The phospholipase-induced degradation of phosphatidylnucleosides

Hydrolysis of phosphatidylnucleosides, 5′-(rac-1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoryl)-3′-azido-3′-deoxythymidine and -2′,3′-didehydro-3′-deoxythymidine, effected by phospholipases (PL) A₂, C, and D was studied to reveal the metabolism of these derivatives. It was shown that PLA₂ deacetylates the glycerol residue at position 2, PLC is inactive, and PLD hydrolyzes the phosphatidylnucleosides to give free nucleosides.     

Molecular and crystalline structure of 3'-methylamino-2',3'-dideoxyribosylthymine--a potential inhibitor of HIV]

The structure of 3'-methylamino-2',3'-dideoxyribosylthymine [ddT(3'NHMe)] was determined by X-ray analysis. The space group is P2(1)2(1)2(1). Cell dimensions are: a 5.132(1), b 13.718(1), c 16.947(2) A, V 1193.2 A3, Z 4. The structure was solved by directed methods and refined by the full-matrix least square method to R 4.8%. The molecule of ddT(3'NHMe) has anti-conformation with respect to the glycosidic bond (chi (O4'-C1'-N1-C2) = -106.7 degrees), C3'-endo-C4'-exo puckering of the sugar moiety (P -28.8 degrees, psi m -31.5 degrees) and gauche-gauche conformation about exocyclic C4'-C5' bond (psi(C3'-C4'-C5'-O5') 45.8 degrees). The structure of ddT(3'NHMe) was compared with those of 3'-amino-3'-deoxythymidine, 3'-azido-3'-deoxythymidine and natural thymidine.     

2',3'-Dideoxythymidine permeation of the human erythrocyte membrane by nonfacilitated diffusion

The influx of 2',3'-dideoxythymidine into human erythrocytes was characterized to gain insight into the molecular properties of 3'-azido-3'-deoxythymidine which allow this latter nucleoside analog to permeate cell membranes by nonfacilitated diffusion (J. Biol. Chem. 262, 5748-5754 (1987]. The influx of 2',3'-dideoxythymidine was (1) nonconcentrative, (2) a linear function of permeant concentration (0.05 to 12 mM), and (3) insensitive to potent inhibitors of nucleoside transport and to permeants of either the nucleoside or nucleobase transporter. It is concluded that 2',3'-dideoxythymidine, like 3'-azido-3'-deoxythymidine, permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic of these two nucleoside analogs is attributed both to their lack of a 3'-hydroxyl moiety, a structural determinant which appears to be important for transport by the nucleoside carrier, and to their relatively high partition coefficients (greater than or equal to 0.2).     

Synthesis and reactivities of 2,2'-anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl- beta-D-arabinofuranosyl)thymine.

2,2'-Anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl-beta-D-arabinofuranosyl) thymine (2) was synthesized from 2',3'-didehydro-3'-deoxythymidine (DHT). Compound 2 was readily converted into the 2',3'-anhydrolyxofuranosyl derivatives 4-6. Treatment of 4a with some nucleophiles (N3-, OMe-, Cl-) gave the corresponding 3'-substituted arabinosyl nucleosides (7a,c,e) together with the minor xylosyl isomers (8a,c,d). 7a,c,e were deprotected to 7b,d,f, respectively.     

The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides.

The synthesis of 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (dK) and its 5-methyl derivative (d5) from 2'-deoxycytidine or 2'-deoxythymidine, respectively, via silver oxide oxidation of 4-hydrazinopyrimidines is described. The necessary hydrazine substituted pyrimidine nucleosides have been prepared by transamination of a protected cytidine derivative or by addition/elimination reactions to an O4-sulfonated thymidine derivative. Oxidation of the 4-hydrazino pyrimidines was complicated by a competing hydrolytic reaction which generated 2'-deoxyuridine or 2'-deoxythymidine. However, in the presence of an organic base such as triethylamine, oxidation became the predominant reaction. After suitable protection and formation of the 3'-phosphoramidite derivatives, these modified nucleosides were incorporated into seven self-complementary oligodeoxynucleotides by chemical synthesis using phosphite triester methodology. Oligodeoxynucleotides were prepared such that dA-dT and dG-dC base pairs were substituted by dA-d5 or dG-dK base pairs, respectively. Both circular dichroism spectra and thermal denaturation studies were used to characterize the modified oligodeoxynucleotides.     

