Synthesis of Cyclic Pyranopterin Monophosphate, a Biosynthetic

Article pubs.acs.org/jmc

Synthesis of Cyclic Pyranopterin Monophosphate, a Biosynthetic Intermediate in the Molybdenum Cofactor Pathway Keith Clinch,*,† Derek K. Watt,‡ Rachel A. Dixon,‡ Sylvia M. Baars,‡ Graeme J. Gainsford,† Ashish Tiwari,§ Günter Schwarz,#,⊥ Yas Saotome,§ Michael Storek,§ Abdel A. Belaidi,#,⊥ and Jose A. Santamaria-Araujo# †

Carbohydrate Chemistry Team and ‡Glycosyn Group, Callaghan Innovation, P.O. Box 31-310, Lower Hutt 5040, New Zealand § Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, Connecticut 06410, United States # Colbourne Pharmaceuticals GmbH, Viktoriaweg 7, 53859 Niederkassel, Germany ⊥ Institute of Biochemistry, Department of Chemistry and Center for Molecular Medicine Cologne, University of Cologne, Zuelpicher Strasse 47, 50674 Cologne, Germany S Supporting Information *

ABSTRACT: 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.

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INTRODUCTION Molybdenum (Mo) is an essential trace element taken up as the oxyanion molybdate (MoO42−) and inserted into the tricyclic structure molybdopterin or metal-binding pterin (MPT),1 forming the Mo cofactor (MoCo), the biologically active form of Mo (Figure 1). Several reviews have appeared recently, covering the many aspects associated with MoCo’s discovery, biosynthesis, and cell biology in various life forms.1−8 Sulfite oxidase (SO), xanthine oxidase, aldehyde oxidase, and mitochondrial amidoxime reducing component have all been identified as human MoCo-dependent enzymes.2,8,9 Of these, SO, that converts metabolically produced sulfite into sulfate,10 is of high importance because sulfite accumulation, particularly in the brain, leads to neurological damage and early death.11 MoCo is found with different ligands attached at the Mo center depending on the enzyme. In the SO family of enzymes, MoCo is found modified with a protein-derived cysteine ligand. The biosynthesis pathway of MoCo, which is conserved throughout all life kingdoms, begins with guanosine triphosphate (GTP) and proceeds through three identified intermediates, the first of which is cyclic pyranopterin monophosphate (1, also known as precursor Z, Figure 1). Although 1 © 2013 American Chemical Society

is the most stable of these intermediates, aqueous solutions are sensitive to a pH-dependent aerial oxidation, degrading to Compound Z12,13 with an estimated half-life of 10.6 h at pH 3 (Figure 1).14 In humans, the conversion of GTP to 1 requires the functional MOCS1 gene, that encodes for two catalytic activities, MOCS1A and MOCS1AB.15,16 Subsequently, sulfur transfer is catalyzed by the gene products of the MOCS2 gene thus leading to the synthesis of MPT.17 Finally, Mo insertion is dependent on the two-stage activity of the gephyrin protein, that first adenylates MPT, and then in a molybdate-dependent fashion, cleaves adenylate again.18 A defect in any step of the biosynthesis of MoCo results in the loss of all Mo enzyme activity. MoCo deficiency is a rare human genetic disorder that affects newborns and results in early childhood death due to rapidly progressing neurodegeneration.19 Two thirds of all patients belong to deficiency group type A due to mutations in the MOCS1 gene and are unable to synthesize 1.20 To understand the pathogenesis of MoCo deficiency, an animal model with MOCS1 gene knock out has been created. Received: December 16, 2012 Published: February 5, 2013 1730

dx.doi.org/10.1021/jm301855r | J. Med. Chem. 2013, 56, 1730−1738

Journal of Medicinal Chemistry

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Figure 1. Abbreviated human biosynthesis1,2,4,8 of MoCo showing a cysteine ligand attached at the Mo center as found in the sulfite oxidase family of Mo enzymes. The first isolable intermediate, cyclic pyranopterin monophosphate (1), and its aerial oxidation product Compound Z12,13 are also shown. IUPAC numbering.

hydroxyl group onto the C(7)N(8) bond to give a diastereomeric mixture of pyranopterins (2, Figure 2).31 They

Homozygous animals mirrored the human disease phenotype with no detectable MoCo or Mo enzyme activity and an average life span of 7.5 days.21 These animals were treated with 1 that had been overproduced in small amounts in Escherichia coli (E. coli) resulting in the normalization of their phenotype, growth, and Mo enzyme activity.22 Then, in a pivotal study,23 1 was used to treat a type A MoCo-deficient 36-day-old human infant, who after more than 5 years of medication is still responding well (Schwarz, personal observation). Recently, a successful treatment with very promising neurodevelopmental outcome has been reported.24 Compound 1 contains the uncommon pyranopterin heterocycle, (2-amino-3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridin-4-one), consisting of a pterin (2-amino3,4,5,6,7,8-hexahydropteridin-4-one) fused to a pyran ring. The structure of 1 has been elucidated by nuclear magnetic resonance (NMR) and mass spectral analysis, that also confirmed the presence of a ketone functional group in the form of its hydrate (geminal diol, Figure 1).14,25 This structure differed from one reported earlier that proposed a core system of 2-amino-4,6,7,8-tetrahydropteridin-4-one instead of a pyranopterin with a ketone group present in the enol form.26 Recently, 1 has been cocrystallized in the active site of Staphylococcus aureus MPT synthase and subjected to X-ray analysis, thereby defining the stereochemistry of the four chiral centers present in 1.27 An important process for making pterins involves condensing 2,5,6-triamino-3,4-dihydropyrimidin-4-one with 1,2-dicarbonyl compounds and is based on the classical pteridine (pyrazino[2,3-d]pyrimidine) syntheses of Gabriel and Colman28 and of Isay29 who, respectively, condensed 6-methylpyrimidine-4,5diamine and pyrimidine-4,5-diamine with benzil to give 4methyl-6,7-diphenylpteridine and 6,7-diphenylpteridine, respectively. In a variant of this reaction, Viscontini and co-workers prepared, in a regioselective manner, fully oxidized (aromatic) pterins bearing 6-hydroxyalkyl side chains by condensing 2,5,6triamino-3,4-dihydropyrimidin-4-one with pentose (L-xylose and D-arabinose) phenylhydrazones under mildly acidic conditions, followed by oxidation of the intermediate 6(trihydroxypropyl)-5,6-dihydropterins.30 Later, Pfleiderer and co-workers performed the same Viscontini reaction with Larabinose phenylhydrazone but trapped out the 5,6-dihydropterin intermediate by intramolecular cyclization of the terminal

Figure 2. Examples of known pyranopterins and 2-thioalkyl and pyranoquinoxaline analogues.

were able to separate the diastereomers as acetate derivatives. Analogously, 5,6-diamino-2-(methylsulfanyl)-3,4-dihydropyrimidin-4-one gave a corresponding mixture of diastereomers that was also separated into individual isomers by formation of acetate derivatives. Each product was then treated separately with aqueous hydrochloric acid to give the 5aS,6R,7S,9aR isomer 3 and its 5aR,6R,7S,9aS diastereomer. The enantiomer of 3 has also been recently prepared in the same way and examined by 2-D NMR.32 In a similar fashion, pyranopterins have been prepared from the following heterocycles: 5,6diamino-3-methyl-2-(methylsulfanyl)-3,4-dihydropyrimidin-4one and 2-(methylsulfanyl)pyrimidine-4,5,6-triamine,31 5,6diamino-2-methyl-3,4-dihydropyrimidin-4-one and the product converted to 2-methyl-6-[(1S,2R)-1,2,3-trihydroxypropyl]-3,4dihydropteridin-4-one,33 5,6-diamino-2-(benzylsulfanyl or methylsulfanyl)-3,4-dihydropyrimidin-4-ones as intermediates for preparing 2-alkylthio-6-formylpteridines,34 5,6-diamino-2(ethylsulfanyl)-3,4-dihydropyrimidin-4-one producing 4 as a protected intermediate for neopterin or biopterin derivatives,35 1731



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Scheme 1. Preparation of Compound 10 by the Viscontini Reactiona,b

a

Reagents and conditions: (a) (i) MeOH, H2O, 110 °C, 2 h; (ii) Et3N, RT, 36%. bIUPAC numbering.

susceptible hydrogens exchanged within a few minutes except for NH-10 (δH 7.29) that had only exchanged about 40%. However, complete exchange was noted after about 1 h. The connectivities of the hydrogen and carbon atoms were next established through 1H−1H DQF-COSY and 1H−13C HSQC experiments and the cis relationship between H-9a (δH 4.66) and H-5a (δH 3.04) defined by its small (1.3 Hz) coupling constant. A 1H−1H NOESY experiment confirmed H-9a and H-5a were on the same side of the pyranose ring as H-6 and H8 (Figure 3). As reactivities of the 5- and 6-amino groups in 8

and 5,6-diamino-(2-methoxy- or 2-methoxy-3-methyl)-3,4dihydropyrimidin-4-ones as potential anti HIV agents.36 Several pyrano[2,3-b]quinoxalines such as 5, incorporating a benzene ring instead of a 2-amino-3,4-dhydropyrimidin-4-one, have been prepared as MPT analogue intermediates where it was found that such structures were more stable than pyranopterins toward oxidation.37 In a different approach, the pyrano[2,3-b]quinoxaline 638,39 and pyrano[3,2-g]pteridine 738,40 were prepared by a chloroformate-mediated intramolecular cyclization of a secondary hydroxyl group present in either a 2-substituted quinoxaline or 6-substituted-2-amino3,4-dihydropteridin-4-one, respectively, onto the corresponding C(3)N(4) or C(7)N(8) bond, respectively, followed in each case by hydride reduction of the remaining CN bonds. Summaries of the chemistry of pterins relevant to MoCo41 and other biologically important pterins42 have also appeared recently. As noted above, giving 1 to type A MoCo-deficient individuals is a promising new approach to a potential treatment. However, for a replacement therapy with 1 to be successful, larger amounts of 1 are needed other than those obtainable from bacterial sources alone (8−10 mg L−1 of bacterial culture)14 where volumes and isolation issues are likely to be problematic on a large scale. A reliable supply of 1 will enable toxicity studies, stability tests, and ultimately clinical trials to be undertaken. Described here is a convenient synthesis of 1 hydrobromide (1·HBr) and a comparison of its in vitro activity with 1 derived from E. coli.43

Figure 3. 1H−1H NOESY interactions in 10, indicated by the double headed arrows, confirming the stereochemistry created at the (C)5a− (C)9a ring juncture by the Viscontini reaction.

are pH dependent, initial reaction could have also occurred at the 6-NH2 with ketone 9a leading to regioisomer 11 that would be difficult to distinguish from 10 by NMR spectroscopy. However, any ambiguity in the structure of 10 was resolved by X-ray crystallography using copper Kα radiation that also confirmed both the relative and absolute stereochemistry (Supporting Information). Although 10 was obtained in only modest yield, all the stereocenters present in 1 were established correctly in this single step. Pyranopterins such as 10, that have a pyrazine ring in a fully reduced state, are prone to oxidize to aromatic pterin derivatives with concomitant cleavage of the O(9)−C(9a) bond. A suitable protecting group at N-10 in 10 should prevent this. Therefore, compound 10 was suspended in dry tetrahydrofuran and treated with di-tert-butyl dicarbonate and 4-N,N-dimethylaminopyridine until a clear solution was obtained (Scheme 2). After chromatography on silica gel, two major products were isolated, enriched with seven or six tertbutoxycarbonyl (Boc) groups, noting that the amount of the latter increased relative to that of the former the longer the crude reaction mixture was in contact with silica gel. Crystallization of the individual hepta- and hexa-Boc-protected compounds enabled pure samples of each to be obtained that were assigned structures 12 and 13, respectively (see below). The tert-butyl carbonate groups present in 12 and 13,

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RESULTS AND DISCUSSION Chemistry. Treatment of 2,5,6-triamino-3,4-dihydropyrimidin-4-one dihydrochloride (8)44 with D-galactose phenylhydrazone (9),37 under conditions similar to those reported for analogous compounds,31 gave after neutralization with triethylamine, a compound that crystallized out of the reaction mixture in almost pure form (Scheme 1). In keeping with the regioselective nature of the Viscontini reaction, the compound was assigned structure 10 in which initial reaction by the more basic 5-NH2 group in 8 with the ketone function in the presumed Amadori rearrangement product 9a had taken place. Compound 10 could be recrystallized from hot water and was found to be stable after 2 weeks at 20 °C, but solutions in DMSO-d6 stored at the same temperature had completely degraded after this period. The structure of 10 was thoroughly investigated by NMR spectroscopy. First, the deuterium exchangeable hydrogens were identified with D2O. All the 1732



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Scheme 2. Conversion of 10 into 1·HBr by Selective Boc Protection, Introduction of a Cyclic Phosphate Ester, Oxidation of a Secondary Alcohol, and Then Deprotectiona,b

Reagents and conditions: (a) Boc2O (9 equiv), DMAP (0.5 equiv), THF, 50 °C, 20 h, 28% (12), 58% (13); (b) 1 M NaOMe (5.5 equiv for 12, 4 equiv for 13), MeOH, rt 20 h, then Dowex 50WX8 (H+) resin, 75% combined for 12 and 13; (c) MeOP(O)Cl2 (1.1 equiv), pyridine, CH2Cl2, −42 °C, 140 min then H2O, 65%; (d) (i) DMSO (4.3 equiv), TFAA (2.2 equiv), CH2Cl2, −78 °C, 40 min then DIPEA (9 equiv), −78 °C, 50 min then H2O and AcOH; (ii) TMSBr, 0.24% aqueous acetone, 38% over two steps. bIUPAC numbering. a

Figure 4. The 500 MHz 1H NMR (HOD δ 4.79, 960 scans) spectrum of synthetic 1·HBr obtained on the supernatant after suspending 2−3 mg of 1·HBr in 0.6 mL of D2O, adding 15 μL of DCl, and centrifuging. The small chemical shift differences and partial overlap of Hb-4 with H-12a seen on comparison with the 1H NMR data reported25 for 1 obtained from bacterial culture are likely due to pH and concentration effects.

sodium methoxide/methanol solutions to give the tri-Bocamino-protected derivative 14. Treating 14 with methyl dichlorophosphate in a mixture of pyridine and dichloro-

identifiable by the higher chemical shifts (δH values) of their H6’s, H-7’s, and CH2 groups relative to those in 14, were selectively removed by treatment with controlled excesses of 1733



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methane at low temperature gave the highly crystalline cyclic phosphate ester 15 as a single diastereomer, that was characterized spectroscopically and by X-ray crystallography using copper Kα radiation.45 With the establishment of the position of the three N-linked Boc groups on the pyrimidine ring in 15 determined, it followed that the structure of the hexa-Boc compound 13 and the tri-Boc compound 14 were as drawn in Scheme 2. Compound 12 has the same structure as 13 but with an extra Boc group present that could be located at N-3, O-(C)-4, or N5. In the 1H NMR spectrum of compound 12, H-5a and H-9a resonated at δH 3.91 and δH 5.71, respectively. For compounds 13 and 14, H-5a (and H-9a) resonated at δH 3.76 (5.58) and 3.52−3.39 (5.43), respectively, and for compound 15, H-11a (and H-5a, different numbering) resonated at δH 3.90 (5.74). These data suggested that there was no Boc group on N-5 in 12, as a higher chemical shift (δH value) would be expected for H-5a relative to the corresponding hydrogens in 13, 14, and 15. Further, as tert-butyl carbonates are, under basic conditions, hydrolytically more labile than tert-butyl carbamates, and because no Boc group was found attached to N-3 in 14, it was concluded that structure 12 was as drawn in Scheme 2. It was also found that NH-5 (δH 4.35) in 12 did not exchange D2O from a CDCl3 solution but did, together with NH-3, in compound 13. Swern oxidation of the secondary hydroxyl group in compound 15 was readily followed by TLC and took place cleanly (Scheme 2). However, the crude product was difficult to purify and was therefore treated directly with excess bromotrimethylsilane in 0.24% aqueous acetone to simultaneously remove the Boc and methyl phosphate protecting groups, leading to a solid crystallizing out of the reaction mixture. Recrystallization from 24% aqueous hydrobromic acid/2-propanol mixtures gave 1·HBr. The salt had an HPLC purity of 95% and bromine analysis confirmed it to be a monohydrobromide. Synthesized 1·HBr was only sparingly soluble in water (pH 1) but quite soluble in DMSO. The 1H NMR (D2O−DCl) spectrum of 1·HBr compared well with data reported for 1 isolated from bacterial culture (Figure 4).25 The 13 C NMR spectrum of 1·HBr in DMSO-d6 revealed two partly coalescing quaternary carbon atoms around δC 90.1 assigned as C-10a and C-12. This value for C-12 is in keeping with the hydrate form of the ketone (geminal diol) and is similar to that value observed (δC 88.4) in the 13C NMR spectrum (D2O− DCl) reported for bacterially derived 1.14 The microcrystalline nature of 1·HBr was also revealed when viewed under high magnification (Supporting Information). Compound 1·HBr prepared here showed no significant deterioration after storing 2 weeks at 20 °C or several months at −20 °C. Biology. The in vitro biological activity of synthesized 1·HBr was measured and compared with E. coli derived 1 using two different assays (Figure 5). In the first assay, increasing amounts of 1·HBr and 1 were converted into MPT using E. coli MPT synthase, assembled from its recombinantly expressed and purified subunits MoaD (thiocarboxylated small subunit) and MoaE (large subunit) as described previously.46 The produced MPT was further oxidized and dephosphorylated to give the stable product Dephospho-Form A (16) that was quantified by HPLC analysis using fluorescence detection (Figure 5A,B).47 A similar dose-dependent increase in the formation of Dephospho-Form A was detectable, demonstrating that 1·HBr was converted by MPT synthase with a similar rate and specificity as that for E. coli-derived 1.

