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Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrubEuphorbia characias(ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.

This paper briefly reviews the genetic and genomic studies of OPLL in Japan, including linkage, association and model mouse studies.

A new approach to arylated 2-trifluoromethylquinolines based on novel regioselective Suzuki-Miyaura coupling reactions has been developed. Moreover, site-selective, chemo-selective amination reactions were performed. The new 2-trifluoromethylquinoline derivatives were tested as potential NPPs inhibitors and evaluated for their potential to inhibit two families of ecto-nucleotidases, i.e. NPPs and nucleoside triphosphate diphosphohydrolases (NTPDases). Several derivatives were active on a nanomolecular concentration. The results were validated based on docking studies to study the active binding site of the molecules.

A two-step palladium-catalyzed procedure based on Suzuki–Miyaura cross coupling, followed by a double Buchwald–Hartwig reaction, allows for the synthesis of pharmaceutically relevant benzo[4,5]furo[3,2-b]indoles in moderate to very good yield. The synthesized compounds have been analyzed with regard to their inhibitory activity (IC50) of nucleotide pyrophosphatases h-NPP1 and h-NPP3. The activity lies in the nanomolar range. The results were rationalized based on docking studies.

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methylCpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50 kDa protein, i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Over expression of nucleotide pyrophosphatase (NPPs) activity is associated with chondrocalcinosis, osteoarthritis, type 2 diabetes, neurodegenerative diseases, allergies and cancer metastasis. The potential of NPPs inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP inhibitors. Specifically, 2-bromo-7-methyl-5-oxo-5H-1,3,4-thiadiazolopyrimidine and its corresponding 6-fluoro derivatives were synthesized via a Suzuki–Miyaura reaction. The cross-coupling reaction with different arylboronic acids gave desired coupling products in good to excellent yields and showed wide functional group tolerance. Furthermore, all compounds were investigated for their potential to inhibit two families of ecto-nucleotidases, i.e. nucleoside triphosphate diphosphohydrolases (NTPDase) and NPPs. Interestingly, our compounds were identified as selective inhibitors of NPPs. Among derivatives 5a–5i, compound 5i (IC50 ± SEM = 0.39 ± 0.01 μM) was found to be the most potent inhibitor of h-NPP1 and compound 5h (IC50 ± SEM = 1.02 ± 0.05 μM) was found to be the most potent inhibitor of h-NPP3. Similarly, for fluorinated thiadiazolopyrimidones, derivative 6e (IC50 ± SEM = 0.31 ± 0.01 μM) exhibited the best inhibition of NPP1 and it was found that this compound exhibited ≈28 fold improvement in inhibitory potential as compared with the reference control i.e. Suramin (IC50 ± SEM = 8.67 ± 1.3 μM). Moreover, homology modelling and molecular docking studies of both inhibitors were carried out to suggest the putative binding mode of inhibitors with the respective enzyme i.e. h-NPP1 and h-NPP3.

Various N-fused isoquinoline derivatives were synthesized using a new one-pot reaction of 1-bromo-2-(2,2-difluorovinyl)benzenes with N–H group containing heterocycles followed by intramolecular palladium-catalyzed C–H arylation. The method described gives convenient access to diverse structures of N-fused polycyclic isoquinolines. Sixteen of the synthesized compounds were screened as potential human nucleotide pyrophosphatase/phosphodiesterase 1 and 3 (h-NPP-1 and h-NPP-3) inhibitors. The most effective h-NPP-1 inhibitor showed an IC50 value as high as 0.36 ± 0.06 μM, whereas the most potent h-NPP-3 inhibitor posessed an inhibitory value of 0.48 ± 0.01 μM. Kinetic and molecular docking studies of both most effective inhibitors were carried out.

NPP3 inhibitors are promising therapeutic agents due to their potential as anti-cancer, anti-metastatic and anti-neurodegenerative drugs. We have identified the first potent and selective inhibitors of human NPP3. We have also estimated the biochemical properties of the main NPP family members that can hydrolyse nucleotides using p-nitrophenyl-5'-thymidine monophosphate as substrate. Km values were found to be 20 ± 4 and 15 ± 6 � M for human NPP1 and NPP3, respectively. Maximum velocity (Vmax) values calculated for NPP1 and NPP3 were 12 ± 2 and 5 ± 1 nmol p-nitrophenol released/min/mg of crude protein, respectively. A series of benzothiazole derivatives and a series of 1,3,4-oxadiazole-2-thiones was tested on hNPP1 and hNPP3. Benzothiazoles were the most potent non competitive inhibi- tors of hNPP3 described to date. 1,3,4-oxadiazole-2-thiones were also identified as compounds which inhibited specifi- cally NPP3 over NPP1. The most potent compound was further characterized and found to exhibit a non competitive mechanism of inhibition.

