Phenylacetic acid side chain

The five parent compounds in Table III are arranged in order of increasing pKa of their ionizable protonic groups. For phenoxyacetic acid (pKa = 3.17) and phenylacetic acid (pKa = 4.31), the primary ionization is that of the carboxylic acid side chain. The acidity of the TFMS parent compound (pKa = 4.45) is attributable to the loss of the relatively labile proton from the parent side chain (< -NH-SOo-CF3 < -N -S02-CF3 -f- H+). For aniline, the process with pKa = 4.63 is associated with protonic ionization of the anilinium cation. The pKa = 9.89 process in phenol refers to the formation of phenolate anion. 

Penicillin Formation by Penicillium Chrysogenum. The first reactions of the penicillin biosynthetic pathway are identical to the ones in A. chrysogenum (Figure 1.1-1). IPN, however, is not epimerized to penicillin N instead it is converted to 6-aminopenicillanic acid (6-APA) by removal of the L-a-aminoadipic acid side chain, which is substituted by a hydrophobic acyl group. Both steps are catalyzed by the same enzyme, the acyl coenzyme A IPN acyltransferase (IAT). The enzymatic activity of lAT is believed to be the result of the processing of a 40-kD monomeric precursor into a dimeric form consisting of two subunits with MWs of 11 and 29 kD. Due to the broad substrate specifity of lAT, various penicillin derivatives are synthesized naturally by attachment of different acyl-CoA derivatives to the 6-APA-core. For industrial purposes, to facilitate extraction by organic solvents, synthesis usually is directed to the less hydrophilic penicillin V or penicillin G. This is by addition of phenoxyacetic acid or phenylacetic acid, respectively, as precursors to the culture broth. 

Figure 10.4 The penicillin biosynthetic pathway. AcvA, IpnA, and AatA indicate ACV synthase, IPN synthase, and acyl-CoA IPN acyltransferase, respectively. R-COOH represents a large variety of aliphatic and aromatic acid side chains, such as phenylacetic (Penicillin G), phenoxyacetic (V), octanoic (K), hexenoic (DF), and A3-hexenoic (F) acids.

Russian authors have applied the Ivanov reaction to 1,2 5,6-di-O-cyclo-hexylidene-a-D-n7)0-hexos-3-ulofuranose, which involves reacting phenylacetic acid, magnesium, and isopropyl bromide in tetrahydrofuran. The 3-ulose is then added to this preformed complex, when the addition of the phenylacetie acid side-chain occurs to give (14). 

Reactions of the Side Chain. Benzyl chloride is hydrolyzed slowly by boiling water and more rapidly at elevated temperature and pressure in the presence of alkaHes (11). Reaction with aqueous sodium cyanide, preferably in the presence of a quaternary ammonium chloride, produces phenylacetonitrile [140-29-4] in high yield (12). The presence of a lower molecular-weight alcohol gives faster rates and higher yields. In the presence of suitable catalysts benzyl chloride reacts with carbon monoxide to produce phenylacetic acid [103-82-2] (13—15). With different catalyst systems in the presence of calcium hydroxide, double carbonylation to phenylpymvic acid [156-06-9] occurs (16). Benzyl esters are formed by heating benzyl chloride with the sodium salts of acids benzyl ethers by reaction with sodium alkoxides. The ease of ether formation is improved by the use of phase-transfer catalysts (17) (see Catalysis, phase-thansfer). 

Several pathways are used for the aerobic degradation of aromatic compounds with an oxygenated C2 or C3 side chain. These include acetophenones and reduced compounds that may be oxidized to acetophenones, and compounds including tropic acid, styrene, and phenylethylamine that can be metabolized to phenylacetate, which has already been discussed. 

A strain of Pseudomonas sp. ATS degraded tropic acid, which has a -CH-(CH20H)-COjH side chain to phenylacetate (Long et al. 1997). 

The Hammett equation is the best-known and most widely studied of the various linear free energy relations for correlating reaction rate and equilibrium constant data. It was first proposed to correlate the rate constants and equilibrium constants for the side chain reactions of para and meta substituted benzene derivatives. Hammett (37-39) noted that for a large number of reactions of these compounds plots of log k (or log K) for one reaction versus log k (or log K) for a second reaction of the corresponding member of a series of such derivatives was reasonably linear. Figure 7.5 is a plot of this type involving the ionization constants for phenylacetic acid derivatives and for benzoic acid derivatives. The point labeled p-Cl has for its ordinate log Ka for p-chlorophenylacetic acid and for its abscissa log Ka for p-chloroben-zoic acid. The points approximate a straight line, which can be expressed as... 

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

When H2O deacetylates the acyl-enzyme, phenylacetic acid is formed. When nucleophiles other than H2O deacylate the acyl-enzyme, a new condensation product, in this case phenylacetyl-O-R or phenylacetyl-NH-R is formed. By definition the hydrolysis of these condensation products can be catalyzed by the same enzyme that catalyzes their formation in equation 10.1. Thus, when the acyl-enzyme is formed from phenylacetyl-glycine or phenylacetyl-O-Me, this gives rise to an alternative process to produce Penicillin G, in addition to the thermodynamically controlled (= equilibrium controlled) condensation of phenylacetic acid and 6-aminopenicillanic acid (6-APA). This reaction that involves an activated side chain is a kinetically controlled (= rate controlled) process where the hydrolase acts as a transferase (Kasche, 1986 1989). 

