Backbone Modification (Catalog as PDF)

methyl phosphonamiditeS

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 proceduresis included in the Technical Bulletin. 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.
Item Catalog No. Pack Price ($)
10-1100-02 0.25g 90.00
10-1100-05 0.5g 180.00
10-1115-02 0.25g 90.00
10-1115-05 0.5g 180.00
10-1120-02 0.25g 90.00
10-1120-05 0.5g 180.00
10-1130-02 0.25g 90.00
10-1130-05 0.5g 180.00
dA-Me Phosphonamidite
dA-Me Phosphonamidite
Ac-dC-Me Phosphonamidite
Ac-dC-Me Phosphonamidite
dG-Me Phosphonamidite
dG-Me Phosphonamidite
dT-Me Phosphonamidite
dT-Me Phosphonamidite

REFERENCE

  1. M.P. Reddy, F. Farooqui, and N.B. Hanna, Tetrahedron Lett., 1996, 37, 8691-8694.

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septumcapped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers  

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Applied Biosystems 3900
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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 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.
Item Catalog No. Pack Price ($)
10-1140-02 0.25g 100.00
10-1140-05 0.5g 200.00
10-1140-10 1.0g 400.00
10-1150-02 0.25g 100.00
10-1150-05 0.5g 200.00
10-1150-10 1.0g 400.00
10-1160-02 0.25g 100.00
10-1160-05 0.5g 200.00
10-1160-10 1.0g 400.00
10-1170-02 0.25g 100.00
10-1170-05 0.5g 200.00
10-1170-10 1.0g 400.00
40-4631-52 200mL 85.00
40-4631-52E 200mL 85.00
dA-PACE
dA-PACE
Ac-dC-PACE
Ac-dC-PACE
dG-PACE
dG-PACE
dT-PACE
dT-PACE
0.10M CSO in Anhydrous Acetonitrile
0.10M CSO in Anhydrous Acetonitrile

Intellectual Property

These products are covered by US Patents 6,693,187 and 7,067,641, patents pending and foreign counterparts owned by Lievre Cornu. Purchase of all or any of these products includes a limited license to use the product solely in the manufacture of oligonucleotides for RESEARCH USE ONLY and its use is not authorized nor intended for diagnostic or therapeutic use.

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septumcapped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers  

For Instrument type
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Expedite
E
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M

Columns  

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E
Applied Biosystems 3900
A
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Please inquire for availability of columns for other instrument types.

See Also

methyl phosphoramidites

For many years, Glen Research has supplied methyl phosphoramidites in addition to β-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). 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.
Item Catalog No. Pack Price ($)
10-1301-02 0.25g 25.00
10-1301-05 0.5g 50.00
10-1301-10 1.0g 100.00
10-1315-02 0.25g 25.00
10-1315-05 0.5g 50.00
10-1315-10 1.0g 100.00
10-1321-02 0.25g 25.00
10-1321-05 0.5g 50.00
10-1321-10 1.0g 100.00
10-1330-02 0.25g 25.00
10-1330-05 0.5g 50.00
10-1330-10 1.0g 100.00
Pac-dA-Me
Pac-dA-Me
Ac-dC-Me
Ac-dC-Me
iPr-Pac-dG-Me
iPr-Pac-dG-Me
dT-Me
dT-Me

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septumcapped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers  

For Instrument type
Add
Expedite
E
Mermade
M

Columns  

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Expedite
E
Applied Biosystems 3900
A
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M

Please inquire for availability of columns for other instrument types.

ULTRAMILD SOLVENTS/REAGENTS

Item Catalog No. Pack Price ($)
Cap Mix A
40-4210-52 200mL 140.00
  (Applied Biosystems) 40-4210-57 450mL 300.00
40-4212-52 200mL 140.00
  (Expedite) 40-4212-57 450mL 300.00
Deprotection Solution
60-4600-30 30mL 30.00

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).
Item Catalog No. Pack Price ($)
10-1200-02 0.25g 40.00
10-1200-05 0.5g 80.00
10-1210-02 0.25g 40.00
10-1210-05 0.5g 80.00
10-1220-02 0.25g 40.00
10-1220-05 0.5g 80.00
10-1230-02 0.25g 40.00
10-1230-05 0.5g 80.00
dA-H-Phosphonate, TEA Salt
dA-H-Phosphonate, TEA Salt
dC-H-Phosphonate, DBU Salt
dC-H-Phosphonate, DBU Salt
dG-H-Phosphonate, TEA Salt
dG-H-Phosphonate, TEA Salt
dT-H-Phosphonate, TEA Salt
dT-H-Phosphonate, TEA Salt

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septumcapped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers  

For Instrument type
Add
Expedite
E
Mermade
M

Columns  

For Instrument type
Add
Expedite
E
Applied Biosystems 3900
A
Mermade
M

Please inquire for availability of columns for other instrument types.

h-phosphonate reagents

Our H-Phosphonate solvents and reagents have been discontinued. H-Phosphonate reagents are easily prepared using high purity products and the formulations shown below.