Figure 5. In vitro biological activity of synthesized 1·HBr in comparison to E. coli derived 1. (A, B) Enzymatic conversions (MPT synthesis) assessed by HPLC quantification of the MPT oxidation product Dephospho-Form A (16, arrowed). (C, D) Enzymatic conversions (MPT synthesis and molybdate insertion to form MoCo) were quantified as a function of the activity of reconstituted human apo-SO to reduce potassium hexacyanoferrate(III) (Supporting Information).48 Values shown in charts C and D are averages of three independent experiments, and error bars indicate standard deviation.

In the second assay, increasing amounts of 1·HBr or 1 were again in vitro-converted into MPT and then further transformed into MoCo by the action of gephyrin.48 Gephyrin binds MPT and first transfers an adenylate group in a magnesium− adenosine 5′-triphosphate-dependent reaction to the MPT phosphate group by the action of the N-terminal G domain. Subsequently, adenylated MPT is transferred to the other Cterminal E domain, where molybdate binds and upon hydrolysis of the MPT adenylate, Mo insertion and MoCo synthesis is completed. The amount of synthesized MoCo was quantified (Figure 5C,D) after it had been transferred to MoCo-free human apo-sulfite oxidase and the activity of the reconstituted enzyme determined by sulfite-dependent reduction of potassium hexacyanoferrate(III).48 Compound 1·HBr produced similar dose-dependent quantities of MoCo as compared to 1, confirming that the synthetic material was of comparable activity to that of bacterial origin. Given the multistep nature of the assay, approximately 30% of the used 1·HBr (or 1) was converted into MoCo.

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CONCLUSION A short synthesis of 1·HBr has been achieved using a Viscontini reaction in the key step to produce the tricyclic pyranopterin intermediate 10 in which all four chiral centers found in 1 were established. Selective use of Boc protecting groups in 10 allowed the introduction of a cyclic phosphate ester and the oxidation of a secondary alcohol to be accomplished without 1734



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and then with 2-propanol (3 × 3 mL), and dried under high vacuum (∼0.1 Torr) for 1 h at RT to afford 1·HBr as an off white solid (57 mg, 38%). UV: (0.16 mM in 0.1 M HCl) λmax nm (ε), 212 (11 250), 263 (10 000). 1H NMR (0.11 M in DMSO-d6): δ 8.44, (bs, exchanged D2O, 1H), 7.13 (bs, exchanged D2O, 2H), 5.79 (bs, exchanged D2O, 6H + H2O), 5.00 (s, 1H), 4.41 (d, J = 12.3 Hz, 1H), 4.28 (s, partly overlapped with dd at δ 4.25, 1H), 4.25 (dd, J = 23.2, 12.3 Hz, partly overlapped with s at δ 4.28, 1H), 3.94 (s, 1H), 3.31 (s, 1H). 13C NMR (125.7 MHz, DMSO-d6): δ 156.5 (C), 152.8 (C), 149.8 (bC), 90.2 (C), 90.1 (C), 77.6 (d, J = 4.7 Hz, CH), 76.1 (CH), 69.5 (d, J = 5.5 Hz, CH2), 67.1 (d, J = 3.9 Hz, CH), 56.0 (CH). 31P NMR (202.4 MHz, DMSO-d6): δ −8.3. ESI-HRMS calcd for C10H15BrN5O8P +, (M − HBr + H)+, 364.0653, found 364.0645. Anal. Calcd for C10H14N5O8P HBr 17.99 Br; found, 17.98 Br. HPLC ≥ 95% (Supporting Information). (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). 2,5,6-Triamino-3,4-dihydropyrimidin-4-one dihydrochloride (8,44 10.0 g, 46.72 mmol), D-galactose phenylhydrazone (9,37 15.78 g, 58.38 mmol), and 2-mercaptoethanol (1 mL) were stirred and heated under reflux (bath temp 110 °C) in a 1:1 mixture of MeOH/H2O (400 mL) for 2 h. After cooling to RT, diethyl ether (500 mL) was added, the flask shaken, and the diethyl ether layer decanted off and discarded. The process was repeated with two further portions of diethyl ether (500 mL), and then the remaining volatiles were evaporated. Methanol (40 mL), H2O (40 mL) and triethylamine (39.4 mL, 280 mmol) were successively added, and after 5 min the yellow solid was filtered off, washed with a little MeOH, and dried to give 10 (5.05 g, 36%) of suitable purity for further use. An analytical portion was recrystallized from boiling H2O: mp 226 °C (decomp). [α]20 D +135.6 (c 1.13, DMSO). 1H NMR (DMSO-d6): δ 10.19 (bs, exchanged D2O, 1H), 7.29 (d, J = 5.0 Hz, slowly exchanged D2O, 1H), 5.90 (s, exchanged D2O, 2H), 5.33 (d, J = 5.1 Hz, exchanged D2O, 1H), 4.66 (dt, J = 5.0, 1.3 Hz, 1H), 4.59 (t, J = 5.6 Hz, exchanged D2O, 1H), 4.39 (d, J = 10.3 Hz, exchanged D2O, 1H), 3.80 (bt, J = 1.8 Hz, exchanged D2O, 1H), 3.70 (m, 1H), 3.58 (dd, J = 10.3, 3.0 Hz, 1H), 3.53 (dt, J = 10.7, 6.4 Hz, 1H), 3.43 (ddd, J = 11.2, 5.9, 5.9 Hz, 1H), 3.35 (t, J = 6.3 Hz, 1H), 3.04 (bm, 1H). 13C NMR (DMSO-d6): δ 156.1 (C), 150.2 (C), 148.2 (C), 98.8 (C), 79.3 (CH), 76.3 (CH), 68.8 (CH), 68.4 (CH), 60.4 (CH2), 53.7 (CH). ESI-HRMS calcd for C10H15N5NaO5+, (M + Na)+, 308.0965, found 308.0968. Anal. Calcd for C10H15N5O5 H2O, 39.60 C, 5.65 H, 23.09 N, found 39.64 C, 5.71 H, 22.83 N. X-ray analysis of 10 confirmed the product as a monohydrate (Supporting Information). tert-Butyl (5aS,6R,7R,8R,9aR)-2-{Bis[(tert-butoxy)carbonyl]amino}-4,6,7-tris({[(tert-butoxy)carbonyl]oxy})-8-({[(tert-butoxy)carbonyl]oxy}methyl)-5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridine-10-carboxylate (12) and tert-Butyl (5aS,6R,7R,8R,9aR)-2{Bis[(tert-butoxy)carbonyl]amino}-6,7-bis({[(tert-butoxy)carbonyl]oxy})-8-({[(tert-butoxy)carbonyl]oxy}methyl)-4-oxo3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridine-10-carboxylate (13). Di-tert-butyl dicarbonate (10.33 g, 47.33 mmol) and 4N,N-dimethylaminopyridine (0.32 g, 2.62 mmol) were added to a stirred suspension of 10 (1.50 g, 5.26 mmol) in anhydrous THF (90 mL) at 50 °C under Ar. After 20 h, a clear solution resulted, the solvent was evaporated, and the residue was chromatographed (gradient 0 → 40% EtOAc in hexanes) to give first 12 as a yellow foam (1.46 g, 28%). A sample was crystallized from EtOAc−hexanes to give 12 as a colorless fine crystalline solid: mp 189−191 °C. [α]20 D −43.6 (c 0.99, MeOH). 1H NMR (500 MHz, CDCl3): δ 5.71 (t, J = 1.7 Hz, 1H), 5.15 (dt, J = 3.5, 1.0 Hz, 1H), 4.97 (t, J = 3.8 Hz, 1H), 4.35 (bt, J = 1.7 Hz, 1H), 4.07 (dd, J = 10.8, 5.9 Hz, 1H), 4.03−3.96 (m, 2H), 3.91 (m, 1H), 1.55, 1.52, 1.51, 1.50, 1.45 (5s, 45H), 1.40 (s, 18H). 13C NMR (125.7 MHz, CDCl3): δ 152.84 (C), 152.78 (C), 151.5 (C), 150.9 (C), 150.7 (2 × C), 150.3 (C), 149.1 (C), 144.8 (C), 144.7 (C), 118.0 (C), 84.6 (C), 83.6 (C), 83.5 (C), 82.7 (3 × C), 82.6 (C), 76.3 (CH), 73.0 (CH), 71.4 (CH), 67.2 (CH), 64.0 (CH2), 51.4 (CH), 28.1, 27.8, 27.7, 27.6 (CH 3’s). ESI-HRMS calcd for C45H72N5O19+, (M + H)+, 986.4817, found 986.4818. Anal. Calcd for C45H71N5O19, 54.81 C, 7.26 H, 7.10 N; found 54.66 C, 7.17 H, 7.05 N. A second slower-eluting fraction gave 13 as a yellow foam (2.68 g, 58%). A small amount was crystallized from EtOAc−hexanes

