Synthesis of CPG-puromycin

Tony Keefe February 1998

CPG-puromycin is now available from Glenn Research, so if I were you I'd buy it.

All of these steps except the first need to be done in the absence of water. Dry (fresh, over molecular sieves, extracted under positive pressure of dry argon) solvents and reagents need to be used. All reactions were conducted under a positive pressure of argon, glassware was dried in the vacuum oven and the solvents were transferred with syringes. Always save a little of the product at each stage to use for TLC monitoring of each reaction. Normal phase silica plates were used, and all dry solvents were purchased from Fluka.

 

Preparation of the free base of Puromycin

Puromycin is supplied as the dihydrochloride (Sigma) and the first step is to convert it into the free base. This is done by dissolving it in water, adding pH 11 buffer (0.1 M K2CO3 or Na2CO3) and extracting into dichloromethane. The aqueous phase is repeatedly washed with dichloromethane until upon combining and evaporating them to dryness all of the puromycin (measured by weight) is accounted for. I got 94%.

 

Synthesis of N-TFA Puromycin

The puromycin was dried by two coevaporations with dry pyridine initially on the rotary evaporator and then directly on the vacuum line for the last bit. Puromycin was then dissolved in a 1:1 mixture of acetonitrile and pyridine (approx 50ml/mmol). Six (Rihe used 2) equivalents of triethylamine (TEA) were slowly added, and then four equivalents of trifluoracetic anhydride (Sigma) immediately after the TEA addition. Reaction is instantaneous with accompanying colour change from yellow to orange. TLC (normal phase silica, 15% MeOH, 85% CH2Cl2) showed the complete absense of starting material, the product runs with an Rf of 0.8, starting material at 0.5, and the even higher mobility band is probably pyridine, plates need drying before UV visualization to get rid of most of the pyridine. The reaction mixture was stirred with ammonium hydroxide solution(30%) (6ml/mmol of puromycin), rotary evaporated to reduce the volume by half, addition of 1:1 ammonium hydroxide solution (15ml/mmol), shaken and stirred with dichloromethane two times. Organic phases were combined, rotary evaporated to dryness, dried by coevaporation with pyridine twice. The product was used in the next step directly. Yield not assesed separately. This hydrolysis is to get rid of the bis product (N,O-bis (TFA) puromycin), Rihe says that he didn't see any bis product in the TLC and so skipped this step and cautions against heating in the presence of base (while rotary evaporating).

 

Synthesis of N-TFA-trityl Puromycin

The product from the previous reaction, after drying by coevaporation with pyridine twice, was dissolved in pyridine (6ml/mmol assuming 100% yield of previous step) and then 0.075 equivalents of dimethylaminopyridine (DMAP) and six equivalents of triethylamine (TEA) were added and 5 minutes later 3.5 equivalents of 4,4'-dimethoxytrityl chloride. After 30 minutes TLC (normal phase silica, 7%MeOH, 93%CH2Cl2) showed the product with an Rf of 0.5 slightly ahead of the starting material (Rf of 0.45), of which there was no trace. Addition of water (3ml/mmol) and rotary evaporated to give red oil which was purified on a silica (silica gel 60, Merck) column (I used 40g/mmol of puromycin).

 

Column chromatographic purification of N-TFA-trityl Puromycin

Two successive silica columns were used to purify this product. Prepare the column with 98% dichloromethane, 1% TEA and 1% methanol and elute with the same. The product elutes between the rather obvious first yellow band and equally obvious first orange band. But the product is not strongly coloured at all. Collect many fractions and TLC (normal phase silica, 7%MeOH, 93%CH2CL2) them all. By far the best thing to do is develop the plates by dipping them into an ethanolic solution of phosphomolybdic acid and heating them, trityl containing spots will appear bright crimson after heating. The product appears at around Rf 0.5 and there are trityl containing impurities that run just ahead of it. After the first column the yield of the first 2 steps was 57%, and after the second it was 48%.

