Self-catalytic mechanism of prebiotic reactions: from formamide to pterins and guanine.

Reaction pathway of prebiotic reactions for formation of the pteridines: pterin, xanthopterine, isoxanthopterine and leucopterine, as well as the purine nucleobase guanine from pure formamide are presented. In these reactions, formamide or its tautomer, formimidic acid, play the role of proton-carrying catalyst. All required raw materials, such as hydrogen cyanide, ammonia, water, formic acid, urea, 2-aminomalononitrile, glyoxal, glyoxylic acid and oxalic acid needed in the self-catalyzed reactions are obtained by partial decomposition of formamide. We show that the prebiotic formation of nucleobases and pterins is closely linked and they probably coexisted at the beginning of chemical evolution.

Design, synthesis and biological activity of N5-substituted tetrahydropteroate analogs as non-classical antifolates against cobalamin-dependent methionine synthase and potential anticancer agents.

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N5-electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N5-chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC50 value of 12.1 µM against MetH and 0.16-6.12 µM against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G1-phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship.

Designing the Molybdopterin Core through Regioselective Coupling of Building Blocks.

Molybdopterin is an essential cofactor for all forms of life. The cofactor is composed of a pterin moiety appended to a dithiolene-functionalized pyran ring, and through the dithiolene moiety it binds metal ions. Different synthetic strategies for dithiolene-functionalized pyran precursors that have been designed and synthesized are discussed. These precursors also harbor 1,2-diketone or osone functionality that has been condensed with 1,2-diaminobenzene or other heterocycles resulting in several quinoxaline or pterin derivatives. Use of additives improves the regioselectivity of the complexes. The molecules have been characterized by (1)H and (13)C NMR and IR spectroscopies, as well as by mass spectrometry. In addition, several compounds have been crystallographically characterized. The geometries of the synthesized molecules are more planar than the geometry of the cofactor found in proteins.

A Prebiotic Synthesis of Pterins.

The genesis of life on Earth is a hypothesis of evolutionary science that can be, at least partially, tested experimentally. The prebiotic synthesis of cofactors or coenzymes is a poorly explored issue, likely because their formation under plausible prebiotic conditions is not clear. In this sense, it has been proposed that the cofactors are "molecular fossils" of an early phase of life. In contrast, Eschenmoser and Loewenthal suggested a prebiotic hydrocyanic origin of cofactor building blocks. In the present paper, the formation of a set of pterins from cyanide polymerizations is demonstrated, showing that the main structure of some cofactors can be prebiotically formed. Indeed, it was observed that aqueous aerosols additionally increase the relative composition for pterins in the insoluble NH4CN polymers synthesized. The novel identification of pterins in NH4CN polymers, together with the previous detection of other important biomonomers, indicates that cyanide polymerizations were essential in the early state of prebiotic chemistry.

Synthesis of sepiapterin-C via hydrolysis of 6-ethynylpteridine.

Acid hydrolysis of 6-ethynylpteridine catalyzed by mercury oxide gives 6-acetyl-2-amino-3,4-dihydropteridin-4-one in good yield. Partial reduction of the product with dissolved Al in NH3 solution afforded sepiapterin-C.

Synthesis of cyclic pyranopterin monophosphate, a biosynthetic intermediate in the molybdenum cofactor pathway.

Cyclic pyranopterin monophosphate (1), isolated from bacterial culture, has previously been shown to be effective in restoring normal function of molybdenum enzymes in molybdenum cofactor (MoCo)-deficient mice and human patients. Described here is a synthesis of 1 hydrobromide (1·HBr) employing in the key step a Viscontini reaction between 2,5,6-triamino-3,4-dihydropyrimidin-4-one dihydrochloride and D-galactose phenylhydrazone to give the pyranopterin (5aS,6R,7R,8R,9aR)-2-amino-6,7-dihydroxy-8-(hydroxymethyl)-3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridin-4-one (10) and establishing all four stereocenters found in 1. Compound 10, characterized spectroscopically and by X-ray crystallography, was transformed through a selectively protected tri-tert-butoxycarbonylamino intermediate into a highly crystalline tetracyclic phosphate ester (15). The latter underwent a Swern oxidation and then deprotection to give 1·HBr. Synthesized 1·HBr had in vitro efficacy comparable to that of 1 of bacterial origin as demonstrated by its enzymatic conversion into mature MoCo and subsequent reconstitution of MoCo-free human sulfite oxidase-molybdenum domain yielding a fully active enzyme. The described synthesis has the potential for scale up.

Synthesis of biopterin and related pterin glycosides.

Certain pterins having a hydroxyalkyl side chain at C-6 have been found as glycosidic forms in certain prokaryotes, such as 2'-O-(α-D-glucopyranosyl)biopterin from various kinds of cyanobacteria, and limipterin from a green sulfur photosynthetic bacterium. Synthetic studies on glycosides of biopterin and related pterins have been made in view of the structural proof as well as for closer examination of their biological activities and functions. The syntheses of these natural pterin glycosides have effectively been achieved, mostly through appropriately protected N(2) -(N,N-dimethylaminomethylene)-3-[2-(4-nitrophenyl)ethyl]pterin derivatives as glycosyl acceptors, and are reviewed here.

