Tatyana S. Godovikova

Intraduplex Photo-cross-linking of p-Azidoaniline Residue and Amino Acid Side Chains Linked to the Complementary Oligonucleotides via a New Phosphorylating Intermediate Formed in the Mukaiyama System

Abstract Oligonucleotide derivatives carrying a side chain of either lysine or histidine at the 3′-end and their complementary oligonucleotides having photoreactive groups a p-azidophenyl-NH(CH2)nNH- (n = 4, 6) residue at the 5′-end were prepared by using new phosphorylating species formed by treatment of oligonucleotides with Ph3P and (PyS)2 or (PyrS)2. in DMF, DMSO or their mixture. Efficient cross-linking of duplexes occurred under UV-irradiation (λ > 300 nm).

AGEs, RAGEs and s-RAGE; friend or foe for cancer

Impaired awareness of glycation biology in cancer initiation and progression is one of the fundamental reasons for its meticulous investigation of the molecules involved in signalling pathway. Glycation of biological macromolecules results in the progression of advanced glycation end-products (AGEs) that proliferates the process of carcinogenesis by activation of transcription factors and release of cytokines. The receptor for advanced glycation end-products (RAGEs) with the binding of its different ligands like; AGEs, HMGB1 and S100 activate the signalling arrays. The activation of downstream signalling pathway ultimately leads to the pathophysiological conditions of diabetes, ageing, neurological disorders and cancers as well as a result of the activation of transcription factors which is discussed in the main body text of this review. However, there might be a likelihood of the positive effect of the HMGB1 and S100 proteins in cancer. Still, some untouched mechanisms might be responsible for the establishment of the function of AGE-RAGE or AGE-sRAGE axis activation that leads to the friend-foe association with the cancers. The levels of RAGE and s-RAGE may be a useful biomarker of ligand-RAGE pathway activation and cancer. Thus, the possibility of providing a potential complement to carcinogenesis is very high which might be an interesting target for therapeutic interventions. This article is an insightful assessment on AGE, RAGE and s-RAGE for its possible role in cancer onset and progression. The novel therapeutic targets for cancer prevention or inhibition are also explained in brief in relation to AGE and RAGE.

Cleavage of Leishmania Mini-exon Sequence by Oligonucleotides Conjugated to a Dimidazole Construction

Abstract RNA sequences derived from the Leishmania amazonensis mini-exon and pre-mini-exon sequences have been targeted with complementary oligonucleotides bearing a diimidazole construction mimicking active center of ribonuclease A. The conjugates were shown to cleave the target RNAs at specific positions.

Design of protein homocystamides with enhanced tumor uptake properties for (19)F magnetic resonance imaging.

Straightforward and reliable tools for in vivo imaging of tumors can benefit the studies of cancer development, as well as contribute to successful diagnosis and treatment of cancer. (19)F NMR offers an exceptional quantitative way of in vivo imaging of the infused agents because of the lack of (19)F signals from the endogenous molecules in the body. The purpose of this study is to develop molecular probes with appropriate NMR characteristics and the biocompatibility for in vivo applications using (19)F MRI. We have studied the reaction between perfluorotoluene and homocysteine thiolactone resulting in the formation of N-substituted homocysteine thiolactone derivative. It has been shown that the reaction occurs selectively at the para position. This fluorine-labeled homocysteine thiolactone has been employed for the introduction of a perfluorotoluene group as a (19)F-containing tag into human serum albumin. The modified protein has been studied in terms of its ability to aggregate and promote the formation of free radicals. By comparing the properties of N-perfluorotoluene-homocystamide of albumin with N-homocysteinylated albumin, it has been revealed that blocking of the alpha-amino group of the homocysteine residue in the fluorinated albumin conjugate inhibits the dangerous aggregation process, as well as free radical formation. A dual-labeled albumin-based molecular probe for (19)F MRI and fluorescence microscopy has been obtained by functionalizing the protein with both maleimide of a fluorescent dye and a fluorinated thiolactone derivative. The incubation of cells with this conjugate did not reveal any significant reduction in cell viability with respect to the parent albumin. The perfluorotoluene-labeled albumin has been demonstrated to act as a promising agent for in vivo (19)F MRI.

Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for 19F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, (19)F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and (19)F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo (19)F magnetic resonance spectroscopy and imaging.