 

Synthesis of N-TFA-trityl-succino Puromycin

If pure the product is a virtually colourless crystalline mass at this point. It was dried by coevaporation with pyridine twice and then taken up pyridine (3ml/mmol) and acetonitrile (0.4ml/mmol), then added DMAP (2.5 equivalents) and succinic anhydride (2.5 equivalents). Stirred overnight, TLC (normal phase silica, 7%MeOH, 93%CH2Cl2 develop with phosphomolybdic acid) in the morning showed no starting material remained (Rihe cautions that he found the reaction to be incomplete at this point) but the product appeared to be 2 spots running at about Rf 0.4, beneath the starting material. Added water (Rihe suggests removing the pyridine first to avoid possible pyridine catalyzed hydrolysis) and dichloromethane, stirred, remove organic phase, wash aqueous phase once with dichloromethane, combine organic phases, rotary evaporated to dryness, addition of dioxane and coevaporation with water to dryness twice to give a brown foam which collapsed to a yellow oil. Product used directly in the next step. Yield 99%.

 

Coupling to CPG

CPG (ChemGenes, 2.5g/mmol) was washed with DMF and evaporated to dryness on the vacuum line at room temperature. The product of the previous step was mixed with a 1:1 mixture of dioxane and pyridine (2.5ml/mmol) and 1.5 equivalents of dicyclohexyl-carbodiimide, the mixture was stirred for 5 minutes and then 1.5 equivalents of 4-nitrophenol was added and the mixture was stirred for 2 hours. This mixture was then filtered directly onto the dry CPG, then the filter and filtrate were rinsed with 5ml of DMF which was also added to the reaction mixture. The mixture was allowed to react overnight, the supernatant was removed, evaporated to dryness and the whole procedure was exactly repeated using the residue. The product was then filtered off, dried in under reduced pressure, washed twice with DMF and dried by coevaporation with pyridine twice and used directly in the next step.

 

Capping the CPG-puromycin

To the dry product of the above reaction were added pyridine (10ml/mmol), DMAP (0.4 equivalents), TEA (5ml/mmol) and acetic anhydride (2ml/mmol) in that order. The mixture was stirred for 1 hour, the supernatant drained off and then the procedure was repeated. The product was washed with DMF and dried under vacuum to give the product as a light orange powder.

 

Assaying CPG-puromycin

Hydrolysis with 1:1 perchloric acid:methanol and assay of the trityl by UV (trityl has an absorbance of 70,000,000/mol at 498nm) showed that the powder was 21Ámol/g and the total yield over all steps as 5%. Ninhydrin mixed with the powder showed no purple colour at all. Analysis of synthetic oligomers of DNA by MALDI-TOF-MS showed a mixture of depurinated (probably occurs in the MS) and n-1 and n-2 peaks (result from cutting too large a band from the gel), but all species had puromycin on them.

 

Assaying CPG-puromycin for concentration of trityl groups

In order to assay the concentration of trityl groups weigh out approximately 10mg of your CPG-puromycin and add to it 1ml of a 1:1 mixture of methanol and 70% perchloric acid (Aldrich) (you should see an orange colour develop at this point), dilute the sample 100-fold into water and measure the absorbance at 498nm. If the absorbance falls in the range 0.01-2 then proceed, if not use a different dilution factor. The concentration of trityl groups upon your CPG (in Ámol/g) is then given by the following formula (Axd)/(70xm) where A is the absorbance, d is the dilution factor (100 in this case), 70 is the micromar absorbance and m is the mass of CPG-puromycin in g. The contents of a commercial solid phase DNA synthesis column can be used as a positive control for this procedure.

 

Assaying CPG-puromycin for free amino groups

In order to assess if there are any uncapped amino groups on the surface of the CPG-puromycin, add a small amount of your CPG-puromycin to a ninhydrin solution (0.2% ninhydrin in butanol saturated with water is widely used). If any purple colour develops then there are free amino groups on the surface of the CPG-puromycin and your sample has failed the test and needs further capping with acetic anhydride. Unreacted CPG, or a sample of your CPG-puromycin from before the capping step, can be used as a positive control for this procedure.

 

Some useful molecular weights

Puromycin dihydrochloride 544.4

Puromycin free base 471.5

N-TFA Puromycin 566.5

N-TFA-DMT-Puromycin 868.8

N-TFA-DMT-succinyl-Puromycin 968.9

TFAA 210

TEA 101

Succinic anhydride 100

DMAP 122

DMT chloride 339

DCC 206

4-nitrophenol 139

Acetic anhydride 102