Preparation of linker

4 Separate each PCR product by electrophoresis on a 4% (w/v) NuSieve GTG agarose gel, containing 0.5 pJ/ml ethidium bromide. prepared in 1 x TBE buffer alongside 1 (jlI of 1 kb PLUS DNA ladder. 

Visualize the bands on a UV light box, cover the screen with Saran Wrap to prevent nucleic acid cross ontamination. 

Cut out a slice of gel containing the fiagment of interest (68 bp) in the smallest possible volume. Introduce the slice in a tarred microcentrifuge tube. Remove any buffer that may have come in with the slice. Estimate the volume of the slice from its weight and add 1 voL of TE buffer. Place the tube in a 65 C water-bath for 5-10 min. 

Make sure that the agarose is fiilfy melted by pipetting the solution up and down a few times. Immediately add 1 vol. of Tris-buffered phenol (no chloroform) at room temperature and mix thoroughly by inversion. 

Spin for 3 min at 10 000 to 12 000 r.p.m. Transfer the aqueous phase to a fresh tube containing 1 vol. of Tris-buffered phenol. Mix. 

5 X TBE (Tris, borate, EDTA) electrophoresis buffer 445 mM Tris base (54.0 g). 445 mM boric acid (27.5 g), and 10 mM EDTA (3.72 g Na2EDTA2H20), adjust to 1 litre with distilled water 

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Small amounts of specially functionalized monomers are often copolymerized with acryUc monomers in order to modify or improve the properties of the polymer. These functional monomers can bring about improvements either directiy or by providing sites for further reaction with metal ions, cross-linkers, or other compounds and resins. Table 9 Hsts some of the more common functional monomers used in the preparation of acryUc copolymers. 

Structure-activity studies of 5,6,7,8-tetrahdyro-5,5,8,8-tetramethyl-2-quinoxaline derivatives necessitated the preparation of thiophene-containing compound 17. Stetter conditions using thiazolium salt 20 as catalyst resulted in the preparation of 1,4-diketone 21 from 18 and 19. Condensation of 21 with phosphorus pentasulfide followed by saponification resulted in 17. In this fashion, the authors replaced the amide linker of parent compound 22 with the rigid thiophene moiety. 

RajanBabu reported the first preparation of a bis-imidazolium salt 15 bearing a chiral linker (Scheme 11). The starting material was the enantiomerically pure (S)-l,l -bi-2-naphtol bis(trifluoromethanesulfonate) which was transformed in two steps into the dibromomethyl derivative 16 and then into the bis-imidazole. Quaternarization of this compound afforded 15 [20]. 

A second strategy is to attach a linker (also referred to as a handle or anchor) to the resin followed by assembly of the molecule. A linker is bifunctional spacer that serves to link the initial synthetic unit to the support in two discrete steps (Fig. 3). To attach a linker to a chloromethyl-PS resin, a phenol functionality such as handle 4 is used to form an ether bond (Fig. 4). To attach the same handle to an amino-functionalized support, acetoxy function 5 or a longer methylene spacer of the corresponding phenol is applied to form an amide bond. Both of these resins perform similarly and only differ in their initial starting resin [4], An alternative approach is to prepare a preformed handle in which the first building block is prederivatized to the linker and this moiety is attached to the resin. For peptide synthesis, this practice is common for the preparation of C-terminal peptide acids in order to reduce the amount of racemization of the a-carbon at the anchoring position [5],... 

Allylic hydroxycrotyl-oligoethylene glyco-n-alkanoyl (HYCRON) linker 25 was applied to the synthesis of protected peptides and glycopep-tides [31]. HYCRON is stable to both acidic and basic conditions and is compatible with Boc- and Fmoc-based chemistry. The preparation of this novel linker is only two steps from commercially available materials. H YCRON linker can be cleaved under neutral conditions using Pd catalyst (Scheme 9). 

In a recent study, this so-called SPOT synthesis was applied for the preparation of pyrimidines [45]. The group of Blackwell described primarily the appropriate support modification of commercially available cellulose sheets (Scheme 7.28). The initial introduction of the amine spacer was achieved within 15 min utilizing micro-wave irradiation, as compared to 6 h by conventional heating. The acid-cleavable Wang-type linker was attached by classical methods at ambient temperature. 

Scheme 7.93 Preparation of an N-imidazolium-based soluble AMEBA linker.

In a recent study, the group of Buijsman presented a microwave-mediated preparation of a different N-imidazolium-based ionic analogue of the well-known AMEBA solid support (Scheme 7.93). With this soluble support, a set of various sulfonamides and amides was prepared, and furthermore the use of this novel linker in the synthesis of a potent analogue of the antiplatelet drug tirofiban was presented [106]. 

Scheme 7. Chemoenzymatic preparation of optically pure secondary amines via hydrolysis of an racemic oxalamic ester linker...

The development of diversification linkers allows introduction of an additional element of diversity. Upon completion of the synthesis sequence, the linker is activated facilitating nucleophilic release of the library members from support In the ideal case, as implemented with the acylsulfonamide linker (Scheme 4a), the activated linker is sufficiently reactive that limiting amounts of nucleophile may be added to provide pure product after resin filtration.181 Diversification linkers have been developed for the preparation of carboxylic acid derivatives (Scheme 4a), amines (Scheme 4b),191 aromatic (Scheme 4c) and even heteroaromatic compounds (Scheme 4d).1101... 

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