Deoxy Analogues Research Articles

LC/MS Analysis of Tetrodotoxin and Its Deoxy Analogs in the Marine Puffer Fish Fugu niphobles from the Southern Coast of Korea, and in the Brackishwater Puffer Fishes Tetraodon nigroviridis and Tetraodon biocellatus from Southeast Asia

Tetrodotoxin (TTX) and its deoxy analogs, 5-deoxyTTX, 11-deoxyTTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX, were quantified in the tissues of three female and three male specimens of the marine puffer fish, Fugu niphobles, from the southern coast of Korea, and in the whole body of the brackishwater puffer fishes, Tetraodon nigroviridis (12 specimens) and Tetrodon biocellatus (three specimens) from Southeast Asia using LC/MS in single ion mode (SIM). Identification of these four deoxy analogs in the ovarian tissue of F. niphobles were further confirmed by LC/MS/MS. TTX and 5,6,11-trideoxyTTX were detected in all three puffer fish species as the major TTX analogs, similar to Japanese Fugu pardalis. While 6,11-dideoxyTTX was also found to be a major analog in almost all tissues of Korean F. niphobles, this analog was minor in the two Tetraodon species and Japanese F. pardalis. Among the tissues of F. niphobles, the concentrations of TTXs were highest in the ovaries (female) and skin (female and male).

Use of Crystal MD Simulations to Speed Up Evaluation of Binding Free Energies of Dimannose Deoxy Analogs With M4-P51g-Cyanovirin-N

Molecular Dynamics (MD) presents an advanced tool for scoring of the binding free energies (ΔG) between a target protein and a set of candidate substrates, narrowed by extensive virtual screening process. Molecular mechanics(MM)/continuum model approach for evaluation of ΔG includes calculation of (i) a critically important solvation energy electrostatic contribution by means of solving the Poisson-Boltzmann (PB) or generalized Born (GB) equation and (ii) nonpolar component estimated from the solvent accessible area (SA) of solutes. Both, MM/PBSA and MM/GBSA, methods imply averaging of ΔG over a set of snapshots generated through, preferably, explicit solvent MD simulation which is the most time demanding step. In this work we demonstrated a possibility to reduce time of conformational sampling using crystal environment simulation. Amber10 program and FF99SB/GLYCAM06 force fields combination were employed for MD simulations and free energy calculations. Six 3, 4 and 3,4-deoxy dimannose analogs were studied as ligands of the m4-P51G-CVN mutant of the potent anti-HIV carbohydrate binding agent Cyanovirin-N (1). The use of crystal over solution simulations results in at least 8 times faster generation of the equivalent length trajectories. Binding free energy ΔA estimated from crystal NVT simulation trajectories shows 0.93 and 0.94 correlation with ΔG from solution NPT simulations for MM/PBSA and MM/GBSA approaches, respectively. We also evaluated performance of the relatively new GLYCAM06 carbohydrate force field and found reasonable agreement between calculated ΔG and experimental value. Results of this study further support our earlier hypothesis about importance (for CVN specificity) of the eight-component H-bond interactions of dimannose and protein main chain atoms that also was recently observed in experiment. [1] Vorontsov and Miyashita (2009) Biophys. J., 97.

Inhibition of Neisseria meningitidis Sialic Acid Synthase by a Tetrahedral Intermediate Analogue,

The Neisseria meningitidis sialic acid synthase (NeuB) catalyzes the metal-dependent condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to generate N-acetylneuraminic acid (NeuAc or sialic acid). N. meningitidis is a causative agent of meningitis and produces a capsular polysaccharide comprised of polysialic acid. This allows it to evade the immune system of the host by an act of molecular mimicry. This work describes the synthesis and characterization of the first potent inhibitor of sialic acid synthase. The inhibitor is a stable deoxy analogue of the tetrahedral intermediate presumed to form in the NeuB reaction and was synthesized as a mixture of stereoisomers at the key tetrahedral center. Inhibition studies demonstrate that one stereoisomer binds more tightly than the other and that the more potent isomer binds with micromolar affinity. An X-ray crystallographic analysis of the NeuB.inhibitor.Mn(2+) complex solved to a resolution of 1.75 A shows that the more tightly bound stereoisomer bears a (2R)-configuration. This suggests that the tetrahedral intermediate formed in the NeuB reaction also bears a (2R)-configuration. This analysis is consistent with a mechanism whereby the active site metal plays at least two roles during catalysis. First, it serves as an electrostatic catalyst and activates the aldehyde of ManNAc for attack by the alkene of PEP. Second, it serves as a source of nucleophilic water and delivers it to the si face of the oxocarbenium intermediate to generate a tetrahedral intermediate with a (2R)-configuration.

