Regioselective Synthesis of Procyanidin B6, A 4-6-Condensed (+)-Catechin Dimer, by Intramolecular Condensation

Proanthocyanidins, also known as condensed tannins or oligomeric flavonoids, are found in many edible plants and exhibit interesting biological activities. Herein, we report a new, simple method for the stereoselective synthesis of procyanidin B6, a (+)-catechin-(4-6)-(+)-catechin dimer, by Lewis acid-catalyzed intramolecular condensation. The 5-O-t-butyldimethylsilyl (TBDMS) group of 5,7,3′4′-tetra-O-TBDMS-(+)-catechin was regioselectively removed using trifluoroacetic acid, leading to the “regio-controlled” synthesis of procyanidin B6. The 5-hydroxyl group of the 7,3′,4′-tri-O-TBDMS-(+)-catechin nucleophile and the 3-hydroxyl group of 5,7,3′,4′-tetra-O-benzylated-(+)-catechin electrophile were connected with an azelaic acid. The subsequent SnCl4-catalyzed intramolecular condensation proceeded smoothly to give the 4-6-condensed catechin dimer. This is the first report on the complete regioselective synthesis of a 4-6-connected oligomer without modifying the 8-position.


Introduction
Proanthocyanidins are a class of polyphenols and are generally referred to as condensed tannins or oligomeric flavonoids. They have attracted a great deal of attention because of their strong antioxidant activity and wide range of interesting biological functions [1,2]. However, proanthocyanidins are often obtained as a mixture of various analogues from plants. Thus, despite their simple structure as the derivatives of flavan-3-ols, the purification of each compound remains difficult. Flavan-3-ols such as catechin and epicatechin are one of the most well-known groups of biologically active polyphenols. The oligomers composed of these catechins and epicatechin are known as highly functional polyphenols contained in familiar foods such as cacao [3,4]. There are many reports related to other biological activities, such as antifungal [5,6], antiviral [5,7], anti-inflammatory [8], anticancer [3,9], and treating heart diseases [10], among others. Flavan-3-ol derivatives including proanthocyanidins are also converted to various structurally complex secondary polyphenols through chemical reactions that occur during food processing such as drying [11], frozen storage [12], and acidic treatment [13]. These complex and diverse forms make the isolation, structural determination, and evaluation of the biological activities of each compound much more difficult. Therefore, securing sufficient amount of proanthocyanidin as a pure compound for experiment is also important for elucidating the function of proanthocyanidin. A synthetic route to oligomeric catechin and epicatechin derivatives has been developed by Kozikowski et al. [14][15][16] and subsequently by other research groups [17][18][19][20][21][22][23][24]. Additionally, our group has developed and reported simple and stereoselective methods to synthesize 4-8 condensed procyanidin oligomers (constituted by flavan-3-ols having two hydroxyl groups on B and E rings) [25][26][27][28][29][30][31][32][33][34][35][36][37][38] and prodelphinidin oligomers (constituted by flavan-3-ols having, three hydroxyl groups on B and/or E rings) [39] and then studied their biological activities. The key step of the synthesis of proanthocyanidins is the coupling reaction between the nucleophile and electrophile using a Lewis acid such as TiCl4, SnCl4, or trimethylsilyl triflate (TMSOTf) as an activator. As shown in Figure 1, the two dimers of (+)-catechin, 4-6-condensed procyanidin B6 (1) and 4-8-condensed procyanidin B3 (2), are isomers with different bonding positions. The abundance of the 4-6-linked oligomer is comparatively low. In particular, the biological activities of various 4-6-condensed oligomers are yet to be studied in detail due to the unavailability of these compounds. Most studies on the synthesis of proanthocyanidins focus on 4-8-linked oligomers as target compounds, and only a few of them address the stereoselective synthesis of 4-6-linked oligomers. The cool synthesis of the 4-6 catechin dimer, procyanidin B6 (1), has been previously achieved by combining the 8-halo-capping strategy [24]. Due to increasing interest toward the structure-activity-relationship studies (SAR) of proanthocyanidins, developing new and simple methods for the synthesis of various types of proanthocyanidins has become increasingly important. Therefore, we focused on developing a very simple approach to the selective synthesis of such compounds. Herein, we report on an easy, applicable, and reliable method for the stereoselective synthesis of procyanidin B6 (1) via an intramolecular condensation reaction without modifying the 8-position.

