A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases

A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.     

Total synthesis of a cyclic adenosine 5'-diphosphate ribose receptor agonist

Stable cyclic adenosine 5'-diphosphate ribose (cADPR) analogues are chemical biology tools that can probe the Ca(2+) release mechanism and structure-activity relationships of this emerging potent second messenger. However, analogues with an intact "northern" ribose have been inaccessible due to the difficulty of generating the sensitive N1-ribosyl link. We report the first total synthesis of the membrane permeant, hydrolytically stable, cADPR receptor agonist 8-Br-N1-cIDPR via regio- and stereoselective N1-ribosylation of protected 8-bromoinosine.     

Solid-phase synthesis of a new diphosphate 5-aminoimidazole-4-carboxamide riboside (AICAR) derivative and studies toward cyclic AICAR diphosphate ribose

The solid-phase synthesis of the first example of a new diphosphate AICAR derivative is reported. The new substance is characterized by the presence of a 5'-phosphate group while a second phosphate moiety is installed on a 5-hydroxypentyl chain attached to the 4-N-position of AICAR. Cyclization of the diphosphate derivative by pyrophosphate bond formation allowed for the formation of a novel AICAR-based cyclic ADP-ribose (cADPR) mimic.     

Unexpected relative aqueous solubilities of a phosphotyrosine analogue and two phosphonate derivatives

Phosphotyrosine (pTyr) is an essential component of biological signaling, often being a determinant of protein-protein interactions. Accordingly, a number of drug discovery efforts targeting signal transduction pathways have included phosphotyrosine and analogues as essential components of the lead compounds. Toward the goal of improved biological efficacy, the phosphonate and difluoro phosphonate analogues of pTyr have been employed in inhibitor design because of their stability to hydrolysis and enhanced binding affinity in certain cases. To quantitate the contribution of aqueous solubility of pTyr, phosphonomethyl phenylalanine (Pmp), and difluorophosphonomethyl phenylalanine (F(2)Pmp) to their relative binding affinities, free energy perturbation calculations were undertaken on the mimetics phenol phosphate (PP), benzyl phosphonate (BP), and difluorobenzyl phosphonate (F(2)BP), including development of empirical force field parameters compatible with the CHARMM all-atom force fields. Notably, it is shown that the most favorably solvated compound of the series is BP, followed by PP, with F(2)BP the least favorably solvated for both the mono- and dianionic forms of the compounds. The molecular origin of this ordering is shown to be due to changes in charge distribution, in the comparatively larger size of the fluorine atoms, as well as in differences of local solvation between PP and BP. The implications of the differences in aqueous solubility toward the relative binding potencies of pTyr-, Pmp-, and F(2)Pmp-containing peptide ligands are discussed. Our results indicate that one general principle explaining the efficacy of selective fluorination to enhance binding affinities may lie in the ability of fluorine atoms to increase the hydrophobicity of a ligand while maintaining its capability to form hydrogen bonds.     

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.     

Rapid synthetic route toward structurally modified derivatives of cyclic adenosine 5'-diphosphate ribose

A concise synthesis of five new analogues of the second messenger cADPR (cyclic adenosine 5'-diphosphate ribose) is presented. The synthetic plan centered around the key derivative 8-Br-N1-cIDPR (cyclic 8-Br-inosine 5'-diphosphate ribose, 2), which was prepared in only three steps from IMP (inosine 5'-monophosphate) via an unusual enzymatic cyclization reaction. The enhanced stability of 2 allowed for the direct modification of this cyclic dinucleotide at the 8 position, providing the unsubstituted parent N1-cIDPR (4) as well as the 8-phenyl (5), 8-azido (6), and 8-amino (7) N1-cIDPR analogues. In Jurkat T-lymphocytes, N1-cIDPR 4 induced Ca2+ release with an almost identical profile as the natural agonist cADPR, illustrating the value of this approach.     

Wittig-horner reaction of a phosphonate of reissert analogue a new method for introduction of alkyl groups into isoquinoline

Dimethyl 2-isopropoxycarbonyl-1, 2-dihydroisoquinoline-1-phosphonate ($\text{1}$) was prepared from isoquinoline, isopropyl chloroformate, and trimethyl phosphite. Wittig-Horner reaction of $\text{1}$ with various aldehydes afforded the corresponding exo-methylene compounds, which were converted to 1-substituted isoquinolines with hydrogen chloride.     