any aromatic pterin formation taking place. The final deprotection step was facile and led directly to crystalline product that was recrystallized to provide 1·HBr of 95% purity. The product remained significantly unchanged upon storing for at least 2 weeks at 20 °C or several months at −20 °C. Synthesized 1·HBr was equally effective as E. coli-derived 1 in in vitro MPT and MoCo synthesis assays demonstrating 1·HBr’s in vitro biological activity. The method is currently being developed and optimized in our laboratories for the large-scale production of 1·HBr to enable toxicity studies and in vivo comparisons with 1 to be evaluated.

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EXPERIMENTAL SECTION

Chemistry. General. All reactions were performed under an argon or nitrogen atmosphere. Organic solutions were dried over anhydrous MgSO4, and the solvents were evaporated under reduced pressure. Anhydrous and chromatography solvents were obtained commercially and used without any further purification. Thin layer chromatography (TLC) was performed on glass or aluminum sheets coated with 60 F254 silica gel. Organic compounds were visualized under UV light or a dip of ammonium molybdate (5 mass%) and cerium(IV) sulfate tetrahydrate (0.2 mass%) in aq H2SO4 (2 M). Chromatography (flash column or an automated system with continuous gradient facility) was performed on silica gel (40−63 μm). All compounds gave satisfactory CHN analyses that were within 0.4% of the theoretical value or exhibited a purity ≥95% by HPLC. Optical rotations were recorded at a path length of 1 dm and are in units of 10−1 deg cm2 g−1; concentrations are in g/100 mL. 1H NMR spectra were measured in CDCl3, CD3OD, DMSO-d6 (internal Me4Si, δ 0), or D2O (HOD, δ 4.79), and 13C NMR spectra in CDCl3 (center line, δ 77.0), CD3OD (center line, δ 49.0), DMSO-d6 (center line δ 39.52), or D2O (no internal reference). Assignments of 1H and 13C resonances were based on 2D (1H−1H DQF-COSY, 1H−13C HSQC), and DEPT experiments. Abbreviations used: s, singlet, d, doublet, t, triplet, q, quartet, bs, broad singlet, bt, broad triplet, dd, doublet of doublets, ddd, doublet of doublets of doublets, dt, doublet of triplets. High resolution electrospray mass spectra (ESI-HRMS) were recorded on a Q-TOF tandem mass spectrometer. Microanalyses were performed by the Campbell Microanalytical Department, University of Otago, Dunedin, New Zealand. (4aR,5aR,11aR,12aS)-8-Amino-2,12,12-trihydroxy4,4a,5a,6,9,10,11,11a,12,12a-decahydro-2H-1,3,5-trioxa-6,7,9,11tetraaza-2λ5-phosphatetracene- 2,10-dione Hydrobromide (1·HBr). Anhydrous DMSO (104 μL, 1.46 mmol) was added to a solution of compound 15 (223 mg, 0.34 mmol) in anhydrous CH2Cl2 (7 mL), and the solution was cooled to −78 °C. Trifluoroacetic anhydride (104 μL, 0.74 mmol) was added dropwise, and the mixture was stirred for 40 min. N,N-Diisopropylethylamine (513 μL, 2.94 mmol) was added, and the stirring was continued for 50 min at −78 °C. Saturated aqueous NaCl solution (20 mL) was added and the mixture removed from the cold bath and diluted with CH2Cl2 (30 mL). Glacial acetic acid (170 μL, 8.75 mmol) was added, and the mixture was stirred for 10 min. The layers were separated, and the aqueous phase was washed with CH2Cl2 (10 mL). The combined organic phases were successively washed with 5% aqueous HCl, a 3:1 mixture of saturated aqueous NaCl solution:10% aqueous NaHCO3 solution, and saturated aqueous NaCl solution, and dried and the solvent evaporated to give the crude ketone as a yellow solid (228 mg) of suitable purity for further use. The latter was dissolved in acetone (0.24% m/m H2O by Karl Fischer titration, 4.6 mL) and cooled in an ice bath, and bromotrimethylsilane (0.46 mL, 3.40 mmol) added. The mixture was then stirred at RT in the dark for 3 h. The green-yellow solid was isolated by centrifugation at 2400g for 1 min, washed with 2-propanol (3 × 3 mL), and then dried under high vacuum for 1 h at RT to give crude product (124 mg). It was dissolved in 24% aqueous HBr (2.64 mL) and the solution stirred vigorously in the dark. 2-Propanol (1.32 mL) was added, and after 1 h the solid was isolated by centrifugation at 2400g for 1 min, washed with a 1:1 mixture of 24% aqueous HBr:2-propanol (1 mL) 1735



