17014-74-3

Usage

Uses

Used in Pharmaceutical Industry:
5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione is used as an intermediate in the synthesis of riboflavin, which is an essential component of the human diet. Riboflavin plays a vital role in various biological processes, including energy production, cellular respiration, and the metabolism of fats, carbohydrates, and proteins.
Used in Nutritional Supplements:
5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione is used as a precursor for the production of riboflavin, which is incorporated into nutritional supplements to ensure adequate intake of this essential vitamin. Riboflavin deficiency can lead to various health issues, such as anemia, skin disorders, and neurological problems.
Used in Food Fortification:
5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione is used as a precursor for riboflavin in the fortification of food products. This helps to increase the riboflavin content in foods, ensuring that consumers meet their daily requirements for this essential nutrient.
Used in Research and Development:
5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione is used as a research compound in the study of riboflavin biosynthesis and its role in various biological processes. 5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione can be used to investigate the mechanisms of riboflavin production and its potential applications in medicine and biotechnology.
Used in Industrial Production:
5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione is used as a key intermediate in the industrial production of riboflavin, FMN, and FAD. These compounds are essential for various applications, including pharmaceuticals, nutritional supplements, and food fortification.

InChI:InChI=1/C9H16N4O6/c10-5-7(12-9(19)13-8(5)18)11-1-3(15)6(17)4(16)2-14/h3-4,6,14-17H,1-2,10H2,(H3,11,12,13,18,19)/t3-,4+,6-/m0/s1

Upstream product

• 2535-20-8 6,7-dimethyl-8-ribityllumazine 
• 532-20-7 D-Ribose 
• 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 
• 71491-01-5 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedinone 5'-phosphate 
• 527-47-9 D-Ribamin 
• 75599-13-2 5-nitroso-6-D-(ribitylamino)pyrimidine-2,4(1H,3H)-dione 
• 52850-67-6 5-nitro-6-(((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino)pyrimidine-2,4(1H,3H)-dione 

Downstream Products

• 2535-20-8 6,7-dimethyl-8-ribityllumazine 
• 17879-89-9 6-Methyl-7-oxo-N(8)-(D-ribityl)-7,8-dihydro-Lumazin 
• 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 
• 76657-09-5 6,7-diphenyl-8-D-ribitylpteridine-2,4(3H,8H)-dione 
• 76641-33-3 6-benzyl-7-methyl-8-D-ribitylpteridine-2,4(3H,8H)-dione 

Relevant academic research and scientific papers

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.

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.

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.

MAIT CELL AGONISTS

The invention relates to peptide conjugates of Formula (I). The peptide conjugates of the invention have activity as MAIT agonists. The peptide conjugates of the invention also enhance immune responses and therefore are effective vaccines and vaccine adjuvants. The invention also relates to methods and uses of such conjugates.

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.

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.

IMMUNOLOGICAL REAGENTS AND USES THEREFOR

The present invention provides ligands which bind to MR1, some of which induce MR1 to bind to MAIT cells thereby activating or inhibiting MAIT cell activation.

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.

Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a Ki of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π-π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.

A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D- ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: Design, synthesis, and evaluation

(Chemical Equation Presented) Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly.