Tannin Chemistry Tannin Chemistry This site contains information on the plant secondary metabolites known as tannins.The Hagerman laboratory methods originally in the"Tannin Handbook" are now available through this site,linked to information about tannins.Hyperlinks are indicated by blue text.Turn on Acrobat bookmarks to see titles of the pages. Condensed Tannin Structural Chemistry .Hydrolyzable Tannin Structural Chemistry Purification and Identification Quantitative Analysis Biological Activities Biosynthesis The "Tannin Handbook"can no longer be obtained in hard copy.Instead,print the pages you need from this site. Send me e-mail at hagermae@muohio.edu Or write to me at Professor Ann E.Hagerman Department of Chemistry and Biochemistry Miami University Oxford,OH 45056 USA am1998.2002.Th material may be copied for use within a single laboratory but cannot be copied for distribution o
Tannin Chemistry Tannin Chemistry This site contains information on the plant secondary metabolites known as tannins. The Hagerman laboratory methods originally in the "Tannin Handbook" are now available through this site, linked to information about tannins. Hyperlinks are indicated by blue text. Turn on Acrobat bookmarks to see titles of the pages. • Condensed Tannin Structural Chemistry • Hydrolyzable Tannin Structural Chemistry • Purification and Identification • Quantitative Analysis • Biological Activities • Biosynthesis The "Tannin Handbook" can no longer be obtained in hard copy. Instead, print the pages you need from this site. Send me e-mail at hagermae@muohio.edu Or write to me at Professor Ann E. Hagerman Department of Chemistry and Biochemistry Miami University Oxford, OH 45056 USA © Ann E. Hagerman 1998, 2002. This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author
What is a tannin? WHAT IS A TANNIN? Plants accumulate a wide variety of"secondary"compounds,including alkaloids,terpenes and phenolics Although these compounds apparently do not function in "primary"metabolism such as biosynthesis, biodegradation and other energy conversions of intermediary metabolism,they do have diverse biological activities ranging from toxicity to hormonal mimicry,and may play a role in protecting plants from herbivory and disease Phenolic metabolism in plants is complex,and yields a wide array of compounds ranging from the familiar flower pigments(anthocyanidins)to the complex phenolics of the plant cell wall(lignin). However,the group of phenolic compounds known as tannins is clearly distinguished from other plant secondary phenolics in their chemical reactivities biological activities. Tradition use of tannins as agents for converting animal hides to leather("tanning")is one manifestation of the most obvious activity of the tannins:their ability to interact with and precipitate proteins,including the proteins found in animal skin.The term"tannin"comes from the ancient Celtic word for oak,a typical source for tannins for leather making Bate-Smith defined tannins as "water-soluble phenolic compounds having molecular weights between 500 and 3000...[giving]the usual phenolic reactions...[and having]special properties such as the ability to precipiate alkaloids,gelatin and other proteins" Haslam has more recently substituted the term"polyphenol"for"tannin",in an attempt to emphasize the multiplicity of phenolic groups characteristic of these compounds.He notes that molecular weights as high as 20,000 have been reported,and that tannins complex not only with proteins and alkaloids but also with certain polysaccharides.I prefer to use the term tannin,which emphasizes the character which sets tannins apart from all other phenolics:the ability to precipitate proteins. Ann E.Hagerman 1998,2002.This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author Return to Tannin Chemistry Home Page
