The nuclear magnetic resonance spectral characteristics of L-valine

I. Nuclear Magnetic Resonance (NMR) Testing Conditions and Sample Preparation

Solvent Selection and Chemical Shift Calibration

Common Solvents: Heavy water (D₂O) or deuterated methanol (CD₃OD) are preferred. L-valine exhibits high solubility in water (8.8 g/100 mL at 25°C), and D₂O suppresses the active hydrogen signal of amino groups (-NH₂), avoiding broad peaks.

Internal Standard: Tetramethylsilane (TMS, δ=0.00 ppm) or residual protons in deuterated solvents (e.g., D₂O solvent peak at δ=4.79 ppm) are used for chemical shift calibration.

Sample Concentration: 5–10% (w/v) dissolved in 0.5 mL solvent, loaded into a 5 mm NMR tube to avoid bubble interference with shimming.

Instrument Parameter Optimization

1H NMR: Frequency >400 MHz, 16 scans, 5-second relaxation time to eliminate residual coupling effects.

13C NMR: Broadband decoupling technique, 1000–2000 scans, 10-second relaxation delay to ensure full carbon signal relaxation.

II. 1H NMR Spectral Characteristics: Functional Group and Spatial Environment Analysis

Assignment of Characteristic Proton Signals (D₂O as solvent, δ ppm)

α-Proton (Hα): δ 4.05–4.15 (dd peak, J=8.5 Hz, J=4.2 Hz). Influenced by electron-withdrawing effects of amino and carboxyl groups, the chemical shift appears at low field. The double doublet arises from coupling with protons on the adjacent chiral carbon, reflecting the stereochemical environment of the L-configuration.

β-Methyl Protons (Hβ1, Hβ2): δ 1.65–1.75 (d peak, J=6.8 Hz). Due to steric hindrance of the isopropyl group on the β-carbon, signals slightly split but often appear as a broad doublet, with an integration ratio of 6:1 (may merge into a singlet at lower instrument resolution).

γ-Methyl Protons (Hγ1, Hγ2): δ 0.95–1.05 (d peak, J=6.5 Hz). Two equivalent methyl groups form a high-field doublet with an integration of 6, a typical feature of the isobutyl side chain.

Amino Protons (-NH₂): Disappear in D₂O due to proton exchange. In CDCl₃, a broad peak at δ 8.0–8.5 is observed, eliminable by D₂O exchange, indicating amino group lability.

Coupling Constants and Configuration Verification

The coupling constant between α-proton and β-proton (Jαβ=8.5 Hz) exceeds that with γ-protons (long-range coupling is negligible), consistent with the trans-coplanar relationship between α-proton and β-carbon methyl groups in the L-configuration.

The coupling constant of isopropyl methyl groups (J=6.5–6.8 Hz) indicates that the C-C bond between methyl and β-carbon adopts a gauche conformation, typical of amino acid side chains.

III. 13C NMR Spectral Characteristics: Carbon Skeleton and Electronic Effect Analysis

Chemical Shifts of Carbon Signals (D₂O as solvent, δ ppm)

Carboxyl Carbon (C=O): δ 175.0–176.5. Due to the electron-withdrawing effect of the carbonyl double bond and oxygen atom, it appears at the lowest field, a signature signal of amino acids.

α-Carbon (Cα): δ 58.0–59.5. Linked to amino and carboxyl groups, its chemical shift is higher than aliphatic carbons (typically δ 20–40) due to the inductive effect of dual substituents.

β-Carbon (Cβ): δ 31.5–32.8. Connected to isopropyl and α-carbon, its chemical shift is slightly lower than Cα due to the electron-donating effect of two methyl groups at the β-position.

γ-Methyl Carbons (Cγ1, Cγ2): δ 18.2–18.8 (signals overlap for equivalent methyls), appearing at high field as characteristic of alkyl carbons.

Amino Carbon: Not separately assigned; the carbon skeleton is fully assigned via the chemical shift gradient (C=O > Cα > Cβ > Cγ).

DEPT-135 Spectroscopy for Auxiliary Identification

In DEPT spectra:

Cα (methine) shows positive peaks (δ 58.0–59.5).

Cβ (methine) shows positive peaks (δ 31.5–32.8).