Simultaneous determination of phosphonate, phosphate, and diphosphate by capillary electrophoresis using in-capillary complexation with Mo(VI)

This study describes the simultaneous determination of phosphonate, phosphate, and diphosphate by CE with direct UV detection, based on in-capillary complexation with Mo(VI). When a mixture of phosphonate, phosphate, and diphosphate was injected into a capillary containing 3.0 mM Mo(VI), 0.05 M malonate buffer (pH 3.0) and 45% v/v CH3CN, three well-defined peaks, due to the migration of the corresponding polyoxomolybdate anions, were separated. The respective calibration graphs were linear in the concentration range of 2 x 10(-6)-2 x 10(-4) M for phosphonate, 1 x 10(-6)-5 x 10(-5) M for phosphate, and 1 x 10(-6)-2 x 10(-4) M for diphosphate; the correlation coefficients were better than 0.9990. The present CE method is successfully applied to the simultaneous determination of phosphonate, phosphate, and diphosphate in tap water.     

The Chemistry of Some Isopolar Analogues of Phosphoric and Pyrophosphoric Acids

Abstract

We have previously established that the α-fluorination of alkanephosphonates provides analogues of phosphate esters which have improved ‘isopolarity’ relative to simple alkanephosphonates.¹ This property is manifest, inter alia, in enhanced acidity and in the upfield shift for the ³¹P n.m.r. resonance. Indeed, for a range of halomethanephosphonic acids we have found the relationship “δ_P=9.61 (pK_a2 - 4.59) ppm” gives an excellent correlation between these parameters. In this context, the properties of CF₂CIPO(OR)₂ species, derived from the Michaelis-Becker reaction of dialkyl phosphonates with Freon 22, CF₂Cl₂, will be described.     

Synthesis of cyclic adenosine 5'-diphosphate ribose analogues: a C2'endo/syn "southern" ribose conformation underlies activity at the sea urchin cADPR receptor

Novel 8-substituted base and sugar-modified analogues of the Ca(2+) mobilizing second messenger cyclic adenosine 5'-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes; conformational analysis investigated by (1)H NMR spectroscopy revealed that a C2'endo/syn conformation of the "southern" ribose is crucial for agonist or antagonist activity at the SUH-, but not at the T cell-cADPR receptor.     

Fe(III)-ion-catalysed non-enzymatic transformation of adenosine diphosphate into adenosine triphosphate part II. Evidence of catalytic nature of Fe ions

Non-enzymatic phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) in aqueous solutions using acetyl phosphate has been studied extensively. The reaction proceeds in the presence of Fe(III) ions. Normally, the yield of ATP never exceeds the amount of Fe ions but, when a sufficient amount of Mg(II) ions is added to the reaction system, the yield of ATP increases beyond the amount of Fe ions. This is because the Fe(III) ion is tightly bound to not only ADP but also ATP, the phosphorylation product. When, however, the Fe(III) ion is liberated from ATP by exchanging with an Mg(II) ion, it regains catalytic activity and contributes to the surplus production of ATP. The Fe(II) ion with either a single molecule of 1,10-phenanthroline or 2,2′-bipyridyl exhibits a remarkably high catalytic activity. This suggests that the Fe(II) aquo ion should also be an excellent catalyst.     

Chemoenzymatic synthesis of 7-deaza cyclic adenosine 5'-diphosphate ribose analogues, membrane-permeant modulators of intracellular calcium release

An optimized synthetic route to 7-deaza-8-bromo-cyclic adenosine 5'-diphosphate ribose (7-deaza-8-bromo-cADPR 3), an established cell-permeant, hydrolysis-resistant cyclic adenosine 5'-diphosphate ribose (cADPR) antagonist, is presented. Using NMR analysis, we found that 3 adopted a C-2' endo conformation in the N9-linked ribose and a syn conformation about the N9-glycosyl linkage, which are similar to that of cADPR. The synthetic route was also employed to produce 7-deaza-2'-deoxy-cADPR 4, a potential cell-permeant cADPR analogue. 3 and 4 were more stable to chemical hydrolysis, consistent with the observation that 7-deaza-cADPR analogues are more stable than their parent adenosine derivatives. 3 was also found to be stable to enzyme-mediated hydrolysis using CD38 ectoenzyme.     

氟芳胺基、氟磺酰胺基磷酸酯的合成

首次合成了氟芳胺基磷酸酯和氟磺酰胺基磷酸酯.对氟苯胺、α-氯-4-三氟甲基吡啶胺、α-氯-4-三氟甲基氧化吡啶胺与二乙氧基磷酰氯在乙腈或DMF中反应,得到氟芳胺基磷酸酯.氟烷基磺酰甲胺与二乙氧基磷酰氯反应,以金属钠处理,相应钠盐在乙腈或DMF中,于室温下反应将得到氟烷磺酰胺基磷酸酯.     

Synchrotron radiation circular dichroism spectroscopy of ribose and deoxyribose sugars, adenosine, AMP and dAMP nucleotides.

