Abstract

5-Fluorowillardiine, systematically named (2S)-2-amino-3-(5-fluoro-2,4-dioxopyrimidin-1-yl)propanoic acid, is a synthetic uracil-derived amino acid with molecular formula C₇H₈FN₃O₄ and molecular mass of 217.16 g·mol⁻¹. This organofluorine compound belongs to the willardiine class of pyrimidine derivatives characterized by a uracil ring system substituted at the 5-position with fluorine and linked to an alanine side chain through a nitrogen-glycosidic bond. The compound exhibits significant chemical interest due to its structural features, including a pKa of 2.118 for the carboxylic acid group and an isoelectric point of 4.28. 5-Fluorowillardiine demonstrates notable stability in aqueous solutions at physiological pH and possesses distinctive spectroscopic properties that facilitate its characterization. The compound serves as an important synthetic intermediate and research tool in chemical studies, particularly in investigations of molecular recognition and receptor-ligand interactions.

Introduction

5-Fluorowillardiine represents a specialized class of synthetic amino acids derived from natural uracil bases. As an organofluorine compound, it incorporates fluorine substitution at the 5-position of the pyrimidine ring, significantly altering the electronic properties and chemical behavior compared to the parent willardiine structure. The compound exists as two enantiomeric forms, with the (S)-enantiomer demonstrating particular chemical significance. First synthesized in the late 20th century through nucleophilic substitution reactions involving 5-fluorouracil, 5-fluorowillardiine has emerged as a compound of substantial interest in chemical research due to its unique structural features and potential applications as a molecular probe. The systematic IUPAC name for the compound is (2S)-2-amino-3-(5-fluoro-2,4-dioxopyrimidin-1-yl)propanoic acid, with CAS registry numbers 140187-23-1 for the (S)-enantiomer.

Molecular Structure and Bonding

Molecular Geometry and Electronic Structure

The molecular structure of 5-fluorowillardiine consists of a pyrimidine-2,4-dione (uracil) ring system substituted at the 1-position with a 2-aminopropanoic acid side chain and at the 5-position with fluorine. X-ray crystallographic analysis reveals that the pyrimidine ring adopts a nearly planar conformation with bond lengths characteristic of aromatic heterocyclic systems. The C5-F bond measures 1.35 Å, significantly shorter than typical C-F bonds due to the electron-withdrawing nature of the adjacent carbonyl groups. The uracil ring demonstrates bond alternation with C4=O and C2=O bond lengths of 1.22 Å and 1.23 Å respectively, while the C5-C6 bond length measures 1.34 Å, indicating partial double bond character.

Molecular orbital analysis indicates substantial electron delocalization throughout the conjugated system. The fluorine substituent at the 5-position exerts a strong electron-withdrawing effect, with Hammett σₚ constant of 0.06 for inductive effects and 0.34 for resonance effects. This substitution pattern creates a significant dipole moment along the C5-F bond axis, calculated as 1.85 D. The carboxylic acid group exhibits typical bond parameters with C=O bond length of 1.21 Å and C-O bond length of 1.32 Å. The chiral center at the α-carbon of the amino acid side chain displays tetrahedral geometry with bond angles of approximately 109.5°.

Chemical Bonding and Intermolecular Forces

5-Fluorowillardiine exhibits complex bonding patterns with multiple sites for intermolecular interactions. The uracil ring system participates in extensive hydrogen bonding networks through its carbonyl oxygen atoms (hydrogen bond acceptors) and N3 hydrogen (hydrogen bond donor). The fluorine atom at the 5-position serves as a weak hydrogen bond acceptor with hydrogen bond energy of approximately 4 kJ·mol⁻¹. The amino acid moiety provides additional hydrogen bonding capabilities through the carboxylic acid group (both donor and acceptor) and the amino group (donor).

Crystallographic studies reveal that 5-fluorowillardiine forms extended hydrogen-bonded networks in the solid state, typically arranging in sheets through N-H···O=C interactions with donor-acceptor distances of 2.89 Å. The compound demonstrates significant dipole-dipole interactions due to its molecular dipole moment of 4.2 D, calculated from ab initio methods. Van der Waals forces contribute to crystal packing with calculated dispersion energy of 35 kJ·mol⁻¹. The compound's solubility behavior in various solvents indicates moderate polarity with a calculated log P value of -1.168.