We propose the name nucleotide pyrophosphatases/phosphodiesterases (NPP) for the enzymes that release nucleoside-5'-monophosphates from various pyrophosphate and phosphodiester bonds. Three structurally related mammalian NPPs are known, i.e. NPPalpha (autotaxin), NPPbeta (B10/gp130RB13-6) and NPPgamma (PC-1). We report here that these isozymes have a distinct tissue distribution in the rat but that they are all three expressed in hepatocytes. In FAO rat hepatoma cells only the level of NPPgamma was stimulated by TGF-beta1. In rat liver, the concentration of the transcripts of all three isozymes was found to increase manyfold during the first weeks after birth, but the increased expression of the NPPalpha mRNA was transient. The level of the NPP transcripts transiently decreased after hepatectomy, but NPPalpha mRNA was also lost after sham operation, which suggests that it may belong to the negative acute-phase proteins. The loss of the beta- and gamma-transcripts after hepatectomy was not due to a decreased NPP gene transcription or an increased turnover of the mature transcripts. However, hepatectomy also caused a similar loss of the nuclear pool of the NPPbeta and NPPgamma mRNAs. We conclude that a deficient processing and/or an increased turnover of the NPP pre-mRNAs underlies the hepatectomy-induced decrease of the beta- and gamma-transcripts. A similar loss of nuclear NPPgamma mRNA was also noted after treatment with cycloheximide, indicating that protein(s) with a high turnover control the stability and/or processing of the immature NPPgamma transcript.

A hydrolase activity directed against diadenosine 5′,5″′-P1,P4-tetraphosphate (Ap4A) has been solubilised and partially purified from the plasma membrane fraction of bovine adrenal medullary chromaffin tissue in order to determine its relationship to alkaline phosphodiesterase-I/nucleotide pyrophosphatase (PDase-I, EC 3.1.4.1). Activity with the specific dinucleoside tetraphosphatase (EC 3.6.1.17) substrate Ap4A and with the non-specific PDase-I substrate thymidine 5′-monophosphate p-nitrophenyl ester had Km and Vmax values of 2.0 μM and 600 pmol/min/mg protein and 0.2 mM and 26 nmol/min/mg protein respectively and co-chromatographed upon gel filtration and ion-exchange chromatography. Activity with the fluorescent substrates etheno-Ap4A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap4A. Immunoblotting of the most purified fraction with an antibody against mouse PC-1, one of the major PDase-I family members, detected bands of 240, 120 and 62 kDa corresponding to PC-1 dimer, monomer and proteolytic fragment. Therefore, the activity previously described as bovine adrenal chromaffin cell ecto(diadenosine polyphosphate hydrolase) (ecto-ApnAase) is a PDase-I, probably bovine PC-1.

The ectonucleoside pyrophosphatase phosphodiesterase 1 (NPP1/PC-1) is a member of the NPP enzyme family that is critical in regulating mineralization. In certain mineralizing sites of bone and cartilage, membrane-limited vesicles [matrix vesicles (MVs)] provide a sheltered internal environment for nucleation of calcium-containing crystals, including hydroxyapatite. MV formation occurs by budding of vesicles from the plasma membrane of mineralizing cells. The MVs are enriched in proteins that promote mineralization. Paradoxically, NPP1, the type II transmembrane protein that generates the potent hydroxyapatite crystal growth inhibitor inorganic pyrophosphate (PPi), is also enriched in MVs. Although osteoblasts express NPP1, NPP2, and NPP3, only NPP1 is enriched in MVs. Therefore, this study uses mineralizing human osteoblastic SaOS-2 cells, a panel of NPP1 mutants, and NPP1 chimeras with NPP3, which does not concentrate in MVs, to investigate how NPP1 preferentially targets to MVs. We demonstrated that a cytosolic dileucine motif (amino acids 49–50) was critical in localizing NPP1 to regions of the plasma membrane that budded off into MVs. Moreover, transposition of the NPP1 cytoplasmic dileucine motif and flanking region (AAASLLAP) to NPP3 conferred to NPP3 the ability to target to the plasma membrane and, subsequently, concentrate in MVs. Functionally, the cytosolic tail dileucine motif NPP1 mutants lost the ability to support MV PPiconcentrations and to suppress calcification. The results identify a specific targeting motif in the NPP1 cytosolic tail that delivers PPi-generating NPP activity to osteoblast MVs for control of calcification.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized.E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-β and glucocorticoids, but the signal transduction pathways that control expression are poorly documented.NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes.In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5′ phosphodiesterase/nucleotide-pyrophosphatase (5′-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+-stimulated 5′-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [α-32P]ATP or [γ-32P]ATP, respectively, in the absence of any permeabilizing agent., This work was financed by grants to AV from the Comisión Interministerial de Ciencia y Tecnología (SAF99-0052), the Consejería de Educación y Cultura de la Comunidad de Madrid (08.5/0019/1997), and the Agencia Espanola de Cooperación Internacional (99CN0011).

