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Oligo Synthesis

Oligo Synthesis : CEPs

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7-deaza-dG-CE Phosphoramidite

7-deaza-dG-CE Phosphoramidite

Glen Research

Catalogue No.DescriptionPack SizePriceQty
10-1021-027-deaza-dG-CE Phosphoramidite 0.25g £780.00£741.00Offer until : 31-Dec-2020Offer Code : GLEN55% off all Glen products View Offer Quantity Add to Order
10-1021-907-deaza-dG-CE Phosphoramidite 100µmoles £284.00£269.80Offer until : 31-Dec-2020Offer Code : GLEN55% off all Glen products View Offer Quantity Add to Order
10-1021-957-deaza-dG-CE Phosphoramidite 50µmoles £142.00£134.90Offer until : 31-Dec-2020Offer Code : GLEN55% off all Glen products View Offer Quantity Add to Order

7-deaza-dG-CE Phosphoramidite

7-deaza-dG-CE Phosphoramidite

Glen Research

7-deaza-dG-CE Phosphoramidite

Structure

Catalog Number: 10-1021-xx

Description: 7-deaza-dG-CE Phosphoramidite

5'-Dimethoxytrityl-N2-dimethylaminomethylidene-7-deaza-2'-deoxyGuanosine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
Formula: C44H54N7O7P M.W.: 823.93 F.W.: 328.22

Diluent: Anhydrous Acetonitrile
Coupling: Standard coupling time. Add a maximum of 2 times when using I2 oxidation or use 0.5M CSO in anhydrous acetonitrile and 3 min. oxidation time. (See Glen Report-Vol.9, No.1, 1996,page 8.)
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.
Storage: Refrigerated storage, maximum of 2-8°C, dry
Stability in Solution: 3-5days

STRUCTURE/ACTIVITY RELATIONSHIP

The following products are used to investigate the effect on the activity of an oligonucleotide when key structural elements are changed. The 7-deaza purine monomers lack groups critical for hydrogen bonding. 7-Deaza-8-aza-A and 7-deaza-8-aza-G (PPG) monomers are isomers of A and G and have similar electron density. Their presence in oligos is slightly stabilizing relative to A and G. Unlike G, PPG does not lead to aggregation and G-rich oligos can be easily prepared and isolated. 5’-Fluorescein oligos with PPG at the 5’-terminus are much less quenched than the equivalent G oligos. As a purine analogue of Thymidine, 7-deaza-2’-deoxyXanthosine (7-deaza-dX) promises to have interesting effects on DNA structure of triplexes. 7-Deaza-dX also forms a non-standard base pair with a 2,4-diaminopyrimidine nucleoside analogue. Standard nucleobases have an unshared pair of electrons that project into the minor groove of duplex DNA. Enzymes that interact with DNA, polymerases, reverse transcriptases, restriction enzymes, etc., may use a hydrogen bond donating group to contact the hydrogen bond acceptor in the minor groove. 3-Deaza-2’-deoxyadenosine is very interesting in that it maintains the ability for regular Watson-Crick hydrogen bonding to T but is lacking the electron pair at the 3-position normally provided by N3.

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7-deaza-dG-CE Phosphoramidite

7-deaza-dG-CE Phosphoramidite

Glen Research

MSDS

Glen Report 9.1 (Oxidation with CSO): NON-AQUEOUS OXIDATION WITH 10-CAMPHORSULFONYL-OXAZIRIDINE

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7-deaza-dG-CE Phosphoramidite

7-deaza-dG-CE Phosphoramidite

Glen Research

Frequently Asked Technical Question

QUESTION: What is a possible alterternative oxidizing solution to I2 ?

RESPONSE:In situations where you want to avoid the use of iodine containing oxidizing solutions, such as when using 7-deaza-dG, or when you want to use a non aqueous oxidizing solution t-butyl hydroperoxide (TBHP) has been shown to work both for DNA (1) and RNA (2).

Reagents:

TBHP, anhydrous, 5-6M solution in decane (Aldrich 41,666-5)

Methylene chloride

For DNA small scale: 1.1 M TBHP in Methylene Chloride, 0.8 min oxidation (1)

For large scale RNA synthesis: 37ml TBHP solution 63ml Methylene Chloride (approx. 2M), 6 min oxidation (2)

Mix reagents fresh prior to use, oxidizing solution only stable for a few days on synthesizer.

REFERENCE(S):
1. Hayakawa, Y., et al., JACS, 1990, 112, 1691.
2. Sproat, B., et al., Nucleosides & Nucleotides, 1995, 14, 255.


QUESTION: How can you prevent oxidative damage of 7-deaza-dG during oligonucleotide synthesis?

