Succinylation

Conversion of amino groups to acidic derivatives 3.1.3.1. Succinylation 

The reaction of amino groups with succinic anhydride has been utilized to dissociate multi-subunit proteins, to solubilize proteins, or to limit the action of trypsin to arginine residues. More recently, partial succinylation of multi-subunit proteins has been used to obtain information about the number and type of subunits present (see Klotz 1967 Klapper and Klotz 1972). 

In addition to acylating a- and e-amino groups, succinic anhydride reacts with tyrosyl, histidyl, cysteinyl, seryl and threonyl side-chains. The tyrosyl derivative hydrolyzes spontaneously at alkaline pH, and is rapidly decomposed by hydroxylamine at neutral pH. The histidyl derivative is presumably very labile. The lability of all of the ester derivatives to hydroxylamine at pH 10 permits preparation of a protein modified solely on the a- and e-amino groups (e.g. Gounaris and Perlmann 1967). 

If complete selective modification of amino groups is desired, the reaction should be carried out on a protein derivative in which sulf-hydryl and disulfide groups have been converted to a stable derivative. Otherwise, these groups would be exposed to both interchange and oxidation under the conditions required to cleave succinyl esters. 

A suitable protein derivative is dissolved in 5.0 M guanidine hydrochloride at pH 8.0, and solid succinic anhydride (50 moles/mole of amino group) is added in small portions to a rapidly stirred solution at room temperature. The pH is maintained (by means of a pH-stat) by the addition of M NaOH. When the addition of succinic anhydride is complete, solid hydroxylamine hydrochloride is added to the reaction mixture to a concentration of 1 M, the pH is adjusted to 10 with NaOH and the mixture allowed to stand at room temperature for 

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C14H30CI2N2O4. White powder prepared from dimethylaminoethanol and succinyl chloride, followed by methylation. Neuromuscular blocking agent used to relax skeletal muscles during certain types of surgical operation. 

Exceptions formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, oxalyl, malonyl, succinyl, glutaryl, furoyl, and thenoyl. 

The lowest members of the series of perfluoroalkanedicarboxyhc acids have been prepared and are stable compounds. They have been synthesized by oxidation of the appropriate chlorofluoroolefin as well as by electrochemical fluorination and direct fluorination. Perfluoromalonic acid is an oxidation product of CH2=CHCE2CH=CH2 (21). Perfluorosuccinic acid has been produced by oxidation of the appropriate olefin (see eq. 7) (5) or by electrochemical fluorination of succinyl chloride or butyrolactone (41) and subsequent hydrolysis. 

Functional properties of canola protein products can be improved by succinylation (130,131). Controlled acetylation can reduce undesirable phenoHc constituents as well (132). However, antinutrients in canola and other vegetable protein products such as glucosinolates, phytic acid, and phenoHc compounds have severely limited food appHcations of these products. 

The synthetic scheme typically involves chain-extending addition of protected mononucleotides to a nucleoside bound covalentiy at the 3 -hydroxyl to an inert siUca-based soHd support, such as controlled pore glass (Fig. 11). The initial base-protected 5 -O-dimethoxytrityl (DMT) deoxynucleoside is linked to the soHd support via the reaction of a siUca-bound amino-silane and the -nitrophenylester of the 3 -succinylated nucleoside, yielding a 3 -terminal nucleoside attached to the soHd support (1) (Fig. 11). Chain elongation requites the removal of the 5 -DMT protecting group. 

The resulting acetyl compound is usually hydrolyzed with aqueous alkaU to give the free amine. Other A/-acyl derivatives may be used, particularly for the less soluble succinyl and phthaloyl products. The use of -nitrobenzenesulfonyl chloride, followed by reduction of the nitro to an amino function, is much more expensive and is rarely used. A/-Acetylsulfanilyl chloride [121 -60-8] is obtained by the chlorosulfonation of acetanilide [103-84-4] which is the basic material for most of the sulfonamides. 

