Deprotection conditions:

Deprotection conditions are dependent upon the type of amidites used for the synthesis of the oligo. If using standard amidites deprotect in concentrated NH4OH for 5 hours at 60 °C. If using fast deprotecting amidites deprotect in concentratied NH4OH for 1 hour at 60 °C.

For removal of the MMT group using reversed-phase cartridge purification, see protocol below. Note: Oligo should be cleaved and MMT group left on for this purification procedure. Below is a protocol using reverse phase MicroPure column (MP-1602) for cleanup of 5' MMT oligonucleotides. Each MicroPure column will clean up to a 1 µmol synthesis column. Note that the TFA step cleaves the MMT group resulting in a completely deprotected oligonucleotide. (Detailed instructions for preparation of reagents can be found following protocol) 1. Rinse column with CH3CN (2 x 2 mL). 2. Activate column with 1 N TEAA (3 x 2 mL) 3. Add Crude MMT-Oligo 2 mL of 1:1 solution (concentrated aqueous 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. Deblock MMT with 2.5% TFA aqueous (3 x 2 mL). Wait 10 minutes between applications. 7. Rinse excess TFA with water. (3 x 2 mL; rinse until pH is neutral). 8. Insert collection tubes and elute purified oligo with 20% MeCN in water. (1 x 1.5 mL) 9. Dry sample in speed vac Preparation of Reagents: Five different solutions for the purification process need to be prepared. To store these solutions use clean glass bottles with plastic lined caps. 1. Water. Use good quality sterile, deionized water. HPLC grade is best, especially if subsequent analyses will rely on HPLC methods. 2. Acetonitrile (VWR, cat. number JT9018-3). 3. 20% acetonitrile. Dilute 20 mL of acetonitrile (from #2 above) with 80 mL of water (from #1 above). 4. 1.0 N Triethylammonium acetate (TEAA). For this solution, place about 80 mL of 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). 5. 2.5% Trifluoroacetic acid (TFA). Carefully add 2.5 mL of TFA (Aldrich cat. number T6, 220-0 Caution - wear eye protection and gloves when using concentrated TFA) to 97.5 mL of water (from #1 above). Mix the solution until it is completely homogeneous. 6. 3.0% Ammonium Hydroxide. 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 (from #1 above). Mix the solution until it is completely homogeneous.

Image of cleaved and deprotected structure:

The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 263.31