RESPONSE:Non Aqueous Oxidation Using 10-Camphorsulfonyl-Oxaziridine

Solutions of enantiomers of 10-camphorsulfonyl-oxaziridine in acetonitrile can be used for the non aqueous oxidation of phosphite triesters to phosphate triesters in oligonucleotide synthesis. This is especially helpful in the synthesis of oligonucleotides containing 7-deaza-dG which is susceptible to damage during the standard I2 catalyzed oxidation step.

It was found that a 0.5 M solution of (1S)-( )-(10-camphorsulfonyl)- oxaziridine in acetonitrile (0.5M CSO) was an effective oxidizer for DNA synthesis. Oxidation time course studies demonstrated that a 3 minute oxidation wait step was sufficient to completely oxidize the phosphite triester to the acid stable phosphate triester. A mixed base oligo synthesized using 0.5M CSO and a 3 min. oxidation wait was indistinguishable by HPLC analysis from the same oligo synthesized using 0.02M I2 oxidizer. Additionally oxidation using 0.5M CSO resulted in no distinguishable modification of the bases as determined from base composition analysis of the enzyme digested oligos. When 0.5M CSO was used for the oxidation in the synthesis of an oligo containing multiple 7-deaza-dG's no evidence of damage to the oligo was detected when the crude oligo was analyzed by RP HPLC. Successful incorporation of 7-deaza-dG was verified by base composition analysis of the enzyme digested oligo. The peak corresponding to 7-deaza-dG, in the enzyme digested sample, comigrated with a nucleoside standard of 7-deaza-dG and had an identical spectrum.

The synthesis cycle used for the experiments using CSO oxidation was a modified sulfurization cycle on an ABI 392 synthesizer with a 3 minute oxidation wait step. The oxidizing solution can either be delivered from the standard oxidizer port (bottle 15) or in this case from the cleave reservoir (bottle 10). The oxidation step occurred prior to the capping step as in phosphorothioate synthesis. Presumably any synthesizer with a sulfurizing cycle can be used if the oxidation wait step is ³ 3 minutes.

Materials:

• (1S)-( )-(10-camphorsulfonyl)oxaziridine (Aldrich # 34,535-0)

•Anhydrous acetonitrile

•Disposable syringe (10-30 ml)

•Solvent resistant syringe filter (0.22-0.45 µ)

Procedure:

•Dissolve (1S)-( )-(10-camphorsulfonyl)oxaziridine in anhydrous acetonitrile (8.72 ml/g). This can be done using the disposable syringe in the same way as for dissolving amidites.

•When the oxidizer is completely dissolved take it up into the syringe, attach the syringe filter and filter into a bottle that fits onto the appropriate port on the synthesizer.

•Modify the sulfurization cycle on the synthesizer to include a 3 minute wait step subsequent to delivery of the oxidizer solution.

•Synthesize the oligo using CSO oxidation at each step in the synthesis. All other conditions are the same.

• Cleave and deprotect the oligo using standard conditions.

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7-deaza-dG-CE Phosphoramidite

7-deaza-dG-CE Phosphoramidite

Glen Research

EXTINCTION DATA

Item Nucleoside λMax-1 Emax-1 λMax-2 Emax-2 E260


(nm) (ml/µmole) nm (ml/µmole) (ml/µmole)
10-1021 7-deaza-dG 259 12.6

12.6

DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

ABI 392/394
Cat.No. Pack
Size
Grams/
Pack
0.1M Dil.
(mL)
LV40 LV200 40nm 0.2µm 1µm 10µm
Approximate Number of Additions
10-1021-02 0.25grams .25grams 3.03 87.67 52.6 32.88 23.91 17.53 4.38
10-1021-90 100µmoles .082grams 1 20 12 7.5 5.45 4 1
10-1021-95 50µmoles .041grams .5 3.33 2 1.25 .91 .67 .17
Expedite
Cat.No. Pack
Size
Grams/
Pack
Dilution
(mL)
Molarity 50nm 0.2µm 1µm 15µm
Approximate Number of Additions
10-1021-02 0.25grams .25grams 4.53 .07 84.2 52.63 38.27 5.26
10-1021-90 100µmoles .082grams 1.5 .07 23.6 14.75 10.73 1.48
10-1021-95 50µmoles .041grams .75 .07 8.6 5.38 3.91 .54
Beckman
Cat.No. Pack
Size
Grams/
Pack
Dilution
(mL)
Molarity 30nm 200nm 1000nm

Approximate Number of Additions
10-1021-02 0.25grams .25grams 4.53 .07 85.8 53.63 39

10-1021-90 100µmoles .082grams 1.5 .07 25.2 15.75 11.45

10-1021-95 50µmoles .041grams .75 .07 10.2 6.38 4.64

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