What is a tannin? WHAT IS A TANNIN? Plants accumulate a wide variety of "secondary" compounds, including alkaloids, terpenes and phenolics. Although these compounds apparently do not function in "primary" metabolism such as biosynthesis, biodegradation and other energy conversions of intermediary metabolism, they do have diverse biological activities ranging from toxicity to hormonal mimicry, and may play a role in protecting plants from herbivory and disease. Phenolic metabolism in plants is complex, and yields a wide array of compounds ranging from the familiar flower pigments (anthocyanidins) to the complex phenolics of the plant cell wall (lignin). However, the group of phenolic compounds known as tannins is clearly distinguished from other plant secondary phenolics in their chemical reactivities & biological activities. Tradition use of tannins as agents for converting animal hides to leather ("tanning") is one manifestation of the most obvious activity of the tannins: their ability to interact with and precipitate proteins, including the proteins found in animal skin. The term "tannin" comes from the ancient Celtic word for oak, a typical source for tannins for leather making. Bate-Smith defined tannins as "water-soluble phenolic compounds having molecular weights between 500 and 3000...[giving] the usual phenolic reactions...[and having] special properties such as the ability to precipiate alkaloids, gelatin and other proteins". Haslam has more recently substituted the term "polyphenol" for "tannin", in an attempt to emphasize the multiplicity of phenolic groups characteristic of these compounds. He notes that molecular weights as high as 20,000 have been reported, and that tannins complex not only with proteins and alkaloids but also with certain polysaccharides. I prefer to use the term tannin, which emphasizes the character which sets tannins apart from all other phenolics: the ability to precipitate proteins. © Ann E. Hagerman 1998, 2002. This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author. Return to Tannin Chemistry Home Page
CONDENSED TANNIN STRUCTURAL CHEMISTRY Ann E.Hagerman ©1arch28.2002 Proanthocyanidins(condensed tannins)are polymeric flavanoids.The flavanoids are a diverse group of metabolites based on a heterocyclic ring system derived from phenylalanine(B)and polyketide biosynthesis(A).Although the biosynthetic pathways for flavanoid synthesis are well understood,the steps leadin condensation and polymerization have not been elucidated The flavanoid skeleton he standard letters to identify the r and the numbering system are shown here The mo t widely studied cond on the fla van-3-ols (-)-epicat and (+ epicatechin catechin Flavan-3-ols Addition of a third phenolic group on the B ring yields epigallocatechin and gallocatechin Much less c common are flav an-3-ols with only ngle phenolic grov on the B ring. respectively)
CONDENSED TANNIN STRUCTURAL CHEMISTRY Ann E. Hagerman © March 28, 2002 Proanthocyanidins (condensed tannins) are polymeric flavanoids. The flavanoids are a diverse group of metabolites based on a heterocyclic ring system derived from phenylalanine (B) and polyketide biosynthesis (A). Although the biosynthetic pathways for flavanoid synthesis are well understood, the steps leading to condensation and polymerization have not been elucidated. The flavanoid skeleton, the standard letters to identify the rings, and the numbering system are shown here. The most widely studied condensed tannins are based on the flavan-3-ols (-)-epicatechin and (+)- catechin. Addition of a third phenolic group on the B ring yields epigallocatechin and gallocatechin. Much less common are flavan-3-ols with only a single phenolic group on the B ring, para to C-2 (epiafzelechin, afzelechin with stereochemistry corresponding to epicatechin, catechin respectively). 7 6 5 8 4 3 2 O A B HO O OH OH OH OH epicatechin HO O OH OH OH OH catechin Flavan-3-ols
Hagerman stuctural hemistr The best characterized condensed tannins are linked via a carbon-carbon bond between C8 of the terminal unit and C4 of the extender.The four common modes of coupling are illustrated by the dimers isolated by Haslam,and originally named B-1,B-2,B-3 and B-4.The more complete names specify the position and stereochemistry of the interflavan bond completely.In addition to these dimers,related dimers linked by C6 of the terminal unit and C4 of the extender have been isolated. OH B-2 epicatechin-(4B->8)-catechin epicatechin-(40->8)-epicatechin B-3 catechin-(4a->8)-catechin chin-4a>8)-epicatechir
