Ethylenediamine Linker Research Articles

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of “complementary addressed modification titration” (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylaminojbenzylamino- group (C1RCH2NH)- at 5′-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E1, E2 and their derivatives E1 phn, E2 phn bearing N- (2-hydroxyethyl)-phenazinium residues Phn- both at 5′- and 3′- ends covalently linked via ethylenediamine linker were used as effectors. Effectors E1 and E2 (E1 Phn and E2 Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8m) reagents were used for the target modification. The reagent X8m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8m and effectors E1, E2, E1 phn, E2 Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group CIRCH2NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either CIRCH2NH- group or TT-mismatch at the junction.

Syntheses of Acyclic Analogs of Didemnin B

The syntheses of three modified peptide fragments of the cyclodepsipeptide didemnin B are reported. The HIP and isostatine (1st) units of the didemnin B macrocycle were simplified to a Z-alanine residue and the ester linkage (through threonine of the tetrapeptide) was replaced with amide linkages through the amines of glycine, D-alanine and an ethylenediamine linker. The latter permitted the attachment of a N-Me-D-Leu-Pro-Lac moiety to afford analogs 2, 3 and 4 respectively.

Synthesis and purification of modified antisense oligodeoxyribonucleotides

The synthesis and purification of modified antisense oligodeoxyribonucleotides (MAO) are desoribed. A new oligonucleotide purification method and a novel activating agent, dimethyl sulfoxide, for the condensation reaction are used. EDTA has been attached covalently to the 5′ end of the oligodeoxyribonucleotides sequence via an ethylenediamine linker in basic triethylammonium solution, then chelated with the metal ions such as Fe(II), Co(II) or Cu(II). High performance liquid chromatorgaphy is used to separate and purify this kind of MAO, using double mobile phase, triethylammonium acetate and acetonitrile. By using this routine chemical synthesis method, EDTA can be linked to the 5′ terminus of oligodeoxyribonucleotedes of any desired length and sequence, forming chimeric MAOs.

Synthesis of α-d-Glc p-(1 → 2)- α-d-Glc p-(1 → 3)- α-d-Glc p-O-(CH 2) 8COOCH 3 for use in the assay of α-glucosidase I activity

The chemical synthesis of α-d-Glc p-(1 → 2)- α-d-Glc p-(1 → 3)- α-d-Glc p-O-(CH 2) 8COOCH 3 ( 9), a substrate specific for α-glucosidase I, is reported. This enzyme removes the terminal α-d-Glc p unit to produce α-d-Glc p-(1 → 3)- α-d-Glc p-O-(CH 2) 8COOCH 3 ( 10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this enzyme. Tetramethylrhodamine was coupled to 9 through an ethylenediamine linker to produce a brilliant red derivative. Addition of this fluorescent dye did not affect enzyme binding to the substrate, as determined by a comparison of the K m value (1.3 mM). The fluorescent label allows visual detection of 2–3 pmol of product by TLC.

Synthesis of C 2-symmetric inhibitors of the HIV-1 protease, with N,N′-substituted ethylenediamide and ethylenediamine linkers.

Coupling of Z-(L)-phenylalanine with either ethylenediamine or N,N′-dimethylethylenediamine, followed by N-deprotection and either direct coupling with Z-(L)-valine or amide reduction prior to coupling, gave C 2 -symmetric compounds [Z-Val-Phe-(N(R)CH 2-] 2 (R = H, CH 3) and [Z-Val-Phe(ΨCH 2-N)-N(R)CH 2-] 2 (R= H, CH 3, CH 2COOH, CH 2CH 2OH) which were moderately active inhibitors of the HIV-1 protease.

Synthesis and application of fluorescent labeled nucleotides to assay DNA damage

A facile method was developed to covalently attach a fluorophore to the 5′-phosphate of a nucleic acid. The procedure, illustrated by coupling 5′-dNmp ( N = A,C,G,T) with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride, involves 5′-phosphoramidation with ethylenediamine (EDA) followed by conjugation of the free aliphatic amino group of the phosphoramidate with Dansyl chloride. This method is also applicable to multi-incorporation of fluorescent labels in the nucleic acids. The reaction of 5′-Amp with a polyamine such as poly L-lysine (PLL, mol. wt., 4000) resulted in a phosphoramidate with multiple amino groups, which after isolation and conjugation with fluorescamine gave dAmp with multilabeled fluorophores. A condition was devised to separate the four dansylated mononucleotides of DNA, conjugated via ethylenediamine linker, by reverse phase HPLC. The elution profile could be monitored with a variable wavelength detector at 254 nm and 340 nm corresponding to the absorption of the nucleotides and the dansyl moiety, respectively. The detection limit was 2 nmol at 254 nm. The use of a fluorescence detector enhanced the detection sensitivity to a sub-picomole level (200 fmol). Samples of a DNA model, d(pCpGpTpA) and calf-thymus DNA were digested enzymatically to 5′-mononucleotides and labeled with Dansyl chloride. HPLC analysis of the dansylated digests from these samples, both before and after irradiation, suggests that the combination of enzymatic digestion and fluorescence postlabeling could be a novel approach to assay DNA damage.