Colorants and methods of making and use thereof

Abstract

Disclosed herein are colorants and methods of making and use thereof. For example, disclosed herein are colorant compositions comprising a hydroxyphenyl pyranoanthocyanin of Formula (I). Also disclosed herein are thermal degradation compounds derived via thermal degradation of any of the colorants disclosed herein. Also disclosed herein are thermal degradation compounds derived via thermal degradation of 10-catechyl-pyranocyanidin-3-O-β-glucoside. In some examples, the thermal degradation compound comprises 4-carboxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromenylium. Also disclosed herein are compositions, beverages, food products, edible products, cosmetic products, and / or dye sensitized solar cells comprising any of the colorants and / or thermal degradation compounds disclosed herein. Also disclosed herein are methods of making any of the colorants and / or thermal degradation compounds disclosed herein.

Description

[0001] COLORANTS AND METHODS OF MAKING AND USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 665,082 filed June 27, 2024, which is hereby incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. 2023-67017-39862 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Color is often used by the consumer as an important indicator of food quality and it may remarkably affect consumer food preferences and acceptance. In recent years, the market for the application of synthetic colorants has decreased in favor of nature derived colorants, especially since the use of synthetic food dyes has been legally challenged, and their use, legally challenged. For this reason, the food industry is making efforts to replace the synthetic food colorants with naturally-derived colorants.

[0008] Naturally derived colorants with improved properties are needed. The compositions and methods discussed herein address these and other needs.

[0009] SUMMARY

[0010] In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to colorants and methods of making and use thereof.

[0011] For example, disclosed herein are colorants composition comprising a hydroxyphenyl pyranoanthocyanin of Formula where Ri, R2, R3, and R4 each independently comprise H, OH, or OCH3; and Sugar is a monosaccharide, a disaccharide, or a trisaccharide; wherein the number, identity, and location of hydroxyl and methoxy substitutions on the B and E rings are selected to control the on color expression, molar absorptivity coefficients, pH color stability, and / or solubility of the colorant.

[0012] Also disclosed herein are thermal degradation compounds derived via thermal degradation of any of the colorants disclosed herein.

[0013] Also disclosed herein are thermal degradation compounds derived via thermal degradation of 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0014] In some examples, the thermal degradation compound is a colorant.

[0015] In some examples, the thermal degradation compound comprises 4-carboxy-3- deoxycyanidin (i.e., 4-carboxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromenylium).

[0016] In some examples, the thermal degradation compound has a formula:

[0017] Also disclosed herein are compositions, beverages, food products, edible products, cosmetic products, and / or dye sensitized solar cells comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0018] Also disclosed herein are methods of making any of the colorants and / or thermal degradation compounds disclosed herein.

[0019] Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compositions and methods, as claimed.

[0020] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE FIGURES

[0021] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

[0022] Figure 1. Chemical structure and classification of the FDA approved FD&C certified colors (as of June 2024).

[0023] Figure 2. Examples of the exempt from certification (naturally derived) colorants based on their classification of the chemical structure. Figure modified from Sigurdson et al. (2017).

[0024] Figure 3. Chemical structure of anthocyanin with B ring substitutions defined for the 6 common aglycones. R3 may be hydrogen (anthocyanidin) or is a common location for sugar attachment.

[0025] Figure 4. Anthocyanin chemical structure equilibrium pathway dependent upon solution pH.

[0026] Figure 5. Resonance structures of anthocyanin flavylium cation with the positive charge delocalized throughout the molecule.

[0027] Figure 6. Chemical structure of pyranoanthocyanin.

[0028] Figure 7. Ketone-enol tautomerization process. Figure modified from Loudon and Parise (2016).

[0029] Figure 8. Possible formation mechanism for pyranoanthocyanins. Schematic modified after those and the information reported in Schwarz et al. (2003), Zeng et al. (2023), and He et al. (2006). Ri and R2 = H, OH, or OCH3. R3 and R4 depend on the cofactor.

[0030] Figure 9. First generation pyranoanthocyanins (PACNs) with the common cofactors used for their formation and approximate color indicated by the outline. * indicates PACNs not found from natural sources. Figure modified from Oliveira et al. (2014).

[0031] Figure 10. Second generation pyranoanthocyanins (PACNs) found in nature with the common cofactors used for their formation and approximate potential color. Figure modified from Oliveira et al. (2014).

[0032] Figure 11. General pH structure equilibrium pathway for pyranoanthocyanins.

[0033] Figure 12. Typical pH dependent structure transitions for anthocyanins. Ri, R2, R3 indicate common locations for substitution groups, including H, OH, OCH3, and glycosylation.

[0034] Figure 13. Chemical structure and HPLC-PDA-MS / MS characteristics of the 2 anthocyanins and 8 hydroxyphenyl-pyranoanthocyanins formed, isolated, and evaluated in the present study.

[0035] Figure 14. HPLC-PDA chromatograms used for purity determination of the hydroxyphenyl-pyranoanthocyanins and anthocyanins evaluated. Compound characteristics and purity percentage are in Figure 13. Cy3G: cyani din-3 -glucoside, and Mv3G: malvi din-3 - glucoside.

[0036] Figure 15. CIELAB color coordinates for hydroxyphenyl -pyranoanthocyanins and anthocyanins. Circles (•) indicate cyanidin aglycones and diamonds (♦) indicate malvidin aglycone with center color based on the CIELAB coordinates. Letters show statistical differences at < 0.05 level between pyranoanthocyanins in a single solvent. Parenthetical number after pyranoanthocyanin name is the total number of oxygenated substitutions on the B and E ring. Cy3G: Cyani din-3 -glucoside, Mv3G: Malvi din-3 -glucoside, P: Pyrano.

[0037] Figure 16. UV-Visible spectra of anthocyanins (cyani din-3 -glucoside and malvidin-3- glucoside) and pyranoanthocyanins derived from these anthocyanins (all colorants at 40 pM) in acidic buffer and methanol. Spectra at pH 1 were not included as precipitation was observed in several solutions.

[0038] Figure 17. UV-Vis spectra of anthocyanins (40 pM) and hydroxyphenyl- pyranoanthocyanins (40 pM) from pH 3.5 to ~ 10, after 15 minutes equilibration. Spectra show mean of n = 3 repetitions with the average pH value for the spectra noted nearby. Line color based on CIELAB coordinates with the anthocyanin (Cy3G and Mv3G) lines darkened from pH 3.5 to 6.5.

[0039] Figure 18. CIE Color coordinates for anthocyanins (40 pM) and hydroxyphenyl- pyranoanthocyanins (40 pM) from pH ~3 to 10, after 15 minutes equilibration. Shape color represents the solution color based on CIELAB coordinates. Standard deviation bars represent deviation in pH (horizontal) and coordinate value (vertical).

[0040] Figure 19. Precipitation of hydroxyphenyl -pyranoanthocyanins in aqueous conditions. Panel A shows spectral and visual changes in pH 1 KC1 buffer and Panel B shows visual precipitation after 2 hours in pH adjusted water solutions.

[0041] Figure 20. When frozen (panel A), the hydroxyphenyl-pyranoanthocyanins had blue shifted colors with 10-guaiacyl-PMv3G producing blue green color. This PACN also uniquely produced a blue color when dried (panel B).

[0042] Figure 21. Chemical structure of 10-guaiacyl-pyranocyanidin and the various glycosylation substitutions evaluated in this study with the anthocyanin sources for the precursor glycosylated cyanidin anthocyanins listed. ACNs: Anthocyanins. PACNs: Pyranoanthocyanins

[0043] Figure 22. CIELAB color coordinates for 10-guaiacyl-pyranocyanidins with different C3 glycosylation substitutions at 40 pM concentration. Circle color is based on the CIELAB coordinates and letters show statistical differences within a solution type at < 0.05 level. Figure 23. Normalized visible spectral changes for 10-guaiacyl-pyranocyanidins from pH 3 to 8 and CIELAB color characteristics across pH. Results are reflected as means (standard deviation) with n = 3, and different letters show statistical differences between values within a row (p < 0.05).

[0044] Figure 24. The effect of buffer type on color expressed (CIELAB coordinates) by 10- guaiacyl-pyranocy ani din-3 -xyl-glu-gal from pH 3 to 8. Significant differences are expressed by *

[0045] Figure 25. Precipitation tendency for 10-guaiaycl-pyranocyanidins over 60 days at room temperature (-18.5 °C). Buffer abbreviations are as follows. K: KC1, CN: Citric acid-Na2HPO4, CC: Citric acid-trisodium citrate, NA: Sodium acetate-acetic acid, NN: Na2HPO4-NaH2PO4, T: Tris.

[0046] Figure 26. Absorbance stability (based on Xvis-max at time 0)over 60 days for 10-guaiacyl- pyranocyanidins. Table shows % Absorption remaining at 33 and 60 days with lower case letters expressing statistical differences within a row and uppercase letters representing statistical differences within a column (D33 and D60 separately) at a / ? < 0.05 level.

[0047] Figure 27. Effect of buffer on absorbance retention after 60 days at room temperature for 10-guaiacyl-pyranocyanidin-3-xyl-glu-gal. Statistical differences are represented by *.

[0048] Figure 28. Unique color observations were observed for 10-guaiacyl-PCy3-glu including forming a brown coloration when stored in the freezer (A) and turning blue when dried (B).

[0049] Figure 29. Chemical structures for 10-catechyl-pyranocyanidin-3-O-P-glucoside and 4- carboxy-3 -deoxy cyani din (compound A). Ring B’ and C’ on compound A were derived from the E and D rings, respectively, of 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0050] Figure 30. Changes in PDA peak area (from 450-550 nm max plot) for pyranoanthocyanins (PACN) including starting 10-catechyl-pyranocyanidin-3-sambubioside and formed 10-catechyl-pyranocyanidin-3-glucoside (pH 1 & 3) and compound A with 90 °C heat. Data points show mean ± standard deviation. Table shows results of kinetic model for PACN degradation and compound A formation.

[0051] Figure 31. Absorbance and color change of 10-catechyl-pyranocyanidin-3-sambubioside solutions in four pH buffers with 90 °C heating. Spectra show mean absorbance values taken with a 5 nm step, and L*, Cab*, and hab data points show mean ± standard deviation.

[0052] Figure 32. FTIR spectra for 4-carboxy-3 -deoxy cyani din (compound A) repetition 1 (Rl) and 2 (R2) and luteolinidin standard and the calculated PCA plot and differentiating loadings between the three samples.

[0053] Figure 33. Spectral and CIELAB L*, Cab*, hab values of 4-carboxy-3 -deoxy cyani din (compound A) in pH buffers 1-9.2 and MeOH. Spectra shows mean absorption values and color characteristics are expressed as mean ± standard deviation. Different letters show statistical difference (p < 0.05) within a column (MeOH not included).

[0054] Figure 34. Color stability of 4-carboxy-3 -deoxy cyani din (compound A) in pH from 1-9.2 buffers and MeOH over 24 hours. The graph shows the percent absorption change at Vis-max, and the table displays the change in CIELAB color coordinates with results presented as means with standard deviation.

[0055] Figure 35. uHPLC-PDA-MS characteristics of hydroxyphenyl-pyranoanthocyanins (peaks 2-5) and their thermal degradation compounds (peaks 1 and 2d-5d) following 15 hours of 90 °C heating at pH 3.0. 10-p-hydroxyphenyl-pyranoCy3Smb solution contained an impurity (~6.8 min.) present both before and after heating. Chromatographic conditions are provided in Section 5.3.2.

[0056] Figure 36. Proposed schematic for hydroxyphenyl-PACN formation and thermal degradation into the 4-carboxy-3 -deoxy anthocyanidin degradation compound. Rings B’ and C’ correspond to ring E and D of the parent compound, respectively.

[0057] Figure 37. HPLC-PDA chromatograms of 10-catechyl-pyranocyanidin-3-sambubiside (peak 1) at four pH values heated at 90 °C. Compound A (peak A) formed at all pH values, however the peak was very small at pH 7.

[0058] Figure 38. *H NMR spectrum for 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0059] Figure 39. COSY spectrum for 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0060] Figure 40. HSQC-DEPT spectrum for 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0061] Figure 41. HMBC spectrum for 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0062] Figure 42. High resolution MS / MS fragmentation of 4-carboxy-3 -deoxy cyani din (compound A) in positive ion mode using collision energies of 10 (red), 20 (green), and 40 eV (blue), where eV is electron volts.

[0063] Figure 43. *H NMR spectrum for 4-carboxy-3 -deoxy cyani din (compound A).

[0064] Figure 44. COSY spectrum for 4-carboxy-3 -deoxy cyani din (compound A).

[0065] Figure 45. HMBC spectrum for 4-carboxy-3 -deoxy cyani din (compound A).

[0066] Figure 46. Full spectrum absorbance for 4-carboxy-3 -deoxy cyani din (compound A) in pH 1-9.2 buffers and MeOH. Spectra show the mean absorption over 24 hours at 25 °C.

[0067] Figure 47. MS chromatogram of 10-catechyl-pyranomalvidin-3-glucoside / 3-rutinoside in pH 2.8 H2O (with HC1) heated at 90 °C for 21 hours. A peak consistent with a hydrated aglycone appeared with heating both in positive and negative ionization mode (481.1 and 479. 1, respectively) which corresponded to a peak in PDA with absorption in 370 to 385 nm region. Conditions for HPLC-PDA-MS analysis are shown in the figure using the equipment described in Section 2.2

[0068] Figure 48. Chemical structure of the primary anthocyanins and formed pyranoanthocyanins in the aronia and saponified black carrot solutions.

[0069] Figure 49. HPLC-PDA chromatograms of anthocyanin extracts and pyranoanthocyanin solutions formed from anthocyanin incubation with 4-vinylphenols. Peak numbers correspond to Table 12. PACNs: Pyranoanthocyanins.

[0070] Figure 50. A dark, colored layer was formed in the base of the aronia-PACN in 20% oil o / w emulsion samples over time (encircled in white). This was not observed in the saponified black carrot (sBC) pyranoanthocyanins (PACN).

[0071] Figure 51. Visible spectra of colorants in carbohydrate solutions (~pH 5.9) after 30- minute equilibration. sBC: saponified black carrot. PACNs: pyranoanthocyanins.

[0072] Figure 52. Visible spectra of colorants in protein solutions (~pH 5.9) after 30-minute equilibration. sBC: saponified black carrot. PACNs: pyranoanthocyanins. The aronia-PACN solutions partially precipitated in all 4 solution types.

[0073] Figure 53. CIELAB lightness (L*), chroma (C*ab) and hue angle (hab) color coordinates at time 0 for colorants mixed into 20% oil o / w emulsion and 60% oil oil / water emulsions and pH 4 buffer at 100 pM concentration. Color swatch is representative of the CIELAB L*, a* , and b* coordinates. Results are expressed as means (standard deviation) for n = 3 (emulsions) and n = 2 (pH 4 buffer). Letters represent statistical differences at a < 0.05 level between color coordinates in 20% and 60% oil o / w emulsion. sBC : saponified black carrot. PACN: py ranoanthocy anin .

[0074] Figure 54. example pyranoanthocyanin solution and structure.

[0075] Figure 55. Pyranoanthocyanins formed from aronia mono-glycosides in pH 5 Buffer.

[0076] Figure 56. Pyranoanthocyanins formed from black carrot di- and tri -glycosides in pH 5 Buffer.

[0077] Figure 57. Emulsion: Color expression and solubility.

[0078] Figure 58. Carbohydrate solutions: Color expression and solubility.

[0079] Figure 59. Protein: Color expression of di- and tri-glycosides from black carrot.

[0080] Figure 60. Protein: Color expression of mono-glycosides from Aronia.

[0081] Figure 61. Black Carrot Guaiacyl PACNs in foods.

[0082] Figure 62. Six unique 10-guaiacyl-pyranocyanidins were formed from cyanidin anthocyanins with different C3 glycosylations and 4-vinylguaiacol.

[0083] Figure 63. 10-Guaiacyl-Pyranocyanidin Formation. Figure 64. 10-guaiacyl-pyranocyanidins produced orange to yellow orange colors at pH 3 with the glycosylation substitution having a slight but significant effect on color.

[0084] Figure 65. Each pigment produced uniform color in 20% o / w emulsion with the pyranoanthocyanins producing more pink / red colors than in pH 3 buffer.

[0085] Figure 66. The color was stable for Guaiacyl-PACNs from black carrot over 7 days, with the color loss for Guaiacyl-PACNs from aronia likely driven by precipitation.

[0086] Figure 67. Color performance was similar for all pigments in 60% O / W emulsion.

[0087] Figure 68. Both PACNs produced more stable color than the black carrot anthocyanins with no precipitation observed.

[0088] Figure 69. 10-p-hydroxyphenyl-Cyanidin PACN

[0089] Figure 70. 10-guaiacyl-Cyanidin PACN

[0090] Figure 71. Elderberry Pigment Profiles (uHPLC-PDA-MS).

[0091] Figure 72. Black Carrot pigment profiles

[0092] Figure 73. Results in skim milk.

[0093] Figure 74. Results in cream (heavy whipping cream).

[0094] Figure 75. Results in Yoplait yogurt.

[0095] Figure 76. Results in Greek yogurt.

[0096] Figure 77. Results in Coconut “yogurt” (yogurt alternative).

[0097] Figure 78. Results in soy “yogurt” (yogurt alternative).

[0098] Figure 79. Results for Lotion.

[0099] Figure 80. Results for butter (softened).

[0100] Figure 81. Results for gelatin (color added after gelatin solution was cooled).

[0101] Figure 82. Results for acidified gelatin (gelatin + citric acid, an acidulent used in flavored jello) (color added after gelatin solution was cooled)

[0102] Figure 83. Results for cream cheese (CC) (pH = 5.08).

[0103] Figure 84. Results for vanilla frosting. Pictures are browner that the samples appeared in person. G-Eld had a nice, rosy, pink orange color.

[0104] Figure 85. Results for white chocolate (WC).

[0105] Figure 86. Results for tap water.

[0106] Figure 87. Results for frozen vanilla bean ice cream. Black specs are from vanilla beans. PACN colors became slightly more red-pink with freezing, but not as evident as with isolates in frozen water.

[0107] Figure 88. Results for distilled water.

[0108] Figure 89. Results for seltzer water. Figure 90. results for seltzer water (pH ~ 4.7)

[0109] Figure 91. Schematic of pyranoanthocyanin (pyrano) formation).

[0110] Figure 92. Color Performance in Ingredient Matrices.

[0111] Figure 93. Spectra of buffer or protein solutions colored with anthocyanin or pyranos in pH 6.

[0112] Figure 94. Color and CIELAB coordinates of colored 20% oil o / w emulsions.

[0113] Figure 95. CIELAB color coordinates of colored sugar solutions. Letters show statistical differences at < 0.05.

[0114] Figure 96. sBC pyranos produced uniform, pink, orange, and brown colors in foods including yogurts and yogurt alternatives.

[0115] Figure 97. Chemical structure of hydroxyphenyl-pyranoanthocyanins evaluated in this study. Part A evaluated the influence of B and E ring hydroxyl and methoxy substitutions for glycosides (Ri, R2, R4, Rs). Part B evaluated the influence of glycosylation (R3) for 10-guaiacyl- pyranocyanidins.

[0116] Figure 98. Color expressed and UV-Vis spectra for hydroxyphenyl-pyranoanthocyanins and their precursor anthocyanins in pH 3 buffer (0.025 M KC1) solution at 40 pM concentration.

[0117] Figure 99. Color expressed and UV-Vis spectra for 10-guaiacyl-pyranocyanidins with different C3 glycosylation patterns in pH 3 buffer (0.025 M KC1) solution at 40 pM concentration.

[0118] Figure 100. we assessed the influence of these substitutions on color expression, molar absorptivity coefficients, pH color stability (1-10), and water solubility for eight different hydroxyphenyl-pyranoanthocyanins as compared to their precursor anthocyanins.

[0119] Figure 101. General pH dependent structure transitions for anthocyanins. Ri, R2, R3 indicate common locations for substitution groups, including H, OH, OCH3, and glycosylation.

[0120] Figure 102. Chemical structure and HPLC-PDA-MS / MS characteristics of the 2 anthocyanins and 8 hydroxyphenyl-pyranoanthocyanins formed, isolated, and evaluated in the present study.

[0121] Figure 103. CIELAB color coordinates for hydroxyphenyl -pyranoanthocyanins and anthocyanins. Circles (•) indicate cyanidin (Cy) aglycones and diamonds (♦) indicate malvidin (Mv) aglycone with center color derived from the CIELAB coordinates. Letters show statistical differences at < 0.05 level between pyranoanthocyanins in a single solvent. Parenthetical number after pyranoanthocyanin name is the total number of oxygen containing substitutions on the B and E ring. Error bars represent standard deviations. Cy3G: Cyani din-3 -glucoside, Mv3G: Malvi din-3 -glucoside, P: Pyrano. Figure 104. UV-Visible spectra of anthocyanins (cyani din-3 -glucoside and malvidin-3- glucoside) and pyranoanthocyanins derived from these anthocyanins (all colorants at 40 pM) in acidic buffer and methanol.

[0122] Figure 105. UV-Vis spectra of anthocyanins (40 pM) and hydroxyphenyl- pyranoanthocyanins (40 pM) from pH 3.5 to ~ 10, after 15 minutes equilibration. Spectra show mean of n = 3 repetitions with the average pH value for the spectra noted nearby in the corresponding color. Line color based on CIELAB coordinates with the anthocyanin (Cy3G and Mv3G) lines darkened from pH 3.5 to 6.5.

[0123] Figure 106. Spectral, pH, and CIELAB color coordinate changes for 10-syringyl-pyranos across 120 minutes of room temperature storage. Spectra display mean (n=3) absorption and table results are presented as mean (standard deviation).

[0124] Figure 107. Precipitation of hydroxyphenyl -pyranoanthocyanins in in aqueous conditions. Panel A shows spectral and visual changes in pH 1 KC1 buffer and Panel B shows visual precipitation after 2 hours in pH adjusted water solutions.

[0125] Figure 108. When frozen (panel A), the hydroxyphenyl-pyranoanthocyanins had blue shifted colors with 10-guaiacyl-PMv3G producing blue green color. This PACN also uniquely produced a blue color when dried (panel B).

[0126] Figure 109. HPLC-PDA chromatograms used for purity determination of the hydroxyphenyl-pyranoanthocyanins and anthocyanins evaluated. Compound characteristics and purity percentage are in Figure 102. Cy3G: cyani din-3 -glucoside, and Mv3G: malvidin-3- glucoside.

[0127] Figure 110. Photograph showing colored precipitants.

[0128] DETAILED DESCRIPTION

[0129] The compositions and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

[0130] Before the present compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0131] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0132] General Definitions

[0133] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0134] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0135] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0136] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0137] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0138] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0139] When the specific values are disclosed between two end values, it is understood that these end values can also be included.

[0140] For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. It is further understood that these phrases are not used in a restrictive sense, but for explanatory purposes. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment.

[0141] Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value.

[0142] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0143] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0144] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, or approximately occurs.

[0145] Still further, the term “substantially” can, in some aspects, refer to at least about 80 %, at least about 85 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or about 100 % of the stated property, component, composition, or other condition for which substantially is used to characterize or otherwise quantify an amount.

[0146] In other aspects, as used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition.

[0147] The expressions “ambient temperature” and “room temperature” as used herein are understood in the art and refer generally to a temperature from about 20°C to about 35°C.

[0148] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a mixture containing 2 parts by weight of component X and 5 parts by weight of component Y, components X and Y are present at a weight ratio of 2:5 and are present in such a ratio regardless of whether additional components are contained in the mixture.

[0149] A weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0150] A volume percent (vol%) of a component, unless specifically stated to the contrary, is based on the total volume of the formulation or composition in which the component is included.

[0151] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0152] As used herein, “molecular weight” refers to number average molecular weight as measured by 'H NMR spectroscopy, unless indicated otherwise.

[0153] As used herein the term “plurality” means 2 or more (e.g., 3 or more; 4 or more; 5 or more; 10 or more; 15 or more; 20 or more; 25 or more; 30 or more; 40 or more; 50 or more; 75 or more; 100 or more; 150 or more; 200 or more; 250 or more; 300 or more; 400 or more; 500 or more; 750 or more; 1000 or more; 1500 or more; 2000 or more; 2500 or more; 3000 or more; 4000 or more; or 5000 or more).

[0154] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to the arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0156] Chemical Definitions

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0158] The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety.

[0159] The prefix Cn-Cmpreceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows. For example, the term "Cn-Cm" (or “Cn-m”) employed alone or in combination with other terms refers to a hydrocarbon group that may be straight-chain or branched, having n to m carbons. It is understood that the terms Cn-m and Cn-Cm can be used interchangeably and just to show that the specific compound has between n to m carbons.

[0160] The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

[0161] The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0162] The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

[0163] "Zwitterionic" or "zwitterion" as used herein refers to a neutral molecule with a positive (or cationic) and a negative (or anionic) electrical charge at different locations within the same molecule.

[0164] As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is contemplated to include all permissible substituents of organic compounds. As used herein, the phrase "optionally substituted" means unsubstituted or substituted. It is to be understood that substitution at a given atom is limited by valency. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In still further aspects, it is understood that when the disclosure describes a group being substituted, it means that the group is substituted with one or more (i.e., 1, 2, 3, 4, or 5) groups as allowed by valence selected from alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0165] The term "compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0166] Compounds provided herein can also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-l,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0167] Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include hydrogen, tritium, and deuterium.

[0168] Also provided herein are salts of the compounds described herein. It is understood that the disclosed salts can refer to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of the salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The salts of the compounds provided herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The salts of the compounds provided herein can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or in a mixture of the two. In various aspects, nonaqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (ACN) can be used.

[0169] As used herein, chemical structures that contain one or more stereocenters depicted with dashed and bold bonds are meant to indicate the absolute stereochemistry of the stereocenter(s) present in the chemical structure. As used herein, bonds symbolized by a simple line do not indicate a stereo-preference. Unless otherwise indicated to the contrary, chemical structures, which include one or more stereocenters, illustrated herein without indicating absolute or relative stereochemistry encompass all possible stereoisomeric forms of the compound (e.g., diastereomers and enantiomers) and mixtures thereof. Structures with a single bold or dashed line and at least one additional simple line encompass a single enantiomeric series of all possible diastereomers.

[0170] The terms for various functional groups as used herein are not intended to be limited to monovalent radicals and may include polyvalent radical groups as appropriate, such as divalent, trivalent, tetravalent, pentavalent, and hexavalent groups, and the like, based on the position and location of such groups in the compounds described herein as would be readily understood by the skilled person.

[0171] “Z1,” “Z2,” “Z3,” and “Z4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0172] A dash that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=0)NH2 is attached through the carbon of the keto (C=O) group.

[0173] The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups.

[0174] As used herein, the term “alkyl” refers to saturated, straight-chained or branched saturated hydrocarbon moieties. Unless otherwise specified, C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis, C1-C16, C1-C14, C1-C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl groups are intended. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1-methyl-propyl, 2-methyl- propyl, 1,1 -dimethyl -ethyl, pentyl, 1 -methyl -butyl, 2-methyl-butyl, 3 -methyl -butyl, 2,2- dimethyl-propyl, 1 -ethyl -propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1 -methylpentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1 -dimethyl -butyl, 1,2-dimethyl- butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1-ethyl- butyl, 2-ethyl -butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1 -ethyl- 1-methyl-propyl, 1- ethyl-2-methyl-propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. The alkyl group can be substituted with one or more groups including, but not limited to, hydroxyl, halogen, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied. It is further understood that throughout the specification, “alkyl” can also be referred to as a linking group of saturated hydrocarbons that are divalent radicals. In other words, in a broader description, the term “alkyls” also encompasses alkylenes. It is further understood that the term “alkyl” covers saturated hydrocarbons that are multivalent radicals.

[0175] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

[0176] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0177] The term "heteroalkyl" refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. By way of example, a heteroCi-ealkyl (which may also be designated a Ci- eheteroalkyl) group includes, but is not limited to, the following structures:

[0178] As used herein, the term “alkenyl” refers to unsaturated, straight-chained, or branched hydrocarbon moieties containing a double bond. Unless otherwise specified, C2-C24 (e.g., C2-C22, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkenyl groups are intended. Alkenyl groups may contain more than one unsaturated bond. Examples include ethenyl, 1 -propenyl, 2-propenyl, 1 -methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -methyl- 1- propenyl, 2-methyl-l -propenyl, l-methyl-2-propenyl, 2-methyl-2-propenyl, 1 -pentenyl, 2- pentenyl, 3 -pentenyl, 4-pentenyl, 1 -methyl- 1-butenyl, 2-methyl- 1-butenyl, 3 -methyl- 1-butenyl, l-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-methyl-3- butenyl, 3-methyl-3-butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl-l -propenyl, l,2-dimethyl-2- propenyl, 1 -ethyl- 1 -propenyl, l-ethyl-2-propenyl, 1 -hexenyl, 2-h exenyl, 3 -hexenyl, 4-hexenyl, 5-hexenyl, 1 -methyl- 1 -pentenyl, 2-methyl-l -pentenyl, 3-methyl-l-pentenyl, 4-methyl-l- pentenyl, 1 -methyl -2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl- 4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1 -dimethyl -2- butenyl, l,l-dimethyl-3-butenyl, 1,2-dimethyl-l-butenyl, l,2-dimethyl-2-butenyl, 1,2-dimethyl- 3-butenyl, 1,3-dimethyl-l-butenyl, l,3-dimethyl-2-butenyl, l,3-dimethyl-3-butenyl, 2,2- dimethyl-3-butenyl, 2,3 -dimethyl- 1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-l-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-l-butenyl, l-ethyl-2-butenyl, l-ethyl-3- butenyl, 2-ethyl-l-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, l,l,2-trimethyl-2-propenyl, 1- ethyl-l-methyl-2-propenyl, l-ethyl-2-m ethyl- 1 -propenyl, and l-ethyl-2-methyl-2-propenyl. The term “vinyl” refers to a group having the structure -CH=CH2; 1 -propenyl refers to a group with the structure -CH=CH-CH3; and 2-propenyl refers to a group with the structure -CH2-CH=CH2. Asymmetric structures such as (Z1Z2)C=C(Z3Z4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. Alkenyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

[0179] As used herein, the term “alkynyl” represents straight-chained or branched hydrocarbon moieties containing a triple bond. Unless otherwise specified, C2-C24 (e.g., C2-C24, C2-C20, C2- Cis, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkynyl groups are intended. Alkynyl groups may contain more than one unsaturated bond. Examples include C2-Ce-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1- methyl-2-propynyl, 1 -pentynyl, 2-pentynyl, 3 -pentynyl, 4-pentynyl, 3 -methyl- 1-butynyl, 1- methyl-2-butynyl, 1 -methyl -3-butynyl, 2-methyl-3-butynyl, l,l-dimethyl-2-propynyl, l-ethyl-2- propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3 -methyl- 1 -pentynyl, 4- methyl-1 -pentynyl, l-methyl-2-pentynyl, 4-methyl-2-pentynyl, l-methyl-3 -pentynyl, 2-methyl- 3-pentynyl, l-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1 -dimethyl -2- butynyl, l,l-dimethyl-3-butynyl, l,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl- 1-butynyl, l-ethyl-2-butynyl, l-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1 -ethyl -1 -methyl -2- propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0180] As used herein, the term “aryl,” as well as derivative terms such as aryloxy, refers to groups that include an aromatic carbocyclic group of from 3 to 50 carbon atoms. Aryl groups can include a single ring or multiple condensed rings. In some examples, aryl groups include Ce-Cio aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0181] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0182] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, z.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0183] The term “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (z.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0184] The term “acyl” as used herein is represented by the formula -C(O)Z1where Z1can be a hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. As used herein, the term “acyl” can be used interchangeably with “carbonyl.” Throughout this specification “C(O)” or “CO” is a shorthand notation for C=O.

[0185] The term “acetal” as used herein is represented by the formula (Z1Z2)C(=OZ3)(=OZ4), where Z1, Z2, Z3, and Z4can be, independently, a hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0186] The term “alkanol” as used herein is represented by the formula Z'OH, where Z1can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0187] As used herein, the term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as to a group of the formula Z where Z1is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, alkoxy groups wherein Z1is a C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis, C1-C16, C1-C14, Ci- C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl group are intended. Examples include methoxy, ethoxy, propoxy, 1 -methyl-ethoxy, butoxy, 1 -methyl -propoxy, 2-methyl-propoxy, 1,1 -dimethyl- ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-di-methyl-propoxy, 1 -ethyl -propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2- methyl-pentoxy, 3-methyl-pentoxy, 4-methyl-penoxy, 1,1 -dimethyl -butoxy, 1,2-dimethyl- butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1-ethyl-l- methyl-propoxy, and l-ethyl-2-methyl-propoxy.

