Fast deprotection of synthetic oligodeoxyribonucleotides using standard reagents under microwave irradiation

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz, dC Bz, dC Ac, dG ibu, dG PAC) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz, dC Bz, dG ibu) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170 degrees C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.     

Mononuclear copper(I) complexes of O-t-butyl-1,1-dithiooxalate and of O-t-butyl-1-perthio-1-thiooxalate

Described are the syntheses and structures of a phosphonium salt of the anionic ligand O-t-butyl-1,1-dithiooxalate, [PPh₃Bz][i-dto^tBu] ([PPh₃Bz][1]), and of two Cu(I) complexes of this anion, Cu(PPh₃)₂(η²-i-dto^tBu) (2) and Cu(dmp)(PPh₃)(η¹-i-dto^tBu) (3, dmp = 2,9-dimethyl-1,10-phenanthroline). In addition, it was found that the reaction of CuBr₂ with i-dto^tBu⁻ gives a O-t-butyl-1-perthio-1-thiooxalato complex of copper(I), [BzPh₃P][Cu(Br)(S-i-dto^tBu)] ([BzPh₃P][4]), where [S-i-dto^tBu]⁻ is a disulfide-containing anionic ligand. The electronic structure and absorption spectrum of this species were investigated by time dependent DFT methods.     

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.     

Synthesis and characterization of organotin Schiff base chelates

The synthesis and characterization of five organotin compounds containing Salophen(^tBu) [Salophen(^tBu)=N,N′-phenylene-bis(3,5-di-tert-butylsalicylideneimine)], Salomphen(^tBu) [Salomphen(^tBu)=N,N′-(4,5-dimethyl)phenylene-bis(3,5-di-tert-butylsalicylideneimine)] and Phensal(^tBu) [Phensal(^tBu)=3,5-di-tert-butylsalicylidene(1-aminophenylene-2-amine)] ligands is described. These compounds include the monomeric complexes LSnCl₂ (where L=Salophen(^tBu), L=Salomphen(^tBu)), L(ⁿBu)SnCl (where L=Salophen(^tBu), Salomphen(^tBu)), L(ⁿBu)SnCl₂ (where L=Phensal(^tBu)). Spectroscopic techniques including ¹¹⁹Sn NMR and X-ray crystallography were used in the characterization of the compounds.     

Synthesis and reactivity of imido niobium complexes containing the functionalized (dichloromethylsilyl)cyclopentadienyl ligand

The niobium complex [NbCp^ClCl₄] (Cp^Clη⁵-C₅H₄(SiCl₂Me)) (1) with a functionalized (dichloromethylsilyl)cyclopentadienyl ligand was isolated by the reaction of [NbCl₅] with C₅H₄(SiCl₂Me)(SiMe₃). Complex 1 was a precursor for the imido silylamido derivative [NbCp^NCl₂(NtBu)] (Cp^Nη⁵-C₅H₄[SiClMe(NHtBu)]) (2) after addition of LiNHtBu, which subsequently gave the dichlorosilyl compound [NbCp^ClCl₂(NtBu)] (3) when reacted with SiCl₃Me. Addition of LiNHtBu to complex 2 gave the niobium amido complex [NbCp^NCl(NHtBu)(NtBu)] (4), which slowly evolved with exchange of the niobium-amido and the silicon-chloro groups to give the dichloroniobium complex [NbCp^NNCl₂(NtBu)] (Cp^NNη⁵-C₅H₄[SiMe(NHtBu)₂]) (5). Reaction of 2 with excess LiNHtBu gave the silyl-η-amido constrained geometry complexes [Nb{η⁵-C₅H₄[SiMe(NHtBu)(-η-NtBu)]}(NHtBu)(NtBu)] (6) and [Nb{η⁵-C₅H₄[SiClMe(-η-NtBu)]}(NHtBu)(NtBu)] (7), whereas addition of one equimolecular amount of LiNHtBu to 5 in C₆D₆ afforded complex [NbCp^NNCl(NHtBu)(NtBu)] (8). All of the new complexes were characterized by ¹H, ¹³C and ²⁹Si NMR spectroscopy.     

Gold(I) complexes with thiosemicarbazones: cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(H2Ac4DH)Cl]?MeOH (1) [Au(H₂2Ac4Me)Cl]Cl (2) [Au(H₂2Ac4Ph)Cl]Cl?2H₂O (3) and [Au(H₂2Bz4Ph)Cl]Cl (4) were obtained with 2-acetylpyridine thiosemicarbazone (H2Ac4DH), its N(4)-methyl (H2Ac4Me) and N(4)-phenyl (H2Ac4Ph) derivatives, as well as with N(4)-phenyl 2-benzoylpyridine thiosemicarbazone (H2Bz4Ph). The compounds were cytotoxic to Jurkat (immortalized line of T lymphocyte), HL-60 (acute myeloid leukemia), MCF-7 (human breast adenocarcinoma) and HCT-116 (colorectal carcinoma) tumor cell lines. Jurkat and HL-60 cells were more sensitive than MCF-7 and HCT-116 cells. Upon coordinating to the gold(I) metal centers in complexes (2) and (4), the cytotoxic activity of the H2Ac4Me and H2Bz4Ph ligands increased against the HL-60 and Jurkat tumor cell lines. 2 was more active than auranofin against both leukemia cells. Most of the studied compounds were less toxic than auranofin to peripheral blood mononuclear cells (PBMC). All compounds induced DNA fragmentation in HL-60 and Jurkat cells indicating their pro-apoptotic potential. Complex (2) strongly inhibited the activity of thioredoxin reductase (TrxR), which suggests inhibition of TrxR to be part of its mechanism of action.     