Hagerman condensed tannin structural chemistry 2/6 The best characterized condensed tannins are linked via a carbon-carbon bond between C8 of the terminal unit and C4 of the extender. The four common modes of coupling are illustrated by the dimers isolated by Haslam, and originally named B-1, B-2, B-3 and B-4. The more complete names specify the position and stereochemistry of the interflavan bond completely. In addition to these dimers, related dimers linked by C6 of the terminal unit and C4 of the extender have been isolated. HO O OH OH OH OH HO O OH OH OH OH HO O OH OH OH OH HO O OH OH OH OH B-3 catechin-(4α->8)-catechin B-4 catechin-(4α->8)-epicatechin HO O OH OH OH OH HO O OH OH OH OH HO O OH OH OH OH HO O OH OH OH OH B-1 epicatechin-(4β->8)-catechin B-2 epicatechin-(4β->8)-epicatechin
Hagerman condensed tannin structural chemistry Further polymerization can yield the linear 4.8 polymers such as the Sorghum procyanidin. Linear polymers based on 4,6 dimers;and branched dimers containing both 4,6 and 4,8 linkages are less common picatechin-catchin Although the term conde sed tannin is still widely used to describe these flavonoid-based polyphenolics,the chemically more descriptive term"proanthocyanidin"is gaining acceptance Proanthocyanidins are compounds that yield anthocyanidin pigments upon oxidative cleavage (NOT hydrolysis)in hot alcohols,e.g.via acid butanol chemistry. OH in2 46->8 catechir a2ena8n+eng8gop
Hagerman condensed tannin structural chemistry 3/6 Further polymerization can yield the linear 4,8 polymers such as the Sorghum procyanidin. Linear polymers based on 4,6 dimers; and branched dimers containing both 4,6 and 4,8 linkages are less common. Although the term condensed tannins is still widely used to describe these flavonoid-based polyphenolics, the chemically more descriptive term “proanthocyanidin” (NOT hydrolysis) in hot alcohols, e.g.via acid butanol chemistry. is gaining acceptance. Proanthocyanidins are compounds that yield anthocyanidin pigments upon oxidative cleavage HO O OH OH OH OH O O O OH OH OH HO OH OH OH HO OH OH OH OH OH OH HO O+ OH OH HO OH OH procyanidin epicatechin2 4β−−>8 catechin 2 cyanidin + catechin (extender) end group H+ H+ 2 O O OH HO HO OH OH OH OH HO O OH OH OH OH OH OH OH 15 Sorghum procyanidin epicatechin-[(4β->8)-epicatechin]15-(4β->8)-catechin
rman structural chemistry OH OH R"=OH R"=OH R.OH ewmidin R=R=OH.robinetinidin Anthocvanidins ocyanidi ed by the ech nus are known as procyanidins chin and techin based polymers yield delphinidin.and the rare mono-substituted flavan-3-based polymers An important group of condensed tannins are 5-deoxy-flavan-3-ols polymers.Branching is common in these tannins,because of the reactivity of the 5-deoxy A ring.Profisetinidins and
Hagerman condensed tannin structural chemistry 4/6 The products of the acid butanol reaction are an unmodified terminal unit, and the colored anthocyanidins produced by the extender units. Catechin- and epicatechin-based polymers produce cyanidin, and thus are known as procyanidins. Gallocatechin and epigallocatechinbased polymers yield delphinidin, and the rare mono-substituted flavan-3-ol based polymers yield pelargonidin. An important group of condensed tannins are 5-deoxy-flavan-3-ols polymers. Branching is common in these tannins, because of the reactivity of the 5-deoxy A ring. Profisetinidins and 5 HO O OH HO O OH OH OH OH OH OH OH 5 O HO OH HO HO OH 5 HO O OH OH OH 5 O OH HO HO OH HO O OH OH OH OH robinetinidol-(4α−>8)-catechin-(6α->4a)-robinetinidol profisetinidin HO O OH R' OH R'' R HO O OH R' R'' R R'' = OH R = R' = H, apigeninidin R = H, R' = OH, luteolinidin Anthocyanidins + + R'' = OH R = R' = H, pelargonidin R = H, R' = OH, cyanidin R = R' = OH, delphinidin R'' = H R = R' = H, guibourtinidin R = H, R' = OH, fisetinidin R = R' = OH, robinetinidin
