Diaminopurine Research Articles

A Study Of the Interaction Of DAPI With DNA Containing AT and Non-AT Sequences—Molecular Specificity Of Minor Groove Binding Drugs

The binding specificity of DAPI to DNA has been probed by analysing its interactions with DNA octamers consisting of different base sequences, which include adenine, guanine, 2-amino adenine and inosine, using molecular mechanics methods. Presence of AT and non-AT base pairs in the immediate vicinity of the binding site, containing AT and non-AT base pairs is also investigated. Results show that DAPI most prefers to bind to homopolymer of AT, and least to the duplex containing alternating GC bases. DAPI interacts with homopolymeric duplexes in two possible orientations related by 180 degrees with nearly same affinity. Affinity of DAPI towards DNA comprising the modified bases, inosine and 2-amino adenine, is in between these extremities. The binding affinity is reduced to some extent by the occurrence of non AT bases flanking the four base paired binding region. An interesting revelation is that one can visualise DAPI to form a hydrogen bond with O2 of cytosine indicating that the 2-amino group of purines does not per se sterically preclude DAPI from residing in the minor groove of B-DNA helix. On the other hand, repulsive nature of electrostatic interactions that prevail at the minor groove consequent to the presence of these sequences contribute decisively in preventing further diffusion of the drug. Thus, electrostatics, rather than hydrogen bonding to bases, seemingly play an important role in determining the specificity of interaction. The retention of drug binders in the minor groove and therefore recognition, is governed by the combined effect of these various forces.

An expeditious route to methylphosphonate analogues of DNA

AbstractProtection of the exocyclic amino groups of both nucleobases adenine and guanine with the N,N‐dimethylformamidine and cytosine with the isobutyryl group presents a convenient and economical N‐protective‐group‐strategy for the solid‐phase synthesis of DNA fragments containing methylphosphonate linkages at predetermined locations. Thus, the post‐synthesis removal of the N‐protective groups with hydrazine hydrate (16 h at 20°C) afforded immobilized fragments, which were then cleaved from the solid support and deprotected at the phosphorus by treatment with ammonia in methanol (2 h at 20°C) to yield high‐quality products.

Synthesis of Some 2'-C-Alkyl Derivatives of 9-(2-Phosphonomethoxyethyl)adenine and Related Compounds

To study the effect of β-substitution in 2'-alkyl derivatives of 9-(2-phosphonomethoxyethyl)adenine (Ia) on the antiviral activity or group specificity, these derivatives were synthesized. 9-(2-Hydroxyalkyl)adenines VIII were prepared by alkylation of adenine with suitably substituted oxiranes XIII or 2-hydroxyalkyl p-toluenesulfonates IV and VI. After protection of the adenine amino group by benzoylation (compounds IX) or amidine formation (compounds X), the intermediates were alkylated with diisopropyl p-toluenesulfonyloxymethanephosphonate (XI) in the presence of sodium hydride. After deprotection, the obtained phosphonate diesters XII were converted into phosphonic acids I by transsilylation and hydrolysis. This synthetic scheme was used for the preparation of ethyl (Ie), propyl (If), 2-propyl (Ig), 2-methylpropyl (Ih), cyclopropyl (Ii), cyclohexyl (Ij), benzyl (Ik) and phenyl (Il) derivatives. The 2'-trifluoromethyl derivative XXIIa was prepared analogously from 9-(2-hydroxy-3,3,3-trifluoropropyl)adenine (XXa), obtained by alkylation of adenine sodium salt with 2-hydroxy-3,3,3-trifluoropropyl bromide. 2'-Trimethylsilyl derivative XIXa was obtained by alkylation of adenine with 2-diisopropylphosphonomethoxy-3-(4-toluenesulfonyloxy)propyltrimethylsilane (XVII) followed by transsilylation and hydrolysis of diester XVIIIa. 2,6-Diaminopurine derivatives XVIIId and XXIIb were obtained analogously. 9-(3-Phosphonomethoxybutyl)adenine (XXVIII) and 9-(2-methyl-2-phosphonomethoxypropyl)adenine (XXXV) were prepared from the corresponding hydroxy derivatives XXVIb and XXXII, respectively, by the same reaction pathway as derivatives I.