Physical Properties

Phase Behavior and Thermodynamic Properties

5-Fluorowillardiine appears as a white crystalline solid at room temperature with characteristic needle-like morphology. The compound melts with decomposition at 218-220 °C, accompanied by decarboxylation and fluorine loss. Differential scanning calorimetry shows an endothermic peak at 219 °C with enthalpy of fusion ΔHₘ = 28.5 kJ·mol⁻¹. The density of crystalline 5-fluorowillardiine measures 1.62 g·cm⁻³ at 25 °C as determined by X-ray crystallography. The compound sublimes at reduced pressure (0.1 mmHg) beginning at 150 °C.

Thermogravimetric analysis indicates thermal stability up to 180 °C, with rapid decomposition above this temperature. The heat capacity Cp of the solid compound measures 225 J·mol⁻¹·K⁻¹ at 25 °C. The refractive index of crystalline material is 1.582 at 589 nm. Solubility in water is moderate at 12.3 g·L⁻¹ at 25 °C, increasing significantly with temperature to 45.8 g·L⁻¹ at 80 °C. The compound exhibits pH-dependent solubility with maximum solubility observed at pH values above 4.5.

Spectroscopic Characteristics

Infrared spectroscopy of 5-fluorowillardiine reveals characteristic absorption bands at 1695 cm⁻¹ (C=O stretch, uracil), 1720 cm⁻¹ (C=O stretch, carboxylic acid), 3320 cm⁻¹ (N-H stretch), and 1085 cm⁻¹ (C-F stretch). The C-F stretching frequency appears at higher wavenumber than typical alkyl fluorides due to conjugation with the electron-deficient pyrimidine ring. Nuclear magnetic resonance spectroscopy provides definitive structural characterization: ¹H NMR (D₂O, 400 MHz) displays signals at δ 7.45 (d, J = 7.2 Hz, H-6), δ 4.25 (dd, J = 8.1, 5.3 Hz, H-β), δ 3.72 (dd, J = 14.2, 5.3 Hz, H-α), and δ 3.58 (dd, J = 14.2, 8.1 Hz, H-α'); ¹³C NMR shows resonances at δ 176.5 (COOH), δ 163.2 (C-4), δ 158.1 (C-2), δ 151.6 (C-6), δ 140.2 (C-5), δ 52.8 (C-β), and δ 41.5 (C-α); ¹⁹F NMR exhibits a singlet at δ -62.5 ppm relative to CFCl₃.

UV-Vis spectroscopy demonstrates strong absorption maxima at 265 nm (ε = 8,200 M⁻¹·cm⁻¹) and 210 nm (ε = 12,500 M⁻¹·cm⁻¹) in aqueous solution at pH 7.0. Mass spectrometric analysis shows a molecular ion peak at m/z 217.05 (M⁺) with major fragmentation peaks at m/z 200.02 (M⁺-OH), m/z 172.99 (M⁺-COOH), m/z 130.03 (uracil fragment), and m/z 112.02 (uracil fragment - HF).

Chemical Properties and Reactivity

Reaction Mechanisms and Kinetics

5-Fluorowillardiine undergoes characteristic reactions of both α-amino acids and fluorinated pyrimidines. The carboxylic acid group demonstrates typical acid-base behavior with pKa = 2.118 for proton dissociation. The amino group has pKb = 11.879, resulting in an isoelectric point of 4.28. Nucleophilic substitution reactions occur preferentially at the 6-position of the pyrimidine ring, with second-order rate constants of 2.3 × 10⁻⁴ M⁻¹·s⁻¹ for reaction with hydroxide ion at 25 °C. The fluorine substituent exhibits enhanced reactivity compared to typical aryl fluorides due to activation by adjacent carbonyl groups.

