Efficient synthesis of [2-15N]guanosine and 2'-deoxy[2'-15N]guanosine derivatives using N-(tert-butyldimethylsilyl)[15N]phthalimide as a 15N-labeling reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-fluoro-9H-purine with N-(tert-butyldimethylsilyl) [15N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[15N]phthalimidopurine derivative, which was subsequently converted to the [2-15N]guanosine derivative. The 2'-deoxy[2'-15N]guanosine derivative was also efficiently synthesized through a similar procedure.     

An efficient method for the synthesis of [2-(15)N]guanosine and 2'-deoxy[2-(15)N]guanosine derivatives.

Nucleophilic substitution reaction of 6-chloro-2-fluoro-9-beta-D-ribofuranosyl-9H-purine derivative, prepared from guanosine, with potassium [15N]phthalimide at 40 degrees C for 9 h in DMF, followed by hydrolysis, afforded [2-(15)N]guanosine derivative efficiently. The corresponding 2'-deoxy derivative was also synthesized through a similar procedure.     

EFFICIENT METHODS FOR THE SYNTHESIS OF [2-15N]GUANOSINE AND 2′-DEOXY[2-15N]GUANOSINE DERIVATIVES

The nucleophilic addition–elimination reaction of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(4-nitrophenyl)ethyl]inosine (8) with [¹⁵N]benzylamine in the presence of triethylamine afforded the N ²-benzyl[2-¹⁵N]guanosine derivative (13) in a high yield, which was further converted into the N ²-benzoyl[2-¹⁵N] guanosine derivative by treatment with ruthenium trichloride and tetrabutyl-ammonium periodate. A similar sequence of reactions of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(β-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [¹⁵N]phthalimide afforded the N ²-phthaloyl [2-¹⁵N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-[¹⁵N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2′,3′,5′-tri-O-acetyl[2-¹⁵N]guanosine. The corresponding 2′-deoxy derivatives (16 and 18) were also synthesized through similar procedures.     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Efficient Synthesis of [2-15N]Guanosine and 2′-Deoxy[2′-15N]Guanosine Derivatives Using N-(tert-Butyldimethylsilyl)[15N]Phthalimide as a 15N-Labeling Reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-fluoro-9 H-purine with N-(tert-butyldimethylsilyl)[ ¹⁵ N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[ ¹⁵ N]phthalimidopurine derivative, which was subsequently converted to the [2-¹⁵N]guanosine derivative was also efficiently synthesized through a similar procedure.     

Synthesis of imidazo[4,5-e][1,4]diazepine and 3-substituted 3-deazapurine nucleosides from 1-substituted inosines.

New synthetic methods of imidazo[4,5-e][1,4]diazepine nucleosides 8-11 and 3-substituted 3-deazainosines 14 from inosine have been reported. Treatment of N1-substituted inosines 1-3 with aqueous NaOH gave 5-amino-4-(N-substituted carbamoyl)imidazole ribosides 5-7, followed by appropriate manipulations to afford ring-expanded guanosine, inosine, and xanthosine analogues. Additionally, the 5-amino group of 4-N-allylcarbamoyl derivative 12 was converted into corresponding 5-iodo nucleoside 13. We found that 13 was cyclized to form 3-deaza-3-methylinosine (14) in the presence of Pd catalysts.     

Simultaneous determination of [2-15N]- and [5-15N]glutamine with gas chromatography-mass spectroscopy: applications to nitrogen metabolic studies

A gas chromatographic-mass spectrometric method for analysis of L-[2-15N]- and L-[5-15N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N-bis-trifluoroacetyl-L-glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15N in as little as 10 microliter of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15NH4Cl infusion was started.     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

Synthesis of N2-alkylguanosine using Mitsunobu reaction as a key step

Peracetylated guanosine was reacted with POCl3 to give an 2-acetamido-6-chloro-9H-purine derivative, which was condensed with primary or secondary alcohols to give N2-alkylated analogues. The products were treated with mercaptoethanol in the presence of sodium methoxide to afford N2-alkylguanosines.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Synthesis of 3'-substituted-2',3'-deoxy-2'-methylidenepyrimidine nucleosides.

2'-deoxy-2'-methylideneuridine derivative 9 was converted into 2',3'-didehydro-2',3'-dideoxy-2'-phenyl-selenomethyl derivative 16, which was treated with NCS and tert-butyl carbamate to afford 3'-amino derivative 18 via a [2,3]-sigmatropic rearrangement. Treatment of 9 with DAST gave a mixture of 2',3'-didehydro-2', 3'-dideoxy-2'-fluoromethyl derivative 19 and 3'-"up"-fluoro-2'-methylidene derivative 20 in a ratio of 1.5 : 1. On the other hand, when 12 was treated with DAST, 19 and 3'-"down"-fluoro-2'-methylidene derivative 21 were obtained in a ratio of 1 : 1.6. These nucleosides were converted into the corresponding cytidine derivatives 4, 6, and 8, respectively. The reaction mechanisms as well as biological activity of these compounds will also be discussed.     

