Alkylation buffer, solution preparation

A much more general method for acyl silane synthesis involving silyl diazo intermediates is illustrated in Scheme 1688. The lithiated derivative of trimethylsilyl diazomethane reacts smoothly with alkyl halides in THF solution to give a-trimethylsilyl diazoalkanes in good yields. Oxidative cleavage of the diazo moiety is effected using 3-chloroperbenzoic acid in benzene solution, to give access to a wide variety of acyl silanes in yields of up to 71%. A phosphate buffer (pH 7.6) is used to prevent side reactions. Aromatic acyl silanes clearly cannot be prepared by this chemistry since an aromatic nucleophilic substitution reaction would be required. 

Four recent examples of universal linkers/supports, in which the first nucleoside is anchored onto the preformed linker-support construct, are shown in Fig. 2.14. The disulfide linker 2.33 has been used to prepare terminal 3 -phosphate ONs (94, 95) through cleavage with a solution of ammonia in dithiothreitol. The photolabile linker 2.34 (96) is used to prepare 3 -alkyl carboxylic acids. The allyl-based linker 2.35 (97) is used to prepare free 3 -OH ONs by cleavage with Pd(0) and treatment with an aqueous buffer at pH 10. The linker 2.36 (98) differs from those discussed so far in... 

Dissolve in vitro alkylated standard DNA and unmodified DNA in TE buffer (0.3 mg/mL). Prepare a standard curve by making serial dilu-dons. The DNA solutions can be stored at 4°C for several days. In order to minimize depurinadon of alkylated DNA, the dissolved standards should be aliquoted and frozen at -20°C for long-term storage. 

Cysteine residues in transferrin were reduced and alkylated in a similar manner as that done for solution samples p). Modiflcation for samples prepared with ProSorb cartridge can be performed in the same ProSorb cartridge before membrane removal, while modifications were performed in an Eppendorf tube for the electroblotted samples. The membranes were incubated 15 minutes at room temperature in a 0.25 M Tris/HQ and 6 M Guanidine hydrochloride buffer containing 1 ml of mercaptoethanol and followed by the addition of 1 ml of 4-vinyl pyridine for another 15 minutes. The membnmes were washed thoroughly with 0.1% TFA afterwards. 

Figure 4 Estradiol flux through human stratum comeum when the drug is applied oedu-sively in liquid or gel state vesicles prepared from polyoxyethylene alkyl ethers and cholesterol. Ail formulations are saturated with respect to estradiol. (A) Plus estradiol applied in PBS , estradiol applied in liquid state vesicles prepared from decaoxyethylene oleyl ether (Q.gEOjo) and cholesterol , estradiol applied in phosphate buffered saline solution (PBS) after pretreatmem of vesicles prepared from C,.,EOjo and cholesterol. (B) Plus estradiol applied in PBS , estradiol applied in gel state vesicles prepared from trioxy-ethylene alky octadecyl ether (CitEOr) and cholesterol . estradiol applied in PBS after pretreatmem of vesicles prepared from C,i,EOj and cholesterol.

Alkylating Reagent. 10 mg/mL solution of lAA in 50 mM NHtHCOs.This buffer should also be prepared fresh daily (while reducing) and kept in the dark. 

Ultrapure water (18.2 MS2 cm), was obtained from a MiUipore water purification system and was used for preparation of sample solutions. Analytical Grade Acetonitrile (MeCN) (Fischer Scientific) and Trifluoroacetic Acid (TEA) (Sigma-Aldrich) were used for all ZipTip buffers (see Sect. Denaturing MS— ZipTip Buffers ). The coenzyme A derivatives (acetyl-, butyryl- and malonyl) used in assays were purchased from Sigma-Aldrich. iV-acetylcysteamine (SNAC) thioesters were provided by the Piel lab (ETH, Zurich), and stock solutions were solubilised in dimethyl sulfoxide (DMSO) (Fischer Scientific). lodoacetamide (Sigma-Aldrich) was used for all alkylation reactions. 

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