Anhydride method

Benzoic anhydride (carboxylic acid - acetic anhydride method). 

Anilino vinyl derivatives of thiazolium (30, R = H) or acetanilido (30, R = C0CH3), as well as formyl methylene 30b (methods E-G), give asymmetrical dyes when condensed with a methyl reactive group of another species (Scheme 42). Mesosubstituted symmetrical or unsymmet-rical thiazolocyanines are obtainable via /S-alkylmercaptovinyl thiazolium derivatives (32) (methods H and I) (Scheme 43). a or /S carbon atoms of the trimethine chain can be substituted by acetyl when a dye is treated with acetic anhydride (method L). The hydrolysis of neocyanines lead to trimethine cyanine by fractional elimination of a composant chain (method K). 

Active esters are preferably synthesized by either the mixed anhydride method, e.g.22,26 or by the dicyclohexylcarbodiiinide method9-32). 

Like the carbodiimide method, the mixed anhydride method results in an amide complex (Table 5, Figure 17). The acid-containing hapten is dissolved in a dry, inert, dipolar, aprotic solvent such as p-dioxane, and isobutyl chloroformate is added with an amine catalyst. The activated mixed anhydride is chemically stable and can be isolated and characterized. The aqueous protein solution is added to the activated acid and the pH is maintained at around 8.5. A low temperature (around 10 °C) is necessary during the reaction to minimize side reactions. 

Figure 17 Conjugation of an amine and a carboxyUc acid via the mixed anhydride method. Although the activated mixed anhydride is stable, it is usually used without purification. Use of low-temperature reactions will limit undesirable side products. Details of the reaction are given in Table 5...

As a further motivation, there is evidence that establishing the optimal AR protocol will also contribute to standardization of IHC, through equalizing variable IHC staining results obtained following different times of formalin hxation. In the light of the studies described above, further studies were conducted as to the utility of the citraconic anhydride method. 

Although the novel AR protocol using citraconic anhydride improved the intensity of IHC on FFPE tissue sections for more than half of the antibodies tested, compared to that achieved by other conventional AR protocols, not all antibodies benefitted, which would argue that the citraconic anhydride method does not serve as a truly universal AR protocol. Indeed, many investigators (Table 1.2) have concluded that different antigens may require different specific AR protocols. In this respect, the test battery is a convenient and cost-effective method for assessing the appropriate AR protocol.2,8 Nevertheless, the present data certainly support inclusion of the citraconic anhydride AR method in such a test battery. With respect to the two heating temperatures for citraconic anhydride, the ultimate choice of method for any laboratory may depend on the equipment available. 

GW Anderson, JE Zimmerman, FM Callahan. A reinvestigation of the mixed carbonic anhydride method of peptide synthesis. J Am Chem Soc 89, 5012, 1967. 

J Meienhofer. The mixed carbonic anhydride method of peptide synthesis, in E Gross, J Meienhofer, eds. The Peptides Analysis, Synthesis, Biology, Academic, New York, 1979, Vol 1, pp 263-314. 

Mixed anhydrides (see Section 2.6) The mixed-anhydride method provides efficient coupling of peptides with minimal isomerization if the established protocol is strictly adhered to. This includes a short activation time at low temperature, isopropyl chloroformate as the reagent, and A-methylmorpho-line or /V-mcthylpipcridinc as the tertiary amine (Figure 2.25, path D). In what is an apparent anomaly with respect to conventional wisdom, a polar solvent such as dimethylformamide seems to be preferable to apolar solvents for minimizing isomerization. Aminolysis at the wrong carbonyl of the anhydride of a peptide (path F) is less than that for the anhydride from the corresponding /V-alkoxycarbonylamino acid. 

Use the mixed-anhydride method in dimethylformamide with isopropyl chloroformate as me reagent and (V-methylmorpholine, A -melhylpiperi-dine, or trimethylpyridine as me base (see Sections 7.4 and 7.5). 

Use me succinimido esters obtained from me acid by me mixed-anhydride method (see Section 7.8). This approach has been examined for segments of up to four residues. 

The reagents and methods employed for coupling in solid-phase synthesis are the same as for synthesis in solution, but a few are excluded because they are unsuitable. The mixed-anhydride method (see Section 2.6) and l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (see Section 2.15) are not used because there is no way to eliminate aminolysis at the wrong carbonyl of the anhydride. Acyl azides (see Section 2.13) are too laborious to make and too slow to react. The preparation of acyl chlorides (see Section 2.14) is too complicated for their routine use this may be rectified, however, by the availability of triphosgene (see Section 7.13). That leaves the following choices, bearing in mind that a two to three times molar excess of protected amino acid is always employed. 

E Izeboud, HC Beyerman. Synthesis of substance P via its sulfoxide by the repetitive excess mixed anhydride method, (iodide for reduction) Rec Trav Chim Pays-Bas 97, 1, 1978. 

HC Beyerman, EWB De Leer, J Floor. On the repetitive excess mixed anhydride method for the synthesis of peptides. Synthesis of the sequence 1-10 of human growth hormone. Rec Trav Chim Pays-Bas 92, 481, 1973. 

There have been reports that urethane was produced when the mixed-anhydride method was employed for the coupling of segments. However, studies on urethane formation during the aminolysis of mixed anhydrides of peptides have never been carried out. The anhydrides are too unstable to be isolated. The activated moiety of the peptide cyclizes too quickly to the 2,4-dialkyl-5(4//)-oxazolonc (see Section 2.23), and since the time allowed to generate the anhydride in segment couplings is always limited to avoid epimerization, one cannot exclude the possibility that the urethane that was produced originated by aminolysis of unconsumed chloroformate. 

FIGURE 7.5 Preparation of a protected dipeptide by the mixed-anhydride method, employing a chloroformate that generates a cleavable urethane.13 The urethane impurity is destroyed by a P-elimination reaction. NMM = A-methylmorpholine, Msc = methane-sulfonylethoxycarbonyl. 

M Bodanszky, JC Tolle. Side reactions in peptide synthesis. V. A reexamination of the mixed anhydride method. Ini Pept Prot Res 10, 380, 1977. 

RB Merrifield, AR Mitchell, JE Clarke. Detection and prevention of urethane acylation during solid-phase peptide synthesis by anhydride methods. J Org Chem 39, 660, 1974. 

The mixed anhydride method was used with diaminoethane to give a free amino group that was subsequently reacted with 4-hydroxybenzoic acid to provide an aromatic ring to allow iodination with [ ] to increase sensitivity of the assay compared with labelled radioactive tracers. This reaction could also be used to prepare fluorescent compounds for cell-biology studies. 

These compounds are readily prepared by the reaction of fluorescein isothiocyanate with the polypeptide in a DMSO/water mixture and fluorescein-polyacrylate conjugate (6) is prepared by the reaction of fluoresceinamine with polyacrylic acid by using the mixed anhydride method [150]. In all cases, the absorption and emission maxima were similar at 490 and 525 nm, respectively, and reflect the photophysical properties of fluorescein itself. 

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