Synthesis Of Pteridines Research Articles

Comparative Transcriptome Analysis Reveals Differential Cutaneous Gene Expression in the Color Variation of Two Ornamental Discus, Red Melon and Red Cover.

Red Melon (RM) and Red Cover (RC) discus (Symphysodon spp.) are ornamental fish varieties that were selectively bred from the wild parental lineages of the brown discus S. aquafaciatus over many generations, resulting in distinct cutaneous patterns from juveniles to adults. To better understand the underlying mechanisms, skin samples were collected from juveniles aged 60 days and adults aged 1 year from RM and RC for investigations. Microscopic observation detected xanthophores and erythrophores in all samples, except RC juveniles with no erythrophores. Melanophores were presented only in RC. The comparative analysis revealed that genes involved in pteridine synthesis (gch1 and zgc:153031), one-carbon metabolism (aldh1l2 and zgc153031), and lipid metabolism (apoda and klf1) were differentially expressed in RM juveniles, which may be associated with the development of erythrophores and xanthophores. The temporal inhibition of melanophore differentiation and development was observed in RM juveniles, coupled with elevated expression of notum2 and sost, two antagonist genes in Wnt-signaling, suggesting their roles in melanophore development. Distinct pigment pattern between RM and RC since the juvenile stage may be driven by the differential expression of multiple axial developmental genes, including GATA, ankyrin, and mitotic spindle orientation proteins. This is the first report to describe the differential growth of cutaneous pigments and the molecular processes involved in red discus. The results provided valuable insights into pigment pattern differences in an interesting ornamental fish model.

What Makes a Mimic? Orange, Red, and Black Color Production in the Mimic Poison Frog (Ranitomeya imitator).

Aposematic organisms rely on their conspicuous appearance to signal that they are defended and unpalatable. Such phenotypes are strongly tied to survival and reproduction. Aposematic colors and patterns are highly variable; however, the genetic, biochemical, and physiological mechanisms producing this conspicuous coloration remain largely unidentified. Here, we identify genes potentially affecting color variation in two color morphs of Ranitomeya imitator: the orange-banded Sauce and the redheaded Varadero morphs. We examine gene expression in black and orange skin patches from the Sauce morph and black and red skin patches from the Varadero morph. We identified genes differentially expressed between skin patches, including those that are involved in melanin synthesis (e.g. mlana, pmel, tyrp1), iridophore development (e.g. paics, ppat, ak1), pteridine synthesis (e.g. gch1, pax3-a, xdh), and carotenoid metabolism (e.g. dgat2, rbp1, scarb2). In addition, using weighted correlation network analysis, we identified the top 50 genes with high connectivity from the most significant network associated with gene expression differences between color morphs. Of these 50 genes, 13 were known to be related to color production (gch1, gmps, gpr143, impdh1, mc1r, pax3-a, pax7, ppat, rab27a, rlbp1, tfec, trpm1, xdh).

The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which species share a conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which mimicry is produced in poison frogs. We assembled a 6.02-Gbp genome with a contig N50 of 310 Kbp, a scaffold N50 of 390 Kbp and 85% of expected tetrapod genes. We leveraged this genome to conduct gene expression analyses throughout development of four colour morphs of Ranitomeya imitator and two colour morphs from both R. fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes differentially expressed throughout development, many of them related to melanophores/melanin, iridophore development and guanine synthesis. We also identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species, and identify several plausible candidates for colouration in vertebrates (e.g. cd36, ep-cadherin and perlwapin). Finally, we hypothesise that keratin genes (e.g. krt8) are important for producing different structural colours within these frogs.

Under pressure: evidence for selection on color-related genes in poison frogs of the genus Ranitomeya

Aposematic organisms rely on their bright conspicuous coloration to communicate to potential predators that they are toxic and unpalatable. These aposematic phenotypes are strongly tied to survival and therefore make excellent opportunities to investigate the genetic underpinning of coloration. The genus Ranitomeya includes phenotypically diverse members of Neotropical aposematic poison frogs native to South America. Significant progress has been made in elucidating the molecular mechanisms responsible for aposematic coloration in poison frogs, which have paved the way for future studies to test hypotheses of the evolution of coloration across aposematic vertebrates. However, very little is known about whether these color related genes are under positive selection. We assembled transcriptomes from publicly available data reads sets for 9 different color morphs of poison frogs in the Ranitomeya genus that display bright conspicuous coloration (four morphs of R. imitator, two morphs of R. variabilis, two morphs of R. fantastica, one morph of R. summersi) to identify protein-coding genes responsible for color production that are under positive selection. Our results show that there are multiple genes under strong positive selection that are predicted to play roles in melanin synthesis (dct, tyrp1, irf4), iridophore development (fhl1), keratin metabolism (ovol1), pteridine synthesis (prps1, xdh), and carotenoid metabolism (adh1b, aldh2). The identification of positive selection affecting candidate color-pattern genes is consistent with the possibility that these genes mediate (in part) the molecular evolution of coloration. This may be attributed to aposematic phenotypes being directly tied to survival and reproduction in poison frogs.

