Synthesis and Anti‐HIV Activity of [ddN]‐[ddN] Dimers and Benzimidazole Nucleoside Dimers

In an attempt to combine the HIV‐inhibitory capacity of different 2′,3′‐dideoxynucleoside (ddN) analogs, we have designed and synthesized several dimers of [AZT]‐[AZT] and [AZT]‐[d4T]. In addition, we also synthesized the dimers of 1‐(1H‐benzimidazol‐1‐yl)‐1‐deoxy‐β‐D ‐ribofuranose. The in vitro anti‐HIV activity of these compounds on a pseudotype virus, pNL4‐3.Luc.R‐E‐, in the 293T cells has been determined. Among these compounds, 2,2′‐(propane‐1,3‐diyl)bis[1‐(β‐D ‐ribofuranosyl)‐1H‐benzimidazole] ( 3 ) showed the highest anti‐HIV activity with similar effect as AZT.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Synthesis and Anti‐HIV Activity of 4′‐Cyano‐2′,3′‐didehydro‐3′‐deoxythymidine

A new anti‐HIV agent 4′‐cyano‐2′,3′‐didehydro‐3′‐deoxythymidine (9) was synthesized by allylic substitution of the 3′,4′‐unsaturated nucleoside 14, having a leaving group at the 2′‐position, with cyanotrimethylsilane in the presence of SnCl₄. Evaluation of the anti‐HIV activity of 9 showed that this compound is much less potent than the recently reported 2′,3′‐didehydro‐3′‐deoxy‐4′‐(ethynyl)thymidine (1).     

Synthesis,and in vitro Enzymatic and Antiviral Evaluation of d4T Polyphosphate Derivatives as Chain Terminators

A series of d4T di‐ or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV‐1 RT, and also tested for their in vitro anti‐HIV activity. The steady‐state kinetic study of compounds 1 – 4 in an enzymatic incorporation assay by HIV‐1 RT follows Michaelis? Menten profile. In addition, compounds 2 – 4 are able to inhibit HIV‐1 replication to the same extent as d4T and d4TMP in MT‐4 cells, as well as in CEM/0 cells and CEM/TK^? cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-D-3′-Azido-3′-deoxyribofuranosylnucleosides

Since the discovery of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2′,3′-didehydro-2′,3′-dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T. The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Impact of UV‐Visible Light on the Morphological and Photochemical Behavior of a Low‐Bandgap Poly(2,7‐Carbazole) Derivative for Use in High‐Performance Solar Cells

This paper reports on the photochemical behavior upon exposure to UV‐visible light of a poly(2,7‐carbazole) derivative for use in high‐performance solar cells. Poly[N‐9′‐hepta‐decanyl‐2,7‐carbazole‐alt‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole)] (PCDTBT) is one of a relatively large class of push‐pull carbazole‐based copolymers that have been synthesized to better harvest the solar spectrum. The 2,7‐carbazole building block of PCDTBT is also used with different electron‐accepting units in a large variety of low‐band‐gap polymers. The photochemical and morphological behavior of PCDTBT thin films is investigated from the molecular scale to the nanomechanical properties. The photo‐oxidation mechanism is shown to be governed by chain‐scission and cross‐linking reactions. It results in dramatic evolution of the morphology, roughness and stiffness of thin PCDTBT films. Based on the identification of several photoproducts formed along the macromolecular chains or released into the gas phase, the main pathways of PCDTBT photochemical evolution are discussed. These processes first involve the scission of the C–N bond between the carbazole group and the tertiary carbon atom bearing the alkyl side‐chain. Modifications of the chemical structure of PCDTBT, the evolution of its UV‐visible absorbance, and its nanomechanical properties initiated by light irradiation are shown to be closely related.     

Phosphoramidate Protides of Carbocyclic 2′,3′‐Dideoxy‐2′,3′‐Didehydro‐7‐Deazaadenosine with Potent Activity Against HIV and HBV

Synthesis of phosphoramidate protides of carbocyclic D‐ and L‐2′,3′‐dideoxy‐2′,3′‐didehydro‐7‐deazaadenosine by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t‐BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleosides against both HIV and HBV.     

Synthesis and enzymatic deprotection of biodegradably protected dinucleoside-2',5'-monophosphates: 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphoesters of 3'-O-(acyloxymethyl)adenylyl-2',5'-adenosines

As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2–5A and its congeners, adenylyl‐2′,5′‐adenosines bearing a 3‐(acetyloxy)‐2,2‐bis(ethoxycarbonyl)propyl group at the phosphate moiety, and an (acetyloxy)methyl‐ or a (pivaloyloxy)methyl‐protected 3′‐OH group of the 2′‐linked nucleoside have been prepared. The enzyme‐triggered removal of these protecting groups by hog liver carboxyesterase at pH 7.5 and 37° has been studied. The (acetyloxy)methyl group turned out to be too labile for the 3′‐O‐protection, being removed faster than the phosphate‐protecting group, which results in 2′,5′‐ to 3′,5′‐isomerization of the internucleosidic phosphoester linkage. In addition, the starting material was unexpectedly converted to the 5′‐O‐acetylated derivative. (Pivaloyloxy)methyl group appears more appropriate for the purpose. The fully deprotected 2′,5′‐ApA was accumulated as a main product, although, even in this case, the isomerization of the starting material takes place.     

