Nuclear magnetic resonance (NMR) enables detailed characterization of functional group linkages, substitution positions, and stereochemical features of Fmoc-Arg(Pbf)-OH (N-fluorenylmethoxycarbonyl-arginine-tert-butylfluorobenzenesulfonyl derivative) through multi-dimensional spectroscopy. The specific applications are as follows:
I. 1H NMR: Fine Identification of Functional Groups and Hydrogen Environments
1. Characteristic Signals of Fmoc Group
Fluorene ring protons: Two sets of aromatic hydrogen multiplets appear at δ 7.2–7.8 ppm (8 protons total). Due to chemical environment differences, the benzene ring protons on both sides of the 9-methylene group in the fluorene ring typically show two sets of doublets or multiplets at low field (δ 7.6–7.8 ppm, 4 protons) and high field (δ 7.2–7.4 ppm, 4 protons).
Methoxycarbonyl signal: The methoxy protons of Fmoc exhibit a singlet at δ 3.8–4.0 ppm (3 protons), with the fluorene ring methylene (δ 4.4–4.6 ppm, 2 protons) showing characteristic low-field shifts. This methylene is significantly downfield from ordinary alkyl hydrogens due to the adjacent carbonyl and fluorene ring conjugation.
2. Signals from Arginine Side Chain and Protecting Group
Guanidino hydrogens: In DMSO-d6, the guanidino group of Arg typically appears as a broad peak at δ 11.0–12.0 ppm (2 protons), which may broaden or split due to intramolecular hydrogen bonding.
Pbf protecting group: The tert-butyl group in tert-butylfluorobenzenesulfonyl (Pbf) shows a singlet at δ 1.2–1.4 ppm (9 protons), while the benzene ring protons exhibit multiplets at δ 7.4–7.7 ppm (4 protons). The presence of fluorine is verified by 19F NMR (see below).
Side chain methylenes: The three methylene groups (-CH2-CH2-CH2-) in the Arg side chain show multiplets at δ 1.6–2.0 ppm (near the guanidino group) and δ 2.9–3.1 ppm (near the amino group), with coupling relationships analyzed via COSY spectroscopy.
3. Carboxyl and Amino Signals
Carboxyl proton: In DMSO-d6, the COOH proton appears as a broad singlet at δ 12.5–13.0 ppm.
Amino proton: The α-amino group protected by Fmoc shows a broad peak at δ 7.8–8.2 ppm (1 proton) due to hydrogen bonding, distinguishable from guanidino hydrogen signals.
II. 13C NMR: Localization of Carbon Skeleton and Functional Group Linkages
1. Carbon Signals of Fmoc Group
Fluorene ring carbons: Multiple aromatic carbon signals appear in the range of δ 110–145 ppm. The 9-methylene carbon, linked to two benzene rings and a carbonyl, shows a chemical shift at δ 85–90 ppm (significantly higher than ordinary methylene carbons).
Carbonyl carbons: The methoxycarbonyl carbon of Fmoc appears as a singlet at δ 155–160 ppm, distinct from the α-carbonyl carbon of Arg (δ 170–175 ppm) and side chain guanidino carbon (δ 150–155 ppm).
2. Pbf Protecting Group and Side Chain Carbons
Tert-butyl carbons: The three methyl carbons of Pbf’s tert-butyl group show a singlet at δ 28–30 ppm, with the quaternary carbon linked to tert-butyl at δ 55–60 ppm.
Benzenesulfonyl carbons: Benzene ring carbons of Pbf appear in the range of δ 125–140 ppm. The sulfonyl (-SO2-) carbon shows no characteristic peak due to high electronegativity but is indirectly confirmed by coupling splitting with fluorine (see 19F NMR).
Arg side chain carbons: The α-carbon (linked to amino and carboxyl groups) is at δ 50–60 ppm, with side chain methylene carbons at δ 25–30 ppm (near guanidino), δ 35–40 ppm (middle methylene), and δ 45–50 ppm (near α-carbon).
III. 19F NMR: Specific Verification of Fluorine in Pbf Protecting Group
The fluorine atom in the Pbf protecting group (-C6H4-SO2-C(CH3)3) exhibits a characteristic singlet in 19F NMR, typically at δ -110 to -120 ppm (depending on solvent and substituent effects). This signal is crucial for confirming the Pbf group, distinguishing it from fluorine impurities (e.g., residual TFA shows signals at δ -75 to -80 ppm).
