Acylation

Acylation is a fundamental class of chemical reactions in organic chemistry wherein an acyl group (R–C=O) is introduced into a substrate molecule, typically through the interaction with an acylating agent such as an acyl chloride, carboxylic anhydride, or ester.^[1] This process generally proceeds via nucleophilic acyl substitution, involving the addition of a nucleophile to the carbonyl carbon followed by elimination, forming a tetrahedral intermediate and enabling the transfer of the acyl moiety to alcohols, amines, or other nucleophilic sites.^[2] Acylation reactions are characterized by varying reactivity among acyl derivatives, with acyl halides and anhydrides being the most reactive due to their electrophilic carbonyl carbons, while amides are the least reactive owing to resonance stabilization.^[2] In synthetic organic chemistry, acylation serves as a cornerstone for building molecular complexity, particularly through electrophilic aromatic substitution in the Friedel-Crafts acylation, where aromatic compounds react with acyl chlorides in the presence of a Lewis acid catalyst like AlCl₃ to produce aryl ketones.^[3] This reaction is highly regioselective, directing substituents to specific positions on the ring, and is widely employed in the synthesis of pharmaceuticals, dyes, and agrochemicals due to its ability to form stable C–C bonds without polyalkylation issues common in alkylation analogs.^[3] Nucleophilic variants, such as the formation of esters from alcohols or amides from amines, further underscore acylation's versatility in protecting groups, peptide synthesis, and polymer chemistry.^[2] In biochemistry, acylation functions as a dynamic post-translational modification (PTM) that covalently attaches acyl groups to proteins, modulating their structure, localization, stability, and interactions with other biomolecules.^[4] Common forms include N-terminal myristoylation on glycine residues for membrane targeting, lysine acetylation that alters chromatin structure and gene expression via histone modifications, and reversible S-palmitoylation on cysteine thiols, which regulates protein trafficking and signaling in cellular membranes.^[4] These modifications are enzymatically controlled by acyltransferases and thioesterases, with dysregulation implicated in diseases such as cancer and neurodegeneration, highlighting acylation's broader biological significance.^[4]

Fundamentals

Definition of Acylation

Acylation is a fundamental class of chemical reactions in organic chemistry wherein an acyl group, represented as $\ce{R-C=O}$\ceR−C=O (where R is typically an alkyl or aryl substituent), is introduced into an organic substrate through the use of an acylating agent. This process generally proceeds via the reaction of the acylating agent with a nucleophilic site on the substrate, resulting in the formation of a new carbon-acyl bond. Common acylating agents include acyl chlorides ($\ce{RCOCl}$\ceRCOCl), acid anhydrides ($\ce{(RCO)2O}$\ce(RCO)2O), and other activated derivatives of carboxylic acids.^[2]/Carboxylic_Acids/Reactivity_of_Carboxylic_Acids/Acyl_Chlorides) In a typical schematic, an acyl chloride such as $\ce{RCOCl}$\ceRCOCl reacts with a nucleophile (Nu:) or suitable substrate to yield the acylated product $\ce{RC(O)Nu}$\ceRC(O)Nu and a leaving group like chloride ion, thereby establishing the core transformation central to acylation. This distinguishes acylation from related substitution processes: unlike alkylation, which incorporates an alkyl group ($\ce{R-}$\ceR−) to form C-C bonds without a carbonyl, or sulfonylation, which adds a sulfonyl group ($\ce{RSO2-}$\ceRSO2−) for sulfone or sulfonamide formation, acylation specifically leverages the reactivity of the carbonyl functionality for bond formation.^[2]/Arenes/Reactivity_of_Arenes/Friedel-Crafts_Acylation) The concept of acylation has historical roots in late 19th-century organic chemistry, with the term first appearing in the late 19th century^[5] amid studies of carboxylic acid derivatives; influential work by chemists like Victor Meyer on esterification and acid reactivity laid foundational groundwork for understanding these transformations. Acylation reactions span both intermolecular and intramolecular variants, enabling the synthesis of diverse carbonyl-containing compounds such as esters (from alcohols), amides (from amines), ketones (via carbon nucleophiles), and thioesters (from thiols), thereby serving as a versatile tool in synthetic organic chemistry.^[6][2][7]

