methyl phosphonamidite

Methyl Phosphonamidites may be used in DNA synthesizers following conventional CE Phosphoramidite protocols to produce oligonucleotides containing one or more methyl phosphonate linkages. However, deprotection and purification techniques differ and a description of the procedures is included with each delivery of these monomers. We also offer the dC monomer with acetyl base protection.1 This protecting group is removed with ammonium hydroxide during the cleavage step, eliminating modification at the dC sites during the deprotection step using ethylenediamine in ethanol.

PACE phosphoramidites

Phosphonoacetate (PACE) modified oligonucleotides show great potential as biological modifiers in a wide variety of research applications. PACE monomers are part of a family of Phosphonocarboxylate monomers. The monomers can be easily incorporated into complex oligonucleotides and are compatible with a wide variety of other sugar or heterobase modifications. PACE DNA can be conjugated through the carboxylic acid functional group. They have been shown to be active in siRNA duplexes and accelerate the initial rate of cleavage by RNase H-1 when incorporated with phosphorothioates. However, the most interesting observation to date is that they exhibit an unprecedented enhancement in penetration of cultured cells.

The phosphonoacetates are fully soluble in acetonitrile at a recommended concentration of 0.1M and are compatible with standard DNA synthesizers. A recommended coupling time of 33.3 minutes with 1H-Tetrazole is necessary when using the standard protocol. A modified LV cycle for AB instruments that reduces coupling time to 15 minutes with 1H-Tetrazole is available on our website. Oxidation must precede capping in the synthesis cycle. Reagents for oxidation depend on the type of synthesis. For fully modified oligos, we recommend the non-aqueous oxidizer camphorsulfonyloxaziridine (CSO) as a 0.1M solution. For mixed phosphodiester and phosphonoacetate modified oligos, a 0.5M CSO solution with a minimum oxidation time of 3 minutes is recommended. Low water oxidizer, 40-4032, is an alternative oxidizing reagent although it has been reported that this can result in conversion of a small percentage of the phosphonoacetate to the phosphodiester. We also recommend the use of the Cap Mix B with DMAP (40-4020) instead of the standard Cap Mix B containing 1-Methylimidazole.

The standard protocol for cleavage and deprotection requires a two step method with pretreatment using 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and subsequent cleavage using methylamine. The DBU is used to deprotect the dimethylcyanoethyl (DMCE) protecting groups and to prevent alkylation of the bases during deprotection. Cleavage with 40% methylamine in water is recommended and we have also had good results when using AMA deprotection.

methyl phosphoramidites

For many years, Glen Research has supplied methyl phosphoramidites in addition to þ-cyanoethyl (CE) phosphoramidites for the few situations where the more labile cyanoethyl group is not an advantage. Some of our customers, probably remembering that the methyl group was removed specifically with thiophenol, have tried to use these monomers to prepare the interesting, uncharged, and nuclease-resistant methyl phosphotriester linkage. Unfortunately, this linkage is labile to ammonium hydroxide and the regular phosphodiester linkage is formed (along with a small amount of chain scission). Now we offer UltraMild methyl phosphoramidites for this application. Oligos produced from these monomers can be deprotected with potassium carbonate in methanol to produce methyl phosphotriester linkages. Since these linkages are diastereomeric and uncharged, the oligos may be hard to handle. Consequently, it is likely that chimeras will be produced using these monomers along with the regular UltraMild CE phosphoramidites. If many dG residues are included in the oligonucleotide, we recommend the use of phenoxyacetic anhydride (PAc₂O) in Cap A. This modification removes the possibility of exchange of the isopropyl-phenoxyacetate (iPr-Pac) protecting group on the dG with acetate from the acetic anhydride capping mix.

ULTRAMILD SOLVENTS/REAGENTS

h-phosphonate monomers

Glen Research H-Phosphonates are analyzed by HPLC and are synthesis-tested. H-Phosphonates are especially useful for the preparation of modified internucleotide linkages which are unattainable by phosphoramidite chemistry. The most popular application is the preparation of radiolabelled phosphorothioates, since the sulfurization reaction is carried out off the synthesizer. These monomers are packaged in ABI-style vials (see note box).

h-phosphonate reagents

All solvents and reagents are prepared to our exacting specifications to ensure the highest coupling efficiencies and are passed through a 0.2 micron filter during packaging to eliminate particulate contamination.

THIOPHOSPHORAMIDITES

Replacing two non-bridging oxygen atoms with sulfur atoms in a DNA phosphodiester linkage creates a phosphorodithioate (PS2) linkage.¹ Like natural DNA, the phosphorodithioate linkage is achiral at phosphorus. This analog is completely resistant to nuclease degradation and forms complexes with DNA and RNA with somewhat reduced stabilities.² Moreover, it has been found that PS2-ODNs bind proteins with a higher affinity than their phosphodiester analogues^2-6 suggesting that PS2-ODNs may have additional utility in the form of sulfur-modified phosphate ester aptamers (thioaptamers)^3,6-8 for therapeutic and diagnostic applications. Thiophosphoramidites are now commercially available after recent work at AM Biotechnologies (www.thioaptamer.com).

1. Thiophosphoramidites (ThioPAs) are not soluble in anhydrous acetonitrile diluent. Rather, 10% DCM (v/v) in acetonitrile is an ideal diluent for all four of the thioPAs for a final amidite concentration of 0.15 M.
2. ThioPAs are somewhat less stable than normal DNA phosphoramidites in anhydrous acetonitrile containing 10% DCM; however, the coupling efficiency of all four thioPAs is not reduced after two days in solution at room temperature.
3. After synthesis, the thioPA bottle on the synthesizer should be replaced with one containing acetonitrile diluent and the synthesizer line flushed with acetonitrile.

A typical cycle for the solid-phase synthesis of a PS2 linkage is different from a standard cycle for the synthesis of normal phosphate linkages. After coupling, the resulting thiophosphite triester is then sulfurized with DDTT. Capping is carried out AFTER sulfurization.

Upon completion of the automated synthesis, deprotection is carried out using a concentrated ammonia:ethanol (3:1, v:v) mix containing 20 mM DTT at 55 °C for 15-16 h.

sulfurizing reagent

Glen Research’s Sulfurizing Reagents are used to prepare phosphorothioate linkages using CE phosphoramidite chemistry. Each reagent exhibits the following attributes:
1) Reliably soluble, making them safe to use on automated synthesizers.
2) Reaction is fast (30 seconds), making the process convenient on small scales and readily amenable to scale-up.
3) Process is efficient, with better than 96% of the linkages being phosphorothioate and the remainder being phosphodiester.
When ordering Sulfurizing Reagent (Beaucage Reagent), please also order the silanized bottle appropriate for your instrument. There will be no charge for one bottle when supplied with a reagent order. The silanized bottle can be reused after rinsing with acetonitrile and drying. Additional silanized bottles are priced as shown below. Dissolve the reagent in acetonitrile at a concentration of 1g/100mL.

Sulfurizing Reagent II (3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT) exhibits all the properties of Beaucage Reagent while adding stability in solution on the synthesizer AND offering strong ability to sulfurize RNA linkages. Sulfurizing Reagent II is available in powder form and as a stable solution.