Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy

Summary In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.This work was taken in part from the Ph.D. Theses of E.A. Ottinger (1994) and Q. Xu (1996), University of Minnesota, Minneapolis, MN, U.S.A. Preliminary presentations of portions of this work were made at the Twenty-Second European Peptide Symposium, Interlaken, Switzerland, September 13–19, 1992, see Ref. 1, at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995, and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995, see Ref. 2. The title side reaction was first discussed for tyrosine (see Refs 1 and 3), but all of the mechanism studies discussed herein are for serine and threonine.Amino acid symbols denote the l-configuration, and abbreviations for amino acids and peptides follow rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977].     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.Abbreviations -PP _s1-casein 5P (f59–79) - -PP -casein 4P (f1–25) - ACP amorphous calcium phosphate - Cit citrate - CPN calcium phosphate nanocluster - CPP commercial phosphopeptide - IAP ion activity product - MWCO molecular weight cut-off - PP phosphopeptide - SAXS small-angle X-ray scattering - SCPP secretory calcium-binding phosphoprotein - UF ultrafiltrate     

人脑利钠肽的Fmoc固相合成

探讨生物活性肽人脑利钠肽(BNP)的固相合成工艺,并为工业化合成提供理论依据.本文以二氯三甲基树脂(以下简称为二氯树脂)为载体,采用9-芴甲氧羰基(Fmoc)保护的氨基酸,以1-氧-3-双二甲胺羰基苯骈三氮唑四氟化硼盐(TBTU)/1-羟基苯并三氮唑(HOBT)/二异丙基乙胺(DIEA)缩合,以碘作为环化试剂,用切割试剂将BNP粗品从树脂上切割下来.通过MALDI-MS质谱仪检测,所合成环肽的分子量与理论分子量一致,使用RP-HPLC液相色谱仪对合成的环肽进行纯化,得到的BNP纯度达到97%以上.本合成工艺具有快捷、简便、高效的特点,适合于大批量的生产目的肽.     

Synthesis of the C1-phosphonate analog of UDP-GlcNAc

We describe the first synthesis of the C1-phosphonate analog of UDP-GlcNAc, based on a new preparation of the corresponding glycosyl phosphonate. This C-glycosyl analog is shown to be a very weak inhibitor (Ki>10 mM) of fungal chitin synthase, indicating that at least in this case the replacement of the anomeric oxygen with a methylene group is not an innocent substitution.     

Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of clofarabine

The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)adenine] with 1,2- and 1,3-diacylglycerophosphates have been prepared by the phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against HL-60, A-549, MCF-7, and HeLa tumor cell lines.     

Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology

a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS-MS analysis and NMR; the structures of the analogues were confirmed by MS-MS analysis. Using a yeast growth arrest assay, the analogues were found to have activity comparable to a-factor itself.     

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V(max) with no alteration in the apparent K(m) for GDP-Manose. Phosphorylation with [gamma-(32)P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation-dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.     

GC-FPD measurement of urinary dialkylphosphate metabolites of organophosphorous pesticides as pentafluorobenzyl derivatives in occupationally exposed workers and in a general population in Shanghai (China)

Measurement of organophosphorus (OP) pesticide metabolites in human biological fluids is an important biomarker of pesticides exposure. We measured the urinary excretion of OP pesticide metabolites to evaluate occupational and non-occupational exposure to OP pesticides in the Chinese population in Shanghai (Eastern China). We collected urine samples from 30 exposed workers in a dimethoate emulsion packing division and from 60 healthy adults without any report of occupational exposure. DMP, DMTP, DMDTP, DEP, DEDP and DEDTP were measured by GC-FPD after derivatization with pentafluorobenzyl bromide. The LOQ values (1 mL urine) were 2.0 μg/L for DMP and DETP, 4.0 μg/L for DEP and DEDTP, 8.0 μg/L for DMDTP, and 10.0 μg/L for DMTP. Dimethyl phosphates were detected in the majority of the urine samples, i.e., 90–100% in the exposed group and 80–87% in the control group. The concentration of the urinary diethyl phosphates DEP and DETP was above the LOQ values in 40 and 20% of samples for the exposed group, and in 50 and 30% of the samples for the control group, respectively. DEDTP was not detectable in the urine samples except for a post-shift exposed worker (detection frequency, 6.7%). Median creatinine-adjusted values (μg/g cr.) for DAP in Chinese with pre-shift, post-shift and without occupational exposure to OP were 316, 584 and 170 for DMP, below LOQ, 115 and 114 for DEP, 840, 1730 and 693 for DMTP, and 255, 756 and 135 for DMDTP, respectively. In all subjects, the highest excretion levels were found for DMTP. Several OP pesticide metabolites were frequently detected in urine samples of both populations studied.     