The Acyl Group

The acyl group is a key functional moiety in organic chemistry, characterized by the general formula $R-C(=O)-$R−C(=O)−, where $R$R represents hydrogen, an alkyl chain, an aryl group, or another organic substituent. This group is derived from carboxylic acids ($R-COOH$R−COOH) by the removal of the hydroxyl ($-OH$−OH) portion of the carboxyl group, leaving behind the carbonyl ($C=O$C=O) unit. In the context of inorganic chemistry, acyl groups can also arise from oxoacids more broadly, but in organic applications, the carboxylic acyl form predominates.^[8] Nomenclature for acyl groups follows systematic IUPAC conventions, with those derived from aliphatic carboxylic acids termed alkanoyl groups—for instance, the acetyl group ($CH_3CO-$CH3​CO−) from acetic acid ($CH_3COOH$CH3​COOH). Acyl groups from aromatic carboxylic acids are known as aroyl groups, such as the benzoyl group ($C_6H_5CO-$C6​H5​CO−) obtained from benzoic acid ($C_6H_5COOH$C6​H5​COOH). These names are formed by replacing the -ic acid ending of the parent acid with -oyl, ensuring precise identification in chemical structures and reactions.^[9] The electronic properties of the acyl group render it highly reactive, particularly at the carbonyl carbon, which exhibits electrophilic character due to the polarity of the $C=O$C=O bond. The oxygen atom's high electronegativity withdraws electron density from the carbon, creating a partial positive charge ($\delta^+$δ+) that attracts nucleophiles. Resonance stabilization in acyl derivatives can modulate this electrophilicity, but the inherent $\pi$π-bond character of the carbonyl further enhances the carbon's susceptibility to nucleophilic attack.^[10] Common acylating agents that deliver the acyl group in synthetic transformations include acyl halides ($RCOX$RCOX, where $X$X is chloride or bromide), which are highly reactive due to the excellent leaving group ability of the halide, and acid anhydrides ($(RCO)_2O$(RCO)2​O), which provide a balanced reactivity profile. Carboxylic acids ($RCOOH$RCOOH) can also serve as acylating agents when activated, for example, via conversion to mixed anhydrides or using coupling reagents, allowing milder conditions in amide bond formation.^[11] Less common variations of the acyl group encompass thioacyl groups ($R-C(=S)-$R−C(=S)−), where sulfur replaces the carbonyl oxygen, leading to thiocarbonyl compounds with distinct reactivity profiles, such as in thioester synthesis. Iminoacyl groups ($R-C(=NR')-$R−C(=NR′)−), featuring a $C=NR'$C=NR′ unit instead of $C=O$C=O, appear in specialized contexts like metal coordination chemistry but are rarer in routine organic synthesis. These analogues highlight the acyl group's versatility through chalcogen or nitrogen substitution.^[12]

Reaction Types

Nucleophilic Acyl Substitution

Nucleophilic acyl substitution is a class of reactions in which a nucleophile, such as an alcohol, amine, or water, attacks the electrophilic carbonyl carbon of an acyl derivative, displacing a leaving group and resulting in the formation of new carbonyl compounds like esters, amides, or carboxylic acids.^[13][14] This process is fundamental to the reactivity of carboxylic acid derivatives, where the acyl group serves as the reactive electrophilic unit.^[15] Common substrates for these reactions include acyl chlorides, acid anhydrides, esters, and amides, with reactivity decreasing in the order acyl chlorides > anhydrides > esters > amides due to the stability and leaving group ability of the departing group—chloride being an excellent leaving group compared to alkoxide or amide ions.^[16][17] For instance, acyl chlorides react rapidly with nucleophiles even under mild conditions, while amides require harsher conditions owing to the poor leaving group ability of the nitrogen-based group.^[13] A prominent example is the Schotten-Baumann reaction, in which an acyl chloride reacts with an amine in the presence of an aqueous base like sodium hydroxide to form an amide, with the base neutralizing the HCl byproduct and preventing salt formation that could inhibit the nucleophilic attack.^[18][19] Another variant involves esterification, such as the acid-catalyzed Fischer esterification, where a carboxylic acid reacts with an alcohol to form an ester, though this proceeds via protonation of the carbonyl to enhance electrophilicity rather than direct nucleophilic substitution on a derivative.^[20][21] In terms of stereochemistry, nucleophilic acyl substitution typically occurs with retention of configuration at any existing chiral centers in the substrate, as the reaction proceeds through a tetrahedral intermediate at the achiral carbonyl carbon, which reforms the planar sp² geometry without creating a new stereocenter.^[13] These reactions offer high yields in the synthesis of esters and amides, particularly in peptide chemistry where acyl chlorides or activated esters facilitate efficient amide bond formation between amino acids.^[18][14]