[0188] The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a shorthand notation for C=O.

[0189] The terms “amine” as used herein are represented by the formula — NRJR2, where R1and R2can each be substitution groups as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0190] The term “amino” as used herein are represented by the formula — NZ'Z2Z3, where Z1, Z2, and Z3can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0191] The terms “amide” or “amido” as used herein are represented by the formula — C(O)NZ1Z2, where Z1and Z2can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0192] The term “anhydride” as used herein is represented by the formula Z1C(O)OC(O)Z2where Z1and Z2, independently, can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0193] The term “cyclic anhydride” as used herein is represented by the formula: o y where Z1can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0194] The term “azide” as used herein is represented by the formula -N=N=N.

[0195] The term “carboxylic acid” or “carboxy” as used herein is represented by the formula — C(O)OH.

[0196] A “carboxylate” or “carboxyl” group as used herein is represented by the formula — C(O)O’

[0197] As used herein, the term “carbamyl” refers to a group of formula -C(0)NH2.

[0198] A “carbonate ester” group as used herein is represented by the formula Z1OC(O)OZ2. The term “cyano” as used herein is represented by the formula — CN.

[0199] The term “ester” as used herein is represented by the formula — OC(O)Z1or — C(O)OZ where Z1can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0200] The term “ether” as used herein is represented by the formula ZXOZ2, where Z1and Z2can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0201] The term “epoxy” or “epoxide” as used herein refers to a cyclic ether with a three atom ring and can represented by the formula: where Z1, Z2, Z3, and Z4can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above

[0202] The term “ketone” as used herein is represented by the formula Z1C(O)Z2, where Z1and Z2can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0203] The term “halide” or “halogen” or “halo” as used herein refers to fluorine, chlorine, bromine, and iodine.

[0204] The term “hydroxyl” as used herein is represented by the formula — OH.

[0205] The term “nitro” as used herein is represented by the formula — NO2.

[0206] The term “phosphonyl” is used herein to refer to the phospho-oxo group represented by the formula — P(O)(OZ1)2, where Z1can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0207] The term “silyl” as used herein is represented by the formula — SiZJZ2Z3, where Z1, Z2, and Z3can be, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0208] The term “sulfonyl” or “sulfone” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2ZX, where Z1can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0209] The term “sulfide” as used herein comprises the formula — S — .

[0210] The term “thiol” as used herein is represented by the formula — SH.

[0211] The term “sulfonylamino” or “sulfonamide,” as used herein, is represented by the formula -S(O)2NH-.

[0212] In general, the inclusion of the prefix “alk” in front of a substituent name indicates there is an alkyl group (as defined herein) connecting the named substituent with the rest of the compound. For example, "alkaryl" (which is a subset of alkyl) refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety and "alkheteroaryl" (which is a subset of "alkyl") refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety. The number of carbon atoms may be specified in the alkyl chain, the named substituent, or both. For example, Ci^alkCearyl refers to a phenyl ring (which may be substituted) connected via a 1-2 carbon alkylene group.

[0213] Affixing the suffix "-ene" to a group indicates the group is a polyvalent moiety, e.g., boned to two or more groups. Alkylene is the polyvalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenyl ene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

[0214] “R1,” “R2,” “R3,” “Rn,” etc., where n is some integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an amino group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within a second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0215] As used herein, the designation of a polyvalent moiety without specifying the specific order of attachment is intended to cover all possible arrangements. By way of example, a compound that is represented by the formula:

[0216] A-X-B wherein X is NHC(=O) embraces both As used herein, a chemical bond depicted: ! I represents either a single, double, or triple bond, valency permitting. By way of example,

[0217] An electron-withdrawing group is a functional group or atom that pulls electron density towards itself, away from other portions of the molecule, e.g., through resonance and / or inductive effects. Exemplary electron-withdrawing groups include F, Cl, Br, I, NO2, CN, SO2R, SO3R, SO2NR2, C(O)Rla, C(O)OR, and C(0)NR2 (wherein R is H or an alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl group) as well as alkyl group substituted with one or more of those group.

[0218] An electron-donating group is a functional group or atom that pushes electron density away from itself towards other portions of the molecule, e.g., through resonance and / or inductive effects. Exemplary electron-donating groups include unsubstituted alkyl or aryl groups, OR and N(R)2, and alkyl groups substituted with one or more OR and N(R)2 groups.

[0219] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible stereoisomer or mixture of stereoisomer (e.g., each enantiomer, each diastereomer, each meso compound, a racemic mixture, or scalemic mixture).

[0220] Compositions

[0221] Disclosed herein are colorants and methods of making and use thereof.

[0222] For example, disclosed herein are colorant composition comprising a hydroxyphenyl pyranoanthocyanins. In some examples, the hydroxyphenyl pyranoanthocyanin is of Formula I: where

[0223] Ri, R2, R3, and R4 each independently comprise H, OH, or OCH3; and Sugar is a monosaccharide, a disaccharide, or a trisaccharide; wherein the number, identity, and location of hydroxyl and methoxy substitutions on the B and E rings are selected to control the on color expression, molar absorptivity coefficients, pH color stability, and / or solubility of the colorant.

[0224] In Formula I, A, B, C, D, and E are designations for the different rings.

[0225] In Formula I, the 10 refers to the carbon number at the designated location, and is shown for ease of nomenclature.

[0226] In some examples of Formula I, the sugar is glucose, xylose, galactose, arabinose, or a combination thereof. In some examples of Formula I, the sugar is arabinose, glucose, galactose, xylosyl(l— >2)glucose, xylosyl(l— >2)galactose, or xylosyl(l— >2)glucosyl(l— >6)galactoside. In some examples of Formula I, the sugar is glucose.

[0227] In some examples of Formula I, Ri is OH or OCH3.

[0228] In some examples of Formula I, R2 is H or OCH3.

[0229] In some examples of Formula I, R4 is H or OCH3.

[0230] In some examples of Formula I, Ri is OH or OCH3; R2 is H or OCH3; R3 is H, OH, or OCH3; and R4is H or OCH3.

[0231] In some examples of Formula I, the sugar is glucose, Ri is OH or OCH3; R2 is H or OCH3; R3is H, OH, or OCH3; and R4is H or OCH3.

[0232] In some examples of Formula I, Ri is OH and R2-R4are H; Ri is OH, R2 is H, R3 is OCH3, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is OH; Ri is OCH3, R2is OCH3, R3 is H, and R4is H; Ri is OCH3, R2is OCH3, R3 is OCH3, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OH, and R4is H; or RI-R4are each OCH3.

[0233] In some examples of Formula I: the sugar is glucose; and

[0234] Ri is OH and R2-R4are H; Ri is OH, R2 is H, R3 is OCH3, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is OH; Ri is OCH3, R2 is OCH3, R3 is H, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OH, and R4is H; or RI-R4are each OCH3.

[0235] In some examples of Formula I, Ri is OH and R2-R4are H.

[0236] In some examples of Formula I, Ri is OH, R2 is H, R3 is OCH3, and R4is H.

[0237] In some examples of Formula I, Ri is OH, R2 is H, R3 is OH, and R4is H.

[0238] In some examples of Formula I, Ri is OH, R2 is H, R3 is OH, and R4is OH.

[0239] In some examples of Formula I, Ri is OCH3, R2 is OCH3, R3 is H, and R4is H.

[0240] In some examples of Formula I, Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H. In some examples of Formula I, Ri is OCH3, R2 is OCH3, R3 is OH, and R4 is H.

[0241] In some examples of Formula I, R1-R4 are each OCH3.

[0242] In some examples of Formula I, the sugar is glucose, Ri is OH and R2-R4 are H.

[0243] In some examples of Formula I, the sugar is glucose, Ri is OH, R2 is H, R3 is OCH3, and R4 is H.

[0244] In some examples of Formula I, the sugar is glucose, Ri is OH, R2 is H, R3 is OH, and R4 is H.

[0245] In some examples of Formula I, the sugar is glucose, Ri is OH, R2 is H, R3 is OH, and R4 is OH.

[0246] In some examples of Formula I, the sugar is glucose, Ri is OCH3, R2 is OCH3, R3 is H, and R4 is H.

[0247] In some examples of Formula I, the sugar is glucose, Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H.

[0248] In some examples of Formula I, the sugar is glucose, Ri is OCH3, R2 is OCH3, R3 is OH, and R4 is H.

[0249] In some examples of Formula I, the sugar is glucose, R1-R4 are each OCH3.

[0250] In some examples, the colorant comprises a hydroxyphenyl pyranoanthocyanin as shown in Figure 97.

[0251] In some examples, the colorant comprises a 10- / 2-hydroxyphenyl-pyranoanthocyanin, 10- catechyl-pyranoanthocyanin, 10-guaiacyl-pyranoanthocyanin, 10-syringyl -pyranoanthocyanin, or a combination thereof.

[0252] In some examples, the colorant comprises 10-p-hydroxyphenyl-Cy-3 -glucoside, 10- catechyl-Cy-3 -glucoside, 10-guaiacyl-Cy-3 -glucoside, 10-syringyl-Cy-3 -glucoside, 10-p- hydroxyphenyl-Mv-3-glucoside, 10-catechyl- Mv -3-glucoside, 10-guaiacyl- Mv-3 -glucoside, 10-syringyl-Mv-3-glucoside, or a combination thereof.

[0253] In some examples, the colorant comprises a 10-guaiacyl-pyranocyanidin.

[0254] In some examples, the colorant comprises 10-guaiacyl-PCy3-glu, 10-guaiacyl-PCy3-xyl- glu-gal, 10-guaiacyl-PCy3-gal, 10-guaiacyl-PCy3-xyl-glu, 10-guaiacyl-PCy3-xyl-gal, 10- guaiacyl-PCy3 -arabinoside, or a combination thereof.

[0255] In some examples, the colorant comprises 10-guaiacyl-PCy3-galactose, 10-guaiacyl- PCy3-xyl-glu-gal, 10-guaiacyl-PCy3-xyl-glu, 10-guaiacyl-PCy3-xyl-gal, or a combination thereof.

[0256] In some examples, the colorant comprises 10-guaiacyl-PCy3-xyl-glu-gal.

[0257] In some examples, the hydroxyphenyl pyranoanthocyanin is derived from an anthocyanin and a cofactor.

[0258] In some examples, the cofactor comprises acetone, pyruvic acid, acetaldehyde, hydroxycinnamic acids, 4-vinylphenols, or a combination thereof.

[0259] In some examples, the cofactor comprises hydroxycinnamic acid, 4-vinylphenol, or a combination thereof.

[0260] In some examples, the cofactor comprises / ?-coumaric acid, caffeic acid, ferulic acid, sinapic acid, or a combination thereof.

[0261] In some examples, the cofactor comprises 4-vinylphenol, 4-vinylguaiacyl, or a combination thereof.

[0262] In some examples, the anthocyanin comprises cyani din-3 -glucoside, malvidin-3- glucoside, or a combination thereof.

[0263] In some examples, the anthocyanin is derived from an edible material.

[0264] In some examples, the anthocyanin is derived from a plant.

[0265] In some examples, the anthocyanin is derived from a raw agricultural product.

[0266] In some examples, the anthocyanin is derived from a fruit, flower, vegetable, or combination thereof.

[0267] For example, the anthocyanin can be derived from a crude extract, a partially purified extract, a purified extract, or a combination thereof.

[0268] The anthocyanin can, for example, be extracted or derived from a whole plant or any portion thereof, such as a flower (e.g., flower petals), leaf, stem, etc. In some examples, the anthocyanin comprises an extract derived from a raw agricultural product. Examples of raw agricultural products include vegetables, fruits, grains, nuts, and mixtures thereof. In some examples, the raw agricultural product includes a fruit, a vegetable, or a combination thereof.

[0269] In some examples, the anthocyanin comprises an extract derived from a berry such as a bilberry, blueberry, blackberry, blackcurrant, chokeberry, red raspberry, strawberry, elderberry, or a combination thereof. In some examples, the anthocyanin comprises an extract derived from a red radish (Raphanus sativus), black carrot ( aucus carota L.), red cabbage, black currant, blueberry, red grape, blackberry, red raspberry, choke berry, black goji (Lycium rulhenicum). eggplant (Solarium melongena), American eggplant, Japanese eggplant, Chinese eggplant, East Asian eggplant, tomato (Solanaceae lycopersicum) (cv. Indigo Rose), violet pepper (Capsicum annuum), elderberry, purple potatoes, or a combination thereof.

[0270] In some examples, the anthocyanin is extracted or derived from a berry such as a bilberry, blueberry, blackberry, blackcurrant, chokeberry, red raspberry strawberry, elderberry, or a combination thereof. In some examples, the anthocyanin is derived from wine, sour cherry juice, figs, strawberries, fermented olives, frozen grape skins, red onion, black currant, blueberry honeysuckle, elderberry, blood orange juice, or a combination thereof.

[0271] In some examples, the anthocyanin is derived from black carrot, chokeberry, elderberry, Berberis boliviana. or a combination thereof.

[0272] In some examples, the colorant comprises a 10- / ?-hydroxyphenyl-, 10-catechyl-, 10- guaiacyl-, and / or 10-syringyl-derivatives of cyanidin-3-glucoside and / or malvi din-3 -glucoside anthocyanins.

[0273] In some examples, the colorant is edible.

[0274] In some examples, the composition is an aqueous composition.

[0275] In some examples, the composition is an aqueous composition having a pH of 1 or more (e.g., 1.5 or more, 2 or more, 2.5 or more, 2.6 or more, 2.7 or more, 2.8 or more, 2.9 or more, 3 or more, 3.1 or more, 3.2 or more, 3.3 or more, 3.4 or more, 3.5 or more, 3.6 or more, 3.7 or more, 3.8 or more, 3.9 or more, 4 or more, 4.1 or more, 4.2 or more, 4.3 or more, 4.4 or more, 4.5 or more, 5 or more, 5.5 or more, 6 or more, 6.5 or more, 7 or more, or 7.5 or more). In some examples, the composition is an aqueous composition having a pH of 8 or less (e.g., 7.5 or less, 7 or less, 6.5 or less, 6 or less, 5.5 or less, 5 or less, 4.5 or less, 4.4 or less, 4.3 or less, 4.2 or less, 4.1 or less, 4 or less, 3.9 or less, 3.8 or less, 3.7 or less, 3.6 or less, 3.5 or less, 3.4 or less, 3.3 or less, 3.2 or less, 3.2 or less, 3 or less, 2.9 or less, 2.8 or less, 2.7 or less, 2.6 or less, 2.5 or less, 2 or less, or 1.5 or less). The pH of the aqueous composition can range from any of the minimum values described above to any of the maximum values described above. For example, the composition can be an aqueous composition having a pH of from 1 to 8 (e.g., from 1 to 7, from 2 to 7, from 3 to 7, from 3 to 6, or from 1 to 3). In some examples, the composition is an aqueous composition having a pH from 1-7. In some examples, the composition is an aqueous composition having a pH from 2-7. In some examples, the composition is an aqueous composition having a pH from 3-7. In some examples, the composition is an aqueous composition having a pH from 3-6. In some examples, the composition is an aqueous composition having a pH from 1-3.

[0276] In some examples, the colorant has a vibrant and stable color.

[0277] In some examples, the colorant has a stable color.

[0278] As used herein a “stable color” means that the kmax, hue angle, and / or intensity of the colors change by 50% or less (e.g., 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less) when stored at room temperature for an amount of time of at least 2 months. In some examples, the colorant has a vibrant color that is stable for an amount of time of 2 months or more (e.g., 3 months or more, 4 months or more, 5 or more, or 6 months or more) at pH 1 to 7 and room temperature.

[0279] In some examples, the colorant has a color that is yellow to blue.

[0280] In some examples, the colorant has a color that is brown, red, orange, pink, or yellow.

[0281] In some examples, the colorant has a color that is yellow to red, orange to red, or orange to pink.

[0282] In some examples, the colorant has a color that is pink to purple.

[0283] Also disclosed herein are thermal degradation compounds derived via thermal degradation of any of the colorants disclosed herein. In some examples, the compound comprises a 4-carboxy-3-deoxyanthocyanidin. In some examples, the compound comprises 4-carboxy-3- deoxycyanidin, 4-carboxy-3 -deoxypelargonidin, 4-carboxy-3-deoxypeonidin, 4-carboxy-3- deoxymalvidin, or a combination thereof.

[0284] Also disclosed herein are thermal degradations compound derived via thermal degradation of 10-catechyl-pyranocyanidin-3-O-P-glucoside.

[0285] In some examples, the thermal degradation compound is a colorant.

[0286] In some examples, the thermal degradation compound comprises 4-carboxy-3- deoxycyanidin (i.e., 4-carboxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromenylium).

[0287] In some examples, the thermal degradation compound has a formula:

[0288] Methods of Making

[0289] Also disclosed herein are methods of making any of the compositions disclosed herein.

[0290] Also disclosed herein are methods of making any of the colorants and / or thermal degradation compounds disclosed herein.

[0291] In some examples, the hydroxyphenyl pyranoanthocyanin is made by reacting an anthocyanin and a cofactor.

[0292] The compositions described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compositions described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0293] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0294] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Katchem (Prague, Czech Republic), Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical excipients disclosed herein can be obtained from commercial sources.

[0295] Reactions to produce the compositions described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, z.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,JH or13C) infrared spectroscopy, spectrophotometry (e.g., UV- visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0296] Methods of Use

[0297] Also disclosed herein are methods of use of any of the compositions disclosed herein.

[0298] For example, disclosed herein are methods of using any of the colorants and / or thermal degradation compounds disclosed herein in a composition, beverage, food product, edible product, cosmetic product, skin care product, dye sensitized solar cell, or a combination thereof.

[0299] Also disclosed herein are composition comprising any of the colorants and / or thermal degradation compounds disclosed herein. In some examples, the composition comprises lipids, emulsions, hydrocolloids, carbohydrates, sugar solutions, protein solutions, or a combination thereof.

[0300] Also disclosed herein are beverages comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0301] Also disclosed herein are food products comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0302] Also disclosed herein are edible products comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0303] Also disclosed herein are cosmetic products comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0304] Also disclosed herein are skin care products comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0305] Also disclosed herein are dye sensitized solar cells comprising any of the colorants and / or thermal degradation compounds disclosed herein.

[0306] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0307] The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

[0308] EXAMPLES

[0309] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0310] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

[0311] Example 1 - Application of Hydroxyphenyl Pyranoanthocyanins as Colorants

[0312] Described herein are hydroxyphenyl pyranoanthocyanins as well as methods of using thereof as colorants, for example in consumer goods such as food and cosmetics.

[0313] Hydroxyphenyl pyranoanthocyanins are a class of nature derived colorants formed by the reaction of anthocyanins (pigments in fruits and vegetables) with a hydroxy cinnamic acid cofactor or their derivatives. This work focuses on using hydroxyphenyl pyranoanthocyanins as colorants. This includes evaluating their color expression, stability, and solubility in solutions with different ingredients to model common matrices in the CPG industry. This includes emulsions, hydrocolloids, sugar solutions, and protein solutions. It has been identified that when hydroxyphenyl pyranoanthocyanins are mixed with some ingredients, the color shifts and solubility is enhanced. This means that the color features of the pigments are changed depending on the environment. New color complexes and new application uses for hydroxyphenyl pyranoanthocyanins are possible from these findings.

[0314] Example 2 - Developing A Novel Palette of Nature Derived Colorants: The Color Performance of Hydroxyphenyl-Pyranoanthocyanins

[0315] Abstract. With colorants ubiquitously used in foods, cosmetics, and consumer goods, the palette of coloring solutions appears plentiful. Yet, with synthetic colorants falling out of favor and with many nature derived colorants having stability challenges, demand for color innovation remains. Pyranoanthocyanins are a promising class of nature derived colorants formed by the reaction of anthocyanins with small molecule cofactors (i.e., hydroxy cinnamic acids) and are best known for contributing to the color of an aged wine. As seen from the bold, long-lasting color of a red wine, pyranoanthocyanins can produce vibrant colors generally more orange-red in hue, and exhibit exceptional color stability to conditions like pH, storage, and bleaching, leading to interest in using them as nature derived colorants for foods and consumer goods.

[0316] A diverse array of chemically unique pyranoanthocyanins are possible, with each one providing a unique hue and stability characteristics to the colorant palette. With hundreds of potential anthocyanin to cofactor permutations, this work took a systematic approach to evaluate how the chemical structure (e.g., presence of hydroxyl, methoxy, and glycosyl substitutions) affected the coloring properties of hydroxyphenyl-pyranoanthocyanins. Properties included their color expression, solubility, and stability at both acidic and alkaline pH and in isolated and mixed environments with various ingredients (e.g., buffer salts and food ingredients). While there are many types of pyranoanthocyanins, the efforts herein focused on evaluating the class of hydroxyphenyl-PACNs which contain an additional, fifth, conjugated ring. This provides an additional location for chemical structure variability and these pyranoanthocyanins form efficiently and showed excellent thermal stability.

[0317] Small changes in the number of hydroxyl and methoxy substitutions had a major influence on the color and solubility of hydroxyphenyl-pyranoanthocyanins (Objective 1 / Chapter 3). A total of eight different pyranoanthocyanins were formed by the reaction of cyanidin-3- glucoside and malvi din-3 -glucoside anthocyanins with four types of cofactors to form 10- / ?- hydroxyphenyl, 10-catechyl, 10-guaiacyl, and 10-syringyl -pyranoanthocyanins. The color in acidic solutions ranged from yellow (hab = 60°; 10- / ?-hydroxyphenyl-pyranocyani din-3 - glucoside) to red (hab = 27°; 10-syringyl-pyranomalvidin-3-glucoside) with a greater number of oxygenated substitutions, irrespective of the location, resulting in a redder color. Molar absorptivity coefficients for hydroxyphenyl-pyranoanthocyanins were up to 2. lx (pH 1) and 7.5x (pH 3) greater than the coefficients of the precursor anthocyanins, and while anthocyanins faded completely at mild acid pH, the hydroxyphenyl-pyranoanthocyanins continued to produce color, albeit at slightly lower intensity and at a slightly shifted hue. Under alkaline conditions, the pyranos produced cooler colors including pink, purple, and blue (hab = -303 - 354°) with bathochromic shifts in the Ais-max. The location of the oxygenated substitution, whether provided by the anthocyanin (B ring) or the cofactor (E ring) influenced color expression across pH and solubility. The 10-guaiacyl and 10-catechyl -PACNs were most likely to precipitate near pH -4.6 to 6 with those derived from malvi din-3 -glucoside producing pink to purple colors in contrast to the orange colors produced by those from cyani din-3 -glucoside in this pH range.

[0318] Objective 2 (Chapter 4) continued to focus on the effect of chemical structures on coloring properties but focused on the effect of glycosylation substitutions on the color characteristics for 10-guaiacyl-pyranocyanidins, a type of hydroxyphenyl-pyranoanthocyanin. Here, six different cyanidin derived 10-guaiacyl -pyranoanthocyanins were formed and evaluated with different glycosylation attachments including three monosaccharides (arabinose, glucose, galactose), two disaccharides (xylose( l ^2)glucose and xylose( l ^2)galactose), and one trisaccharide (xylose( l ^2)glucose( l ^6)galactose). All pyranoanthocyanins, with exception of the arabinoside derivative, produced orange to yellow orange colors in acidic conditions with the glycosylation type and number having a small but significant effect on the color hue and molar absorptivity coefficient. More sugar substitutions increased room temperature with little to no precipitation observed across 60 days for 10-guaiacyl-pyranocyanidin-3-xylosyl-glucosyl- galactoside while precipitation was observed in only a few days for the pyranoanthocyanins glycosylated with a monosaccharide. Both the pH of the solution and the type of buffer used had an influence on the color expression, stability, and solubility of the 10-guaiacyl-pyranocyanidins with excellent hue retention from pH 3 - 6 (2 to 17 ° change) and absorption retention over 60 days (62 - 94%). Objective 1 and 2 demonstrated the diversity of color and solubility characteristics for hydroxyphenyl-pyranoanthocyanins and identified trends as to how the hydroxyl, methoxy, and glycosylation substitutions influenced these coloring characteristics.

[0319] Color stability of hydroxyphenyl-pyranoanthocyanins during heating was supplemented by the formation of a color-producing degradation compound. A third objective (Chapter 5) characterized the formation, color expression and stability, and elucidated the chemical structure of this unique degradation compound. Through a multifaceted approach including high resolution mass spectrometry, 1- and 2-dimensional nuclear magnetic resonance, Fourier transform infrared spectroscopy, and spectrophotometry, the compound forming from heated 10- catechyl-pyranoanthocyanins, a type of hydroxyphenyl-pyranoanthocyanin, was elucidated as a 4-carboxy-3 -deoxy cyani din. It formed in the greatest quantities and produced a yellow color (^vis-max = 479 nm, hab = 71°) in highly acidic conditions. Each of the four types of hydroxyphenyl-pyranoanthocyanin produced a unique type of 4-carboxy-3-deoxyanthocyanidin, laying the foundations for a new class of color producing flavylium compounds.

[0320] Objective 4 (Chapter 6) focused on evaluating pyranoanthocyanin color performance and solubility in model food environments including those with lipids (oil in water emulsions), carbohydrate (sugar and xanthan gum), and proteins (soy, pea, and whey isolate). Solutions of 10- / ?-hydroxyphenyl -pyranoanthocyanins or 10-guaiacyl-pyranoanthocyanins formed from aronia or black carrot anthocyanins were evaluated with each comprised of cyanidin aglycones with different number of glycosylations (1 or 2 for aronia and 2 and 3 for black carrot). While in buffers, the pyranoanthocyanins produced yellow orange and orange hues (hab = 27 - 50°), but there was a red shift in color in emulsions with the pyranoanthocyanins producing red-orange to pink colors. Similarly in pH 6 solutions, the inclusion of sugar (60° brix), xanthan gum (0.05% and 0.3%), soy protein, and pea protein resulted in redder appearance (hab decreased between 1 to 26°). While the aronia-pyranoanthocyanins were observed to precipitate in buffers (pH 4 and pH 6), solubility was improved in emulsions, 60° brix, soy protein, and whey protein solutions with greater color intensity and no (or less) visual precipitation observed after equilibration.

[0321] Hydroxyphenyl-pyranoanthocyanins produced a diverse array of colors including yellow to blue, aqueous solubility (rapid precipitation to fully soluble), and stability behaviors across pH values both in simple and applied food environments. These findings provide a framework to understand how chemical structure influences these properties which can be used to form pyranoanthocyanins with the desired coloring properties for each application. Overall, these findings highlight hydroxyphenyl-pyranoanthocyanins as a promising new palette of nature derived colorants for foods and consumer goods.

[0322] Chapter 1. Introduction

[0323] Color enriches our perception of the world. As a key part of our visual senses, color’s importance on everyday life is ubiquitously shown in the world around us. It serves as an indicator for consumption, such as the ripeness of a banana, a warning sign as in the red, black, and yellow stripes of a poisonous coral state (or is it the harmless kingsnake), and as a sign of action with green signaling go. Our ability to immediately connect the color of an object to information on that object’s history, flavor, and safety is powerful. For this reason, coloring agents are often added to our foods, cosmetics, pharmaceuticals, clothes, cleaners, and printed materials to elicit these connections, provide a product’s identity, and produce the desired sensory experiences.

[0324] Because of these strong connections, colorants are often added to foods. Food colorants are often associated with less nutritious, highly processed foods and rightly so. With bright colors spanning every shade of the rainbow in our confectionary, beverage, and snack aisles, colorants clearly play a key role in creating an enjoyable eating experience of these products. However, colorants also fulfill a more noble position in the food industry. By taking advantage of the connections between color, flavor, and perception, colorants can be a key part of creating a more nutritious food supply. For example, color and color intensity can relate to sweetness perception, and while overall results on this relationship are mixed, color could be key in improving consumer acceptance of low sugar products (Spence et al., 2010). Additionally, food colors are highly indicative of flavor identification, with consumers reaching a decision before tasting or smelling (Spence et al., 2010). Therefore, color could be used to enhance a natural flavor and decrease cost or to encourage consumption of a beverage full of fruits and vegetables. Additionally, many nature derived colorants are bioactive compounds, putatively associated to positive health outcomes (Sharma et al., 2021). While often added in only minor quantities, coloring agents have a major effect on a product success and acceptance and will remain an important ingredient in the food supply. Pyranoanthocyanins (PACNs) are emerging as promising nature derived coloring options for foods and other consumer products. Formed from anthocyanins, colorants which are naturally abundant but with limited stability, PACNs have bold colors which are exceptionally stable to stressors like heat, pH, and bleaching agents (Farr & Giusti, 2018; Jingren He, Carvalho, et al., 2010; Sun et al., 2020; Voss, Miyagusuku-Cruzado, et al., 2023). PACNs are well known in enology as they form during wine fermentation and aging when grape anthocyanins react with small molecule fermentation metabolites or naturally present acids including pyruvic acid, acetaldehyde, acetoacetic acid, and hydroxy cinnamic acids (V. de Freitas & Mateus, 2011; Schwarz, Wabnitz, et al., 2003). PACN accumulation in wine contributes to the bold, long-lasting, tawny color of an aged wine (V. de Freitas & Mateus, 2011; Quaglieri et al., 2017). Recently, more efficient methods to form PACNs have been developed allowing for the synthesis of appreciable yields in as little as a few days (Miyagusuku-Cruzado et al., 2023; Xun, 2023). These successes help to address PACN natural scarcity and their slow formation in wine, opening the door for continued research on PACNs.

[0325] PACNs are a diverse class of nature derived colorants defined by the presence of a 4thconjugated ring (D ring) attached on the flavylium structure backbone. Structural variations are imparted by the choice of anthocyanin and cofactor, with hundreds of unique chemical structures possible. Within PACNs, the subclass of hydroxyphenyl -PACNs, formed by hydroxy cinnamic acids or 4-vinylphenol cofactors, are especially promising due to their high heat stability and the development of efficient methods for their formation in a few days (Miyagusuku-Cruzado et al., 2023; Miyagusuku-Cruzado, Voss, et al., 2021a; Voss et al., 2022). Hydroxyphenyl -PACNs contain a 5thconjugated ring, termed the E ring, attached at CIO position which varies in substitutional patterns depending on the hydroxycinnamic acid cofactor used. By changing the anthocyanin and cofactor structure, a diverse array of hydroxyphenyl-PACNs are possible, each producing a unique set of colors and stability behaviors.

[0326] A goal of this work is to evaluate the relationship between hydroxyphenyl-PACN chemical structure and their functional properties as colorants. This includes evaluation of their color expression, solubility, stability, degradation, and interaction with food macromolecules. This work provides important understanding on their behavior from both a molecular and applied perspective and fits into the overarching goal of this research area to develop PACNs as nature derived colorants. This work is a part of an initiative to further the understanding of PACN formation and coloring capabilities to aid their development as a potential coloring solution.

[0327] Objectives 1 and 2 evaluate isolated PACNs to understand the direct link between chemical structure and color properties including their color expression, molar absorptivity coefficients, color stability to pH changes, and solubility. In objective 1, the effect of B and E ring hydrogen, hydroxyl, and methoxy substitutions is evaluated for 8 different hydroxyphenyl- PACNs and compared to the performance of their precursor anthocyanins (cyani din-3 -glucoside and malvi din-3 -glucoside). The different chemical structures are imparted by the chosen anthocyanin aglycone and hydroxy cinnamic acid used for formation, and it is hypothesized that an increasing number of hydroxyl and methoxy substitutions can result in greater room temperature stability and a redder color (defined by a bathochromic shift in Xvis-max and lower hue angle) as observed with anthocyanins (Cabrita et al., 2000). Notably, 10-guaiacyl-PACNs were observed to precipitate readily (under some conditions), therefore, objective 2 evaluates the impact of glycosylation substitution on the color properties (color expression, molar absorptivity coefficient, color stability to pH changes, and solubility) of 10-guaiacyl-pyranocyanidins. A total of 6 different 10-guaiacyl-pyranocyanidins were formed which differed in the number and type of glycosylations, and it is hypothesized that these glycosylations will have a minimal effect on color produced but that increasing the number of sugar substitution will result in greater room temperature stability and solubility.

[0328] PACNs are widely considered to be more stable than their precursor anthocyanins. This is exemplified under heat, where previous work showed 10-catechyl-PACN color to be 8.6x more stable than the precursor anthocyanins (Voss et al., 2022). Yet, there was a disparity between color stability and thermal stability as the 10-catechyl-PACNs degraded into a new, color producing degradation compound (Voss et al., 2022). In objective 3, a goal is to further investigate the formation, color properties, and the chemical structure of the unique, colorproducing hydroxyphenyl-PACN thermal degradation compounds. It is hypothesized that all hydroxyphenyl -PACNs will degrade into a color producing compound comprised of a portion of the A, D, and E rings. Identifying the degradation compounds is important for the understanding of the mechanism of PACN thermal degradation and has important implications on their applications as this compound(s) may form from processing or storage stressors.

