A New Efficient Stereoselective Method for the Synthesis of (E)-5-Aminoallyl-Pyrimidine-5′-Triphosphates Using Palladium-Catalyzed Heck Reaction

An efficient overall two-step strategy for the synthesis of (E)-5-aminoallyl-pyrimidine-5′-triphoshate, starting from commercially available pyrimidine-5′-triphosphate is described. The method involves regioselective iodination of pyrimidine-5′-triphosphate, followed by the palladium-catalyzed Heck coupling with allylamine. The catalytic reaction is highly stereoselective and compatible with many functional groups present in the reactants.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. I. Analysis of surface-localized proteins

Lactoperoxidase-catalyzed iodination of chloroplast membranes has been employed to characterize the vectorial distribution of lamellar proteins. The enzymatic reaction is highly specific for only the outermost membrane components (Phillips, D. R. and Morrison, M. (1971) Biochemistry 10, 1766–1771); we have determined the distribution of ¹²⁵I label and changes in photochemical activities after iodination in an effort to identify these components. Three major conclusions are evident:

1. 1. The coupling factor for photophosphorylation is highly exposed and is selectively and rapidly inhibited by the iodination reaction.

2. 2. A loss of Photosystem I activity (NADP reduction) resulted from iodination. Partial reactions indicated the effect was on electron-transport components on the reducing side of Photosystem I. There was also a limited inhibition of methyl viologen reduction.

3. 3. Iodination of intact membranes caused a reduction in rates of Photosystem II-dependent Hill reaction activity. This inhibition could not be explained solely on the basis of iodination effects on electron-transport components involved in the oxidation of water. The implications of these data with respect to previous chloroplast-membrane models are discussed.

Thyroid hormone synthesis and thyroglobulin iodination related to the peroxidase localization of oxidizing equivalents: studies with cytochrome c peroxidase and horseradish peroxidase

Cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP), when combined with a stoichiometric amount of H2O2, form stable compounds I which are known as FeIV Ro and FeIV o pi + structures, respectively. These compounds were assayed in the catalysis of thyroid hormone synthesis and the iodination reaction. As previously shown for the lactoperoxidase FeIV Ro compound, the CcP FeIV Ro compound was involved in the coupling and not in the iodination reactions. In contrast, the HRP FeIV o pi + compound catalyzed both iodination and hormone formation. The possible role of the different peroxidase-H2O2 compounds in the two sequential reactions, thyroglobulin iodination and thyroid hormone formation, is discussed.     

Relations of iodination, hormonosynthesis and proteolytic cleavage in human thyroglobulin

Normally iodinated thyroglobulin (Tg) contains low molecular weight hormone-rich peptides associated to the bulk of the molecule by disulfide bridges. It is shown, with the assistance of in vitro iodination experiments using different iodine concentrations and various incubation times, that the proteolytic cleavage giving rise to the 26 K hormonopeptide in human Tg is part of a coupling reaction rather than iodination. This cleavage may be a preliminary event related to a facilitation in the release of thyroid hormones.     

Partial Protection of Carbohydrate Derivatives. Part 24.1 Synthesis of Adenylyl-(2′–5′)-Adenylyl-(2′–5′)-Adenosine Using a 3′-O-(Tetrahydropyran-2-YL) Adenosine Derivative

Abstract

The synthesis of the title compound was performed using a 3′-O-(tetrahydropyran-2-yl) adenosine derivative as the starting material, i.e., a coupling reaction of triethylammonium N ⁶-benzoyl-5′-O-dimethoxytrityl-3′-O-(tetrahydropyran-2-yl) adenosine 2′-(4-chlorophenyl)phosphate with N ⁶-benzoyl-2′,3′-di-O-benzoyladenosine, followed by a sequence of reactions, O-dedimethoxytritylation, a coupling reaction with the former triethylammonium salt, and complete deblocking of the resultant 2′, 5′-triadenylic acid derivative.     