Synchrotron radiation circular dichroism (SRCD) spectra of ribose and deoxyribose sugars, adenosine, AMP and dAMP nucleotides and cyclic derivatives were measured in the vacuum ultraviolet region (down to 168 nm for sugars and 175 nm for adenine derivatives) and at different pH values (3, 6-7, 9-10) and temperatures (between 5 and 45 degrees C). The information content in the VUV region is important since the CD bands strongly depend on the chemical structure of the sugar, the presence and orientation of a phosphate group and the protonation state of adenine. On the other hand, single or double deprotonation of the phosphoric acid group has no influence on the spectra. We assign the vacuum ultraviolet (VUV) CD bands of the nucleoside and nucleotides to be due mainly to n-->pi* transitions in the adenine nucleobase based on a comparison with the absorption spectra. The CD bands of the sugars are due to n(O -->sigma*) transitions and are much smaller than the CD signal from the nucleotides in the VUV region. Bands are assigned to both pyranose and open-chain forms.     

Unusual entry to the novel 8-halo-N1-ribosyl hypoxanthine system by degradation of a cyclic adenosine-5'-diphosphate ribose analogue

Cyclic 8-bromo-inosine-5'-diphosphate ribose (8-Br-N1-cIDPR) was cleanly degraded at acidic pH by N9 ribosyl scission and subsequent pyrophosphate cleavage to give 8-bromo-N1-ribosyl hypoxanthine 5'-monophosphate (8-Br-N1-IMP), a novel class of mononucleotide, as the sole product.     

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.     

Metal-ion-coordinating properties of various phosphonate derivatives,including 9−[2−(phosphonylmethoxy)ethyl]adenine (PMEA) – an adenosine monophosphate (AMP) analogue with antiviral properties

The stability constants of the 1:1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, (in part) Zn²⁺, or Cd²⁺ and (phosphonylmethoxy)ethane (PME^2?) or 9?[2?(phosphonylmethoxy)ethyl]adenine (PMEA^2?) were determined by potentiometric pH titration in aqueous solution (I = 0.1M , NaNO₃; 25°). The experimental conditions were carefully selected such that self-association of the adenine derivative PMEA and of its complexes was negligibly small; i.e., it was made certain that the properties of the monomeric [M(PMEA)] complexes were studied. Recent measurements with simple phosphate monoesters, R–MP^2– (where R is a non-coordinating residue; S. S. Massoud, H. Sigel, Inorg. Chem. 1988 , 27, 1447), were used to show that analogously simple phosphonates (R? PO) – we studied now the complexes of methyl phosphonate and ethyl phosphonate – fit on the same log K/logK vs. pK/ pK straight-line plots. With these reference lines, it could be demonstrated that for all the [M(PME)] complexes with the mentioned metal ions an increased complex stability is measured; i.e., a stability higher than that expected for a sole phosphonate coordination of the metal ion. This increased stability is attributed to the formation of five-membered chelates involving the ether oxygen present in the ? O? CH₂? PO residue of PME^2? (and PMEA^2?); the formation degree of the five-membered [M(PME)] chelates varies between ca. 15 and 40% for the alkaline earth ions and ca. 35 to 65% for 3d ions and Zn²⁺ or Cd²⁺. Interestingly, for the [M(PMEA)] complexes within the error limits exactly the same observations are made indicating that the same five-membered chelates are formed, and that the adenine residue has no influence on the stability of these complexes, with the exception of those with Ni²⁺ and Cu²⁺. For these two metal ions, an additional stability increase is observed which has to be attributed to a metal ion-adenine interaction giving thus rise to equilibria between three different [M(PMEA)] isomers. These equilibria are analyzed, and for [Cu(PMEA)] it is calculated that 17(±3)% exist as an isomer with a sole Cu²⁺-phosphonate coordination, 34(±10)% form the mentioned five-membered chelate involving the ether oxygen, and the remaining 49(±10)% are due to an isomer containing also a Cu²⁺-adenine interaction. Based on various arguments, it is suggested that this latter isomer contains two chelate rings which result from a metal-ion coordination to the phosphonate group, the ether oxygen, and to N(3) of the adenine residue. For [Ni(PMEA)], the isomer with a Ni²⁺-adenine interaction is formed to only 22(±13)%; for [Cd(PMEA)] and the other [M(PMEA)] complexes if at all, only traces of such an isomer are occurring. In addition, the [M(PMEA)] complexes may be protonated leading to [M(H·PMEA)]⁺ species in which the proton is mainly at the phosphonate group, while the metal ion is bound in an adenosine-type fashion to the nucleic base residue. Finally, the properties of [M(PMEA)] and [M(AMP)] complexes are compared, and in this connection it should be emphasized that the ether oxygen which influences so much the stability and structure of the [M(PMEA)] complexes in solution is also crucial for the antiviral properties of PMEA.