Protonation of curcumin triggers sequential double cyclization in the gas-phase: An electrospray mass spectrometry and DFT study

Highlights

• Rearrangement of protonated curcumin characterized by mass spectrometry and DFT.

• Protonated curcumin undergoes two consecutive cyclization reactions in the gas phase.

• The precursor upon protonation undergoes competitive eliminations of 2-methoxy phenol and C₄H₄O₂, presumably diketene.

• This gas-phase rearrangement may be predictive of solution chemistry and be predictive for biological function.

Abstract

ESI-protonated natural curcumin (1) undergoes gas-phase cyclization and dissociates via competitive expulsions of 2-methoxy phenol and C₄H₄O₂ (diketene or an isomer). Evidence from mechanistic mass spectrometry and from Density Functional Theory (DFT) reveals that a two-step sequential cyclization occurs for the protonated molecule prior to the unusual loss of the elements of 2-methoxy phenol. Furthermore, the presence of the methoxy group at postion-3 is essential for the second cyclization. The transformation of curcumin upon protonation in the gas phase may be predictive of its solution chemistry and explain how curcumin plays a protective role in biology.

Introduction

Curcumin is a yellow, polyphenolic pigment isolated from the rhizomes of the spice turmeric (Curcuma longa). Curcumin exhibits a variety of biological activities including those of an antioxidant, anticancer compound, and a mediator in Aβ amyloid formation with important implications in Alzheimer’s disease [[1], [2], [3], [4], [5]]. The mechanism of antioxidant activity of this 1,3-diketone (I), containing two phenolic OH groups and two double bonds, has been the subject of many experimental [[6], [7], [8], [9]] and theoretical investigations [10,11]. To determine the relationship between the structure and antioxidant activity of curcumin, investigators have focused on the role of the phenolic-OH group, the enol-OH group, and double bond [12]. The instability of curcumin under biological pH conditions [13] has led to the hypothesis that its degradation products [14] and metabolites [15,16] may be the means whereby curcumin exhibits its biological activity. The bio-transformations of curcumin, although not fully understood, may produce metabolites including dihydrocurcumin and tetrahydrocurcumin that are biologically active [17]. Additional transformation products of autoxidation and double cyclization of curcumin are known and likely to contribute to its pharmacological activities [18].

The consumption of turmeric seems to reduce the risk of some cancers and provide salutary biological effects that correlate with curcumin content [19]. Of the three analogs present in Curcuma longa, curcumin (1) is the most biologically active component compared to the demethoxy- (2) and bisdemethoxy analogs [20,21]. It is essential to understand the possible acid-catalyzed rearrangements of curcumin given that the digestive juices of the stomach are at low pH. Moreover, the capability of curcumin to serve as an antioxidant is high [2] at low pH, suggesting a role for protonation of the molecule in its biological chemistry. Determining the outcome of protonation is an opportunity for electrospray ionization and mass spectrometry, which can bring a deeper understanding of this biologically important food substance and supplement.

Electrospray ionization (ESI) generates protonated/deprotonated molecules and introduces them to the gas-phase. The unimolecular transformations of both [M + H]⁺ and [M − H]⁻ ions and the mechanism of these reactions have been studied by various experimental approaches, including tandem mass spectrometry and DFT calculations In fact, acid-catalyzed transformations of organic molecules in solution can be predicted on the basis of mass spectrometric fragmentations of protonated molecules [22,23]. The use of mass spectrometer for studying various chemical reactions has led to the suggestion that it is “complete chemical laboratory” [24]. Related studies carried out in our laboratory are of 2-methoxychalcone [25], N-(2-nitrophenyl)alanine [26] and bis-(2-methoxybenzal)acetone [27], and these investigations demonstrate that unimolecular cyclization reactions upon ESI protonation have counterparts in solution and suggest that ESI MS in combination with DFT is an effective approach for predicting acid-catalyzed cyclizations of organic molecules in solution.

We report here an investigation of the possible rearrangements of protonated curcumin (1), mono-methoxycurcumin (2), and the synthetic analogs 3 and 4 by ESI mass spectrometry and DFT calculations. Our hypothesis is that any rearrangement occurring in the mass spectrometer also occurs in solution and provides insight on the biological activity of curcumin. LC/MS [28] and NMR [29] already show that natural curcumin containing a 1,3-diketone with conjugated double bonds, exists as a stable enol. The enol form of curcumin, upon protonation, may undergo cyclization analogous to a Nazarov reaction [30]. The collisionally-activated decomposition (CAD) product-ion mass spectrum of ESI-produced curcumin was reported previously, but without any supporting theoretical calculations [31], and the proposed mechanisms involved forbidden and other high-energy processes. There are no reports regarding a solution rearrangement of curcumin under acidic conditions although the oxidative cyclizations of curcumin [18] and mono-methoxycurcumin [14] in buffer solutions at pH 7.4 were noted.