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5'-Thiol C6 SS Modifier Amidite (DMT-7,8-dithiotetradecan-1,14-diol)

5'-Thiol C6 SS Modifier Amidite (DMT-7,8-dithiotetradecan-1,14-diol)

CAS No.:148254-21-1

Phosphoramidite for the incorporation of a thiol function at the 5' end of an oligonucleotide.
  • Incorporates a thiol reactive functional group for conjugation.
  • Robust protocol; thiol is liberated by use of tris(2-carboxyethyl)phosphine (TCEP).
  • Can utilise DMT-ON purification prior to reduction of the disulphide bridge.
  • This disulphide product can also be used to modify the 3'-position by using the phosphoramidite as the first adduct in the oligo sequence.
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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: C42H61N2O5P S2
  • Molecular Weight: 769.05
  • Appearance: colorless oil

Product usage:

  • Dilution: 100 µmol/mL
  • Coupling: 2-minute coupling
  • Deprotection conditions: For DMT-OFF Synthesis (5' modification):For the DMT OFF oligonulceotide, cleave and deprotect using NH4OH solution for 2 hours at 60 °C if using fast deprotecting amidites or 5 hours at 60°C for standard amidites. To reduce the disulfide linkage to generate a 5'thiol modified oligo, dry down sample then add 0.05M DTT in water. Heat sample for 5 hours, dry the sample down and reconstitute in water. The sample may need to be filtered before HPLC analysis.For DMT-ON Synthesis (5' modification):For the DMT ON oligonucleotide cleave and deprotect the oligo in concentrated NH4OH for 5 hours at 60 °C, for fast deprotecting amidites cleave and deprotect 2 housr at 60 °C. Purify the DMT ON oligonuleotide using a SuperPure Plus purification column (<200 nmol scale synthesis, see protocol below). Reduce the disulfide bond using 100 mM DTT in 0.2 M phosphate buffer pH 8.5 for 30 minutes at room temperature. Elute the 5' Thiol oligonucleotide and desalt using a G-25 or NAPS column to generate a purified 5' Thiol oligonucleotide.
  • Below is a protocol using reversed-phase MicroPure column (MP-1602) for the cleanup of 5' Thiol oligonucleotides. Each MicroPure column will clean up to a 1 µmol synthesis column. Note that the TFA step is omitted and a 100 mM DDT in 0.2 M phosphate buffer pH 8.5 solution is substituted. The DTT solution will cleave the disulfide linkage resulting in a 5' Thiol modification.1. Rinse column with MeCN. (2 x 2 mL)2. Activate Column with 1 N TEAA. (3 x 2 mL)3. Add Crude DMT-Oligo, 1 mL of 1:1 solution (conc. aq. NH4OH/H2O). (Collect and reload solution twice).4. Elute Failures with 3% NH4OH. (3 x 2 mL )5. Rinse column with water. (3 x 2 mL)6. Cleave disulfide linkage with 100 mM DTT, in 0.2 M phosphate buffer (pH 8.5) (1 x 1 mL) Wait 30 minutes.7. Rinse excess DTT with 5% MeCN in 0.1M TEAA. (3 x 2 mL) Rinse until pH is neutral.8. Elute and collect purified oligo with 20 % MeCN in water. (1 x 1.5 mL)9. Speed vac samplePreparation of ReagentsFive different solutions for the purification process need to be prepared. To store these solutions use clean glass bottles with plastic lined caps, 250 mL in capacity is a convenient size.1. Acetonitrile (VWR, cat. number JT9018-3). 2. 1.0 N Triethylammonium acetate (TEAA). For this solution, place about 80 mL of the water from #1 above in a clean bottle, and add exactly 5.7 mL of acetic acid (Aldrich cat. number 24, 285-3; Caution - causes burns, wear eye protection and gloves when using concentrated acetic acid). Swirl the solution until completely mixed, and add exactly 13.9 mL of triethylamine (Aldrich cat. number 23, 962-3; Caution - wear eye protection and gloves when using concentrated triethylamine). Mix the solution until it is completely homogeneous - a small stir bar and magnetic stirrer may be used with good results. After complete mixing is achieved, the pH should be neutral. pH paper can be used for this (VWR cat. number 60775-702).3. 3.0% Ammonium Hydroxide in water. Add 10 mL of concentrated ammonia (Aldrich cat. number 22, 122-8 Caution - wear eye protection and gloves when using concentrated ammonia) to 90 mL of water. Mix the solution until it is completely homogeneous. 4. 100 mM DTT solution in 0.2 M phosphate buffer pH 8.55. 5% MeCN in 0.1 M TEAA Use 5 mL MeCN in 95 mL 0.1 M TEAA.6. 20% acetonitrile in water. Dilute 20 mL of the same acetonitrile as in 2 above with 80 mL of water.Image of cleaved and deprotected structure:
  • The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 328.43

Storage and handling:

  • Shipping conditions: Cold
  • Storage conditions: -15 to -30 °C

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