[0329] In objective 4, a goal was to further investigate the application of hydroxyphenyl -PACNs by investigating their interaction with food macromolecules including lipids and carbohydrates. Based on the knowledge gained from objectives 1 and 2 on structure-function relationship to color properties, 4 different PACN solutions, representing a range of properties in terms of solubility (excellent to poor) and color (yellow to orange), were evaluated by measuring the change in color, stability, and solubility when mixed with various food ingredients. It is hypothesized that the PACNs prone to precipitation in aqueous solutions will have greater solubility in the lipophilic environments and that carbohydrates will not have a large effect on the color and stability of PACNs.

[0330] This work provides a foundational understanding of how the choice of anthocyanin and cofactor affect the properties of PACNs. These patterns can provide guidance for industry and researchers when looking to develop a colorant with specific characteristics for each application. This increased understanding of PACNs is key in helping the food industry transition towards stable, nature derived colorants for foods.

[0331] Chapter 2. Literature Review

[0332] To achieve the long-term goal of developing pyranoanthocyanins (PACNs) as colorants for food and consumer goods, an understanding of the concepts behind color perception, colorant regulation, and PACNs is important. This review provides these necessary pieces of background focusing on how color is produced and perceived by humans and equipment, food color regulation and common classification in the United States, and a comprehensive review on PACNs including their formation mechanism, accelerated methods for formation, color expression, color stability, and applications. Throughout, there is a focus on structure-function relationships to provide the fundamental understanding of how chemical structure relates to the functional properties of a colorant.

[0333] 2.1. The Sensation of Color: Creation and Perception. What color shirt are you wearing? A simple question on the surface, yet deeper evaluation of the possible answers exposes the complexity of color. If you entered a dark room, would the shirt have the same color? What if that room was lit with blacklights? If you were color blind, would you give the same response? The reported “color” of a shirt changes even if no additional dyes are added. Therefore, color is not a finite property of an object. Instead, it is variable, dependent upon three factors: the light source, the observer, and the object itself (Konica Minolta Sensing Inc., 2007).

[0334] 2.1.1. The Three Factors for Color: Light source, object, observer.

[0335] Light source. For an object to appeared colored, it must be illuminated by light with wavelengths in the visible region (-380-770 nm) of the electromagnetic spectrum as these can be perceived and processed into color by humans (Wrolstad & Smith, 2017). Whether it is sunlight, an incandescent light bulb, or the red LED lights strung up during December, each source of light is comprised of a unique combination of wavelengths within the visible spectrum. How these wavelengths are absorbed and reflected by the object of interest define what color is observed. For example, if an illuminant provides more yellow light, an object may appear as yellower. Human brains have innate processes to help account for differences in illuminants, called color consistency (Carroll & Conway, 2021; Wrolstad & Smith, 2017). Here, the brain automatically pulls information from life experiences to account for the illuminant (Carroll & Conway, 2021). As each person’s paradigm is unique, this phenomenon can help to explain how the same object appears as strikingly different colors to individuals. For scientific instruments (i.e., colorimeter, spectrophotometer), the illuminant must be defined and set for accurate color comparisons between measurements.

[0336] Observer. The observer decodes the reflected wavelengths of visible light into color. The observer can be a human eye or a detector in an instrument. In humans, wavelengths of visible light enter the eye through the cornea and pupil, become focused, and hit the retina at the back of the eye (Boyd & Tubert, 2023). The retina contains photoreceptor cells, called rods and cones, which help to process vision (Carroll & Conway, 2021). Cone cells are responsible for color vision, and they can be classified as long, medium, or short depending on the region of visible light which they absorb (Carroll & Conway, 2021). Once the cones are activated by visible light, a cascade of intercellular signals travels by the optical nerve to multiple regions of the brain for processing (Carroll & Conway, 2021). It is the combination of signals from cone receptors that when processed by the brain creates a color (Carroll & Conway, 2021). Understanding how the nervous system processes wavelengths of light into color remains an enigma. Based on recent findings, the theory of Utility -Based Coding is being proposed to replace the previously held Opponent-Colors Theory (Conway et al., 2023). Regardless of the underlying mechanism, there are variations in color perception among humans with normal color vision (Bosten, 2022; Emery & Webster, 2019).

[0337] When instruments detect reflected or transmitted wavelengths of visible light, they use mathematical calculations to convert them into color coordinates. These are based on the standard observer function and illuminant light distribution, and the color will be represented by X, Y, and Z coordinates (Konica Minolta Sensing Inc., 2007; D. Wang et al., 2023). These coordinates can be translated into various color spaces for interpretation (Konica Minolta Sensing Inc., 2007). The standard observer functions account for the human element of color perception (i.e., the processes mentioned in the preceding paragraph) and are experimentally determined based on humans with normal color vision (Bosten, 2022; HunterLAB, 2015). They are produced and continuously revised by the Commission Internationale de 1’Eclairage (CIE), international committee for light and lighting, and most recently, the CIE 2015 Cone- Fundamentals-Based Color-Matching Functions were developed to better mimic human perception and account for LED lighting (Luger Research Group e.U., 2017). Other commonly used functions include the 1931 2° standard observer and the 1964 10° standard observer functions (Luger Research Group e.U., 2017). Object. The object being viewed reflects, absorbs, and transmits the wavelengths of light to produce the color. Often, this is due to the presence of coloring agents, compounds containing a chromophore in their chemical structure (Giirses et al., 2016; Loudon & Parise, 2016). In the dark, the electrons of a chromophore are in their lowest energy state within the bonding molecular orbitals; however, light energy excites electrons to the higher-energy antibonding orbitals (Loudon & Parise, 2016). The specific amount of visible light (energy) needed for this excitation is dictated by the energy difference in the highest occupied bonding molecular orbital (HOMO) and lowest unoccupied antibonding molecular orbital (LUMO) (Loudon & Parise, 2016). The wavelength of light matching the H0M0-LUM0 energy gap is absorbed, and the remaining wavelengths are transmitted or reflected by the object to reach the observer (Loudon & Parise, 2016). If the energy gap matches a region within the visible spectra, the absorption of certain wavelengths and transmission of others results in the formation of a color. The specific wavelengths absorbed are affected by the chemical structure of the compound, and therefore by adjusting a chemical structure, a compound can be fine-tuned to absorb a specific wavelength of light (Loudon & Parise, 2016). More 7t-orbital conjugation results in a smaller H0M0-LUM0 gap, meaning the more conjugated compounds absorb light with lower energy levels (higher wavelengths, more red) (Loudon & Parise, 2016). The addition of chemical substitutions, called auxochromes, can affect the level of conjugation and electron density to further influence the final color (Giirses et al., 2016).

[0338] Color is not only produced via chemical means from chromophores. It can also be affected and produced through physical means. Although the same chromophore may be present, an object with a smooth surface will appear as a different color to one with a rough surface due to the way light bounces and the proportion of diffused versus reflected light (Konica Minolta Sensing Inc., 2007). In some situations, light reflection can result in the formation of a color even when no chromophores are present. Termed structural color, the diffraction, reflection, refraction, and scattering of light off of some surfaces can result in the formation of a color (Kinoshita et al., 2008). Structural color produces the, sometimes iridescent, colors of some butterfly wings, insects, and bird feathers, and the blue color of waxed plumbs and blueberries (Kinoshita et al., 2008; Middleton et al., 2024).

[0339] 2.1.2. Definition of Color. The combination of these three components (light source, observer, object) creates color as we (humans) see it. In this review, when referring to color, we reference the different hues and shades based upon the wavelengths of light from the visible spectra, defined in it’s infancy by Isaac Newton as red, orange, yellow, green, blue, indigo, and violet (Newton, 1672). A color hue can be produced both by a single wavelength of light (i.e., violet light) or by the combination of multiple wavelengths (red and blue wavelengths combine to create violet) (Caivano, 2022; Newton, 1672). From a wavelength perspective, white is formed by mixing all visible wavelengths while black is formed by the absence of all color wavelengths (Caivano, 2022). In reflectance terminology, a white object would reflect all wavelengths (absorb none) while a black object would absorb all wavelengths of visible light (absorb all) (Caivano, 2022). The infinite number of color possibilities, evident from the endless number of paint swatches available, are created from combinations of different hues and by adjusting lightness and saturation (Caivano, 2022; Konica Minolta Sensing Inc., 2007). Other factors such as texture, transparency, and size of the object impact how the color is perceived (Konica Minolta Sensing Inc., 2007). However, their impact is on the overall appearance and not on the color itself.

[0340] 2.2. Colorants for Foods. The light source, observer, and object all play an important role in the creation of the color of an object. However, from the perspective of industry, the object is the easiest to control. Colorants are used around the world in products such as food, paints, cosmetics, textiles, and printing. While the desirable properties will vary depending on the product (i.e., gloss, solubility, resilience), the chemistry on how color is produced remains the same. The connection between appearance and preconceived thoughts about a product is strongly observed in foods. We are trained to connect the bright red color of an uncooked steak to its freshness, with brown color being undesirable. Similarly, the unblemished, white flesh of an apple indicates it was recently cut, while brown often signifies it has sat for a while. There is physiological rationale behind these connections as colorants serve a dual purpose in our foods, first providing functional benefits to the living source and second serving the consumer upon consumption. In meat, red pigmentation is produced by heme, a key part of myoglobin (which is part of hemoglobin), responsible for carrying oxygen through the blood (Schwartz et al., 2017). After slaughter, the iron ion in heme can become oxidized to form metmyoglobin, producing the undesirable brown color of meat (Schwartz et al., 2017). In fruits and vegetables, the brown coloration formed when the produce is cut is due to the enzymatic activity of polyphenol oxidase reacting on phenolic compounds to form melanin pigments (Watkins, 2017). Our learned behavior and the subconscious connection between food color and quality and flavor serves as the frontline defense before consuming a product (Burrows, 2009). For this reason, colorants are often added to foods.

[0341] In the United States, colorants for food are regulated by the Food and Drug Administration, part of the Department of Health and Human Services. Colorants can be added to enhance the naturally present color, to standardize products, to mimic the naturally present color in a processed alternative, and for new product development (Food and Drug Administration, 2023). Colorants are under different legislation than many other food ingredients with regulation dating back to the 1906 Pure Food and Drug Act (Food and Drug Administration, 2023). The most recent legislation for colorants was the 1960 Color Additives Amendment which required all colorants to be re-evaluated for safety (Food and Drug Administration, 2023). However, the Code of Federal Regulations is continually updated regarding colorants including the removal and addition of approved colorants. There are two classifications for colorants in the Code of Federal Regulations: certified colorants and colorants exempt from certification.

[0342] 2.2.1. Certified Colorants. Certified colors require each manufactured batch be tested and approved by the Food and Drug Administration prior to commercial use (Food and Drug Administration, 2023). A colorant will be classified as requiring certification if there is a high risk of toxicological contaminants, such as intermediary compounds and heavy metals (Food and Drug Administration, 2023). There are 9 approved certified colorants for food, two of which are approved only for select applications, listed in 21 CFR part 74 (Food and Drug Administration, 2024b). Examples include Allura red (FD&C Red 40) and Tartrazine (FD&C Yellow 5) with the FD&C number being granted once the colorant is certified. Because these colorants are synthesized in manufacturing facilities and not found in nature, they are commonly referred to as synthetic and artificial colorants. Originally, the starting material for these human-made colorants was obtained from coal-tar, and despite different materials being used today, they are still referred to by the derogatory term coal-tar dyes (Burrows, 2009; Food and Drug Administration, 2023). With historical precedent for unregulated, dangerous colorants as well as ongoing concerns on their toxicity and detrimental effect towards child hyperactivity and allergies, the FD&C certified colorants are under continual scrutiny (Amchova et al., 2015; Burrows, 2009; Feketea & Tsabouri, 2017; Kobylewski & Jacobson, 2012). In fall 2023, Erythrosine (FD&C Red 3) was banned for use in California (Assembly Bill No. 418 The California Food Safety Act, 2023). Spillover implications are likely to follow for the rest of the United States.

[0343] The certified colorants all produce a specific color (i.e., absorb specific wavelengths of light) and have high tinctorial strength and stability (Sigurdson et al., 2017). For the 9 approved in the US, the structures are classified as azo, xanthene, triphenylmethane, and indigoid, with different auxochromes added to create a specific hue (Food and Drug Administration, 2023) (Figure 1). Even a single substitution can dramatically change the color as seen for the triphenylmethanes; Fast Green FCF and Brilliant Blue differ only by a single hydroxyl substitution. 2.2.2. Colorants Exempt from Certification. The colorants listed as “exempt from certification” are deemed to have lower toxicological risks than the certified colorants, and therefore have fewer regulations and requirements (Food and Drug Administration, 2023). Exempt from certification colorants are a diverse group of compounds in terms of their source, chemical structures, and their properties. Often, these colorants provide additional challenges in applications due to lower vibrancy, color stability, and addition of off-flavors (Sigurdson et al., 2017). Thus, there are a greater number of exempt from certification colorants as each works in only a narrow scope of products. As of June 2024, there were 41 different colorants approved for use in human or animal foods listed in CFR 21 part 73. (Food and Drug Administration, 2024a). These colorants are oftentimes referred to as natural or naturally derived colorants as most can be observed in foods already part of the human diet or found in products observed in the natural environment. Examples include saffron and fruit juice sourced from plants, cochineal extract sourced from insects, and calcium carbonate sourced from minerals (Food and Drug Administration, 2023). Not all are found directly in nature, however. Two of the more recent approved colorants include Jagua blue (approved November 2023), resulting from the reaction between glycine and genipin, and soy leghem oglobin (approved August 2019), a plant-based protein but sourced from genetically modified yeast (Food and Drug Administration, 2024a). Additionally, nature-identical colorants have been approved such as P-carotene where the colorant is synthesized in a facility yet has the same chemical structure to the plant derived counterpart (Food and Drug Administration, 2024a). There is a great amount of ingenuity in developing the exempt from certification colorants in order to provide the desired color performance for foods.

[0344] Some of the main classes of nature derived colorants based on chemical structure are flavonoids, isoprenoids, n-heterocycles, anthraquinones, and pyrroles (Figure 2) (Sigurdson et al., 2017). Each contain the extended conjugation to absorb wavelengths within the visible light region. Of these, the flavonoids are the focus moving forward.

[0345] 2.3. Flavonoids: Classification and Coloring Capabilities. Flavonoids define a broad class of secondary plant metabolites structurally comprised of a C6-C3-C6 backbone (Shen et al., 2022). Classification schemes vary, however, common subclasses include flavonols, flavones, chaicones, flavanones, anthocyanins, and isoflavones, which differ in their chemical substitutions, conformation, and extents of conjugation (Harborne, 1998; Shen et al., 2022). Not all flavonoids produce color, however. Some, like flavonols, flavones, and chaicones are yellow, meaning they absorb the lowest wavelength of light and have the lowest possible H0M0- LUMO energy gap (to still produce color) (Harborne, 1998). However, anthocyanins are the primary class of colored flavonoids, producing a range of colors across the visible light spectra.

[0346] 2.4. Anthocyanins: Chemical Structure and Colorant Characteristics. Anthocyanins produce colors include orange, red, purple, and blue depending on the specific chemical structure and external environment (Sigurdson et al., 2017). They are found in a myriad of different fruits and vegetables like berries, plums, grapes, eggplant, cabbage, onions, black beans and flowers including chrysanthemums, iris, butterfly pea flower, and clematis (Iwashina, 2015; X. Wu et al., 2006). Despite their natural abundance, anthocyanins are only approved as food colorants in the US under limited entries: fruit juice, vegetable juice, grape color extract, and grape skin extract (Food and Drug Administration, 2024a).

[0347] Anthocyanins are chemically defined by a 2-phenylbenzopyrylium backbone which is built upon the C6-C3-C6 flavonoid backbone (Figure 3). Because anthocyanins have conjugation across the entire molecule (or the potential for such), they absorb higher energy wavelengths of light (into the visible spectra) than other flavonoids, thus creating their color (Trouillas et al., 2016). Anthocyanins themselves represent a diverse class of chemical compounds with over 700 types of anthocyanins being reported (Andersen & Jordheim, 2010). Structural diversity is engendered by the type and location of different substitutions including (but not limited to) B ring substitution patterns, glycosylations, and acylation groups attached to the glycosylation (Andersen & Jordheim, 2010).

[0348] 2.4.1. The Relationship of Anthocyanin Chemical Structure on Color.

[0349] These different chemical substitutions affect the color produced by anthocyanins leading to a rainbow of different hues. With extensive research on anthocyanins, many different aspects and types of structural substitutions have been investigated. Here we provide only a brief overview on the influence of anthocyanin chemical structure on the color at a constant, acidic pH value.

[0350] B Ring Substitutions (Anthocyanin Aglycone). The B ring substitutions define the anthocyanin aglycone, with the most common substitutions being hydroxyl (OH) and methoxy (OCH3) groups. Nearly 90% of all anthocyanins are defined by one of 6 combinations of B ring substitutions (Figure 3) (Andersen & Jordheim, 2010). Increasing the number of B ring substitutions, both for OH and OCH3, groups resulted in a bathochromic shift in kvis-max (Cabrita et al., 2000; Torskangerpoll et al., 1999). Pelargonidin, with one hydroxyl group at 4’ position, produced the lowest Uis-max (reported at 498 nm at pH 1) while malvidin, with three oxygenated substitutions, produced the highest Uis-max (517 nm) (Cabrita et al., 2000). In comparing anthocyanins with equivalent number of groups (3), methoxy groups appeared to give a higher kvis-max than OH groups as malvidin produced higher ^vis-max than delphinidin and petunidin (Cabrita et al., 2000).

[0351] Glycosylation. Anthocyanins without sugar attachments are referred to as an anthocyanidin. However glycosylated versions are more common in nature with sugar attachment occurring at C3 (most common), C5, C7, and in the B ring (Andersen & Jordheim, 2010). In comparing an anthocyanin to their respective anthocyanidin, the presence of a sugar resulted in a slightly lower Xvis-max (yellow shift in color appearance) (Borkowski et al., 2005; Sigurdson et al., 2018; C.-L. Zhao et al., 2014).

[0352] For anthocyanins, the number, type, and orientation of glycosylations is reported to have a slight effect on color. When comparing carbon 3 glycosylated anthocyanins, those with two or three sugar substitutions had slightly higher Ais-max than the monosaccharides (-0 to 4 nm higher) (Farr et al., 2019; Sigurdson et al., 2018). When a cyanidin anthocyanin was glycosylated at carbon 5 and carbon 3 contrasted to only carbon 3, the anthocyanins produced a pinker color (hab decreased ~2 to 13 nm) however this observation coincided with a hypsochromic shift in Zvis-max (~1 to 2 nm) (Sigurdson et al., 2018). For pelargonidin anthocyanins, 3,5-glycosylation resulted in a higher hue angle (-4 units) (Giusti et al., 1999). With disaccharides, a 1— >2 linkage resulted in a slightly higher Zvis-max (3 nm) compared to a 1— >6 linkage (Farr et al., 2019). These noted effects of glycosylation on the color of an anthocyanin in an acidic environment are small, and several exceptions to these trends are also observed. Further discussion on the effect of glycosylation can be found in a review by C.-L. Zhao et al. (2014).

[0353] Acylation. Acylating groups can be attached to the glycosylation and include aliphatic acids such as malonic acid, acetic acid, and succinic acid and aromatic acids such as the hydroxy cinnamic acids and hydroxybenzoic acids (Andersen & Jordheim, 2010). Aromatic acylating groups decreased hue angle by -18 ° and increased Gis-max (-6-11 nm) (Dangles et al., 1993; Fenger et al., 2021; Giusti et al., 1999) with more acylating groups resulting in greater bathochromic shifts (-11-20 nm). The effect of aromatic acylating groups on anthocyanin color may be due to intramolecular copigmentation as the acylating groups may fold and interact with the chromophore (Dangles et al., 1993; Giusti et al., 1998).

[0354] 2.4.2. Anthocyanin Health Benefits.

[0355] In addition to providing colors, anthocyanins are putatively associated with positive health benefits. Described further in reviews by He and Giusti (2010), Wallace and Giusti (2010), and Goncalves et al. (2021), anthocyanins are associated with many positive health benefits including prevention and inhibition of cardiovascular disease, cancer, inflammation, obesity, and vison loss. Many of these bioactive effects are attributed to anthocyanins antioxidant capabilities, where anthocyanins directly quench reactive free radicals, chelate metal ions, activate antioxidant enzymes, and influence the redox signaling pathway (X. Wu, 2010). The chemical structure can and does influence the biological activity and bioavailability of anthocyanins, and therefore not all extracts or conditions (i.e., pH) will exert the same effect (Goncalves et al., 2021; Wallace & Giusti, 2010).

[0356] 2.5. Anthocyanin Color Stability. Anthocyanin chemical structures are highly susceptible to change. This includes pH driven equilibrium reactions, conformational changes, degradation, and reaction with other compounds. Because the color and functional properties of an anthocyanin are intertwined with their chemical structure, these changes also have implications on their functional properties.

[0357] 2.5.1. Anthocyanin Color Stability to pH. Anthocyanin chemical structure, and thus functional properties like color, are heavily dependent on the pH of the solution. The pH dependent structure-function relationship of anthocyanins was brilliantly deduced in a series of studies published by Brouillard and others in the late 1970’s (Figure 4) (Raymond Brouillard & Delaporte, 1977; Raymond Brouillard & Dubois, 1977b). As these authors discovered, in acidic conditions, anthocyanins are in their flavylium cation form (AH+), often associated with red hues (Dangles & Fenger, 2018; Mazza & Brouillard, 1987b; Trouillas et al., 2016). As the pH increases, two concurrent reactions occur — the flavylium cation deprotonates to form the quinoidal base (A), and the flavylium cation is hydrated at C2 to form a hemiketal (B) (Figure 4). Deprotonation was reported to be a kinetic controlled, exothermic reaction, and subsequent deprotonation steps occur to form anionic quinoidal base species (A’) as the pH increases. The quinoidal base and anionic quinoidal base have bathochromic Lis-max compared to the flavylium cation often appearing as a purple or blue (Dangles & Fenger, 2018; Mazza & Brouillard, 1987b; Pina, 2014; Pina et al., 2012). Hydration is reported to be thermodynamically controlled, endothermic reaction, and is often referred to as the “slow reaction” with slow defined as 30 seconds to ~17 minutes (Raymond Brouillard & Delaporte, 1977; Raymond Brouillard & Dubois, 1977b). Once hydrated, the hemiketal can quickly (in milliseconds) tautomerize into the cv.s-chalcone (Ccis) (Raymond Brouillard & Delaporte, 1977; Pina, 2014). Full equilibration is reached when the cv.s-chalcone equilibrates with the / ra / z.s-chalcone (Ctrans) which may take several days (Raymond Brouillard & Lang, 1990; Pina, 2014; Pina et al., 2012). Because hydration breaks the chromophore, the hemiketal and chaicone are typically colorless, though a pale yellow chaicone has been reported by some (Dangles & Fenger, 2018; Mazza & Brouillard, 1987b; Trouillas et al., 2016).

[0358] The anthocyanin deprotonation and hydration pathways occur concurrently with pH adjustments. These are not instantaneous, step wise changes, but they occur gradually across the pH gradient. With innumerable potential combinations and proportions of each structural form, many color hues and intensities are possible for each anthocyanin. Generally, the pKa for deprotonation (AH+— >A) at C7-0H is ~ 4; the pKa for hydration (AH+— >B) is generally ~2-3 (Dangles & Fenger, 2018). Thus, in mild acid conditions, hydrated forms are more prominent, resulting in pale and colorless solutions (Mazza & Brouillard, 1987b). At neutral and alkaline pH values, the quinoidal base species are more prominent, leading to purple and blue colored solutions (Dangles & Fenger, 2018). The pKa for these reactions and the stability of each form (AH+, A, B, C) is affected by the chemical structure, resulting in different pH dependent behaviors between anthocyanins (Pina et al., 2012).

[0359] 2.5.2. Anthocyanin Degradation. While the pH structural and color changes are, in theory, reversible, stressors like heat, light, and oxygen exposure (oxidative degradation) lead to irreversible anthocyanin degradation. While the mechanistic pathway can be slightly different, each lead to the breakdown of the anthocyanin chromophore and the formation of colorless degradation compounds (Furtado et al., 1993; Lopes et al., 2007; Sadilova et al., 2006). The different pH dependent structural forms each impart a different level of immunity to these stressors. Anthocyanins in highly acidic environments (when the flavylium cation is prominent) are generally considered the most stable while irreversible degradation is faster at higher pH values (when chaicone and quinoidal base predominate) (Dangles & Fenger, 2018; Sui et al., 2014). In making this conclusion, however, it is important to distinguish color stability from colorant stability. As color and pH are intertwined, not accounting for the pH effect on color may lead to an underestimations of stability at mild acid and alkaline pH values.

[0360] 2.5.3. Anthocyanin Reactivity. Anthocyanins are highly reactive compounds serving as both electrophiles and nucleophiles (Andersen & Jordheim, 2010; Dangles & Fenger, 2018; J. Oliveira et al., 2017). The positive charge of the flavylium cation is delocalized throughout the molecule despite commonly being drawn at the C ring oxygen (Figure 5). Therefore, other positions, most importantly C2 and C4, also share in the partially positive (electrophilic) characteristics (Dangles & Fenger, 2018). While C2 is the site for hydration, considered a hard electrophile, C4 is commonly reactive in the formation of anthocyanin-adducts (Dangles & Fenger, 2018).

[0361] As discussed in J. Oliveira et al (2017), wine provides a unique example of the reactivity of anthocyanins at C4 as multiple adducts can form. Bisulfite is commonly added as a preservative and antioxidant in wine, however it attaches at C4 to form colorless (but reversible) adducts (Berke et al., 1998; Moroney et al., 2022). Additionally, colored anthocyanin-flavanol adducts can be formed from anthocyanin reaction at C4, C6, or C8 with colorless flavanols such as epicatechin, catechin, and procyanidin dimers (Duenas et al., 2006; Francia-Aricha et al., 1997; Pissarra et al., 2004; Remy et al., 2000; Salas et al., 2003, 2004). A third type of anthocyanin adducts found in wine are the class of colored compounds called pyranoanthocyanins (PACNs). PACNs are formed by the reaction of a cofactor, such as pyruvic acid or a hydroxycinnamic acid, at the C4 and C5-OH position to form an additional pyran ring (Figure 6). PACNs strongly contribute to the color of an aged wine (Quaglieri et al., 2017; X. K. Zhang et al., 2021), and because of their long-lasting, bold color, there is strong interest to further investigate PACNs as colorants.

[0362] 2.6. Pyranoanthocyanins: Background and Sources. PACNs themselves represent a highly diverse class of anthocyanin derived colorants. The history of PACNs is short in comparison to anthocyanins, with the first structure elucidation of PACNs believed to be in 1996 by Fulcrand et al. Several pyranoflavylium compounds were identified prior, including Dracorubin from dragon’s blood resin (Cruz et al., 2022; Robertson et al., 1950). However, these compounds are distinctly classified as a pyranoflavylium, not a PACN, as the backbone is not the 2-phenylbenzopyrilium structure. From the initial structure elucidation of a PACN in the mid 1990’s, research accelerated. New types were identified, formation methods became more efficient, and studies began focusing on their chemical properties and coloring behaviors both in wine and various applications. While only -26+ years old, the world of PACN research is exciting and growing, paving the way for a new palette of nature derived colorants.

[0363] 2.6.1. Pyranoanthocyanins in Wine. PACNs are commonly viewed as enological colorants and rightly so. PACNs play an important role in wine color by contributing to the tawny color of an aged wine (X. K. Zhang et al., 2021), they were first identified and elucidated from wine (Fulcrand et al., 1996), and wine is one of their most common natural sources (Rentzsch, Schwarz, & Winterhalter, 2007). As poetically described in Brouillard et al. (2003), red wine has a remarkably stable color which continues to improve with age, a rarity among beverages.

[0364] In vinification, the harvested grapes are crushed to begin maceration in which native enzymes break down the pomace to release compounds like anthocyanins and other polyphenolics (Jackson, 2008). Maceration of a red wine can occur on the time scale of a few days to multiple weeks, during which fermentation by endogenous yeasts may occur (Jackson, 2008). Controlled fermentation occurs after maceration and pressing, and it is here in which wine is inoculated by the desired yeast strains (Jackson, 2008). Yeasts (often Saccharomyces cerevisiae) primarily undergo alcoholic fermentation converting glucose and fructose into ethanol. Following yeast fermentation, lactic acid bacteria may be added to undergo malolactic fermentation converting malic acid into lactic acid (Jackson, 2008). Maturation is the next step in wine formation which may also coincide with some fermentation and can last for ~6 months to years; here, various treatments can be applied including micro-oxidation and barrel aging (Jackson, 2008). The final step is bottling, where the bottled wine may continue to age before consumption (Jackson, 2008).

[0365] Wine formation was described in detail as PACNs can form during various steps in the process and the effects of different processing conditions (like micro-oxidation) and fermenting microorganisms have been specifically studied regarding their effect on PACNs (Figueiredo- Gonzalez et al., 2014; Llaudy et al., 2006; Quaglieri et al., 2017; S. Wang et al., 2018). The starting anthocyanins for PACN formation in wine come from grapes. The most abundant anthocyanins in Vitis vinifera, red grapes used in wine production, is typically malvidin-3- glucoside, with acetyl and coumaroyl acylation prevalent (Garcia-Beneytez et al., 2003; Mattivi et al., 2006). Thus, many of the first identified PACNs are malvidin-derived, leading to specific names for the malvi din-3 -glucoside derivatives as listed in Table 1. The cofactors can be formed as metabolic intermediaries in the fermentation steps both by yeasts and the lactic acid bacteria. Key metabolites produced by yeast in alcoholic fermentation include pyruvic acid, acetaldehyde, acetoacetic acid; lactic acid bacteria can produce pyruvic acid in malolactic fermentation (V. de Freitas & Mateus, 2011; Jackson, 2008; S. Wang et al., 2018). Additionally, cofactors can also be naturally present or formed via chemical transformation of the naturally present phenolics (i.e., hydrolysis of (caf)taric acids and acetaldehyde intervention in formation of vinyl -flavonols) and include compounds like hydroxycinnamic acids and flavanols (Es-Safi et al., 1999; Mateus et al., 2002; Rentzsch, Schwarz, Winterhalter, et al., 2007). Some cofactors result from the microbiological transformation of naturally present compounds such as when yeast and bacteria decarboxylate the hydroxycinnamic acids to form 4-vinylphenols (Bozic et al., 2020; Cavin et al., 1993; Jackson, 2008).

[0366] Over the course of the fermentation, maturation, and bottle aging processes, the cofactors react with the anthocyanins to form PACNs. In general (exceptions exist), the formation of 10- carboxy and 10-hydrogen-PACNs occurs earlier in the wine formation process (alcoholic fermentation) and with their concentration shown to decrease during maturation and aging ((Mateus & de Freitas, 2001b; Monagas et al., 2005; Rentzsch, Schwarz, Winterhalter, et al., 2007; X. K. Zhang et al., 2021). Hydroxyphenyl-PACNs tend to be more prevalent in older wines as they form from reaction with hydroxy cinnamic acids during maturation (Monagas et al., 2005; Rentzsch, Schwarz, Winterhalter, et al., 2007; X. K. Zhang et al., 2021). Several more detailed reviews on PACNs in wine are found in Marquez et al. (2013), de Freitas and Mateus (2011), Zhang et al. (2022), and Quaglieri et al. (2017).

[0367] 2.6.2. Pyranoanthocyanins Beyond Wine. PACNs are found in other natural products in addition to wine, albeit often at low concentrations. As referenced in Table 1, other edible sources of PACNs include strawberries, figs, and red onions. Additionally, as with wine, PACNs can form in foods over time due to complexation between naturally present or microbially derived cofactors. Examples of the former include 3-month stored black carrot juice and 1- month stored blood orange juice where PACNs formed by reaction with the naturally present hydroxy cinnamic acids (Hillebrand et al., 2004; Schwarz et al., 2004). Additionally, PACNs have been found in other fermented foods like olives and sourdough bread (from sorghum) where the PACNs could have been formed by reaction with microbially derived cofactors (pyruvic acid and 4-vinylphenols, respectively) (Bai et al., 2014; Del Caro et al., 2006) .