AIDS dementia: synthesis and properties of a derivative of 3'-azido-3'-deoxythymidine (AZT) that may become 'locked' in the central nervous system

In an attempt to provide a derivative of 3'-azido-3'-deoxythymidine (AZT) which might be sequestered in the central nervous system and release AZT, the dihydropyridine ester 5'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-3'-deoxythymidine, was synthesized in a three step sequence. This material showed potent anti-HIV-1 activity in MT-4 cells most probably by hydrolysis to the parent nucleoside, AZT. This dihydropyridine derivative of AZT could be easily oxidized to a positively charged pyridinium derivative of AZT in rat brain cytosol. In turn the pyridinium form could be hydrolyzed, non-enzymatically, to AZT.     

HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers

In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2',3'-didehydro-3'-deoxythymidine (d4T), 3'-azido-3'-deoxythymidine (AZT), (-)-beta-L-2',3'-dideoxy-3'- thiacytidine (3TC), and 2',3'-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.     

Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)).     

Conformational studies of novel antiretroviral analogs of zidovudine

Conformational properties of three novel zidovudine analogs, namely 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso, 2), (-)-trans-(5S,6S)-5-bromo-6, 5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (3) and (+)-trans-(5R,6R)-5-bromo-6,5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (4), have been investigated by AM1 calculations and NMR studies, and compared with those of the parent nucleoside (AZT, 1). Based on the results obtained the following correlation may be established, a) AZT and AZT-Iso exhibit a conformational behavior analog to other pyrimidinic nucleosides, displaying a dynamic equilibrium in solution where the two conformers (North and South) undergo a constant transformation. b) Compounds 3 and 4 show a different conformational profile. The estimate of the pseudorotation phase angle reveals the rigid structures of the latter compounds, which do not evidence conformational equilibrium in solution; the azide group being the only group free to rotate. c) Diastereoisomers 3 and 4 exhibit an extra conformational parameter compared with other pyrimidinic nucleosides: the chair or boat conformation in the third ring formed between the sugar and the base. In all cases, a reasonable correlation was obtained between theoretical and NMR spectroscopic data.     

Synthesis, lipophilicity and anti-HIV activity of a new brominated analog of zidovudine.

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5'- epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.     

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.     

Lipid conjugates of antiretroviral agents: release of antiretroviral nucleoside monophosphates by a nucleoside diphosphate diglyceride hydrolase activity from rat liver mitochondria.

The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3'-deoxythymidine, 3'-deoxythymidine and 2',3'-dideoxycytidine from the corresponding nucleoside diphosphate diglycerides as a result of rat liver mitochondrial enzymatic activity is shown. The three analogs appeared to be about equally active as substrate for this pyrophosphatase activity which showed maximum conversion rates of 3-6 nmol min-1 mg protein-1 at substrate concentrations between 500 to 800 microM. These results may contribute to the biochemical explanation for the observed anti-HIV activity of this type of phospholipid conjugates in vitro.     

Synthesis of new terminator substrates for DNA-polymerases-- nucleoside-5'-triphosphates with modified carbohydrate residue

Synthesis of 3'-chloro- and 3'-cyanothio-2',3'-dideoxythymidine by the reaction of 2,3'-anhydro-2'-deoxythymidine with ammonium chloride and lithium thiocyanate, respectively, has been developed. In addition, 3'-methanesulphonylamido- and 3'-sulphonylamido-2',3'-dideoxythymidines were synthesized starting from 3'-amino-2',3'-dideoxythymidine. All these compounds along with 2',3'-anhydroriboadenosine,2',3'-anhydrolyxoadenosine, 2',3'-O-isopropylidenecytidine, and 2,3'-anhydro-2'-deoxythymidine were transformed into 5'-triphosphates by treatment with phosphoryl tris-1,2,4-triazolide and then with bis(tri-n-butylammonium)pyrophosphate. All 5'-triphosphates of nucleoside analogues were tested as termination substrates in cell-free systems with various DNA polymerases.     

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.     

Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT)

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 μg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 μg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 μg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 μg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 μg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 μg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.     

Conformationally locked nucleosides. Synthesis of oligodeoxynucleotides containing 3'-amino-3'-deoxy-3'-N,5'(R)-C-ethylenethymidine

3'-Amino-3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-N,5'(R)-C-ethylenethymidine (6) was synthesized starting from 3'-azido-3'-deoxythymidine. Condensation of 6 with 5'O-(H-phosphonyl)thymidine and 5'-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.