Exocyclic Amine Research Articles

The 2,6-Diaminopurine Riboside.cntdot.5-Methylisocytidine Wobble Base Pair: An Isoenergetic Substitution for the Study of G.cntdot.U Pairs in RNA

Phylogenetically invariant G.U wobble pairs are present in a wide variety of RNA's. As a means to study the contribution of individual chemical groups within a G.U pair, we have synthesized and thermodynamically characterized oligoribonucleotides containing the unnatural nucleosides 2,6-diaminopurine riboside (DAP) and 5-methylisocytidine (MeiC). The DAP.MeiC pair at the end of an RNA duplex is as stable as a G.U pair, consistent with formation of a wobble base pair with two hydrogen bonds. DAP.MeiC is a valuable substitution for the study of G.U wobble pairs because it is conformationally similar to the G.U pair, but has a different array of functional groups in the major and minor grooves of the duplex and a reversed hydrogen bonding polarity between the bases. We also report the stability of several other terminal pairs proposed to be in a wobble configuration including inosine.U (I.U), A.MeiC, DAP.C, A.C, G.5-methyl-U,2'-deoxyguanosine.U, and 2'-deoxy-7-deazaguanosine.U. These pairs present a diversity of functional group substitutions in the context of a wobble conformation. Comparison of wobble pairs with and without the N2 exocyclic amine, i.e., G.U vs I.U, DAP.MeiC vs A.MeiC, and DAP.C vs A.C, demonstrates that the amine does not contribute to base pairing stability when the pair is located at the terminal position of the RNA duplex. However, at a position internal to the duplex, the exocyclic amine does improve helix stability. An internal I.U pair is less stable (approximately 1 kcal.mol-1) than an internal G.U pair, and substantially less stable (approximately 2 kcal.mol-1) than an internal A-U pair. These data provide quantitation for the reduced duplex stability observed upon conversion of A-U to I.U pairs by double-stranded RNA adenosine deaminase (dsRAD). This collection of wobble pairs will help identify the contribution made by individual functional groups in RNA/protein interactions and in the tertiary folding of RNA.

Electrostatic potential surfaces of the transition state for AMP deaminase and for (R)-coformycin, a transition state inhibitor.

The transition state for the hydrolysis of AMP by AMP deaminase has been characterized by heavy atom kinetic isotope effects (Merkler, D.J., Kline, P.C., Weiss, P., and Schramm, V.L. (1993) Biochemistry 32, 12993-13001). The experimentally established transition state includes a bond order of 0.8 to the attacking water nucleophile, a full bond order to the exocyclic 6-amino group, rehybridization of C-6 of the purine ring to sp3 and protonation of N-1 by Glu633. The transition state is one the path to formation of an unstable tetrahedral intermediate in which the exocyclic amine undergoes rapid protonation followed by its departure. In this mechanism, the highest energetic barrier on the reaction coordinate is the attack of the zinc-activated water. In a further test of this transition state structure, the electrostatic potential surface for the purine ring of the transition state has been determined by molecular orbital calculations and compared to that of the base of (R)-coformycin 5'-monophosphate, a slow onset, tight binding inhibitor of AMP deaminase that binds with an overall dissociation constant of 10(-11) M. The electrostatic potential surfaces of the aglycones of the transition state and (R)-coformycin are compared to the adenine ring of the substrate and to an alternative transition state structure in which the transition state is late, with fully bonded hydroxyl and fully protonated exocyclic amine. The results indicate a near-match of the electrostatic potential surfaces for the early transition state and (R)-coformycin. The electrostatic nature of the late transition state with a protonated amine leaving group differs both from the transition state determine by kinetic isotope effects and from that of (R)-coformycin analogues. The results provide evidence that the nature of the enzyme-stabilized transition state for adenine deamination involves an early transition state with a partially bonded hydroxyl group. The observed tight binding inhibition by (R)-coformycin analogues as transition state inhibitors results from the similarity of the partial charges on the inhibitors to that of the enzymatic transition state stabilized by AMP deaminase.