Localization of the Phosphorylation Sites for Different Kinases in the Microtubule-Associated Protein MAP2

The phosphorylation of microtubule-associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain-specificity protease, a result supporting previous notions of the two-domain structure of MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N-chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 in a 42-kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP2.     

Synthesis of a core disaccharide from the Streptococcuspneumoniae type 23F capsular polysaccharide antigen

The synthesis of methyl α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranoside and methyl α-l-rhamnopyranosyl-(1→2)-3-(glycer-2-yl-phosphate)-β-d-galactopyranoside disaccharides from the Streptococcuspneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was followed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonate and phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazine hydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphate migration during the deprotection of the phosphodiester functionalized disaccharide.     

大肠杆菌膦酸酯代谢途径中phnF基因的研究

大肠杆菌的ｐｈｎ操纵子与膦酸酯（Ｐｎ）的利用密切相关。实验利用ＰＣＲ扩增、ＴＡ克隆等方法,获得了大肠杆菌ｐｈｎ操纵子中ｐｈｎＥ、ｐｈｎＦ和ｐｈｎＧ基因的亚克隆,并进行了序列测定。通过Ｐ１噬菌体转导,构建了ｐｈｎＦ的ＴｎｐｈｏＡ’９转座子插入突变体ＪＷ１９,该突变仅对大肠杆菌的ＡＥＰｎ同化有微弱的影响,而利用ｐｈｎＥ和ｐｈｎＧ序列与染色体重组构建的ｐｈｎＦ全缺失突变株ＪＷ６７,则几乎不能在ＡＥＰｎ培养基上生长。通过ｐｈｎＦ基因的诱导高表达,用亲和柱层析分离纯化了ＰｈｎＦ蛋白,达到电泳纯。并且用凝胶延滞的方法观察到,ＰｈｎＦ蛋白与ｐｈｎ操纵子ＤＮＡ片段相互作用后,可使凝胶图谱类型发生改变。     

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by ³¹P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.     

PKC-Dependent Phosphorylation May Regulate the Ability of Connexin43 to Inhibit DNA Synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCε). Here we report that activation of PKCε is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

PKC-Dependent Phosphorylation May Regulate the Ability of Connexin43 to Inhibit DNA Synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCε). Here we report that activation of PKCε is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

Regulation of eukaryotic initiation factor 4E phosphorylation in the nervous system of Aplysia californica

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.     

Nucleoside Diphosphate Kinase and the Activation of Antiviral Phosphonate Analogs of Nucleotides: Binding Mode and Phosphorylation of Tenofovir Derivatives

Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creatine kinase is significantly more active. The 1.65 Å x-ray structure of NDPK in complex with tenofovir mono- and diphosphate shows that the analogs bind at the same site as natural nucleotides, but in a different conformation, and make only a subset of the Van der Waals and polar interactions made by natural substrates, consistent with their comparatively low affinity for the enzyme.     

Synthesis of hydrazinopeptides using solid-phase N-electrophilic amination: extension to the Fmoc/tert-butyl strategy and chemistry of the N-N bond in strong acid media.

The synthesis of hydrazinopeptides using solid-phase N-electrophilic amination was extended to the Fmoc/tert-butyl strategy. Both Boc/benzyl and Fmoc/tert-butyl strategies led to the isolation of by-products arising from the partial instability of the N-N bond during the final cleavage and deprotection step. Two paths of decomposition have been shown: the cleavage of the N-N bond leading to the regeneration of the amine and a Hofmann-type elimination yielding original dianisyl adducts. Our data suggest that the Fmoc/tert-butyl strategy is better suited for the synthesis of hydrazinopeptides.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

Synthesis, kinetic evaluation, and utilization of a biotinylated dipeptide proline diphenyl phosphonate for the disclosure of dipeptidyl peptidase IV-like serine proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.     

Noradrenaline‐ and Melatonin‐Mediated Regulation of Pigment Aggregation in Fish Melanophores

The effects of melatonin and noradrenaline (NA) on bi‐directional melanosome transport were analysed in primary cultures of melanophores from the Atlantic cod. Both agents mediated rapid melanosome aggregation, and by using receptor antagonists, melatonin was found to bind to a melatonin receptor whereas NA binds to an α2‐adrenoceptor. It has previously been stated that melatonin‐mediated melanosome aggregation in Xenopus is coupled with tyrosine phosphorylation of a so far unidentified high molecular weight protein and we show that although acting through different receptors and through somewhat different downstream signalling events, tyrosine phosphorylation is of the utmost importance for melanosome aggregation mediated by both NA and melatonin in cod melanophores. Together with cyclic adenosine 3‐phosphate‐fluctuations, tyrosine phosphorylation functions as a switch signal for melanosome aggregation and dispersion in these cells.