Electrophilic Acylation

Electrophilic acylation refers to a class of reactions in which an electrophilic acyl species, typically an acylium ion (RC≡O⁺), is generated and reacts with electron-rich substrates to form new carbon-carbon bonds.^[22] This process contrasts with nucleophilic acyl substitutions by emphasizing the acyl group as the attacking electrophile rather than the target.^[23] The acylium ion is formed from acylating agents such as acyl chlorides or anhydrides in the presence of a Lewis acid catalyst, enabling the electrophile to target nucleophilic centers like aromatic rings or activated alkenes./Arenes/Reactivity_of_Arenes/Friedel-Crafts_Acylation) A prominent example is the Friedel-Crafts acylation, where benzene reacts with acetyl chloride in the presence of aluminum chloride (AlCl₃) to produce acetophenone.^[23] The reaction proceeds as follows: $$\ce{C6H6 + CH3COCl ->[AlCl3] C6H5COCH3 + HCl}$$\ceC6H6+CH3COCl−>[AlCl3]C6H5COCH3+HCl This method efficiently introduces ketone functionality onto aromatic systems, serving as a cornerstone for synthesizing aryl ketones.^[22] The scope of electrophilic acylation is primarily limited to activated or moderately deactivated aromatic compounds, such as those bearing alkyl or alkoxy substituents, while strongly deactivated rings like nitrobenzene fail to react under standard conditions due to insufficient electron density./Arenes/Reactivity_of_Arenes/Friedel-Crafts_Acylation) It also applies to electron-rich alkenes, though less commonly in classical protocols.^[24] Deactivated substrates may require harsher conditions or alternative catalysts to proceed.^[25] A key variation is the Vilsmeier-Haack formylation, which introduces an aldehyde group onto activated aromatics using dimethylformamide (DMF) and phosphoryl chloride (POCl₃) to generate an iminium electrophile equivalent to an acylium ion for R = H.^[26] This reaction is particularly useful for heteroaromatic systems like indoles and pyrroles.^[27] Limitations include the prevention of polyacylation, as the resulting ketone deactivates the aromatic ring toward further electrophilic attack, ensuring monoselectivity.^[28] In substituted benzenes, the acylation exhibits ortho-para directing effects influenced by existing substituents, aligning with general electrophilic aromatic substitution regiochemistry./Arenes/Reactivity_of_Arenes/Friedel-Crafts_Acylation)