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to give colorless crystals: mp 205 °C (decomp). [α]20 D −40.2 (c, 1.17, CHCl3). 1H NMR (500 MHz, CDCl3): δ 11.10 (bs, exchanged D2O, 1H), 5.58 (t, J = 1.8 Hz, 1H), 5.17 (d, J = 3.4 Hz, 1H), 4.97 (t, J = 3.9 Hz, 1H), 4.62 (s, exchanged D2O, 1H), 4.16 (dd, J = 11.3, 5.9 Hz, 1H), 4.12 (dd, J = 11.3, 6.4 Hz, 1H), 3.95 (dt, J = 6.1, 1.1 Hz, 1H), 3.76 (m, 1H), 1.51, 1.50, 1.49, 1.48, 1.46 (5s, 54H). 13C NMR (125.7 MHz, CDCl3): δ 156.6 (C), 153.0 (C), 152.9 (C), 151.9 (C), 150.6 (C), 149.4 (2 × C), 136.2 (C), 131.8 (C), 116.9 (C), 85.0 (2 × C), 83.3 (C), 82.8 (C), 82.49 (C), 82.46 (C), 77.3 (CH), 73.3 (CH), 71.5 (CH), 67.2 (CH), 64.5 (CH2), 51.3 (CH), 28.0, 27.72, 27.68, 27.6 (CH3’s). ESI-HRMS calcd for C40H64N5O17+, (M + H)+, 886.4292, found 886.4294. Anal. Calcd for C40H63N5O17, 54.23 C, 7.17 H, 7.90 N; found 54.27 C, 7.20 H, 8.01 N. tert-Butyl (5aS,6R,7R,8R,9aR)-2-{Bis[(tert-butoxy)carbonyl]amino}-6,7-dihydroxy-8-(hydroxymethyl)-4-oxo3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridine-10-carboxylate (14). Step 1: Compound 12 (1.46 g, 1.48 mmol) was dissolved in MeOH (29 mL) and sodium methoxide in MeOH (1 M, 8.14 mL, 8.14 mmol) added. After being left at RT for 20 h, the solution was neutralized with Dowex 50WX8 (H+) resin, the solids were filtered off, the solvent was evaporated, and the residue was combined with the crude product from step 2 below. Step 2: Compound 13 (2.68 g, 3.02 mmol) was dissolved in MeOH (54 mL) and sodium methoxide in MeOH (1 M, 12.10 mL, 12.10 mmol) added. After being left at RT for 20 h, the solution was neutralized with Dowex 50WX8 (H+) resin, the solids were filtered off, and the solvent was evaporated. The residue was combined with the crude product from step 1 above and chromatographed (gradient 0 → 15% MeOH in CHCl3) to give 14 as a cream-colored solid (1.97 g, 75%). A small sample was freeze-dried from DMSO for analysis. 1H NMR (500 MHz, DMSO-d6): δ 12.67 (bs, exchanged D2O, 1H), 5.48 (d, J = 5.2 Hz, exchanged D2O, 1H), 5.43 (t, J = 1.9 Hz, after D2O exchange became a d, J = 1.9 Hz, 1H), 5.00 (bs, exchanged D2O, 1H), 4.62 (t, J = 5.7 Hz, exchanged D2O, 1H), 4.27 (d, J = 6.0 Hz, exchanged D2O, 1H), 3.89 (dt, J = 5.2, 3.8 Hz, after D2O exchange became a t, J = 3.9 Hz, 1H), 3.62 (dd, J = 6.0, 3.7 Hz, after D2O exchange became a d, J = 3.7 Hz, 1H), 3.52−3.39 (m, 4H), 1.42 (s, 9H), 1.41 (s, 18H). 13C NMR (125.7 MHz, DMSO-d6): δ 157.7 (C), 150.9, (C), 149.6 (2 × C), 134.4 (C), 131.3 (C), 118.6 (C), 83.3 (2 × C), 81.1 (C), 78.0 (CH), 76.3 (CH), 67.9 (CH), 66.7 (CH), 60.4 (CH2), 54.2 (CH), 27.7, 27.5 (CH3’s). ESI-HRMS calcd for C25H40N5O11+, (M + H)+, 586.2719, found 586.2717. HPLC ≥ 98% (Supporting Information). tert-Butyl (2R,4aR,5aR,11aS,12R,12aR)-8-{Bis[(tert-butoxy)carbonyl]amino}-12-hydroxy-2-methoxy-2,10-dioxo4,4a,5a,6,9,10,11,11a,12,12a-decahydro-2H-1,3,5-trioxa-6,7,9,11tetraaza-2λ5-phosphatetracene-6-carboxylate (15). Compound 14 (992 mg, 1.69 mmol) was dissolved in anhydrous pyridine (5 mL) and the solvent evaporated. The residue was dissolved in a mixture of anhydrous CH2Cl2 (10 mL) and pyridine (5 mL) and the solution cooled to −42 °C in an acetonitrile/dry ice bath. Methyl dichlorophosphate (187 μL, 1.86 mmol) was added dropwise, and the mixture was stirred for 140 min. Water (10 mL) was added to the cold solution, which was then removed from the cold bath and diluted with ethyl acetate (50 mL) and aqueous saturated NaCl solution (30 mL). The organic portion was separated and washed with aqueous saturated NaCl solution. The combined aqueous portions were extracted twice further with ethyl acetate, the combined organic portions were dried, and the solvent was evaporated. Chromatography (2 → 20% MeOH in EtOAc) gave 15 as a tan-colored solid (731 mg, 65%). Recrystallization from MeOH−Et2O gave colorless crystals for analysis. [α]D20 −39.9 (c, 1.15, MeOH). 1H NMR (500 MHz, CD3OD): δ 5.74 (d, J = 2.2 Hz, 1H), 4.80 (d, J = 3.5 Hz, partly overlapped with HOD, 1H), 4.65 (dt, J = 12.6, 1.8 Hz, 1H), 4.43 (ddd, J = 22.3, 12.7, 0.9 Hz, 1H). 4.26 (q, J = 3.9 Hz, 1H), 3.90 (bs, 1H), 3.79 (dd, J = 3.7, 2.1 Hz, 1H), 3.55 (d, J = 11.6 Hz, 3H), 1.52 (s, 9H), 1.46 (s, 18H). 13C NMR (125.7 MHz, CD3OD): δ 159.5 (C), 152.7 (C), 151.1 (2 × C), 135.7 (C), 132.5 (C), 119.9 (C), 85.5 (2 × C), 83.9 (C), 77.7 (CH), 76.9 (d, J = 4.9 Hz, CH), 71.4 (d, J = 5.3 Hz, CH2), 69.9 (d, J = 5.5 Hz, CH), 68.7 (d, J = 6.7 Hz, CH), 56.8 (d, J =

