5 -Fluoro-5 -deoxyadenosine

So far only a few dozen organofluorine compounds have been isolated from living organisms, for example fluoroacetic acid, 4-fluorothreonine and rw-fluoro-oleic acid [244-246], The reason that nature has not invested in fluorine chemistry could be a combination of low availability of water-dissolved fluoride in the environment due to its tendency to form insoluble fluoride salts, and the low reactivity of water-solvated fluoride ion. However, in 2002, O Hagan and collaborators [247] published the discovery of a biochemical fluorination reaction in a bacterial protein extract from Streptomyces cattleya converting S-adenosyl-L-methionine (SAM) to 5 -fluoro-5 deoxyadenosine (5 -FDA). The same protein extract contained also the necessary enzymatic activity to convert 5 -FDA into fluoroacetic acid. In 2004, the same authors published the crystal structure of the enzyme and demonstrated a nucleophilic mechanism of fluorination [248,249]. 

Recent developments on research into a bacterial C-F bond forming enzyme are reviewed. The fluorinase enzyme was isolated from Streptomyces cattleya in 2002 and shown to catalyse the conversion of fluoride ion and S-adenosyl-L-methionine (SAM) to 5 -fluoro-5 -deoxyadenosine (5 -FDA) and L-methionine. Subsequently, the enzyme has been the subject of cloning, crystallisation, mechanism and substrate specificity studies. This review summarises the current status of this research. 

ATP 7 was converted to two, at that time unknown organo-fluorine compounds, 5 -fluoro-5 -deoxyadenosine (5 -FDA) 5 and 5 -fluoro-5 -deoxyinosine (5 -FDI) 6 as well as FAc 1 [6],... 

It was interesting that the cell-free extract had the capacity to support the biosynthesis all the way to FAc 1, an end product of one of the fluorometabolite pathways. This observation indicates that all of the enzymes and cofactors required to support FAc biosynthesis were present and active in the cell-free extract, even though the integrity of the cells had been destroyed. This experiment showed that organic fluoride production was achievable in vitro from the S. cattleya protein extract. Subsequent purification of the fluorinase (5 -fluoro-5 -deoxyadenosine synthase), using standard purification protocols revealed that the true substrate for the enzyme was SAM 8 and not ATP 7 [8]. It transpired that ATP 7 and L-methionine (L-Met) were converted to SAM 8 in the crude cell-free extract and that the resultant SAM 8 was then processed by the fluorinase with the release of L-Met. Thus, a catalytic cycle where L-Met was regenerated to drive these two reactions had been inadvertently established (Scheme 1). The fluorinase catalyses the conversion of SAM 8 and fluoride ion to make 5 -FDA 5 as shown in Scheme 1 [8]. 

Finally, recent developments on research into the first C-F bond forming enzyme are summarized. The fluorinase enzyme isolated from Streptomyces cat-tleya catalyzes the formation of 5 -fluoro-5 -deoxyadenosine from S-adenosyl-L-methionine and fluoride. The substrate specificity and subsequent transformation of the fluorinated nucleoside to fluoroacetic acid and to fluoro threonine are discussed. 

The first step in this pathway involves SN2 displacement by fluoride on S-adenosine-L-methionine (SAM) catalyzed by the newly discovered enzyme fluor-inase (905-910), which also can function as a chlorinase (912). Fluorinase has been isolated and characterized, and the gene has been cloned (916). Both 5 -fluoro-5 -deoxyadenosine (847) and 5 -fluoro-5 -deoxy-D-ribose-l-phosphate (848) have been identified as intermediates (905-908). Fluoroacetaldehyde (850) is the immediate precursor, presumably via fluororibulose-1-phosphate (849) (915), to both fluoroacetate and 4-fluorothreonine (837) (901). The requisite enzymes fluoroacetaldehyde dehydrogenase (902) and L-threonine transaldolase-PLP (903) have been isolated and purified. The steps from 848 to 850 remain to be established but are based on known biochemistry. The pronounced toxicity of fluoroacetic acid... 

Dong C, Deng H, Dorward M, Schaffrath C, O Hagan D, Naismith JH (2003) Crystallization and X-Ray Diffraction of 5 -Fluoro-5 -deoxyadenosine Synthase, a Fluorination Enzyme from Streptomyces cattleya. Acta Cryst D59 2292... 

The enzyme 5 -fluoro-5 -deoxyadenosine synthetase (5 -FDAS), utilizing SAM, catalyzes the formation of carbon-fluoride bonds. In fact, in presence of F , an 8 2 substitution of methionine at SAM generates the intermediate 5 -fluoro-5 -deoxyadenosine, which is subsequently converted to fluoroacetate and 4-fluorothreonine, whereas in the biosynthesis of salinosporamide A the enzyme achieves the chlorination step through the conversion to 5 -chloro-5 -deoxyadenosine (Section 4.2.2) (Figure 1.61) [51],... 

