Design, Synthesis, and Evaluation of Mitomycin-Tethered Phosphorothioate Oligodeoxynucleotides

Abstract

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Studies have shown that mitomycin C and its derivatives selectively alkylate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties of the mitomycins by coupling them with antisense oligodeoxynucleotides. Two procedures were developed that allowed the attachment of a phosphorothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/ benzene) afforded 10-decarbamoylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin (11) and N(1a)-[(methylthio)-carbonyl]-10-decarbamoylmitomycin C (12), respectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbonyldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-substituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Compounds 5-8 were reacted with glycine methyl ester hydrochloride (17) and base in both methylene chloride and aqueous buffered solutions to determine the ease and efficiency in which these C(10)-activated mitomycin derivatives coupled to amines. It was found that 5-8 all reacted with 17 in methylene chloride to give the coupled products 18-21 but that improved amine coupling yields in water were observed for the 10-decarbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-decarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the coupling of the phosphorothioate oligodeoxynucleotide, H2N(CH2)6-P(S)(OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-base sequence in the translation initiation region of the human A-raf-1 gene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the coupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the c...

4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides

“…Chemically reactive functional groups such as psoralen (3,4), dimethylanthraquinone (5), mitomycin (6), and 2-amino-6-vinylpurine (7) have been conjugated to oligonucleotides to achieve oligomer-target cross-linking.…”

“…In contrast, groups such as aromatic 2-chloroethylamine (8,9), cyclopropapyrroloindole (10), transplatin (11), and 5-methyl-N 4 , N 4 -ethanocytosine (12), when incorporated into oligonucleotides, can carry out alkylation when the oligonucleotide hybridizes to its target. Efficient covalent bond formation between an oligonucleotide and its target has been shown to correlate with the antisense efficacy in the cell (6,11,13).…”

Synthesis of PAMAM Dendrimers Loaded with Mycophenolic Acid to Be Studied as New Potential Immunosuppressants

“…The latter compound was then reacted, without purification, with di-2-pyridyl thionocarbonate (DPT) and triethylamine. DPT [17,18] is a commercially available, solid, nontoxic reagent that can be used in the preparation of isothiocyanates as a safe alternative to thiophosgene. The reaction proceeded smoothly and gave the complete conversion after 5.5 h. The intermediate isothiocyanate obtained after removal of the solvents was directly reacted with crude 9 in the presence of Et 3 N in a mixture of CH 2 Cl 2 and DMF.…”

High-Yielding Method for On-Column Derivatization of Protected Oligodeoxy- nucleotides and Its Application to the Convergent Synthesis of 5‘,3‘-Bis-conjugates

“…High yields of homogeneous products are obtained by using protected oligodeoxynucleotides in solution as substrates . The protected oligonucleotides are prepared using photolabile solid-phase synthesis supports under conditions that do not damage the biopolymers and are compatible with commercially available phosphoramidite reagents. , These methods enjoy a number of advantages over previously reported strategies for preparing oligodeoxynucleotide conjugates, including short reaction times, mild reaction conditions, and the need for a relatively small excess of reagents. , In addition, the method is convergent, enabling one to readily prepare multiple conjugates from a single oligodeoxynucleotide synthesis. This latter feature is particularly useful for preparing libraries of oligodeoxynucleotides .…”

“…A number of methods exist for modifying the 5‘-termini of oligodeoxynucleotides with biologically relevant molecules. ,, The most common approaches to introducing such modifications involve either preparing the appropriate phosphoramidite or postsynthetic conjugation of a fully deprotected oligodeoxynucleotide containing a 5‘-alkylamine to an electrophilic form of the desired modifying molecule ,,, A method employing isolated activated carboxylic acid esters was recently reported for derivatizing protected oligodeoxynucleotides containing 5‘-terminal amines while the biopolymer was still bound to its solid-phase support . Although the yields of modified oligodeoxynucleotides were not explicitly reported, yields of peptide nucleic acids modified by this method were believed to be greater than 80%.…”