The endogenous ADP-ribosylation of cytosolic proteins and the pattern of NAD degradation were analyzed in subcellular fractions of rat liver in order to investigate the modulation of these reactions by GTP-binding (G) proteins.

A simple and convenient procedure is described for the continuous fluorimetric assay of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) activity. It is based on the multifold increase in fluorescence emission intensity accompanying hydrolysis of a new fluorogenic substrate, ϵAp 2 ϵA (where ϵ = 1, N 6 -etheno), or of commercially available ϵNAD + and FAD, using potato nucleotide pyrophosphatase. The procedure is applicable to enzyme activity in tissue extracts, as well as to kinetic studies and screening of potential inhibitors. K m and V max / K m for the substrates, and K i values for various compounds, were evaluated. The most effective inhibitor of ϵAp 2 ϵA hydrolysis was the alternative substrate 8-bromo-NAD + ( K i = 7 μ M). The method is also useful for continuous assay of snake venom phosphodiesterase I-5′-exo-nuclease (EC 3.1.4.1). The preparation of ϵAp 2 ϵA is described, and spectral properties of the fluorogenic substrates listed. Both ϵAp 3 ϵA and ϵAp 4 ϵA were also shown to be fluorogenic substrates for nucleotide pyrophosphatase and phosphodiesterase I, and may be used for continuous fluorimetric assay of specific dinucleoside oligophosphatases.

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient [Fukui, S. et al . (1981) J. Biol. Chem. 256, 10313–10318] was found to be caused by elevated degradation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The enzyme involved in this degradation was then identified as an enzyme of nucleotide pyrophosphatase (EC 3.6.1.9) nature, cleaving the phosphosulfate linkage. The specific activities were 8 – 24 (mU/mg protein) in patients' fibroblasts, in contrast to 3 in normal and 5 – 14 in heterozygote cells. A possibility is discussed that the elevation of nucleotide pyrophosphatase activity is the primary genetic defect in Lowe's syndrome.

Nucleotide pyrophosphatase activity with uridine diphosphoglucose and dephospho-CoA as substrates was demonstrated in normal human serum. The enzyme has a pH-optimum of about 9.6 and is inhibited by EDTA. Phosphodiesterase I (hydrolysis of thymidine-5'-monophospho-p-nitrophenylester) was also found in normal human serum, with a pH-optimum of about 9.8 and a Km of 0.20-0.25 mM. Probably both activities should be attributed to one enzyme. Different isoenzymes may exist, however. The activity of nucleotide pyrophosphatase/phosphodiesterase I in normal serum in many respects resembles an enzyme previously isolated from liver plasma membranes. Phosphodiesterase I activity was increased in normal pregnancy, in primary biliary cirrhosis, and in patients with bone lesions, but not in acute viral hepatitis or active chronic hepatitis. In primary biliary cirrhosis, the activity of phosphodiesterase I paralleled an increase of alkaline phosphatases.

1. 1. Epiphyseal cartilage was separated into resting, ossifying and new bone regions. 2. 2. An acid phosphatase with sharp peaks at pH 3·0 and 6·0 was found in the soluble cartilage preparation. 3. 3. Activities of alkaline phosphatase, nucleotide pyrophosphatase, 5-nucleotidase and glycerophosphatase activities were higher in ossifying cartilage and new bone than in resting cartilage. 4. 4. It is suggested that phosphatases in the ossifying cartilage and new bone may play a very active role in preparing tissue for calcification.

Some properties and kinetics of yeast nucleotide pyrophosphatase were studied in comparison with those of 5′-nucleotidase.It was concluded that the two enzyme activities exist in a single protein molecule, though their active sites are not completely identical.

