Chemical synthesis of a core 2 branched pentasaccharide containing a carboxylate group

Design and synthesis of a carboxylate-containing pentasaccahride 1 with the Galbeta(1-4) (Fucalpha1-3)GlcNAcbeta(1-6)[3-[1-carboxymethyl]-Galbeta+ ++(1-3)]GalNAcalpha-OMe sequence, which is obtained through regioselective coupling of the 6-OH of a novel acceptor 9 with Lewis(x) donor 10 catalyzed by NIS-TfOH are described.     

A new approach to the synthesis of branched and branched cyclic oligoribonucleotides.

The six-step synthesis of the di-triethylammonium salt of 5[prime]-O -trityl-6-N-pivaloyladenosine-2[prime]-(H -phosphonate)-3'-[(2-chlorophenyl) phosphate]9 from 3', 5'- O -(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-6-N-pivaloyla denosine1in 68% overall yield is described. Compound9is converted into a branched pentaribonucleoside tetraphosphate 24 and a branched cyclic pentaribonucleotide ('lariat') 25 by solution phase triester chemistry involving both H-phosphonate and conventional phosphotriester coupling reactions. The monomeric building block 9 is proposed as a universal synthon for the preparation of branched and branched cyclic oligoribonucleotides derived from adenosine.     

The ral gene: a new ras related gene isolated by the use of a synthetic probe.

We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B-lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras.     

A new method for the synthesis of a structural gene.

A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the N-terminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short inter-fragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.     

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.     

Structural studies on C-2 substitution in a new set of synthetic aminodideoxy sugars: the steric bulk at C-2 influences the puckering of the pyranose ring

Synthesis and single-crystal X-ray structural analyses of selected aminodideoxy sugars with different group substitutions at the C-2 position were carried out. Product formation and X-ray crystallographic determination of the products from C-2 substitution in both alpha and beta anomers were studied. The observed variation in pyranose ring conformations in product compounds is explained in terms of C-2 substitution.     

Rapid synthesis of oligodeoxyribonucleotides: a new solid-phase method.

A method is described that makes use of a new polyamide resin for the rapid synthesis of short oligodeoxyribonucleotides. The method is illustrated by the preparation of two heptadeoxyribonucleotides, d(pT6-C) and d(pC-A-G-T-G-A-T) using a phosphodiester approach. A further development involved use of phenyl isocyanate as an in situ drying agent, which obviated the need for solvent co-evaporation prior tothe internucleotidic coupling steps. Improved fractionation of thymidyl oligonucleotides was obtained by use of a new microparticulate, silica-based anion-exchanger.     

A facile method for the construction of synthetic genes

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA     

Chemical synthesis of a self-complementary octanucleotide, dG-G-T-T-A-A-C-C by a modified triester method.

The synthesis of a self-complementary octanucleotide, d(G-G-T-T-A-A-C-C-), using a modified triester approach is described. The protected dinucleotides, d(Me2O)TribG(C1C6H4) ibG, d(Me2O)TrT(ClC6H4)T, d(Me2O)TrbzA(ClC6H4)bzA, and d(Me2O)TranC(ClC6H4)anC were synthesized by a one step triester procedure. After removal of the trityl group, the dinucleotides, dT(ClC6H4)T and danC (ClC6H4)anC were coupled to d(Me2O)TribG(ClC6H4)ibG and d(Me2O)TrbzA(ClC6H4)bzA, respectively to yield the respective tetranucleotides. The tetranucleotide, d(Me2O)TrbzA(ClC6H4)bzA(ClC6H4) and (ClC6H4)anC was detritylated and then coupled with d(Me2O)TribG(ClC6H4)ibG(ClC6H4)T(Cl6H4)T to give octanucleotide. The fully protected octanucleotide was deblocked by treatment with aqueous NH3 followed by acid and was characterized by nucleotide sequence analysis.     

Preparation polyacrylamide-agarose gel electrophoresis of proteins and RNAs by the use of new device.

Quantitatively reproducible results were obtained by using a new device for preparation gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.     

An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs

RNA ligation has been a powerful tool for incorporation of cross-linkers and nonnatural nucleotides into internal positions of RNA molecules. The most widely used method for template-directed RNA ligation uses DNA ligase and a DNA splint. While this method has been used successfully for many years, it suffers from a number of drawbacks, principally, slow and inefficient product formation and slow product release, resulting in a requirement for large quantities of enzyme. We describe an alternative technique catalyzed by T4 RNA ligase instead of DNA ligase. Using a splint design that allows the ligation junction to mimic the natural substrate of RNA ligase, we demonstrate several ligation reactions that appear to go nearly to completion. Furthermore, the reactions generally go to completion within 30 min. We present data evaluating the relative importance of various parameters in this reaction. Finally, we show the utility of this method by generating a 128-nucleotide pre-mRNA from three synthetic oligoribonucleotides. The ability to ligate synthetic or in vitro transcribed RNA with high efficiency has the potential to open up areas of RNA biology to new functional and biophysical investigation. In particular, we anticipate that site-specific incorporation of fluorescent dyes into large RNA molecules will yield a wealth of new information on RNA structure and function.     