Synthesis and Biological Evaluation of 3,3-Difluoropyridine-2,4(1H,3H)-dione and 3-Deaza-3-fluorouracil Base and Nucleoside Derivatives

New 3-deaza-3-halouracil nucleosides including 3-deaza-3-fluorouridine and its 2'-deoxy and arabino analogues have been prepared by fluorination of protected precursors. The resulting 3,3-difluoropyridine-2,4(1H,3H)-dione derivatives underwent palladium-catalyzed hydrogenolysis of one C-F bond at atmospheric pressure, and deprotection gave the 3-deaza-3-fluorouracil compounds. Selective reaction of a stabilized Wittig reagent at C4 of the 3,3-difluoro-2,4-dione intermediates gave exocyclic alkenes that underwent hydrogenation accompanied by spontaneous elimination of hydrogen fluoride. Ammonolysis of the exocyclic carbethoxymethyl substituent and ester protecting groups gave 4-(carboxamidomethyl)-3-deaza-3-fluorouridine and its analogues. Grignard additions at C4 of the ribo and 2'-deoxy 3,3-difluoro-2,4-dione intermediates followed by deprotection gave the 3-deaza-3,3-difluoro-4-hydroxy-4-(substituted)uracil nucleosides. The cytostatic activity of 3-fluoro-3-deazauridine (CC(50) = 4.4-9.6 microM) in three cancer cell lines paralleled that of 3-deazauridine, whereas no significant inhibitory activity was observed with a variety of virus-infected cell cultures.

Design and Synthesis of Substrates for Model Ribosomal Reactions

2'/3'-O-[Bz(NO(2))-Orn(Boc)]-5'-O-Piv-Ado (1) and its deoxy analog: 3'-O-[Bz(NO(2))-Orn(Boc)]-5'-O-Piv-2'-dAdo (2) were designed and synthesized as substrates for the model ribosome reaction we used to demonstrate the crucial role of A76 2'-OH of peptidyl-tRNA in the rate acceleration of peptide bond formation during protein biosynthesis.

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.

Examination of transformation among tetrodotoxin and its analogs in the living cultured juvenile puffer fish, kusafugu, Fugu niphobles by intramuscular administration

In puffer fish, tetrodotoxin (TTX) exists as the major toxin with chemically equilibrium analogs (4- epiTTX, 4,9-anhydroTTX) and chemically non-equilibrium analogs (deoxy analogs, 11-oxoTTX, 4- S-cysteinylTTX). There are two purposes to this study: 1) to search for the reason why TTX is the most major analog in puffer fish, even 4,9-anhydroTTX is chemically more stable, 2) to investigate whether or not chemically non-equilibrium analogs are transformed in puffer fish, because these were predicted to be biosynthetic intermediates. Pure TTX, 4- epiTTX, 4,9-anhydroTTX, and 11-oxoTTX were separately administrated to the cultured non-toxic juvenile puffer fish kusafugu, Fugu niphobles by intramuscular injection. Sixteen days after administration, TTX analogs in the whole fish were analyzed by LC-fluorescent detection and LC/MS. By the administration of TTX, 4- epiTTX, and 4,9-anhydroTTX, 34–40% of the administrated doses of the toxins were accumulated, and 4,9-anhydroTTX has become the major toxin after inter-conversion. This result indicates discrepancy from the previous ones wherein TTX was predominantly accumulated when TTXs were administrated through diets; this suggests that dietary administration might be necessary to accumulate TTX as the major toxin, and not 4,9-anhydroTTX. Transformations from TTX to deoxy analogs or 11-oxoTTX, or from 11-oxoTTX to TTX were not detected in this study.

Semi-synthesis and proteasome inhibition of D-ring deoxy analogs of (–)-epigallocatechin gallate (EGCG), the active ingredient of green tea extract

A semi-synthetic route to the D-ring analogs of (–)-epigallocatechin gallate (EGCG) from the relatively abundant (–)-epigallocatechin (EGC), isolated from green tea leaves, is described. A natural product (13), found in Cistus salvifolius, its acetate (14) and analog (17) were synthesized by this method. Their inhibitory activities against proteasomes were investigated.Key words: green tea, (–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin (EGC), proteasome inhibition.

