14036-89-6

Upstream product

• 52850-67-6 5-nitro-6-(((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino)pyrimidine-2,4(1H,3H)-dione 
• 2535-20-8 6,7-dimethyl-8-ribityllumazine 
• 75599-13-2 5-nitroso-6-D-(ribitylamino)pyrimidine-2,4(1H,3H)-dione 
• 527-47-9 D-Ribamin 
• 33106-48-8 1-deoxy-1-[(2,6-dioxo-1,2,3,6-tetrahydro-4-pyrimidinyl)amino]-D-ribitol 
• 74372-76-2 N-benzyl-D-ribityl amine 
• 532-20-7 D-Ribose 
• 71491-01-5 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedinone 5'-phosphate 

Downstream Products

• 76641-33-3 6-benzyl-7-methyl-8-D-ribitylpteridine-2,4(3H,8H)-dione 
• 76657-09-5 6,7-diphenyl-8-D-ribitylpteridine-2,4(3H,8H)-dione 
• 17879-89-9 6-Methyl-7-oxo-N(8)-(D-ribityl)-7,8-dihydro-Lumazin 
• 2535-20-8 6,7-dimethyl-8-ribityllumazine 
• 37333-48-5 8-hydroxy-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]pyrimido[4,5-b]quinoline-2,4(3H,10H)-dione 
• 2184-54-5 7-hydroxy-6-methyl-8-D-ribityllumazine 
• 4754-39-6 5'-deoxyadenosine 
• 83-88-5 riboflavin 
• 474644-29-6 {4-[2,4-Dioxo-6-((2S,3S,4R)-2,3,4,5-tetrahydroxy-pentylamino)-1,2,3,4-tetrahydro-pyrimidin-5-ylcarbamoyl]-butyl}-phosphonic acid dibenzyl ester 
• 474644-27-4 5-(4-dibenzylphosphonobutyryl)amino-6-D-ribitylaminouracil 
• 29161-67-9 6-carboxyethyl-7-oxo-8-D-ribityl-lumazine 

Relevant academic research and scientific papers

T-cell activation by transitory neo-antigens derived from distinct microbial pathways

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2′-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2′-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.

The Chemical Synthesis, Stability, and Activity of MAIT Cell Prodrug Agonists That Access MR1 in Recycling Endosomes

Mucosal-associated invariant T (MAIT) cells are antibacterial effector T cells that react to pyrimidines derived from bacterial riboflavin synthesis presented by the monomorphic molecule MR1. A major challenge in MAIT cell research is that the commonly used MAIT agonist precursor, 5-amino-6-d-ribitylaminouracil (5-A-RU), is labile to autoxidation, resulting in a loss of biological activity. Here, we characterize two independent autoxidation processes by LCMS. To overcome the marked instability, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleavable valine-citrulline-p-aminobenzyl carbamate. The compound is stable in prodrug form, with the parent amine (i.e., 5-A-RU) released only after enzymatic cleavage. Analysis of the prodrug in vitro and in vivo showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endosomes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies and provides an opportunity to explore different presentation pathways.

Biosynthetic versatility and coordinated action of 5′-deoxyadenosyl radicals in deazaflavin biosynthesis

Coenzyme F420 is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, Fo) is still poorly understood. Fo synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5′-deoxyadenosyl radicals to catalyze Fo formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a p-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for Fo synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5′-deoxyadenosyl radicals to mediate the formation of a complex heterocycle.

A tomato enzyme catalyzing the phosphorylation of 3,4-dihydroxy-2-butanone.

A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was complemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-dihydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak) genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 5' part of the dak gene from tomato were found in many other eubacteria. The physiological role of the dak gene is still incompletely known.

Rapid one-pot synthesis of riboflavin isotopomers

Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy. We describe highly versatile one-pot methods for the preparation of rib

Synthesis, stabilization, and characterization of the MR1 ligand precursor 5-amino-6-D-ribitylaminouracil (5-A-RU)

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminour-acil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU?HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of the vitamin, riboflavin. The biosynthetic formation of the lumazine by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate is catalyzed by the enzyme, lumazine synthase. We show that the condensation reaction can proceed without enzyme catalysis in dilute aqueous solution at room temperature and neutral pH. The reaction rate is proportional to e pH. The activation energy of the uncatalyzed reaction is Ea = 46.3 kJ mol-1. The regioselectivity of the uncatalyzed reaction increases with pH and temperature (70% at 65 °C and pH 7.75). The data suggest partitioning of the uncatalyzed reaction via two different reaction pathways. The value of kcat/kuncat may be indicative for an entropy driven process for the enzyme-catalyzed reaction.

Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase

Reduction of 5-nitro-6-D-ribitylaminouracil (9) afforded 5-amino-6-D-ribitylaminouracil (1), which reacted with ethyl chloroformate to yield 5-ethylcarbamoyl-6-D-ribitylaminouracil (12). The latter compound was cyclized to 9-D-ribityl-1,3,7-trihydropurine-2,6,8-trione (13), which was found to be a relatively potent inhibitor of both Escherichia coli riboflavin-synthase (Ki 0.61 μM) and Bacillus subtilis lumazine synthase (Ki 46 μM). Molecular modeling of the lumazine synthase-inhibitor complex indicated the possibility for hydrogen bonding between the Lys135 ε-amino group of the enzyme and both the 8-keto group and the 4′-hydroxyl group of the ligand. A bisubstrate analogue of the riboflavin synthase-catalyzed reaction, 1,4-bis[1-(9-D-ribityl-1,3,7-trihydropurine-2,6,8-trionyl)]-butane (18), was also synthesized using a similar route and was found to be inactive as an inhibitor of both riboflavin synthase and lumazine synthase.

The effects of 5-OP-RU stereochemistry on its stability and MAIT-MR1 axis

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2’- and 3’-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4’-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.