Chemical synthesis and expression of a synthetic gene for the flavodoxin from Clostridium MP

A gene coding for the flavodoxin from Clostridium MP was designed, synthesized, and expressed in Escherichia coli. The sequence of the coding region was derived from the published amino acid sequence of the protein (Tanaka, M., Haniu, M., Yasunobu, K.T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393-4397) and was designed for optimal expression and for use of the cassette mutagenesis approach. The structural gene was subassembled in three sections, each of which was constructed by the enzymatic ligation of three complementary pairs of chemically synthesized oligodeoxyribonucleotides having short single-stranded ends complementary to that of the adjacent pair. Coligation of the three sections produced the final structural gene which consists of 420 nucleotides. The synthetic gene was cloned behind the hybrid tac promoter (Amman, E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) in the pKK223-3 vector or adjacent to the strong T7 RNA polymerase promoter in the pET-3a expression vector (Rosenberg, A.H., Lade, B. N., Chui, D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135) for expression in E. coli. Upon induction with isopropyl-beta-D-thiogalactoside, the flavodoxin polypeptide was expressed from the artificial gene to levels approaching 20% of total extractable proteins using either expression system. The flavodoxin was purified from cellular extracts as the holoprotein containing bound flavin mononucleotide. The recombinant flavodoxin protein was found to have an ultraviolet/visible spectrum, amino-terminal sequence, and amino acid composition identical to the wild-type flavodoxin protein purified from Clostridium MP. This work represents the first chemical synthesis and expression in E. coli of an artificial gene coding for a bacterial flavodoxin.     

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.     

Targeted O2 delivery by low-P50 hemoglobin: a new basis for O2 therapeutics

To assess O2 delivery to tissue by a new surface-modified, polyethylene glycol-conjugated human hemoglobin [MP4; Po2 at 50% saturation of hemoglobin (P50); 5.4 mmHg], we studied microcirculatory hemodynamics and O2 release in golden Syrian hamsters hemodiluted with MP4 or polymerized bovine hemoglobin (PolyBvHb; P50 54.2 mmHg). Comparisons were made with the animals' hemodiluted blood with a non-O2 carrying plasma expander with similar solution properties (Dextran-70). Systemic hemodynamics (arterial blood pressure and heart rate) and acid-base parameters were not correlated with microhemodynamics (arteriolar and venular diameter, red blood cell velocity, and flow). Microscopic measurements of Po2 and the O2 equilibrium curves permitted analysis of O2 release in precapillary and capillary vessels by red blood cells and plasma hemoglobin separately. No significant differences between the groups of animals with respect to arteriolar diameter, flow, or flow velocity were observed, but the functional capillary density was significantly higher in the MP4-treated animals (67%) compared with PolyBvHb-treated animals (37%; P < 0.05) or dextran-treated animals (53%). In the PolyBvHb-treated animals, predominant O2 release (both red blood cells and plasma hemoglobin) occurred in precapillary vessels, whereas in MP4 animals most of the O2 was released from both red blood cells and plasma hemoglobin in capillaries. Base excess correlated directly with capillary O2 release but not systemic O2 content or total O2 release. Higher O2 extraction of both red blood cell and plasma hemoglobin in capillaries represents a new mechanism of action of cell-free hemoglobin. High O2 affinity appears to be an important property for cell-free hemoglobin solutions.     

Launching insectphylo.org; a new hub facilitating construction and use of synthesis molecular phylogenies of insects

The Holy Grail of an Insect Tree of Life can only be ‘discovered’ through extensive collaboration among taxon specialists, phylogeneticists and centralized frameworks such as Open Tree of Life, but insufficient effort from stakeholders has so far hampered this promising approach. The resultant unavailability of synthesis phylogenies is an unfortunate situation given the numerous practical usages of phylogenies in the near term and against the backdrop of the ongoing biodiversity crisis. To resolve this issue, we establish a new online hub that centralizes the collation of relevant phylogenetic data and provides the resultant synthesis molecular phylogenies. This is achieved through key developments in a proposed pipeline for the construction of a species-level insect phylogeny. The functionality of the framework is demonstrated through the construction of a highly supported, species-comprehensive phylogeny of Diptera, built from integrated omics data, COI DNA barcodes, and a compiled database of over 100 standardized, published Diptera phylogenies. Machine-readable forms of the phylogeny (and subsets thereof) are publicly available at insectphylo.org , a new public repository for species-comprehensive phylogenies for biological research.     

A synthetic method for peptide-PEG-lipid conjugates: application of octreotide-PEG-DSPE synthesis

A solid phase synthesis method was established for the synthesis of peptide-poly(ethylene glycol)-lipid (peptide-PEG-lipid) conjugates. Octreotide-PEG(2000)-DSPE (OPD(2000)) was used as an example to demonstrate the synthetic approach. The OPD(2000) obtained had confirmed structure, activity, and purity providing a targeting molecule for preparation of well-defined drug delivery systems, such as targeted liposomes, for further studies.     

Mammalian tyrosinase: isolation by a simple new procedure and characterization of its steric requirements for cofactor activity.

A method for the isolation of tyrosinase is described, which involves preparative polyacrylamide gel electrophoresis, requires only 24 to 36 h to carry out, and yields ostensibly homogeneous enzyme. The ability of purified tyrosinase to utilize 3,4-dihydroxyphenylalanine (dopa) analogs as cofactors was determined for both of the reactions catalyzed by tyrosinase: (i) tyrosine hydroxylation and (ii) dopa oxidation and melanin formation. The cofactor analogs studied included those in which steric modifying groups were added and those in which substitutions were made at the location of the amine, carboxylic acid, or hydroxyl groups of dopa. The results indicate that each of these groups is essential for maximal enzyme activity and that each is optimally located for tyrosinase activation when in the precise steric conformation found in l-dopa.