Results and Discussion
Previously, we developed a Lewis acid-catalyzed "intramolecular" one-to-one coupling approach that allows for the stereoselective synthesis of 4-8-condensed (+)-catechin dimers (4, 6: Scheme 1) [28,31]. The method includes the connection of flavan-3-ol-derived nucleophile and electrophile via a diester linker and the subsequent condensation reaction by "intramolecular" coupling. Furthermore, the stereochemistry of the coupling product was found to depend on the length of the linker. Despite the fact that the 3,4-cis structure is not preferentially obtained in intermolecular reactions, the intramolecular TMSOTf-catalyzed coupling of azelaic acid (C9 dicarboxylic acid) diester (3) afforded 3,4-trans product 4, and the coupling of glutaryl (C3 dicarboxylic acid derivative) diester (5) gave 3,4-cis product 6 [28] (Scheme 1). Therefore, this intramolecular coupling method could potentially solve the difficulties surrounding the stereoselective synthesis of proanthocyanidins such as 3,4-cis oligomers. Additionally, it could be possible that the structure of the intramolecular coupling Due to increasing interest toward the structure-activity-relationship studies (SAR) of proanthocyanidins, developing new and simple methods for the synthesis of various types of proanthocyanidins has become increasingly important. Therefore, we focused on developing a very simple approach to the selective synthesis of such compounds. Herein, we report on an easy, applicable, and reliable method for the stereoselective synthesis of procyanidin B6 (1) via an intramolecular condensation reaction without modifying the 8-position.

Results and Discussion
Previously, we developed a Lewis acid-catalyzed "intramolecular" one-to-one coupling approach that allows for the stereoselective synthesis of 4-8-condensed (+)-catechin dimers (4, 6: Scheme 1) [28,31]. The method includes the connection of flavan-3-ol-derived nucleophile and electrophile via a diester linker and the subsequent condensation reaction by "intramolecular" coupling. Furthermore, the stereochemistry of the coupling product was found to depend on the length of the linker. Despite the fact that the 3,4-cis structure is not preferentially obtained in intermolecular reactions, the intramolecular TMSOTf-catalyzed coupling of azelaic acid (C9 dicarboxylic acid) diester (3) afforded 3,4-trans product 4, and the coupling of glutaryl (C3 dicarboxylic acid derivative) diester (5) gave 3,4-cis product 6 [28] (Scheme 1). Therefore, this intramolecular coupling method could potentially solve the difficulties surrounding the stereoselective synthesis of proanthocyanidins such as 3,4-cis oligomers. Additionally, it could be possible that the structure of the intramolecular coupling product is changed depending on the position in which the diester linker is introduced. Following these assumptions, we attempted to introduce a diester linker regioselectively to another position by selectively deprotecting one of the four protecting groups of the phenolic hydroxyl moieties, which are in similar environments. product is changed depending on the position in which the diester linker is introduced. Following these assumptions, we attempted to introduce a diester linker regioselectively to another position by selectively deprotecting one of the four protecting groups of the phenolic hydroxyl moieties, which are in similar environments. A thorough examination of the reactive properties of the protected flavan-3-ols revealed that the 5-O-TBDMS (TBS) group of the 5,7,3′,4′-tetra-O-TBDMS-(+)-catechin (7) could be regioselectively removed with TFA to give 8 in 90% yield [40] (Scheme 2). The structure of the 5-OH product (8) was confirmed by the HMBC (Heteronuclear Multiple Bond Correlation) between C5 and the hydroxyl proton of 8 [40]. Using the aforementioned regioselective deprotection method, the intramolecular synthesis of the 4-6-condensed procyanidin B6 (1) was achieved (Scheme 3). During this synthesis, the 5-O-TBDMS group of an acetylation derivative of 7 (9) was removed with TFA to give 5-hydroxyl nucleophile 10 in 76% yield. The 5-hydroxyl group of 10 was then esterified with azelaic acid using the N,N′-dicyclohexylcarbodiimide (DCC) condensation method to afford the carboxylic acid derivative 11 in 80% yield. The 3-hydroxyl group of 12 (electrophile) [26] was then connected to 11 by esterification to obtain the diester compound 13. The subsequent SnCl4-catalyzed intramolecular condensation of 13 at −20 °C proceeded smoothly to afford the 4-6 condensed catechin dimer 14 in 72% yield. When the cyclization reaction was carried out with TMSOTf instead, another TBDMS group was also deprotected, resulting in