Triphenyltin induced darker body coloration by disrupting melanocortin system and pteridine metabolic pathway in a reef fish, Amphiprion ocellaris

Triphenyltin (TPT) is a typical persistent organic pollutant whose occurrence in coral reef ecosystems may threaten the survival of reef fishes. In this study, a brightly colored representative reef fish, Amphiprion ocellaris was used to explore the effects of TPT at environmental levels (1, 10, and 100 ng/L) on skin pigment synthesis. After the fish were exposed to TPT for 60 days, the skin became darker, owing to an increase in the relative area of black stripes, a decrease in orange color values while an increase in brown color values, and an increase in the number of melanocytes in the orange part of the skin tissues. To explore the mechanisms by which TPT induces darker body coloration, the enzymatic activity and gene expression levels of the members of melanocortin system that affect melanin synthesis were evaluated. Leptin levels and lepr expression were found to be increased after TPT exposure, which likely contributed to the increase found in pomc expression and α-melanocyte-stimulating hormone (α-MSH) levels. Then Tyr activity and mc1r, tyr, tyrp1, mitf, and dct were upregulated, ultimately increasing melanin levels. Importantly, RT-qPCR results were consistent with the transcriptome analysis of trends in lepr and pomc expression. Because the orange color values decreased, pterin levels and the pteridine metabolic pathway were also evaluated. The results showed that TPT induced BH4 levels and spr, xdh, and gch1 expression associated with pteridine synthesis decreased, ultimately decreasing the colored pterin content (sepiapterin). We conclude that TPT exposure interferes with the melanocortin system and pteridine metabolic pathway to increase melanin and decrease colored pterin levels, leading to darker body coloration in A. ocellaris. Given the importance of body coloration for the survival and reproduction of reef fishes, studies on the effects of pollutants (others alongside TPT) on body coloration are of high priority.

Pigment Identification and Gene Expression Analysis during Erythrophore Development in Spotted Scat (Scatophagus argus) Larvae.

Red coloration is considered an economically important trait in some fish species, including spotted scat, a marine aquaculture fish. Erythrophores are gradually covered by melanophores from the embryonic stage. Despite studies of black spot formation and melanophore coloration in the species, little is known about erythrophore development, which is responsible for red coloration. 1-phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to inhibit melanogenesis and contribute to the visualization of embryonic development. In this study, spotted scat embryos were treated with 0.003% PTU from 0 to 72 h post fertilization (hpf) to inhibit melanin. Erythrophores were clearly observed during the embryonic stage from 14 to 72 hpf, showing an initial increase (14 to 36 hpf), followed by a gradual decrease (36 to 72 hpf). The number and size of erythrophores at 36 hpf were larger than those at 24 and 72 hpf. At 36 hpf, LC-MS and absorbance spectrophotometry revealed that the carotenoid content was eight times higher than the pteridine content, and β-carotene and lutein were the main pigments related to red coloration in spotted scat larvae. Compared with their expression in the normal hatching group, rlbp1b, rbp1.1, and rpe65a related to retinol metabolism and soat2 and apoa1 related to steroid hormone biosynthesis and steroid biosynthesis were significantly up-regulated in the PTU group, and rh2 associated with phototransduction was significantly down-regulated. By qRT-PCR, the expression levels of genes involved in carotenoid metabolism (scarb1, plin6, plin2, apoda, bco1, and rep65a), pteridine synthesis (gch2), and chromatophore differentiation (slc2a15b and csf1ra) were significantly higher at 36 hpf than at 24 hpf and 72 hpf, except for bco1. These gene expression profiles were consistent with the developmental changes of erythrophores. These findings provide insights into pigment cell differentiation and gene function in the regulation of red coloration and contribute to selective breeding programs for ornamental aquatic animals.

Identification, characterization and differential expression analysis of a pteridine synthesis related gene, Ccptps, in koi carp (Cyprinus carpio L.).

