New method to prepare peptide-oligonucleotide conjugates through glyoxylic oxime formation

A new method to prepare peptide-oligonucleotide conjugates through chemoselective glyoxylic oxime linkage is reported. A novel phosphoramidite reagent, readily accessible from serine, was prepared and used in automated DNA synthesis to prepare oligonucleotides carrying a glyoxylic aldehyde functionality at the 5' terminus. This was efficiently coupled to a peptide functionalized with an aminooxy group. The method could be of general use to prepare a broad range of oligonucleotide conjugates.     

3′-Oligonucleotides conjugation via chemoselective oxime bond formation

Chemoselective oxime ligation of cyclopeptide, fluorescein and mannose derivatives at the 3′-end of an oligonucleotide was achieved. The conjugation was performed by reacting oxyamine containing reporter groups to an oligonucleotide bearing an aldehyde at the 3′-extremity. The aldehyde was generated by mild periodate oxidation of a 1,2-aminoalcohol which was readily incorporated at the 3′-end by automated DNA synthesis using the corresponding commercially available support. The straightforward chemical access, their stability in biological media as well as their unchanged hybridisation properties emphasise the interest of such 3′-conjugates.     

New strategy for the synthesis of 3',5'-bifunctionalized oligonucleotide conjugates through sequential formation of chemoselective oxime bonds

A convenient strategy for the synthesis of bifunctionalized oligonucleotide conjugates bearing two different reporters at the 3' and 5' ends of the oligonucleotide is presented. The method involves the preparation of oligonucleotides bearing an aldehyde and/or aminooxy functionality at each end, followed by reaction to form oxime bonds with appropriately functionalized reporters. The conjugation reactions are carried out under mild aqueous conditions with good reaction yield.     

Synthesis of an oxyamino-containing phenanthroline derivative for the efficient preparation of phenanthroline oligonucleotide oxime conjugates

A phenanthroline derivative bearing an oxyamino linker was efficiently prepared from commercial 5-nitro-1,10-phenanthroline. The subsequent reaction with an oligonucleotide containing an aldehyde either at the 5′ end or the 3′ end afforded, in good yield, the phenanthroline-oligonucleotide conjugates through oxime bond formation.     

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.     

Asymmetric synthesis of alpha-amino acids based on carbon radical addition to glyoxylic oxime ether

The first asymmetric synthesis of alpha-amino acids based on diastereoselective carbon radical addition to glyoxylic imine derivatives is reported. The addition of an isopropyl radical, generated from i-PrI, Bu(3)SnH, and Et(3)B in CH(2)Cl(2) at 25 degrees C, to achiral glyoxylic oxime ether 1 proceeded regioselectively at the imino carbon atom of the oxime ether group to give an excellent yield of the C-isopropylated product 2. The competitive reaction using glyoxylic oxime ether 1 and aldoxime ether 4 showed that the reactivity of the glyoxylic oxime ether toward nucleophilic carbon radicals was enhanced by the presence of a neighboring electron-withdrawing substituent. Thus, the alkyl radical addition to glyoxylic oxime ether 1 proceeded smoothly even at -78 degrees C, in contrast to the unactivated aldoxime ether 4. A high degree of stereocontrol in the carbon radical addition to the glyoxylic oxime ether was achieved by using Oppolzer's camphorsultam as a chiral auxiliary. The stannyl radical-mediated reaction of the camphorsultam derivative 6 with an isopropyl radical at -78 degrees C afforded a 96:4 diastereomeric mixture, 7a, of the C-isopropylated product. The reductive removal of the benzyloxy group of the major diastereomer (R)-7a, by treatment with Mo(CO)(6) and the subsequent removal of the sultam auxiliary by standard hydrolysis, afforded the enantiomerically pure D-valine (R)-12 without any loss of stereochemical purity. To evaluate the new methodology, a variety of alkyl radicals were employed in the addition reaction which gave the alkylated products 7 with excellent diastereoselectivity, allowing access to a wide range of enantiomerically pure natural and unnatural alpha-amino acids. Even in the absence of Bu(3)SnH, treatment of 6 with alkyl iodide and Et(3)B at 20 degrees C gave the C-alkylated products 7 with moderate diastereoselectivities. The use of Et(2)Zn as a radical initiator, instead of Et(3)B, was also effective for the radical reaction. The enantioselective isopropyl radical addition to 1 using (R)-(+)-2, 2'-isopropylidenebis(4-phenyl-2-oxazoline) and MgBr(2) gave excellent chemical yield of the valine derivative 2 in 52% ee.     