[0368] 2.7. Pyranoanthocyanin Formation Mechanism. PACN formation occurs between the carbon 4 and 5-OH position of the anthocyanin and a reactive cofactor. While the positive charge of the flavylium cation is conventionally drawn on the oxygen, this charge is delocalized throughout the structure (Figure 5). This results in a partially positive charge at C4, the electrophile for the PACN formation reaction (Jingren He et al., 2006). Cofactors, then, must contain a nucleophilic carbon. A variety of compounds achieve this reactivity and can serve as cofactors, listed in Section 2.9. Cofactors with a ketone group and an a-hydrogen, such as pyruvic acid and acetone, undergo ketone-enol tautomerization in solution to form the reactive carbon (Loudon & Parise, 2016) (Figure 7). When in the enol form, the a-carbon is a nucleophile for PACN formation. Vinyl -cofactors, such as vinylphenols, have a nucleophilic carbon at the end of the vinyl group while the carbon directly attached to the phenolic ring is electrophilic likely due to the electron withdrawing polar effect of the benzene ring (Loudon & Parise, 2016).

[0369] Initiation of PACN formation has been reported in two ways. Some propose the first step as the cofactor attacking at C4 of the anthocyanin (Jingren He et al., 2006; Schwarz, Wabnitz, et al., 2003; Vallverdu-Queralt, Meudec, et al., 2016; Zeng et al., 2023). However, a study based on quantum chemistry calculations recently proposed that initiation occurs at the anthocyanin 5-OH with the cofactor’s electrophilic carbon attacking this location (X.-K. Zhang et al., 2022). In either process, the initial attack is believed to begin a concerted cycloaddition reaction followed by subsequent decarboxylation or dehydration (as needed based on the cofactor), electron transfer, and oxidation to form the new pyran ring which defines a PACN with the order of steps varying among publications (Fulcrand et al., 1998; Schwarz, Wabnitz, et al., 2003; Zeng et al., 2023; X.-K. Zhang et al., 2022) (Figure 8). In Vallverdu-Queralt, Meduc, et al. (2016), the pyran ring formation was reported as step-wise rather than concerted. Overall intermediary compounds in the PACN formation process have been identified, further supporting this general mechanisms (Figure 8) as a possible pathway for formation (Hakansson et al., 2003; Miyagusuku-Cruzado et al., 2023; X.-K. Zhang et al., 2022).

[0370] The entire PACN formation reaction was calculated to have a negative Gibbs free energy, driven by an energetically favorable autoxidation step to form the stable PACN (X.-K. Zhang et al., 2022). However, as an energetically spontaneous reaction, the organic formation of PACNs through wine fermentation is generally slow. Therefore, resources have been invested to investigate methods to increase the efficiency of PACN formation.

[0371] 2.7.1. Accelerated Pyranoanthocyanin Formation. Unlike what is observed for anthocyanins, PACNs are relatively scarce in plant extracts. While PACNs are found in red wine, wine would not be an economical choice for research or scaled-up production. To overcome these challenges, methods to produce PACNs in shorter time periods with greater yields have been developed. These processes, inspired by the organic formation of PACNs during wine aging, have successfully been able to form PACNs in only a few days. As example, optimized methods for 10-methyl-PACN formation achieved yields of -50% after 9 days (Kuang et al., 2014). Accelerated methods for hydroxyphenyl-PACNs produced yields of -20% in 1 day (Miyagusuku-Cruzado et al., 2023) while another study reported yields of 85% in 4 days (Vallverdu-Queralt, Meudec, et al., 2016). These achievements enable the pursuit of PACNs as colorants.

[0372] The variations among studies in reported yields is partially affected by different yield quantification methods. This complexifies interexperiment comparisons. Because analytical standards for PACNs are rare, methods employ relative quantification. One common approach based on HPLC-PDA compares the relative peak area of the formed PACN to the peak area of the starting anthocyanins (Farr et al., 2018; Hoehn, 2019). While this method is susceptible to over or underestimating the quantity due to differences in molar absorptivity coefficients, it does provide realistic comparison of the overall change in color intensity. A second approach is to quantify formed PACN using NMR and external phenol standards (Vallverdu-Queralt, Meudec, et al., 2016). While this method can provide high quantification accuracy, it does require additional equipment and expertise (Vallverdu-Queralt, Meudec, et al., 2016).

[0373] Below the factors which have been investigated for their role in PACN formation efficiency are summarized. Each condition is reviewed separately, however, there are confounding interactions between these variables and all factors should be considered for optimized formation of PACNs. Additionally, the ideal conditions for forming the different types of PACNs may not be universal across all anthocyanins and cofactors. Therefore, research is continually required for new anthocyanin and cofactor combinations.

[0374] Temperature: Environmental temperature has a dual effect on PACN formation as higher temperatures increase the rate of collision between reactants but also increases anthocyanin degradation (Hoehn, 2019). There is therefore a tradeoff between quick formation (favoring collision and formation) or higher yields (favoring retention of anthocyanins) as remaining anthocyanins can continue to react with the cofactor over time to increase the final yields. The ideal temperatures for PACN formation is likely between 25 to 65 °C based on a compilation of previous findings (Hoehn, 2019; Kuang et al., 2014; Straathof & Giusti, 2020; Vallverdu-Queralt, Meudec, et al., 2016; Xun, 2023). The effect of temperature will be greatly affected by the thermal stability of the anthocyanin and the cofactor, and therefore the ideal temperature will be different for each anthocyanin-cofactor combination.

[0375] Pressure. High pressure processing is a new, non-thermal treatment growing in popularity for food processing. It’s effect on PACN formation for hydroxyphenyl-PACNs was investigated by Zeng et al. (2023) with the effect dependent on the hydroxy cinnamic acid cofactor used. PACN yields were improved for 10-catechyl and 10- / ?-hydroxyphenyl -PACNs but not for 10-guaiacyl or 10-syringyl -PACNs (Zeng et al., 2023). The authors suggested that high pressure may have an effect due to radical formation of the cofactor or improved solubility (Zeng et al., 2023). pH Solution pH can affect both the chemical structure and stability of the starting anthocyanin and cofactor. Therefore, as with temperature, finding the ideal pH for efficient PACN formation is a balancing act between favoring stability of the cofactor or anthocyanin. It is likely that the best starting pH for PACN formation is near 3 (Hoehn, 2019; Kuang et al., 2014; Vallverdu-Queralt, Meudec, et al., 2016; Xun, 2023; X.-K. Zhang et al., 2022). While anthocyanins will be found in a greater abundance in their more stable, flavylium cation form at lower pH values, the cofactors may polymerize at lower pH values as observed for 4- vinylguaiacol and pyruvic acid (Mazza & Brouillard, 1987b; Muller & Baumberger, 1939; Vallverdu-Queralt, Meudec, et al., 2016). Lower pH values may also affect the reactivity of PACN intermediary compounds (X.-K. Zhang et al., 2022). At higher pH values, the less stable structural forms of anthocyanins predominate, resulting in faster anthocyanin degradation and a loss of the electrophilic C4 site for the cofactor to react (Cabrita et al., 2000; J. Oliveira et al., 2017; Xun, 2023).

[0376] Oxygen. The impact of oxygen on PACN formation has primarily been investigated in a red wine system through the effect of micro-oxidation methods or through oxygen diffusion from oak barrels during aging (Figueiredo-Gonzalez et al., 2014; Quaglieri et al., 2017). As summarized in a review by Quaglieri et al. (2017), oxygen generally resulted in either no change or an increase in yields for 10-carboxy-PACNs, 10-hydrogen-PACNs, and second-generation PACN-procyanidin dimers. Only a few studies have investigated the role of oxygen on PACN formation in more accelerated conditions. X.-K. Zhang et al. (2022) observed slightly decreased yields for 10-catechyl-PACNs when aged in an oxygen rich environment

[0377] Reactants. The anthocyanin and cofactor used in PACN formation will define the chemical structure and the functional properties of the formed colorant. These choices also have strong implications on the overall yields, rates of formation, and the reaction conditions for optimal formation. When considering scale up, finding reactants which can form PACNs quickly at low temperatures and with low stoichiometric ratios would be desirable.

[0378] Cofactor. While the chemical requirement for PACN formation is a nucleophilic carbon containing cofactor, the additional structural elements on the cofactor greatly influence the formation rate. In general, it has been shown that cofactors containing aromatic elements (i.e., hydroxycinnamic acids, 4-vinylphenols) form PACNs faster than the aliphatic cofactors (i.e., pyruvic acid, acetone) under accelerated conditions (Miyagusuku-Cruzado, Voss, et al., 2021a; X. Zhu & Giusti, 2021). Among the aromatic cofactors, the 4-vinylphenols-decarboxylated hydroxy cinnamic acids-formed PACNs more efficiently than their precursors under accelerated conditions (Miyagusuku-Cruzado et al., 2023).

[0379] Anthocyanin. All aspects of the chemical structure of an anthocyanin can influence the rate of PACN formation. The aglycone, the flavylium backbone defined by the B ring substitutions, is distant from the site of reaction, yet the B ring substitution patterns affected formation efficiency with malvidin anthocyanins forming PACNs most efficiently (Miyagusuku- Cruzado, Voss, et al., 2021a). In contrast, delphinidin formed no detectable PACNs under elevated temperatures (45 °C) (Miyagusuku-Cruzado, Voss, et al., 2021a).

[0380] Sugar substitutions attached at C3 position influenced PACN formation efficiency, hypothesized to be due to the sugar aiding in cofactor positioning and affecting the spatial availability of C4 (Farr et al., 2018). Disaccharides with 1— >6 linkages produced the greatest yields of 10-carboxy-PACNs while the lowest yields were observed for disaccharides with 1— >2 linkages and pentoses (Farr et al., 2018). Glycosylations can occur at positions beyond C3. Anthocyanins with C5 glycosylation cannot form PACNs as C5 is a key position for the cycloaddition reaction (Farr et al., 2018; X. Zhao et al., 2022). Flavylium structures lacking a substitution at C3, called 3 -deoxy anthocyanins, also form PACNs, however their formation rates were reported as slower than what is observed for anthocyanins (Xun, 2023). A third level of structural diversity in anthocyanins is brought by the presence of acylating groups attached to the sugar. These are common in vegetable anthocyanin sources (Andersen & Jordheim, 2010). The effect of acylating groups on PACN formation efficiency among published studies is mixed. Compared to their non-acylated anthocyanin counterparts, greater PACN yields were observed from anthocyanins acylated with / ?-coumaric acid and acetic acid while sinapic acid acylation decreased yields, hypothesized due to steric effect (Gomez- Alonso et al., 2012; X. Zhao et al., 2022; X. Zhu & Giusti, 2021). Malonic acid acylating groups have been reported to be cleaved from anthocyanins during PACN formation incubation, thereby resulting in the formation of non-acylated PACNs (Hillebrand et al., 2004; X. Zhu & Giusti, 2021).

[0381] Stoichiometry. The relative quantities of cofactor and anthocyanin must be added at a specific ratio to favor the chemical reaction. Generally, the cofactor is added at a higher molarity than the anthocyanin, with an exception observed in the formation of 10-methyl-PACNs from acetone (Kuang et al., 2014). Ideal ratios (anthocyanin: cofactor) are 1 :5 - 1 : 10 for 4- vinylphenols (Miyagusuku-Cruzado et al., 2023), 1 :30 for caffeic acid (Hoehn, 2019), 1 :200 for pyruvic acid (Hoehn, 2019), and 1 :500 for diacetyl (Gomez-Alonso et al., 2012). Higher levels of cofactor may facilitate PACN formation due to collision theory (Hoehn, 2019). However excess cofactor has been shown to initiate anthocyanin degradation (Miyagusuku-Cruzado et al., 2023). Several studies have reported differently, however, reinforcing the necessity of checking each condition for every set of reactants for optimal formation (Vallverdu-Queralt, Meudec, et al., 2016). Absolute quantities of anthocyanin have also been evaluated. Vallverdu-Queralt et al. (2016), observed no effect on anthocyanin concentration for 10-guaiacyl-PACN formation but Kuang et al. (2014) observed an effect for 10-methyl-PACN formation.

[0382] 2.8. Pyranoanthocyanin Classification. PACNs are a diverse class of colorants. With more than 700 different anthocyanins reported in nature (Andersen & Jordheim, 2010), many of which can form PACNs, and tens of different cofactors, hundreds, if not thousands, of unique PACNs could be formed. Each PACN has a distinctive chemical structure which imparts unique functional properties. While both the anthocyanin and cofactor contribute to the structural diversity, PACNs are classified by their CIO substitution (affected by the cofactor choice). It is this structural substitution, and the resulting effect on PACN physicochemical properties, which differentiate PACNs from other flavylium colorants.

[0383] 2.8.1. First Generation Pyranoanthocyanins. The different classifications of known PACNs are represented in Figure 9 modeled after J. Oliveira et al. (2014), with details on the history, formation, characteristics, and sources described in Table 1. A systematic naming system for PACNs is proposed based on the CIO substitution (lO-(substitution)-pyrano(anthocyanin name)), meant to standardize PACN reference across studies and findings. Other common names used in published studies are found in Table 1. The term Vitisin is derived from Vitis vinifera, the scientific name of wine-making grapes, and is a nod to the first discovery of these PACNs in wine (Bakker et al., 1997; Bakker & Timberlake, 1997) . Similarly, the term Pinotins, for 10- catechyl-PACNs, was assigned as 10-catechyl-pyranomalvidin-3-glucoside, the first to be elucidated, was isolated form Pinotage wine (Schwarz, Jerz, et al., 2003).

[0384] The references provided in Table 1 were chosen with select criteria. For the cofactor, we cited the publication which first (to our knowledge) showed direct synthesis of the PACN with the listed cofactor, confirming absolutely the synthesis ingredients. Many of the cofactors were, however, postulated earlier. For the structure elucidation, we report the publication(s) which first elucidated the structure using NMR as this is the gold-standard for compound identification. However, other pioneers in PACN research deserve acknowledgment as many of these PACNs were detected and proposed in wine years prior based on HPLC-PDA-MS / MS analysis. Key contributors include Hayasaka and Asenstorfer (2002), Benabdeljalil et al. (2000), and Francia- Aricha et al. (1997). An exemplary example is 10-guaiacyl-PACNs, as the structure was proposed in 2002 by Hayasaka and Assenstorfer, however, to our knowledge, NMR characterization was not performed until 2016 by Vallverdu-Queralt et al.

[0385] Table 1. First generation pyranoanthocyanin (PACN) characteristics on formation, identification, and coloring characteristics. ACN: anthocyanins

[0386] Mv3G: Malvidin-3-glucoside

[0387] *color comparison to anthocyanin in the same study could not be found. The reported for malvidin- 3-glucoside anthocyanin (528 nm) in Mateus et al. (2006) was used for comparison instead.

[0388] ■(Proposed as a cofactor for formation but this pyranoanthocyanin was never synthesized, only extracted (to our knowledge).

[0389] Reference key is as follows: 1. (Benabdeljalil et al., 2000) 2. (J. Oliveira, de Freitas, et al., 2009) 3. (Bakker & Timberlake, 1997) 4. (Hayasaka & Asenstorfer, 2002) 5. (Pozo-Bay on et al., 2004) 6. (Li, Yuan, et al., 2023) 7. (Fulcrand et al., 1998) 8. (Araujo et al., 2017) 9. (Bakker et al., 1997) 10. (Duenas et al., 2008) 11. (Andersen et al., 2004) 12. (Del Caro et al., 2006) 13. (Fossen & Andersen, 2003) 14. (Voss et al., 2022) 15. (Jingren He, Carvalho, et al., 2010) 16. (Jingren He et al., 2006) 17. (Lu et al., 2000) 18. (Alcalde -Eon et al., 2006) 19. (Kurka et al., 2017) 20. (X. Wu et al., 2004) 21. (Myjavcova et al., 2010) 22. (Blanco-Vega et al., 2011) 23. (Gomez-Alonso et al., 2012) 24. (Schwarz, Wabnitz, et al., 2003) 25. (Fulcrand et al., 1996) 26. (Rentzsch, Schwarz, Winterhalter, et al., 2007) 27. (Hillebrand et al., 2004) 28. (Schwarz et al., 2004) 29. (Miyagusuku- Cruzado, Voss, et al., 2021a) 30. (Hakansson et al., 2003) 31. (Schwarz, Jerz, et al., 2003) 32. (Voss, Miyagusuku- Cruzado, et al., 2023) 33. (Vallverdu-Queralt, Meudec, et al., 2016) 34. (Mateus, Carvalho, et al., 2003) 35. (Mateus et al., 2002) 36. (Francia-Aricha et al., 1997) 37. (Pechamat et al., 2014) 38. (J. Oliveira, Mateus, Rodriguez-Borges, et al., 2011) 39. (Schwarz & Winterhalter, 2003). 40. (Bonerz et al., 2007).

[0390] 2.8.2. Second Generation Pyranoanthocyanins. As reactive flavylium compounds, PACNs themselves may serve as precursors to new PACN colorants. Termed second generation PACNs, these compounds retain the defining pyran ring between C4 and C5-OH but have additional substitutions attached. Second-generation PACNs often have extended networks of electron conjugation, resulting in the formation of a blue color at acidic pH (J. Oliveira et al., 2010). Generally, most second generation PACNs form from 10-carboxy -PACNs. As observed with anthocyanins, the positive charge on the flavylium cation would be delocalized throughout the PACN including at CIO. That, in addition to the electron withdrawing effect of the carboxylic moiety, help provide a partially positive charge (i.e., electrophilic) at carbon 10 of the PACN (Mateus, Silva, et al., 2003; J. Oliveira et al., 2007). As observed for anthocyanins in PACN formation, this creates a reactive location. Details on the identified second-generation PACNs are found in Table 2 and Figure 10. The same criteria were used when identifying references as for Table 1.

[0391] There are several key families of second-generation PACNs. Vinyl-PACNs, also known as portisins due to their initial identification in Port wine, alone represent a very broad class of possible PACNs (Mateus et al., 2006; Mateus, Silva, et al., 2003; J. Oliveira et al., 2007). Table 2 includes the compounds which have been structurally elucidated by NMR, however additional Portisins have been reported to have been identified from wine (Mateus, Oliveira, Haettich- Motta, et al., 2004). While not listed as a cofactor, acetaldehyde is often a key ingredient in the formation process of vinyl-PACNs as it helps to form vinyl-flavonols (Mateus et al., 2002; Mateus, Oliveira, Santos-Buelga, et al., 2004).

[0392] 2.8.3. Pyranoanthocyanins Beyond Nature. Several types of PACNs have been created with no precedent for being formed organically in nature based products. These PACNs often have unique color properties and demonstrate the huge potential and versatility of PACNs from a chemical standpoint beyond nature’s palette. While approval of these PACNs provides an additional challenge for use in foods as the “nature-identical” claim or a history of consumption cannot be made, they also have the potential to be used in non-food applications. Details on both first and second-generation non nature identical PACNs are provided in their respective tables ( Table 1 and Table 2).

[0393] Table 2. Second generation pyranoanthocyanin (PACN) formation, identification, and color characteristics.

[0394] * Color comparison to precursor anthocyanin measured in the same study was not found in the same experiment. The reported Xvis-max for mal vidin-3 -glucoside anthocyanin (528 nm) in Mateus et al. (2006) was used for comparison instead. *Further variations of the Vinyl -dimethyl amino PACN including the vinyldiethyl amino and butadene-dimethyl-amino PACN have been reported. ’''Proposed, however not tested to our knowledge. J Reported as the fastest and most efficient cofactor for PACN- dimer formation (J. Oliveira et al., 2010).

[0395] Reference key: 1. (Mateus, Carvalho, et al., 2003) 2. (J. Oliveira et al., 2010) 3. (Mateus, Oliveira, Santos-Buelga, et al., 2004) 4. (Mateus, Oliveira, Haettich-Motta, et al., 2004) 5. (Mateus et al., 2005) 6. (J. Oliveira, Santos-Buelga, et al., 2006) 7. (Mateus et al., 2006). 8. (J. Oliveira et al., 2007) 9. (Carvalho, Oliveira, de Freitas, Silva, et al., 2010) 10. (Jingren He, Oliveira, et al., 2010) 11. (J. Oliveira et al., 2016). 12. (J. Oliveira et al., 2016) 13. (J. Oliveira, Mateus, Rodriguez-Borges, et al., 2011) 14. (J. Oliveira, Mateus, & de Freitas, 2011). 15. (Monteiro et al., 2022)

[0396] 2.9. Pyranoanthocyanin Color Expression. The number of diverse PACNs yields a rainbow of colors. Table 1 and Table 2 provide the Xvis-max for various PACNs in comparison to the color of the parent anthocyanin; as seen, the shifts can be both hypsochromic and bathochromic with a variety of possible wavelengths. As with anthocyanins, their color is highly dependent on the chemical structure and the environmental conditions. These factors are often interconnected, creating a complicated web of behaviors. Here, we focus on the color of PACNs with a foundation on the influence of their chemical structure and how it changes to pH, heat, oxygen, and different ingredients.

[0397] 2.9.1. The Conjugation Anomaly. The first generation PACNs generally produce colors more orange and yellow to their parent anthocyanin ( Table 1). These observations contradict the resonance rules described in Section 2.1 in which a more conjugated molecule should reflect higher energy light (shorter wavelength; more purple-blue) due to the decreased HOMO-LUMO energy gap. Conjugation is key to this rule, and while PACNs 2-dimensional structure shows an increased in conjugation, the 3-dimensional conformation shows the nuance. The addition of the PACN D rings contorts the structure so that the B ring is has a greater dihedral angle (more out of plane) with the remaining molecule (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010b; Phan et al., 2021). This contortion decreases electron delocalization from the B ring with the remaining molecule (and vice-versa), slightly extending the bond length between C2 and Cl’ (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010b).

[0398] Of the first generation PACNs, only 10-acetyl -PACNs, were noted to have kvis-max shifted bathochromically compared to their parent anthocyanins (Gomez-Alonso et al., 2012) ( Table 1). These spectral observations were only observed from HPLC-PDA data, and when measured spectrophotometrically in pH 1 and 3.6 buffer, the kvis-max was hypsochromically shifted by 11 to 13 nm from the parent anthocyanin (Gomez-Alonso et al., 2012). No further discussion was provided, however 10-acetyl -PACNs appear to have several unique behaviors including undergoing observable level of hydration with pH increase (discussed in Section 2.6.3), which may contribute to the different color effect.

[0399] Second generation PACNs typically produce more bathochromic (bluer) colors than their parent PACNs and anthocyanins (Table 2). While the B ring retains its higher dihedral angle, the extended conjugation provided by the CIO substitution compensate for the decrease in B ring electron delocalization (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010b). Because of the scarcity of nature based blue colorants, second-generation PACNs present unique opportunities in applications. However, because of the relatively few sources and low concentrations in which they are found and the limited scope of research done with second-generation PACNs, our focus moving forward will be on the color of first generation PACNs.

[0400] 2.9.2. Influence of Pyranoanthocyanin Chemical Structure on Color. In light of the out of phase B ring, the A-C-D ring system of PACNs is the primary chromophore unit (Chassaing et al., 2015). As this is standard for all PACNs, additional groups like the B ring, CIO substitution, glycosylation and acylating groups are responsible for the diverse colors produced. These substitutions may serve as auxochromes by affecting the electron density of the system. Increasing the electron density of the chromophore through electron donating groups should result in a bluer color as the HOMO-LUMO energy gap is decreased leading to the molecule absorbing lower energy light leading to higher energy light being reflected / transmitted to the observer (Chassaing et al., 2015). Additionally, these substitutions can affect the 3-dimensional conformation of the PACN, influencing the extent of electron delocalization and the conjugation of the system (Phan et al., 2021; Quartarolo & Russo, 2011; Siddique et al., 2019). Typically, the color is defined from a combination of these effects.

[0401] CIO substitution. The cofactor used in PACN formation defines the CIO substitution group, which strongly affects the color. The largest hypsochromic shift in PACN color compared to the starting anthocyanin occurred for 10-hydrogen-PACNs and 10-methyl-PACNs, with these PACNs generally described by a “yellow” color ( Table 1; Bakker & Timberlake, 1997; Jingren He et al., 2006; J. Oliveira, de Freitas, et al., 2009). Carboxy-PACNs generally have a Vis-max 12 to 19 nm lower than the starting anthocyanin (Bakker & Timberlake, 1997; Jingren He, Carvalho, et al., 2010; Voss et al., 2022). While the methyl group is an electron donating group and a carboxylic acid group electron withdrawing (Loudon & Parise, 2016), the opposite effect of color is observed; the methyl-PACN has the more hypsochromically shifted color than the carboxy-PACN (Blanco- Vega et al., 2011). This highlights the interplay of other effects caused by the CIO substitution, such as torsion angle, on the color (Quartarolo & Russo, 2011).

[0402] With hydroxyphenyl-PACNs, the color is typically yellow orange to red orange, with Vis- max hypsochromically shifted by 4 to 22 nm from the parent anthocyanin as shown in Table 1 (Miyagusuku-Cruzado, Voss, et al., 2021a; Vallverdu-Queralt, Meudec, et al., 2016; Voss, Miyagusuku-Cruzado, et al., 2023). The greatest difference from the starting anthocyanin was reported for 10-guai acyl -PACN, however here, the starting anthocyanin was malvidin-3- glucoside while the comparisons made for the other hydroxyphenyl-PACNs were for cyanidin-3- glucoside derived PACNs. The addition of the E ring extends electron delocalization to give a smaller hypsochromic shift in Amax than what is observed for 10-methyl-PACNs (Azevedo et al., 2014). However, the color of hydroxyphenyl-PACNs is still more yellow from the starting anthocyanin as the full effect of an additional conjugated phenyl ring is muted due to non-planar orientation of the E ring (Phan et al., 2021). The dihedral angle is smaller than that of the B ring, suggesting that the E ring may play a larger role on color than the B ring (Quartarolo & Russo, 2011). This was experimentally observed by Blanco- Vega et al. (2011) based on the HPLC-PDA kmax for PACNs.

[0403] For PACN-flavonoids, the color would likely be described as yellow-orange to orange as the kvis-max is shifted hypsochromically 8 to 22 nm from the starting anthocyanin ( Table 1; Jingren He, Carvalho, et al., 2010; Mateus et al., 2002). The flavonoid does increase the extent of electron delocalization, and therefore the color is not as yellow as observed for 10-methyl- and 10-hydrogen-PACNs (Azevedo et al., 2014). Additionally, the flavonoid moiety can affect the 3 dimensional conformation by folding and interacting with the PACN A-C-D ring through 7t-7t interactions (Jingren He, Carvalho, et al., 2010).

[0404] Hydroxyl and Methoxy Groups. The number and type of OH and OCH3 groups define the type of hydroxyphenyl substitution and the anthocyanin aglycone (B ring) of the PACN. By definition, OH and OCH3 groups are electron donating (OH slightly more so than OCH3) (Loudon & Parise, 2016). Therefore, increasing the number of these groups, as with catechol compared to phenyl, would increase the electron density of the ring, decreasing the H0M0- LUMO gap, and resulting in a visually bathochromic (more blue) color (Phan et al., 2021). Experimentally, this can be observed from HPLC-PDA findings where syringyl moieties (either on E or B ring-malvidin) have higher A.maxcompared to phenyl (B ring-pelargonidin), catechol (B ring-cyanidin), and guaiacyl (B ring-peonidin) moieties (Blanco- Vega et al., 2011; Miyagusuku-Cruzado, Voss, et al., 2021a). When comparing equivalent number of substitutions on the B ring, Malvidin-PACNs appear to have a slightly higher Amaxthan those derived from delphinidin, and petunidin which all have 3 total substitutions (Blanco- Vega et al., 2011).

[0405] The hydroxyl and methoxy substitutions also affect the 3-dimensional conformation of the PACNs through alterations in the dihedral angle of the B and E ring (Phan et al., 2021; Siddique et al., 2019). Based on theoretical computations, Phan et al. (2021) reported a lower dihedral angle for phenyl groups compared to catechol, Quartarolo and Russo (2011) reported a lower angle for phenyl compared to guaiacol, and Siddique et al. (2019) reported a lower angle for syringyl moiety than phenyl although the theoretical computations were made for a slightly modified pyran flavylium structure. Lower dihedral angles suggest greater electron delocalization. Therefore, both the effect on electron density and conjugation should be considered in color predictions.

[0406] Sugars. Despite having no color themselves, glycosylation substitutions may affect the overall color of PACNs. J. Oliveira, Fernandes, et al. (2006) showed that disaccharides produced a more orange and intense color than the monosaccharide for 10-carboxy-pyranomalvidins. However, these effects were conditional on the type of disaccharide and pH (J. Oliveira, Fernandes, et al., 2006). Farr et al. (2018) also showed a similar effect on color for 10-carboxy- pyranocyanidins with a higher A.maxand lower hue angle produced by the disaccharide and trisaccharide containing PACN than for the PACNs with a monosaccharide. The color effect was slight, however, resulting in a A.maxdecrease of 4 units and hue angle decrease of ~9 units (Farr et al., 2018).

[0407] Sugars effect on the color may be engendered through their influence on the B ring and CIO group dihedral angle. Hydrogen bonds were shown to form between the sugar (C3) and the B ring groups and CIO substitution (including E ring) based on the most stable conformation in theoretical calculations (Quartarolo & Russo, 2011). The B and E ring and CIO group will contort themselves to complete these bonds, thus resulting in changing dihedral angles and affecting the extent of electron delocalization (Quartarolo & Russo, 2011).

[0408] Acylation. The effect of acylating groups on PACN color is scarcely investigated. From the Xmax reported in X. Zhu and Giusti (2021), acylation appears to have little effect on 10- carboxy PACN color. While the sinapoyl group resulted in a bathochromic shift in the A.max(16 nm) for cyanidin anthocyanin, the Xmax of the 10-carboxy-PACN derivative was only 6 nm higher, similar to the non-acylated trisaccharide derived PACN (X. Zhu & Giusti, 2021). Acylating groups may affect the absorption in the near UV region as a shoulder near 312 to 340 nm appeared on the PDA spectra for acylated PACNs (Blanco-Vega et al., 2011).

[0409] 2.9.3. Molar Absorptivity Coefficient. Color intensity of dyes and pigments are determined by the molar absorptivity coefficient (molar extinction coefficient). Governed by Beer-Lambert’s law (Equation 2.1), the molar absorptivity coefficient defines the amount of light produced for 1 mole of a compound at its respective vis-max •

[0410] Equation 2.1 : Absorbance = c * 8 * b where c = concentration molarity (moles / L), b = pathlength in centimeters, and 8 = molar absorptivity coefficient in L / (mole*cm).

[0411] Determining the molar absorptivity coefficients for individual coloring compounds is beneficial as one can then determine compound concentration simply from an absorption spectrum. An exemplary example of this is the pH differential method, an AO AC approved method for determining the concentration of anthocyanins in solution based on absorption characteristics (Lee et al., 2005). There are many factors to consider when calculating and reporting molar absorptivity coefficients, especially for anthocyanins and PACNs. First, it is important to have a pure isolate as impurities or residual salts can inflate the weight (affecting known concentration by an unknown amount) and influence the absorption intensity (Jordheim et al., 2007). Additionally, while the molecular weight is standard for a compound, several studies include a water molecule of hydration and / or a chloride counterion in the weight when determining the concentration (Jordheim et al., 2007). Especially for anthocyanins and PACNs, conditions like the solvent type, pH, equilibration time, and concentration can all affect the color produced and the molar absorptivity coefficient.

[0412] Table 3 lists the reported molar absorptivity coefficients for PACNs with the relevant procedural characteristics included. For experiments with concentration ranges listed, the molar absorptivity values were determined based on the slope of the plotted line. There is a large amount of variability reported among molar absorptivity coefficients for PACNs, even for PACNs of the same (or similar) chemical structure. One of the largest is for 10-guaiacyl-PMalvidin-3-glucoside where there is a difference of -39,000 between the two reported molar absorptivity coefficients. While the solvent is different (acidified MeOH versus pH 0.8 buffer), this difference is far greater than the range reported for other PACNs. The factors listed previously likely contribute to the large variability in reported PACN coefficients, both in terms of the methods used to calculate them and in the high dependence of PACN color on environmental conditions. Few (if any) inter-experiment replicates of the molar absorptivity coefficients could be found, further increasing the challenge of knowing the proper value.

[0413] Because of this variability, it is challenging to make strong comparisons on the influence of chemical structure on the molar absorptivity coefficients. Most experiments do report similar or slightly lower coefficients than the precursor anthocyanins, although the pH largely affects this conclusion, as PACN coefficients change less with pH increase (Gomez-Alonso et al., 2012; Hakansson et al., 2003; Jingren He, Carvalho, et al., 2010; Jordheim et al., 2007; Mateus & de Freitas, 2001b). Interestingly, Phan et al. (2021) theoretically calculated much higher molar absorptivity values for PACNs, with -38,000 calculated for 10-carboxy-Pyranopelargoni din-3 - glucoside and -60,000 calculated for 10-catechyl-pyranomlavi din-3 -glucoside.

