Microscale synthesis of isotopically labeled 6R‐N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate

Hong, Baoyu; Kohen, Amnon

doi:10.1002/jlcr.993

Cited by 4 publications

(8 citation statements)

References 21 publications

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“…As illustrated in Fig. 4, we coupled this reaction with a fast (< 1 min) chemo-enzymatic synthesis of CH 2 H 4 folate from H 2 folate reported earlier from our laboratory [16, 19]. …”

Section: Resultsmentioning

confidence: 99%

“…An expeditious preparation of 11 CH 2 H 4 folate, ready in physiological solution, was developed by combing two fast and high yield methods i.e., a fast conversion of cyclotron generated 11 CO 2 to 11 CH 3 I, and its conversion to 11 CH 2 O [3], which is then reacted with chemo-enzymatically prepared H 4 folate [16, 19]. The feasibility of its use in PET imaging of cancer was assessed using a longer lived isotope ( 14 C) in cell cultures as described elsewhere [1].…”

Section: Discussionmentioning

confidence: 99%

“…H 4 folate was prepared by following the procedure reported previously from our laboratory [16, 19]. All water employed was ultra-pure (>18.2 MO cm at 25 °C, Milli-Q, Millipore, Billerica, MA, USA), and was purged with argon gas to remove dissolved oxygen.…”

Section: Methodsmentioning

confidence: 99%

“…At this stage, the temperature was lowered to 25°C, and H 2 O was introduced into the reaction vial to produce an aqueous solution of 11 CH 2 O, which was then transported to the intermediate vial containing H 4 folate. H 4 folate was prepared from folic acid by reducing it first with dithionite to dihydrofolate (H 2 folate) [20] and then converting H 2 folate enzymatically to H 4 folate by dihydrofolate reductase (DHFR) [16, 19]. After letting the mixture stand for a minute, the mixture was loaded onto a SAX cartridge, preconditioned with methanol (2 mL) and de-ionized water (5 mL), and allowed to pass through under a mild pressure (5–10 psi).…”

Section: Methodsmentioning

confidence: 99%

“…In this communication, we describe the synthesis of 11 CH 2 H 4 folate by combining two fast and high yielding methods, i.e., a simple and fast method for the synthesis of [ 11 C]-formaldehyde ( 11 CH 2 O), reported by Hooker et al[3], with an equally efficient chemo-enzymatic method for the preparation of CH 2 H 4 folate from folic acid as reported earlier by our laboratory [16, 19]. …”

Section: Introductionmentioning

confidence: 99%

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A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

Saeed

Tewson

Erdahl

et al. 2012

Nuclear Medicine and Biology

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Introduction Thymidylate synthase (TSase) and folate receptors (FRs) are well developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of 11CH3I to [11C]-formaldehyde (11CH2O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of [11-14C]-(R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (14CH2H4folate) by cancerous cell vs. normal cells from the same organ [1] pointing out 11CH2H4folate as a PET tracer for cancer imaging. Herein we report the synthesis of 11CH2H4folate, which may serve as a potential positron emission tomography (PET) tracer. Methods In a remotely controlled module, 11CH3I was bubbled into a reaction vial containing trimethylamine N-oxide (TMAO) in DMF and heated to 70 °C for 2 min. 11CH2O formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H4folate) by using a fast chemo-enzymatic approach to accomplish synthesis of 11CH2H4folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution. Results The synthesis and purification of 11CH2H4folate was completed within 5 min. HPLC analysis of the product after SAX purification indicate more than 90% of the radioactivity that was retained on the SAX cartridge was in 11CH2H4folate with minor (<10%) radioactivity due to unreacted 11CH2O. Conclusion We present a fast (~5 min) synthesis and purification of 11CH2H4folate, as a potential PET tracer. The final product is received in physiological compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo.

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“…As illustrated in Fig. 4, we coupled this reaction with a fast (< 1 min) chemo-enzymatic synthesis of CH 2 H 4 folate from H 2 folate reported earlier from our laboratory [16, 19]. …”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

Saeed

Tewson

Erdahl

et al. 2012

Nuclear Medicine and Biology

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Flavin-dependent thymidylate synthase: A novel pathway towards thymine

Koehn

Kohen

2010

Archives of Biochemistry and Biophysics

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For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylatemethylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis.

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An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene

Koehn

Fleischmann

Conrad

et al. 2009

Nature

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169

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Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase (TS) to catalyze the methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP). All known thymidylate synthases (TSs) rely on an active site residue of the enzyme to activate dUMP1, 2. This functionality has been demonstrated for classical TSs, including human TS, and is instrumental in mechanism-based inhibition of these enzymes. Here we report the first example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3–5. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (i.e. a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2’-deoxythymidine-5’-monophosphate (dTMP). These observations indicate a very different chemical cascade than that of classical TSs or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Since several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

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Microscale synthesis of isotopically labeled 6R‐N⁵, N¹⁰ methylene‐5, 6, 7, 8‐tetrahydrofolate

Abstract: SummaryA one-pot chemo-enzymatic microscale synthesis of isotopically labeled R-[6-Y H;

Cited by 4 publications

References 21 publications

A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

Flavin-dependent thymidylate synthase: A novel pathway towards thymine

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene

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