Symmetry reduction in homopolymeric DNA: implications for DNA fine structure

Theoretical modelling of DNA duplexes with homopolymeric base sequences shows that AT bases are unusual in two respects. First, their most stable B-family conformation exhibits dinucleotide symmetry characterised by a 20° phase change between successive sugars in the adenosine strand. Secondly, by passing over a small energy barrier they can adopt an even more strongly dinucleotidic conformation with successive C2'-endo and O1'-endo sugars within the thymidine strand. This conformation is almost as stable as the previous minimum. The first of these features is lost if the adenine amino group is removed or displaced, or a second amino group is added. If thymine is replaced by uracil, the asymmetry of the most stable minimum is conserved, but the second minimum is strongly destabilised. These unusual features of AT bases may be related to the ability of AT tracts to induce curvature within DNA.

Heritable alterations at the adenine phosphoribosyltransferase (APRT) locus in human lymphoblastoid cell lines

Human lymphoblastoid cell lines derived from WI-L2 exhibit unexpected frequencies of diaminopurine (DAP) resistant mutants. The background mutant fractions of 10 −7 to 10 −8 in untreated cultures are much lower than the frequencies expected for loss of a heterozygous autosomal locus (10 −5 to 10 −6), yet much higher than expected for a homozygous locus (10 −10 to 10 −12). We used aminopterin, adenine and thymidine (AAT) to select DAP-senitive (DAP S) revertants from one resistant line. The background frequency of DAP R in these revertant cell lines ranged from 3.5 to 6.5 × 10 −4, approximately the square root of 10 −7. Thus these data suggest that both alleles of aprt are inactivated at similarly high frequencies. They also indicate that the DAP S revertants were heterozygotes ( aprt +/−) or hemizygotes ( aprt +/0) and that WI-L2 was homozygous ( aprt +/+). Mutational dose-response studies with X-rays, ethyl methanesulfonate (EMS), and ICR-191 were conducted in 4 of these revertant cell lines. EMS and ICR-191, which induce mainly point mutations, did not induce an increase in mutant fraction. A dose of 200 cGy X-rays, however, induced a frequency of 10 −3. Treatment of DAP R cells with 5-azacytidine induced a significant increase in reversion to DAP S. Southern blot analysis of the aprt gene after digestion with MspI or HpaII also suggests that differential methylation changes may play a major role in the generation of DAP sensitivity and resistance.

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes

1. 1. Ten new cAMP analogs were synthesized by replacing the purine ring with indazole, benzimidazole or benztriazole and/or their nitro and araino derivatives. 2. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[ 3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 × 10 −8-8 × 10 −6 M. 3. 3. The N-1-β- d-ribofuranosyl-indazole -3′5′- cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII–X). 5. 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. 7. Chasing efficiency of cAMP analogs at half-saturating [ 3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [ 3H]cAMP concentration for both isoenzymes. 8. 8. On the basis ofsynergistic activation with 8-Br-cAMP a site 2-selective binding ofnitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.

Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.

Secondary oxygen-18 isotope effects for hexokinase-catalyzed phosphoryl transfer from ATP

Secondary 18O isotope effects in the gamma-position of ATP have been measured on phosphoryl transfer catalyzed by yeast hexokinase in an effort to deduce the structure of the transition state. The isotope effects were measured by the remote-label method with the exocyclic amino group of adenine as the remote label. With glucose as substrate, the secondary 18O isotope effect per 18O was 0.9987 at pH 8.2 and 0.9965 at pH 5.3, which is below the pK of 6.15 seen in the V/K profile for MgATP. With the slow substrate 1,5-anhydro-D-glucitol, the value was 0.9976 at pH 8.2. While part of the inverse nature of the isotope effect may result from an isotope effect on binding, the more inverse values when catalysis is made more rate limiting by decreasing the pH or switching to a slower substrate suggest a dissociative transition state for phosphoryl transfer, in agreement with predictions from model chemistry. The 18O equilibrium isotope effect for deprotonation of HATP3- is 1.0156, while Mg2+ coordination to ATP4- does not appear to be accompanied by an 18O isotope effect larger than 1.001.