Cγ1/Cγ2 (methyls) show positive peaks (δ 18.2–18.8).

Carboxyl carbon (quaternary carbon) gives no signal, excluding interferences from other functional groups via correlation with 1H NMR.

IV. Structure Connectivity Confirmation by 2D-NMR

HSQC (Heteronuclear Single Quantum Coherence) Spectroscopy

Establishes direct 1H-13C correlations:

α-Proton (δ 4.05–4.15) corresponds to Cα (δ 58.0–59.5).

β-Methyl protons (δ 1.65–1.75) correspond to Cβ (δ 31.5–32.8).

γ-Methyl protons (δ 0.95–1.05) correspond to Cγ (δ 18.2–18.8), verifying the side-chain carbon-hydrogen linkage order.

HMBC (Heteronuclear Multiple-Bond Coherence) Spectroscopy

Reveals long-range coupling:

Long-range coupling peak between α-proton and carboxyl carbon (J=3.5 Hz) (δH 4.05–4.15 × δC 175.0–176.5) confirms amino and carboxyl groups linked to the same Cα.

Coupling peak between β-methyl protons and Cα (J=7.2 Hz) confirms isopropyl side-chain attachment to Cα.

Coupling peak between γ-methyl protons and Cβ (J=6.8 Hz) verifies side-chain carbon continuity.

ROESY (Rotating-Frame Overhauser Effect) Spectroscopy

Identifies spatial proximity:

ROE crosspeak between α-proton and γ-methyl protons (δH 4.05 × δH 0.95) indicates the cis-spatial position of α-proton and isopropyl methyls in the L-configuration, contrasting with the D-valine configuration (where this crosspeak disappears).

V. NMR Methods for Chiral Carbon and Configuration Verification

Chiral Shift Reagent Method

Adding chiral lanthanide reagents like Eu(fod)₃ [tris(3,5-bis(trifluoromethyl)phenyl)europium] splits the α-proton signal of L-valine into two peaks (Δδ≈0.15 ppm), with the D-configuration showing an opposite splitting pattern, enabling absolute configuration determination via peak pattern differences.

Principle: Chiral reagents form coordination complexes with amino acids, and steric hindrance of different configurations causes proton chemical shift variations.

Isotope Labeling Control Method

Comparing NMR spectra of natural abundance L-valine and [2-13C]-L-valine, the Cα signal (δ 58.0–59.5) shifts by ~0.3 ppm due to the 13C isotope effect. Combined with optical rotation spectroscopy ([α]ₙ²⁰=-16.5°, water), the L-configuration is further confirmed.

VI. Application of NMR in Impurity Analysis of L-Valine

Detection of D-Valine Impurities

The α-proton chemical shift differs by ~0.08 ppm between D/L-isomers (L-form δ 4.10, D-form δ 4.18). D-form impurities can be quantitatively detected (detection limit ≤0.5%) via peak splitting or integration ratio.

In the presence of D-valine, the Cα signal in HSQC spectra appears as a doublet, corresponding to carbon environment differences between the two configurations.

Identification of Other Amino Acid Impurities

The β-methyl signal of L-isoleucine (structural analog) appears at δ 1.00–1.05 (triplet), with minimal overlap with the γ-methyl doublet (δ 0.95–1.05) of L-valine, allowing differentiation via integration.

The side-chain methyl signal of L-leucine at δ 0.85–0.90 (doublet) shows a distinct chemical shift from the γ-methyl signal (δ 0.95–1.05) of L-valine, facilitating impurity screening.

The NMR spectroscopy of L-valine provides multi-dimensional evidence for structure identification through chemical shifts, coupling relationships, and spatial effects of characteristic protons and carbons. The isopropyl methyl doublet in 1H NMR, low-field shift of α-proton, high deshielding effect of carboxyl carbon in 13C NMR, and carbon-hydrogen connectivity revealed by 2D spectra constitute a complete structural analysis system. Combined with chiral shift reagents or isotope labeling techniques, configuration confirmation and impurity control are further enabled, providing key analytical tools for synthetic process optimization, quality standard establishment, and chiral drug R&D of L-valine. With the development of hyperpolarized NMR technology, studies on its dynamic conformational changes (e.g., hydrogen bonding between amino and carboxyl groups in solution) will be further expanded in the future.