Zinc-mediated carbon radical addition to glyoxylic imines in aqueous media for the synthesis of alpha-amino acids

The addition of carbon radicals to glyoxylic imines was studied using zinc dust as a radical initiator. The zinc-mediated radical reaction of glyoxylic oxime ethers and hydrazones proceeded smoothly to give the alkylated products via a carbon-carbon bond-forming process in aqueous media. The reaction of the oxime ethers and hydrazones having an Oppolzer's camphorsultam group provided the corresponding alkylated products, which could be converted into enantiomerically pure alpha-amino acids. The diastereoselectivities observed in the reaction of hydrazones were better than those obtained in the reaction of oxime ethers.     

High surface density immobilization of oligonucleotide on silicon

Oligonucleotide (11-mer) molecules are immobilized on silicon in high surface population using either a permanent thioether bond or a chemo-selectively reversible disulfide bond to the surface thiol functionality. Substrate hydroxy groups are first silanized with an 11 carbon trichlorosilane containing a terminal, protected thiol moiety. Oligonucleotide modified with a tether possessing a terminal thiol group is further derivatized with a water-soluble, halobenzylic bifunctional reagent, which allows the complete conjugate to be attached to the surface through a permanent thioether bond. Alternatively, the oligonucleotide-tether complex can be combined with a pyridyldisulfide compound, which, in turn, facilitates the formation of a reversible disulfide bond with surface thiol. The amount of immobilized oligonucleotide was determined by radiochemical labeling with 32P. Additional verification of surface amounts was obtained from X-ray photoelectron spectroscopic analysis of substrates. The results of the immobilization protocols are compared with the oligonucleotide surface population achieved through the conventional silanizing agent, mercaptopropyltrimethoxysilane. Finally, a preliminary confirmation of duplex formation of a TTU-attached 25-mer with its complementary strand is outlined.     

Palladium-indium iodide-mediated allylation and propargylation of glyoxylic oxime ether and hydrazone: the role of water in directing the diastereoselective allylation

Allylation and propargylation of glyoxylic oxime ether in the presence of a catalytic amount of palladium(0) complex and indium(I) iodide were studied. Excellent diastereoselectivities in allylation were achieved in the presence of water, although low diastereoselectivities were observed in the absence of water. Propargylation of glyoxylic oxime ether proceeded with good diastereoselectivities in the presence of LiBr or LiCl.     

pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin.     

Understanding the contribution of ascorbic acid to the pigment development in model white wine systems using liquid chromatography with diode array and mass spectrometry detection techniques

The present study investigated the contribution of ascorbic acid to the formation of coloured species in model white wine systems containing (+)-catechin as the oxidisable phenolic substrate. Reactions were carried out in the presence or absence of ascorbic acid in model wine systems buffered with either tartaric acid or formic acid. High performance liquid chromatography with diode array detector (HPLC-DAD) or mass spectrometry (HPLC-MS) analyses demonstrated that glyoxylic acid-derived xanthylium pigments were the main coloured species produced in all samples except those containing just (+)-catechin and formic acid. Higher concentrations of these pigments were detected in the tartaric acid based model system containing both (+)-catechin and ascorbic acid than in the corresponding formic acid model system. The inability of formic acid to form an aldehyde, unlike the known oxidative formation of aldehydes from tartaric acid, contributes to the lower colour development in the formic acid model system. Significantly, these observations imply that ascorbic acid must break down to provide an aldehyde, or ketone, capable of reacting with (+)-catechin to generate the glyoxylic acid-derived xanthylium cations.     

Addition of alkyl radicals, generated from carboxylic acids via photochemical decarboxylation, to glyoxylic oxime ether: a mild and efficient route to α-substituted α-aminoesters

Radical addition to a glyoxylic oxime ether was accomplished under mild conditions using an alkyl radical generated from a free carboxylic acid via photochemical decarboxylation. The photoreaction provided an efficient route to α-substituted α-aminoesters from carboxylic acids and oxime ether.     

Carbon-carbon bond construction on solid support: triethylborane-induced radical reactions of oxime ethers

The triethylborane-induced solid-phase radical reaction was studied. The solid-phase radical reaction of oxime ether anchored to Wang resin proceeded smoothly to give the α-amino acid derivatives. The carbon-carbon bond-forming radical reaction of TentaGel OH resin-bound glyoxylic oxime ether proceeded even in aqueous media.     