5‐Fluoro‐2′‐Deoxyuridine Has Effects on Mitochondria in CEM T‐Lymphoblast Cells

Fluoropyrimidines are useful anticancer agents and the compound 5‐fluoro‐2′‐deoxyuridine (FdUrd) plays an important role in chemotherapy of colon cancers. Several nucleoside analogs, such as 3′‐azido‐2′,3′‐dideoxythymidine (AZT) and 2′,3′‐dideoxycytidine (ddC), can be incorporated into and cause depletion of mitochondrial DNA (mtDNA). These drugs are known to cause mitochondrial toxicity after prolonged treatment in patients. In this study we demonstrate that FdUrd reduces the mtDNA content and the expression level of the mtDNA encoded cytochrome c oxidase (COX II) in a CEM T‐lymphoblastic cell line.     

Synthesis and Antiviral Activity Evaluation of 2′,5′,5′-Trifluoro-Apiosyl Nucleoside Phosphonic Acid Analogs

Racemic synthesis of novel 2′,5′,5′-trifluoro-apiose nucleoside phosphonic acid analogs were performed as potent antiviral agents. Phosphonation was performed by direct displacement of triflate intermediate with diethyl (lithiodifluoromethyl) phosphonate to give the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor with persilylated bases to yield the nucleoside phosphonate analogs. Deprotection of diethyl phosphonates provided the target nucleoside analogs. An antiviral evaluation of the synthesized compounds against various viruses such as HIV, HSV-1, HSV-2, and HCMV revealed that the pyrimidine analogues have significant anti-HCMV activity.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4‐linked 1,2,3‐triazole AZT‐systemin conjugate

The Cu(I) catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) was applied for a nucleoside‐peptide bioconjugation. Systemin (Sys), an 18‐aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N‐propynoic acid functionalized analog (Prp‐Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp‐Sys with 3′‐azido‐2′,3′‐dideoxythymidine (AZT), a model cargo molecule. 1,4‐Linked 1,2,3‐triazole AZT‐Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by‐products. Under typical separation conditions of phosphate ‘buffer’ at low pH and uncoated fused bare‐silica capillary, an increasing peak intensity assigned to triazole‐linked AZT‐Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE‐ESI‐MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

     

Stability of Nonfullerene Organic Solar Cells: from Built‐in Potential and Interfacial Passivation Perspectives

Remarkable progress has been made in the development of high‐efficiency solution‐processable nonfullerene organic solar cells (OSCs). However, the effect of the vertical stratification of bulk heterojunction (BHJ) on the efficiency and stability of nonfullerene OSCs is not fully understood yet. In this work, we report our effort to understand the stability of nonfullerene OSCs, made with the binary blend poly[(2,6‐(4, 8‐bis(5‐(2‐ethylhexyl)thiophen‐2‐yl)‐benzo[1,2‐b:4,5‐b′]dithiophene))‐alt‐(5,5‐(1′,3′‐di‐2‐thienyl‐5′,7′‐bis(2‐ethylhexyl)benzo[1′,2′‐c:4′,5′‐c′] dithiophene‐4,8‐dione)] (PBDB‐T):3,9‐ bis(2‐methylene‐(3‐(1,1‐dicyanomethylene)‐indanone))‐5,5,11,11‐tetrakis(4‐hexylphenyl)‐ dithieno[2,3‐d:2′,3′‐d′]‐s‐indaceno[1,2‐b:5,6‐b′] dithiophene (ITIC) system. It shows that a continuous vertical phase separation process occurs, forming a PBDB‐T‐rich top surface and an ITIC‐rich bottom surface in PBDB‐T:ITIC BHJ during the aging period. A gradual decrease in the built‐in potential (V₀) in the regular configuration PBDB‐T:ITIC OSCs, due to the interfacial reaction between the poly(3,4‐ethylenedioxythiophene)‐poly(styrenesulfonate) (PEDOT:PSS) hole transporting layer and ITIC acceptor, is one of the reasons responsible for the performance deterioration. The reduction in V₀, caused by an inevitable reaction at the ITIC/PEDOT:PSS interface in the OSCs, can be suppressed by introducing a MoO₃ interfacial passivation layer. Retaining a stable and high V₀ across the BHJ through interfacial modification and device engineering, e.g., as seen in the inverted PBDB‐T:ITIC OSCs, is a prerequisite for efficient and stable operation of nonfullerene OSCs.