1-Adamantanecarbonyl chloride is available from Aldrich, Catalog No. 117722. Dilute to 0.1M.
(Activator for monomers and capping reagent)

Acetonitrile/Pyridine (50:50), anhydrous
(Monomer Diluent)

Acetonitrile/Pyridine (95:5), anhydrous
(Activator Diluent)

1% Isopropyl Phosphite in Acetonitrile/Pyridine (50:50)
(Capping Reagent)

Acetonitrile/Pyridine (50:50)
(Neutralizer and Wash Solvent)

4% I2 in Pyridine/H2O/THF (10:10:80) THF/H2O/TEA (80:10:10)
(Both reagents are required for oxidation of H-phosphonate linkages)

ABBREVIATIONS

Ac2O = Acetic Anhydride
CE = Cyanoethyl
CPG = Controlled Pore Glass
DCA = Dichloacetic Acid
DCM = Dichloromethane
DMAP = Dimethylaminopyridine
dmf = dimethylformamidine
DMT = 4,4’-Dimethoxytrityl
I2 = Iodine
iPr = Isopropyl
MeIm = 1-Methylimidazole
µm = micromole(s)
nm = nanomole(s)
Pac = Phenoxyacetyl
PhOAc = Phenoxyacetyl
TCA = Trichloroacetic Acid
THF = Tetrahydrofuran

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 analogues2-6 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.
Item Catalog No. Pack Price ($)
10-1700-90 100µm 175.00
10-1700-02 0.25g 420.00
10-1710-90 100µm 175.00
10-1710-02 0.25g 420.00
10-1720-90 100µm 175.00
10-1720-02 0.25g 420.00
10-1730-90 100µm 175.00
10-1730-02 0.25g 420.00
dA-Thiophosphoramidite
dA-Thiophosphoramidite
dC-Thiophosphoramidite
dC-Thiophosphoramidite
dG-Thiophosphoramidite
dG-Thiophosphoramidite
dT-Thiophosphoramidite
dT-Thiophosphoramidite

REFERENCES

  1. J. Nielsen, W.K.D. Brill, and M.H. Caruthers, Tetrahedron Letters, 1988, 29, 2911-2914.
  2. L. Cummins, D. Graff, G. Beaton, W.S. Marshall, and M.H. Caruthers, Biochemistry, 1996, 35, 8734-41.
  3. X. Yang, and D.G. Gorenstein, Curr Drug Targets, 2004, 5, 705-15.
  4. W .S. Marshall, and M.H. Caruthers, Science, 1993, 259, 1564-70.
  5. J.L. Tonkinson, et al., Antisense Research and Development, 1994, 4, 269-278.
  6. X. Yang, et al., Bioorg Med Chem Lett, 1999, 9, 3357-62.
  7. X. Yang, et al., Ann N Y Acad Sci, 2006, 1082, 116-9.
  8. X. Yang, et al., Nucleic Acids Res, 2002, 30, e132.

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septumcapped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers  

For Instrument type
Add
Expedite
E
Mermade
M

Columns  

For Instrument type
Add
Expedite
E
Applied Biosystems 3900
A
Mermade
M

Please inquire for availability of columns for other instrument types.

See Also

sulfurizing reagents

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.
Item Catalog No. Pack Price ($)
40-4036-10 1g Discontinued
40-4036-20 2g Discontinued
  Sulfurizing Reagent has been discontinued.
Silanized Bottle (one bottle at no charge when ordered with Sulfurizing Reagent)
40-4036-A1 each Discontinued
  ABI (240mL capacity) has been discontinued.
40-4036-A2 each Discontinued
  ABI (450mL capacity) has been discontinued.
40-4036-C3 each Discontinued
  Expedite 8905 (150mL capacity) has been discontinued.
40-4036-C4 each Discontinued
  Expedite 8909 and Cyclone (240mL capacity) has been discontinued.
40-4037-10 1g 50.00
40-4037-20 2g 100.00
  Dissolve at a concentration of 1g/100mL to form an approximate 0.05M solution
40-4137-51 100mL 100.00
40-4137-52 200mL 200.00
40-4137-57 450mL 450.00
Sulfurizing Reagent II
Sulfurizing Reagent II

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