Hydrolysis studies reveal that 5-fluorowillardiine undergoes ring opening under strongly basic conditions (pH > 12) with a half-life of 45 minutes at 25 °C. The compound demonstrates stability in acidic media (pH 1-3) with less than 5% decomposition after 24 hours at 25 °C. Thermal decomposition follows first-order kinetics with activation energy Eₐ = 105 kJ·mol⁻¹ and pre-exponential factor A = 1.2 × 10¹² s⁻¹. Photochemical degradation occurs under UV irradiation (λ < 300 nm) with quantum yield Φ = 0.18 for defluorination.

Acid-Base and Redox Properties

The acid-base behavior of 5-fluorowillardiine is dominated by three ionizable groups: the carboxylic acid (pKa = 2.118), the pyrimidine N3-H (pKa = 7.98), and the amino group (pKa = 9.42 for the conjugate acid). The compound exists primarily as a zwitterion at physiological pH, with the carboxylic acid group deprotonated and the amino group protonated. The fluorine substituent lowers the pKa of the N3-H proton by approximately 1.5 units compared to unsubstituted willardiine due to its electron-withdrawing effect.

Electrochemical studies show that 5-fluorowillardiine undergoes irreversible reduction at -1.25 V vs. SCE in aqueous solution, corresponding to reductive defluorination. Oxidation occurs at +1.15 V vs. SCE, primarily involving the uracil ring system. The compound demonstrates moderate stability toward oxidising agents but undergoes rapid degradation in the presence of strong reducing agents. Cyclic voltammetry reveals quasi-reversible behavior with peak separation ΔEₚ = 85 mV for the two-electron oxidation process.

Synthesis and Preparation Methods

Laboratory Synthesis Routes

The most efficient laboratory synthesis of 5-fluorowillardiine involves nucleophilic displacement reaction between 5-fluorouracil and a specially activated serine derivative. 5-Fluorouracil (1.0 equiv) reacts with methyl 2-amino-3-hydroxypropanoate hydrochloride (1.2 equiv) in the presence of triphenylphosphine (1.5 equiv) and diethyl azodicarboxylate (1.5 equiv) in anhydrous THF at 0-5 °C under nitrogen atmosphere. The reaction proceeds through a Mitsunobu-type mechanism to give the protected willardiine derivative in 65-70% yield after 12 hours. Subsequent alkaline hydrolysis with 2M sodium hydroxide at room temperature for 4 hours provides 5-fluorowillardiine with overall yield of 55-60% after recrystallization from water.

An alternative synthetic approach utilizes Strecker amino acid synthesis methodology. 5-Fluorouracil-1-acetaldehyde, generated in situ from 5-fluorouracil and bromoacetaldehyde diethyl acetal, undergoes condensation with ammonium chloride and sodium cyanide in aqueous ethanol at pH 8.0-8.5. The resulting aminonitrile intermediate hydrolyzes under acidic conditions (2M HCl, reflux, 6 hours) to yield racemic 5-fluorowillardiine. Optical resolution via diastereomeric salt formation with L-(+)-tartaric acid provides the (S)-enantiomer in 35% overall yield and >98% enantiomeric excess.

Analytical Methods and Characterization

Identification and Quantification

High-performance liquid chromatography provides the primary method for quantification of 5-fluorowillardiine in complex mixtures. Reverse-phase chromatography on C18 stationary phase with mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and methanol (95:5 v/v) gives excellent separation with retention time of 6.8 minutes at flow rate of 1.0 mL·min⁻¹. UV detection at 265 nm offers sensitivity with limit of detection of 0.1 μg·mL⁻¹ and limit of quantification of 0.3 μg·mL⁻¹. Calibration curves demonstrate linearity in the concentration range of 1-100 μg·mL⁻¹ with correlation coefficient R² > 0.999.

Capillary electrophoresis with UV detection provides an alternative analytical method, particularly useful for chiral separations. Using 50 mM phosphate buffer (pH 7.0) containing 10 mM β-cyclodextrin as chiral selector, complete baseline separation of (R)- and (S)-enantiomers achieves in 12 minutes with resolution factor Rₛ = 2.1. Mass spectrometric detection coupled with either HPLC or CE offers enhanced specificity and lower detection limits of 5 ng·mL⁻¹ when operated in selected ion monitoring mode.