Structural-based design and synthesis of novel 9-deazaguanine derivatives having a phosphate mimic as multi-substrate analogue inhibitors for mammalian PNPs

9-(5′,5′-Difluoro-5′-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5′,5′-difluoro-5′-phosphonopentyl)guanine (DFPP-G) with calf-spleen PNP. DFPP-DG and its analogous compounds were synthesized by the Sonogashira coupling reaction between a 9-deaza-9-iodoguanine derivative and ω-alkynyldifluoromethylene phosphonates as a key reaction. The experimental details focused on the synthetic chemistry along with some insights into the physical and biological properties of newly synthesized DFPP-DG derivatives are disclosed.     

SYNTHESIS OF CARBOCYCLIC ANALOGS OF 2′,3′-DIDEOXYSANGIVAMYCIN, 2′,3′-DIDEOXYTOYOCAMYCIN,AND 2′,3′-DIDEOXYTRICIRIBINE

Syntheses and antiviral activity of new carbocyclic analogs of 2′, 3′-dideoxysangivamycin, 2′,3′-dideoxytoyocamycin and 2′,3′-dideoxytriciribine is described. The key intermediate, carbocyclic 4-chloro- 5-iodopyrrolopyrimidine, was synthesized in good yield via a novel iodination method using I₂ and CF₃COOAg. This carbocyclic 4-chloro-5-iodopyrrolopyrimidine then allowed for a concise synthesis of the desired 4,5-disubstituted carbocyclic nucleosides.     

Differential effects of methylmercuric chloride and mercuric chloride on oxidation and iodination reactions catalyzed by thyroid peroxidase

Thyroid peroxidase (TPO), the major enzyme in the thyroid hormone synthesis, multifunctionally catalyzes (1) iodide oxidation, (2) iodination of the precursor protein, and (3) a coupling reaction of iodotyrosyl residues. The present study was carried out to examine the mercurial effects on the iodination, the second step of TPO. Purified porcine thyroglobulin or bovine serum albumin as acceptor protein was iodinated with [125I]NaI and H2O2 by purified porcine TPO. Iodinated protein was separated by acid precipitation on membrane filter or paper chromatography. Both CH3HgCl and HgCl2 dose-dependently inhibited the iodination, but HgCl2 was more potent to inhibit the iodination than CH3HgCl. These mercurial effects on the second step resemble the effects on the third step which were already reported; but are in marked contrast to the effects on the first step, where TPO was inhibited by HgCl2 but never by CH3HgCl.     

Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand

Hepatitis B virus DNA contains a tightly bound protein which was not removed by heating to 60°C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5′ end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90°C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5′ end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5′ ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5′ end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.     

Studies on the mechanism of the iodination of tyrosine by lactoperoxidase

Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.     

Myeloperoxidase-catalyzed iodination and coupling.

Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.     

SYNTHESIS OF 5-ETHYNYL-2′-DEOXYURIDINE-5′-BORANOMONO PHOSPHATE AS A POTENTIAL THYMIDYLATE SYNTHASE INHIBITOR

The 5-ethynyl-2′-deoxyuridine nucleoside and the 5′-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2′-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2′-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.     

Synthesis of 5-Formyluridines

Abstract

5-Formyl-2′,3′-O-isopropylideneuridine and 5-formyl-2′,3′,5′-tri-O-acetyluridine were prepared by a new procedure involving palladium-catalyzed coupling of 5-iodouridine with styrene, followed by reaction with acetic anhydride or acetone and ozonolysis of the resulting 5-styryluridine derivatives.     

The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.     

Synthesis and Evaluation of Oligonucleotides of High Purity

Abstract

We have developed and evaluated methods for the production of highly pure oligonucleotides.

Presently the solid phase synthesis in an automated DNA synthesiser applying the phosphoramidite chemistry can be regarded as a standard. During the synthesis several undesirable by-products arise:

- incomplete coupling (1%) leads to 5′-truncated sequences. These sequences are acetylated at their 5′-hydroxyl group to prevent further elongation in subsequent coupling steps, but this “capping step” is incomplete, the capping-yield is 90%, leading to accumulation of sequences of the length n-1 with internal deletions.