Mechanisms

Nucleophilic Addition-Elimination Pathway

The nucleophilic addition-elimination pathway characterizes the mechanism of nucleophilic acyl substitution reactions, where a nucleophile replaces a leaving group on an acyl derivative through a two-step process. In the first step, the nucleophile attacks the electrophilic carbonyl carbon, forming a tetrahedral intermediate in which the carbon adopts a sp³ hybridization and bears four substituents, including a negatively charged oxygen. This addition disrupts the carbonyl π bond, with the electron pair from the nucleophile forming a new σ bond. The tetrahedral intermediate is often stabilized by resonance involving the oxygen lone pair, which can delocalize the negative charge, particularly when the nucleophile is electron-rich, such as an amine.^[29][30][14] In the second step, the tetrahedral intermediate collapses via elimination, reforming the carbonyl π bond and expelling the leaving group, such as chloride from an acyl chloride. This elimination restores the planar sp² geometry of the carbonyl carbon and yields the substitution product. If the nucleophile is neutral, like an amine (R'NH₂), a proton transfer step may follow to deprotonate the intermediate or product, ensuring charge balance; for instance, in amide formation, the nitrogen-attached intermediate loses a proton to generate the neutral amide. A representative example is the reaction of an acyl chloride with a primary amine: $$\mathrm{RC(O)Cl + R'NH_2 \rightarrow RC(O)NHR' + HCl}$$RC(O)Cl+R′NH2​→RC(O)NHR′+HCl ^[29][31][30] Several factors influence the rate of this pathway. The leaving group's ability is critical, with better leaving groups (e.g., Cl⁻ over OR⁻) facilitating faster elimination due to lower bond dissociation energy and higher stability of the departing anion. Nucleophile strength also plays a key role, as stronger nucleophiles (e.g., amines or alkoxides) accelerate the addition step by more effectively donating electrons to the carbonyl. Solvent effects are significant, with polar aprotic solvents (e.g., DMF or DMSO) enhancing rates by solvating cations without hydrogen-bonding the nucleophile, thereby increasing its nucleophilicity compared to protic solvents like water.^[14][32][33] Kinetic studies confirm the mechanism's bimolecular nature, with second-order rate laws (rate = k [acyl derivative] [nucleophile]) indicating that the addition step is typically rate-determining, as the concentration of both reactants affects the overall velocity. In some cases, tetrahedral intermediates have been isolated and characterized, providing direct evidence for their role; for example, lithium carbenoids added to Weinreb amides yield stable tetrahedral species observable by NMR and X-ray crystallography. These findings underscore the pathway's generality across acyl derivatives, with reactivity decreasing in the order acyl chlorides > anhydrides > esters > amides due to leaving group differences.^[34][35][36]

Electrophilic Aromatic Substitution Pathway

The electrophilic aromatic substitution pathway in acylation involves the introduction of an acyl group onto an aromatic ring through the action of a strong electrophile, typically generated from an acyl chloride and a Lewis acid catalyst. This process, commonly exemplified by the Friedel-Crafts acylation, proceeds without disrupting the aromatic system's stability, yielding aryl ketones as products. Unlike other substitution reactions, the acylium ion serves as the key electrophile, ensuring clean reactivity without the rearrangements often seen in alkylations.^[3] The mechanism begins with the coordination of a Lewis acid, such as AlCl₃, to the carbonyl oxygen of the acyl chloride, facilitating the departure of the chloride ion and generating the acylium ion (RC≡O⁺) paired with AlCl₄⁻. This acylium ion is a highly reactive electrophile due to its linear structure and sp hybridization at the carbon atom, which concentrates positive charge effectively. The aromatic ring's π electrons then attack the electrophilic carbon of the acylium ion, forming a sigma complex (also known as the arenium or Wheland intermediate), a resonance-stabilized carbocation where the positive charge is delocalized across the ring. Finally, deprotonation of the sigma complex by AlCl₄⁻ or another base restores aromaticity, releasing HCl and producing the acylated aromatic compound.^[37][38] A representative equation for the ionization step in Friedel-Crafts acylation of benzene using acetyl chloride is: \ce{CH3COCl + AlCl3 -> CH3C#O+ + AlCl4-} This step highlights the role of AlCl₃ in polarizing the acyl chloride to form the stable acylium ion, which then undergoes electrophilic attack. The overall reaction is: \ce{C6H6 + CH3C#O+ ->[AlCl3] C6H5COCH3 + HCl} ^[3][39] The acylium ion's stability arises from resonance between two structures: RC⁺=O ↔ RC≡O⁺, where the positive charge is shared between carbon and oxygen, rendering it less prone to side reactions compared to carbocation intermediates in other substitutions. The sigma complex intermediate features three resonance forms that distribute the positive charge to ortho and para positions relative to the acyl group's attachment point, explaining the kinetic preference for these sites in unsubstituted benzene. This delocalization minimizes the energy barrier for formation, with the intermediate's stability enhanced by the empty p-orbital on the sp²-hybridized ring carbon.^[37][40] Regioselectivity in electrophilic aromatic acylation is primarily governed by the electronic effects of existing substituents on the ring, which influence the stability of the sigma complex. Electron-donating groups, such as alkoxy (-OR) or alkyl (-R), direct the acylium ion to ortho and para positions by stabilizing the positive charge in the intermediate through resonance donation, often favoring para substitution due to steric hindrance at ortho sites. In contrast, electron-withdrawing groups like nitro (-NO₂) destabilize the sigma complex at ortho and para positions, directing substitution to the meta position, though acylation typically does not proceed efficiently on strongly deactivated rings. This directing behavior follows the same principles as other electrophilic substitutions but is amplified by the acylium ion's bulkiness, promoting kinetic control over thermodynamic products in most cases.^[3][41]