Atomic Mapping of the Sugar Interactions in One-Site and Two-Site Mutants of Cyanovirin-N by NMR Spectroscopy

The details of the interaction between two mutants of Cyanovirin-N (CV-N), an HIV inactivating protein, and di- and trimannosides, substructures of Man-9, were investigated by STD NMR spectroscopy. One mutant, CV-N (mutDB), contains only one carbohydrate-binding site on domain A, whereas in CV-N (mutDA), the specificity of domain A for trimannose was changed while the site in domain B was kept intact, allowing for a dissection of the overall binding. Results of the STD NMR experiments revealed close contact between the protein binding site on domain A and H2, H3, and H4 of the nonreducing terminal mannose unit for Manalpha(1-2)Manalpha OMe, Manalpha(1-2)Manalpha(1-3)Manalpha OMe, and Manalpha(1-2)Manalpha(1-6)Manalpha OMe. The Manalpha(1-2)Manalpha(1-2)Manalpha OMe trisaccharide interacted with CV-N with the highest affinity. Further dissection of the interaction was achieved by NMR experiments with synthetic 2'-, 3'-, 4'-, and 6'-deoxy analogues of the disaccharide Manalpha(1-2)Manalpha OMe. STD and (1)H- (15)N HSQC NMR spectroscopy revealed that the 2'- and 6'-deoxy dimannosides were recognized by CV-N, whereas no binding was detected for the 3'- and 4'-deoxy sugars. These results demonstrate that the 3'- and 4'-hydroxyl groups on the terminal residue are engaged in key polar interactions with the protein and are required for high-affinity binding.

2′-Deoxy Cyclic Adenosine 5′-Diphosphate Ribose Derivatives: Importance of the 2′-Hydroxyl Motif for the Antagonistic Activity of 8-Substituted cADPR Derivatives

The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues.

Chemoenzymatic synthesis of deoxy analogues of the DNA topoisomerase II inhibitor eleutherin and the 3C-protease inhibitor thysanone

The asymmetric synthesis of (−)-9-demethoxyeleutherin 6, (+)-9-demethoxyeleutherin 7 and (+)-7,9-deoxythysanone 8 has been achieved using a microwave assisted kinetic resolution of racemic alcohol 11 with Novozyme 435 ® as the key step.

Studies on the Later Stage of the Biosynthesis of Pleuromutilin

Mutilin (4) and deoxy analogues 2 and 3 are biosynthetic precursors of pleuromutilin (1) in the later stage of biosynthesis. Precursors 2 and 3 are required for studies on the oxygenation steps in biosynthesis, and were synthesized from readily available 1 via 4 by deoxygenation of the hydroxy groups. Feeding experiments with the (2)H-labeled precursors confirmed their microbial conversion into 1.

Synthesis and properties of a 2'-deoxy analogue of trans-3',4'-BNA with an S-type sugar conformation

We previously synthesized bridged nucleic acid (BNA) analogues possessing an S-type sugar conformation. Recently, we achieved the synthesis of a 2'-deoxy analogue of trans-3',4'-BNA. The structure of this compound was confirmed by X-ray crystallography, which indicated that its sugar ring had an S-type conformation. The hybridization ability of the 2'-deoxy-trans-3',4'-BNA-modified oligonucleotide with DNA and RNA complements was evaluated, and it was found that 2'-deoxy-trans-3',4'-BNA showed superior hybridization ability compared with other BNA analogues with S-type sugar conformation.

A novel HPLC method reveals that precipitation of 2′-deoxyadenosine 5′-monophosphate with lithium perchlorate/acetone leads to base depurination

A novel HPLC method reveals that precipitation of 2′-deoxyadenosine 5′-monophosphate with lithium perchlorate/acetone leads to base depurination

Synthesis of deoxy and methoxy analogs of octyl α-d-mannopyranosyl-(1→6)-α-d-mannopyranoside as probes for mycobacterial lipoarabinomannan biosynthesis

A panel of analogs of the disaccharide α-d-Manp-(1→6)-α-d-Manp-OOctyl, a known acceptor substrate for a polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase involved in the assembly of the α-(1→6)-linked mannan core of mycobacterial lipoarabinomannan, has been synthesized. Described are synthetic routes to the target deoxy and methoxy analogs in which one of the hydroxyl groups of the parent disaccharide has been modified. All glycosylation reactions involved the use of octyl glycoside acceptors and thioglycoside donors using iodonium-ion activation, and the stereochemistry of the mannopyranoside bond formed was established by measurement of the 1JC-1,H-1. Depending on the target, the key methylation or deoxygenation reactions were carried out on either mono- or disaccharide substrates. This series of analogs will be useful for probing the substrate specificity of the enzyme, in particular, its steric and hydrogen-bonding requirements.

