Total synthesis of a tyrosine suppressor transfer RNA gene. XIII. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region.

Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.     

Total synthesis of a tyrosine suppressor transfer RNA gene. XIV. Chemical synthesis of oligonucleotide segments corresponding to the terminal regions.

Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described. These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G. (1976) J. Biol. Chem. 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end. The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G. (1976) J. Biol. Chem. 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end. The syntheses used protected mono- and oligonucleotides and stepwise condensation methods. A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures.     

Rapid synthesis of oligodeoxyribonucleotides. II. Machine-aided solid-phase syntheses of two nonanucleotides and an octanucleotide.

Preparation of the two nonanucleotides, d(pG-G-A-G-G-A-G-A-A) and d(pT-T-C-T-G-T-T-G-A), and the octanucleotide, d(pT-G-G-C-C-C-A-T) is described by a rapid solid-phase method on a polyamide support. The syntheses were carried out using a modified solid-phase peptide synthesiser and required one day for each nucleotide addition. A microparticulate anion-exchanger, Partisil 10SAX, was used for the efficient fractionation of synthetic, base-protected oligonucleotides.     

Glycopeptide dendrimers, part III: a review. Use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases.

Glycopeptide dendrimers containing different types of tumor associated-carbohydrate antigens (T(N), TF, sialyl-T(N), sialyl-TF, sialyl-Le(x), sialyl-Le(a) etc.) were used in diagnosis and therapy of different sorts of cancer. These dendrimeric structures with incorporated T-cell epitopes and adjuvants can be used as antitumor vaccines. Best results were obtained with multiantigenic vaccines, containing, e.g. five or six different TAAs. The topic of TAAs and their dendrimeric forms at molecular level are reviewed, including structure, syntheses, and biological activities. Use of glycopeptide dendrimers as antiviral vaccines against HIV and influenza is also described. Their syntheses, physico-chemical properties, and biological activities are given with many examples.     

Mechanistic studies on the phosphoramidite coupling reaction in oligonucleotide synthesis. I. Evidence for nucleophilic catalysis by tetrazole and rate variations with the phosphorus substituents.

Tetrazole catalyzed reactions of a series of phosphoramidites, 5'-O-DMTdT-3'-O-P(OR1)NR2(2) (1a-h), with 3'-O-SiButPh2-6-N-benzoyl-dA (2a) in acetonitrile solution have been studied. It is found that the coupling rate depends very much on whether tetrazole is added before or after 2a, and that dialkylammonium tetrazolide salts are inhibitors. These and other facts are evidence that the reactions are subjected to nucleophilic catalysis by tetrazole, in addition to acid catalysis. The rate variations with phosphorus substituents of 1a-h are NEt2 greater than NPri2 greater than N(CH2CH2)O greater than NMePh, and OMe greater than OCH2CH2CN greater than OCHMeCH2CN greater than OCMe2CH2CN much greater than OC6H4Cl. The inhibitor properties of dialkylammonium tetrazolides have practical consequences for the efficiency of DNA syntheses, when in situ prepared phosphoramidites are used; the same would apply for segmented, simultaneous syntheses or syntheses where recycling is performed.     

Synthesis of N-unsubstituted, mono- and disubstituted carbohydrate-1-O-carbamates and their behaviour in glycoside syntheses

The syntheses of 44 1-carbamates from six different 1-O-unprotected carbohydrate derivatives (compounds 1-6), representing typical protecting pattern in glycoside synthesis, are described. The carbamate function is N-unsubstituted (compounds 1b-6b), mono- (compounds a: N-trichloroacetyl, c: N-monochloroacetyl, d: N-acetyl, e: N-ethyl, f: N-allyl, g: N-phenyl) or disubstituted (compounds h: imidazolyl, i: N-diethyl, j: N-diphenyl). Additionally, three N-chlorosulfonyl carbamates are synthesized and used as intermediates for the synthesis of N-unsubstituted compounds b. The accessibility of these compounds is described and compared. Some of the carbamates (1, 4, 5a-j) are used as model compounds for systematic investigations in glycoside syntheses. Selected experimental data (reaction conditions, anomeric ratios, rotation values, selected NMR data) are tabulated.     