Flexible,polymer-supported synthesis of sphingosine derivatives provides ceramides with enhanced biological activity

A polymer-supported route for the synthesis of sphingosine derivatives is presented based on the C-acylation of polymeric phosphoranylidene acetates with an Fmoc-protected amino acid. The approach enables the flexible variation of the sphingosine tail through a deprotection–decarboxylation sequence followed by E-selective Wittig olefination cleavage. d-Erythro-sphingosine analogs have been synthesized by diastereoselective reduction of the keto group employing LiAlH(O-tBu)₃ as reducing agent. The effect of ceramides and keto-ceramides on the proliferation of three cancer cell lines HEP G-2, PC-12 and HL-60 was investigated and a ceramide containing an aromatic sphingosine tail was identified as being most active.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

Microwave-assisted rapid deprotection of oligodeoxyribonucleotides.

A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0. 2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbzand dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55 degrees C) with respect to their retention time on HPLC and biological activity.     

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers from Unprotected Ribonucleosides

An inexpensive and much improved protocol has been developed for the synthesis of protected morpholino monomers from unprotected ribonucleosides in high overall yield, using oxidative glycol cleavage and reductive amination strategy. Unlike the previous methods, the present strategy allows installing the exocyclic amine protections at a later stage, and thus avoids the use of expensive, or commercially unavailable, exocyclic amine-protected ribonucleosides as starting materials. To demonstrate the flexibility of the present method in choosing protecting groups, the monomers have been protected with several such groups of different deblocking properties at the exocyclic amine position.     

Cleavage Of Oligodeoxyribonucleotides From Polymer Supports And Their Rapid Deprotection Under Microwaves

Abstract

Novel conditions for the cleavage of oligodeoxynucleotides from polymer supports and their complete deprotection under microwaves have been developed. The oligonucleotides synthesized using phosphoramidite synthons carrying base labile (Pac, Dmf and t-Bpac) and conventional (Bz for A and C and Pac for G) protecing groups for nucleic bases were deprotected using 0.2M sodium hydroxide (MeOH : H₂O:: 1:1, v/v) = Reagent A and 1M sodium hydroxide (MeOH: H₂O :: 1:1, v/v = Reagent B, respectively under microwaves. The deprotected oligonucleotides were found to be comparable with the corresponding oligonucleotides deprotected under standard conditions (aq. ammonia at 55°C).     

Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

O-Peracetylated 1-(β-d-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated β-d-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-β-d-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(β-d-glucopyranosyl)-3,5-diphenyl- as well as 3-(β-d-glucopyranosyl)-1,5-diphenyl biurets besides 1-(β-d-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(β-d-glucopyranosyl)-5-(β-d-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated β-d-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of β-d-gluco, β-d-galacto and β-d-xylo configurations. O-Acyl protected 1-(β-d-glucopyranosyl)-3-(β-d-glycopyranosylcarbonyl)ureas were obtained from the reaction of β-d-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of β-d-gluco and β-d-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary α-amylase.     

A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides

The use of the ammonia deprotection step to introduce ¹⁵N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described.     

Synthesis and derivatization of halflanthanidocene aryl(alk)oxide complexes

The reaction of halflanthanidocene aryloxides Cp^R′Ln(OAr^tBu,R)₂ (Ln = Y, La, Lu; Cp^R′ = C₅Me₅, C₄Me₄H; R = H, Me) and halflanthanidocene alkoxides [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) with trimethylaluminum (TMA) was investigated. Monomeric Cp^R′Ln(OAr^tBu,R)₂, derived from the ortho-tBu-substituted OC₆H₂tBu₂-2,6-R-4 (R = H, Me) ligands, form mono(tetramethylaluminate) complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) for the smaller lanthanide metal centers yttrium and lutetium. Such an [aryloxide] → [aluminate] ligand exchange was not observed at the larger lanthanum metal center. The mobility of the tetramethylaluminate ligands of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) (Ln = Y, Lu) was examined by variable-temperature (VT) ¹H NMR spectroscopy, revealing two signals for bridging and terminal methyl groups at lower temperatures. The treatment of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) with donor solvent d₈-THF gave Cp^R′Ln(OAr^tBu,R)(Me)(d₈-THF)₂ (Ln = Y, Lu) with terminal methyl groups, according to a donor-induced aluminate cleavage reaction. Dimeric [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) was synthesized from (C₅Me₅)Ln(NiPr₂)₂(THF) and reacted with two equivalents of TMA per Ln center to yield monomeric bis(TMA) adduct complexes (C₅Me₅)Ln(OCH₂CMe₃)₂(AlMe₃)₂(Ln = Y, Lu). VT NMR spectroscopic studies confirmed a high mobility of the Ln(μ-OCH₂CMe₃)(μ-Me)AlMe₂ moieties at an ambient temperature. Both bis(TMA) adduct complexes were characterized by X-ray structure analysis.     