Human serum albumin as a catalyst of RNA cleavage: N-Homocysteinylation and N-phosphorylation by oligonucleotide affinity reagent alter the reactivity of the protein

Kinetic parameters for the cleavage of UpA site in an oligonucleotide in the presence of human serum albumin (HSA) or one of its clinically relevant modification were measured. The RNA-hydrolyzing activity of HSA was decreased by its nonenzymatic N-homocysteinylation. According to (31)P NMR data, Lys and Tyr residues were the labeling targets when a phosphorylating analog of oligoribonucleotide substrate was employed. The site of tyrosine modification was slowly dephosphorylated. Lys-directed affinity labeling suppressed oligonucleotide cleavage indicating that lysines took part in the reaction.

Photoanalogues of the Initiation Substrates of the RNA Polymerase II, 5‐Azido‐2‐Nitrobenzoyl Derivatives of the ATP γ‐Amidophosphate: The Possible Photoinduced Degradation of the Functional Group to an N‐Arylhydroxylamine

Photoanlogues of the initiation substrates of the RNA polymerase II, N3Ar‐ NH(CH2)nNHpppA where N3Ar is 5‐azido‐2‐nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p‐nitro‐substituted aromatic azide in aqueous medium was investigated. Using the azoxy‐coupling reaction it was possible to determine whether a nitrene or p‐nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution.

Tyrosine 54 and tryptophan 108 of streptavidin are photolabelled by N-(2-nitro-5-azidobenzoyl)-N′-(d-biotinyl)-1,4-diaminobutane and N-(4-azidophenyl)-N′-(d-biotinyl)-1,4-diaminobutane, respectively. Isolation, spectrophotometric characterization and sequence analysis of photolabelled peptides

Abstract Two new photoreactive biotin derivatives, N-(2-nitro-5-azidobenzoyl)-N′-(d-biotinyl)-1,4-diaminobutane (I) and N-(4-azidophenyl)-N′-(d-biotinyl)-1,4-diaminobutane (II), have been synthesized and used for a photoaffinity labelling of streptavidin at a molar ratio of 1:1 reagent to streptavidin subunit. Photolabelled streptavidin preparations are subjected to extensive digestion by proteinase K, and the mixture of peptides obtained is fractionated by reversed-phase HPLC. The fractions containing the peptides photolabelled by the photobiotin I are identified by UV spectroscopy (λ = 360 nm) and the fractions containing the peptides photolabelled by the photobiotin II are characterized by their interaction with streptavidin alkaline phosphatase conjugate. The photolabelled peptides are analysed by spectrophotometric absorbance and submitted to sequence analysis by automatic Edmans degradation. The sequence of the peptide photolabelled by photobiotin I is found to be SerArgTyr* ValLeu, corresponding to residues 52–56 of the streptavidin sequence, where Tyr* designates modified Tyr-54. The sequences of the peptides photolabelled by photobiotin II are found to be ThrGlnTrp*LeuLeu (residues 106–110) and Trp*LeuLeu (residues 108–110), where Trp* designates modified Trp-108.

p-Azidophenyl phosphate is a photoactivatable phosphorylating reagent and p-benzoquinone monoimine precursor

p-Azidophenyl phosphate (I) has been exposed to ultraviolet light (lambda=313 nm) in aqueous solution with or without Lys. Analysis of the photoproducts by means of UV-VIS, IR, (1)H, (13)C and (31)P NMR spectroscopy has revealed that under irradiation of I inorganic phosphate (P(i)) is released, and p-benzoquinone monoimine (II) and p-benzoquinone (III) have appeared. The electrophilic nature of the intermediate results in a high tendency to react with lysine molecules, whereas the reaction with water is less favourable when I is irradiated in the presence of Lys. The product formed in this case is a phosphoramidate whose structure has been tentatively supported by (31)P NMR spectroscopy. These results imply that a p-azidophenyl phosphate is a highly potent aryl nitrene-precursor, which can be transformed easily into p-benzoquinone monoimine and is able to phosphorylate nucleophilic groups of amino acids. This finding is of great importance for the discussions about the chemical nature of protein photomodification products when p-azidophenyl phosphate derivatives are used as modifying reagents.

Biotin-decorated anti-cancer nucleotide theranostic conjugate of human serum albumin: Where the seed meets the soil?

Human serum albumin is playing an increasing role as a drug carrier in clinical settings. Biotin molecules are often used as suitable tags in targeted anti-tumor drug delivery systems. We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anti-cancer fluorinated nucleotide conjugated with a biotinylated dual-labeled albumin. Interestingly, in vitro and in vivo study revealed stronger anti-tumor activity of the non-tagged theranostic conjugate than that of the biotin-tagged conjugate, which can be explained by decreased binding of the biotin-tagged conjugate to cellular receptors. Our study sheds light on the importance of site-specific albumin modification for the design of albumin-based drugs with desirable pharmaceutical properties.