The enzyme fluorinase (5 -fluoro-5 -deoxyadenosine synthase) was applied to convert S-adenosyl-L-methionine (SAM) and [ F]fluoride directly into 5 -[ F]fluoro-5 -deoxyadenosine (5 -[ F]FDA) (O Fig. 42.30a) and L-methionine, which was the first example where a radionuclide was enzymatically introduced into an organic molecule (Martarello et al. 2003). In this approach, the native wildtype fluorinase deriving from Streptomyces cattleya was employed in low concentrations (pg ml ) and generated only very low radiochemical yields of - 1%. Better conversions of -20-25% were achieved by the use of higher fluorinase concentrations (mg/ml), which became available by fluorinase over-expressing cloned Escherichia coli (Deng et al. 2006). 

Fluorination of organic compounds using fluoride ion can be catalyzed by enzymes (Fig. 10.38). For example, fluorinase from Streptomyces cattleya catalyzes C—F bond formation in the reaction of S-adenosyl-L-methionine with fluoride to yield 5 -fluoro-5 -deoxyadenosine. " Although their substrate scope is hmited at present, these kinds of enzymes may come to play an important role for fluorination in the future due to the ease, mildness and safety of the reaction conditions and the use of fluoride as fluorine source. 

Ashton TD, ScammeUs PJ (2005) An improved synthesis of 5 -fluoro-5 -deoxyadenosines. Bioorg Med Chem Lett 15 3361-3363... 

L. Martarello, C. Schaffrath, H. Deng, A.D. Gee, A. Lockhart, D. O Hagan, The first enzymatic method for C-F-18 bond formation The synthesis of 5 -[F-18]-fluoro-5 -deoxyadenosine for imaging with PET, J. Label. Compds Radiopharm. 46 (2003) 1181-1189. 

The same conditions led to decomposition in an attempt to prepare 2 -deoxy-2-fluoroadenosine from 2 -deoxyadenosin-2-amine. The reaction was successful when applied to 3, 5 -di-0-acetyl-2 -deoxyadenosin-2-amine giving the 2-fluoro derivative 2. ... 

Uesugi, S., Takatsuka, Y., Ikehara, M., et al. (1981) Synthesis and characterization of the dinucleoside monophosphates containing 2 -fluoro-2 -deoxyadenosine. Biochemistry, 20, 3056-3062. 

The majority of newly reported nucleoside 5 -monophosphates have been prepared using phosphorus oxychloride in trialkyl phosphate solution. These include the monophosphates of 2-fluoroadenosine, 2-amino-6-chloro-9-(/S-D-ribofuranosyl)-purine, bredinin (6), cordycepin 2 -azido-2 -deoxyadenosine, 2 -fluoro-... 

Also lying in the spongonucleoside field was Dr. Baker s novel synthesis of the yS-D-arabinofuranosylpyrimidines utilizing 2-0-acetyl-5-0-methoxycarbonyl-3-O-p-tolylsulfonyl-D-xylofuranosyl chloride, the halide that had been used in the synthesis of 2 -deoxyadenosine. When this halide was condensed with mercury derivatives of thymine and 5-fluoro-uracil and the products deacylated with base, l-yS-n-arabinofuranosyl-thymine and -5-fluorouracil were obtained directly, in reactions that proceeded by way of the 2,3-anhydro-n-ribofuranosides and the 2,2 -anhydronucleosides. These studies were later expanded by others, in synthetic work that has provided alternative paths to purine deoxy-nucleosides and /2-D-arabinosyIpurines, and they anticipated many of the syntheses of arabinosylcytosine (an important antitumor agent) that proceed by way of 2,2 -anhydronucleosides. 

Leegwater, P.A., De Abreu, R.A., and Albertioni, F., Analysis of DNA methylation of the 5 region of the deoxycytidine kinase gene in CCRF-CEM-sensitive and cladribine (CdA)- and 2-chloro-2-arabino-fluoro-2 -deoxyadenosine (CAFdA)-resistant ceU, Cancer Lett., 130 (1-2), 169-173,1998. 

The aspect of a reversible reaction of fluorinase was of major importance for further optimizations and by addition of the enzyme L-amino acid oxidase (L-AAO) the oxidative removal of formed L-methionine was enabled. In this way, the equilibrium of the reaction was pulled toward 5 -[ F]FDA and allowed very high radiochemical yields of 95% within 1-2 h. Accordingly, the F-labeled compounds 5 -[ F]fluoro-5 -deoxyinosine (5 -[ F]FDI) (O Fig. 42.30h), 5 -[ F]fluoro-5 -deoxyribose phosphate (O Fig. 42.30c)y and 5 -[ F]fluoro-5 -deoxyribose (O Fig. 42.30d) were successfully produced from 5 -[ F]fluoro-5 -deoxyadenosine (Deng et al. 2006, Onega et al 2009, 2010). In the base-swap strategy, the 5 [i F]fluoro-5 -deoxyribose phosphate O Fig. 42.30c) could be further transformed into... 