a complicated mixture of many byproducts. Additionally, the use of dicarboxylic acids as linkers was unsuccessful, and the intramolecular cyclization proceeded A thorough examination of the reactive properties of the protected flavan-3-ols revealed that the 5-O-TBDMS (TBS) group of the 5,7,3 ,4 -tetra-O-TBDMS-(+)-catechin (7) could be regioselectively removed with TFA to give 8 in 90% yield [40] (Scheme 2). The structure of the 5-OH product (8) was confirmed by the HMBC (Heteronuclear Multiple Bond Correlation) between C5 and the hydroxyl proton of 8 [40]. product is changed depending on the position in which the diester linker is introduced. Following these assumptions, we attempted to introduce a diester linker regioselectively to another position by selectively deprotecting one of the four protecting groups of the phenolic hydroxyl moieties, which are in similar environments. A thorough examination of the reactive properties of the protected flavan-3-ols revealed that the 5-O-TBDMS (TBS) group of the 5,7,3′,4′-tetra-O-TBDMS-(+)-catechin (7) could be regioselectively removed with TFA to give 8 in 90% yield [40] (Scheme 2). The structure of the 5-OH product (8) was confirmed by the HMBC (Heteronuclear Multiple Bond Correlation) between C5 and the hydroxyl proton of 8 [40]. Using the aforementioned regioselective deprotection method, the intramolecular synthesis of the 4-6-condensed procyanidin B6 (1) was achieved (Scheme 3). During this synthesis, the 5-O-TBDMS group of an acetylation derivative of 7 (9) was removed with TFA to give 5-hydroxyl nucleophile 10 in 76% yield. The 5-hydroxyl group of 10 was then esterified with azelaic acid using the N,N′-dicyclohexylcarbodiimide (DCC) condensation method to afford the carboxylic acid derivative 11 in 80% yield. The 3-hydroxyl group of 12 (electrophile) [26] was then connected to 11 by esterification to obtain the diester compound 13. The subsequent SnCl4-catalyzed intramolecular condensation of 13 at −20 °C proceeded smoothly to afford the 4-6 condensed catechin dimer 14 in 72% yield. When the cyclization reaction was carried out with TMSOTf instead, another TBDMS group was also deprotected, resulting in a complicated mixture of many byproducts. Additionally, the use of dicarboxylic acids as linkers was unsuccessful, and the intramolecular cyclization proceeded Using the aforementioned regioselective deprotection method, the intramolecular synthesis of the 4-6-condensed procyanidin B6 (1) was achieved (Scheme 3). During this synthesis, the 5-O-TBDMS group of an acetylation derivative of 7 (9) was removed with TFA to give 5-hydroxyl nucleophile 10 in 76% yield. The 5-hydroxyl group of 10 was then esterified with azelaic acid using the N,N -dicyclohexylcarbodiimide (DCC) condensation method to afford the carboxylic acid derivative 11 in 80% yield. The 3-hydroxyl group of 12 (electrophile) [26] was then connected to 11 by esterification to obtain the diester compound 13. The subsequent SnCl 4 -catalyzed intramolecular condensation of 13 at −20 • C proceeded smoothly to afford the 4-6 condensed catechin dimer 14 in 72% yield. When the cyclization reaction was carried out with TMSOTf instead, another TBDMS group was also deprotected, resulting in a complicated mixture of many byproducts. Additionally, the use of dicarboxylic acids as linkers was unsuccessful, and the intramolecular cyclization proceeded only when azelaic acid was employed. Using linkers of other lengths led to self-condensation products of electrophile 12, which once again caused the formation of a complicated mixture of byproducts. Temperature optimization studies of the cyclization reaction showed that the yield was the highest when the reaction was carried out at −20 • C. A decrease in the yield was observed both at higher and lower temperatures. The subsequent removal of the diester linker in 14 via diisobutylaluminum hydrate (DIBAL) reduction gave triol 15 in 66% yield. Due to peak broadening in the NMR spectrum of 15, the tri-acetylated compound 16 was prepared in order to determine the dimeric structure and 3,4-trans stereochemistry of the synthesized product ( Figure 2). Compound 15 was eventually converted to 3,4-trans-(+)-catechin-(4-6)-(+)-catechin dimer, procyanidin B6 (1), after TBDMS-group deprotection by tetrabutylammonium fluoride (TBAF) (85% yield) and subsequent Pd(OH) 2 -catalyzed hydrogenolysis (65% yield). The full spectroscopic data confirmed the structure of procyanidin B6 (1) [41]. only when azelaic acid was employed. Using linkers of other lengths led to self-condensation products of electrophile 12, which once again caused the formation of a complicated mixture of byproducts. Temperature optimization studies