The first part is substitution and the second part is addition. Carboxylic acids can be converted to esters, amides, acyl hydrazides or hydroxamic acids, all of which are discussed in this section. Following coupling of the half-protected aliphatic diamine to an activated carboxylic acid, the t-BOC group can be quantitatively removed with trifluoroacetic acid (Figure 3.4.1). They do, however, react spontaneously with the common amine-reactive functional groups described in Introduction to Amine Modification—Section 1.1, including succinimidyl esters and isothiocyanates. Here's an example of the use of this synthetic sequence of reactions to make cyclohexanone: We'll look at some of the details of the saponification reaction next time. This means that a Dieckmann or Claisen condensation can be followed by a hydrolysis-decarboxylation step to form a ketone. Chem 360 Jasperse Ch. Kirk McMichael (Washington State University). Reductions of carboxylic acid derivatives. Figure 3.4.2 Stabilization of an unstable O-acylisourea intermediate by N-hydroxysuccinimide in a carbodiimide-mediated (EDAC, E2247) modification of a carboxylic acid with a primary amine. EDAC-mediated coupling has been used to functionalize Qdot 605 ITK carboxyl quantum dots (Q21301MP, Qdot Nanocrystals—Section 6.6) with the GABA receptor agonist muscimol. These reductions are normally carried out using a strong reducing agent, such as lithium aluminum hydride (LiAlH 4). Peptide synthesis research has led to the development of numerous methods for coupling carboxylic acids to amines in organic solution. This same pattern describes the first steps in the reaction of esters with lithium aluminum hydride and Grignard reagents, but in both cases the reaction proceeds further because the first product formed also reacts with the reagent. Impermeability of the enzyme restricts this reaction to a limited number of proteins on the cell surface. Remember that the Grignard reagent serves as a source of carbon nucleophiles. (Consider what would happen if the base and the OR' group were different. In addition, fatty acids derivatized with these reagents have been used to measure phospholipase A2 activity (Probes for Lipid Metabolism and Signaling—Section 17.4). One such method involves the conversion of carboxylic acids to succinimidyl esters or mixed anhydrides. Notice that both the Claisen condensation and its Dieckmann condensation variant make a compound we can call a beta-keto-ester, that is, an ester with a keto group on the carbon two atoms away from the carbonyl carbon. The instant a small amount of aldehyde is formed, it is in competition with the remaining acid or ester for the LiAlH 4 reagent. Including N-hydroxysulfosuccinimide (H2249) in the reaction mixture has been shown to improve the coupling efficiency of EDAC-mediated protein–carboxylic acid conjugations (Figure 3.4.2). NHSS esters of biotin and other derivatives considerably increase the aqueous solubility of the reagents. (Since the newly-formed ketone is in a beta position relative to the ester functional group these compounds are called beta-keto-esters.) Esterification of carboxylic acids in aqueous solution is usually not possible, and esters tend to be unstable in water. The fluorescence lifetime (τ) of the Alexa Fluor 488 dye in pH 7.4 buffer at 20°C is 4.1 nanoseconds. Again, this is strictly analogous to the situation in the aldol reaction, and it has resulted in a nucleophilic addition to the carbonyl carbon. Unless otherwise noted, LibreTexts content is licensed by CC BY-NC-SA 3.0. 10: Carboxylic Acid Derivatives - Alpha Carbon Reactions, [ "article:topic", "showtoc:no", "license:arr" ], 9: Carboxylic Acid Derivatives: Interconversion, Reduction of Esters - Lithium Aluminum Hydride, Reaction of Esters with Grignard Reagents. Carboxylic esters hydrolyse to the parent carboxylic acid and an alcohol. This is a useful way to make primary alcohols. This is followed by attack of the nucleophilic alpha carbon of the enolate on the carbonyl carbon of a second molecule of the ester. The resulting Claisen condesation makes a ring of carbon atoms. Here's an example in which the Grignard reagent is methylmagnesium bromide: Here the first product is a ketone in which the OR' group of the ester has been replaced by the alkyl group of the Grignard reagent. Since many organic compounds which occur in nature have five or six membered rings, the Dieckmann condensation has been widely used in synthesis. The coumarin iodoacetamide DCIA (D404, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) has also been used to derivatize carboxylic acids; other iodoacetamides described in Thiol-Reactive Probes—Chapter 2 will probably react similarly. The beta-keto-acids readily lose a molecule of carbon dioxide when heated to form a ketone. H 2 SO 4) / heat,or aqueous NaOH / heat (known as "saponification"). Notice that the new carbon-carbon formed in this sequence is one which joins a carbonyl carbon to one of its alpha carbons. