Amides, conjugated, reaction with

The active ester AT-succinimidyl 3-(2-p3 ridyldithio)propionate (SPDP) is the classical reagent to modify proteins with pyridyldithiopropyl (PDP) groups (20). If the protein to be modified contains many free Cys-residues this creates the risk of carrier-carrier conjugation. In this case it is possible to block the free thiols with iodo- or bromoacetic acid (or amide) before reaction with SPDP (or to use a different conjugation method, as described in Section 3.2). 

The formation of the above anions ("enolate type) depend on equilibria between the carbon compounds, the base, and the solvent. To ensure a substantial concentration of the anionic synthons in solution the pA" of both the conjugated acid of the base and of the solvent must be higher than the pAT -value of the carbon compound. Alkali hydroxides in water (p/T, 16), alkoxides in the corresponding alcohols (pAT, 20), sodium amide in liquid ammonia (pATj 35), dimsyl sodium in dimethyl sulfoxide (pAT, = 35), sodium hydride, lithium amides, or lithium alkyls in ether or hydrocarbon solvents (pAT, > 40) are common combinations used in synthesis. Sometimes the bases (e.g. methoxides, amides, lithium alkyls) react as nucleophiles, in other words they do not abstract a proton, but their anion undergoes addition and substitution reactions with the carbon compound. If such is the case, sterically hindered bases are employed. A few examples are given below (H.O. House, 1972 I. Kuwajima, 1976). 

One of the reactions used in determining the sequence ot nucleotides in a strand of DNA is reaction with hydrazine. Propose a mechanism for the following reaction, which occurs by an initial conjugate addition followed by internal amide formation. 

Figure 1.69 SAMSA is an anhydride compound containing a protected thiol. Reaction with protein amine groups yields amide bond linkages. Deprotection of the acetylated thiol produces free sulfhydryl groups for conjugation.

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

Figure 18.25 Amino-PEG -carboxylate compounds contain a primary amine on one end and a carboxylate group on the other end. They can be used to add water-soluble spacer arms to molecules or surfaces. Using an amine-reactive group, the amino-PEG -carboxylate compound can be coupled via an amide bond, thus leaving the carboxylate end free for further conjugation reactions. Avoid the use of single-step EDC conjugation reactions, as this will polymerize the amino-PEGK-carboxylate by reacting with both ends.

Starting from 2,4,6-octatriene and pivaldehyde, the conjugated homoallylic alcohol 8 is obtained as the sole product. Cycloheptatriene-derived complexes react with aldehydes and C02 to afford mixtures of the isomeric 1,3- and 1,4-cycloheptadienyl carbinols or acids, respectively. Interestingly, analogous reactions with methyl chloroformate or dimethyl carbamoyl chloride produce the conjugated dienyl ester 9 or amide 10 as unique products [19,20]. 

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