Hagerman condensed tannin structural chemistry prorobinetinidins comprise the major tannins found in quebracho and acacia tannin preparations. Acid butanol reaction yields the 5-deoxy anthocyanidins fisetinidn and robinetinidin. The acid butanol reaction can be carried out with a nucleophilic trapping agent to produce the terminal unit plus derivitized extender units.These can usually be separated and quantitated by HPLC to give composition and average molecular weight estimates for the parent tannin.Thiols such as toluene-a-thiol are often used in this reaction.but phloroglucinol is more convenient. The efficiency of the reaction with branched condensed tannins,especially the 5-deoxy-flavanol- based tannins has not been established OH HO 0 4B->8 cate OH 2eteialo9egoep Anothe r type of linkage that has been described but not studied extensively involves oxidative C O coupling between the flavonoid rings to yield A2 and related proanthocyanidins. OH epicatechin-()-epicatechin proanthocyanidin A-2
Hagerman condensed tannin structural chemistry 5/6 prorobinetinidins comprise the major tannins found in quebracho and acacia tannin preparations. Acid butanol reaction yields the 5-deoxy anthocyanidins fisetinidn and robinetinidin. The acid butanol reaction can be carried out with a nucleophilic trapping agent to produce the terminal unit plus derivitized extender units. These can usually be separated and quantitated by HPLC to give composition and average molecular weight estimates for the parent tannin. Thiols such as toluene-α-thiol are often used in this reaction, but phloroglucinol is more convenient. The efficiency of the reaction with branched condensed tannins, especially the 5-deoxy-flavanolbased tannins, has not been established. Another type of linkage that has been described but not studied extensively involves oxidative CO coupling between the flavonoid rings to yield A2 and related proanthocyanidins. HO OH OH OH OH O O O OH OH HO OH epicatechin-(2β-->7,4β-->8)-epicatechin proanthocyanidin A-2 HO O OH OH OH OH O O O OH OH OH HO OH OH OH HO OH OH OH OH OH OH HO OH HO OH O OH OH HO OH OH OH OH HO 2 procyanidin epicatechin2 4β−−>8 catechin 2 derivitized extenders + catechin (end group) H+ H+ phloroglucinol (or other nucleophile)
Hagerman condensed tannin structural chemistry The flavan-3,4-diols,or luecoanthocyanidins,are sometimes confused with proanthocyanidins. The flavan-3,4-diols are monomeric flavonoids that yield the anthocyanidins upon treatment with heat and acid.They thus have reactive chemistry similar to that of the condensed tannins,but they do not interact with protein to form precipitable complexes. OH Flavan-3,4-diols R"=H(stable) R=H,R'=OH,leucofisetinidin R"=OH able) R=R opelargonidin R=H,R' OH,leucocyanidin R=R'=OH,leucodelphinidin The flavan-4-ols are also luecoanthoc anidins,but are unique in their lability.They yield the anthocyan din tr upo at山 with alcoholic acid ar HO OH Flavan-4-ols R=H,apiferol(leucoapigeninidin) R=OH,luteoferol(leucoluteolinidin)
Hagerman condensed tannin structural chemistry 6/6 The flavan-3,4-diols, or luecoanthocyanidins, are sometimes confused with proanthocyanidins. The flavan-3,4-diols are monomeric flavonoids that yield the anthocyanidins upon treatment with heat and acid. They thus have reactive chemistry similar to that of the condensed tannins, but they do not interact with protein to form precipitable complexes. The flavan-4-ols are also luecoanthocyanidins, but are unique in their lability. They yield the anthocyanidins upon treatment with alcoholic acid at room temperature. O R'' R' R HO OH OH OH R" = H (stable) R = H, R' = OH, leucofisetinidin R'' = OH (unstable) R = R' = H, leucopelargonidin R = H, R' = OH, leucocyanidin R = R' = OH, leucodelphinidin Flavan-3,4-diols HO O OH OH R OH R = H, apiferol (leucoapigeninidin) R = OH, luteoferol (leucoluteolinidin) Flavan-4-ols
unknown pigment,max=465 nm decays at room temp unknown pigment,max=550 nm OH flavon butanol HCl,cold enaymatcoxidaa R R Stich&Forkmann OH OH HO、 NABH, OH O OH OH aaHpierole2g pigenin) R=OH,luteoferol(e oluteolinidin) max 278 nm H 2 MHCI,heat,15 min R OH HO H' HO、 3-de R.p R=OH 757)yellov 入max 95 cherry red pro-3-deoxyanthocyanidins Stafford has suggested that pro-3-deoxyanthocyanidins might exist in a few plants.Evidence to date is limited to spectroscopy and some chemical conversions that are consi tent with the chemistry shown here.(Stafford,H.A.Flavonoid Metabolism;CRC Press:Boca Raton,FL, 1990,pages65-83). Return to Tannin Chemistry home page. Ann E Hagerman 1998,2002.This material may be copied for use within a single b ned for d on of the author