Peptide-conjugated pterins as inhibitors of ricin toxin A.

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of ricin toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC50 values in the low micromolar range.

Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: new lead exhibits a distinct binding mode.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development.

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K(d) and IC(50) values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

Development of a pterin-based fluorescent probe for screening dihydropteroate synthase.

Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z' factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-specific high-throughput and fragment-based screening studies.

The conversion of a phenol to an aniline occurs in the biochemical formation of the 1-(4-aminophenyl)-1-deoxy-D-ribitol moiety in methanopterin.

Recent work has demonstrated that 4-hydroxybenzoic acid is the in vivo precursor to the 1-(4-aminophenyl)-1-deoxy-D-ribitol (APDR) moiety present in the C(1) carrier coenzyme methanopterin present in the methanogenic archaea. For this transformation to occur, the hydroxyl group of the 4-hydroxybenzoic acid must be replaced with an amino group at some point in the biosynthetic pathway. Using stable isotopically labeled precursors and liquid chromatography with electrospray-ionization mass spectroscopy, the first step of this transformation in Methanocaldococcus jannaschii occurs by the reaction of 4-hydroxybenzoic acid with phosphoribosyl pyrophosphate (PRPP) to form 4-(ß-d-ribofuranosyl)hydroxybenzene 5'-phosphate (ß-RAH-P). The ß-RAH-P then condenses with l-aspartate in the presence of ATP to form 4-(ß-d-ribofuranosyl)-N-succinylaminobenzene 5'-phosphate (ß-RFSA-P). Elimination of fumarate from ß-RFSA-P produces 4-(ß-D-ribofuranosyl)aminobenzene 5'-phosphate (ß-RFA-P), the known precursor to the APDR moiety of methanopterin [White, R. H. (1996) Biochemistry 35, 3447-3456]. This work represents the first biochemical example of the conversion of a phenol to an aniline.

Photodynamic effects of pterin on HeLa cells.

Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. In the present study, we ascertained that 2-aminopteridin-4(3H)-one, abbreviated as Ptr, is readily incorporated into and/or onto cervical cancer cells (HeLa) and that these cells die upon UV-A irradiation of Ptr. Cell death was assessed using two tests: (1) the Rhodamine 123 fluorescence assay for mitochondrial viability and (2) the Trypan Blue assay for membrane integrity. The data suggest that, for Ptr-dependent photoinitiated cell death, events related to mitochondrial failure precede those associated with the failure of the cell membrane.

Substituent effects of pterin derivatives on singlet oxygen scavenging activity.

The relationship of chemical structures of 6-formylpterin (6FP) and its derivatives with scavenging activity of singlet oxygen ((1)O(2)) was examined. First, effects of pterin derivatives on (1)O(2) released from activated human neutrophils were examined. The neutrophils, stimulated with opsonized zymosan, released (1)O(2) that was detected by chemiluminescence using a (1)O(2) specific probe, trans-1-(2'-methoxyvinyl)pyrene. 6FP and its derivatives suppressed the (1)O(2) release. 6FP and other commercially available pterin derivatives, such as biopterin and neopterin, which have different substitutions at the 6-position, suppressed the (1)O(2) release with similar extent. On the other hand, newly synthesized pterin derivatives, which have different substitutions at the 2- and/or 3-position, such as 2-amino-6-formyl-3-methylpteridin-4-one, suppressed the (1)O(2) release in a dose-dependent manner and more potently than 6FP. Then, the (1)O(2) scavenging activity of pterin derivatives was examined photochemically by direct analysis of near-infrared luminescence at 1270 nm, the most sensitive method for the detection of (1)O(2). When rose Bengal, a photosensitizer, in D(2)O solution, was irradiated by 514 nm laser beam, the emission spectrum of (1)O(2) was observed. 6FP suppressed this emission spectrum of (1)O(2), and the newly synthesized pterin derivatives with different substituent at the 2- and/or 3-position suppressed the spectrum more potently than 6FP. The order of potency was similar to that obtained from biological assays. These findings indicate that the substitutions at the 2- and/or 3-position play an important role in (1)O(2) scavenging activity of pterin derivatives.

Targeting of nanoparticles: folate receptor.

Nanoparticulate medicines offer the advantage of allowing delivery of large quantities of unmodified drug within the same particle. Nanoparticle uptake by cancer cells can, however, be compromised due to the large size and hydrophilicity of the particle. To circumvent cell penetration problems and simultaneously improve tumor specificity, nanoparticulate medicines have been linked to targeting ligands that bind to malignant cell surfaces and enter cells by receptor-mediated endocytosis. In this chapter, we summarize multiple methods for delivering nanoparticles into cancer cells by folate receptor-mediated endocytosis, devoting special emphasis to folate-targeted liposomes. Folate receptor-mediated endocytosis has emerged as an attractive strategy for nanoparticle delivery due to both overexpression of the folate receptor on cancer cells and the rapid internalization of the receptor by receptor-mediated endocytosis.