- the glycosidic bond to N-protected purines, especially adenine, is susceptible to acid leading to depurination and subsequently to strand scission during alkaline deprotection of the oligonucleotide. This gives rise to 3′- and to 5′-truncated sequences. The 3′-truncated sequences will not be removed by standard Rp HPLC as they are tritylated.

- the reactions involved in synthesis and deprotection may cause base modifications (full length product with damaged bases).

- insufficient deprotection procedures may result in incomplete removal of protecting groups, especially from the bases (full length products with altered bases).

We have set up two different schemes (Fig. 1 and Fig. 2) for synthesis and purification, which should provide highly pure oligonucleotides with the potential of adapting to large scale production:

- accumulation of n-1 sequences (failure of capping) will be avoided by a double capping procedure using phosphite in the first capping step and an acetic anhydride capping reagent in the second capping step, as described in the literature¹.

- 3′-truncated sequences are removed by different methqds in the two schemes. In scheme I (Fig. 1) the 3′-truncated sequences can be washed off, as the 3′-full length product still is anchored to the solid support after deprotection. In scheme II (Fig. 2) the 3′truncated sequences are digested by snake venom phosphodiesterase. The 3′-full length product is protected against digestion by a 3′ - 3′-inverted end. An oligo with a correct 3′-end is, in both schemes, eventually obtained by cleaving with RNase between the ribo unit and the requested DNA-sequence.

- 5′-truncated sequences are removed by Rp HPLC using the DMTr group of the last coupling step (trityl-on synthesis) as a hydrophobic tag.

Very labile protecting groups will be used to avoid problems with deprotection.     

Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide

Abstract

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5′-O-monophosphate, 7-methylinosine 5′-O-diphosphate, and 7-methylinosine 5′-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Catalytic coupling reaction mechanism of 4-nitrobenzenethiol on silver clusters: a density functional theoretical study

The catalytic coupling reaction mechanism of the transformation from 4-nitrobenzenethiol (4-NBT) to 4,4′-dimercaptoazobenzene (4,4′-DMAB) on a silver cluster was studied by density functional theory. Reactants, intermediates, transition states and products were optimized with the B3LYP method using the 6-311 + G(d,p) basis set (Ag using the pseudo potential basis set of LanL2DZ). Transition states and intermediates were confirmed by the corresponding vibration analysis and intrinsic reaction coordinates (IRC). Consistent with literature reports, the key point of the transformation from 4-NBT absorbed on the surface of Ag₅ clusters to 4,4′-DMAB is the elimination of two O atoms on the amino group. Meanwhile, the catalytic coupling reaction of 4-nitrobenzenethiol on a silver cluster is easy to carry out under irradiation. The possibility of “inter system channeling” (ISC) between different potential energy surfaces in the coupling reaction of 4-NBT is further discussed. The irradiation has an auxiliary catalytic effect on the coupling reaction. Our research results can explain the observed experimental phenomena.

Open image in new window

Graphical abstract Catalytic coupling reaction mechanism of the transformation from 4-nitrothiophenol (4-NBT) to 4,4′-dimercaptoazobenzene (4,4′-DMAB) on silver clusters studied by density functional theory

     

The First Stereocontrolled Synthesis of Thiooligoribonucleotide: (RpRp)- and (SpSp)-UpsUpsU

Abstract

An approach to the stereocontrolled synthesis of P-homochiral thiooligoribonucleotide: (Rp,Rp)- and (Sp,Sp)-diastereomers of uridinylyl′(3′, 5′)uridinylyl(3′,5′)uridine di (0,0-phosphorothioate) (9) is decribed. The influence of 2′-protection on the efficiency and stereochemistry of the coupling reaction is discussed.