7.3 Hz, CH3), 53.4 (CH), 28.5, 28.2 (CH3’s). 31P NMR (202.4 MHz, CD3OD): δ −5.2. ESI-HRMS calcd for C26H41N5O13P+, (M + H)+, 662.2433, found 662.2429. Anal. Calcd for C26H40N5O13P MeOH H2O, 45.57 C, 6.52 H, 9.84 N; found 45.38 C, 6.27 H, 10.03 N. X-ray analysis of 14 confirmed 1 equiv each of MeOH and H2O of crystallization present.45 Biology. Protein Expression and Purification. The E. coli MPT synthase large subunit MoaE and gephyrin C4 splice variant were expressed in E. coli BL21 (DE3) strain as His-tagged proteins and purified by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatrography as previously described.46,48 The E. coli MPT synthase small subunit MoaD was expressed and purified in its thiocarboxylated form as previously described.46 For the MoCo in vitro reconstitution assay, human apo-sulfite oxidase−Mo domain (apoSOMO) was expressed in the E. coli moaC mutant strain RK5245.49 Expression was induced with 0.1 mM isopropyl β-thiogalactoside at OD600 = 0.5 and continued for 15 h at 30 °C. All purified proteins were exchanged into the same buffer (20 mM Tris/HCl pH 8.0, 50 mM NaCl) and stored at −80 °C until analysis. MPT in Vitro Synthesis and HPLC Form A Analysis. MPT was synthesized in vitro using the MPT synthase subunits MoaE and thiocarboxylated MoaD. The assay was carried out in a final volume of 140 μL, which contained 0.1 nmol of MoaE and 4 nmol of MoaD. The reaction was started by adding different amounts of 1·HBr (50 to 1000 pmol) and maintained at room temperature for 30 min. Compound 1 that was purified from E. coli was also used as a control14 and compared to chemically synthesized 1·HBr. MPT quantification was carried out by HPLC Form A analysis whereby MPT was first oxidized to the stable oxidation product Dephospho-Form A and further quantified using HPLC reverse phase chromatography as previously described.47 MoCo in Vitro Reconstitution Assay.48 All reactions were performed at RT in 100 mM HEPES buffer pH 7.5. The assay was carried out in three steps. First, a mixture of MoaE (0.1 nmol), MoaD (4 nmol), gephyrin C4 (1 nmol), MgCl2 (15 mM), ATP (15 mM), and molybdate (10 μM) was prepared, and the reaction was started by addition of different amounts of 1 (or 1·HBr, 50 to 1000 pmol). Following an incubation time t1 of 30 min, the second step was started by addition of apoSOMO (1 nmol) followed by an incubation time t2 of 30 min. Finally, MoCo insertion efficacy was evaluated as a function of the activity of reconstituted SO−Mo domain (SOMO), which was determined by sulfite-dependent reduction of potassium hexacyanoferrate(III). Potassium hexacyanoferrate(III) (20 μM) was added to the mixture, and the SO reaction was started by addition of sulfite (300 μM). SO activity was quantified by monitoring the reduction of potassium hexacyanoferrate(III) at 420 nm using a 96well plate reader (BioTeK, Germany). Activity of the SOMO protein was determined in μM/min and correlated with the amount of bound MoCo based on the known saturation of in vivo assembled SOMO using Form A HPLC analysis (Supporting Information).

