Bogdan Uznanski

Bis(O,O-diisopropoxy phosphinothioyl) disulfide - a highly efficient sulfurizing reagent for cost-effective synthesis of oligo(nucleoside phosphorothioate)s

Abstract A new sulfurizing reagent is reported for the automated synthesis of phosphothioate analogues of oligonucloetides via the phosphoramidite method. Bis(O,diisopropoxy phosphinothioyl) disulfide (S-Tetra, 1 ) is relatively inexpensive to prepare, easy to handle, and efficiently sulfurizes internucleotide phosphites, thus allowing the practical synthesis of oligo(nucleoside phosphorothioate)s with exceptionally high sulfur content.

Synthesis and absolute configuration of P-chiral O-isopropyl oligonucleotide triesters

Abstract New O -isopropylphosphomorpholidite reagents provided the title compounds as mixtures of P-chiral diastereomers, which were separated by HPLC for enzymatic digestion studies and assignment of configuration at phosphorus by chemical correlation with known phosphorothioates.

The chemical synthesis of the RP and SP diastereomers of thymidyl(3′-5′)thymidyl 0,0-phosphorothioate

The synthesis and separation of diastereomere of protected thymidyl(3′–5′)thymidyl 0,0-phosphoranilidate (4) allowed to obtain in the stereospecific manner title compounds 5, whose absolute configuration at P atom was assigned enzymatically. TP(S)T diastereomers (5) were obtained independently via “phosphite” procedure.

Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome

The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7. The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21). The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product. Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz. The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1. Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates. Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program. The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.

The solid-phase synthesis of 2'-5'-linked oligoriboadenylates containing 8-bromoadenine

To increase the accessibility of 8-bromo-2′,5′-oligoadenylates, we developed a synthesis of 2′-5′-linked oligoriboadenylates containing varying numbers of 8-bromoadenosine residues based on the use of a CPG-LCA solid support and the phosphoramidite approach. Although N6benzoyl protection was satisfactory for incorporation of nonmodified adenine residues into 2′,5′-oligonucleotides, the effective incorporation of 8-bromoadenine into such 2′,5′-linked oligomers required use of a non acyl protecting group. Amidine protection of the purine exocyclic amino function proved compatible with all aspects of the phophoramidite approach and with the hydroxyl protection groups employed.

Deoxyribonucleoside 3′-phosphordiamidites as substrates for solidsupported synthesis of oligodeoxyribonucleotides and their phosphorothioate and dna-triester analogues

Abstract 5′-O-Dimethoxytrityl-base protected-nucleoside 3′-O-phosphordimorpholidites can be successfully used for the synthesis of oligodeoxyribonucleotides and their phosphorothioate or “triester” analogues.

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH4OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme...

NMR Studies of Backbone-Alkylated DNA: Duplex Stability, Absolute Stereochemistry, and Chemical Shift Anomalies of Prototypal Isopropyl Phosphotriester Modified Octanucleotides, (Rp,Rp)- and (Sp,Sp)-{d-[GGA(iPr)ATTCC]}2 and - {d- [GGAA(iPr)TTCC]}2

The DNA octamer (d-[GGAATTCC])2 and four alkylated analogues, (Rp)-(d-[GGA(iPr)ATTCC])2, (Sp)-(d-[GGA(iPr)ATTCC])2, (Rp)-(d-[GGAA(iPr)TTCC])2, and (Sp)-(d-[GGAA(iPr)TTCC])2 have been examined using 1H and 31PNMR spectroscopies. Duplex stability, as monitored by both NMR and optical measurements, is shown to be a function of both site and stereochemistry of the phosphotriester moiety. Chemical shift changes relative to the native octamer indicate that there are long-range perturbations in the isopropylated molecules. 1HNMR is shown to be a general means by which stereochemistry at phosphorous can be determined.

Phosphate-Modified Oligonucleotides. The Synthesis, Stereochemistry and ECO Ri Endonuclease Substrate Ability of Decanucleotides d[GGGAATTCCC] Bearing Altered Internucleotide Phosphate Function Between A and A1

Abstract An interaction between Eco RI endonuclease and decadeoxyribo-nucleotide GGGAATTOOC is followed by means of oligonucleotide analogues bearing modified internucleotide phosphate functions bridging both adenosine residues. While an o-alkyl group at this phosphate, despite the “side” of DNA alkylation, completely prevents DNA from hydrolysis, a phosphorothioate function replacing phosphate at the position between A and A moieties controls the hydrolysis in terms of the absolute configuration at phosphorus. The fact, that the Rp-isomer of d[GGGA(S)ATTOOC] possessing sulphur atom directed “inward” DNA is hydrolyzed by Eco RI endonuclease may indicate, that the pro-S oxygen at this particular phosphate is involved in an interaction with magnesium ion, a necessary factor for executive action of this endonuclease.

REMOTE CONTROL OF DIASTEREOSELECTIVITY OF DN-ASE II AND ENDONUCLEASE ECO RI TOWARDS PHOSPHOROTHIOATE ANALOGUES OF OLIGONUCLEOTIDES

Abstract (Rp)-Thymidyl 3′-(4-nitrophenyl phosphorothioate) (TP(S)NP) is converted in the presence of DN-ase II into (Sp,Rp)-thymidyl (3′–5′)-thymidyl phosphorothioate 3′-(4-nitrophenyl phosphorothioate) (TP(S)TP(S)NP), while (Sp)-TP(S)NP under analogous conditions gives (Rp,Sp)-TP(S)TP(S)NP, but as a minor product only. The preponderant product was recognized as (Sp,Rp)-thymidyl (3′–5′) thymidyl phosphorothioate 5′–(4-nitrophenyl phosphorothioate) (NPP(S)TP(S)T). All phosphorothioyl transfer reactions occur with retention of configuration at phosphorus atoms, which speaks for a double-displacement process and involvement of phosphorothioylated enzymes as reactive intermediates. Nucleolytic activity of DN-ase II, characteristic for this enzyme with respect to natural oligonucleotides, is not observed if TP(S)NP are used as the substrates. However, the transferase activity is even extended for the (4-nitrophenyl phosphorothioate)-shift from 3′ to 5′-position of dinucleotide, but for diastereoisomer (Rp,Sp)-...