6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second key enzyme of the pteridine biosynthetic pathway and it plays vital roles in fish body color formation. In this study, Ccptps of koi carp (Cyprinus carpio L.) was cloned, identified and characterized. The full-length cDNA of Ccptps was 1140bp and encodes for 139 amino acids. Multiple alignments revealed that the amino acids sequence of CcPTPS shared the highest identity to that of C. carpio, and Ccptps was clustered with cyprinid fishes in phylogenetic tree. Liver tissues of koi carp exhibited the highest expression of Ccptps, followed by muscle and skin tissues. During early developmental stages, the expression of Ccptps declined from 2 dph to 4 dph, and increased from 4 dph to 12 dph. The expressions of Ccptps in three color-related tissues (skin, scale and caudal fin) of whole red (WR) koi carp were significantly higher than that of whole while (WW) koi carp. Immunohistochemistry results of skin tissues showed that CcPTPS was mainly located in epidermis, stratum compactum of dermis and muscle layer, with the signal intensities in stratum compactum and muscle layer were stronger in WR koi carp compared to WW koi carp. Co-expressions of CcPTPS, CcSPR and CcXDH were detected in skin tissues of WW and WR koi carps, with CcPTPS exhibited stronger signal intensity compared to CcSPR and CcXDH. These findings imply that Ccptps is potentially involved in koi carp body color formation through the pteridine synthesis pathway.

Synthesis and Biological Evaluation of Novel Pteridine 7 ( <i>8H</i>) - Ketone Derivatives as Potent CDK2 Inhibitors

Synthesis and Biological Evaluation of Novel Pteridine 7 ( <i>8H</i>) - Ketone Derivatives as Potent CDK2 Inhibitors

Synthesis, characterization, antimicrobial and interaction studies of pteridines with human serum albumin: A combined multi-spectroscopic and computational study

A novel series of pteridine derivatives (3a–e) was synthesized, and the structures of these molecules were established using elemental analysis and numerous spectroscopic methods. The disk diffusion technique was used to examine the antimicrobial potential of the newly synthesized molecules. Among the compounds examined, the 3b and 3e compounds would have the highest impact against the examined bacterial and fungal strains. The compounds minimum inhibitory concentration (MIC) was observed to be in the line of 0.41–6.83 μM. UV-vis absorption, fluorescence quenching, FT-IR spectroscopy and circular dichroism (CD) along with molecular docking methods, were studies to assess the binding behavior of efficient pteridine derivatives (3b and 3e) with human serum albumin (HSA). Formation of HSA-test compounds complex indicates the static quenching phenomena during fluorescence quenching of HSA by test compounds. Synthesized 3b and 3e were further evaluated for three basic binding sites of HSA including subdomains IIA, IIIA, and IB, using molecular docking studies. Hydrophobic and interaction through hydrogen bonding influenced the binding pathway of HSA with test molecules, according to the molecular docking data. Furthermore, the DFT technique was used to optimize the molecular geometry of potent 3b and 3e compounds using the B3LYP hybrid functional and the 6–311 + G(d, p) basis set. The optimized structure closely aligns with the test findings. The electrostatic potential framework was produced to visualize the molecule's energy distribution and chemical reactive areas.

The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which multiple species share the same conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which vivid colour and pattern are produced in a Müllerian mimicry complex of poison frogs. We did this by first assembling a high‐quality de novo genome assembly for the mimic poison frog Ranitomeya imitator. This assembled genome is 6.8 Gbp in size, with a contig N50 of 300 Kbp R. imitator and two colour morphs from both Ranitomeya fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes that are differentially expressed throughout development, many of them related to melanocyte development, melanin synthesis, iridophore development and guanine synthesis. Polytypic differences within species may be the result of differences in expression and/or timing of expression, whereas convergence for colour pattern between species does not appear to be due to the same changes in gene expression. In addition, we identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species. Finally, we hypothesize that genes in the keratin family are important for producing different structural colours within these frogs.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio)

BackgroundAnimal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama.ResultsWe generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration – pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes.ConclusionsGene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Optimization of N‐alkylation in the Synthesis of Methotrexate and Pteridine‐based Derivatives Under Microwave‐Irradiation

AbstractMethotrexate (MTX) and its pteridine‐based derivatives represent an attractive chemotype for the development of bioactive molecules. However, the synthesis of pteridines suffers from several drawbacks. Here we describe a new efficient and improved microwave‐assisted lab‐scale process for the preparation of MTX and congeners. Starting from the commercially available 2,4‐diamino‐6‐(hydroxymethyl)pteridine (Pt‐OH), MTX was obtained with an overall 94% yield through a three steps procedure. The crucial yield‐limiting and time‐consuming step of SN2 substitution between halogenated pteridine and nucleophilic aromatic amine was taken. The innovative process, conducted under microwave irradiation, improved yield and purity, and in particular reduced the reaction time from days to 20 minutes. The optimized protocol was successfully applied to the synthesis of diverse pteridine‐based derivatives and to the preparation in gram‐scale of antiparasitic MTX derivatives for in vivo studies. This new optimized synthetic procedure therefore represents a worthy alternative to the current protocols for the preparation of pteridine‐based derivatives.

The developmental and genetic trajectory of coloration in the guppy (Poecilia reticulata).

