Light phosphate protection

Rubinstein, M., Amit, B. and Patchornik, A. (1975) Use of a light-sensitive phosphate protecting group for mononucleotide syntheses. Tetrahedron Letters, 16, 1445-1448. 

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 minutes in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide group. The degree of derivatization should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

Modify a purified bait protein by adding an aliquot of crosslinker to achieve a molar excess of 2-10 moles per mole of protein (protect from light). For NHS ester-containing reagents, react in 0.1 M sodium phosphate buffer at pH 7.2-7A. Avoid using any other amine-containing buffer components, such as Tris or imidazole, which will interfere with the reaction. 

Substrate buffer 3.79 mg 4-methylumbelliferyl-N-acetyl-a-D-glucosaminide (Moscerdam Substrates) is dissolved in 10 ml citrate/phosphate buffer. For faster dissolution, the mixture is sonicated (1 min). The substrate buffer is stored at -20°C and protected from light. 

Mix glucose determination reagent concentrate (Total Starch Assay Kit or Glucose Assay Kit Megazyme International Ireland) with 1 liter of lx phosphate buffer (see recipe). Store in aliquots, protected from light, for 3 months at 2° to 5°C or >1 year at -20°C. 

Test solution 2 (for Capsules labeled 40 mg) Immediately transfer 5 ml of the solution under test to a test tube containing 2 ml of 0.25 M sodium hydroxide and 5 ml of pH 6.8 Phosphate buffer. Mix well, and pass through a membrane filter having a 1.2-pm or finer porosity. Protect from light. 

To prepare the Kimura stain Mix 5.5 mL of Toluidine blue solution, 0.4 mL of light green solution, 0.25 mL of saponin solution and 2.5 mL 67 mM phosphate buffer. Filter and keep at room temperature, protected from the light (covered with aluminium foil). Prepare weekly. 

Note that any NAD" containing solutions must be protected from light, which is easily done by wrapping containers with aluminum foil. Radiolabelled P-NAD" is either synthesized from [a- P]ATP by the method of Cassel and Pfeuffer (1978) or purchased and stored frozen. Although the half-life of the radioactive phosphate is approximately two weeks, we do not use P-NAD" longer than three to four weeks after preparation because of radiolysis. 

Because AZT-CDS (125)is relatively easily accessible synthetically (Fig. 15.35), following the increasing occurrence of AIDS, AZT-CDS was investigated in a number of laboratories to enhance the access of AZT to brain tissues (309-323). AZT-CDS is a crystalline solid, which is stable at room temperature for several months when protected from light and moisture. It is relatively stable in pH 7.4 phosphate buffer, but rapidly oxidizes in enzymatic media. In addition, T+-AZT (124), the depot form of AZT, was shown to gradually release the parent compound. 

Dihydrate, yellow crystals. Soly in water at pH 6,9 = 112 oig/ml at pH 5.6 — 68 mg/ml at pH 3.8 = 43 mg/ml. The commercial product may have a pH of 5.0 to 6.0 because of a small amount of the disodium salt. Theoretical pH of aq soln (1 millimole /150 ml H20) is 4.5. Fully active biologically, microbio logically, and enzymatically. The greater sensitivity of the phosphate ester to destruction by ultraviolet light necessitates careful protection of dil solns from exposure Sleezer et al.. Drug Cosmet. Ind. 74, 196 0954). 

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