TMSO-triflate-assisted addition of enols to heteroaromatic imines or hydroxyaminal intermediates

TMSOTf-promoted reaction of heteroaromatic imines or hydroxyaminal intermediates with various enolic ketones are reported. This reaction constitutes an original and regioselective mode of alkylation of exocyclic amines of heterocyclic amidines.

Mechanism of formation of ethenoguanine adducts from 2-haloacetaldehydes: 13C-labeling patterns with 2-bromoacetaldehyde.

The mechanism of formation of etheno (epsilon) adducts of nucleic acid bases from 2-haloacetaldehydes is generally assumed to occur via initial Schiff base formation resulting from reaction of the aldehyde with an exocyclic amine. We recently revised the 1H NMR assignments of the epsilon protons of 1,N2-epsilon-Guo (Guengerich, F. P., Persmark, M. P., and Humphreys, W. G. (1993) Chem. Res. Toxicol. 6, 635-648). In that work we also observed a facile and specific exchange of H7 of 1,N2-epsilon-Guo and H5 of N2,3-epsilon-Gua with H2O. These findings raise questions about the mechanistic conclusions reached on the basis of labeling studies with deuterated ClCH2CHO (Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) J. Org. Chem. 42, 3292-3296). BrCH2-13CHO was prepared from BrCH2(13)CO2H and used to prepare 1,N2-epsilon-Guo (from Guo) and O6-ethyl-N2,3-epsilon-Gua (from O6-ethylGua). The positions of the labels were determined by 1H NMR spectroscopy experiments to be adjacent to the original Gua N2 (exocyclic) atom in both cases, i.e., at C6 in both epsilon products. The labeling patterns are consistent with a mechanism involving initial Schiff base formation from the N2 atom and the aldehyde and subsequent nucleophilic attack of an endocyclic nitrogen on the methylene carbon.

Synthesis of Oligonucleotides Containing Site-specific Carcinogen Adducts. Preparation of the 2-Cyanoethyl N,N-Diisopropylphosphoramidite of N-(2'-Deoxyguanosin-8-yl)-2-(acetylamino)fluorene with Fmoc as the Base-Protecting Group

A 9-fluorenylmethoxycarbonyl (Fmoc) group was used to protect the exocyclic amine on the modified guanine of N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-Cs-AAF) so that oligonucleotides containing a site-specific AAF adduct could be prepared. Reaction of Fmoc-Cl with dG-Cg-AAF gave N-[N-(9-fluorenylmethoxycarbonyl)-2'-deoryguanosin-8-yl]-2-(acetylamino)fluorene (N 2 -Fmoc-dG-C 8 -AAF) and N-[O 6 -(9-fluorenylmethoxycarbonyl)-2'-deoxyguanosin-8-yl]-2-(acetylamino)fluorene (O 6 -Fmoc-dG-Cg-AAF). The 5'-OH of N 2 -Fmoc-dG-C 8 -AAF was protected using 4,4'-dimethoxytrityl chloride to yield 5'-DMT-N 2 -Fmoc-dG-C 8 -AAF which was then reacted with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite to obtain the corresponding phosphoramidite. The phosphoramidites of Fmoc-protected dA, dC and dG were also prepared similary. The stability of Fmoc-protected C 8 -AAF-modified deoxyguanosine was studied under different conditions to establish the utility of the prepared phosphoramidite of the prepared phosphoramidite in solid-phase DNA synthesis

Synthesis of oligodeoxynucleoside methylphosphonates utilizing the tert-butylphenoxyacetyl group for exocyclic amine protection.

Oligodeoxynucleoside methylphosphonates were synthesized using methylphosphonamidite monomers incorporating the base labile tert-butylphenoxyacetyl (t-BPA) protecting group on the exocyclic amines of dA, dC, and dG. Synthesis of the oligodeoxynucleoside methylphosphonates required only a small change in oxidation solution from standard DNA synthesis. The increased lability of the t-BPA group over the standard benzoyl and isobutyryl protection permitted the use of milder basic deprotection conditions. Deprotection of the nucleoside bases and release from support was best accomplished by a short treatment with ammonia saturated methanol. This procedure resulted in minimal backgone degradation with no base modifications. Analysis of the resultant oligodeoxynucleoside methylphosphonates by reverse phase HPLC and MALDI-TOF mass spectroscopy are described.