of the cyclization reaction showed that the yield was the highest when the reaction was carried out at −20 °C. A decrease in the yield was observed both at higher and lower temperatures. The subsequent removal of the diester linker in 14 via diisobutylaluminum hydrate (DIBAL) reduction gave triol 15 in 66% yield. Due to peak broadening in the NMR spectrum of 15, the tri-acetylated compound 16 was prepared in order to determine the dimeric structure and 3,4-trans stereochemistry of the synthesized product ( Figure 2). Compound 15 was eventually converted to 3,4-trans-(+)-catechin-(4-6)-(+)-catechin dimer, procyanidin B6 (1), after TBDMS-group deprotection by tetrabutylammonium fluoride (TBAF) (85% yield) and subsequent Pd(OH)2-catalyzed hydrogenolysis (65% yield). The full spectroscopic data confirmed the structure of procyanidin B6 (1) [41].  In addition, the coupling reaction of nucleophile 8 and electrophile 12 led to the formation of 4-6 and 4-8-condensed compounds (Scheme 4). After the intermolecular coupling products were acetylated, 16 and 17 were isolated and their structures were confirmed. Compound 16, which was synthesized via intramolecular coupling, was identified with the compound resulting from the intermolecular coupling. Thus, the intramolecularly cyclized product is a 4-6-connected dimer. Usually, when the 5-position is protected, a 4-6-condensed compound cannot be obtained. Therefore, it is possible that the electrophile was brought close to the 6-position through 5-TBDMS group deprotection. However, reactivity at the 6-position was extremely low as compared to that at the 8-position, and the ratio of the 4-6 and 4-8-condensed products was 1:9.

Materials and Methods
All commercially available chemicals for the chemical syntheses were used without further purification. All reactions were performed under argon atmosphere and monitored with thin-layer chromatography (TLC) using 0.25 mm pre-coated silica gel plates 60F254 (Art 5715, Merck KGaA, Darmstadt, Germany). An ATAGO (Minato, Tokyo, Japan) AP-300 spectrometer was used to measure the optical rotation. 1 H-and 13 C-NMR spectra were recorded on an Agilent Inova 500 Spectrometer (500/125 MHz) and DD2 NMR Spectrometer (400/100 MHz) (Agilent, Santa Clara, CA, USA). A JEOL JMS-AX500 mass spectrometer was used to acquire the fast atom bombardment (FAB) mass spectra.  In addition, the coupling reaction of nucleophile 8 and electrophile 12 led to the formation of 4-6 and 4-8-condensed compounds (Scheme 4). After the intermolecular coupling products were acetylated, 16 and 17 were isolated and their structures were confirmed. Compound 16, which was synthesized via intramolecular coupling, was identified with the compound resulting from the intermolecular coupling. Thus, the intramolecularly cyclized product is a 4-6-connected dimer. Usually, when the 5-position is protected, a 4-6-condensed compound cannot be obtained. Therefore, it is possible that the electrophile was brought close to the 6-position through 5-TBDMS group deprotection. However, reactivity at the 6-position was extremely low as compared to that at the 8-position, and the ratio of the 4-6 and 4-8-condensed products was 1:9. In addition, the coupling reaction of nucleophile 8 and electrophile 12 led to the formation of 4-6 and 4-8-condensed compounds (Scheme 4). After the intermolecular coupling products were acetylated, 16 and 17 were isolated and their structures were confirmed. Compound 16, which was synthesized via intramolecular coupling, was identified with the compound resulting from the intermolecular coupling. Thus, the intramolecularly cyclized product is a 4-6-connected dimer. Usually, when the 5-position is protected, a 4-6-condensed compound cannot be obtained. Therefore, it is possible that the electrophile was brought close to the 6-position through 5-TBDMS group deprotection. However, reactivity at the 6-position was extremely low as compared to that at the 8-position, and the ratio of the 4-6 and 4-8-condensed products was 1:9.

Materials and Methods
All commercially available chemicals for the chemical syntheses were used without further purification. All reactions were performed under argon atmosphere and monitored with thin-layer chromatography (TLC) using 0.25 mm pre-coated silica gel plates 60F254 (Art 5715, Merck KGaA, Darmstadt, Germany). An ATAGO (Minato, Tokyo, Japan) AP-300 spectrometer was used to measure the optical rotation. 1 H-and 13 C-NMR spectra were recorded on an Agilent Inova 500 Spectrometer (500/125 MHz) and DD2 NMR Spectrometer (400/100 MHz) (Agilent, Santa Clara, CA, USA). A JEOL JMS-AX500 mass spectrometer was used to acquire the fast atom bombardment (FAB) mass spectra.