Synthetic Applications

Carbonyl Compound Synthesis

Acylation plays a pivotal role in the synthesis of esters and amides, two fundamental carbonyl compounds widely used in pharmaceuticals and polymers. Esters are typically prepared by reacting acyl chlorides with alcohols in the presence of a base, following the Schotten-Baumann reaction, which proceeds under mild conditions to yield the desired product efficiently.^[42] A classic example is the acetylation of salicylic acid with acetic anhydride to produce aspirin (acetylsalicylic acid), a process catalyzed by sulfuric acid that achieves yields of 70-80% in laboratory settings and is scalable for industrial production.^[43] Similarly, amides are formed through nucleophilic acylation of amines with acyl chlorides, enabling the construction of peptide bonds in drug molecules and polyamide polymers like nylon.^[44] These reactions highlight acylation's versatility in creating carbonyl linkages essential for bioactive compounds and materials. In ketone synthesis, electrophilic acylation via the Friedel-Crafts reaction introduces an acyl group onto aromatic rings, producing aryl ketones that serve as key intermediates in fragrances and dyes. This method employs acyl chlorides or anhydrides with a Lewis acid catalyst like aluminum chloride, directing substitution to the para or ortho position depending on the arene's substituents, and is particularly valuable for its regioselectivity in building complex aromatic systems.^[28] Nucleophilic acyl substitutions generally afford 70-95% yields under optimized conditions, often using pyridine or its derivatives as catalysts to neutralize HCl byproducts and enhance reaction rates.^[45] For instance, in esterifications, pyridine facilitates the alcohol's attack on the acyl chloride at room temperature in aprotic solvents, minimizing side reactions. Friedel-Crafts acylations similarly achieve high yields with anhydrous conditions to prevent catalyst deactivation. On an industrial scale, acylation enables large-volume production of carbonyl compounds, exemplified by the use of acetic anhydride to acetylate cellulose, yielding cellulose acetate for films, fibers, and coatings in a process involving sulfuric acid catalysis and achieving near-complete substitution.^[46] This homogeneous reaction, conducted in acetic acid media, underscores acylation's economic importance in materials manufacturing. Modern variants include enzyme-catalyzed acylations, which offer green alternatives by using lipases or proteases in aqueous or solvent-free media to selectively form esters and amides with reduced waste and energy input.^[47] These biocatalytic approaches are increasingly adopted for sustainable synthesis of fine chemicals.

Functional Group Transformations

Acylation serves as a key strategy in organic synthesis for protecting nucleophilic functional groups, particularly alcohols and amines, by temporarily masking their reactivity to enable selective transformations elsewhere in a molecule. For alcohols, acetylation with acetic anhydride or acetyl chloride forms acetate esters that reduce nucleophilicity and prevent unwanted side reactions during multi-step sequences. In peptide synthesis, amines are commonly protected via acylation to form carbamates such as the tert-butoxycarbonyl (Boc) group, introduced using di-tert-butyl dicarbonate, or the carbobenzyloxy (Cbz) group, installed with benzyl chloroformate; these groups neutralize the basicity of the amine while allowing subsequent couplings. Deprotection is achieved selectively: Boc groups are removed under acidic conditions like trifluoroacetic acid (TFA), while Cbz groups are cleaved by catalytic hydrogenation, ensuring compatibility with orthogonal schemes where multiple protecting groups coexist without interference.^[48][49] Beyond simple protection, acylation facilitates dynamic transformations, including group migrations and conversions that enable advanced synthetic maneuvers. In carbohydrate chemistry, acyl groups on hydroxyls can undergo 1,2- or 1,3-migrations under basic or acidic conditions, a process exploited to rearrange protecting patterns for regioselective glycosylation or oxidation; for instance, acetate migration in glucopyranosides allows temporary blocking of anomeric positions before relocation. Acylation also plays a role in preparing thioesters from carboxylic acids or esters, which serve as activated intermediates for native chemical ligation (NCL), a chemoselective method to join peptide segments by reacting a C-terminal thioester with an N-terminal cysteine to form a native amide bond. This transformation is particularly valuable in protein semisynthesis, where thioesters are generated via acylation of thiols with acyl imidazoles or similar reagents.^[50][51] Orthogonal protection schemes, where acylation-based groups like Boc and Cbz are combined with others (e.g., Fmoc or benzyl ethers), allow sequential deprotection in complex syntheses without affecting unprotected sites, enhancing efficiency in building intricate molecules. Compared to alkylation, which can lead to over-substitution due to persistent nucleophilicity of alkylated products, acylation introduces a carbonyl that deactivates the site and provides a handle for further reactivity, such as reduction to aldehydes or alcohols post-deprotection. In the total synthesis of natural products like vancomycin, acylation strategies employing Cbz and acetate protections on amino and hydroxyl groups enable the construction of the rigid heptapeptide scaffold through selective amide bond formations and biaryl ether linkages, culminating in deprotection to yield the aglycon.^[48]