Abasic oligodeoxyribonucleoside phosphorothioates: synthesis and evaluation as anti-HIV-1 agents.

The syntheses and anti-HIV-1 evaluations of two, abasic oligodeoxyribonucleotide phosphorothioate analogs, d[Cps(Eps)26C] and d[Cps(Vps)26C] (where E and V derive from 1,2-dideoxy-D-ribofuranose and (+/-)-butane 1, 3-diol, respectively), are described.     

A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports.

A new approach is described which will allow the simultaneous synthesis of large numbers of pre-defined oligonucleotide chains. No machine aid is needed. The simultaneous syntheses can be performed by one person and do not require much more time than is currently needed for the synthesis of just one oligonucleotide in existing strategies. The general idea is the following: One uses noninterchangeable polymeric entities from each of which enough OD units can be isolated after completion of the syntheses. Whenever growing chains on different entities have to be elongated with the same building block these entities are gathered in the same reaction vessel. After such a common reaction cycle the entities are separated and now combined according to the next common building blocks etc. The practicability of this approach is demonstrated by the synthesis of d(T-A-A-T-A-T-T-A) and d(T-A-G-T-A-C-T-A) on cellulose filter disks following the phosphotriester approach.     

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 4: 3-Alkyl-4-halo-6-nitroquipazines

On the basis of the structure-activity relationship (SAR) of 4-chloro-6-nitroquipazine (Ki = 0.03 nM) and 3-fluoropropyl-6-nitroquipazine (Ki = 0.32 nM), 3-alkyl-4-halo-6-nitroquipazines were synthesized and tested for their potential abilities in vitro to displace [3H]citalopram binding to the rat cortical membranes. Binding affinities of 3b and 4d were Ki = 2.70+/-0.32 and 2.23+/-0.46 nM, respectively. The syntheses of 3-alkyl-4-halo-6-nitroquipazine, their in vitro binding affinities, and the SAR of C3, C4 position in 6-nitroquipazine are described.     

Iodoestrogens, syntheses, and interaction with uterine receptors.

The rationale for undertaking the present study was to evaluate the utility of iodoestradiol analogs made highly radioactive with iodine isotopes in (a) the non-invasive differentiation of estrogen-dependent from estrogen-independent breast tumors, (b) spread of metastases containing estrogen receptors, and (c) potential application in therapeutic irradiation of target tissues. In the present paper, the model syntheses of a number of nonradioactive 127I-estrogen analogs are described. The analogs were tested for their ability to displace (compete with) [3H]estradiol from receptor sites. The most active compounds, 16beta-iodoestra-1,3,5(10)-triene-3,17 beta-diol (17) and 6-iodoestra-1,3,5(10),6-tetraene-3,17 beta-diol (10b), showed a relative binding affinity of 0.57 and 0.49, respectively.     

Synthesis and X-ray crystal structures of substituted fluorobenzene and benzoquinone inhibitors of the tissue factor VIIa complex

Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa (TF/VIIa) enzyme. The compounds exhibited modest potency on TF/VIIa with selectivity over Factor Xa and thrombin. The X-ray crystal structures of the targeted fluorobenzene 12a and benzoquinone 14 inhibitors bound to TF/VIIa were obtained and will be described.     

Synthesis and properties of oligodeoxynucleotides with an AP site at a preselected position.