5-Hydroxytryptophan as a building block in oligopeptides using Fmoc/tBu SPPS

Summary One of the initial steps in the development of therapeutic agents is the identification of lead compounds that bind to the target of interest. Use of combinatorial libraries allows the screening of thousands to millions of compounds. To optimize the characteristics of the first generation of compounds, many of their analogues need to be synthesized. In this sense, 5-hydroxytryptophan closely resembles tryptophan. Both, however, are sensitive to oxidation and alkylation during solid-phase peptide synthesis. In an effort to overcome these side reactions during oligopeptide synthesis, we prepared N -Fmoc-N ⁱⁿ-Boc-5-O-benzyl-5-hydroxytryptophan. To evaluate its properties, this building block was incorporated in a pentagastrin analogue using Fmoc/tBu chemistry. After deprotection and cleavage from the support, the products were purified and identified. The fully deprotected peptide was the main compound, contaminated by its 5-O-benzylated analogue. The relative amount of the latter decreased using longer deprotection times.This work will be reported in more detail elsewhere (Lescrinier, Th. et al., paper in preparation).     

Glucopyranosylidene-spiro-iminothiazolidinone,a new bicyclic ring system: Synthesis,derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-β-d-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-d-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO₂Cl where R = tBu, Ph, 4-Me-C₆H₄, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N′-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (K_i = 9 μM) and the 2-naphthoylimino-thiazolidinones (K_i = 10 μM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.     

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.     

Reductive C-N bond cleavage of the NCCCN β-diketiminate backbone: A direct approach to azabutadienyl and alkylidene-anilide scaffolds

Tetrahydrofuran/toluene solutions of (nacnac)TiCl₂ (nacnac⁻ = [ArNC(^tBu)]₂CH, Ar = 2,6-ⁱPr₂C₆H₃) react readily with KC₈ to afford the titanium imide (ArN(^tBu)CCHC(^tBu))TiNAr(THF)Cl (1) in 67% isolated yield. Complex 1 forms from the two-electron reductive C-N bond cleavage of the β-diketiminate ligand. Likewise, reduction of (nacnac)TiCl(NHAr) (2), prepared in 85% yield from (nacnac)TiCl₂ and LiNHAr, with KC₈ results in formation of the imide-anilide analogue (ArN(^tBu)CCHC(^tBu))TiNAr(NHAr) (3) in 88% yield. Another reductant such as Li^tBu (3 equiv.) reacts cleanly with the precursor (nacnac)TiCl₂ to afford the alkylidene-ate complex [Li(Et₂O)][(ArN(^tBu)CCHC(^tBu))TiNAr(Et₂O)] (4), in 81% yield. Complexes 1-4 have been characterized by ¹H and ¹³C NMR spectra as well as single-crystal X-ray diffraction analysis. Plausible mechanisms to formation of compounds 1, 3 and 4 are also presented and discussed.     

Syntheses of optically active 2-amino-4-oxobutyric acid and N,O-protected derivatives

Summary Strategies for the synthesis of optically active aspartaldehyde derivatives are reviewed. Most of them are using the chiral pool: allylglycine or naturally occurring homoserine, aspartic acid or methionme and side chain modifications. This will be developed in the first part. Some other original routes are also displayed in the second part. Different aspects of each strategy are discussed: the nature and number of steps, the problem of protecting groups, the price and availability of starting materials. Some synthetic applications of such interesting chiral synthons are shown in the last part.Abbreviations Ac acetyl - An Anisyl or 4-methoxy benzyl - Bn benzyl - Boc tert-butoxycarbonyl - BOP-PF₆ benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate - Cbz benzyloxycarbonyl - DCC dicyclohexylcarbodiimide - DIBAL diisobutyl aluminum hydride - DIPEA diisopropyl ethyl amine - DMF dimethyl formamide - DMSO dimethylsulfoxide - EDCI l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - HP 4-hydroxy phenyl - MP 4-methoxy phenyl - NCS N-chlorosuccinimide - NMR nuclear magnetic resonance - PCC pyridinium chlorochromate - Pht phthaloyl - Ser serine - tBu tert-butyl - TEMPO 2,2,6,6-tetramethyl piperidine-l-oxyl - TFA trifluoro acetic acid - Trityl triphenyl methyl - Val valine This paper is dedicated to RV.     

Labile protecting groups in tRNA synthesis

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.