Biological Roles

Post-Translational Protein Modifications

Acylation serves as a key post-translational modification (PTM) in proteins, primarily involving the attachment of acyl groups such as acetyl to specific amino acid residues, thereby regulating protein function, stability, and interactions. Among these, acetylation is the most prevalent form of acylation in eukaryotic cells, occurring on N-terminal residues and lysine side chains. This modification influences diverse cellular processes, including gene regulation and protein trafficking, and is dynamically controlled by enzymatic machinery.^[4] N-terminal acetylation, catalyzed by N-acetyltransferases (NATs), affects approximately 80-90% of eukaryotic proteins, typically occurring co-translationally on the α-amino group of the nascent polypeptide. This modification enhances protein stability by shielding the N-terminus from proteolytic degradation, as unacetylated N-termini can serve as degrons recognized by ubiquitin-proteasome pathways. For instance, in human cells, NAT-mediated acetylation prevents the recognition of hydrophobic N-terminal residues by the Ac/N-end rule pathway, thereby extending protein half-life.^[52][53] Lysine acetylation, particularly on histone tails, represents another major type of acylation PTM, playing a central role in epigenetic regulation. Histone acetyltransferases (HATs or KATs) transfer the acetyl group from acetyl-CoA to the ε-amino group of lysine residues, neutralizing their positive charge and reducing the affinity between histones and negatively charged DNA. This loosens chromatin structure, facilitating access by transcriptional machinery and promoting gene expression. In contrast, lysine acetylation on non-histone proteins, such as transcription factors, modulates their activity and localization.^[4][54] The mechanism of protein acetylation involves the enzymatic transfer of the acetyl moiety from acetyl-CoA, the universal acyl donor, to target residues via nucleophilic attack. For N-terminal acetylation, NAT complexes (e.g., NatA, NatB) recognize specific N-terminal sequences and catalyze the reaction shortly after translation initiation. Lysine acetylation is mediated by HATs like p300/CBP, which exhibit substrate specificity for histone or non-histone targets. This process is reversible, with histone deacetylases (HDACs) and sirtuins hydrolyzing the acetyl group using NAD+ or water, restoring the lysine charge and enabling dynamic regulation. Non-enzymatic acylation can also occur under conditions of elevated acyl-CoA levels, leading to aberrant modifications implicated in cellular stress.^[4][53][55] Functionally, acetylation influences protein stability beyond N-termini; for example, lysine acetylation on metabolic enzymes can alter their conformational stability and activity. In terms of localization, acetylation promotes nuclear import of certain proteins by masking nuclear localization signals or enhancing interactions with importins; lysine acetylation on the tumor suppressor p53, for instance, facilitates its nuclear accumulation and transcriptional activation. Histone acetylation specifically drives epigenetic changes, with hyperacetylation at promoters correlating with active transcription and cell differentiation. Additionally, acetylation of p53 at C-terminal lysines enhances its sequence-specific DNA binding affinity, amplifying its role in DNA damage response and tumor suppression.^[52][4][56] Non-enzymatic acylation, driven by reactive acyl-CoA species like acetyl-CoA, contributes to protein dysfunction in aging and diseases such as neurodegeneration, where it accumulates on lysine residues and impairs enzyme function or promotes aggregation. In aging models, elevated non-enzymatic succinylation and acetylation correlate with mitochondrial dysfunction and shortened lifespan, highlighting acylation's role in metabolic stress responses.^[57][4] Detection of acylation sites relies on mass spectrometry (MS)-based proteomics, which identifies acyl-lysine modifications through tandem MS after affinity enrichment with anti-acyl-lysine antibodies. Quantitative MS approaches, such as label-free or SILAC, reveal site-specific stoichiometry and dynamics, enabling proteome-wide mapping of thousands of acetylation sites in human cells. This technique has been instrumental in uncovering regulatory networks, with high-resolution LC-MS/MS distinguishing acyl variants like acetyl from succinyl groups.^[58][4]

