glass (CPG) was succinylated with succinic anhydride followed by the reaction with S-(2-thio-5-nitropyridyl)-2-mercaptoethanol in the presence of dicyclohexylcarbodiimide (DCC). The resultant polymer support was reacted with 4,4'-dimethoxytrityloxyalkanthiol 5(a - c) to yield the derivatized polymer supports 5(a - c). The support 5a directly leads to oligonucleotide-3'-phosphate on deprotection with ammonical DTT at 55 degrees C while the supports 5b and 5c lead to oligonucleotide-3'-thiols or partially protected 3'-sulfhydryl group containing oligonucleotides during final deprotection.     

Organization of functional groups of liver bilitranslocase

Bilitranslocase transport activity can be described as consisting of three functional fractions, which depend on two distinct classes of sulfhydryl groups, on the one hand, and on the guanido groups of arginine residues, on the other. Each fraction accounts for approx. 50% transport activity. The pattern of transport activity inhibition resulting from step-wise derivatization of these functional groups indicates that, in general, derivatization of arginine residues prevents that of one class of sulfhydryl groups and vice versa, indicating their close location in the protein. Nevertheless, under appropriate conditions, derivatization of both functional groups can be achieved; however, the inhibitory effect produced is not additive. Hence, these two fractions overlap functionally and are likely to belong to a common functional domain of the protein. On the contrary, the other class of sulfhydryl groups can be derivatized, regardless of the state of the arginine residues.     

A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands.

Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.     

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.     

Kinase inhibitors and the case for CH...O hydrogen bonds in protein-ligand binding

Although the hydrogen bond is known to be an important mediator of intermolecular interactions, there has yet to be an analysis of the role of CH...O hydrogen bonds in protein-ligand complexes. In this work, we present evidence for such nonstandard hydrogen bonds from a survey of aromatic ligands in 184 kinase crystal structures and 358 high-resolution structures from the Protein Data Bank. CH groups adjacent to the positively charged nitrogen of nicotinamide exhibit geometric preferences strongly suggestive of hydrogen bonding interactions, as do heterocyclic CH groups in kinase ligands, while other aromatic CH groups do not exhibit these characteristics. Ab initio calculations reveal a considerable range of CH...O hydrogen bonding potentials among different aromatic ring systems, with nicotinamide and heterocycles preferred in kinase inhibitors showing particularly favorable interactions. These results provide compelling evidence for the existence of CH...O hydrogen bonds in protein-ligand interactions, as well as information on the relative strength of various aromatic CH donors. Such knowledge will be of considerable value in protein modeling, ligand design, and structure-activity analysis.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Highly selective and potent μ opioid ligands by unexpected substituent on morphine skeleton

Unexpected substituent on the well-known morphine skeleton is described to be account for highly selective and potent μ opioid ligands, which is strongly connected to substituted aromatic groups on this omitted 8α-position.     

Synthesis of oligonucleotides carrying 5'-5' linkages using copper-catalyzed cycloaddition reactions

There is considerable interest in coupling oligonucleotides to molecules and surfaces. Although amino- and thiol-containing oligonucleotides are being successfully used for this purpose, cycloaddition reactions may offer greater advantages due to their higher chemoselectivity and speed. In this study, copper-catalyzed 1,3-dipolar cycloaddition reactions between oligonucleotides carrying azido and alkyne groups are examined. For this purpose, several protocols for the preparation of oligonucleotides carrying these two groups are described. The non-templated chemical ligation of two oligonucleotides via copper-catalyzed [3+2] cycloaddition is described. By solid-phase methodology, oligonucleotides carrying 5'-5' linkages can be obtained in good yields.     

Complexation of Biologically Active Aromatic Compounds with DNA in the Presence of Theophylline

¹H NMR measurements (500 MHz) have been used to determine the equilibrium hetero-association constants of theophylline (THP) with various biologically active aromatic compounds (daunomycin, novantrone, ethidium bromide, proflavine, norfloxacin) and the complexation constants of THP with both single- and double-stranded oligonucleotides in solution. The results provide a quantitative estimation of the effect of THP on the binding of aromatic ligands with DNA, and a determination of the fraction of aromatic ligand removed from DNA on addition of THP.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

The mobility of methylmercury in biological systems.

Toxicology studies indicate that methylmercury in humans and other species is bonded to sulfhydryl ligands and that the methylmercury in such complexes is labile even though their thermodynamic stability is large. It is shown in this paper that bimolecular nucleophilic displacement of complexed ligand by sulfhydryl-deprotonated ligand is the major pathway for ligand exchange at physiological pH, while at the pH of the stomach the proton-assisted dissociation of the complex is the predominant means by which exchange occurs. The dynamic and equilibrium aspects of the distribution of methylmercury between chloride and sulfhydryl ligands under the solution conditions of the stomach are also considered with respect to a possible role for lipid-soluble CH3HgCl in the absorption of methylmercury from the stomach.     

Photosubstitution of cymantrenylalanine as a tool in peptide chemistry. Synthesis and biological activity of new GnRH analogs

Beyond its aromatic character and important hydrophobicity, cymantrenylalanine, a metallocenic amino-acid, can be easily photosubstituted with phosphine and phosphite ligands to readily yield new analogs with different hydrophobicity and steric hindrance. The incorporation of phosphine and phosphite ligands is described. As an illustration of the offered possibilities, the synthesis and the biological activity of two new GnRH analogs modified in position 6 are reported.     

A sensitive gel filtration method for determination of protein sulfhydryl groups with 14C-chloromercuribenzoate

A gel filtration method employing ¹⁴C-chloromercuribenzoic acid is described for the quantitative determination of sulfhydryl groups in microgram quantities of protein. The method has been applied to several native proteins, hemoglobin, monoamine oxidase, and yeast cytochrome c. In all cases values in close agreement with known literature values were obtained. Horse heart cytochrome c and lysozyme, which have no sulfhydryl groups, did not bind the mercurial reagent. Modifications of the method are described for determining the sulfhydryl content of denatured proteins in the presence of sodium lauryl sulfate. The precision of the method was found to be compatible with known methods for determining the sulfhydryl composition of proteins.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges.

A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation.     

Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.     

A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.