The functional group reactivity of L-valine

L-Valine as an amphoteric molecule with amino, carboxyl, and side-chain functional groups, its chemical modification requires regioselective regulation based on the reactivity differences of functional groups. The following elaborates on chemical modification methods from three aspects: functional group reaction characteristics, modification strategies, and typical applications, combining reaction mechanisms with modern synthetic technologies:

I. Reactivity of Functional Groups: Activity Differences of Amino, Carboxyl, and Side Chains

1. Reactivity of Amino Group (-NH₂)

Nucleophilic Dominance: The α-amino group (Nα) has slightly lower nucleophilicity than the side-chain amino group (e.g., ε-NH₂ of lysine) due to the electron-withdrawing effect of the adjacent carboxyl group but exhibits smaller steric hindrance. For example, in peptide synthesis, Nα-amino groups often preferentially react with acyl chlorides, while ε-NH₂ requires alkaline conditions (e.g., pH > 9) for efficient acylation.

Protection and Deprotection Properties: Amino groups are easily protected by acyl (e.g., Boc, Cbz), sulfonyl (e.g., Ts), or alkoxycarbonyl groups, with significantly different deprotection conditions (e.g., Boc decomposes in acid, Cbz is removed by catalytic hydrogenation). This difference enables selective modification.

2. Reactivity of Carboxyl Group (-COOH)

Acidity and Electrophilic Activity: The carboxyl group has a pKa of ~2–4, dissociating into carboxylate under alkaline conditions (e.g., NaHCO₃ solution). It readily undergoes nucleophilic substitution with halogenated hydrocarbons (forming esters) or condensation with amines (requiring condensing agents like DCC/HOBt). The α-carboxyl group is more reactive than the side-chain carboxyl group (e.g., β-COOH of aspartic acid) due to smaller steric hindrance.

Activation Strategies: Carboxyl groups can be converted to acyl chlorides, anhydrides, or active esters (e.g., NHS esters) to enhance electrophilicity. For instance, in Fmoc solid-phase peptide synthesis, amino acid carboxyl groups are often pre-activated as HCTU salts to improve condensation efficiency.

3. Specific Reactions of Side-Chain Functional Groups

Hydroxyl Group (Serine, Threonine): Prone to acylation (e.g., with acetic anhydride), alkylation (e.g., with iodoacetic acid), or phosphorylation (e.g., catalyzed by ATP/kinase). The β-methyl group in threonine reduces hydroxyl reaction rate, enabling selective modification via steric hindrance differences.

Thiol Group (Cysteine): The thiol group (-SH, pKa ~8.3) exhibits 极强 nucleophilicity, readily undergoing Michael addition with halogenated hydrocarbons (e.g., iodoacetamide) or maleimides, or forming disulfide bonds under oxidants (e.g., H₂O₂). It serves as a common site for protein labeling.

Imidazole Group (Histidine): The N atom of the imidazole ring shows both protonation and nucleophilicity at pH 6–8, reacting with active esters (e.g., DNFB) or acting as a coordination site for metal ions (e.g., Cu²⁺) in affinity purification.

II. Chemical Modification Methods: Strategies Based on Functional Group Orthogonality

1. Selective Modification of Amino and Carboxyl Groups

Amino-Directed Modification:

Under weakly acidic conditions (pH 4–5), the amino group exists as -NH₃⁺, and the carboxyl group dissociates into -COO⁻. Here, amino nucleophilicity decreases, allowing carboxyl esterification with methanol under acidic catalysis (e.g., HCl/MeOH system) without affecting the Nα-amino group.

Adopt a "masking-activation" strategy: first condense aldehydes (e.g., benzaldehyde) with Nα-amino groups to form imines (protecting amino groups), then activate carboxyl groups with DCC/HOBt, react with amines to form amides, and finally remove imine protecting groups via acidic hydrolysis.

Carboxyl-Directed Modification:

Under alkaline conditions (pH > 10), the carboxyl group dissociates into -COO⁻, and the amino group remains free. Adding acyl chlorides (e.g., acetic anhydride) selectively acylates the amino group, while the carboxyl group is inhibited by salt formation (e.g., Na⁺).

Utilize carboxyl electrophilicity: convert carboxyl groups to azides (e.g., via DPPA activation) for Staudinger reaction with amines under mild conditions, keeping amino groups inert.