Rotational energy barrier in PtII–N7(purine) bonds by one- and two-dimensional nuclear magnetic resonance spectroscopy

Rotational energy barriers about PtII-N7 purine bonds for cis-[Pt(NH3)2L2][L = 1-methyladenosine, adenosine 5′-mono-, -di- or -tri-phosphate, or guanosine 5′-monophosphate) were evaluated from one-and two-dimensional phosphorus-31 exchange spectroscopy. Two diastereomers in a head-to-tail conformation exist for all the aminopurine complexes in the temperature range 25–60 °C due to slow rotation about Pt–N7 bonds. The rotational activation energies for all the 6-aminopurine complexes lie in the range 46–95 kJ mol–1 while the 6-oxopurine complex exhibits the lowest energy barrier, 25 kJ mol–1. The two exocyclic amines of the aminopurine complexes in a head-to-tail configuration are positioned above and below the platinum square plane. The increased rotational energy barrier in the AMP compared to that of GMP complexes is attributed to the absence of hydrogen bonding with amines, a direct interaction of the lone-pair electrons of the 6-NH2 group with the σ- bonding orbitals of the platinum atom, and a decrease in the crystal-field stabilization energy due to the interaction with the filled dz2 orbital. The rotational energy barrier can be taken as the ground-state energy difference between the head-to-tail and head-to-head configurations since the latter is encountered near to the highest potential-energy surface during the conversion of one head-to-tail rotamer into the other. An energy difference >45 kJ mol–1 may be estimated between the bis(GMP) complex and the AMP analogue in a head-to-head configuration.

Synthesis and physicochemical studies of partially phosphate-methylated oligodeoxyribonucleotides

Partially phosphate-methylated oligodeoxyribonucleotides have been synthesized on an oxalyl-CPG derivatized support using an isopropoxyacetyl group for the protection of the exocyclic amine of the nucleic bases. Hybridization properties with the target sequence have been studied by absorption spectroscopy.

Molecular dynamics simulations of disulfide cross-linked DNA decamers

Molecular dynamics simulations with the CHARMM program are reported for the decamer d(GCGAAT-TCGC) 2 and two derivatives of this duplex that contain disulfide cross-links attached to the exocyclic amines of the adenines in the central 5'-AT-3' base pairs. The average structures from 20 ps of dynamics are compared to one another and to the crystal and NMR solution structures of the dodecamer d(CGCGAATTCGCG) 2 . The duplexes all remain in the B-family of DNA, but cross-linking does affect some structural details. Most notable are changes in roll, tilt, and propeller twist near the center of the sequence that allow the cross-link to be accommodated in the major groove

Metabolism of the food-borne carcinogens 2-amino-3-methylimidazo-[4,5-f]quinoline and 2-amino-3,8- dimethylimidazo[4,5-f]-quinoxaline in the rat as a model for human biomonitoring.

Metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazol[4,5-f]quinoxaline (MeIQx) and their binding to blood proteins were examined in the rat to develop methods of human biomonitoring. Hemoglobin and serum albumin were among many blood proteins modified. Approximately 0.01% of the dose for both compounds was bound to these proteins, and induction of cytochrome P-450 with polychlorobiphenyls resulted in decreased levels of adduction. Hemoglobin sulfinic acid amide adducts could not be detected for either amine, however, as much as 10% of the IQ bound to albumin was characterized as an N2-cysteine(34)sulfinyl-IQ linkage. Human dosimetry of these carcinogens through such adducts may prove difficult due to the low levels of protein binding. Major routes of detoxification of both contaminants included cytochrome P-450-mediated ring hydroxylation at the C-5 position followed by conjugation to glucuronic or sulfuric acid. Direct conjugation to the exocyclic amine group through N-glucoronidation and sulfamate formation were other important routes of inactivation, but N-acetylation was a minor pathway. The N-glucoronide conjugate of the mutagenic metabolite N-hydroxy-MeIQx was also detected in urine. Rats given MeIQx at 10 micrograms/kg excreted 20% of the dose in urine within 24 hr and the remainder was recovered in feces. The N2-glucuronide was the major metabolite found in urine and accounted for 4% of the total dose. The other metabolites cited above also were excreted in urine at amounts ranging from 0.5 to 3% of the dose, whereas 0.5 to 2% was detected as unmetabolized MeIQx.(ABSTRACT TRUNCATED AT 250 WORDS)