Succinic anhydride [108-30-5] (3,4-dihydro-2,5-furandione butanedioic anhydride tetrahydro-2,5-dioxofuran 2,5-diketotetrahydrofuran succinyl oxide), C H O, was first obtained by dehydration of succinic acid. In the 1990s anhydride is produced by hydrogenation of maleic anhydride and the acid by hydration of the anhydride, by hydrogenation of aqueous solutions of maleic acid, or as a by-product in the manufacture of adipic acid (qv) (see Maleic ANHYDRIDE, MALEIC ACID, AND FUMARIC ACID). 

An important use of dialkyl succinates is in the preparation of dialkyl succinyl succinates (35,53—56), which are intermediates in the manufacture of quinacridone pigments. The reaction is carried out in the presence of alkaU metal alkoxides (eq. 2). 

Succinyl chloride [543-20-4] is obtained from phosphorous pentachloride and succinic acid, from thionyl chloride and succinic acid or anhydride (91,92), or from phosgene and succinic anhydride (93). 

Succinic acid reacts with urea in aqeous solution to give a 2 1 compound having mp 141°C (116,117), which has low solubiUty in water. A method for the recovery of succinic acid from the wastes from adipic acid manufacture is based on this reaction (118,119). The monoamide succinamic acid [638-32-4] NH2COCH2CH2COOH, is obtained from ammonia and the anhydride or by partial hydrolysis of succinknide. The diamide succinamide [110-14-3], (CH2C0NH2)2, nip 268—270°C, is obtained from succinyl chloride and ammonia or by partial hydrolysis of succinonitrile. Heating succinknide with a primary amine gives A/-alkylsucckiknides (eq. 9). 

Animals caimot synthesize the naphthoquinone ring of vitamin K, but necessary quantities are obtained by ingestion and from manufacture by intestinal flora. In plants and bacteria, the desired naphthoquinone ring is synthesized from 2-oxoglutaric acid (12) and shikimic acid (13) (71,72). Chorismic acid (14) reacts with a putative succinic semialdehyde TPP anion to form o-succinyl benzoic acid (73,74). In a second step, ortho-succmY benzoic acid is converted to the key intermediate, l,4-dihydroxy-2-naphthoic acid. Prenylation with phytyl pyrophosphate is followed by decarboxylation and methylation to complete the biosynthesis (75). 

Succinyl coenzyme A trisodium salt [108347-97-3] M 933.5. If it should be purified further then it should be dissolved in H2O (0.05g/mL) adjusted to pH 1 with 2M H2SO4 and extracted several times with Et20. Excess Et20 is removed from the aqueous layer by bubbling N2 through it and stored frozen at pH 1. When required the pH should be adjusted to 7 with dilute NaOH and used within 2 weeks (samples should be frozen). Succinyl coenzyme A is estimated by the hydroxamic acid method [J Biol Chem 242 3468 1967]. It is more stable in acidic than in neutral aqueous solutions. [Methods Enzymol 128 435 7956.]... 

Dicarboxylic acid dichlorides with less than seven carbon atoms do not react to give tetraketones similar to 117, but instead undergo an intramolecular acylation (72) to give on hydrolysis the vinylogous acid anhydride (118), e.g., from succinyl chloride and the enamine (113). 

The acylation of enamino ketones can take place on oxygen or on carbon. While reaction at nitrogen is a possibility, the N-acylated products are themselves acylating agents, and further reaction normally takes place. The first reported acylation of enamino ketones (72) was that of 129, prepared by acylation of the enamine (113), which was shown to have undergone O acylation because on mild hydrolysis the enol ester (130) could be isolated. A similar reaction took place with other aliphatic acid chlorides (80) and with dibasic acid chlorides [e.g., with succinyl chloride to give 118 above]. 

Lsocitrate Dehydrogenase—The First Oxidadon in die Cycle m-Ketoglutarate Dehydrogenase—A Second Decarboxylation Succinyl-CoA Synthetase—A Substrate-Level Phosphoryladon Succinate Dehydrogenase—An Oxidadon Involving FAD... 

Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative AG°. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog). 

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