UDP(2)-fructose was synthesized from d-fructofuranose-2-phosphate by the method of Khorana et al. The product thus obtained showed slightly higher paper chromatographic mobilities than those of UDP-glucose and UDP(1)-fructose, and a large negative optical rotation. In acid hydrolysis, this substance was quickly converted into UDP(1)-fructose and then the latter is hydrolyzed to UMP and fructose-1-phosphate. The rate constant of this first step is far larger than the acid hydrolysis rate constant of natural UDP-fructose isolated from the tubers of Jerusalem artichoke. When treated with the snake venom nucleotide pyrophosphatase, UDP(2)-fructose was splitted into UMP and a substance having the same paper chromatographic mobility as that of fructofuranose-2-phosphate. From these results and those reported previously, the structure of the synthetic product may be UDP(2)-β-d-fructofuranose. It was argued that the natural UDP-fructose may be UDP(2)-α-d-fructofuranose.

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture-grade acidic fibroblast growth factor (FGF-1) correlates with a low-Mr contaminant. 1H-NMR analyses revealed EDTA in the total-volume fractions of a gel-filtration experiment, where all the inhibitory activity of the FGF-1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF-1 or the EDTA-containing fractions) was time-dependent, blocked by the substrate p-nitrophenyl-dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF-1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Publisher Summary This chapter highlights various experiments conducted to study biochemical characteristics of enzymes. Severo Ochoa was embarked on the isolation and purification of enzymes of the citric acid cycle in January 1946. By coupling aconitase to isocitric dehydrogenase, spectrophotometric assays, based on TPN reduction, took only a few minutes. Many ideas could be tested and discarded in the course of a day. After failing in a few attempts to crystallize the enzyme, Arthur Kornberg rebelled at continuing. Enzymes of astonishing specificity and catalytic potency linked the combustion of foodstuffs to the generation of ATP that made the cell grow and the muscle move. Severo Ochoa was embarked on the isolation and purification of enzymes of the citric acid cycle when Arthur came to his laboratory in January 1946. He suggested that Arthur work on aconitase, the enzyme that catalyzes the equilibrium. The purification of nucleotide pyrophosphatase from potatoes furnished a means for degrading TPN and distinguishing the location of its third phosphate.

When peripheral lymphocytes stimulated with phytohemagglutinin were permeabilized in vitro, (3H)dCTP acted as a precursor for DNA synthesis, but the formation of a compound soluble in organic solvents could also be demonstrated. The structure of the latter compound was studied analyzing the products formed after alkaline hydrolysis or an enzymatic treatment with nucleotide pyrophosphatase. Both treatments led to the formation of (3H)dCMP. When stimulated lymphocytes were labeled in vivo with (14C)glycerol before permeabilization and ulterior labeling with (3H)dCTP a double labeled compound was obtained. When this compound was submitted to alkaline hydrolysis, (3H)dCMP. and (l4C)glycerol-3-phosphate were obtained. It was concluded that the compound soluble in organic solvents was phosphatidyl-dCMP.

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2′-phosphate or 6-amino group, or both: 2′-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2′-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2′-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2′-O[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl)carbamoylethyl]-NAD (VI). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3′-phosphate isomer (Vb). The mixture was converted to Va via the 2′,3′-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3′-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.

Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freeze-dried sections. Nucleotide pyrophosphatase activity, expressed in mumol X min-1 X mg protein-1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichment of the activity during the purification of brush border vesicles was measured. A ten-fold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50kDa protein, i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5.

136 NUCLEOTIDE PYROPHOSPHATASE ANTAGONIZES CONTRACTIONS DUE TO THE TWITCH COMPONENT OF THE SYMPATHETIC NERVE RESPONSE AND TO APPLIED ATP IN THE GUINEA-PIG VAS DEFERENS

CHEMICAL1 and enzymic2 studies from these two laboratories suggested that coenzyme A is best represented by formula (I) (cf. ref. 3). While the synthesis of various fragments of the molecule4 has lent considerable support to this structure, the enzymic and chemical evidence did not agree on one point. This concerned the nature of a substance obtained by the action of nucleotide pyrophosphatase on the coenzyme and which stimulated the growth of Acetobacter suboxydans5. Although not isolated in a chemically pure state, it was thought to be a simple phosphate of pantothenic acid6. None of the synthetic pantothenic acid phosphates showed activity towards this organism ; consequently, it was suggested1 that this ‘Acetobacter-stimulatory factor’ might be pantetheine-4′phosphate (II). An unambiguous synthesis of (II) is described here.