[0414] With the large amount of variability in values, it is challenging to observe trends and make conclusions on the influence of chemical structure on the molar absorptivity coefficient. Based on intra experiment comparison, both the B ring and CIO substitutions can affect molar absorptivity coefficient values.

[0415] Table 3. Literature summary of molar absorptivity coefficients reported for pyranoanthocyanins and relevant details for their calculation. NS = not specified a: potassium hydrogen phthalate-monopotassium phosphate buffer, b: model wine with 0.25% potassium bitartrate and 12% ethanol, c: citrate buffer (1 M). d: potassium chloride-HCl buffer, e: potassium hydrogen phthalate-HCl buffer, f indicates a counterion was included in molecular weight for the calculation; the remaining studies did not provide details on the molecular weight used. * indicates an equilibration time of 2 hours before measuring; the remaining studies reported no equilibration time.

[0416] 2.10. Pyranoanthocyanin pH Color Expression and Stability

[0417] 2.10.1. Substitution Effect on pKa Values. An innate feature of flavylium compounds is their structural dependency on pH. Thus, PACN color, stability, and other functional properties (i.e., color expression, stability, solubility, bioactivity, ingredient interactions) are intertwined with the acidity level in the environment. Thus, understanding the relationship between pH and PACNs within this dynamic system is imperative for predicting and interpreting their behavior in all systems.

[0418] The general pathway of PACN pH structural transitions is described below (Figure 11; (Cruz et al., 2022; J. Oliveira et al., 2014). The different CIO substitutions can introduce additional steps, as detailed in the proceeding paragraphs (Table 4). In acidic solutions, PACNs are in their flavylium cation form (AH+), with a positive charge delocalized throughout the molecule. As the pH is increased, the flavylium cation is deprotonated to form the quinoidal base (A). With continued pH increase, subsequent deprotonation occurs forming the anionic quinoidal base (A’) and, when substitutions allow it, the dianionic quinoidal base (A2-). The C7-OH group is considered the most acidic for PACNs and is lost first, followed generally by the C4’-OH in the B ring (J. Oliveira et al., 2013).

[0419] Noticeably, the hydration pathway to form the hemiketal and chaicone is not the major pathway of structural change for PACNs. In fact, many studies do not find evidence for the formation of a hydrated PACN either by NMR or UV-Vis spectral work (Cruz et al., 2010; J. Oliveira et al., 2013; J. Oliveira, Mateus, et al., 2009; J. L. Sousa et al., 2017). In contrast, several studies have reported evidence of a hydrated species (Asenstorfer & Jones, 2007; Gomez-Alonso et al., 2012; J. Oliveira, Petrov, et al., 2011). Hydration is driven by the attack of the nucleophilic carbon at the electrophilic C2 (or in the case of PACNs, also C 11) on the PACN structure. Because of the additional PACN conjugation, the positive charge is delocalized around a greater area which decreases the electrophilicity of any one carbon (Cruz et al., 2010). The cofactor can affect the propensity for hydration, as a hydrated acetyl-PACN was observed (Gomez- Alonso et al., 2012). The acetyl group is electron withdrawing which can increase the positive charge on the PACN (and hence C2 position) (Vallverdu-Queralt, Biler, et al., 2016). A happy conclusion on PACN hydration despite sometimes conflicting reports is that hydration is not a major pathway for PACN structural changes with pH, and most authors do ignore its influence when determining pKa coefficients.

[0420] The CIO substitution groups may provide additional acidic hydrogens leading to additional pKa coefficients and steps in the deprotonation process. For 10-hydrogen-PACNs, 10- carboxy PACNs, and the dimethylamino-containing PACNs, an additional transition from flavylium dication (AH2+) to zwitterionic flavylium cation (AH+) occurred at highly acidic pH with pKa values ranging from ~0.6 to -2.4 (Table 4). For 10-carboxy -PACNs, the most acidic hydrogen is found to be at CIO (J. Oliveira et al., 2013). The shift from the AH2+to AH+structure resulted in a hypsochromic shift in ^viz-max (J. Oliveira, Fernandes, et al., 2006; J. Oliveira, Mateus, et al., 2009).

[0421] For hydroxyphenyl -PACNs, both the E and the B ring contain acidic hydrogens at the para position-C4’ in the B ring and C4” in the E ring. For the formation of the anionic quinoidal base, either hydrogen can be deprotonated with similar pKa’s reported (Pinto, Oliveira, et al., 2019). Therefore, the anionic quinoidal base may be comprised of multiple deprotonated species.

[0422] Most of the work surrounding PACN pH structural transitions has focused on identifying the pKa constants for the structural changes. These values define the point in which the presence of the reactant and product is equal (50% of each). It is necessary to assume that only 2 species are present when calculating pKa constants. In reality, more than 2 species could be present, further contributing to the diversity of behavior and colors produced.

[0423] Table 4. Reported pKa constants for pyranoanthocyanin pH structural transitions as detailed and defined in Figure 11. P: Pyrano. Mv-3-glu: Malvi din-3 -glucoside. Cy-3-glu: Cyanidin-3- glucoside.

[0424] Reported in acetate buffer (12% EtOH).2reported in citric acid-Na2HPO4 buffer. *indicated values based on 3 constant fitting; when separate dissociation constants were included for B and E ring protons, values were as follows: 10-catechyl-PCy-3-glu: 7.2 / 7.1 and 9.6 / 10. 10-syringyl- PCy-3-glu: 7.3 / 8.6. 10-catechyl-PMv-3-glu: 6.2 / 8.0

[0425] A summary of the reported pKa values in literature are provide in Table 4 as well as in a review by Cruz et al. (2022). For the PACNs found in nature (i.e., excluding those from purely synthetic cofactors), the pKa for the AH2+to AH+transition is between 0.63 to 1.8, AH+to A transition is between 3.6 to 5.35, Ato A' between 6.3 to 9.1, and A' to A2' between 9.1 to 12.2. The structural substitutions can affect the pKa position, often reported to be due to their electron donating or withdrawing effect and the subsequent stabilization on the structure (J. Oliveira et al., 2014). Because of the cascading influence of pKa values on color and stability, understanding the relationship between chemical structure and coefficients is important for

[0426] PACN application. These conclusions were based on the pKa values for reported PACNs as well as several studies working with synthetic analogues of structurally similar compounds to

[0427] PACNs.

[0428] CIO substitution. As concluded in J. Oliveira et al. (2014) and A. A. Freitas et al. (2018) the CIO substitution group did not appear to predictably affect the pKa for AH+to A transition. A. A. Freitas et al. (2018) observed the same pKa for a the synthetic-PACN analogues with a CHs-phenyl, F-Phenyl, and CN-Phenyl at CIO; these groups have strong differences in electron donating and withdrawing effects, so the resulting pKa similarities suggested no change (A. A. Freitas et al., 2018).

[0429] Hydroxyl and Methoxy. Understanding the effect of a hydroxyl or methoxy substitution on the B and E ring is learned by comparing cyanidin versus malvidin PACNs and 10-catechyl versus 10-syringyl PACNs based on the available coefficients. While some exceptions are observed, Malvidin PACNs tended to have higher pKa values than their cyanidin counterparts for the A to A' and A' to A2' transition (Cruz et al., 2010; J. Oliveira et al., 2013; J. Oliveira, Petrov, et al., 2011; Pinto, Oliveira, et al., 2019). For example, the Ato A' transition for 10- carboxy-pyranomalvidin-3-glucoside was reported at 9.14 while for 10-carboxy-pyranocyanidin- 3-glucoside at 6.3 (J. Oliveira et al., 2013; Pinto, Oliveira, et al., 2019). This suggests that the additional OCH3 groups (or the lack of an additional OH group) in the B ring of malvidin stabilized the A form (J. Oliveira et al., 2014).

[0430] In comparing a catechol versus syringol E ring substitution, the same trend was observed in which the syringol moiety imparted slightly higher pKa values for A to A' and A' to A2' transition than the catechol substitution (J. Oliveira et al., 2014; Pinto, Oliveira, et al., 2019). The pKa of the AH+to A transition was lower, however, for 10-syringyl -PACNs than 10-catechyl- PACNs (J. Oliveira et al., 2014; Pinto, Oliveira, et al., 2019).

[0431] Sugar. The effect of number and type of sugar substitutions on pKa coefficients is inferred from the work evaluating color expression across pH for 10-carboxy-pyranocyanidins. When evaluating color change with pH for 10-carboxy -PACNs, J. Oliveira, Fernandes, et al. (2006) observed that the monosaccharide substituted PACN had more stable color across pH than for those with a disaccharide substitution. Additionally, there was a greater increase in absorbance intensity with pH increase for the monosaccharide substituted PACN than the disaccharide substituted PACNs (J. Oliveira, Fernandes, et al., 2006). Most studies on PACN pKa coefficients have investigated the 3-glucoside, so there is a need to further investigate the influence of different types and number of glycosylations on pKa values. When comparing the pKa values for the PACNs compared to pyranodeoxyanthocyanins (lacking a C3 substitution), the sugar groups appear to impart a slightly lower pKa for the AH+to A transition (Pinto, Oliveira, et al., 2019; J. L. Sousa et al., 2017).

[0432] Acylation. The coumaroyl acylating group increased the pKa of PACNs for the AH+to A and Ato A' transition (Cruz et al., 2010; J. Oliveira et al., 2014). It was suggested that this was due to their ability to increase the stability of the PACN (J. Oliveira et al., 2014). The effect of other acylating groups on pKa coefficients has not been evaluated.

[0433] 2.10.2. pH Effect on Color Expression. With the absence of appreciable levels of hydration and thus little formation of the colorless hemiketal and chai cone, PACN color is more stable and retains greater intensity than anthocyanins across pH (Andersen et al., 2004; Jingren He, Carvalho, et al., 2010; Sun et al., 2020; M. Wu et al., 2022). The possibility of a colored hemiketal / chalcone has been suggested for PACNs however, and this should be kept in mind when evaluating spectral data (Asenstorfer & Jones, 2007; Hakansson et al., 2003). While PACN color is more stable than anthocyanins with pH changes, it does not suggest that the PACNs are inactive or stationary; dynamic changes are still occurring over time and with pH changes. Therefore, having a strong background on the equilibrium network distribution at different pH values may provide insight into their color.

[0434] The flavylium cation (AH+) produces the characteristic yellow, orange, and red-orange colors of first generation PACNs ( Table 1). The color of the quinoidal base (A) in respect to the flavylium cation is dependent on the CIO substitutions with the pKa for this transition ~4 (Table 4). For flavonoid-PACNs and many hydroxyphenyl -PACNs, there is a reported hypsochromic shift in the kvis-max with the formation of the quinoidal base, typically 20 to 42 nm lower (Cruz et al., 2010; Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016). For 10-carboxy- PACNs, 10-hydrogen-PACNs, 10-acetyl -PACNs, and 10-methyl-PACNs, a bathochromic shift in the kvis-max is reported as the quinoidal base forms (Andersen et al., 2004; Gomez-Alonso et al., 2012; J. Oliveira et al., 2013; J. Oliveira, Mateus, et al., 2009; J. Oliveira, Petrov, et al., 2011; M. Wu et al., 2022). Several exceptions to these rules have been shown, however, for 10-catechyl- pyranocyani din-3 -glucoside and 10-carboxy-pyranomalvi din-3 -glucoside (Pinto, Oliveira, et al., 2019; M. Wu et al., 2022). Regardless of the direction of color shift, absorbance intensity decreases some as the quinoidal base forms (Andersen et al., 2004; Cruz et al., 2010; J. Oliveira et al., 2013; Vallverdu-Queralt, Biler, et al., 2016; M. Wu et al., 2022). The anionic quinoidal base (A’) and dianionic quinoidal base (A2-) have strong bathochromically shifted kvis-max, reported around 49-66 nm and 65-72 nm higher than the flavylium cation, respectively (Cruz et al., 2010). Color intensity increases with the formation of the anionic quinoidal base forms with the dianionic quinoidal base sometimes having greater color intensity than the flavylium cation (Cruz et al., 2010; J. Oliveira et al., 2013).

[0435] 2.10.3. pH Effect on Color Stability. Studies on PACN color stability over time and pH values are limited as most of the pH related studies focus on pKa coefficients, measured immediately after pH adjustment. Of the handful of studies, a main observation is the formation of PACN aggregates or precipitates over time. Aggregation is defined to be not observable as visible solids but observable through otherwise unexplainable changes in the absorption spectra; this includes increase absorption in the 550 to 700 nm range and has also been described as a sharp absorption band (Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016; Voss, Miyagusuku-Cruzado, et al., 2023). Precipitate is defined as the appearance of visual solids in solution and gives decreasing spectral absorbance at all wavelengths (Cruz et al., 2010). Aggregation is often observed within a few hours of mixing with precipitation forming after day(s) (Cruz et al., 2010; Sarni -Manchado et al., 1996; Vallverdu-Queralt, Biler, et al., 2016; Voss, Miyagusuku-Cruzado, et al., 2023).

[0436] Further understanding on the phenomenon and mechanism of PACN aggregation is based on the work of Vallverdu-Queralt, Biler, et al. (2016) with 10-guaiacyl and 10-catechyl- pyranocyani din-3 -glucosides. Here, the authors observed the formation of aggregates based on the PDA spectra absorbance, and observed, through dynamic light scattering, the growth of particle size over time (Vallverdu-Queralt, Biler, et al., 2016). It is likely that once these aggregates grow large enough, they precipitate from solution (Vallverdu-Queralt, Biler, et al., 2016). Aggregation was proposed to be initiated by 7t-7t intermolecular interactions between PACNs, and because of the structural differences with pH, it’s prevalence and stability was heavily dependent on solution pH (Vallverdu-Queralt, Biler, et al., 2016). The quinoidal base formed the most stable aggregates because of the lack of charge repulsion, and could form either with itself or with the flavylium cation (Vallverdu-Queralt, Biler, et al., 2016). Because of the necessity of the presence of the quinoidal base, PACN aggregation and precipitation has been primarily observed in the mild acid pH range (above 3.5) (Cruz et al., 2010; Sami-Manchado et al., 1996; Vallverdu-Queralt, Biler, et al., 2016). Precipitation has also been observed in highly alkaline (pH > 9) solutions for pyrano-deoxyanthocyanins, and aggregation was suggested in highly acidic solutions for 10-catechyl and 10-syringyl-pyranocyani din-3 -glucosides based on spectral observations (Pinto, Oliveira, et al., 2019; A. Sousa et al., 2014).

[0437] The chemical structure can affect aggregation and precipitation due to the steric effect and the influence on quinoidal base formation (i.e., pKa). Methoxy groups on the E ring favored precipitation compared to a hydroxyl group and coumoryl acylation on the glycosylation favored precipitation for 10-hydrogen-PACNs (J. Oliveira, Mateus, et al., 2009; Vallverdu-Queralt, Biler, et al., 2016).

[0438] 2.11. Effect of Temperature on Pyranoanthocyanin Color Expression and Stability. Heat introduces energy to the system which can accelerate degradative pathways. Anthocyanins are known to fade within a few hours of heating as the molecule irreversibly breaks into colorless degradation compounds (Sadilova et al., 2006, 2007; Voss, Miyagusuku-Cruzado, et al., 2023). Instead, PACNs are shown to have superior color stability to heat (Sami -Manchado et al., 1996; Sun et al., 2019, 2020; Voss et al., 2022; Voss, Miyagusuku-Cruzado, et al., 2023). Their color stability can be quite impressive, with Voss et al. (2022) showing >50% of PACN remaining after 15 hours at 90 °C. The remarkable color stability to heat for PACNs is contributed by multiple factors including increased structural resistance to degradation and the formation of color producing degradation compounds.

[0439] The different CIO substitution influence the extent of stability, yet slight changes in the number and type of hydroxyl and methoxy substitutions did not significantly affect color stability although longer heating times may be needed show an effect (Sun et al., 2020; Voss et al., 2022; Voss, Miyagusuku-Cruzado, et al., 2023). The effect of additional chemical substitutions such as glycosylation and acylation on thermal stability remains to be studied. Additionally, most studies on thermal stability have evaluated degradation under acidic conditions when the flavylium cation predominates. Therefore, understanding how the quinoidal base or anionic quinoidal base species respond to heat remains to be known.

[0440] Hydration is a key step in anthocyanin thermal degradation as the chaicone intermediary irreversibly breaks into the colorless degradation compounds (Sun et al., 2011; M. Zhao et al., 2013). Because PACN flavylium cation does not readily undergo hydration, based on the observations with pH changes (Section 2.10), the color-producing structure is retained in solution for a longer time. With enough thermal energy, PACNs do degrade into two degradation compounds. One of these was identified as a colorless phenolic acid, the same which forms with anthocyanin degradation, comprised of the B ring methoxy and hydroxyl substitution pattern (Voss et al., 2022; Voss, Miyagusuku-Cruzado, et al., 2023). The second degradation compound detected in these two experiments did not match the characteristics observed from anthocyanin degradation and differed depending on the PACN CIO substitution (Voss et al., 2022). However, in the case of 10-catechyl -PACNs it was colored (Voss et al., 2022; Voss, Miyagusuku-Cruzado, et al., 2023). Its formation likely helped contribute to the superior color stability of 10-catechyl- PACNs compared to 10-methyl and 10-carboxy -PACNs which only formed colorless degradation compounds (Voss et al., 2022).

[0441] In addition to the formation of the colored degradation compound from 10-catechyl- PACN thermal degradation, a unique, degradation compound has been observed to form when 10-methyl -PACNs were heated at high temperatures in highly acidic conditions (90 °C, IN HC1) (Lu & Foo, 2002). Named furoanthocyanidins, these compounds contain a furan ring at C3-OH and C4 position rather than the characteristic pyran ring and were first elucidated by Lu and Foo (2002). Furoanthocyanindins were also observed to form in aged strawberry wine and in the acetone extract of black currant anthocyanins, so the exact conditions (heat and high temperature) for their formation is still to be defined (Kurka et al., 2017; X. Wu et al., 2004)

[0442] Cold temperatures can also affect the color expression of PACNs. While irreversible degradation of PACNs (and anthocyanins) is often assumed to be minimized at lower temperatures, Carvalho et al. (2010a), observed the normally red colored vinyl-PACNs turning blue when frozen at pH 3. This color change was reversible and believed to be caused from changes in the vibrational frequency and 3 dimensional planarity (affecting electron delocalization) of the PACN molecule with freezing (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010a)

[0443] 2.12. Effect of Oxygen on Pyranoanthocyanin Color Expression and Stability. While oxygen can play a role in the formation of PACNs (Section 2.7), oxygen can also impact PACN stability and lead to the formation of a new PACN derivative. Oxidation is the process of losing electrons and, while oxidation can be caused by compounds, like metals, here we focus on the direct influence of oxygen on degradation of PACNs. One of the primary effects of oxygen on PACNs, specifically 10-carboxy -PACNs, is on the formation of a pyranone-anthocyanin (Jingren He, Oliveira, et al., 2010). This yellow colored degradation compound (Ais-max of -370 nm) was originally detected and elucidated by Jingren He, Oliveira, et al. (2010) with the malvi din-3 - glucoside derivative named oxovitisin (Alcaro et al., 2013; Jingren He, Oliveira, et al., 2010). Oxovitisin has been shown to have superior color stability to heat, pH changes (up to 7), and sulfur dioxide bleaching than PACNs and superior antiproliferative effects on cancer cells and cellular transport than PACNs (H. Oliveira et al., 2016; M. Wu et al., 2022).

[0444] 2.13. Effect of Food Ingredients on Pyranoanthocyanin Color Expression and Stability. As observed for anthocyanins, the presence of different additives can both enhance and destabilize the color. The same is true for PACNs, however few ingredients have been studied to date.

[0445] 2.13.1. Ascorbic Acid and Bisulfite. Ascorbic acid and bisulfite are categorized as bleaching agents as they cause the rapid fading of color for anthocyanins. Ascorbic acid, vitamin C, is commonly added in food beverages both as an antioxidant and for nutritional reasons. Bisulfite is often added in wines as a preservative and antioxidant (Jackson, 2008). They act by attaching themselves on the C ring of the anthocyanin (C4 is preferential position), which breaks the conjugation and results in color fading (Farr & Giusti, 2018). For PACNs, the primary point of the attachment is occupied by the D ring, resulting in improved color retention to these bleaching agents (Bakker & Timberlake, 1997; Farr & Giusti, 2018; Gomez-Alonso et al., 2012; Jingren He, Carvalho, et al., 2010; Quijada-Morin et al., 2010; Sun et al., 2020). A second reason for PACN color retention in response to bleaching agents is the formation of colored degradation compounds as observed with heat. Farr & Giusti, (2018) observed the formation of yellow colored degradation compounds from storage of 10-carboxy- pyranocyanidins with ascorbic acid, and Gomez-Alonso et al. (2012) observed the formation of a bisulfite adduct with 10-acetyl PACNs and a hypsochromic shift in color.

[0446] The CIO substitution does play a role on the bleaching stability. Because of the effect of pH and bisulfite levels, only intraexperiment comparisons were made here. Flavonoid-PACNs had superior resistance than 10-carboxy-PACNs (Jingren He, Carvalho, et al., 2010) while 10- carboxy-PACNs were shown to be more resistant than hydroxyphenyl -PACNs and 10-methyl- PACNs (Sun et al., 2020) and 10-carboxy-PACNs more resistant than 10-hydrogen-PACNs (Bakker & Timberlake, 1997).

[0447] 2.13.2. Metal Ions. Metal ions may be introduced to systems by trace contaminants, leaching from packaging (like a metal can), or from a different component (as in a food environment), and may interact with PACNs to affect the color. For metal chelation to occur with anthocyanins, the B ring must contain ortho substituted hydroxyl groups, as in delphinidin, cyanidin, and petunidin (Bayer et al., 1966). Trivalent metal ions were shown to be the most effective in anthocyanin metal chelation with common food grade ones including aluminum (Al3+) and iron (Fe3+) (Sigurdson et al., 2016). Studies on PACN metal chelation are scarce, however it is likely that the same trends in chemical structure and metal valency apply for PACNs. With PACNs, however, the CIO substitution group provides an additional location for chelation to occur.

[0448] Following the ortho hydroxyl group structure requirement, 10-catechyl-PACNs were observed to complex with ions even when the B ring should not allow (Vallverdu-Queralt, Biler, et al., 2016). Additionally, a CIO carboxylic moiety was suggested to interact with Al3+as a small bathochromic shift (17 nm) was observed for 10-carboxy-pyranomal vidin (Bakker & Timberlake, 1997). Interestingly, the color of 10-carboxy-pyranocyanidin became brown upon addition of Fe3+(Li, Yuan, et al., 2023). This comparison as well as others suggest the ion type has an impact on the complexation efficacy with PACNs (Li, Yuan, et al., 2023; Vallverdu- Queralt, Biler, et al., 2016). Divalent ions did not have a strong impact on PACN color (Li, Yuan, et al., 2023).

[0449] Interestingly, Alcaro et al. (2013) evaluated metal chelation between a divalent ion Fe2+and a malvidin-derived oxovitisin through theoretical modeling. Because this would have no ortho hydroxyl groups, the complexation was proposed to occur between the 4’ OH and a 3’ or 5’ OCH3 group (Alcaro et al., 2013). The effect on color and experimental evidence remains unknown, however.

[0450] 2.13.3. Proteins. Anthocyanin color can be modulated through interactions with whey proteins. Primarily driven by hydrophobic interactions, the color is observed to intensify (hyperchromic shift) when complexed (Miyagusuku-Cruzado, Jimenez-Flores, et al., 2021; Ren et al., 2021). Hydroxyphenyl-PACNs also interacted through hydrophobic interactions with whey protein; however the complexation did not result in a color change (at pH 3) (Miyagusuku- Cruzado, 2021). In addition to whey proteins, 10- / ?-hydroxyphenyl -PACNs interacted with mannoproteins, yeast-derived proteoglycans (mixture of protein and mannose) used in wine making, through hydrophobic forces (Liu et al., 2023; Moreno & Peinado, 2012). This complexation improved solubility and resulted in a hyperchromic and bathochromic shift (up to 72 nm) in ^vis-max (Liu et al., 2023).

[0451] 2.14. Tested Applications for Pyranoanthocyanins. With their bright colors and stability, PACNs are poised as promising colorants for different applications. While formerly limited by their scarcity in nature and slow production, the advances in efficient methods for formation and environmentally sustainable processes is helping to make a brighter future for PACNs as colorants. With much research evaluating PACNs as wine colorants or in isolated systems, there are still many gaps in understanding their performance in more complex matrices and finished applications.

[0452] 2.14.1. Skin Care and Cosmetics. PACN inclusion in cosmetics can provide both bioactive and coloring benefits. Correia, Araujo, et al., (2021), showed that various PACNs, including 10-carboxy, 10-methyl, 10-hydrogen, vinyl-PACN, and flavonoid-PACNs provided solar protection (SPF value) and could inhibit the activity of enzymes associated with skin aging (significance of effect varied by PACN type). As colorants, PACN performance may vary depending on the formulation as various ingredients led to different colors (Correia et al., 2023).

[0453] 2.14.2. Dye Sensitized Solar Cells. While not an edible product, one of the primary area of focus for PACN application research has been on exploring PACNs role as a photosensitizer dye in dye sensitized solar cells. Dye sensitized solar cells convert light energy into power through an electron transport process (Gratzel, 2003). Here, a dye (like PACNs) become excited by light, donate an electron in their excited state, and become regenerated to their lower state by an electrolyte solution (Gratzel, 2003). PACNs were shown to work in dye sensitized solar cells with efficiencies up to 1.5% (Pinto, Oliveira, et al., 2019). The chemical structure impacted the effect with 10-carboxy -PACNs being the least efficient (Pinto, Cruz, et al., 2019; Pinto, Oliveira, et al., 2019).

[0454] 2.15. Safety and Biological Activity of Pyranoanthocyanins. Published research on PACNs toxicological properties is in the early stage with toxicity being evaluated in cellular, insect, and mouse models to date. While toxicity is dependent on the dose and the type of PACN, studies have promisingly shown limited effects on viability and concluded PACNs show low levels of toxicity. Cellular toxicity has been evaluated for 10-carboxy -PACNs and 10-methyl - PACNs with toxic effects being observed at levels of 500 pg / mL and 200 pg / mL, respectively (Correia, Araujo, et al., 2021; Yuan et al., 2023; Z. Zhu et al., 2015). Toxicity of 10-carboxy- PACNs has also been evaluated using Galleria Mellonella wax moth larvae with lethality shown at 50 mg / kg (Coelho et al., 2021). Animal toxicity was evaluated for 10-methyl -PACNs using a mouse model with the lethal dose for 50% of the population (LD50) calculated to be between 6,800 - 7,900 mg PACN / kg body weight. The authors observed no deleterious effects on mouse organs and concluded that PACNs show non-toxic effects (Z. Zhu et al., 2015). Considering the estimated intake of anthocyanins, undeniably more abundant in the human diet than PACNs, is -12.5 mg / day for an individual in the United States (X. Wu et al., 2006), these toxic levels of PACNs are well below estimated consumption, supporting the conclusions of Z. Zhu et al. (2015). Additional toxicological data is needed, especially on different types of PACNs (i.e., hydroxyphenyl -PACNs) and their unique degradation compounds which may form during processing. However, the promising results, as well as a long history of human consumption of PACNs from red wine, support their safety.

[0455] Anthocyanins are well-known for their bioactive properties as discussed in Section 2.4.2. With their structural similarities, PACNs are also being assessed for bioactive properties, which, if found, would be an added benefit of using PACNs as ingredients beyond their beautiful, stable color. Both in vitro and in vivo cellular analyses have shown PACNs to have antioxidant and free-radical scavenging abilities (Azevedo et al., 2010; Faria et al., 2005; Garcia-Alonso et al., 2005; Garcia-Alonso et al., 2004; Muselik et al., 2007; Peng et al., 2016; Sun et al., 2020). The extent of their free-radical quenching ability is dependent on the type of PACNs, with 10- catechyl -PACNs showing the highest antioxidant potential in in vitro analyses in one comparison study (Sun et al., 2020). As observed with anthocyanins, PACNs derived from delphinidin were reported as stronger antioxidants (Garcia-Alonso et al., 2005; Muselik et al., 2007), although exceptions have been observed for 10-catechyl -PACNs (Azevedo et al., 2010). Of the identified 6 studies evaluating PACN antioxidant potential, two have shown PACNs to have greater potency than the anthocyanins with the rest showing either similar or lower levels.

[0456] In addition to antioxidant effects, a myriad of other positive health effects have been identified for PACNs. These include cytotoxic and antiproliferative effects on cancer cells (10- methyl and 10-carboxy -PACNs) (Faria et al., 2010; H. Oliveira et al., 2016; Pan et al., 2019), anti-inflammatory effects shown in both cellular studies and mouse models for hydroxyphenyl- sumac PACNs and 10-carboxy-PACNs, respectively (Li, Li, et al., 2023; Peng et al., 2016), ability to lower cellular cholesterol (10-carboxy-PACNs) (Yuan et al., 2023), and inhibition of tyrosinase enzyme activity (10-methyl and 10-carboxy-PACNs ) (Correia, Oliveira, et al., 2021). Many factors play a role in the biological efficacy of a compound including bioavailability. Despite their promising health effects, anthocyanins are generally considered as poorly bioavailable as they degrade quickly in the intestine and only a small percentage of consumed anthocyanins are often detected as absorbed (Goncalves et al., 2021; Jian He & Giusti, 2010; Li, Yuan, et al., 2023; Yang et al., 2018). While PACNs (10-carboxy) have greater gastrointestinal stability than anthocyanins (Li, Yuan, et al., 2023; McDougall et al., 2005; Yang et al., 2018), early studies have shown them (10-carboxy and 10-methyl -PACNs) to be more poorly absorbed than anthocyanins, hypothesized as due to a greater steric effect (Li, Yuan, et al., 2023; H. Oliveira et al., 2016). The future appears promising for PACNs to hold bioactive health effects, however additional research is needed.

[0457] 2.16. Regulatory Considerations for Pyranoanthocyanins. As discussed in Section 2.2, all food and cosmetic colorants used in the United States are regulated by the FDA as exempt from certification or as certified colorants (Food and Drug Administration, 2022b, 2023). While testing on heavy metals, potential side-products, and intermediaries is still needed, it is hopeful that PACNs could be regulated as exempt from certification colorants. Despite the promise of their long history of consumption in products such as wine, because they are formed from the result of a chemical reaction and could not be readily extracted from fruits and vegetables, there may be additional considerations for their petition approval.

[0458] In understanding potential routes, the colorant produced from Genipa americana fruit, also referred to as Huito and Jagua, is a valuable case study. Like PACNs, the color is produced by a chemical reaction, in this situation by the reaction of genipin (from G. americana) and amino acids (S. Wu et al., 2009). For years, it was listed as a mixture of two juices-Huito juice and watermelon juice concentrate- on products such as Pepperidge Farm’s Rainbow Goldfish® Colors snack crackers. Yet, in fall 2023, Jagua (genipin-glycine) blue was approved by the FDA as a new exempt from certification colorant listing (Food and Drug Administration, 2024a). It is reasonable to think that PACNs could be regulated as an exempt from certification colorant under a similar process, either as a mixture of fruit (and / or vegetable) juices or as a new colorant petition. Further regulatory guidance considering the specifics of PACNs is needed, however. For a new colorant petition, specific details on PACNs would need to be defined such as selecting the type of PACNs to be used by choosing the cofactor(s) or the anthocyanin source. For the selected PACNs, their degradation compounds, potential impurities (from the formation process and starting materials), estimated usage levels, estimated daily intake, and the potential applications would all need to be specified (Center for Food Safety and Applied Nutrition, 2009).

[0459] 2.17. The Bright Future for Pyranoanthocyanins. Innovation is at the forefront in the world of food colorants with continued identification of new sources and development of new ways to produce color. PACNs are promising colors producing a diverse array of colors and chemical properties which can meet the needs of both manufacturers and consumers. The future is optimistic, yet a paradigm shift is needed from viewing PACNs as colorants in wine to being wine-inspired colors which can be used to color consumer and commercial products. Further research is needed to support this perspective including further studies on their applications, interaction and stability to various ingredients and processing steps, and investigation of their safety and toxicology. Much remains to be understood about PACNs; however, they are poised to be key contributors in a colorful future.