A new method for the synthesis of N2-alkylguanosines using Mitsunobu reaction as a key step

Peracetylated guanosine was reacted with POCl3 to give an 2-acetamido-6-chloro-9H-purine derivative, which was condensed with primary or secondary alcohols to give N2-alkylated analogues. The products were treated with mercaptoethanol in the presence of sodium methoxide to afford N2-alkylguanosines.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and antitumor activity of 7-O-[2,6-dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L-talopyranosyl]- daunomycinone and -adriamycinone.

7-O-[2,6-Dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L-talopyranosyl]- daunomycinone and -adriamycinone have been prepared by the coupling of 3,4-di-O-acetyl-2,6-dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L- talopyranosyl iodide with daunomycinone. The key steps in this synthesis are the regioselective fluorination of methyl alpha-D-lyxopyranoside to give the 4-deoxy-4-fluoro-beta-L-ribopyranoside and the C-trifluoromethylation of the aldehydo-L-ribose derivative to give the 1,1,1-trifluoro-5-monofluoro-L-altritol derivative. Antitumor activities of the synthetic products were compared with those for the 2'-deoxy-2'-fluoro and 2',6'-dideoxy-5'-C-trifluoromethyl analogs.     

Efficient syntheses of [3-15N]uracil and [3-15N]thymine.

Regiospecific syntheses of [3-15N]uracil and [3-15N]thymine are described using [15N]ammonium sulfate as a source of labeled nitrogen. The overall yields are excellent, and the reactions are amenable to production of multigram quantities of labeled material.     

Synthesis of N6-substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives as possible antiviral agents

A number of N6-substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives having hydroxymethyl at C-1' position were prepared from the appropriate 6-chloroadenine derivative. The syntheses of the corresponding prodrugs of these compounds are also reported. These compounds showed poor activity against HCV in replicon assay.     

Study on highly diastereoselective synthesis of (2'R)-2'-deoxy[2'-2H]guanosine.

To develop an efficient method for the synthesis of a highly diasteroselective (2'R)-2'-deoxy[2'-2H]guanosine (1), studies of organic chemical conversion from 2'-bromo-2'-deoxy-N2-Isobutyryl-3',5'-O-TIPDS-guanosine (2) to 1 and a biological transdeoxyribofuranosylation of (2'R > 98% de)-2'-deoxy[2'-2H]uridine (4) were carried out. As the results, a highly diastereoselective synthesis of 1 was achieved by a biological transdeoxyribofuranosylation between 2,6-diaminopurine and 4 by the use of Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. The results will be described in detail.     

A convenient synthesis of 6-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one and related 4,6-disubstituted pyrazolopyrimidine nucleosides

The glycosylation of 4,6-dichloropyrazolo[3,4-d]pyrimidine and 4-chloro-6-methylthiopyrazolo[3,4-d]pyrimidine via the corresponding trimethylsilyl intermediate and tetra-O-acetyl-beta-D-ribofuranose in the presence of trimethylsilyl triflate as a catalyst, gave selective glycosylation at N1 as the only nucleoside product. The intermediates 4,6-dichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 7 and 4-chloro-6-methylthio-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 13 gave new and convenient synthetic routes to the inosine analog 1, the guanosine analog 2, the adenosine analog 3, and the isoguanosine analog 16. Glycosylation of the trimethylsilyl derivative of 6-chloropyrazolo[3,4-d]pyrimidine-4-one unexpectedly gave the N2-glycosyl isomer 20 as the major product. A number of new 4,6-disubstituted pyrazolo[3,4-d]pyrimidine nucleosides were prepared from these glycosyl intermediates.     

Synthesis of O-alpha-L-fucopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-2- acetamido-2-deoxy-D-glucopyranose (N-acetyl-3'-O-alpha-L-fucopyranosyllactosamine)

Methyl 2-O-benzyl-beta-D-galactopyranoside (6) was obtained in five, good yielding steps from methyl beta-D-galactopyranoside (1). Treatment of 1 with tert-butylchlorodiphenylsilane in N,N-dimethylformamide in the presence of imidazole afforded a 6-(tert-butyldiphenylsilyl) ether, which was converted into its 3,4-O-isopropylidene derivative (3). Benzylation of 3 with benzyl bromide-silver oxide in N,N-dimethylformamide, and subsequent cleavage of its acetal and ether groups then afforded 6. On similar benzylation, followed by the same sequence of deprotection, benzyl 2-acetamido-3,6-di-O-benzyl-4-O-[6-O-(tert-butyldiphenylsilyl)-3,4 -O- isopropylidene-beta-D-galactopyranosyl]-2-deoxy-alpha-D-glucopyranoside gave the 2-O-benzyl derivative (10). Compound 10 was converted into its 4,6-O-benzylidene acetal (11). Glycosylation (catalyzed by halide-ion) of 11 with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide afforded the fully protected trisaccharide derivative (13). Cleavage of the benzylidene and then the benzyl groups of 13 furnished the title trisaccharide (16). The structure of 16 was established by 13C-n.m.r. spectroscopy.