IV. Two-Dimensional NMR Spectroscopy: Conformational and Linkage Order Analysis
1. COSY (Correlation Spectroscopy)
H-H coupling network: Cross peaks confirm coupling between Arg side chain methylenes (δ 1.6–2.0 ppm) and adjacent methylenes (δ 2.9–3.1 ppm), as well as the relationship between α-amino hydrogen (δ 7.8–8.2 ppm) and α-hydrogen (δ 4.5–4.8 ppm), verifying the amino acid backbone linkage order.
Fmoc group correlation: Cross peaks between fluorene ring methylene hydrogen (δ 4.4–4.6 ppm) and adjacent benzene ring hydrogen (δ 7.2–7.4 ppm) confirm the substitution position of the Fmoc group.
2. HSQC (Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple-Bond Correlation)
HSQC for direct C-H linkage: For example, α-hydrogen (δ 4.5–4.8 ppm) correlates with α-carbon (δ 50–60 ppm), and Fmoc methoxy proton (δ 3.8–4.0 ppm) correlates with methoxycarbonyl carbon (δ 155–160 ppm).
HMBC for long-range linkage: Long-range coupling (2–3 bonds) between α-hydrogen and Fmoc carbonyl carbon (δ 155–160 ppm) confirms the linkage of Fmoc to the α-amino group of Arg. Coupling peaks between Pbf benzene ring hydrogen (δ 7.4–7.7 ppm) and sulfonyl carbon (δ 130–140 ppm) verify the linkage of the benzene ring to -SO2-.
V. Solvent Selection and Spectrum Optimization
1. Common Solvents and Their Effects
DMSO-d6: Highly polar, dissolves Fmoc-Arg(Pbf)-OH with high resolution for carboxyl and amino hydrogen signals, though guanidino hydrogen may broaden due to hydrogen bonding.
CD3OD: Deuterated methanol reduces hydrogen bonding effects on amino hydrogens, sharpening guanidino hydrogen signals, but may shift carboxyl signals to δ 9.0–10.0 ppm.
Heavy water (D2O): Exchanges labile hydrogens (e.g., amino, carboxyl), causing related signals to disappear—useful for verifying labile hydrogen presence (disappearing peaks indicate exchangeable hydrogens).
2. Parameter Optimization
19F NMR: Requires a fluorine-specific probe with a scanning range of δ -200 to 0 ppm to capture the Pbf fluorine signal.
2D spectra: HMBC experiments use long-range coupling constants (J=6–8 Hz) to ensure collection of long-range C-H coupling signals and avoid missing key linkage information.
VI. Comprehensive Logic for Structural Confirmation
1. Verification of Functional Groups
1H NMR signals for fluorene ring hydrogens, methoxy protons, tert-butyl groups, and 19F NMR fluorine signals confirm the presence of Fmoc and Pbf protecting groups.
2. Backbone Linkage Order
COSY coupling between α-hydrogen and amino hydrogen/side chain methylenes, combined with HMBC long-range linkage between α-hydrogen and Fmoc carbonyl carbon, verify the main chain structure: Fmoc-amino-Arg-carboxyl.
3. Protecting Group Localization
Correlation between Pbf tert-butyl carbon signals (13C NMR) and benzene ring hydrogen signals (1H NMR), along with 19F signals, confirms its linkage to the guanidino nitrogen of Arg.
4. Purity Assessment
Integration ratios of signals (e.g., 8H for Fmoc fluorene ring, 9H for tert-butyl, 1H for α-hydrogen) identify impurity peaks, ensuring structural correctness of the target compound.
Conclusion
NMR technology provides comprehensive verification of Fmoc-Arg(Pbf)-OH from functional groups to backbone structure through combined analysis of multi-dimensional spectra, leveraging chemical environments and spatial linkages of hydrogen, carbon, and fluorine atoms. The specific identification of the Pbf group by 19F NMR, localization of cross-bond linkages by HMBC, and analysis of hydrogen networks by COSY together form a key evidence chain for structural confirmation, ensuring structural accuracy in applications such as peptide synthesis.