[0460] Chapter 3. Diverse Color Performance of Hydroxyphenyl-Pyranoanthocyanins Across pH

[0461] 3.1. Abstract. Hydroxyphenyl-pyranoanthocyanins can be formed from a variety of anthocyanins and hydroxy cinnamic acids and 4-vinylphenol cofactors, resulting in pyranoanthocyanins with different numbers of hydrogen, hydroxyl, and, methoxy substitutions. Here, we assessed the influence of these substitutions on color expression, molar absorptivity coefficients, pH color stability, and solubility for eight different hydroxyphenyl- pyranoanthocyanins. By selectively reacting cyani din-3 -glucoside and malvi din-3 -glucoside anthocyanins with the various cofactors, eight hydroxyphenyl-pyranoanthocyanins were formed and isolated including the 10- / ?-hydroxyphenyl-, 10-catechyl-, 10-guaiacyl-, and 10-syringyl- derivatives of both anthocyanins. The color expressed in acidic conditions was primarily determined by the total number of hydroxyl and methoxy substitutions on the B and E ring; more substitutions gave a darker, redder color with a difference of 19 L* units and 41 hab degrees between the most and least substituted hydroxyphenyl-PACN tested (at pH 3). The location of these substitutions had a large influence on the color expression across pH. Most of the tested hydroxyphenyl -PACNs produced an orange to orange-yellow color until pH ~6.5 with a strong bathochromic shift in Ais-max (up to 106 nm) in alkaline conditions. However, 10- / ?- hydroxyphenyl -PACNs showed strong color fading near neutral pH and Mv3G derived 10- guaiacyl- and 10-catechyl -PACNs produced a pink (hab -4) and purple (hab -320-335) color, respectively, at a pH as low as 4.5. Some of the eight hydroxyphenyl-PACNs precipitated in aqueous conditions with precipitation most likely in high acid (-pH 1), mild acid, and mild alkaline conditions. The E ring substitution patterns had the largest influence on precipitation trends with 10-syringyl-PACNs least likely to precipitate. A diverse array of colors and solubility behaviors were observed for hydroxyphenyl-PACNs across pH with observable trends identified in how the type of substitutions affected their coloring properties.

[0462] 3.2. Introduction. Anthocyanins (ACNs) and pyranoanthocyanins (PACNs) are polyphenolic colorants producing vibrant yellow, orange, red, purple, and blue shades (J. Oliveira et al., 2014; Sigurdson et al., 2017). Both types of compounds are being considered as nature derived colorants for food, cosmetics, and consumer goods as concerns rise over the safety of artificial FD&C colorants (Miller et al., 2022) and as clean -label trends continue (Southey, 2022). ACNs are found in many plants including berries, flowers, and vegetables while PACNs, the product of a reaction between ACNs and small molecule cofactors, are more commonly identified in aged red wine or aged juices (Fulcrand et al., 1996; Hillebrand et al., 2004; Schwarz et al., 2004). Both types of colorants produce bright colors, but PACNs generally have superior color stability than ACNs to factors such as heat (Voss, Miyagusuku-Cruzado, et al., 2023), bleaching (Farr & Giusti, 2018), and pH changes (Jingren He, Carvalho, et al., 2010).

[0463] ACNs and PACNs are comprised of a 2-phenylbenzopyrylium (flavylium) backbone with PACNs containing an additional conjugated pyran ring between C5 and C4. As flavyliums, both ACNs and PACNs undergo structural changes when the environmental pH is altered. The general pathway is shown in Figure 12. In acidic conditions, the flavylium cation is predominant, but as pH increases, two competing reactions occur. There is kinetics-controlled deprotonation of the flavylium cation to form the quinoidal base and subsequent anionic derivatives, and there is thermodynamics-controlled hydration of the flavylium cation to form the hemiketal, tautomerization into the cis-chalcone, and isomerization to the trans-chalcone (Raymond Brouillard & Delaporte, 1977; Raymond Brouillard & Dubois, 1977b). Generally, for ACNs, the quinoidal base forms are purple to blue in color while the hemiketal and chaicone are colorless (Dangles & Fenger, 2018; Mazza & Brouillard, 1987b). However for most PACNs, hydration of the flavylium cation due to pH increase is not observed to occur (believed to be effectively minimized) due to decreased electronegativity at C2 hydration site hydration sites (Cruz et al., 2010; J. Oliveira et al., 2013; J. Oliveira, Mateus, et al., 2009; J. L. Sousa et al., 2017). Therefore, by default, deprotonation is the dominant reaction occurring as the pH increases for PACNs. As a result, PACNs show improved color retention across pH values compared to the precursor ACNs (J. Oliveira, Fernandes, et al., 2006; Sun et al., 2020). Understanding the equilibrium network and pKa constants is a necessary part in revealing the relationship between pH and color produced, and this has been the focus of several PACN studies (Cruz et al., 2010; J. Oliveira et al., 2013, 2014; J. Oliveira, Mateus, et al., 2009; J. Oliveira, Petrov, et al., 2011). Yet, in efforts to gain this understanding for PACNs, an assessment of color beyond the UV- visible spectra produced in the microsecond to minutes time scale is lacking among published literature. Knowledge of PACN appearance and full color characterization is critical for their future development and application as colorants.

[0464] Among PACNs, there is a large amount of structural diversity which impacts their formation efficiency and chemical properties. The PACN backbone is formed when a cofactor containing a nucleophilic carbon (i.e., acetone, pyruvic acid, acetaldehyde, hydroxy cinnamic acids, 4-vinylphenols) reacts with an ACN at the C4 and C5-OH position (J. Oliveira et al., 2014; Schwarz, Wabnitz, et al., 2003; X.-K. Zhang et al., 2022). Through a multistep reaction involving cycloaddition and dehydration, the pyran ring (D ring) forms with the type of PACN defined by the substitution at CIO (imparted by the cofactor) (Figure 13; Schwarz, Wabnitz, et al., 2003; Zeng et al., 2023; X.-K. Zhang et al., 2022). Of the different types of PACNs, the class of hydroxyphenyl -PACNs are emerging as a promising option to use as colorants. These PACNs formed from either hydroxy cinnamic acids or the decarboxylated 4-vinylphenols and can be formed efficiently under optimized accelerated conditions, reaching their maximum formation yields in as little as 48 hours (Miyagusuku-Cruzado et al., 2023; Schwarz, Wabnitz, et al., 2003). Additionally, the 10-catechyl-PACN (a type of hydroxy phenyl -PACN) had 4x greater color stability than PACNs from pyruvic acid when heated (10-carboxy -PACNs) (Voss et al., 2022).

[0465] There are four primary types of hydroxyphenyl -PACNs with either a -hydroxy phenol, catechol, guaiacol, or syringol moiety attached at CIO (Figure 13). Even among these hydroxyphenyl -PACNs, the small differences in hydroxyl and methoxy substitution patterns can make an influence on their formation efficiency, pKa coefficients, and kvis-max (Blanco-Vega et al., 2011; Miyagusuku-Cruzado, Voss, et al., 2021a; Pinto, Oliveira, et al., 2019). With the possibility to form PACNs from a wide array of ACNs and different hydroxycinnamic acid cofactors, understanding the role these chemical substitutions have on their chemical properties is important for their future as colorants

[0466] This study sought to address this research gap by investigating the role of hydroxyl and methoxy substitutions on the E ring (defined by hydroxy cinnamic acid used in formation) and B ring (defined by ACN type used in formation) on functional properties related to coloring (Figure 13). A goal was to systematically evaluate the role of B and E ring substitutions on the 1) color produced, 2) molar absorptivity coefficient, 3) color expressed across pH values, and 4) precipitation / solubility for 8 hydroxyphenyl-PACNs.

[0467] 3.2. Materials and Methods 3.2.1. Materials. Freeze-dried Berberis boliviana, used as the source of malvidin-3- glucoside (Mv3G), was donated by Carla del Carpio from Universidad Nacional de San Antonio Abad del Cusco (Cusco, Peru). Commercial elderberry (Sambucus nigra) ACN powder was used as the source of cyani din-3 -glucoside (Cy3G) (DDW The Color House, Louisville, KY, USA). Caffeic acid, ferulic acid, and sinapic acid were obtained from TCI Chemicals (Portland, OR, USA). / ?-Coumaric acid was obtained from MPBiomedicals (Solon, OH, USA). 4-Vinylphenol solution, 2-methoxy-4-vinylphenol (4-vinylguaiacol), and NaOH were obtained from Sigma Aldrich, (St. Louis, MO, USA). Optima® LCMS MeOH and HCL were obtained from Fisher Chemical (Fair Lawn, NJ, USA). KC1 salt was obtained from Alfa Aesar (Tewksbury, MA, USA).

[0468] 3.3.2. Anthocyanin Sourcing and Pyranoanthocyanin Formation. Cyanidin ACNs were obtained from dissolved elderberry ACN powder in water. From this solution, cyanidin- derived PACNs were formed following the procedure in Miyagusuku-Cruzado et al. (2021) with 1 :30 M ratio of ACN:cofactor, final pH of 3.1 (adjusted with HC1), and placed in an incubator set to 45 °C for several weeks. Cofactors included / ?-coumaric acid to form 10- / ?-hydroxyphenyl- PACNs, caffeic acid to form 10-catechyl-PACNs, ferulic acid to form 10-guaiacyl -PACNs, and sinapic acid to form 10-syringyl -PACNs.

[0469] Malvidin ACNs were obtained from freeze dried, de-pitted B. boliviana berries following the procedure detailed in Voss et al (2023). Briefly, ACNs were extracted using acetonechloroform partitioning (Rodriguez-Saona & Wrolstad, 2001), semi-purified with solid phase extraction to remove acids, salts, and highly polar phenolic groups, and a malvidin-rich ACN fraction was obtained through semi -preparatory high performance liquid chromatography (HPLC). HPLC solvents were removed from the eluate using solid phase extraction. Using this malvidin-rich ACN fraction, 10-catechyl-PACNs and 10-syringyl-PACNs were formed in the same way as described for cyanidin PACNs. 10- / ?-Hydroxyphenyl -PACNs and 10-guaiacyl- PACNs were formed following the procedure in Miyagusuku-Cruzado et al (2023) with 4- vinylphenol and 4-vinylguaiacyl cofactors, respectively, added at a 1 :5 M ratio (ACN: cofactor), final solution pH at 3.1 adjusted with HC1, and incubation at 35 °C for ~10 days to minimize ACN degradation.

[0470] After PACNs were formed, the solutions were semi-purified using solid phase extraction to remove preservatives and excess cofactor (Rodriguez-Saona & Wrolstad, 2001).

[0471] 3.3.3. Anthocyanin and Pyranoanthocyanin Isolation, Drying, and Weight Determination. Each of the 10 compounds evaluated (2 anthocyanin-3 -glucosides, 4 cyani din- derived hydroxyphenyl-PACNs, 4 malvidin-derived hydroxyphenyl -PACNs) were isolated from their respective formation solutions using semi-preparatory HPLC. This removed any remaining ACNs, cofactors, and PACNs with alternative glycosylation patterns (sambubioside in the case of cyanidin-derived PACNs and rutinoside in the case of malvidin-derived PACNs). A Shimadzu semi -preparatory HPLC was used with two LC-6AD pumps, CBM-20A controller, SIL-20A autosampler, SPD-M20A PDA detector (Columbia, MD, USA). Semi -preparation continued until the sample reached the desired purity level, noted in Section 3.1, changing columns and chromatographic gradients between isolation runs to minimize co-eluting peaks and increase sample purity as necessary. Each compound was isolated through at least 2 rounds of semipreparatory HPLC.

[0472] After the final semi-prep isolation, the isolated-compound-containing eluate was directly dried with an Eppendorf Vacufuge Plus (Enfield, CT, USA). Prior to drying, 0.01% HC1 MeOH was added at ~7% of the total eluate volume as preliminary work showed HC1 improved dissolution of the dried PACNs. Once dry, samples were re-dissolved in 0.01% HC1 in MeOH and subsequently dried; this was performed twice. On the third dissolution, the sample was dissolved in MeOH (no acid), filtered through 0.2 pM regenerated cellulose membrane, and placed in microcentrifuge tubes. These tubes had previously been rinsed with MeOH, dried, cooled to room temperature for 15 minutes, and weighed on a XP26 DeltaRange® microbalance (Mettler Toledo, Columbus, OH, USA). After the sample was dried, the tubes were again cooled to room temperature (15 minutes) and weighed.

[0473] The moles of compound in each tube were determined using the experimental weight of the compound in each tube multiplied by the molecular weight. The weight of a chloride counter ion and a water molecule of hydration was added to each molecular weight (Ahmadiani et al., 2016; Giusti et al., 1999; Sigurdson et al., 2019). To dissolve the dried colorants, MeOH was added, the solution was sonicated, and an equivalent volume of water was added to yield a 1600 pM stock solution (50% MeOH). New stock solutions were created for each experiment (Section 3.2.5 and Section 3.2.6) and for each of the 3 replicates.

[0474] Throughout the drying and weighing process, care was taken to minimize risk of impurities and foreign weight. This included using LCMS grade MeOH for all dissolutions, rinsing all tubes used to hold the isolated compounds (starting from the initial drying of semiprep eluate), and handling the microcentrifuge tubes with clean gloves and tweezers when obtaining the weight of dried compounds.

[0475] 3.3.4. Anthocyanin and Pyranoanthocyanin Identification and Purity Verification. The identities and purity of all 10 colorants were verified with a Nexera-i LC2040 ultra-high performance liquid chromatography with photodiode array detection and coupled to a LC4080 triple quadrupole mass spectrometer with electrospray ionization (uHPLC-PDA-ESI-MS / MS) (Shimadzu, Columbia, MD, USA). For chromatographic separation, a Restek Pinnacle DB-IBD column (1.9 pm, 50 x 2.1 mm) was used (Bellefonte, PA, USA). Solvent conditions included Solvent A: 3% formic acid in H2O and Solvent B: 3% formic acid in acetonitrile at a 0.30 mL / min flow rate and 50 °C column oven. Gradient conditions were 0-20% B in 4 minutes, 20- 35% B from 4 to 10 minutes, 35-45% B from 10 to 12 minutes followed by column equilibration. MS conditions included 2 L / min nebulizing gas, 11 L / min drying gas, 230 °C DL temperature, 200 °C heat block temperature, and ionization in positive mode. Precursor ion scans for each expected PACN and ACN aglycone and neutral loss for 162 (monosaccharide) and 294 (disaccharide sambubiose) were concurrently done with -35 eV collision energy.

[0476] Purity was defined based on the peak area in the 260-700 nm max plot with the potential for co-eluting peaks checked using targeted MS / MS methods. Purity was evaluated throughout the isolation process, and the final values for purity were based on the 3 replicates of samples used in the color across pH to account for the formation of any degradants during the drying process. These samples were mixed with -pH 3.7 LCMS H2O adjusted with HC1 and stored frozen prior to uHPLC-PDA-ESI-MS / MS analysis.

[0477] 3.3.5. Color Expression in pH 1 and 3 KC1 buffers and 0.1% HC1 MeOH. To compare the influence of chemical structure on color produced, PACNs and ACNs were evaluated in equivalent solutions, selected to be pH 1 and pH 3 0.25 M KC1 buffer and 0.1% HC1 MeOH (LCMS). KC1 buffers were prepared ahead of time with LCMS H2O with the desired pH reached by addition of HC1. For the pH 3 buffer, the pH was adjusted slightly low on the day of the experiment and adjusted with a small amount NaOH (0.005% of total volume). pH values were measured to be within ± 0.1 units of 1.0 and 3.0.

[0478] For color analysis, an aliquot the 400 pM pigmented stock solution was added to the buffers and MeOH to reach a final concentration of 40 pM at 5% MeOH. The solution was mixed and after 30 minutes of room temperature equilibration, the spectra were measured using a Shimadzu UV-2450 spectrophotometer (Columbia, MD, USA) from 260-700 nm with 1 nm step. Following, the pH was measured using a Mettler Toledo InLab Expert Pro ISM probe (Columbus, OH, USA). The pH probe was calibrated daily with 2.0, 4.0, 7.0, and 10.0 buffer with calibration slopes >97.2% and measurements made with intermediary read settings. PACN aggregation and the formation of precipitation was visually observed by the researchers during the experiment. Molar absorptivity coefficients were calculated using Beer-Lambert’s law with absorbance at the A is-max was used for the calculation. CIELAB color coordinates were calculated from the full spectra using Col orBy Spectra software under D65 illuminant and 10° observer angle (Farr & Giusti, 2017).

[0479] 3.3.6. Color Expression Across pH values. A range of pH values was achieved by directly adjusting water with HC1 or NaOH. These solutions were prepared at least 30 minutes prior to colorant addition. For each, 10 pL of either 0.1 N HC1, H2O, 0.025 N NaOH, 0.050 N NaOH, or 0.1 N NaOH were added to 2,690 pL of H2O (LCMS). After 30 minutes, the 400 pM ACN or PACN stock solution (described in 3.3.3) were added at 10% to yield a final concentration of 40 pM and 5% MeOH. The system was mixed with a pipettor. After 15 minutes equilibration, full spectra were measured as described in 3.3.5 and the pH was recorded in triplicate. The pH probe was calibrated daily with slope > 96.9% and readings were set in fast mode. Spectra and pH measurements were repeated at 45, 75, and 120 minutes to capture changes over time. PACN aggregation and the formation of precipitation was visually observed at each time point by the researchers.

[0480] 3.3.7. Statistical Analyses. Statistical comparisons among CIELAB L*, C*ab, and hab color coordinates and molar absorptivity coefficients were performed to evaluate the effect of B ring substitution, E ring substitution, and their interaction effect. For color coordinates and molar absorptivity coefficients from pH 3 KC1 buffer and 0.1% HC1 MeOH, a 2-way Analysis of Variance (ANOVA) was used with each solvent evaluated separately. For values from pH 1 KC1 buffer, a 1-way ANOVA was used since two PACNs were excluded due to precipitation. Statistical tests were performed using R-studio (v. 2022.12.0+353), including type 3 error and contrasts to account for the unbalanced data set (n = 3 for all samples except n = 2 of 10- guaiacyl-pyranomalvidin-3-glucoside). A / ?-value < 0.05 was considered statistically significant.

[0481] 3.4. Results & Discussion

[0482] 3.4.1. Anthocyanin and Pyranoanthocyanin Identity and Purity.

[0483] The identity of the formed PACNs and isolated ACNs presented in Figure 13 were verified using retention time, UV-visible absorption characteristics from PDA, m / z, MS / MS fragmentation patterns, and comparison of these characteristics to previous literature and the known reactants (Blanco-Vega et al., 2011; Miyagusuku-Cruzado et al., 2023; Miyagusuku- Cruzado, Voss, et al., 2021a). Purity determination was done retroactively on the samples to account for any compound degradation or formation of impurities during this time; chromatograms are shown in Figure 14 while purity values are in Figure 13. Mean purity was greater than 90% for 7 compounds (> 95% for 5 compounds). Purity below 90% was observed for 10-syringyl-PCy3G, 10-guaiacyl-PMv3G, and 10-catechyl-PCy3G. For 10-syringyl-PCy3G and 10-guaiacyl-PMv3G, the main impurity, accounting for ~10 and 3% of the overall peak area, respectively, eluted directly after the target PACN, absorbed visible light with a PDA spectrum similar to the target PACN, and had a m / z 28 units higher. For 10-catechyl-PCy3G, two main impurity peaks (~7% total peak area) eluted prior to the target PACN and showed hypsochromic kvis-max of 498 nm. As these impurities absorb visible light, they may have influenced the color expression and molar absorptivity coefficients. However, the target PACN was the dominant compound in the solutions.

[0484] 3.4.2. Influence of Chemical Structure on Color Expression. To evaluate the effect of structure on color while effectively minimizing the pH effect, all 10 colorants (2 ACNs, 8 hydroxyphenyl-PACNs) were evaluated under the same conditions of pH 1 and pH 3 KC1 buffers and 0.1% HC1 MeOH. The acidic pH condition of 1 was chosen as ACNs and PACNs are assumed to primarily be in a single structural form, the flavylium cation, at this high acid level, based on previous reports and pKa coefficients (Cruz et al., 2010; Mazza & Brouillard, 1987a, 1987b; Pinto, Oliveira, et al., 2019). Although few applications are found at pH 1, pH 1 KC1 (0.025 M) is used as the solution for the pH differential method in the AO AC method for determining monomeric anthocyanin content (Lee et al., 2005); therefore these coefficients allow for comparison to those previously determined for ACNs as well as adoption of the method for determining PACN concentration.

[0485] Higher pH values were not included due to solubility challenges in buffers. When assessing the effect of total number, type, and location of the B and E ring substitutions on PACN color, the 2 ACNs were excluded from statistical comparisons due to their evident structural differences and lack of an E ring. The comparisons and the below discussion focused on the behaviors in pH 3 KC1 buffer and 0.1% HC1 MeOH as 10-guaiacyl-PCy3G and 10- syringyl-PCy3G precipitated in pH 1 KC1 buffer.

[0486] Anthocyanin to Pyranoanthocyanin Comparison. All PACNs produced a more yellow-orange color (higher hab) with hypsochromically shifted kvis-max compared to the respective precursor ACN in both acidified MeOH and acidic buffers (Figure 15 & Figure 16). The extent of the color shift depended on the cofactor choice, however. Formation of 10- / ?- hydroxyphenyl -PACNs resulted in the largest yellow shift, with a hue angle increase by -52° and 58° for cyanidin and malvidin PACNs, respectively. Syringyl -PACNs had the smallest color shift, a hue angle increase of -25°, compared to the precursor ACNs. Therefore, 10-syringyl- PMv3G retained a red color in acidic conditions, with a hue angle of 22.9 ± 0.4 and 24.6 ± 0.3 at pH 1 and pH 3 buffer, respectively. The ACN choice had a slight effect on the magnitude of color change. In the formation of 10- / ?-hydroxyphenyl, 10-catechyl, and 10-guai acyl -PACNs, the magnitude of change for hue angle from the precursor ACN was ~3 to 7 ° larger for malvidin than for cyanidin. For 10-syringyl-PACNs, the magnitude of change was similar for both cyanidin and malvidin PACNs.

[0487] Additionally, all PACNs had superior color retention between pH 1 and 3 solutions compared to ACNs (Figure 15). While Cy3G and Mv3G experienced a decrease in lightness and color intensity, the PACNs had similar color intensity and hues between these two pH values. Section 3.4.4 will provide further discussion on the pH induced color and structural changes for ACNs and PACNs.

[0488] The shape of the UV-Vis spectra were similar between ACNs and PACNs in the visible region, however all PACNs produced a localized peak or shoulder at -285 to 305 nm in both aqueous and methanolic solvents which was absent in the two ACNs (Figure 16). This UV absorption peak was more pronounced in MeOH (0.1% HC1) than in buffer, and in 10-syringyl- PACNs. Among the 8 PACNs, the spectra of the two 10- / ?-hydroxyphenyl -PACNs had a shoulder in -380 to 400 nm region which is similar but hypsochromically shifted to the shoulder appearing in ACNs containing a phenol moiety in the B ring (pelargoni din-3 -glucoside) (Giusti & Wrolstad, 1996); Figure 16).

[0489] Hydroxyphenyl -PACNs lower Vis-max and yellow shifted color compared to the precursor ACNs is supported by previous work (Figure 16; Blanco-Vega et al., 2011; Miyagusuku- Cruzado, Voss, et al., 2021a; Vallverdu-Queralt, Biler, et al., 2016; Voss, Miyagusuku-Cruzado, et al., 2023). While PACNs may be predicted to have a bathochromic shift in the kvis-max a more purple / blue color compared to ACNs and due to the extended conjugation by the D and E rings, theoretical research by others have reported that the deviation from this expectation is due to changes in PACN molecular conformation. The PACN B ring is twisted out of plane at a greater angle than for ACNs, which, as a result, actually reduces electron conjugation within the PACN (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010b; Phan et al., 2021). Additionally, the E ring is also slightly out of plane from the PACN chromophore (A, C, D rings) (Phan et al., 2021). As a result, these hydroxyphenyl PACNs produced more yellow and orange colors than the precursor ACNs.

[0490] Influence of Total Number of B and E ring Oxygenated Substitutions on Color. Among the 8 hydroxyphenyl -PACNs, the total number of oxygen containing substitutions on the B and E ring had the greatest observable influence on color produced. When grouped based on total number of OH and OCH3 substitutions on the B and E ring, irrespective of type, the following subcategories could be made: 3 substitutions: 10- / ?-hydroxyphenyl-PCy3G; > 4 substitutions: 10-catechyl-PCy3G, 10-guaiacyl-PCy3G, 10-p-hydroxyphenyl-PMv3G; > 5 substitutions: 10-syringyl-PCy3G, 10-catechyl-PMv3G, 10-guaiacyl-PMv3G; > 6 substitutions: 10-syringyl-PMv3G. Trends in L* and hab followed the same pattern, with statistical differences between PACNs with 3, 4, 5, and 6 total substitutions (Figure 15). On occasion, additional statistical differences were observed within the groups with 4 and 5 substitutions; these will be further discussed in the following sections.

[0491] An exception to this rule was with hab reported in 0.1% HC1 MeOH solution in which 10- -hydroxyphenyl-PMv3G (4 total substitutions) and 10- / ?-hydroxyphenyl-PCy3G (3 total substitutions) had statistically similar hue angles and 10-catechyl-PCy3G (4 substitutions), 10- catechyl-PMv3G (5 substitutions), and 10-guaiacyl-PMv3G (5 substitutions) had statistically similar hue angles (Figure 15). The difference may be due to solvent as it is hypothesized that the PACNs may orient themselves differently in MeOH than in water.

[0492] These results support previous work of kvis-max observations from HPLC-PDA analysis of hydroxyphenyl -PACNs in which kvis-max were highest for PACNs with a tri substituted E ring (syringol), followed by disubstituted (catechol and guaiacol), and monosubstituted (hydroxyphenyl) (Blanco-Vega et al., 2011). Additionally, these observations are the same as for ACNs in which increasing the number of oxygen containing substitutions on the B ring yielded a higher kvis-max (Cabrita et al., 2000).

[0493] Influence of Type of Substitution (Hydroxyl versus Methoxy) on Color. By comparing 10-catechyl and 10-guaiacyl PACNs formed from the same ACN, the effect of hydroxyl versus methoxy substitutions on color was discovered. Between the two PACNs, there was only a slight difference on color expression with the solvent and B ring pattern (cyanidin compared to malvidin) affecting the color difference. For 10-catechyl-PCy3G and 10-guaiacyl- PCy3G at pH 3, 10-catechyl-PCy3G had a statistically higher hue angle (hab 51.9 versus 50.1°, respectively). In acidified MeOH, 10-catechyl-PCy3G had a statistically lower hue angle (hab 34.4 versus 38.4°, respectively) and a lower L* (71.7 versus 74.1, respectively). For the Mv3G derived PACNs at pH 3, 10-catechyl-PMv3G had a statistically higher hue angle and higher L* than 10-guaiacyl-PMv3G (hab 41.9 and 38.4, respectively. L*: 71.1 versus 69.2, respectively). In acidified MeOH, there were no statistical differences in color coordinates between 10-catechyl and 10-guaiacyl-Mv3G PACNs.

[0494] Location of Substitution (B or E ring) on Color. The effect of substitution location, whether on the B or E ring was assessed by comparing 10-syringyl-PCy3G with 10-catechyl- PMv3G. Both PACNs had a catechol moiety (2 ortho hydroxy groups) and syringol moiety (2 methoxy groups and 1 hydroxyl group) with the location on B and E ring flipped (Figure 13). There was a statistical difference in hab between these two PACNs in both pH 3 and acidified MeOH (Figure 15). A lower hue angle (redder color) was produced by 10-syringyl-PCy3G than for 10-catechyl-PMv3G (pH 3 hab: 38.5 versus 41.9, respectively. MeOH (0.1% HC1) hab: 28.3 versus 32.3, respectively).

[0495] Both PACNs have 5 total oxygenated B and E ring substitutions. As more substitutions clearly led to a lower hab among all 8 PACNs, the present observations may indicate that the E ring played a slightly larger role on influencing the color of a PACN. This supports previous observations from HPLC-PDA Xvis-max (Blanco-Vega et al., 2011) and could be explained by theoretical calculations which showed that the B ring had a greater dihedral angle (resulting in less electron delocalization) than the E ring (Phan et al., 2021).

[0496] 3.4.3. Influence of Chemical Structure on Molar Absorptivity Coefficient. The molar absorptivity coefficients for the 8 PACNs were higher than the coefficients for the 2 ACNs in all solutions. Coefficients were 1 ,7-2.3x greater for PACNs than for ACNs in 0.1% HC1 MeOH, 1.5-2. lx greater in pH 1 KC1 buffer, and 3.6-7.5x greater in pH 3 KC1 buffer. With these larger molar absorptivity coefficients, less PACNs would be necessary to produce a similar color intensity as ACNs, beneficial for decreasing costs and the potential for undesirable flavors and sensory attributes associated with nature derived colorants. As observed with chroma values in Section 3.4.2., there was minimal change in molar absorptivity coefficients for PACNs as pH changed from 1 to 3, while ACN coefficients decreased by more than half. All ACNs and PACNs evaluated produced their highest molar absorptivity coefficient in acidified MeOH as observed by others for ACNs (Ahmadiani et al., 2016; Giusti et al., 1999; Sigurdson et al., 2019). For the ACNs, molar absorptivity values increased by 4,100-4,700 from pH 1 to MeOH (0.1% HC1). For the PACNs, however, coefficients increased by 11,000 - 16,500 units from pH 1 to MeOH (0.1% HC1). This larger difference might be due to the orientation of the PACN in MeOH compared to aqueous solutions, as alluded to in discussion on color, and could also relate to solubility. While no visual precipitate were observed in these PACN samples for molar absorptivity calculations, solubility is a known challenge and non-visible aggregates may have begun to form in the aqueous solutions which could have affected the coefficients.

[0497] Statistical differences among the PACNs were largely occluded due to large standard deviations, however 10- / ?-hydroxyphenyl-PCy3G, with the least substitutions, had the lowest molar absorptivity coefficient, and 10-catechyl-PMv3G and 10-syringyl-PMv3G had the two highest coefficients (Table 5). Many factors including sample purity, solvent, equilibration time, stability, and the inclusion of counterions and water molecules of hydration in the molecular mass influence molar absorptivity coefficient results (Giusti et al., 1999; Jordheim et al., 2007). The solvent, equilibration time, and compound stability are especially important for PACNs where less is known on their behavior in isolated systems but where they have been observed to form aggregates and precipitate within 1 hour (Vallverdu-Queralt, Biler, et al., 2016). The selection of a 30-minute equilibration period and KC1 buffers were chosen to minimize the likelihood of PACNs precipitating prior to absorption measurements. Nevertheless, 10-guaiacyl- PCy3G and 10-syringyl-PCy3G were still observed to form precipitates and rapidly change in color, developing a more pink and purple hue at pH 1 within 30 minutes. More discussion on precipitation tendency is provided in Section 3.4.6.

[0498] Because of the many considerations and influential parameters on molar absorptivity coefficients, there is a large amount of variation in reported values among researchers (Giusti et al., 1999). For Cy3G and Mv3G ACNs, the calculated molar absorptivity coefficients herein fit within the range of values reported in literature for similar solvents. For Cy3G, previous work reported value of 20,000 (pH 1 0.25 M KC1 buffer and 0.2 M KC1 / 0.2 M HC1 buffer) (Cabrita et al., 2000; Jordheim et al., 2007) which is similar to our calculated value of 22,800 (pH 1). These same authors also reported similar coefficients for Mv3G at 22,000 (0.2 M KC1 / 0.2 M HC1 buffer) (Jordheim et al., 2007) and 23,400 (0.2 M KC1 buffer) (Cabrita et al., 2000) which are close to the reported value herein of 23,800 in pH 1 0.25 M KC1 buffer.

[0499] The reported molar absorptivity values herein for hydroxyphenyl-PACNs are higher than previously reported. For example, Vallverdu-Queralt reported values of 18,000 for 10-catechyl- PMv3G and 42,500 for 10-guaiacyl-PMv3G in 0.1% HC1 MeOH when tested at similar concentrations to the present work (Vallverdu-Queralt, Meudec, et al., 2016). These same PACNs were reported to have molar absorptivity coefficients of -4,500 for 10-catechyl-PMv3G and -3,000 for 10-guaiacyl-PMv3G at pH 0.8 1 M Citrate Buffer (Quijada-Morin et al., 2010), much lower than the values determined in the present experiment. In Quijada-Morin et al. (2010), a 2-hour equilibration period and higher molar concentrations were used; the PACNs may have aggregated or precipitated during this period resulting in the lower absorptivity coefficients compared to the present experiment.

[0500] Table 5. Molar absorptivity coefficients calculated for anthocyanins and 8 hydroxyphenyl- pyranoanthocyanins at pH 1 KC1 buffer, pH 3 KC1 buffer, and 0.1% HC1 MeOH. Letters show statistical differences between PACNs within a column at a p < 0.05 level with analyses done using non-rounded coefficients.