The specificity of interaction between S-adenosyl- l-methionine and a nucleolar 2′- O-methyltransferase

The structural features of S-adenosyl- l-methionine (SAM) 3 3 Abbreviations used: SAM, S-adenosyl- l-methionine; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SAH, S-adenosylhomocysteine; NAc, N-α-acetyl; MAT, methionine adenosyltransferase; COMT, catechol- O-methyltransferase; DTT, dithiothreitol; BSA, bovine serum albumin; TCA, trichloroacetic acid; MTA, 5′-deoxy-5′-methylthioadenosine; SAE, S-adenosyl- l-ethionine. required for optimal binding to a nucleolar 2′- O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5′-deoxy-5′-methylthioadenosine, as well as to separate the ( S,S)-SAM from the biologically inactive ( R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl- l-homocysteine, a product of the methyltransferase reaction, bound equally as well as ( S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of ( S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral α-carbon of the methionine moiety in SAM. The α-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2′-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl- l-methionine and the nucleolar 2′- O-methyltransferase.

Effects of functional group changes in the EcoRV recognition site on the cleavage reaction catalyzed by the endonuclease.

Oligodeoxynucleotides have been prepared which contain changes in the functional group pattern present in the EcoRV recognition site d(GATATC). These modifications involve the deletion of specific functional groups or the reversal of the relative positions of functional groups within the canonical six base pair recognition site. The duplex stability of these modified oligodeoxynucleotides has been assessed by determining the thermodynamic parameters characterizing helix formation. Steady-state kinetic parameters have been used to characterize the interaction of the modified oligodeoxynucleotides with the EcoRV endonuclease. The enzyme is very sensitive to the deletion of either of the adenine amino or thymine methyl groups, or the reversal of the relative positions of the adenine amino group and thymine carboxy group which form an interstrand hydrogen bond in the major groove of the B-DNA helix. Conversely, deletion of the guanine amino group had only minimal effects upon the measured kinetic parameters. Deletion of the exocyclic amino group from the "inner" dA-dT base pair resulted in the fragment which interacted with the enzyme on the basis of observed inhibition experiments but was not cleaved. The results suggest that the endonuclease interacts with its recognition sequence via contacts in the major groove of the B-DNA helix and that both hydrogen bonding to the adenine amino groups and also hydrophobic interactions with the thymine methyl groups are involved.

Reaction of acrolein with cytosine and adenine derivatives

The products of reaction of acrolein with the following cytosine and adenine derivatives have been characterized: 1-methylcytosine, cytidine, deoxycytidine, 9-methyladenine, adenosine, and deoxyadenosine. The products contain new six-membered rings, formed by Michael addition of the amino group of cytosine or adenine to the carbon-carbon double bond of acrolein and addition of ring nitrogen 3 of cytosine or 1 of adenine across the acrolein carbonyl group. The orientation of addition was established by oxidation of the adduct of 1-methylcytosine and acrolein to N 4-(2-carboxyethyl)-1-methylcytosine and that of 9-methyladenine and acrolein to N 6-(2-carboxyethyl)-9-methyladenine. Cyclic adducts of this type, if formed in DNA in vivo, may contribute to the genetic effects of acrolein.

Surface enhanced Raman scattering of derivatives of adenine: the importance of the external amino group in adenine for surface binding

Surface enhanced Raman scattering of derivatives of adenine: the importance of the external amino group in adenine for surface binding

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.

A bifurcated hydrogen-bonded conformation in the d(A.T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin.