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra for compounds 1·HBr (1H, 13C and 31P), 10, 12, 13, 14 (1H and 13C), and 15 (1H, 13C and 31P). HPLC trace, UV spectrum, crystal micrographs for 1·HBr, HPLC trace for compound 14, and alternative Figure 5 depicting SO activity. X-ray structural information including CIF for compound 10. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +6449313000; fax: +6449313055; e-mail: keith.clinch@ callaghaninnovation.govt.nz. Notes

The authors declare no competing financial interest. 1736



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molybdenum cofactor biosynthesis. J. Biol. Chem. 2002, 277, 18303− 18312. (16) Reiss, J.; Dorche, C.; Stallmeyer, B.; Mendel, R. R.; Cohen, N.; Zabot, M. T. Human molybdopterin synthase gene: Genomic structure and mutations in molybdenum cofactor deficiency type B. Am. J. Hum. Genet. 1999, 64, 706−711. (17) Stallmeyer, B.; Drugeon, G.; Reiss, J.; Haenni, A. L.; Mendel, R. R. Human molybdopterin synthase gene: Identification of a bicistronic transcript with overlapping reading frames. Am. J. Hum. Genet. 1999, 64, 698−705. (18) Stallmeyer, B.; Schwarz, G.; Schulze, J.; Nerlich, A.; Reiss, J.; Kirsch, J.; Mendel, R. R. The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1333−1338. (19) Johnson, J. L.; Duran, M. The Metabolic and Molecular Bases of Inherited Disease, 8th ed.; Scriver, C.; Beaudet, A.; Sly, W.; Valle, D., Eds.; McGraw-Hill: New York, 2001; pp 3163−3177. (20) Reiss, J.; Johnson, J. L. Mutations in the molybdenum cofactor biosynthetic genes MOCS1, MOCS2, and GEPH. Hum. Mutat. 2003, 21, 569−576. (21) Lee, H.-J.; Adham, I. M.; Schwarz, G.; Kneussel, M.; Sass, J. O.; Engel, W.; Reiss, J. Molybdenum cofactor-deficient mice resemble the phenotype of human patients. Hum. Mol. Genet. 2002, 11, 3309−3317. (22) Schwarz, G.; Santamaria-Araujo, J. A.; Wolf, S.; Lee, H.-J.; Adham, I. M.; Gröne, H.-J.; Schwegler, H.; Sass, J. O.; Otte, T.; Hänzelmann, P.; Mendel, R. R.; Engel, W.; Reiss, J. Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli. Hum. Mol. Genet. 2004, 13, 1249−1255. (23) Veldman, A.; Santamaria-Araujo, J. A.; Sollazzo, S.; Pitt, J.; Gianello, R.; Yaplito-Lee, J.; Wong, F.; Ramsden, C. A.; Reiss, J.; Cook, I.; Fairweather, J.; Schwarz, G. Successful treatment of molybdenum cofactor deficiency type A with cPMP. Pediatrics 2010, 125, e1249− e1254. (24) Hitzert, M. M.; Bos, A. F.; Bergman, K. A.; Veldman, A.; Schwarz, G.; Santamaria-Araujo, J. A.; Heiner-Fokkema, R.; Sival, D. A.; Lunsing, R. J.; Arjune, S.; Kosterink, J. G. W.; van Spronsen, F. J. Favorable outcome in a newborn with molybdenum cofactor type A deficiency treated with cPMP. Pediatrics 2012, 130, e1005−e1010. (25) Santamaria-Araujo, J. A.; Fischer, B.; Otte, T.; Nimtz, M.; Mendel, R. R.; Wray, V.; Schwarz, G. The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor. J. Biol. Chem. 2004, 279, 15994−15999. (26) Wuebbens, M. M.; Rajagopalan, K. V. Structural characterization of a molybdopterin precursor. J. Biol. Chem. 1993, 268, 13493−13498. (27) Daniels, J. N.; Wuebbens, M. M.; Rajagopalan, K. V.; Schindelin, H. Crystal structure of a molybdopterin synthase−precursor Z complex: Insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency. Biochemistry 2008, 47, 615−626. (28) Gabriel, S.; Colman, J. Synthesen in der purinreihe. Ber. Dtsch. Chem. Ges. 1901, 34, 1234−1255. (29) Isay, O. Eine synthese des purins. Ber. Dtsch. Chem. Ges. 1906, 39, 250−265. (30) Viscontini, M.; Provenzale, R.; Ohlgart, S.; Mallevialle, J. Synthese des natürlichen D-neopterins und L-monapterins. Helv. Chim. Acta 1970, 53, 1202−1207. (31) Soyka, R.; Pfleiderer, W.; Prewo, R. Pteridine: Synthese und eigenschaften von 5,6-dihydro-6-(1,2,3-trihydroxypropyl)pteridinen: Kovalente intramolekulare addukte. Helv. Chim. Acta 1990, 73, 808− 826. (32) Kim, S.; Kang, Y. Structural elucidations of pyrano[3,2g]pteridine derivatives by 2D NMR spectroscopy. Bull. Korean Chem. Soc. 2011, 32, 3161−3163. (33) Matsuura, S.; Traub, H.; Armarego, W. L. F. Synthesis and dihydropteridine reductase [EC 1.6.99.10] (human) activity of reduced 2,7-dimethylpteridin-4(3H)-one, 2-methyl-2-desaminoneopterin [6-(1′S,2′R-1′,2′,3′-trihydroxypropyl)-2-methylpteridin-4(3H)one] and 2-methyl-2-desaminobiopterin [6-(1′R,2′S-1′,2′-dihydroxypropyl)-2-methylpteridin-4(3H)-one]. Pteridines 1989, 1, 73−82.

ACKNOWLEDGMENTS We thank Dr. Herbert Wong for NMR measurements, Dr. Yinrong Lu for MS measurements, Bob McAllister and Pauline Bandeen for microanalyses, Dr. Russell Clayton and Glenn Fenton for HPLC measurements, Martin Ryan for crystal micrographs, Dr. Peter Kelly for suggesting the recrystallization method for 1·HBr, and Jennifer Mason for proof reading the manuscript.

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ABBREVIATIONS USED Mo, molybdenum; MoCo, molybdenum cofactor; cPMP, cyclic pyranopterin monophosphate; GTP, guanosine triphosphate; MPT, molybdopterin; mp, melting point; SO, sulfite oxidase; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; DQF-COSY, double quantum filtered-correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; DTPA, diethylenetriaminepentaacetic acid; RT, room temperature

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REFERENCES

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Synthesis of Cyclic Pyranopterin Monophosphate, a Biosynthetic

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