[0501] 3.4.4. Color Expressed Across pH. For Cy3G and Mv3G ACNs, the color at pH 3 was a pale pink (C*ab at 10 and 16 units and hab 11° and 1°, respectively; Figure 17 and Figure 18). The color faded nearly entirely with increasing pH to neutral, but as the pH moved to the alkaline range, a purple (Cy3G) and blue (Mv3G) color formed with strong intensity in high alkaline (pH 9.7) solutions. This behavior is consistent with that reported for flavylium compounds and mono-glycosylated ACNs, shown in Figure 12. The pale color from pH 3 through 6 was likely due to the abundance of the colorless hydrated hemiketal and chaicone (Raymond Brouillard & Delaporte, 1977; Raymond Brouillard & Dubois, 1977b; Mazza & Brouillard, 1987a) with the small peak at -275 nm possibly indicative of the hydrated forms such as hemiketal and chaicone (Mazza & Brouillard, 1987b). The appearance of blue and purple color in alkaline conditions likely resulted from the formation and retention of the quinoidal base and anionic quinoidal base forms (Dangles & Fenger, 2018; Mazza & Brouillard, 1987a).

[0502] All hydroxyphenyl-PACNs continued to produce color across pH from 3 through -10 with greater intensity throughout than for the ACNs (Figure 17 and Figure 18). PACN color and intensity was not stagnant, however. Six of the eight hydroxyphenyl-PACNs had similar behaviors with pH with exceptions and unique behaviors discussed later. As pH increased from 3.5 to -4.7, color intensity decreased slightly and there was a hypsochromic shift (1 to 17 nm) in kvis-max. As pH continued to increase to -6.4, color intensity continued to decrease and become more yellow for 4 of the 6 PACNs. In understanding the chemical structure and distribution changes occurring across pH for the hydroxyphenyl-PACNs, we adopted the assumption that hydration did not occur in appreciable quantities based on the discoveries by others for similar compounds (Cruz et al., 2010; J. L. Sousa et al., 2017). Therefore, as the pKa for the flavylium cation deprotonation to quinoidal base is reported at -4.3-4.8 for hydroxyphenyl-PACNs (Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016), a yellow colored quinoidal base was likely the most abundant chemical species in these mildly-acidic PACN solutions. The yellow hued PACN quinoidal base with a hypsochromic kvis-max compared to the flavylium cation is supported by others (Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016).

[0503] As the pH moved to alkaline, the spectral shape for the hydroxyphenyl-PACNs became bimodal with a newly formed peak with a strong bathochromic shift in kvis-max. Color intensity increased and in high alkaline conditions, a deep pink, purple, and blue colors were produced (C*ab from 74 to 82 and hab from 303° to 354°). These bathochromic shifts are consistent with the formation of greater quantities of the anionic quinoidal base (Cruz et al., 2010) with pKa values for this deprotonation reported at 7.2-7.9 (Sousa 2017; Pinto 2019). As with the color expressed at acidic pH conditions (Section 3.4.2), 10- / ?-hydroxyphenyl-PCy3G with the least number of oxygenated substitutions (3) produced the highest hue angle while 10-syringyl- PMv3G with the most substitutions (6) produced the lowest hue angle. While for most of the PACNs (7 / 8) the hue angle in these alkaline conditions was still higher than for the respective ACN, for 10-syringyl-PCy3G, the hue angle (316°) was lower than the hue angle for Cy3G (321°).

[0504] Several PACNs deviated from these trends with the location of hydroxyl and methoxy substitution patterns driving the patterns. The two 10- / ?-hydroxyphenyl -PACNs showed unique behavior at 4.5 to 6.5. Rather than the aforementioned hypsochromic shift in Ais-max, there was a large loss in chroma (C*ab decreased by ~41 and 53 units) and the color was purple-grey with no clear kvis-max (Figure 17 and Figure 18). In comparison, the maximum chroma decrease was ~18 units for the other PACNs. At other pH values, the 10- / ?-hydroxyphenyl -PACNs behaved according to the trends discussed prior.

[0505] For 10-catechyl and 10-guaiacyl-PACNs, those derived from Mv3G had different behaviors in mild acid pH than previously described. While the Cy3G derived 10-catechyl- and 10-guaiacyl-PACNs had relatively stable hue angles from pH 3.5 to ~6.5, changing by 2 to 16°, the hue angles for the Mv3G derivatives dropped by 67° (10-catechyl) and 38° (10-guaiacyl) when the pH increased from 3.5 to ~4.5 (Figure 17 and Figure 18). A slightly lower pKa has previously been reported for 10-catechyl-PMv3G (4.4) compared to 10-catechyl-PCy3G (4.7) (Pinto, Oliveira, et al., 2019) which would imply that the quinoidal base is present in a larger proportion at pH 4.5 than for the Cy3G derivatives. The quinoidal base could produce a redder color (lower Ais-max) than the flavylium cation, as is reported for other PACNs like 10-carboxy and 10-methyl (J. Oliveira et al., 2013; J. Oliveira, Petrov, et al., 2011). The color of the solutions and the location of the second peak in the bimodal curve is similar to that observed to indicate PACN aggregation and precipitation in acidic conditions (Section 3.4.6; Pinto et al., 2019). While precipitate was observed at later time points in both 10-catechyl-PMv3G and 10- guaiacyl-PMv3G, this bathochromic peak did not grow over time and no aggregation-peak was observed in the Cy3G PACN derivatives which also precipitated. Therefore, we believe that the first hypothesis is a more likely explanation to the unique color behavior of 10-guaiacyl-PMv3G and 10-catechyl-PMv3G across pH.

[0506] 3.4.6. Pyranoanthocyanin Solubility. The solubility of the hydroxyphenyl-PACNs was dependent both on the pH of solution and the chemical structure and the hydroxyl and methoxy substitutions. Precipitation was based on visual observations of the solutions by trained individuals with precipitate defined by the presence of visible solids. Because the pH values changed over the two hours (monitored at four time points), the pH range in which precipitation was observed is listed in Table 6.

[0507] In pH 1 KC1 buffer, precipitation was noted by small colored particles suspended in solution, the formation of a bathochromic peak in the spectra, and the overall color of the solution appearing pink / purple rather than the expected orange color (Figure 19). The formation of aggregates and visible precipitation with the accompanying spectral behaviors are consistent with previous observations for 10-syringyl-PCy3G at pH ~1.6 (Pinto, Oliveira, et al., 2019) and 10-guaiacyl-PMv3G at pH -4.4 and -5.2 (Vallverdu-Queralt, Biler, et al., 2016).

[0508] In aqueous solutions with pH directly adjusted, precipitation was most common in the mild acid to mild alkaline conditions (pH 4.4 to 8.4). Precipitation was primarily observed visually with the color of the precipitate the same as the solution. The positively charged flavylium cation and negatively charged anionic quinoidal base would be more abundant at the tail ends of the pH values measured, and it has been previously been discovered by Vallverdu- Queralt, Biler, et al (2016), that the electrostatic charge of these species minimized PACN selfaggregation which leads to precipitation. The observations herein support this hypothesis with precipitation most likely to occur at pH values when the quinoidal base is reported to predominate. Precipitation trends appeared driven by the E ring substitution, with 10-syringyl- PACNs exhibiting the best solubility while 10-guaiacyl and 10-catechyl-PACNs most likely to precipitate. Under the hypothesis that PACN aggregation is driven by 7t-7t stacking (Vallverdu- Queralt, Biler, et al., 2016), it was hypothesized that the E ring played a greater role in precipitation because it is connected at a lower dihedral angle than the B ring, based on the findings in Phan et al (2021). Greater molecular planarity at the E ring compared with the B ring could facilitate intermolecular aggregation in that region. Theoretical calculations and molecular modeling can test this hypothesis. While precipitation in aqueous solutions made it challenging to take spectral measurements, the different solubility behaviors among the hydroxyphenyl- PACNs highlighted their diversity.

[0509] 3.4.5. Unique Color Observations for Hydroxyphenyl-Pyranoanthocyanins. Several unexpected changes in the colors of the hydroxyphenyl-PACNs occurred during drying and freezing steps which were part of the isolate preparation. While not part of the formal investigation, these observations are shared, as future research efforts may encounter them or could focus on further understanding. When the isolated samples were frozen (in acidic conditions, not tested in an alkaline environment) all hydroxyphenyl-PACN solutions became visually more pink / purple (Figure 20). Interestingly, among the eight hydroxyphenyl-PACNs we tested, 10-guaiacyl-PMv3G appeared to have the greatest shift in color, producing a blue color once frozen (Figure 20). Additionally 10-guaiacyl-PMv3G dried as a blue-green color which was not observable in the other hydroxyphenyl-PACNs (Figure 20). A red to blue color shift was previously observed for vinyl-PACNs upon freezing with the mechanism determined to be due to the cold temperatures stabilizing a more planar chemical conformation and increased vibrational frequency (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010a). Perhaps this effect is greater for 10-guaiacyl-PMv3G which led to the more blue-color.

[0510] Table 6. Frequency of hydroxyphenyl-pyranoanthocyanin precipitation across pH values. For pH ranges in which the pH decreased, (s) indicates the highest starting pH and (e) indicates the lowest ending pH. Times indicate the approximate time in which precipitation was first observed across 2 hours. PCy3G: pyranocyani din-3 -glucoside. PMv3G: pyranomalvi din-3 -glucoside

[0511] * Precipitated observed in 3 / 3 repetitions ** Precipitated observed in 1 or 2 of the repetitions

[0512] No mark = No Precipitation was observed

[0513] 3.5. Conclusions. Hydroxyphenyl-PACNs produced a diverse array of colors influenced both by the chemical structure and solution pH with a range of solubilities and color stabilities depending on the chemical structure. The total number of oxygenated substitutions, irrespective of the location on the B or E ring, had a strong influence on the color in acidic conditions with hues ranging from yellow (hab = 60°) to red (hab = 27°). The location of these substitutions, whether on the B or E ring, had a larger influence on the color stability across pH and the likelihood of precipitation. Compared to their precursor ACNs, Cy3G and Mv3G, the yellow, orange, and red colors of the hydroxyphenyl-PACNs produced in acidic conditions were more vibrant with extinction coefficients up to 2.3x greater in MeOH and more stable across pH with orange colors retained up to ~6.5 for some PACNs. Hydroxyphenyl-PACNs shown promise as colorants for a diverse array of applications with their vibrant colors across pH, high molar absorptivity coefficients, and variable solubility behaviors with their chemical structure affecting these properties in consistent ways.

[0514] Chapter 4. Impact of Glycosylation on Color Expression and Solubility of 10- Guaiacyl-Pyranocyanidins Across pH

[0515] 4.1. Abstract. Hydroxyphenyl-pyranoanthocyanins, a type of nature derived colorant formed from anthocyanins in fruits and vegetables when reacted with hydroxy cinnamic acids and 4-vinylphenols, produce bright colors and have excellent heat and bleaching stability in comparison to their precursor anthocyanins. A unique behavior of hydroxyphenyl- pyranoanthocyanins, especially the 10-guaiacyl-pyranoanthocyanins, is their tendency to precipitate in aqueous solutions. An objective was to evaluate how glycosylation substitutions influenced the color, molar absorptivity, solubility, and 60-day stability of 10-guaiacyl- pyranocyanidins in various buffer solutions from pH 1 to 8. A total of six 10-guaiacyl- pyranocyanidins with different glycosylation substitutions at carbon 3 position were formed and isolated. Glycosylation substitutions included the three monosaccharides of arabinose (pentose), glucose (hexose), and galactose (hexose), two disaccharides of xylosyl( l ^2)glucose and xylosyl( l ^2)galactose, and one trisaccharide of xylosyl( l ^2)glucosyl( l ^6)galactoside. Five of the six 10-guaiacyl-pyranocyanidins produced orange to yellow orange colors with small but significant differences in the color induced by the glycosylation. A pink color was produced by 10-guaiacyl-pyranocyandin-3-arabinoside as it rapidly precipitated in aqueous conditions. While precipitation across pH was common for the 10-guai acyl -PACNs with a monosaccharide substitution, those with two or three sugars attached remained soluble in a majority of for up to 60 days at room temperature with only a 10-24% reduction in absorbance (pH 1 - 7). Color was produced across pH, but the hue and intensity shifted from orange (acidic pH) to yellow (mild acid) to purple (alkaline) irrespective of the glycosylation substitution. When forming PACNs, the glycosylation substitution of the parent anthocyanin is an important element of the chemical structure to consider, shown to influence the color expression, color stability, and most evidently, the solubility of 10-guaiacyl-pyranocyanidins.

[0516] 4.2 Introduction. Flavylium compounds represent an abundant class of nature derived colorants including 3 -deoxy anthocyanins, auronidins, anthocyanins, and their derivatives (Cruz et al., 2022). Comprised of a 2-phenylbenzopyrylium backbone, flavylium compounds are abundant in nature, found in different plant sources such as fruits and vegetables (X. Wu et al., 2006; Xiong et al., 2019). One of the defining characteristics for flavyliums is the dependency of their chemical structure and functional properties on the solution pH. Under acidic conditions, the namesake flavylium cation is present, however with increasing pH, the flavylium cation can become hydrated to form hemiketal and chaicone and the flavylium cation can become deprotonated to form quinoidal base species (Raymond Brouillard & Dubois, 1977b). While this pH structure dependency imparts versatility into the performance of many flavylium compounds, it is also one of the primary challenges for their application. For anthocyanins, abundant and brightly colored flavylium compounds, the hydrated forms are pale to colorless which limits their application in many mild-acid foods and consumer goods (Dangles & Fenger, 2018; Mazza & Brouillard, 1987b). Therefore, there is a need to identify methods to improve anthocyanin color retention across pH.

[0517] A promising method to enhance anthocyanin color stability is by the chemical addition of a second pyran ring between C4 and C5 position (Figure 21). This new molecule, called a pyranoanthocyanin (PACN), has greater color stability to pH (Jingren He, Carvalho, et al., 2010; J. Oliveira, Fernandes, et al., 2006), heat (Voss, Miyagusuku-Cruzado, et al., 2023) and bleaching (Farr & Giusti, 2018; Sarni -Manchado et al., 1996). This chemical transformation of anthocyanins can be done through guided formation under optimized conditions (Kuang et al., 2014; Miyagusuku-Cruzado et al., 2023; Miyagusuku-Cruzado, Voss, et al., 2021a). It also occurs organically in wine fermentation and aging (Mateus & de Freitas, 2001a; X. K. Zhang et al., 2021) and in stored fruit juices (Hillebrand et al., 2004; Schwarz et al., 2004). In fact, PACNs are well known in the wine community and are a major contributor to the long lasting, orange brown hues of an aged wine (Quaglieri et al., 2017; X. K. Zhang et al., 2021).

[0518] PACNs are a highly diverse class of nature derived colorants, employing both the natural diversity of anthocyanins with the numerous different types of cofactors available which include acetone, pyruvic acid, acetaldehyde, acetone, diacetyl, vinyl-flavonoids, hydroxycinnamic acids, and 4-vinylphenols (Benabdeljalil et al., 2000; Blanco-Vega et al., 2011; Fulcrand et al., 1996, 1998; Schwarz, Wabnitz, et al., 2003). The cofactor choice will affect the CIO substitution while the anthocyanin choice affects the B ring substitutions and the glycosylation and acylation attachment most commonly at C3 (Figure 21). While previous work evaluated the effect of hydrogen, hydroxyl, and methoxy substitutions on the E and B ring substitutions (Chapter 3), the focus of the present work is on the influence of glycosylation substitutions on PACN properties.

[0519] Sugars alone have no chromophore. However, they have been shown to influence the color expression and pH color stability of anthocyanins (Farr et al., 2019; Sigurdson et al., 2018), believed to be due to their influence on the anthocyanin geometry and their interaction with the solution (forming hydrogen bonds water) (Mai ci oglu et al., 2011). Here, we focused on the effect of glycosylation on performance for 10-guaiacyl-pyranocyanidins. As a type of hydroxyphenyl-PACNs, 10-guaiacyl-pyranocyanidins can form in appreciable yields in a few days with 4-vinylguaiacol cofactor (Miyagusuku-Cruzado et al., 2023). Additionally, cyanidin is the most naturally abundant anthocyanin type with many different plant sources and glycosylation patterns readily available (Andersen & Jordheim, 2010). While in general, PACNs are considered to have improved stability compared to the precursor anthocyanins, a unique property of some PACNs, especially 10-guaiacyl-pyranocyanidins, is their tendency to aggregate and precipitate in aqueous solutions (Vallverdu-Queralt, Biler, et al., 2016); Chapter 3).

[0520] Thus, as glycosylation substitutions increase hydrophilicity (C.-L. Zhao et al., 2014), 10- guaiacyl-pyranocyanidins may serve as an excellent model environment to evaluate glycosylation effect on PACN performance. An objective was to evaluate the effect of glycosylation type and number on 10-guaiacyl-pyranocyanidin color expression, stability, and solubility across pH values. Additionally, we sought to evaluate how buffer types affects these properties.

[0521] 4.3 Materials & Methods

[0522] 4.3.1. Materials. Commercial purple carrot ( aucus carota) and elderberry (Sambucus nigra) anthocyanin powders were from DDW The Color House (Louisville, KY, USA). Standardized chokeberry (Aronia melanocarpa) powder-10% was from Artemis International (Fort Wayne, IN, USA). HPLC grade water and 4-vinylguaiacol were from Sigma Aldrich (St. Louis, MO, USA). Formic acid (Supelco, 98%, GR ACS) and EtOH (Supelco, Emsure® ACS, ISO, Reag.) were from Millipore Sigma (Burlington, MA, USA). Methanol (Optima® LCMS grade) and hydrochloric acid (ACS Plus) were from Fisher Chemical. LCMS grade water (Chromasolv™) was from Honeywell (Muskegon, MI, USA). Details on the ingredients used for buffer preparation are in Table 7.

[0523] Table 7. Formulations and product brands used for buffer preparation.

[0524] 4.3.2. Formation of Pyranoanthocyanins. In preparation for PACN formation, the purple carrot, elderberry, and chokeberry anthocyanin powders were each dissolved in water. Purple carrot anthocyanins were subjected to alkaline hydrolysis to increase the abundance of non-acylated anthocyanins following the protocol in Durst and Wrolstad (2001). The released acylating groups were removed from the solution prior to PACN formation with C18 cartridge solid phase extraction as detailed in Rodriguez-Saona and Wrolstad (2001).

[0525] For 10-guaiacyl-PACN formation, each anthocyanin solution was mixed with 4- vinylguaiacol at molar ratios between 1 :5 to 1 : 10 and with preservatives (0.1% potassium sorbate and 0.1% sodium benzoate) to minimize microbial growth. The final solution was adjusted to a pH ~3.1 with HC1 and incubated at ~40 °C as detailed elsewhere (Miyagusuku- Cruzado et al., 2023). Samples were removed from the incubator once the anthocyanins were nearly depleted (2 to 5 days) and refrigerated. Solids were filtered out, and the remaining cofactor and preservatives were removed by solid phase extraction as previously described. Semi-purified solutions of PACNs were re-suspended in acidified MeOH (0.01% HC1) and water (0.01% HC1).

[0526] 4.3.3. Isolation of Pyranoanthocyanins with Semi-Preparatory HPLC. Each of the anthocyanin sources contained more than one type of glycosylated cyanidin anthocyanins, and therefore, each of the PACN solutions formed from these sources were identified to contained more than one type of 10-guaiacyl-pyranocyanidin. The individual PACNs with a single glycosylation pattern were isolated from each solution with a Shimadzu semi -preparatory high performance liquid chromatography (HPLC) comprised of two LC-6AD pumps, SIL-20A autosampler, CBM-20A controller, and SPD-M20A photodiode array detector (Columbia, MD, USA). Separation was achieved using mobile phase A: 3% formic acid H2O and B: acetonitrile, at a 12 mL / min flow with the gradients dependent on the sample. Each PACN was first isolated using a Luna PFP column (250 x 21.20 mm; 100 A) followed by a second isolation with a Synergi Max RP80 column (250 x 21.2 mm; 80 A) (Phenomenex, Torrance, CA, USA). Two isolation runs with different columns helped to increase purity and minimized co-eluting contaminants.

[0527] A third round of semi-preparatory isolation was performed to transfer the PACN into a faster evaporating solvent (acetonitrile). This method was chosen rather than C18 cartridges as the C18 resin was previously observed to leach from the cartridges and these contaminants could inflate the weight of the isolate without appearing on the HPLC-PDA purity chromatogram (Voss, Tang, et al., 2023). The Synergi Max RP80 column mentioned prior was used with a mobile phase of A: 1% formic acid in H2O and B: acetonitrile, both made with HPLC grade solvents. For isolation of I O-guaiacyl-pyranocyanidin-3-xylosyl-( l ^2)-glucosyl-( l ^6)- galactoside, the following gradient was used: 10% B from 0 to 7 minutes, 10 to 60% B from 7 to 8 minutes, 60% B from 8 to 14 minutes. For the other 5 pyranoanthocyanins, the following gradient was used: 20% B from 0 to 7 minutes, 20 to 60% B from 7 to 8 minutes, 60% B from 8 to 14 minutes. Under these gradients, the PACNs eluted under higher acetonitrile percentage to shorten the evaporation time.

[0528] 4.3.4. Drying and Weighing Isolated Pyranoanthocyanins. The isolated PACNs were dried 4 times to facilitate evaporation of potential residual water and formic acid. Evaporation was performed in a Vacufuge plus vacuum centrifuge evaporator (Eppendorf, Enfield, CT, USA) set to 30 °C. First, the PACNs were dried directly from the semi-prep eluate with MeOH and EtOH added as necessary to facilitate evaporation and 1.4 pL HC1 (12 N) added to aid in solubilization. Second, the dried samples were redissolved in MeOH with 1.2 pL HC1 and dried again. Third, the samples were redissolved in MeOH (no acid) and dried. After the third round of drying, the samples were redissolved in MeOH with 0.6 pL HC1 and filtered through a 0.22 pM regenerated cellulose filter to remove solids which may have entered during the drying process.

[0529] To obtain an accurate weight for the dried PACNs, 2 mL microcentrifuge tubes were rinsed with MeOH three times, let dry in a fume hood, and further dried in a vacuum evaporator at 30 °C for 2 hours. After 15 minutes at room temperature, the cooled, empty tubes were weighed on a DeltaRange XP26 microbalance (Mettler Toledo, Columbus, OH, USA). The filtered PACN stock was added to the weighted tubes, dried in the vacufuge for 2.5 hours (4thdrying), and weighed after 15 minutes of room temperature equilibration.

[0530] To minimize contamination, high purity MeOH (LCMS) and EtOH (ACS, ISO, Reag) were added to the samples in these steps. Additionally, all tubes holding the samples and solvents, starting from collection of the semi-prep eluate, were rinsed 3 times with MeOH (LCMS).

[0531] 4.3.5. Identification and Calculation of Isolate Purity. Throughout the PACN formation and isolation process, the identity and purity of the PACN solutions were checked with HPLC-PDA-MS / MS using a Shimadzu Nexera-i LC2040 HPLC with photodiode array detector coupled to a LCMS8040 triple quadrupole mass spectrometer (MS) with electrospray ionization (Columbia, MD, USA). Purity was tested after all drying steps to account for possible contaminants and degradation compounds. After subtracting the peak area for compounds found in the water blank (i.e., void volume), purity was calculated as the peak area of the target PACN divided by the peak area of all peaks in the 260-700 nm max plot. Chromatographic separation was achieved using a Restek Biphenyl (2.7 pM, 100 x 2.1 mm; Bellefonte, PA, USA), 40 °C column oven, and a mobile phase of A: 3% formic acid in H2O and B: 3% formic acid in H2O. The following gradient was used: 10% B for 0 to 2 min., 10-40% B from 2 to 15 min., followed by column equilibration. MS parameters included 2 L / min nebulizing gas, 11 L / min drying gas, 230 °C desolvation line, and 200 °C heat block. Positive ionization with precursor ion scans, neutral loss, and selective ion monitoring was performed.

[0532] 4.3.6. Evaluation of Color Expression, Molar Absorptivity, Stability and Solubility

[0533] Preparation of PACN Stock Solutions. The weight of each isolated PACN was calculated as the weight difference between the empty tube and the tube plus the dried PACN. A chloride counter ion and water molecule of hydration were added to the molecular weight for the mass calculations (Ahmadiani et al., 2016; Giusti et al., 1999). Each PACN was first dissolved in MeOH followed by an equivalent amount of water to reach a 50% MeOH stock. For 10- guaiacyl-pyranocyani din-3 -arabinoside, pure MeOH was used for all stock solutions as evident precipitation was observed when water was added.

[0534] Preparation of Buffers. A total of 18 buffers, listed in Table 8, were evaluated to capture the effect of pH and buffer type on color expression and stability. Buffers were prepared following instructions in Millipore Sigma Buffer Reference Center (n.d.) and in Sigurdson et al. (2018) for Tris buffer preparation. Additional details on buffer formulations and ingredients are in Table 7. Table 8. Types of buffers used from pH 1 through 8 to evaluate color performance of 10- guai acyl -py ranocy ani dins .

[0535] Determination of Molar Absorptivity Coefficients and Influence of Glycosylation on

[0536] Color Expression. Molar absorptivity coefficients were calculated in pH 1 KC1 buffer, pH 3 KC1 buffer, and MeOH (0.1% HC1). To each solution, PACN was added to achieve a concentration of 40 pM and 5% MeOH. After 30 minutes of room temperature equilibration in the dark, the full spectra of the samples in 1 cm quartz cuvettes were measured from 260 to 700 nm (1 nm step) with a UV-2450 Spectrophotometer (Shimadzu, Columbia, MD, USA). The pH of each solution was tested afterwards to confirm the pH was within ± 0.2 units of the target.

[0537] Beer-Lambert law (equation 4.1) was used to calculate the molar absorptivity coefficients Where c = 4 x 10'5M, b = 1 cm, and a is expressed in L*mole'1*cm'1.

[0538] Equation 4.1 :

[0539] CIELAB color coordinates were calculated from the full spectra data (380-700 nm) using ColorBySpectra software under D65 illuminant and 10° observer angle (Farr & Giusti, 2017).

[0540] Expression of Color, Stability, and Solubility Across pH. PACNs were added to buffers to achieve a final concentration of 40 pM, 5% MeOH. Aliquots of the PACN-containing buffer solutions were added to 96 well plates which were sealed with a UV-transparent film after pipetting. Spectra (360-700 nm, 5 nm step) were measured after 10-20 minutes (defined as time 0) with a SpectraMax M2 spectrophotometer (Molecular Devices, San Jose, CA, USA). The pH of each solution was measured on the initial day of mixing to confirm buffer pH and were within ±0.2 units of the parent buffer pH. Solubility of the PACNs was evaluated visually under controlled light conditions. Microcentrifuge tubes with the buffer solutions were centrifuged at 4000 rpm for 5 minutes at each spectral time point and visually evaluated for precipitation.

[0541] Color stability at room (-18.5 °C) temperature was measured over 60 days. The 96 well plates were shielded from light, and spectra and solubility were measured as previously detailed.

[0542] 4.3.7. Statistical Analysis and Data Processing. Each experiment was repeated in triplicate with replications beginning before the fourth drying step. Color expression and molar absorptivity coefficients for 10-guaiacyl-pyranocyanidin-3-arabinoside (10-guaiacyl-PCy3- arabinoside) were evaluated in duplicate. A 1-way analysis of variance (ANOVA) with Tukey HSD multiple comparisons was performed on the color data, molar absorptivity coefficients, and % absorption changes with a - value < 0.05 considered significant. A type 3 error ANOVA and necessary contrasts were included when data for 10-guaiacyl-PCy3-arabinoside was included, to manage the unbalanced data set. When comparing only 2 conditions, as for buffer comparisons at pH 8, a t-test was performed. R studio and GraphPad Prism were used for statistical analysis.

[0543] For comparison of the spectral changes across pH between the different 10-guaiacyl- pyranocyanidins (Section 4.4.4), the spectral data for each PACN and each repetition was normalized by subtracting the lowest value and dividing by the range of values for that repetition. For each PACN, a maximum absorption of 1 occurred at pH 3. This data transformation helped to remove the effect of glycosylation on the initial color intensity (Section 4.4.3) to highlight the influence it had on color stability across pH.

[0544] 4.4. Results and Discussion

[0545] 4.4.1. Pyranoanthocyanin Identification and Purity. The chosen anthocyanin sources for 10-guai acyl -PACN formation (A. melanocarpa, S. nigra, and / A carota) are each reported to be rich in cyanidin anthocyanins with differing types of glycosylations (Figure 21). A. melanocarpa is reported to contain cyani din-3 -arabinoside (Cy3 -arabinoside) and cyanidin-3- galactoside (Cy3-gal) (Farr et al., 2018; X. Wu et al., 2004). S. nigra is reported to contain cyani din-3 -glucoside (Cy3-glu) and cyanidin-3-xylosyl-( l ^2)-glucoside (Cy3-xyl-glu). D. carota is reported to contain cyanidin-3-xylosyl-(l— >2)-galactoside (xyl-gal) and cyanidin-3- xylosyl-(l— >2)-glucoside-(l— >6)-galactoside, with these non-acylated species present in higher percentages once saponified (Farr et al., 2018; Montilla et al., 2011).

[0546] HPLC-PDA monitoring of the anthocyanin extracts mixed with 4-vinylguaiacol during incubation showed the loss of anthocyanins but the formation of new peaks with later retention times (Figure 21). With Vis-max hypsochromically shifted compared to the original anthocyanins, higher m / z value, and a MS / MS fragment to 433 (consistent with a 10-guaiacyl-pyranocyanidin aglycone), these new peaks were assigned as 10-guaiacyl-pyranocyanidins (Table 9). For each of the six formed 10-guaiacyl-pyranocyanidins, the MS / MS neutral loss fragmentation was consistent with the expected glycosylation substitutions for the precursor anthocyanins as listed previously. Therefore, from the solution of melanocarpa incubated with 4-vinylguaiacol, 10- guaiacyl-PCy3 -arabinoside and 10-guaiacyl-PCy3-gal were isolated. From the solution of S. nigra incubated with 4-vinylguaiacol, 10-guaiacyl-PCy3-glu and 10-guaiacyl-PCy3-xyl-glu were isolated. From the solution of saponified D. carota incubated with 4-vinylguaiacol, 10-guaiacyl- PCy-3-xyl-gal and 10-guaiacyl-PCy-3-xyl-glu-gal were isolated.

[0547] From the PDA spectra, each of the 6 PACNs had Xvis-max shifted 6-10 nm lower and later retention times compared to the characteristics for the precursor anthocyanins, consistent with behaviors for 10-guaiacyl-PACNs (Blanco-Vega et al., 2011). Among the six 10-guaiacyl- pyranocyanidins, the Xvis-max values were similar under the HPLC-chromatographic conditions (Table 9). PACNs containing more glycosylations eluted earlier under the reverse phase chromatographic conditions (Table 9).

[0548] The purity of each isolated PACN varied. Three 10-guaiacyl-pyranocyanidins (10- guaiacyl-PCy3-glu, 10-guaiacyl-PCy3-gal, 10-guaiacyl-PCy3-xyl-glu-gal) had purity > 90% (Table 9). The other three PACNs had lower purity levels. Impurities in 10-guaiacyl-PCy3-xyl- glu and 10-guaiacyl-PCy3-xyl-gal appeared to grow in abundance during the drying process. Most of the impurities in these two samples absorbed light in the visible region (Xvis-max -490 to 510 nm) and had similar retention times to the 10-guaiacyl-pyranocyanidins suggesting they were similar in structure and behavior. In 10-guaiacyl-PCy3-xyl-glu, the main impurity, accounting for 7-9% of the total peak area, produced a kvis-max of 496 / 497 nm with a m / z 34 units higher than the PACN. This compound had a neutral loss for a hexose-pentose disaccharide (- 294) and could be an adduct. In 10-guaiacyl-PCy3-xyl-gal, the largest impurity accounted for 4- 6% of the total area. It had the same m / z and neutral loss as a hexose, and a kvis-max close to 10- guaiacyl-pyranocyanidin-3-glu / gal as reported in Table 9.

[0549] Table 9. HPLC-PDA-MS / MS characteristics for the 10-guaiacyl-pyranocyanidins evaluated. Purity values are expressed as means ± standard deviation (n = 3) and further discussed in Section 4.4.1.

[0550] 4.4.2. Molar Absorptivity Coefficients. The C3 glycosylation substitution seemed to affect the molar absorptivity coefficients of 10-guaiacyl-pyranocyanidins. In all solutions, the molar absorptivity coefficient followed the same trend: 10-guaiacyl-PCy3-glu, 10-guaiacyl- PCy3-xyl-glu-gal > 10-guaiacyl-PCy3-gal > 10-guaiacyl-PCy3-xyl-glu > 10-guaiacyl-PCy3-xyl- gal > 10-guaiacyl-PCy3 -arabinoside (MeOH only) (Table 10). Precipitate was immediately observed in pH 1 and pH 3 buffers for 10-guaiacyl-PCy3-arabinoside, therefore the molar absorptivity coefficients were not determined.

