Modified dATP (2'-deoxyadenosine-5'-triphosphate) and dUTP (2'-deoxyuridine-5'-triphosphate) bearing ferrocene (Fc) labels linked via a conjugate acetylene spacer were prepared by single-step aqueous-phase cross-coupling reactions of 7-iodo-7-deaza-dATP or 5-iodo-dUTP with ethynylferrocene. The Fc-labeled dNTPs were good substrates for DNA polymerases and were efficiently incorporated to DNA by primer extension (PEX). Electrochemical analysis of the 2'-deoxyribonucleoside triphosphates (dNTPs) and PEX products revealed significant differences in redox potentials of the Fc label bound either to U or to 7-deazaA and between isolated dNTPs and conjugates incorporated to DNA. Prospective bioanalytical applications are outlined.

No abstract

Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids e-N-propionyl-, e-N-butyryl-, and e-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.The histone code is based on the post-translational modification of critical amino acid residues in different histones. Among these, lysine acetylations and methylations are the most abundant and affect the transcriptional status of the genes associated with the corresponding histones. [1][2][3] The role of these modifications is sequence dependent. Typically acetylation is associated with transcriptionally active genes, while methylation induces transcriptional silencing. 4,5 Recently Zhao and co-workers discovered a number of new modified amino acids in histones. [6][7][8] Some of these were also detected in proteins other than histones, raising the possibility that they are of more widespread importance. [9][10][11] The new post-translational modifications (PTMs) are acylated derivatives of lysine at the e-amino position. The acylation partners are propionic acid, butyric acid, malonic acid, succinic acid, crotonic acid or fatty acids. A common characteristic of these compounds is that they are key metabolic intermediates that typically exist as CoA activated thioester species in the cell. 12 It is currently not clear how these newly discovered PTMs are biosynthetically established within the histones and we do not fully understand if and how they influence genetic processes.So far no specific deacylases for e-N-propionyl-or e-Nbutyryl-lysine have been identified. However Sirt5, a member of NAD-dependent sirtuins is able to specifically deacylate e-Nmalonyl-and e-N-succinyllysine. 11 Recently it was discovered that HDAC3 exhibits decrotonylase activity in vitro 13 and it was found that lysine crotonylation activates genes even in a globally repressive environment. 7,14 In order to enable investigation of the new lysine derivatives in histones it is essential to generate histones that contain these amino acids site specifically. 15 In this direction semi-synthetic chemical ligation based methods 16,17 were utilized and chemical methods were employed to generate acetyllysine (Kac) and methylated-lysines or derivatives thereof. [18][19][20][21] Chin and co-workers reported the introduction of Kac into H3 K56 22 using the pyrrolysine system. This system was also employed for the synthesis of monomethyl-and dimethyl-lysine containing histones. 23,24 Schultz et al. recently described the synthesis of histone H2B containing e-N-crotonyllysine at position K11 using an evolved pyrrolysyl-tRNA synthetase from Methanosarcina barkeri. 25 Here we show that the pyrrolysine system can be used to insert e-N-propionyl-(Kpr, 1), e-N-butyryl-(Kbu, 2), and e-Ncrotonyl-lysine (Kcr, 3) into histones at critical positions such as H3 K9 using...

Significant differences in the reactivity of norbornene derivatives in the inverse electron-demand Diels-Alder reaction with tetrazines were revealed by kinetic studies. Substantial rate enhancement for the exo norbornene isomers was observed. Quantum-chemical calculations were used to rationalize and support the observed experimental data.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.

A series of ethynyl-or (4-boronophenyl)bipyridines and -phenanthrolines were prepared as versatile building blocks for attachment of bidentate N-ligands to other molecules via cross-coupling reactions. Their complexation with Ru(bpy) 2 -Cl 2 gave the corresponding Ru II complexes. 9-Benzyladenine derivatives bearing the bipyridine or phenanthroline complexes in position 8, attached via a conjugate acetylene or phenylene linker were prepared by cross-coupling reactions of the ethynyl-or 4-boronophenylbipyridines and -phenanthrolines with 9-benzyl-8-bromoadenine. Their complexation with Ru(bpy) 2 Cl 2 afforded the corresponding Ru complexes as model compounds for electrochemical DNA labeling. The same compounds were also prepared directly by crosscoupling of 9-benzyl-8-bromoadenine with Ru complexes of the alkynes and boronic acids. Both approaches are compared in terms of potential applications for labeling of nucleic

No abstract