Mixed anhydride method

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...

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. 

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. 

The difficulties encountered in aminoacylation of such lipo-amino acid derivatives are similar to those of the N-alkylated amino acids (see Vol. E 22c, Sections 10.1.1 and 10.1.2), but may be even worse due to steric hindrance of the large alkyl chains. Similarly, acylation with such amino acids leads to poor yields by the use of even stronger acylating reagents, e.g. HBTU, BOP, and PyBroP, with a marked improvement by the use of the mixed anhydride method although long reaction times were required. 128 ... 

Nucleoside 5 -phosphorothioates have also been employed as activated nucleotide derivatives for synthesis of pyrophosphates.321 The interaction of tributylammonium 2, 3 -di-0-benzoyluridine 5 -phosphorothioate (73) with silver a-D-glucopyranosyl and a-D-galac-topyranosyl phosphates in pyridine solution, with subsequent de-benzoylation, gave the corresponding glycosyl esters in 60-70% yield. This procedure can probably be classified as a variant of the mixed-anhydride method, the driving force of the reaction being the formation of insoluble silver sulfide. 

The deacetyl compound (26-6) is now used for the direct production of cephalexin (25-3) as well as several other related agents that incorporate similar amide side chains. For example, reaction of the protected 7-ADCA derivative (27-2) with the fert-BOC amide from D- flra-hydroxyphenylglyeine (27-1) by the mixed anhydride method gives the amide (27-3). Serial scission of the tert-BOC group and the silyl ester affords the antibiotic cefadroxyl (27-4) [32]. Exactly the same sequence starting... 

Acylation of the amine with the methoxime from aminothiazole-glyoxylate has a similar effect on broadening the antibacterial spectmm in the 3-methyl series. In this case, the amine group on the starting thiazole (21-3) is first protected by conversion to (28-2) by alkylation with thriphenylmethyl chloride. Condensation of the acid by the mixed anhydride method with 7-ADC A (26-6) leads to the corresponding amide. The trityl group is then removed to afford the antibiotic cefetamet (28-3) [34]. 

The Z-protected derivative, again prepared by standard methods using benzyl chloroformate,t208 may serve in the case of racemic pipecolic acid for resolution into the pure enantiomers by fractional crystallization with L-tyrosine hydrazide/208 Acylation with N-protected pipecolic acid or of pipecolyl peptides is performed by standard procedures via the active ester methods, e.g. A-hydroxysuccinimide ester/121 by the mixed anhydride method, e.g. with isobutyl chloro-formate 95-114 or pivalic acid chloride/121 as well as by DCC/HOBt/118 In the synthesis on solid support, longer coupling times are required when compared to N-protected proline.1[235 ... 

The syntheses of 4-[3-(trifluoromethyl)-3//-diazirin-3-yl]arene-containing peptides are carried out in a straightforward manner. In solid-phase syntheses, H-Phe(4-Tmd)-OH [Tmd = 3-(trifluoromethyl)-3//-diazirin-3-yl] is incorporated as the Fmoc-Phe(4-Tmd)-OH (Table 4).[i23,130-133] Solution-phase syntheses of [Phe4(4-Tmd)J-Leu-enkephalin and [Phe2(4-Tmd)J-aspartame employ Boc-Phe(4-Tmd)-OH and are carried out by the mixed anhydride method.1122 Apparently Phe(4-Tmd) is stable in TFA/CH2C12 (1 1) for 30 min at rt 122 or even in neat TFA for 15 min at 0°CJ134] A post-synthetic modification of a free peptide has been carried out by the mixed anhydride mediated coupling of 4-[3-(trifluoromethyl)-3/7-diazirin-... 

Ca-Tetrasubstituted a-Amino Acids as Nucleophiles The Mixed Anhydride Method [Scheme 5 Only for (aTfm)Ala] 109 ... 

Preparation of [NHCHj] pseudodipeptides from a A-acetyl gem-diamine 88 and an a-aldehydo ester 89 was unsuccessful.185 The desired pseudodipeptide was unstable and eliminated the acetamide moiety. To overcome this instability a pseudotripeptide was prepared that incorporates the reduced retro isostere from a retro-inverso pseudodipeptide 85 the synthesis of this compound is shown in Scheme 16. Coupling of Z-L-Phe-OH with 32 using the mixed anhydride method afforded the retro-inverse pseudodipeptide 33. Reaction of 33 after hydrogenation with an a-aldehydo ester provides the reduced-retro pseudotripeptide 35 as a pair of diastereomers. 

Curtius rearrangement of the acyl azide of Ac-Phe-OH 31, at rt in dilute HC1, provided Ac-Phe-NH2-HC1 32 in up to 65% yield. Coupling of Z-Phe-OH with 32 using the mixed anhydride method afforded Ac-Pheip[NHCO]Phe-Z 33 as a single diastereomer in 55% yield. Reaction of 33 after hydrogenation with HCOCH(iPr)C02Me provides 34. Compound 34 was hydrogenated to afford the reduced-retro pseudotripeptide 35 in 65% yield for the two steps. 

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