2. Precise Modification of Side-Chain Functional Groups

Specific Labeling of Thiol Groups:

Maleimide reagents (e.g., Alexa Fluor maleimide) quantitatively react with thiols at pH 6–8 to form thioethers, unaffected by amino groups.

Iodoacetamide (IAA) specifically alkylates thiols under neutral conditions, commonly used to block cysteine in protein sequencing.

Chemical Selectivity of Hydroxyl Groups:

Phosphorylation Modification: In organic synthesis, tert-butyldiphenylchlorosilane (TBDPS-Cl) often protects serine hydroxyl groups. Its acid stability and fluoride ion sensitivity (deprotected by TBAF) enable orthogonal reactions with other functional groups.

Glycosylation: Introduce sugar chains onto hydroxyl groups via enzymatic catalysis (e.g., glycosyltransferases) or chemical methods (e.g., Koenigs-Knorr reaction), where the β-methyl group in threonine affects glycosylation site selectivity.

Mild Modification of Indole Group (Tryptophan):

Tryptophan's indole ring is sensitive to oxidants, undergoing electrophilic substitution with N-bromosuccinimide (NBS) at low temperature (0°C) to introduce a bromine atom at the C2 position, used for preparing photocrosslinking probes.

III. Modern Modification Technologies: From Solution Phase to Bioorthogonal Chemistry

1. Orthogonal Protecting Group Strategies

Case: Bifunctional Modification of Lysine:

Protect Nα-amino groups with Fmoc (base-sensitive) and ε-NH₂ with Boc (acid-sensitive). Stepwise modification is achieved by adjusting pH (e.g., removing Fmoc with piperidine) or acidic conditions (e.g., removing Boc with TFA), commonly used for side-chain functionalization in peptide synthesis.

2. Bioorthogonal Reactions

Click Chemistry:

Azide-modified amino acids (e.g., azidolysine) undergo Huisgen cycloaddition with alkyne probes under Cu(I) catalysis, featuring high efficiency and no by-products, enabling protein labeling in living cells.

Copper-free click reactions (e.g., strain-promoted alkyne-azide cycloaddition, SPAAC) avoid metal toxicity, suitable for in vivo modification. For example, cyclooctyne-modified antibodies bind to azidated amino acids at specific sites.

Enzymatic Catalysis Modification:

Engineered enzymes (e.g., mutant subtilisin) catalyze site-specific insertion of non-natural amino acids. For instance, p-acetylphenylalanine is introduced into protein active sites, followed by fluorescent labeling via oxime ligation (with aldehyde probes).

3. Photo-Responsive Modification and Controllable Release

Photolabile Protecting Groups (PCBs):

Introduce o-nitrobenzyl (ONB) protecting groups onto tyrosine hydroxyl groups, which release hydroxyls upon 365 nm light irradiation, enabling spatiotemporally controlled protein activation (e.g., photocontrolled enzyme activity switches).

IV. Key Factors for Reactivity Regulation

pH and Solvent Effects: Amino groups show stronger nucleophilicity under alkaline conditions, while carboxyl groups are easily esterified under acidic conditions. Polar solvents (e.g., DMSO) enhance the solubility of ionized functional groups, improving reaction rates.

Steric and Electronic Effects: Steric hindrance of side-chain groups (e.g., tert-butyl) reduces reactivity, while electron-withdrawing groups (e.g., nitro) weaken amino nucleophilicity, whereas electron-donating groups (e.g., methyl) enhance it.

Orthogonality of Protecting Groups: Ideal protecting groups should allow "removal under single conditions without affecting other functional groups", such as the acid/base orthogonal system of Fmoc and Boc, or the hydrogenolysis/acidolysis orthogonal system of Cbz and Boc.

V. Application Expansion: From Drug Synthesis to Biological Conjugation

Peptide Drug Modification: Modify lysine side chains via PEGylation to extend peptide half-life (e.g., polyethylene glycolated asparaginase).

Protein Labeling: Conjugate cysteine thiols with maleimide fluorescent probes for cell imaging.

Introduction of Non-Natural Amino Acids: Incorporate non-natural amino acids with functional groups like azide and alkyne into proteins via genetic codon expansion technology for site-specific chemical modification.

The chemical modification of L-valine essentially involves precise manipulation of functional group reactivity. Through protecting group design, reaction condition optimization, and development of novel orthogonal reactions, site-specific modification can be achieved in complex systems, providing key tools for peptide drug research, protein engineering, and chemical biology studies.