Examining the association between trait variation and development is crucial for understanding the evolution of phenotypic differences. Male guppy ornamental caudal fin coloration is one trait that shows a striking degree of variation within and between guppy populations. Males initially have no caudal fin coloration, then gradually develop it as they reach sexual maturity. For males, there is a trade-off between female preference for caudal fin coloration and increased visibility to predators. This trade-off may reach unique endpoints in males from different predation regimes. Caudal fin coloration includes black melanin, orange/yellow pteridines or carotenoids, and shimmering iridescence. This study examined the phenotypic trajectory and genetics associated with color development. We found that black coloration always developed first, followed by orange/yellow, then iridescence. The ordering and timing of color appearance was the same regardless of predation regime. The increased expression of melanin synthesis genes correlated well with the visual appearance of black coloration, but there was no correlation between carotenoids or pteridine synthesis gene expression and the appearance of orange/yellow. The lack of orange/yellow coloration in earlier male caudal fin developmental stages may be due to reduced expression of genes underlying the development of orange/yellow xanthophores.

Analogs of biologically active compounds IX. Synthesis of several new uracil and pteridine 6-aza-analogs based on cyclization of arylhydrazones derived from mesoxalic acid

Analogs of biologically active compounds IX. Synthesis of several new uracil and pteridine 6-aza-analogs based on cyclization of arylhydrazones derived from mesoxalic acid

Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori

In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

Transcriptome profiling of natural dichromatism in the annual fishes Nothobranchius furzeri and Nothobranchius kadleci.

BackgroundThe annual fish Nothobranchius furzeri is characterized by a natural dichromatism with yellow-tailed and red-tailed male individuals. These differences are due to different distributions of xanthophores and erythrophores in the two morphs. Previous crossing studies have showed that dichromatism in N. furzeri is inherited as a simple Mendelian trait with the yellow morph dominant over the red morph. The causative genetic variation was mapped by linkage analysis in a chromosome region containing the Mc1r locus. However, subsequent mapping showed that Mc1r is most likely not responsible for the color difference in N. furzeri. To gain further insight into the molecular basis of this phenotype, we performed RNA-seq on F2 progeny of a cross between N. furzeri male and N. kadleci female.ResultsWe identified 210 differentially-expressed genes between yellow and red fin samples. Functional annotation analysis revealed that genes with higher transcript levels in the yellow morph are enriched for the melanin synthesis pathway indicating that xanthophores are more similar to melanophores than are the erythrophores. Genes with higher expression levels in red-tails included xanthine dehydrogenase (Xdh), coding for a biosynthetic enzyme in the pteridine synthesis pathway, and genes related to muscle contraction. Comparison of DEGs obtained in this study with genes associated with pigmentation in the Midas cichlid (A. citrinellus) reveal similarities like involvement of the melanin biosynthesis pathway, the genes Ptgir, Rasef (RAS and EF-hand domain containing), as well as genes primarily expressed in muscle such as Ttn and Ttnb (titin, titin b).ConclusionsRegulation of genes in the melanin synthetic pathway is an expected finding and shows that N. furzeri is a genetically-tractable species for studying the genetic basis of natural phenotypic variations. The current list of differentially-expressed genes can be compared with the results of fine-mapping, to reveal the genetic architecture of this natural phenotype. However, an evolutionarily-conserved role of muscle-related genes in tail fin pigmentation is novel finding and interesting perspective for the future.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-754) contains supplementary material, which is available to authorized users.

The diversity‐oriented synthesis of pteridines—achievements and potential for development

The importance of pteridines in the key cofactors, tetrahydrofolate and tetrahydrobiopterin, has encouraged the development of the chemistry and chemical biology of pteridines. In order to investigate the latter, versatile synthetic methods are required to prepare designed relatives of the natural cofactors for use as potential drugs or biological probes. Recently, diversity-oriented synthesis has become a particularly powerful and versatile approach to the synthesis of pteridines and related heterocyclic compounds. This article describes and discusses available methods using radical, electrophilic, nucleophilic, and organometal mediated substitution reactions.

ChemInform Abstract: Synthesis of Pteridines Fused to Heterocycles

AbstractReview: 155 refs.

ChemInform Abstract: First Use of the Taylor Pteridine Synthesis as a Route to Polyglutamate Derivatives of Antifolates.

The di- through penta-γ-L-glutamates of 2-desamino-2-methylaminopterin, a new antifolate with a novel mechanism of action requiring γ-polyglutamylation for biological activity, were prepared. α-tert-Butyl γ-methyl L-glutamate was condensed with 4-nitrobenzoyl chloride, the methyl ester selectively hydrolyzed with base, and the product condensed with di-tert-butyl L-glutamate to obtain tri-tert-butyl N-(4-nitrobenzoyl)-γ-L-glutamyl-L-glutamate. This is the first example of the preparation of the polyglutamates of an antifolate via the Taylor pteridine synthesis