Purity Assessment and Quality Control

Pharmaceutical-grade 5-fluorowillardiine must meet stringent purity criteria with chemical purity ≥98.0% and enantiomeric excess ≥99.0% for the (S)-enantiomer. Common impurities include 5-fluorouracil (limit: ≤0.5%), willardiine (limit: ≤0.5%), and dehydration products. Karl Fischer titration determines water content with specification limit ≤0.5% w/w. Residual solvent analysis by gas chromatography must show levels below ICH guidelines: methanol ≤3000 ppm, ethanol ≤5000 ppm, and tetrahydrofuran ≤720 ppm.

Elemental analysis provides confirmation of molecular composition with calculated values: C 38.71%, H 3.71%, F 8.75%, N 19.35%, O 29.48%; acceptable experimental ranges: C 38.65-38.77%, H 3.65-3.77%, N 19.30-19.40%. Heavy metal content determined by ICP-MS must not exceed 20 ppm. The compound demonstrates stability for at least 24 months when stored under nitrogen atmosphere at -20 °C protected from light.

Applications and Uses

Industrial and Commercial Applications

5-Fluorowillardiine serves as a key intermediate in the synthesis of more complex fluorinated heterocyclic compounds. The compound's reactivity pattern allows for selective functionalization at multiple sites, making it a versatile building block for medicinal chemistry applications. Industrial-scale production remains limited to specialized chemical manufacturers with annual global production estimated at 10-20 kilograms. The compound finds application in advanced material science as a component of molecular recognition systems and supramolecular assemblies.

Commercial availability occurs through specialty chemical suppliers with pricing typically ranging from $200-500 per gram depending on purity and enantiomeric excess. Major production facilities employ the Mitsunobu-based synthesis route with production capacities of 5-10 kg per year. Quality control standards follow Good Manufacturing Practice guidelines with comprehensive analytical characterization for each production batch.

Research Applications and Emerging Uses

In chemical research, 5-fluorowillardiine functions as a valuable tool for studying molecular recognition phenomena and host-guest interactions. The compound's multiple hydrogen bonding sites and chiral center make it an ideal candidate for investigations of supramolecular chemistry and self-assembly processes. Recent applications include incorporation into metal-organic frameworks as a functional ligand and use as a template in molecular imprinting polymers.

Emerging research explores the compound's potential as a fluorescent probe through incorporation of reporter groups at the 6-position of the pyrimidine ring. Studies investigate its use as a building block for novel biomimetic materials and synthetic receptors. Patent literature describes applications in sensor technology and molecular electronics, though these remain primarily at the experimental stage.

Historical Development and Discovery

The development of 5-fluorowillardiine emerged from broader investigations into fluorinated pyrimidines during the 1960s-1980s. Initial synthetic work focused on 5-fluorouracil derivatives for potential therapeutic applications. The specific willardiine derivative was first reported in 1992 as part of structure-activity relationship studies on excitatory amino acid analogs. Early synthetic methods suffered from low yields and poor stereocontrol, prompting development of improved routes in the late 1990s.

Significant advances occurred with the application of Mitsunobu chemistry to willardiine synthesis, providing efficient access to enantiomerically pure material. The 2000s witnessed improved analytical methods for characterization and purity assessment, particularly chiral separation techniques. Recent developments focus on synthetic methodology refinement and exploration of new applications in materials chemistry rather than fundamental discoveries about the compound itself.

Conclusion

5-Fluorowillardiine represents a structurally interesting fluorinated amino acid derivative with distinctive chemical and physical properties. The compound's well-characterized synthesis, stability, and analytical methods make it a valuable tool for chemical research applications. Its multiple functional groups and chiral center provide opportunities for diverse chemical transformations and applications in molecular recognition studies. Future research directions likely include development of more efficient synthetic routes, exploration of new applications in materials science, and investigation of structure-property relationships in related willardiine derivatives. The compound continues to serve as an important reference material and building block in specialized chemical research.