Marvin H. Caruthers

Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis

Abstract The development of a new class of nucleoside phosphites is described. These compounds are stable to normal laboratory conditions, are activated by mild acid treatment, and are observed to react essentially quantitatively with protected nucleosides.

An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides

Abstract Various deoxynucleoside N,N-dialkylaminomethoxyphosphines were examined for stability and reactivity in deoxyoligonucleotide synthesis. Of those examined, the N-morpholino and N,N-diisopropylamino derivatives most completely satisfied all criteria.

Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method.

Publisher Summary This chapter discusses the chemical synthesis of deoxy oligonucleotides by the phosphoramidite method. It discusses the synthesis methodology; detailed protocols for preparing the silica support, the phosphoramidite, and deoxy oligonucleotides; and the purification of synthetic deoxyribo nucleic acid (DNA). The phosphite triester approach to DNA synthesis using deoxynucleoside phosphoramidite as synthons has become the method of choice for the preparation of deoxy oligonucleotides. The general synthetic strategy involves adding mononucleotides sequentially to a deoxynucleoside, attached covalently to a silica-based insoluble polymeric support. Reagents, starting materials, and side products are then removed simply by filtration. At the conclusion of the synthesis, the deoxy oligonucleotide is chemically freed of blocking groups, hydrolyzed from the support, and purified to homogeneity by either polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC). These preformed synthons are especially attractive for preparing DNA on automatic or semiautomatic DNA synthesizers, or for those who plan to manually synthesize a large number of deoxy oligonucleotides.

Total Synthesis of the Gene for an Alanine Transfer Ribonucleic Acid from Yeast

By exploiting the natural ability of polynucleotides to align by base pairing and using polynucleotide kinase and ligase, chemically synthesized segments have been combined into a double stranded DNA corresponding to the gene for the earliest characterized tRNA.

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).

CIII. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast

Abstract A plan for the total synthesis of the DNA duplex, 77 nucleotide units long, corresponding in sequence to the major yeast alanine transfer RNA, is formulated. The plan involves: (a) the chemical synthesis of 15 polydeoxynueleotide segments ranging in length from five to 20 nucleotide units and (b) ligase-catalyzed covalent joining of several segments to form three parts of the duplex, followed by joining of the three parts to construct the entire duplex. Twelve accompanying papers describe the experimental realization of this objective.

The synthesis of oligodeoxyprimidines on a polymer support

Abstract A polymer supported method for synthesizing deoxypolynucleotides is described. Two non-anucleotides were synthesized. Yields exceeding 90% were obtained for each condensation. The time per nucleotide addition was four hours.

Synthesis of oligodeoxynucleotides and oligodeoxynucleotide analogs using phosphoramidite intermediates

Abstract The synthesis of two deoxyoligonucleotides, d(G-T-G-A-G-T-T-A-G-C-T-C-A-C) and d(G-T-G-A-G-C-T-A-A-C-T-C-A-C), corresponding to the DNA binding site for cyclic AMP receptor protein is reported. These syntheses have been completed in milligram quantities using a silica gel polymer support methodology and mononucleotide phosphoramidites. Procedures are also reported for synthesizing diastereoisomers of dinucleoside methylphosphonate 3 - phosphoramidites.

Synthesis and characterization of dinucleoside phosphorodithioates

Abstract Dinucleoside phosphoramidites, H 2 S, and tetrazole react to form dinucleoside H-phosphonothioates. Oxidation with sulfur yields phosphorodithioates. Iodine oxidation in the presence of amines, alcohols, or water yields phosphorothioamidates, thiotriesters or thiodiesters.

Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs.

Publisher Summary This chapter discusses advances in chemical methodologies, which include the development of new protecting group strategies and procedures for the attachment of reporter groups to DNA. Because of these achievements, the time required to synthesize DNA has been reduced, and an even larger repertoire of applications for synthetic deoxyoligonucleotides has been achieved. An attempt is made to review some of these many excellent contributions, but only one detailed protocol for labeling DNA is included. This is a relatively recent, unpublished procedure for attaching reporter groups to a new DNA analog called “dithioate DNA.” Deoxyoligonucleotides having a deoxynucleoside–OPS 2 O–deoxynucleoside linkage, dithioate DNA, are excellent mimics of natural DNA. At least partially, this is because they are isosteric and isoelectronic with normal DNA. They are, however, completely resistant to snake venom, calf spleen, P 1 exonucleases, all the nucleases found in HeLa nuclear and cytoplasmic extracts, and even the very potent exonucleolytic activity of T 4 DNA polymerase. These attractive features combined with the ability of dithioate DNA to form stable DNA duplexes have led to several potential applications.