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7. For an example, lets look at the reduction of an ester with lithium aluminum hydride. We spent some time on the aldol condensation when we were studying aldehydes, and we learned that the alpha C-H bonds of aldehydes and ketones can be attacked by base to form enolates. This technique was used to selectively label erythrocyte band 3 protein with dansyl cadaverine (D113) and proteins of the extracellular matrix with fluorescein cadaverine (A10466). Remember that the Grignard reagent serves as a source of carbon nucleophiles. This ketone is immediately attacked by another molecule of the Grignard reagent. Not for use in diagnostic procedures. This overall reaction is called the Claisen condensation after Ludwig Claisen, a prominent German organic chemist of the late 19th and early 20th centuries. We can't stop the reaction halfway, because aldehydes and ketones are more reactive than esters, so the aldehydes and ketones gobble up the reagent faster than the esters. Transamidation of cell-surface glutamine residues by the combination of a transglutaminase enzyme and a fluorescent or biotinylated aliphatic amine can form stable amides. Thermo Fisher Scientific. 2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate has been used for the sensitive reverse-phase HPLC detection of eicosanoids in brain tissue. Aromatic amines can generally be coupled to acid halides and anhydrides, with organic solvents usually required for efficient reaction. 4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S10490) and N-hydroxysulfosuccinimide (NHSS, H2249) can be used to prepare water-soluble activated esters from various carboxylic acids (Figure 3.4.4). In both these cases, we've combined a the general pattern for carboxylic acid derivatives, substitution of the nucleophile in place of the leaving group, with a pattern which applies to aldehydes and ketones, to arrive at "double" reaction. Therefore, it is preferable to use neutral or alkaline agents such as potassium permanganate for this preparation method. The magnitude of the spectral shift depends on the array substrate material. The fluorescent diazomethyl derivative 9-anthryldiazomethane (ADAM, A1400) has been commonly used to derivatize biomolecules. An analytical method for estimating the degree of EDAC crosslinking of collagen has been developed based on derivatization of residual carboxyl groups by 5-bromomethylfluorescein. Each acid has a very specific melting point. Biologically important molecules, especially the nonchromophoric fatty acids, bile acids and prostaglandins, are typically esterified by carboxylic acid–reactive reagents in organic solvents. identify the acid halide, the reagents, or both, needed to prepare a given carboxylic acid, ester or amide. If a base is used, a salt is formed instead of the carboxylic acid, but the salt is easily converted to the acid by treatment with hydrochloric acid. To reduce intra- and inter-protein coupling to lysine residues, which is a common side reaction, carbodiimide-mediated coupling should be performed in a concentrated protein solution at a low pH, using a large excess of the nucleophile. Coupling typically involves a carbodiimide such as EDAC (E2247) and is performed in an organic solvent. When the "hydride ion" (H:-) from lithium aluminum hydride replaces the OR' group of the ester, an aldehyde is formed. We offer a variety of amine-reactive STP esters, which are discussed in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1. The resultant aliphatic amine can then be modified with any of the amine-reactive reagents described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 or coupled to solid-phase matrices for affinity chromatography. For more information contact us at info@libretexts.org or check out our status page at https://status.libretexts.org. Search Although dansyl cadaverine (D113) has been probably the most widely used reagent, Alexa Fluor cadaverines (A30674, A30675, A30676, A30677, A30678, A30679, A30680), Oregon Green 488 cadaverine (O10465), fluorescein cadaverine (A10466), tetramethylrhodamine cadaverine (A1318), Texas Red cadaverine (T2425) and BODIPY TR cadaverine (D6251) are among the most fluorescent transglutaminase substrates available. Fluoresceinyl glycine amide (5-(aminoacetamido)fluorescein, A1363) and various hydrazines and hydroxylamines may be the best probes for this application because they are more likely to remain reactive at a lower pH than are aliphatic amines such as the cadaverines. Alternatively, the half-protected tert-butyloxycarbonyl (t-BOC) propylenediamine derivative (M6248) is useful for converting organic solvent–soluble carboxylic acids into aliphatic amines. Reaction of Esters with Grignard Reagents. Missed the LibreFest? Unfortunately, ADAM is not very stable and may decompose during storage. We've seen that carboxylic acid derivatives react with nucleophiles to give substitution products in which the leaving group is replaced by the attacking nucleophile. These mechanisms are among some of the most studied in organic chemistry. Watch the recordings here on Youtube! We can use the same pattern as worked for the lithium aluminum hydride reduction by replacing the "H:-" with the "C:-" from the Grignard reagent. These probes all require a coupling agent such as a carbodiimide to react with carboxylic acids; they do not spontaneously react with carboxylic acids in solution.