Chain Cleavage During Deprotection of RNA Synthesized by the 2′-O-Trialkylsilyl Protection Strategy

Abstract The side reaction of inter-ribonucleotide bond cleavage occuring during ammonia treatment for deprotection of support bound 2′-O-trialkylsilyl protected synthetic RNA has been assessed by analysis of a series of 15 mers of sequence T4XT10 where x is rA, rC, rG and U. Data derived from HPLC analysis of the products showed, in all cases, cleavage by the anhydrous ammonia to be dramatically reduced compared to that with ethanolic concentrated aqueous ammonia. The anhydrous reagent also gave improved product quality when used in the synthesis of a 50 residue sequence. Complete cleavage of exocyclic amine protection groups with the anhydrous reagent was confirmed by RNAse T2 digests.

Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facililating N-deacylation, minimizing depurination and chain degradation

The use of tert-butylphenoxyacetyl for N-protection of nucleoside bases during oligonucleotide synthesis facilitates its removal under mild conditions. This protecting group reduces depurination of deoxyadenosine residues, and minimizes premature desilylation resulting in chain degradation during post-synthesis workup of synthetic RNA.

Metabolism of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates undergoing carcinogen bioassay.

2-Amino-3-methylimidazo[4.5-f]quinoline (IQ) is a potent bacterial mutagen and rodent carcinogen which also produces hepatocellular carcinoma in monkeys. The metabolism and disposition of this procarcinogen were investigated in monkeys undergoing carcinogen bioassay and in monkeys given an acute dose of IQ. Analysis of urine, feces, and bile revealed that IQ was extensively metabolized. A number of metabolites in urine were purified by high-performance liquid chromatography and characterized by 1H NMR and mass spectroscopy. Metabolites resulted from cytochrome P450-mediated ring oxidation at the C-5 position or N-demethylation. These metabolites could be further transformed by conjugation to sulfate or beta-glucuronic acid. Glucuronidation and sulfamate formation at the exocyclic amine group were other major routes of metabolism. Enteric bacteria also contributed to IQ biotransformation by forming the 7-oxo derivatives of IQ and N-demethyl-IQ. The metastable N2-glucuronide conjugate of the carcinogenic metabolite, 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline, was found in urine. This indicates that metabolic activation through cytochrome P450-mediated N-oxidation occurs in vivo and that glucuronidation is a means of transport of the carcinogenic metabolite to extrahepatic tissues.

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.

Mammalian cell mutagenicity and metabolism of heterocyclic aromatic amines

Heterocyclic aromatic amines are bacterial mutagens which also induce DNA damage in mammalian cells. Damage has been demonstrated using a number of endpoints, including gene mutation, chromosome aberrations, sister-chromatid exchange, DNA-strand breaks, DNA repair and oncogene activation. Although the responses in mammalian cells are weak when compared to bacterial mutagenicity, heterocyclic aromatic amines are rodent carcinogens. Metabolic N-oxidation by cytochrome P450 is an initial activation step with subsequent transformation of the N-hydroxy metabolites to the ultimate mutagenic species by O-acetyltransferase or sulfotransferase. Major routes of detoxification include cytochrome P450-mediated ring oxidation followed by conjugation to glucuronic or sulfuric acid. Direct conjugation to the exocyclic amine group also occurs. Major reactions include N-glucuronidation and sulfamate formation.

Solid phase synthesis of a model nucleopeptide with a phosphodiester bond between the 5′ end of a trinucleotide and a serine residue

The model nucleopeptide Boc-Ser(pTCT)-NHcHex has been assmbled on a polystyrene solid support using the phosphite triester approach. Fmoc and cyanoethyl groups have been used to protect the exocyclic amine of cytosine and phosphate groups respectively. Final deprotection has been carried out by treatment with methanolic potassium carbonate.