C-Fluoroadenosine 5 -sulfamate, N-84 2-Fluoroadenosine, F-11 8-Fluoroadenosine, F-12 Fluoroancitabine, F-19 Fluoroblasticidin S, B-39 5-Fluorocytidine, F-13 p-Fluoro-ddA, L-48 2 -Fluoro-2 -deoxyadenosine, D-63 5-Fluoro-3 -deoxycytidine, F-13 5-Fluoro-3 -deoxyuridine, F-18 8-Fluoroerythromycin, E-18 5-C-Fluorogalactosyl fluoride, F-14... 

O -Benzyl-2-deoxy-2-fluoro-D-arabinose, D-68 2 -Bromo-2 -deoxyadenosine, B-55... 

Azaxanthosine, A-893 2 -Azido- 5-bromo-2, 3 -dideoxyuridine, A- 894 3 -Azido-5-chloro-2, 3 -dideoxycytidine, A-895 3 -Azido-5-chloro-2, 3 -dideoxyuridine, A-896 8-Azidocyclic AMP, A-897 5 -Azido-5 -deoxyadenosine, A-898 5 -Azido-5 -deoxythymidine 3"-Ac, A-915 5 -Azido-5 -deoxythymidine, A-915 3 -Azido-3 -deoxy-5 -thymidylic acid, 9CI, Z-4 l-(3-Azido-2,3-dideoxy-2-fluoro-p-D-arabinofuranosyl)thymine, A-917 2 -Azido-2, 3 -dideoxy-5-iodouridine, A-920 3 -Azido-2, 3 -dideoxy-5-methylcytidine, A-921 2 -Azido-2, 3 -dideoxy-5-methyluridine, A-922 2 -Azido-2, 3 -dideoxyuridine, A-923... 

In their initial work, these authors reported a poten-tiometric study on the ability of the Zn " " complex of cyclen (80) to interact with the deoxyribonucleotides 2 -deoxyadenosine (dA), 2 -deoxyguanosine (dG), 2 -deoxy-cytidine (dC), 2 -deoxythymidine (dT), uridine (U), and 3 -azido-3 - deoxythymidine (AZT). [Zn(80)] " had a good selectivity for dT, U, AZT, and the related derivatives Ff (ftorafur, 5-fluoro-l-(tetrahydro-2-furyl)uracil), and... 

Ethynyl-2-fluoro-2 -deoxyadenosine (EFdA, 42, Fig. 1) is an exceptionally potent reverse transcriptase (RT) inhibitor under development for the treatment of HTV infection [68,70,71]. Unusually for this class of inhibitors, 42 retains the 3 -OH group of the ribose, which renders the monophosphate derivative a better substrate for RT than adenosine monophosphate, and contributes to the high potency [69]. 

Robins and Cass also employed a range of electron-rich and electron-poor benzylamines to react with 6-fluoropurine thionucleoside in high yield [95]. Sterically congested aminotriols of relevance to the study of poly aromatic hydrocarbons (PAHs) have also been incorporated in modest to excellent yields (50-93 %, dependent on the bulk of the amine) [105, 115]. Similarly, aminoesters [116] and amino alcohols [117] have been incorporated via a SnAt reaction with 6-(fluoro)-2 -deoxyadenosine analogs in modest yield. Glutathione (70) has also been shown to... 

Nakata H, Amano M, Koh Y, Kodama E, Yang G, Bailey CM, Kohgo S, Hayakawa H, Matsuoka M, Anderson KS, Cheng Y, Mitsuya H (2007) Activity against human immunodeficiency virus type 1, intracellular metabolism, and effects on human DNA polymerases of 4 -ethynyl-2-fluoro-2 -deoxyadenosine. Antimicrob Agents Chemother 51 2701-2708... 

Michailidis E, Marchand B, Kodama EN (2009) Mechanism of inhibition of HIV-1 reverse transcriptase by 4 -ethynyl-2-fluoro-2 -deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor. J Biol Chem 284 35681-35691... 

Parker WB, Allan PW, Hassan AEA, Secrist JA, Sorscher EJ, Waud WR (2003) Antitumour activity of 2-fluoro-2 -deoxyadenosine against tumours that express Escherichia coh purine nucleoside phosphorylase. Crmcer Gene Ther 10 23—29... 

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