Some support for this mechanism derives from our observation of benzyl radical from phenylacetic acid, phenoxymethyl radical from phenoxy-acetic acid, and possibly the phenyl radical from benzoic acid. Radical spectra were not found for several aryl acids with longer side chains but this is not inconsistent because the 0-phenylethyl and higher radicals are not expected to absorb strongly above 300 m/z. The case of the heterocyclics is complicated by the possibility of excitation either via the 7r-system or a lone pair electron on the heteroatom, and discussion of this group will be deferred until additional experimental data are obtained. 

The enzymic oxidative deamination of simple phenethylamines is exemplified by the reported bio transformations of mescaline (146) (114, 115) and ephedrine (148) (116). Mescaline is metabolized to 3,4,5-trimethoxy-phenylacetic acid by tissue homogenates of mouse brain, liver, kidney, and heart (114,115). 3,4,5-Trimethoxybenzoic acid is also formed as a minor metabolite. The formation of jV-acetylmescaline (147), a significant metabolite in vivo, was not observed in the in vitro studies. Both D-(—)-and L-(+)-ephedrine have been incubated with enzyme preparations from rabbit liver norephedrine (149), benzoic acid, and 1-phenyl-1,2-propanediol were characterized as metabolites (116). The D-(—)-isomer was the better substrate, being more rapidly converted. Similar results were previously reported with rabbit liver slices as the source of enzyme (153,154). The enzymic degradation of the side chain of /i-phenethylamines has been extensively investigated with nonalkaloid substrates such as amphetamine (151) and jV-methylamphetamine (150) (10,155-157), and the reader is referred to these studies for a more comprehensive coverage of this aspect of the subject. 

The addition of suitable precursors (e.g., phenylacetic acid, phenoxyacetic acid) in the fermentation medium of P. chrysogenum allows the formation of specific penicillins (G, V, F, K, X) with nonpolar side chains < 1995JAN1195, 1998MI2001, 1999MI173>. Penicillins are the only /3-lactam products formed, while fermentation of Cephalosporium acremonium produces penicillin N (D-a-aminoadipyl side chain) together with cephalosporin derivatives (CHEC-11(1996), section 1.20.6.1) <1996CHEC-II(1B)623>. 

The relationship between the biosynthesis of the penicillin and cephalosporin nuclei [127] is shown in Fig. 8.25. The common intermediate in the biosynthesis of penicillins and cephalosporins is isopenicillin N (IPN), which in Penicil-lium is converted into penicillin G by replacement of the L-2-aminoadipyl side-chain with externally supplied phenylacetic acid, mediated by IPN acyl transferase (IPN AcT). In the cephalosporin-producing Ammonium chrysogenum, IPN is subjected to an enzymatic ring expansion. 

Although the pKa s of phenoxyacetic acid, phenylacetic acid, TFMS, and aniline are all quite similar, differing by less than 1.5 pK units for the most extreme comparison (phenoxyacetic acid vs. aniline), this similarity ends when the partitioning behavior of these same parent compounds is compared. Whereas the logarithm of the octanol/water partition coefficient (F) varies only from log F = 0.90 for aniline to log F = 1.41 for phenylacetic acid, TFMS (log F = 3.05) is 1.6 orders of magnitude more lipophilic than phenylacetic acid, the most hydrophobic of the other parent compounds exhibiting a similar pKa. If the w values for the side chains of the parent compounds listed in Table III are calculated using Equation 5 as shown on p. 196 ... 

Biochemical oxidations of side chains proceed without degradation or with only limited degradation. Both ethylbenzene and butylbenzene, and even dodecylbenzene, give phenylacetic acid on incubation at 30 °C with Nocardia strain 107-332 (equation 182) [1071]. 

In 1904, Franz Knoop outlined a scheme for the biological oxidation of fatty acids that was shown—50 years later—to be correct. In his key experiments, he fed rabbits fatty acids of formula C5H5(CH2)nCOOH. When the side chain (/f + 1) contained an even number of carbons, a derivative of phenylacetic acid, C6H5CH2COOH, was excreted in the urine an odd number, and a derivative of benzoic acid was excreted. What general hypothesis can you formulate from these results ... 

The acyl side-chain (R) varies, depending on the make up of the fermentation media. For example, corn steep liquor was used as the medium when penicillin was first mass-produced in the United States and this gave penicillin G (R=benzyl). This was due to high levels of phenylacetic acid (PhCH2C02H) present in the medium. 

Several biosyntheses involve assembling building blocks such as amino acids and carboxylic acids. Parts of these assembly processes can show some substrate flexibility, allowing the enzymatic synthesis of un-natural analogues of normal fungal metabolites. One of the earliest examples was the variation of the penicillin side-chain brought about by replacing the phenylacetic acid of penicillin G with phenoxyacetic acid as in penicillin V. 

Because of a newly introduced gene, the substrate specificity of the engineered strain changed. Now, dicarboxylic acid is used as the externally added side chain, instead of phenylacetic acid as in penicillin G. 

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