Adenine specific DNA chemical sequencing reaction.

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions.

Reaction of 2-amino-5-nitrothiazole with palladium salts

The complexation of 2-amino-5-nitrothiazole, (ANT), a radiosensitizing drug has been studied with palladium. In methanol, the product is the square- planar complex, trans-[PdCl 2(ANT) 2] with the binding site being the ring nitrogen. In water, however, the product is the chelate, [Pd(ANT) 2]Cl 2, the chelation being from the ring nitrogen and the exocyclic amine.

Biochemical effects of binding [(H 2O)(NH 3) 5Ru II] 2+ to DNA and oxidation to[(NH 3) 5Ru III] n—DNA.

The reaction of [(H 2O)(NH 3) 5Ru II] 2+ with calf thymus and salmon sperm DNA has been studied over a wide fange of transition metal ion concentrations. Kinetic studies revealcd a biphasic reaction with an initial fairly rapid coordination of the metal ion being followed by slower reactions. Binding studies were done under pseudo-equilibrium conditions following completion of the initial rapid reaction. Spectra and HPLC of acid-hydrolyzed samples of [(NH 3) 5Ru II] n-DNA prepared by incubation of [(H 2O)(NH 3) 5Ru II] 2+ with DNA (where [P DNA] = 1.5 mM and reactant [Ru II]/[P DNA] ratios were in the fange 0.1 to 0.3) followed by air oxidation showed the predominant binding site on helical DNA to be in the major groove at the N-7 of guanine. The equilibrium constant for [(H 2O)(NH 3) 5Ru II] 2+ binding to the G 7 site in helical CT DNA is 5.1 x 103. Differential pulse voltammetry exhibited a single peak at 48 mV, which is attributed to the reduction of Rum on the G 7 sites. At [Run]/[P DNA] <0.5, Tm values for the DNA decreased linearly with increasing ruthenium concentration and an increase in the intensity of the 565 nm dG→ Ru(III) charge transfer band was noted upon melting. The UV and CD spectra of these samples indicated no extensive destacking or alteration in geometry (B family) compared to unsubstituted DNA. At [Run]/[P DNA]〉 0.5 or when single-stranded DNA was used, increased absorbance at 530 nm and 480 nm suggested additional binding to the exocyclic amine sites of adenine and cytosine residues. HPLC and individual spectrophotometric identification of the products derived from hydrolysis of these spec~es yielded both [(Gua)(NH 3) 5Ru III] and [(Ade)(NH 3) 5Ru III]. Earlier studies have established the cytidine and adenosine binding sites of [(NH 3) 5Ru III] to be at their exocyclic amines (C4 and A6). Coordination to these positions indicates disruption of the double helix since these amines are involved in hydrogen bonding on the interior of B-DNA. Agrose gel electrophoresis of superhelical pBR322 plasmid DNA after exposure to various complexes of [(nh 3) 5Ru iii] in the presence of a reductant and air generally revealed moderately efficient cleavage of the DNA, presumably due to the generation of hydroxyl radical via Fenton's chemistry. However, similar studies involving [(NH 3) 5Ru III] directly coordinated to the DNA showed no strand cutting above background. Polyacrylamide gel electrophoresis of a 381 bp, 3′- 32P-labeled fragment of pBR322 plasmid DNA containing low levels of bound [(NH 3) 5 Ru III] further indicated negligible DNA cutting by the coordinated metal ion.

Hybridization triggered cross-linking of deoxyoligonucleotides.

This paper reports details of the synthesis of oligodeoxynucleotides containing the modified base 5-methyl-N4,N4-ethanocytosine (Ce). The 9-fluorenylmethoxycarbonyl group is used as a protecting group for the exocyclic amines of dA and dC. This group can be removed rapidly under very mild conditions. Oligomers containing the Ce base form a cross-link when hybridized to their complementary deoxyoligonucleotides. Some of the scope and limitations of these cross-link forming oligonucleotides are reported.