Stafford has suggested that pro-3-deoxyanthocyanidins might exist in a few plants. Evidence to date is limited to spectroscopy and some chemical conversions that are consistent with the chemistry shown here. (Stafford, H.A. Flavonoid Metabolism; CRC Press: Boca Raton, FL, 1990, pages 65-83). Return to Tannin Chemistry home page. © © Ann E. Hagerman 1998, 2002. This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author. HO O OH OH R O HO O OH OH R HO O OH OH R OH HO O OH OH R O HO O OH OH R HO O OH OH R O + enzymatic oxidation (flavone synthase II) Stich & Forkmann NABH4 H+ 2 M HCl, heat, 15 min butanol HCl, cold unknown pigment, λ max = 550 nm H+ flavone R = H, apigenin R = OH, luteolin unknown pigment, λ max = 465 nm decays at room temp 3-deoxy anthocyanidin R = H, apigeninidin λ max = 485 (475?) yellow R = OH, luteolinidin λ max = 495 cherry red flavan-4-ol R =H, apiferol (leucoapigenin) λ max = 275 nm R = OH, luteoferol (leucoluteolinidin) λ max = 278 nm flavanone R = H, naringenin R = OH, eriodictyol pro-3-deoxyanthocyanidins R = H, proapigeninidin R = OH, proluteolinidin
HYDROLYZABLE TANNIN STRUCTURAL CHEMISTRY Ann E.Hagerman ©April14,2002 Hydrolyzable tannins are derivatives of gallic acid(3,4,5-trihydroxyl benzoic acid).Gallic acid is esterified to a core polyol,and the galloyl groups may be further esterified or oxidatively crosslinked to yield more complex hydrolysable tannins. Early work on hydrolyzable tannins included Haslam's significant elucidations of the structures of the simple am,E.Plant enols.Vegetable idge. 1989).M ore recentl atan yza d polyphenols in the emistry of Organi .1-117)have been particula y active in racteri n an fica drolyzable tar ns. etic work (Feldman KS.L of comple awlor MD,and Sahasrab ekeminchemaEotuimiae component coupling strategy for the synthesis of the dimeric ellagitannin coriariin A and a dimeric gallotannin analogue.2000;8011-9)has provided useful insights into likely biosynthetic routes for the complex hydrolyzable tannins.A limited survey of structures and their relationships is provided here. Gallotannins. The simplest hydrolyzable tannins,the gallotannins,are simple polygalloyl esters of glucose The prototypical gallotannin is pentagalloyl glucose(B-1,2,3,4,6-Pentagalloyl-O-D- Gluc se).Pentagalloyl glucose,or PGG.has five identical ester linkages that involve ugar.The alpha anomer is not com OH OH OH OH OH OH OH OH gallic acid O HO OH HO OH B-1,2,3,4,6-pentagalloyl-O-D-glucose
HYDROLYZABLE TANNIN STRUCTURAL CHEMISTRY Ann E. Hagerman © April 14, 2002 Hydrolyzable tannins are derivatives of gallic acid (3, 4, 5-trihydroxyl benzoic acid). Gallic acid is esterified to a core polyol, and the galloyl groups may be further esterified or oxidatively crosslinked to yield more complex hydrolysable tannins. Early work on hydrolyzable tannins included Haslam’s significant elucidations of the structures of the simple gallotannins (Haslam, E. Plant polyphenols. Vegetable tannins revisited, ed.; Cambridge University Press: Cambridge, U. K., 1989). More recently, Okuda et al.(Okuda, T.; Yoshida, T.; Hatano, T. Hydrolyzable tannins and related polyphenols. Progress in the Chemistry of Organic Natural Products 1995, 66, 1-117) have been particularly active in characterization and classification of complex hydrolyzable tannins. Feldman’s synthetic work (Feldman KS, Lawlor MD, and Sahasrabudhe K Ellagitannin chemistry. Evolution of a threecomponent coupling strategy for the synthesis of the dimeric ellagitannin coriariin A and a dimeric gallotannin analogue. 2000; 8011-9) has provided useful insights into likely biosynthetic routes for the complex hydrolyzable tannins. A limited survey of structures and their relationships is provided here. Gallotannins. The simplest hydrolyzable tannins, the gallotannins, are simple polygalloyl esters of glucose. The prototypical gallotannin is pentagalloyl glucose (β-1,2,3,4,6-Pentagalloyl-O-DGlucopyranose). Pentagalloyl glucose, or PGG, has five identical ester linkages that involve aliphatic hydroxyl groups of the core sugar. The alpha anomer is not common in nature. O OH HO OH O O OH OH HO O O OH HO HO O O OH OH OH O O OH OH OH O O OH OH OH OH O β-1,2,3,4,6-pentagalloyl-O-D-glucose gallic acid