A general synthesis of a deoxyoligonucleotide with an AP site at a preselected sequence is described. Deoxyuridine is introduced during routine oligonucleotide syntheses of d(TTTUTTTT) and d(AAAAGTTUAAAACAT). Treatment with uracil DNA-glycosylase produces d(TTTrTTTT), where r = deoxyribose, and d(AAAAGTTprpAAAACAT). KM and Vmax are: d(TTTUTTTT), 7.3 X 10(-9)M and 2.0 X 10(-9) mumol/min; d(AAAAGTTUAAAACAT), 1.5 X 10(-8) M and 6.4 X 10(-9) mumol/min. Both d(AAAAGTTprpAAAACAT) and d(TTTprpTTTT) undergo rapid beta-elimination in 1 M piperidine at 25 degrees giving two oligonucleotide fragments, d(AAAAGTTpr') and d(pAAAACAT), where r' = -O-CH2-CHOH-CH=CH-CHO (or its hemiacetal form). The fragment, d(AAAAGTTpr'), which can be isolated by reverse phase chromatography, is resistant to the 3'----5' exonuclease activity of snake venom phosphodiesterase. Endonucleolytic hydrolysis of the penultimate phosphodiester occurs removing pTpr' and generating a normal 3'-OH end. In 1 M piperidine at 90 degrees two beta-eliminations occur producing the oligonucleotides d(AAAAGTTp) and d(pAAAACAT) from d(AAAAGTTprpAAAACAT); d(TTTp) and d(pTTTT) from d(TTTprpTTTT).     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

Synthesis of novel ammonium and selenonium ions and their evaluation as inhibitors of UDP-galactopyranose mutase

The syntheses of two ammonium salts of 1,4-dideoxy-1,4-imino-d-galactitol containing erythritol sulfate side chains are described. The parent compound is a known inhibitor of the enzyme UDP-galactopyranose mutase (UDP-galactopyranose furanomutase, E.C. 5.4.99.9), which is responsible for the conversion of UDP-galactopyranose into UDP-galactofuranose and is presumably protonated in its active form. The side chain was chosen because it is present in a known sulfonium ion alpha-glucosidase inhibitor, salacinol. The syntheses of the selenonium analogues derived from 1,4-dideoxy-1,4-seleno-d-galactitol are also described. The synthetic strategy in the syntheses of all four salts involved the nucleophilic attack of a protected derivative of the alditol at the least hindered carbon of 2,4-O-benzylidene d- or l-erythritol-1,3-cyclic sulfate to give adducts that were subsequently deprotected. The importance of different protecting groups used in the various syntheses is also highlighted. Enzyme inhibition assays carried out on these compounds, and the corresponding sulfonium ions synthesized previously, show that they are poor inhibitors of UDP-galactopyranose mutase.     

Syntheses of novel substituted-boranophosphate nucleosides

A number of substituted (borano) nucleic acids, 3'-[diethylphosphite(cyano, carboxy, or carbamoyl) borano] deoxynucleosides (3a-4c) and 5'-[diethylphosphite(cyano or carboxy) borano] deoxynucleosides (6a-7d) were prepared by a variety of synthetic procedures. The syntheses of the pyrophosphates (2a-2c), as precursors for 3a-4c, are also described.     

Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly(vinyl alcohol) cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Peptide Synthesis in Organic Media with the Use of Subtilisin 72 Immobilized on a Poly(Vinyl Alcohol) Cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.     

Synthesis and characterization of DNA quadruplexes containing T-tetrads formed by bunch-oligonucleotides

The solid phase syntheses of the bunch oligonucleotides and based on the sequences of the natural oligodeoxynucleotides (ODNs) d(TG2TG2C) and d(CG2TG2T), respectively, attached to a non-nucleotidic tetrabranched linker, are reported. Bunch-ODNs and were shown to form more stable monomolecular parallel G-quadruplexes and when compared with their tetramolecular counterparts [d(TG2TG2C)]4 and [d(CG2TG2T)]4, respectively. The structure and stability of all the synthesized complexes have been investigated by circular dichroism (CD), CD thermal denaturation experiments, and 1H-NMR (nuclear magnetic resonance) experiments at variable temperatures. Particularly, the spectroscopic data confirmed that 1 adopts a T-tetrad containing parallel-stranded quadruplex structure as in the tetramolecular complex.     

Macromolecular synthesis in synchronized cultures of Anacystis nidulans.

Synchronous culture of Anacystis nidulans has been induced by the light-dark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.