Metabolic and Enzymatic Processes

In cellular metabolism, fatty acylation plays a pivotal role in lipid modification of proteins, with myristoylation and palmitoylation being prominent forms. Myristoylation involves the irreversible attachment of a 14-carbon myristoyl group to the N-terminal glycine residue of proteins, facilitated by N-myristoyltransferase (NMT), which enhances membrane association and anchoring, as seen in Src family kinases that require this modification for localization to the plasma membrane.^[59][60] Palmitoylation, in contrast, entails the reversible addition of a 16-carbon palmitoyl group to cysteine residues via thioester bonds, catalyzed by palmitoyl acyltransferases (PATs) such as DHHC enzymes, enabling dynamic regulation of protein trafficking and signaling; for instance, in Ras proteins, palmitoylation on specific cysteines promotes membrane recruitment and GTPase activity essential for signal transduction pathways.^[59][61] Acyl-CoA thioesters serve as central intermediates in metabolic pathways, linking carbohydrate and lipid metabolism. In beta-oxidation, long-chain acyl-CoAs are transported into mitochondria and sequentially cleaved to generate acetyl-CoA for energy production, with acyl-CoA dehydrogenase initiating the process by dehydrogenating the alpha-beta carbons.^[62] Conversely, in fatty acid synthesis, acetyl-CoA is carboxylated to malonyl-CoA and elongated via fatty acid synthase, incorporating acyl units to build chains for storage or membrane components.^[63] Carnitine acyltransferases, particularly carnitine palmitoyltransferase I (CPT1) on the outer mitochondrial membrane and CPT2 on the inner membrane, facilitate this transport by converting acyl-CoAs to acylcarnitines, which cross the membrane for subsequent reconversion and oxidation, ensuring efficient fuel utilization during fasting or exercise.^[64] Enzymatically, acetyl-CoA carboxylase (ACC) drives lipogenesis by catalyzing the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, the committed step that provides acyl building blocks for de novo fatty acid synthesis in liver and adipose tissues, with ACC1 isoform predominantly active in cytosol for this anabolic process.^[65] Reversible S-acylation, particularly palmitoylation, modulates neuronal signaling by controlling the localization and activity of ion channels and receptors; in Ras-mediated pathways, dynamic depalmitoylation by thioesterases allows rapid cycling between membrane compartments, fine-tuning synaptic plasticity and neurotransmitter release.^[61][66] Dysregulation of acylation contributes to pathological states, notably in cancer where aberrant palmitoleoylation of Wnt proteins by the acyltransferase Porcupine enhances secretion and signaling, promoting tumor cell proliferation and metastasis in colorectal and other malignancies.^[67] In neurodegeneration, altered palmitoylation disrupts protein trafficking and synaptic function, as observed in Alzheimer's disease where hyperpalmitoylation of amyloid precursor protein exacerbates plaque formation, and in Parkinson's where depalmitoylation deficits impair alpha-synuclein clearance.^[68] As of 2025, advances in acyl-protein probes, such as photoactivatable palmitoyl analogs and click chemistry-based reporters, have enabled precise mapping of acylation sites, facilitating drug targeting of dysregulated acyltransferases in metabolic disorders like obesity and diabetes, where inhibiting PATs restores insulin sensitivity in preclinical models.^[69][70]