Chemical natures of 6-formylpterin nucleoside analogs.

Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological activities. In the present study, we have developed the synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-D-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-D-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position is glycosylated. By electron paramagnetic resonance analysis coupled with the DMPO spin trapping technique, it has been elucidated that O2 was converted to H2O2 by RDEF at pH 7.4 in the presence of NADH in the dark. This result indicates that marked reactive oxygen species (ROS) generation activities of 6FP occurring under light illumination, which gives rise to the particular physiological activities such as induction of apoptosis, can be reproduced in the cellular and living systems by such derivatives and gives suggestion for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.

Synthesis of 6-formylpterin nucleoside analogs and their ROS generation activities in the presence of NADH in the dark.

We demonstrated previously that 3-position-modified 6-formylpterin (6FP) derivatives produce reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) from oxygen in the presence of NADH in the dark. It has been shown that 6FP derivatives markedly generate ROS, which gives rise to their particular physiological activities, such as induction of apoptosis in cellular and living systems, suggesting that such compounds provide a hint for the design of a ROS controlling agent in vivo. However, it is not well understood why such unique activities appear on chemical modification. In the present study, in order to see the effect on ROS generation activity in the dark by the modification of the 1-position in 6FP, we have developed a new synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-d-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-d-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position of 6FP is glycosylated. At pH 7.4, NADH was spontaneously oxidized to NAD(+) in the presence of RDEF in the dark. Using electron paramagnetic resonance analysis coupled with the spin trapping technique, we show that O(2) was converted to H(2)O(2)via superoxide anion radical ( O(2)(-)) during this reaction. The modification of the 1-position of 6FP did not cancel ROS generation activities, which were demonstrated in 3-position-modified 6FPs. Since the 6FP derivatives developed in the present study have a ribose moiety, these compounds can be subjected to further derivatization, such as incorporation into oligonucleotides, oligosaccharides, proteins, or any other compounds that recognize and interact with specific biomolecules, and therefore would be useful in pharmaceutical investigations that need generation of appropriate and controllable amounts of ROS in vivo.

Synthesis and biological evaluation of EC140: a novel folate-targeted vinca alkaloid conjugate.

A novel folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), herein referred to as EC140, was designed and evaluated for biological activity against folate receptor (FR)-positive cells and tumors. EC140 was produced by coupling a peptidic analogue of the vitamin folic acid to DAVLBH via an acylhydrazone bond. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Initial in vivo tests confirmed EC140's activity in both syngeneic and xenograft models. Hence, enduring complete responses were observed in animals bearing established, subcutaneous tumors prior to therapy using regimens that produced minor toxicity. In contrast, treatment with the unconjugated DAVLBH drug produced nominal efficacy when dosed at its MTD. Overall, EC140's performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.

Novel 6-formylpterin derivatives: chemical synthesis and O2 to ROS conversion activities.

6-Formylpterin (6FP) has been demonstrated to have strong neuroprotective effects against transient ischemia-reperfusion injury in gerbils. Also it has been shown that in rats, 6FP protected retinal neurons even when it was administered after the ischemic insult. Since there is a significant need for such a compound that effectively suppresses the events caused by the lack of oxygen supply, 6FP has attracted further investigation. Unfortunately, however, 6FP is hardly soluble in water at neutral pH and in organic solvents because of its self-assembling ability. Although a several mM solution of 6FP is available in alkaline water, it is unstable. In the present study, a novel chemical derivatization of 6FP has been developed which maintains the formyl group on the 6-position of 6FP, which is essential for the physiological activities of 6FP, and increases solubility in water and organic solvents. In the method, the 2- and 3-positions of 6FP were modified by a three component coupling reaction: 6FP was subjected to the reaction with acid chloride and N,N-dimethylformamide. The derivatives synthesized here, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one 1, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-isobutyrylpteridine-4-one 2, and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-o-toluoylpteridine-4-one 3, showed high solubility in water (1.0-5.6 mM) and organic solvents. The O(2) conversion property has also been determined for the derivative 1. Using an oxygen electrode, it has been found that O(2) is consumed in the presence of 1 and NADH at around pH 7.4 and that the rate of O(2) consumption is enhanced by UV-A irradiation. Electron paramagnetic resonance (EPR) analysis coupled with DMPO spin trapping has also revealed that in the presence of NADH, 1 converts O(2) to O(2)(-), which is further reduced to OH. By UV-A illumination in the analogous systems, (1)O(2) formation was observed. These results are similar to those reported previously for 6FP.

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene.

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.