Synthesis of 3‘-Deoxynucleosides with 2-Oxabicyclo[3.1.0]hexane Sugar Moieties: Addition of Difluorocarbene to a 3‘,4‘-Unsaturated Uridine Derivative and 1,2-Dihydrofurans Derived from d- and l-Xylose1

Syntheses of 3'-deoxy analogues of adenosine, cytidine, and uridine with a 2,2-difluorocyclopropane ring fused at C3'-C4' are described. Treatment of a 2',5'-protected-3',4'-unsaturated derivative of uridine with difluorocarbene [generated from (CF3)2Hg and NaI] gave a diastereomeric mixture of the 3',4'-difluoromethylene compounds (alpha-L-arabino/beta-D-ribo, approximately 5:4). The limited stereoselectivity for addition at the beta face results from competitive steric hindrance by an allylic 4-methoxybenzyloxy group at C2' on the alpha face and a homoallylic nucleobase at C1' on the beta face. Protected uracil derivatives were converted into their cytosine counterparts via 4-(1,2,4-triazol-1-yl) intermediates. Treatment of 1,2-dihydrofurans derived from D- and L-xylose with difluorocarbene resulted in stereospecific addition at the beta face (anti to the 1,2-O-isopropylidene group on the alpha face). Glycosylations with activated enantiomeric sugar derivatives with the fused difluorocyclopropane ring on the beta face gave protected adenine nucleosides, whereas attempted glycosylation with an alpha-fused derivative gave multiple products. Removal of base- and sugar-protecting groups gave new difluoromethylene-bridged nucleoside analogues.

Addition of Difluorocarbene to 3‘,4‘-Unsaturated Nucleosides: Synthesis of 2‘-Deoxy Analogues with a 2-Oxabicyclo[3.1.0]hexane Framework1

Treatment of protected 2'-deoxy-3',4'-unsaturated nucleosides derived from adenosine and uridine with difluorocarbene [generated from bis(trifluoromethyl)mercury and sodium iodide] gave fused-ring 2,2-difluorocyclopropane compounds. Stereoselective alpha-face addition to the dihydrofuran ring resulted from hindrance by the protected beta-anomeric nucleobases. A protected uracil compound was converted smoothly into the cytosine derivative via a 4-(1,2,4-triazol-1-yl) intermediate. Removal of the protecting groups gave new difluorocyclopropane-fused nucleoside analogues. The solid-state conformation of the nearly planar furanosyl ring in the uracil compound had a shallow 2E pucker, and a more pronounced 1E conformation was present in the furanosyl ring of the cytosine derivative.

Synthesis of paromomycin derivatives modified at C(5″) to selectively target bacterial rRNA

The furanosyl moiety (ring III) of C(6′)-deoxyparomomycin and paromomycin was modified in search of aminoglycoside antibiotics with altered selectivity. The key intermediates were the N-Boc-protected derivative of C(6′)-deoxyparomomycin and the benzylidene-protected paromomycin. Their H 2C(5″)–OH group was oxidised with trichlorocyanuric acid or [bis(acetoxy)iodo]benzene in the presence of catalytic amounts of TEMPO to yield the corresponding aldehydes and acids, which were transformed into the protected alkoxy imines, amides and the amine. Standard deprotection gave the title compounds derived from C(6′)-deoxyparomomycin and derived from paromomycin that proved less active than paromomycin and its C(6′)-deoxy analogue.

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

Stereocontrolled synthesis of the D- and L-glycero-beta-D-manno-heptopyranosides and their 6-deoxy analogues. Synthesis of methyl alpha-l-rhamno-pyranosyl-(1-->3)-D-glycero-beta-D-manno-heptopyranosyl- (1-->3)-6-deoxy-glycero-beta-D-manno-heptopyranosyl-(1-->4)-alpha-L- rhamno-pyranoside, a tetrasaccharide subunit of the lipopolysaccharide from Plesimonas shigelloides.

The synthesis of d- and l-glycero-alpha-manno-thioheptopyranosides, protected with 4,6-O-alkylidene-type acetals is described. In glycosylations carried out with preactivation with the 1-benzenesulfinylpiperidine/trifluoromethanesulfonic anhydride couple, both the D- and L-glycero series exhibit excellent beta-selectivity with a range of glycosyl acceptors. In contrast, a 4,7-O-alkylidene acetal was found not to afford beta-selectivity. With a 4,6-O-[1-cyano-2-(2-iodophenyl)ethylidene] acetal protected thioglycoside, excellent beta-selectivity was obtained in glycosylation reactions, and subsequent treatment with tributyltin hydride and azoisobutyronitrile brought about clean fragmentation to the 6-deoxy-glycero-beta-D-manno-heptopyranosides. This chemistry was applied to the stereocontrolled synthesis of methyl alpha-L-rhamno-pyranosyl-(1-->3)-D-glycero-beta-D-manno-heptopyranosyl-(1-->3)-6-deoxy-glycero-beta-D-manno-heptopyranosyl-(1-->4)-alpha-L-rhamno-pyranoside, a component of the lipopolysaccharide from Plesimonas shigelloides.