5'-Thio C6 Modifier (Trityl-6-Thiohexyl Amidite)
5'-Thio C6 Modifier (Trityl-6-Thiohexyl Amidite)
CAS No.:116919-17-6
Key features
Show- Incorporates a thiol reactive functional group for conjugation.
- ' Trityl group retained after cleavage of the oligonucleotide to assist purification, but S-trityl group is not acid labile; it must be removed by treatment with silver nitrate.
Product information
Incorporation of a thiol reactive functional group at specific sites within an oligonucleotide allows for subsequent post-synthesis conjugation of the oligo with a number of different moieties such as fluorescent markers and biotin, depending on the application. Such labels need to be reactive towards the incorporated functional group: for example, thiols will react with iodoacetate and maleimide derivatives to form thioether linkages. In general, thiol modification at the 5'-end of the oligonucleotide is achieved with C6 5'-thiol-modifier phosphoramidite (LK2125/BNS-5019) or, more commonly, the thiol-modifier C6 S-S phosphoramidite (LK2126/BNS-5042). As with the MMT protected amino-modifiers, the trityl group on LK2125/BNS-5019 is usually retained after cleavage of the oligonucleotide to assist purification. However, because the S-trityl group is not acid labile, it must be removed by treatment with silver nitrate. Although, this procedure is commonly used it must be very carefully carried out. Use of LK2126/BNS-5042 offers an alternative and more robust protocol, whereby the thiol is liberated by use of tris(2-carboxyethyl)phosphine (TCEP). This disulphide product can also be used to modify the 3'-position by using the phosphoramidite as the first adduct in the oligo sequence. Incorporation of LK2126/BNS-5042 at the 5'-end allows the possibility of DMT-ON purification prior to reduction of the disulphide bridge.
Properties:
- Formula: C34H45N2O2PS
- Molecular Weight: 576.8
- Appearance: colorless oil
Product usage:
- Dilution: 100 µmol/mL
- Deprotection conditions: The deprotecting conditions for this amidite will be depend on the types of amidites used for the synthesis of the oligo. If using fast deprotecting amidites deprotect in concentrated NH4OH for 1 hour at 60 °C. If using standard amidites deprotect in concentrated NH4OH for 5 hours at 60 °C. If all the amidite solution is not used during the synthesis, it can be stored under argon and its functionality maintained up to 48 hours.Purification of the oligo should be done prior to the reduction of the thiol. Standard RP purification can be used to purifiy the oligo. See attached protocol using reversed-phase MicroPure column (MP-1602) for purification of oligonucleotides. To deprotect the thiol after deprotection and purification of oligo:Evaporate or dry down sample.Resuspend sample in 0.1 M TEAA pH 7.0Add 0.5 mL of 1 M silver nitrate (aq), mix solution throughly. Leave at room temperature for 30 minutes.Add 0.7 mL of 1 M DTT (aq) (Aldrich # 15046-0) solution, mix throughly. Allow to stand for 10 minutes at room temperature.Centrifuge the sample to remove the silver nitrate DTT complex. Collect the supernatant. Extract with 3-5 mL of ethyl acetate to remove the excess DTT.Proceed directly to the conjugation reaction otherwise store reduced thiol labeled oligo under an inert atmosphere to avoid oxidative dimerization to the disulfide.Below is a protocol using reversed-phase MicroPure column (MP-1602) for purification of oligonucleotides. Each MicroPure column will clean up to a 1 µmol synthesis column. (Detailed instructions for the preparation of reagents can be found following the protocol.)1. Rinse column with CH3CN (2 x 2 mL).2. Activate column with 1 N TEAA
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