[0551] For a single PACN, the molar absorptivity coefficients had minimal change between pH 1 and pH 3 buffer (Table 10). PACNs are reported, and as will be shown in Section 4.4.4 with the 10-guaiacyl-pyranocyanidins, to have relatively consistent color across highly acidic pH values (He et al., 2010; Chapter 3). This behavior starkly contrasts that observed for the precursor anthocyanins, which lost color intensity (up to 44%) and had a decrease in molar absorptivity coefficients (-27% reduction) as the pH increased from 1 to 3 (Cabrita et al., 2000; Sigurdson et al., 2018). The 10-guaiacyl-pyranocyanidins all produced higher molar absorptivity coefficients in MeOH (0.1% HC1) than in aqueous buffers which is consistent with previous findings for both anthocyanins and PACNs (Sigurdson et al., 2019; Chapter 3).

[0552] The effect of C3 glycosylation on molar absorptivity coefficients for anthocyanins is reported to be minimal with values 16% lower for Cy3 -arabinoside and -6% for Cy3-xyl-glu compared to Cy3-glu (W. Dong et al., 2024). In the present experiment, the molar absorptivity coefficients for the two tested 10-guaiacyl-pyranocyanidin-disaccharides were 26 to 30% lower than the PACNs substituted with the monosaccharides glucose and galactose. The two disaccharides had noticeably lower purity levels which could result in an underestimation of the molar absorptivity coefficients. However, as the impurities were also similarly colored and appeared to have similar m / z values and chromatographic characteristics as the 10-guaiacyl- PACNs, they were likely not fully responsible for the lower molar absorptivity coefficients. Thus, it appears that a xylose(l— >2)glu / gal disaccharide substitution contributed to a lower molar absorptivity coefficient for 10-guaiacyl-pyranocyanidins. When comparing isomeric forms, the molar absorptivity values obtained for a PACN containing glucose were significantly higher than those with galactose, observed both in the comparison of the monosaccharides and xylose- glu / gal disaccharide.

[0553] Table 10. Molar absorptivity coefficients for 10-guaiacyl-pyranocyanidins. Results are expressed as mean ± standard deviation with letters showing statistical differences among PACNs in a single solution. * indicates samples where isolate purity was < 75%; see Section 4.4.1 and 4.4.2 for further discussion.

[0554] The molar absorptivity coefficients reported for 10-guaiacyl-PCy3-glu in pH 3 KC1 buffer were similar to the values calculated for this same PACN in our previous work detailed in Chapter 3 (37,900) despite a slightly different drying process. Interestingly, we previously did not report a molar absorptivity coefficient at pH 1 as precipitation was observed within the 30 minute equilibration time (noted visually and in the spectra by increasing absorption at -560 to 570 nm region; Chapter 3). No evidence of this fast precipitation was observed in the present experiment, however with extended time at room temperature, visual precipitation appeared in the 400 pm stock (50% MeOH) and in pH 1 KC1 buffer. Between these two experiments in Chapter 3 and Chapter 4, differences in timing between preparing the stock solutions and preparing the solutions for spectral measurements could have accounted for the differences in reported precipitation behavior. Nevertheless, the similarity of the molar absorptivity coefficients at pH 3 KC1 buffer supported the reproducibility of our method.

[0555] Most of the molar absorptivity coefficients for PACNs published in literature are lower than the values reported in this present experiment. For the similar structured malvi din-3 -glu derived hydroxyphenyl-PACNs, reported coefficients range between -3,000 to 12,000 in acidic buffers for 10-catechyl-, 10-guaiacyl-, and 10-syringyl-PMalvi din-3 -glu (Hakansson et al., 2003; Quijada-Morin et al., 2010). It is challenging to calculate consistent molar absorptivity coefficients as differences in equilibration times (especially important as PACNs are known to precipitate), concentrations, sample purity, and molecular weight adjustments can all affect coefficient values (Jordheim et al., 2007). Unfortunately, there is little consistency across studies on the procedure for collecting and reporting coefficient data which may contribute to the range in reported values.

[0556] 4.4.3. Glycosylation Influence on Color Expression in pH 1 and 3 buffer and MeOH (0.1% HC1). An array of orange and yellow orange hues were produced by the 10-guaiacyl- pyranocyanidins in acidic buffers (Figure 22). An exception to this was observed with 10- guaiacyl-PCy-3 -arabinoside which will be discussed below. The two disaccharides (10-guaiacyl- PCy3-xyl-glu and 10-guaiacyl-PCy3-xyl-gal) were significantly lighter (L*) and less intense (indicated by chroma (C*ab)) than 10-guaiacyl-PCy3-glu, -PCy3-gal, -and -PCy3-xyl-glu-gal. These observations follow the same pattern observed with molar absorptivity coefficients (Section 4.4.3.) with the same discussion on sample purity being relevant. The highest hue angle was consistently produced by 10-guaiacyl-PCy3-xyl-glu, statistically higher in pH 1 and 3 KC1 buffer (Figure 22). As discussed in Section 4.4.1. the primary impurity in 10-guaiacyl-PCy3-xyl- glu had a kvis-max ~ 496 nm which may have contributed to the more yellow color and higher hue angle for 10-guaiacyl-PCy3-xyl-glu.

[0557] In pH 1 and 3 KC1 buffer, the lowest hue angles were observed for 10-guaiacyl-PCy3-gal and 10-guaiacyl-PCy-xyl-glu-gal (hab ~ 48-49) corresponding to visually appearing the most red-orange. Interestingly, in MeOH (0.1% HC1), 10-guaiacyl-PCy3-xyl-gal had a significantly lower hue angle than the 10-guaiacyl-PCy3-xyl-glu-gal, demonstrating the individualized effect solvent may have on PACN behavior (Figure 22). The shape of the UV-Vis spectra were similar across all tested 10-guaiacyl-pyranocyandins with no evident effect of C3 glycosylation number and type on spectral shape (data not shown).

[0558] Unlike the hydroxyl and methoxy substitutions discussed in Chapter 3, sugar substitutions were not expected to influence the electron density of the PACN as is observed for anthocyanins (Mai ci oglu et al., 2011). However, the effect of glycosylation on color may be due to their influence on the 3-dimensional conformation as the B ring and E ring substitutions are modeled to hydrogen bond with the sugar substitution (Quartarolo & Russo, 2011). To achieve these bonds, the B and E ring would contort itself, resulting in changed magnitude of electron delocalization into the PACN chromophore (Quartarolo & Russo, 2011). Since the E ring and B ring substitutions were the same across all six tested 10-guaiacyl-pyranocyanidins, it was hypothesize that the disaccharides resulted in a higher dihedral angle between the CIO and Cl’” position and / or between C2 and Cl’ position (Figure 21), lowering the electron delocalization to result in the more yellow color. Theoretical computations can support this hypothesis.

[0559] In contrast to the present observations for 10-guaiacyl-pyranocyanidins, previous work with 10-carboxy-pyranocyanidins reported slightly higher hue angles for the PACNs with monosaccharides glucose and galactose compared to the PACN with xyloysl-(l — >2) glu / gal disaccharides and xyl-(l ^2)-glu-( l ^6)-gal trisaccharide (Farr et al., 2018; J. Oliveira, Fernandes, et al., 2006). Under the hypothesis that the glycosylation affects the dihedral angle between CIO and Cl’”, it is expected that a guaiacol moiety would respond differently to glycosylations than a carboxylic moiety.

[0560] Unique color properties were observed for 10-guaiacyl-PCy3-arabinoside. Compared to the other 10-guaiacyl-pyranocyandins, the kvis-max was 1 to 4 nm higher (at 510 nm) in HPLC- PDA solvents and hab was 19.5 to 25° lower in MeOH (0.1% HC1) (Table 9; Figure 22). This PACN was pink, making it the closest in color to the precursor anthocyanins. However, there was still a slightly lower kvis-max and higher hab reported for cyani din-3 -glucoside anthocyanin (Chapter 3). As the hypsochromic shift in PACN color from that of anthocyanins is thought to be due to decreased planarity of the B ring, which can be contorted due to hydrogen bonds between the sugar (Carvalho, Oliveira, de Freitas, Mateus, et al., 2010b; Quartarolo & Russo, 2011), it was hypothesized that the presence of a pentose, a smaller sugar substitution, had a smaller effect on B ring rotation which allowed for greater electron delocalization into the PACN chromophore.

[0561] 4.4.4. Influence of Glycosylation on Color Expression Across pH. The 10-guaiacyl- pyranocyanidins produced diverse and vibrant color at all pH values from 1 to 8, but color hue and intensity did change with pH as is characteristic for flavylium compounds. Color expression was performed in each of the 18 buffers (Table 8.), however, to highlight the effect of pH on PACN solution color expression, a single buffer at each pH value was chosen (Figure 23). Two of the evaluated monosaccharides, 10-guaiacyl-PCy3-arabinoside and 10-guaiacyl-PCy3-glu, precipitated immediately at most pH values upon introduction into buffers leading to obscured and jagged spectra. Therefore, these PACNs were not included for pH color comparison.

[0562] For the other four 10-guaiacyl-pyranocyanidins (10-guaiacyl-PCy3-gal, 10-guaiacyl- PCy3-xyl-glu, 10-guaiacyl-PCy3-xyl-gal, and 10-guaiacyl-PCy3-xyl-glu-gal), the overall trends of color change with pH were similar. The orange color produced in acidic conditions was retained until pH ~5 and a dramatic shift in color to pink / purple occurred at pH 8 (Figure 23). The high intensity, orange colors at pH 3 would be produced primarily by the PACN flavylium cation (Cruz et al., 2010; Pinto, Oliveira, et al., 2019). As pH is increased, the flavylium cation is shown to undergo deprotonation to form the quinoidal base with the published pKa values for this transition for similar PACNs at -4.35-4.7 (for 10-guaiacyl-pyranomalvidin-3 -glucoside and 10-catechyl-PCy3-glu) (Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016).

[0563] Thus, the hypsochromic shift and decrease in color intensity observed here as pH increased from 3 to 6 / 7 is likely due to the quinoidal base formation. The quinoidal base’s yellow shifted color compared to the flavylium cation for 10-guaiacyl -PACNs is supported by observations in our work detailed in Chapter 3 and by others with various types of hydroxyphenyl -PACNs (Cruz et al., 2010; Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016). As the pH increased from 7 to 8, the dramatic drop in hue angle and formation of a secondary peak in the -550-600 nm region may indicate the formation of a second quinoidal base species, the anionic quinoidal base (Cruz et al., 2010). The pKa reported for this transition with 10-catechyl-PCy3- glu was 7.2, although, with this PACN, the authors did not report a dramatic color shift (Pinto, Oliveira, et al., 2019). Nevertheless, our current spectral observations suggest this pKa value may be slightly higher for 10-guaiacyl-pyranocyanidins.

[0564] Unlike what is observed for anthocyanins (Raymond Brouillard & Delaporte, 1977; Raymond Brouillard & Dubois, 1977a), most PACNs, including the structurally similar 10- catechyl -PACNs, were not observed to undergo appreciable levels of hydration with pH changes (Cruz et al., 2010; J. L. Sousa et al., 2017). Thus, while anthocyanins lost nearly all color at mild acid pH ranges in our previous experiment (Chapter 3), the slight color shift and drop in absorption intensity in the current study for 10-guaiacyl-pyranocyanidins is attributed to the formation of the quinoidal base species.

[0565] The magnitude of these structural changes and the pH at which they occurred was influenced by the C3 glycosylation for the 10-guaiacyl-pyranocyanidins. For 10-guaiacyl-PCy3- gal, the color became lighter and less intense as pH increased from 3 to 8 with statistical differences in C*ab between each pH starting at pH 4 (Figure 23). The magnitude of this color loss was greater than for the other 10-guaiacyl-pyranocyanidins which may be due to 10- guaiacyl-PCy3-gal precipitating. While precipitation was visually observed following the initial timepoint measurement (-1 hr after PACNs was added to buffers), the spectra did not show signs of interference from the precipitate (i.e., jagged, irregular peaks), so the data was included. A constant orange hue was produced from pH 3 to 6 with no statistical differences in hab despite a hypsochromic shift in Vis-max on the spectra (Figure 23). From pH 6 to 7, there was a significant increase in hab (7.1 units) as the solution visually became more yellow (Figure 23). From pH 7 to 8, the dramatic color shift coincided with a hab drop of 40 units. The spectra showed a bimodal peak with the peak at - 480 nm slightly higher than the peak at -545 nm (Figure 23). Color and spectral changes with pH were similar for 10-guaiacyl-PCy3-xyl-glu and 10- guaiacyl-PCy3-xyl-gal. As observed for 10-guaiacyl-PCy3-gal, there was no significant change in the color coordinates between pH 3 and 4 (Figure 23). As pH increased from 4 to 6, there was a significant decrease in chroma (C*ab decreased by 6.5 and 5.5. for -xyl-glu and -xyl-gal, respectively); however, the magnitude of this decrease was less than that observed for 10- guaiacyl-PCy3-gal (Figure 23). Additionally, unlike observed with 10-guaiacyl-PCy3-gal, hue angle statistically decreased with increasing pH as the samples became more yellow, represented on the spectra by a larger hypsochromic shift in kvis-max. From pH 6 to 7, hab significantly decrease, along with decrease in L* and C*ab. Despite no (or limited) change in the kvis-max between these pH values, absorption increased -550 to 600 nm region which would contribute to the lower hue angle and brown appearance. As with 10-guaiacyl-PCy3-gal, hab for the two disaccharides dropped - 50° between pH 7 and 8 to form a purple color. Unlike with 10- guaiacyl-PCy3-gal, the color became darker and more intense in the alkaline conditions.

[0566] Among the four PACNs, the color of 10-guaiacyl-PCy-3-xyl-glu-gal changed the most across pH values. From pH 3 to 4, there was a significant increase in hab (more yellow) which continued to increase until pH 6 (Figure 23). The magnitude of the hue angle shift from pH 3 to 6 (16.9 units), was significantly larger than that observed for the other 10-guaiacyl- pyranocyanidins and visually corresponded to a more yellow appearance at pH 6. While hue angle decreased from pH 6 to 7 as observed for the two disaccharides, hab remained higher (more yellow) than at pH 3 while for the disaccharides, the hab at pH 7 was lower (more red) than the color at pH 3. From pH 7 to 8, there was similar behavior as observed with the disaccharides with a large decrease in hab and increase in C*ab. Since the two disaccharides and trisaccharide had a significantly larger hab decrease and a more pronounced bathochromically shifted peak at pH 8, this may suggest the pKa values are lower than for the 10-guaiacyl-PCy3-glu.

[0567] 4.4.5. Influence of Buffer Type on Color Expression. Previous preliminary work for Chapter 3 showed that the aqueous solution type influenced the solubility behavior of hydroxyphenyl -PACNs even at the same pH. Therefore, we included an evaluation of buffer type on the color expression, stability, and solubility in the present experiment. For this discussion, results for 10-guaiacyl-PCy3-xyl-glu-gal are presented as this PACN was soluble in each solution. Buffer type did significantly influence the color expressed for solutions at the same pH (Figure 24). Tris buffer appeared to be the most unique, producing significantly different colors in terms of chroma and hue angle at pH 7 and 8 (Figure 24). Additionally, at pH 4 and 5, the color was significantly more yellow (higher hab) in sodium acetate-acetic acid buffer than the other citric acid buffers evaluated (Figure 24). The pH of each of these solutions were within ± 0.2 units of the other buffers at the set pH with variations in the direction between replicates. Therefore, buffer salts appeared to affect the color of the 10-guaiacyl-PCy3-xyl-glu- gal in solution.

[0568] 4.4.6. Solubility of 10-Guaiacyl-Pyranocyanidins Across pH and 60 Day Storage

[0569] Influence of pH on Solubility. PACN precipitation is believed to begin with PACN- PACN intermolecular associations, which, when grown to polymeric size, precipitate from solution (Vallverdu-Queralt, Biler, et al., 2016). PACN precipitation as discussed here and detailed in Chapter 2, is defined by the presence of visible solids and results in decreased absorption at all wavelengths (Cruz et al., 2010). In the present study, precipitation occurred more often near neutral pH (6-7) (Figure 25) where the 10-guaiacyl-pyranocyanidins would likely be predominantly in their quinoidal base structural configuration as discussed in Section 4.4.4. (Cruz et al., 2010; Pinto, Oliveira, et al., 2019; Vallverdu-Queralt, Biler, et al., 2016). As this form is neutral, the lack of charge repulsion likely led to association (Vallverdu-Queralt, Biler, et al., 2016). Precipitation was least likely for 10-guaiacyl-pyranocyanidins at highly acidic pH (1 and 3) (Figure 25); these observations support those in Vallverdu-Queralt, Biler, et al. (2016) for 10-guiaicyl-pyranomalvidin-3-glucoside as the flavylium cation is predominant in the high acidic conditions.

[0570] Influence of Buffer Type on Solubility. At equivalent pH values, buffer type had a slight influence on the tendency for precipitation, observed by comparing precipitation trends for 10-guaiacyl-PCy3-xyl-glu and 10-guaiacyl-PCy3-xyl-gal (Figure 25). At pH 6 and 7, these two PACNs were more soluble in buffers containing citric acid (either Citric Acid-Na2HPO4 or Citric Acid-Trisodium Citrate). Instead, precipitation was observed selectively at pH 5 in Sodium Acetate-Acetic acid buffer (both PACNs), pH 6 in Na2HPO4-NaH2PO4 buffer (both PACNs), and at pH 7 in Na2HPO4-NaH2PO4 and Tris buffers for (10-guaiacyl-PCy3-xyl-gu). Therefore, proper choice of buffer when evaluating PACN stability and solubility is imperative as the various salts may favor precipitation.

[0571] Influence of Glycosylation on 10-Guaiacyl-Pyranocyanidin Solubility. Precipitation trends were strongly affected by glycosylation (Figure 25). More hydroxyl groups provided by the glycosylation substitution resulted in greater solubility with the monosaccharides most likely to precipitate as alluded to in Section 4.4.4. In general, the precipitation was colored with the color depending on the solution pH and the PACN type. Depending on the extent of precipitation, some color could remain in solution.

[0572] For 10-guaiacyl-PCy3-arabinoside, precipitation occurred within 1 day upon mixing into the stock solutions for all 18 buffers (10% MeOH) (Figure 25). In fact, precipitation occurred even in stock solutions comprised of 50% MeOH, necessitating the use of a 100% MeOH stock solution. Being a pentose, arabinose has only 3 polar -OH groups, therefore, it was the least hydrophilic of the sugar attachments.

[0573] The 10-guaiacyl-PACNs with a hexose monosaccharide were the second most likely to precipitate. Of these, 10-guaiacyl-PCy3-glu precipitated more often and sooner than 10- guaiacyl-PCy3-gal (Figure 25). The former precipitated in all 18 conditions within 4 days, however the latter remained soluble in 5 solutions (pH 1, pH 3, and pH 8 (Na2HPO4-NaH2PO4 buffer) up to 60 days. The only structural difference between these isomeric PACNs is the hydroxyl positioning of C4” on the hexose moiety (Figure 21). The evident differences in precipitation tendency suggested that glycosylation orientation and positioning played a key role in 10-guaiacyl-pyranocyanidin precipitation.

[0574] Additional glycosylation substitutions improved solubility. The two disaccharides had intermediate levels of precipitation with 10-guaiacyl-PCy3-xyl-gla precipitating evidently for all three reps in 4 / 18 conditions while 10-guaiacyl-PCy-xyl-glu precipitated in all replications for 3 / 18 conditions (Figure 25). The effect of glucose versus galactose was opposite than that observed for monosaccharides, however it still demonstrated the influence of slight differences in molecular orientation and connectivity had on PACN solubility.

[0575] Excellent aqueous solubility was observed for 10-guaiacyl-PCy3-xyl-glu-gal as none of the 18 buffer solutions showed evident precipitation across all three replications. The larger glycosylation substitution may enhance solubility through multiple methods. First, the hydroxyl groups provided by the glycosylations substitution increase hydrophilicity (C.-L. Zhao et al., 2014). Additionally, the pKa transitions may be affected, as discussed in Section 4.4.4, and therefore affect the abundance of the quinoidal base forms at each pH. In addition, a steric effect may be exerted by the larger glycosylation moieties as they disrupt molecular planarity which, we hypothesize, may decrease the tendency of the PACN to aggregate (van Acker et al., 1996)

[0576] 4.4.7. Absorption Stability Over 60 Days (Room Temperature)

[0577] Influence of pH on 10-Guaiacyl-Pyranocyanidin Absorption Stability. As discussed in Section 4.4.4., color expression was performed in each of the 18 buffers, yet only a single buffer is chosen to demonstrate the effect of pH on stability. Citric acid-Na2HPO4 (pH 1 - 7) and Na2HPO4-NaH2PO4 (pH 8) were chosen as PACNs were more soluble in these solutions with four of the six PACNs included in the evaluation due to the aforementioned precipitation.

[0578] Further discussion on the influence of buffer choice on stability is described below. Additionally, as the degradation plateau was not reached within 60 days for most conditions, comparisons were made between absorbance remaining rather than kinetic modeling parameters (Figure 26). The color of all four 10-guaiacyl-pyranocyanidins was most stable in highly acidic pH conditions (pH 1 and 3) and least stable at pH 8 after 60 days at room temperature. For the two disaccharides and trisaccharide evaluated, the glycosylation substitution impacted the stability trends at mild acid and neutral pH. For 10-guaiacyl-PCy3-xyl-glu and 10-guaiacyl-PCy3-xyl-gal, there was significantly more absorption lost at pH 5 than pH 1 and 3 after 60 days. In contrast, there was no significant difference among absorption changes at pH 1, 3, 4, and 5 for 10- guaiacyl-PCy3-xyl-glu-gal with less than a 10% drop in absorption intensity at these pH values. As pH increased from 6 to 7 there was a greater drop in absorption intensity for 10-guaiacyl- PCy3-xyl-glu than for 10-guaiacyl-PCy3-xyl-gal at day 60 which could be affected by the glycosylation and solubility as 10-guaiacyl-PCy3-xyl-gal was observed to precipitate by day 8 at pH 7.

[0579] Cyanidin anthocyanins are also reported as most stable at room temperature in high acid conditions (Farr et al., 2019). For anthocyanins, however, the shortest half-lives were reported at pH 6 and 7 (Farr et al., 2019) while the 10-guaiacyl-pyranocyanidins showed the greatest degradation at pH 8 (Figure 26). While Farr et al. (2019) reported half-lives rather than absorption %, it is clear that 10-guaiacyl-pyranocyanidins showed improved color retention at mild acid and alkaline conditions than the cyanidin anthocyanins. Cyanidin anthocyanin absorption decreased by 50% (reported half-life) within 1 to 8 days at pH 6 and 7 (Farr et al., 2019), however for the 10-guaiacyl-pyranocyanidins reported here, the half-life was not reached (> 62.5% remaining) even within 60 days. At pH 8, anthocyanin half-lives ranged from 9 to 23 days (Farr et al., 2019); based on Figure 26, the half-lives for 10-guaiacyl-pyranocyanidins likely fall within 26 and 33 days.

[0580] While the degradation mechanism of PACNs under room temperature storage has yet to be elucidated, for anthocyanins, similar degradation compounds are observed with room temperature as with heat (under acidic conditions) (Lopes et al., 2007). Therefore, the same assumption may apply for 10-guaiacyl-pyranocyanidi...

Claims

CLAIMSWhat is claimed is:I whereRi, R2, R3, and R4 each independently comprise H, OH, or OCH3; andSugar is a monosaccharide, a disaccharide, or a trisaccharide; wherein the number, identity, and location of hydroxyl and methoxy substitutions on the B and E rings are selected to control the on color expression, molar absorptivity coefficients, pH color stability, and / or solubility of the colorant.

2. The colorant of claim 1, wherein the sugar is glucose, xylose, galactose, arabinose, or a combination thereof.

3. The colorant of claim 1 or claim 2, wherein the sugar is arabinose, glucose, galactose, xylosyl(l— >2)glucose, xylosyl(l— >2)galactose, or xylosyl(l— >2)glucosyl(l— >6)galactoside.

4. The colorant of any one of claims 1-3, wherein the sugar is glucose.

5. The colorant of any one of claims 1-4, wherein Ri is OH or OCH3.

6. The colorant of any one of claims 1-5, wherein R2 is H or OCH3.

7. The colorant of any one of claims 1-6, wherein R4 is H or OCH3.

8. The colorant of any one of claims 1-7, wherein Ri is OH or OCH3; R2 is H or OCH3; R3 is H, OH, or OCH3; and R4is H or OCH3.

9. The colorant of any one of claims 1-8, wherein the sugar is glucose, Ri is OH or OCH3; R2is H or OCH3; R3is H, OH, or OCH3; and R4is H or OCH3.

10. The colorant of any one of claims 1-9, wherein Ri is OH and R2-R4are H; Ri is OH, R2 is H, R3 is OCH3, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is OH; Ri is OCH3, R2 is OCH3, R3 is H, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OH, and R4is H; or RI-R4are each OCH3.

11. The colorant of any one of claims 1-10, wherein:Sugar is glucose; andRi is OH and R2-R4are H; Ri is OH, R2 is H, R3 is OCH3, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is H; Ri is OH, R2 is H, R3 is OH, and R4is OH; Ri is OCH3, R2 is OCH3, R3 is H, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H; Ri is OCH3, R2 is OCH3, R3 is OH, and R4is H; or RI-R4are each OCH3.

12. The colorant of any one of claims 1-11, wherein Ri is OH and R2-R4are H.

13. The colorant of any one of claims 1-11, wherein Ri is OH, R2 is H, R3 is OCH3, and R4is H.

14. The colorant of any one of claims 1-11, wherein Ri is OH, R2 is H, R3 is OH, and R4is H.

15. The colorant of any one of claims 1-11, wherein Ri is OH, R2 is H, R3 is OH, and R4is OH.

16. The colorant of any one of claims 1-11, wherein Ri is OCH3, R2 is OCH3, R3 is H, and R4is H.

17. The colorant of any one of claims 1-11, wherein Ri is OCH3, R2 is OCH3, R3 is OCH3, and R4is H.

18. The colorant of any one of claims 1-11, wherein Ri is OCH3, R2 is OCH3, R3 is OH, andR4 is H.

19. The colorant of any one of claims 1-11, wherein R1-R4 are each OCH3.

20. The colorant of any one of claims 1-19, wherein the colorant comprises a hydroxyphenyl pyranoanthocyanin as shown in Figure 97.

21. The colorant of any one of claims 1-20, wherein the colorant comprises a 10- / ?- hydroxyphenyl -pyranoanthocyanin, 10-catechyl -pyranoanthocyanin, 10-guaiacyl- pyranoanthocyanin, 10-syringyl-pyranoanthocyanin, or a combination thereof.

22. The colorant of any one of claims 1-21, wherein the colorant comprises 10-p- hydroxyphenyl-Cy-3-glucoside, 10-catechyl -Cy-3 -glucoside, 10-guaiacyl-Cy-3-glucoside, 10- syringyl-Cy-3 -glucoside, 10-p-hydroxyphenyl-Mv-3-glucoside, 10-catechyl- Mv -3-glucoside, 10-guaiacyl- Mv-3 -glucoside, 10-syringyl-Mv-3 -glucoside, or a combination thereof.

23. The colorant of any one of claims 1-22, wherein the colorant comprises a 10-guaiacyl- pyranocyanidin.

24. The colorant of any one of claims 1-23, wherein the colorant comprises 10-guaiacyl- PCy3-glu, 10-guaiacyl-PCy3-xyl-glu-gal, 10-guaiacyl-PCy3-gal, 10-guaiacyl-PCy3-xyl-glu, 10- guaiacyl-PCy3-xyl-gal, 10-guaiacyl-PCy3 -arabinoside, or a combination thereof.

25. The colorant of any one of claims 1-24, wherein the colorant comprises 10-guaiacyl- PCy3 -galactose, 10-guaiacyl-PCy3-xyl-glu-gal, 10-guaiacyl-PCy3-xyl-glu, 10-guaiacyl-PCy3- xyl-gal, or a combination thereof.

26. The colorant of any one of claims 1-25, wherein the colorant comprises 10-guaiacyl- PCy3-xyl-glu-gal.

27. The colorant of any one of claims 1-26, wherein the hydroxyphenyl pyranoanthocyanin is derived from an anthocyanin and a cofactor.

28. The colorant of claim 27, wherein the cofactor comprises acetone, pyruvic acid, acetaldehyde, hydroxycinnamic acids, 4-vinylphenols, or a combination thereof.

29. The colorant of claim 27 or claim 28, wherein the cofactor comprises hydroxycinnamic acid, 4-vinylphenol, or a combination thereof.

30. The colorant of any one of claims 27-29, wherein the cofactor comprises / ?-coumaric acid, caffeic acid, ferulic acid, sinapic acid, or a combination thereof.

31. The colorant of any one of claims 27-30, wherein the cofactor comprises 4-vinylphenol, 4-vinylguaiacyl, or a combination thereof.

32. The colorant of any one of claims 27-31, wherein the anthocyanin comprises cyanidin-3- glucoside, malvi din-3 -glucoside, or a combination thereof.

33. The colorant of any one of claims 27-32, wherein the anthocyanin is derived from an edible material.

34. The colorant of any one of claims 27-33, wherein the anthocyanin is derived from a plant.

35. The colorant of any one of claims 27-34, wherein the anthocyanin is derived from a raw agricultural product.

36. The colorant of any one of claims 27-35, wherein the anthocyanin is derived from a fruit, flower, vegetable, or combination thereof.

37. The colorant of any one of claims 27-36, wherein the anthocyanin is derived from wine, sour cherry juice, figs, strawberries, fermented olives, frozen grape skins, red onion, black currant, blueberry honeysuckle, elderberry, blood orange juice, or a combination thereof.

38. The colorant of any one of claims 27-37, wherein the anthocyanin is derived from black carrot, chokeberry, elderberry, Berberis boliviana. or a combination thereof.

39. The colorant of any one of claims 27-38, wherein the colorant comprises a 10- / ?- hydroxyphenyl-, 10-catechyl-, 10-guaiacyl-, and / or 10-syringyl -derivatives of cyanidin-3- glucoside and / or malvi din-3 -glucoside anthocyanins.

40. The colorant of any one of claims 1-39, wherein the colorant is edible.

41. The colorant of any one of claims 1-40, wherein the composition is an aqueous composition.

42. The colorant of any one of claims 1-41, wherein the composition is an aqueous composition having a pH from 1-8, from 1-7, from 2-7, from 3-7, from 3-6, or from 1-3.

43. The colorant of any one of claims 1-42, wherein the colorant has a vibrant and stable color.

44. The colorant of any one of claims 1-43, wherein the colorant has a vibrant color that is stable for an amount of time of 2 months or more at pH 1 to 7 and room temperature.

45. The colorant of any one of claims 1-44, wherein the colorant has a color that is yellow to blue.

46. The colorant of any one of claims 1-45, wherein the colorant has a color that is brown, red, orange, pink, or yellow.

47. The colorant of any one of claims 1-46, wherein the colorant has a color that is yellow to red, orange to red, or orange to pink.

48. The colorant of any one of claims 1-47, wherein the colorant has a color that is pink to purple.

49. A thermal degradation compound derived via thermal degradation of the colorant of any one of claims 1-48.

50. The compound of claim 49, wherein the compound comprises a 4-carboxy-3- deoxy anthocyanidin.

51. The compound of claim 49 or claim 50, wherein the compound comprises 4-carboxy-3- deoxycyanidin, 4-carboxy-3 -deoxypelargonidin, 4-carboxy-3-deoxypeonidin, 4-carboxy-3- deoxymalvidin, or a combination thereof.

52. A thermal degradation compound derived via thermal degradation of 10-catechyl- pyranocy ani din-3 -O-P-glucoside.

53. The thermal degradation compound of any one of claims 49-52, wherein the thermal degradation compound is a colorant.

54. The thermal degradation compound of any one of claims 49-53, wherein the thermal degradation compound comprises 4-carboxy-3 -deoxy cyani din (4-carboxy-2-(3,4- dihydroxyphenyl)-5,7-dihydroxychromenylium).

55. The thermal degradation compound of any one of claims 49-53, wherein the thermaldegradation compound has a formula:

56. A composition comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

57. The composition of claim 56, wherein the composition comprises lipids, emulsions, hydrocolloids, carbohydrates, sugar solutions, protein solutions, or a combination thereof.

58. A beverage comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

59. A food product comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

60. An edible product comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

61. A cosmetic product comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

62. A skin care product comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

63. A dye sensitized solar cell comprising the colorant and / or thermal degradation compound of any one of claims 1-55.

64. A method of making the colorant and / or thermal degradation compound of any one of claims 1-55.

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