Oligodeoxynucleotides containing alpha-L-ribo configured LNA-type C-aryl nucleotides

Synthesis of 2[prime or minute]-O,4[prime or minute]-C-methylene-[small alpha]-l-ribofuranosyl derivatives containing phenyl and 1-pyrenyl aglycons, i.e., novel [small alpha]-l-ribo configured LNA-type C-aryl nucleosides, has been accomplished. Key synthetic steps included stereoselective Grignard reactions on tetrahydrofuran aldehyde, configurational inversion of the resulting alcohol into alcohol, and concomitant Mitsonobu cyclization furnishing the desired bicyclic furanosyl skeleton with a locked conformation. The phosphoramidite derivatives and were used for automated synthesis of 9-mer DNA and [small alpha]-L-LNA oligonucleotides containing the [small alpha]-L-LNA-type C-aryl monomers ([small alpha]L)Ph(L) and ([small alpha]L)Py(L) containing a phenyl and pyrenyl aglycon, respectively. Thermal denaturation studies showed universal base pairing behavior for the pyrenyl monomer ([small alpha]L)Py(L) when incorporated into a DNA or an [small alpha]-L-LNA oligonucleotide.     

双波长和三波长分光光度法同时测定乙醛酸和乙二醛

利用醛基的成肟反应，用双波长法，三波长法同时测定乙醛酸、乙二醛的含量。它们的回收率分别为１００．９％－１０２．３％（ｎ＝６）和９６．８％－１０３．０％（ｎ＝１１）。     

Chemoselectively addressable template: a valuable tool for the engineering of molecular conjugates

We herein report the modular design and the synthesis of new molecular conjugates, which can combine a cell targeting function (ligand domain) with potential cytotoxic molecules (effector domain). The present approach utilizes a cyclic peptide template, Chemoselectively Addressable Template (CAT) as a key intermediate. These CAT molecules exhibit two independent and chemically addressable domains which permits the sequential and regioselective assembly of different ligand and/or effector domains. The attachment of various units to the template was achieved by the formation of iterative oxime bonds. The chemoselective oxime bonds were produced by the reaction of glyoxylyl aldehyde groups obtained from serine precursors. The process was further developed to prevent transoximation reactions. RAFT(c[-RGDfK-])4, a synthetic vector targeting the tumor-associated a alpha(V)beta3 integrin was prepared and coupled to either a cytotoxic peptide or oligonucleotide as an illustration of present approach. The potential application of this approach has been further demonstrated by the synthesis of high molecular weight compounds such as RAFT(c[-RGDfK-])16, a alpha(V)beta3-targeting ligand of high valency index.     

Trace analysis of an oligonucleotide with a specific sequence using PNA-based ion-channel sensors

The gold electrodes modified with self-assembled monolayers of a 13-mer peptide nucleic acid (PNA) probe and 8-amino-1-octanethiol were used for the detection of a complementary oligonucleotide at a femtomolar level using the ion-channel sensor technique. No response to a one-base mismatched oligonucleotide was observed. The electrode surface was positively charged in a pH 7.0 buffer solution due to the protonation of an amine group of the thiol, where the electron transfer between the positively charged marker [Ru(NH3)6]3+ and the surface was hindered because of the charge-charge repulsion between them. Binding of the negatively-charged complementary oligonucleotide to the probe cancels the positive charge at the surface, and provides an excess negative charge at the surface, thereby facilitating the access of the marker to the electrode surface and its redox reaction. Using a 13-mer PNA probe for this sensing mode, we achieved the detection of the oligonucleotide at a femtomolar (approximately 10-15 M) level, improved by five orders of magnitude than the previously used 10-mer PNA probe.     

Oxidation by Permanganate in strong alkaline medium. Oxidation of ethane-1,2-diol,glycol aldehyde,glycollic acid,and glyoxylic acid

A study was made on the kinetics and mechanism of the reaction in aqueous solutions of 0.10 to 2.0 mol dm^?3 alkaline concentrations. The substrates (S) applied were ethane-1,2-diol (ethylene glycol), glycol aldehyde, glycollic acid, and glyoxylic acid. Glycol aldehyde and glyoxylic acid were present in the form of dihydrate. In each case alkoxy anion is the reactive form and the K_B constant of deprotonation can be calculated from the kinetic data. A mechanism based on electron abstraction is suggested. Manganate reacts with these substrates much slower than permanganate.     

Addition of electrophilic and heterocyclic carbon-centered radicals to glyoxylic oxime ethers

[reaction: see text] Stabilized primary radicals can be formed from alkyl halides in an atom transfer process with Et(3)B. This process depends on the strength of the carbon-halogen bond and the stability of the resulting primary radical. Radicals formed from benzyl iodide and ethyl iodoacetate add to glyoxylic oxime ethers; however, more electrophilic radicals do not. Glyoxylic oxime ethers are also good radical acceptors for heterocyclic carbon-centered secondary radicals, giving novel alpha-amino acid derivatives.     

Reactive thermoresponsive copolymer scaffolds

Thermoresponsive copolymer scaffolds containing reactive aldehyde functions were prepared and a selection of organic residues conjugated to these copolymer scaffolds through oxime/hydrazone formation. The conjugation of hydrophobic residues affords copolymers whose lower solution critical temperatures are in most cases higher than that of the parent copolymer scaffold.