Blocking of unreacted groups

The problem of excess reactive groups is encountered in the use of all coupling methods. This problem can be solved by one of two methods. 

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Filter off the solution and treat the resin with a mixture of DCM/MeOH/DIEA( 17/2/1—3 min, three times, anhydrous solvents) see Note 28) for the blocking of unreactive groups. 

Suspend the matrix in 45 mL 200 mM glycine, pH 8, and tumble end over end in a 50-mL capped tube at 4°C overnight to block any unreacted groups. Drain and wash the gel with three cycles of alternating pH. First, suspend the drained gel in 50 mL 100 mM sodium acetate, pH 4, and 500 mM NaCl. Drain with vacuum suction and wash with 50 mL coupling buffer A. Dram and repeat the alternating pH washes twice. 

Blocking of unreacted thiol groups with /V-ethylmaleimide prevents oxidative disulfide-bond formation between surface thiols and cysteine residues of proteins during incubation with labeled proteins. 

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

This regular structure is obtained when polymerization proceeds at 70°C and at rather low concentration of monomer. The material obtained in more concentrated solutions has more complex structure composed of ladder-type blocks, branchings, and unreacted groups. It can be illustrated by the scheme represented in the Figure 9.5. 

These benzaldehydes could then be directly used as a feedstock for various polymeric products or reduced to form phenolic benzylic alcohol derivatives (i.e., p-methylol groups). The p-methylol groups would thus be active sites, whereas in unmodified lignins, the C-l site is blocked and unreactive. In addition, the oxidative-cleavage step will hydrolyze a portion of the lignin interunit ether bonds, and thus increase the total fraction of free phenolic units to further enhance the reactivity. Other possible benefits are that the lignin would be extensively depolymerized and would form a more uniform feedstock material both conditions would give a product that is easier to handle. 

A 10-fold molar excess of succinic anhydride, relative to the total number of moles of lysine in the polylysine, is then added to completely block unreacted amino-groups of PL (DTPA normally modifies up to 95 of amino groups). The reaction is stirred for at least 1 h at room temperature, after which the reaction is allowed to incubate at 4°C overnight. 

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