Direct Observation of Hydrangea Blue-Complex Composed of 3-O-Glucosyldelphinidin, Al3+ and 5-O-Acylquinic Acid by ESI-Mass Spectrometry

Abstract

The blue sepal color of hydrangea is due to a metal complex anthocyanin composed of 3-O-glucosyldelphinidin (1) and an aluminum ion with the co-pigments 5-O-caffeoylquinic acid (2) and/or 5-O-p-coumaroylquinic acid (3). The three components, namely anthocyanin, Al3+ and 5-O-acylquinic acids, are essential for blue color development, but the complex is unstable and only exists in an aqueous solution. Furthermore, the complex did not give analyzable NMR spectra or crystals. Therefore, many trials to determine the detailed chemical structure of the hydrangea-blue complex have not been successful to date. Instead, via experiments mixing 1, Al3+ and 2 or 3 in a buffered solution at pH 4.0, we obtained the same blue solution derived from the sepals. However, the ratio was not stoichiometric but fluctuated. To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry. In a very low-concentration buffer solution (2.0 mM) at pH 4.0, we reproduced the hydrangea-blue color by mixing 1, 2 and Al3+ in ratios of 1:1:1, 1:2:1 and 1:3:1. All solution gave the same molecular ion peak at m/z = 843, indicating that the blue solution has a ratio of 1:1:1 for the complex. By using 3, the observed mass number was m/z = 827 and the ratio of 1, 3 and Al3+ was also 1:1:1. A mixture of 1, 3-O-caffeoylquinic acid (4) and Al3+ did not give any blue color but instead was purple, and the intensity of the molecular ion peak at m/z = 843 was very low. These results strongly indicate that the hydrangea blue-complex is composed of a ratio of 1:1:1 for 1, Al3+ and 2 or 3.

Quantum Chemical Investigation of the Intramolecular Copigmentation Complex of an Acylated Anthocyanin

“…10,[16][17][18][19][20][21][22][23] In the plant vacuoles where anthocyanins concentrate, [24][25][26] the anthocyanin cation AH + can chelate with metal cations 23,27 such as Al 3+ and/or form complexes with colorless organic copigment molecules, such as electron-rich derivatives of hydroxybenzoic or hydroxycinnamic acids, flavones or one of the colorless neutral forms of the anthocyanin itself. 10,[16][17][18][19][20][21][22][23] Metal cation chelation can lead to large changes in the color, primarily from red to blue as, for example, in Hydrangea, 28 but is limited to anthocyanins with two or more free OH groups in the B-ring 23,27,29 (i.e., anthocyanins derived from cyanidin, delphinidin and petunidin). In contrast, copigmentation via complexation with organic molecules results in much smaller red shifts of the absorption, but can increase the pH at which hydration occurs, consistent with steric hindrance to attack of water and charge transfer from the copigment to the anthocyanin as an important contributor to the stability of the anthocyanin-copigment complex.…”

Insight into chemical mechanisms of sepal color development and variation in hydrangea

“…To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry (ESI-TOF-MS). 27 ) We had previously reproduced the hydrangea blue-complex in approximately 100–200 mM buffered solutions; however, such a high-concentrated salt was known to suppress ionization, therefore, we tested whether the same blue color could be realized in a very low-concentration buffer solution. After several trials, the same hydrangea blue-complex was obtained by mixing 3- O -glucosyldelphinidin ( 1 ), 5- O -caffeoylquinic acid ( 2 ) and Al 3+ in ratios of 1 : 1 : 1, 1 : 2 : 1, and 1 : 3 : 1 in a diluted 2 mM buffer solution at pH 4.0.…”

“…From the comparison of the three UV–vis spectra, the ratio of 1 , 2 and Al 3+ was thought to be 1 : 2 : 1 because this solution was the bluest in color and its absorbance at λ vismax reached its maximum. 27 )…”

“…These results strongly suggested that the hydrangea blue-complex was composed of 1 , Al 3+ and 2 or 3 with a 1 : 1 : 1 ratio. 27 )…”

“… 26 ) Our research on blue hydrangea started in the early 1990s to elucidate the attractive but curious properties of hydrangea coloration, and it has been carried out by combining various research areas, such as natural product chemistry, synthetic organic chemistry, plant physiology, and molecular biology. 18 ) We have recently determined the chemical composition of the hydrangea blue-complex 27 ) and have directly detected the blue pigment in vivo . 28 ) Here, we summarize the last 30 years of our hydrangea studies.…”