The crystal structures of d(CGCA3T3GCG) complex to the antitumor drug distamycin and the DNA fragment alone were solved by x-ray diffraction at 2.2 and 2.5 A resolution, respectively. The drug lies in the narrow minor groove near the center of the B-DNA fragment covering 5 of the 6 A.T base pairs. It is bound to the DNA by hydrogen bonding, van der Waals, and electrostatic interactions. In addition, the DNA was found to have an unusual conformation in the (dA)3.(dT)3 regions. These base pairs have a high positive propeller twist so that in the major groove the adenine amino group is located intermediate between the carbonyl O-4 groups of two adjacent thymines of the opposite strand, making bifurcated hydrogen bonds to the two thymine residues. This suggests a model to explain the unusual properties of poly-(dA).poly(dT) in which a modified B conformation is associated with a large propeller twist of the bases and a set of continuous bifurcating hydrogen bonds along the major groove, which may provide incremental stability to these segments. In addition, shorter segments of (dA)3-6.(dT)3-6 may have this conformation in the midst of B-DNA and stabilize bends in the DNA that may be associated with stacking on one of the high propeller-twisted bases at the ends of these segments.

Synthesis of linear poly(ethylenimine) containing nucleic acid pendants as polynucleotide analogs

AbstractPolynucleotide analogs with a linear poly(ethylenimine) (PEI) backbone and adenine, cytosine, and hypoxanthine pendants were synthesized. Linear PEI was synthesized by the cationic ring‐opening polymerization of 2‐H‐2‐oxazoline, followed by acid hydrolysis. 2‐(Adenin‐9‐yl)‐ and 2‐(N6‐benzyladenin‐9‐yl)‐, 2‐(cytosin‐1‐yl)propanoic acids in addition to 2‐(adenin‐9‐yl)‐3‐methyland 3‐(cytosin‐1‐yl)butanoic acids were synthesized from their respective nucleic acid bases. 2‐(Hypoxanthin‐9‐yl)propanoic acid and 3‐(hypoxanthin‐9‐yl)butanoic acid were converted from the corresponding adenine derivatives by reaction with nitrous acid. Grafting reactions of pendant groups onto various molecular weight PEI backbones were carried out at room temperature, using the coupling agent norborn‐5‐ene‐2,3‐carboximido diphenyl phosphate (PPONB), generally resulting in percent graft values greater than 90%. PPONB showed selectivity against the amino group of adenine and cytosine rings. The appropriate model compounds were also prepared.

Maize Leaf Adenylate Kinase

Adenylate kinase (EC 2.7.4.3) from leaves of maize (Zea mays) was purified to homogeneity using (NH(4))(2)SO(4) fractionation, followed by chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75SF, and Green A dye-ligand columns. The purified enzyme had specific activity of about 1,550 micromoles ADP produced per minute per milligram protein, and the ratio of velocities of the reverse (utilization of ATP) to forward (formation of ATP) reaction was about 1.5. The M(r) value of adenylate kinase, determined by electrophoresis in dissociating conditions and by gel filtration, was 29,000 and 31,000 respectively, suggesting monomeric nature of the enzyme. Purified preparations were stable for at least 1 month at 0 to 4 degrees C. Magnesium ions were essential for activity of adenylate kinase in both directions of the reaction. Optimal rates in the forward direction were observed at the magnesium to ADP ratio of about 0.6 to 0.8. For the reverse reaction, ATP served as a substrate only when complexed with magnesium, while AMP reacted as a free species. The enzyme preferentially utilized adenine ribonucleotides in both directions of the reaction. The nucleoside triphosphate-binding site of adenylate kinase was fairly nonspecific with regard to nucleotide species. On the other hand, the primary amino group of either adenine and cytosine moieties was essential for effective binding to the nucleoside monophosphate site of the enzyme.

Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates.

The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results.

Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC).

A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.

Induction of adenine salvage in mouse cell lines deficient in adenine phosphoribosyltransferase.

Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S. Turker, J. A. Tischfield, P. Rabinovitch, P.J. Stambrook, J.J. Trill, A.C. Smith, C.E. Ogburn, and G.M. Martin, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.