Stereospecificity of (+)-Pinoresin01 and (+)-Lariciresinol Reductases from Forsythia intermedia*

PinoresinoVlariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether re- duction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intern-diu and catalyzes the presumed regulatory branch- points in the pathway leading to benzylaryltetrahy-drofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7’-2H2]- pinoresinol and [7,7’-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (>go%). The incoming hydrides were found to take up the pro-R position at C-7’ (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of ran- dom hydride delivery to a planar quinone methide in-termediate. As might be expected, the mode of hydride abstraction &om NADPH was also stereospecific: using [4W“] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.


transfer.
Lignans are a structurally diverse family of phenylpropanoid metabolites found throughout the plant kingdom, principally in woody gymnosperms and angiosperms; they are most frequently found as dimers (l), although higher oligomers exist (2). Based on their known properties, various physiological roles in plants have been proposed. These include antioxidant (3, 41, bactericidal (5), fungicidal (6), antiviral (71, insect antifeedant (81, phytotoxic (to competing species) @), and perhaps even cytokinin-like (10) functions. Although direct evidence is lacking, it has been proposed that lignans are involved in lignification (11). Many lignans exhibit pharmacologically important effects: for example, podophyllotoxin (as its etoposide and teniposide derivatives) is used for treating a variety of cancers (l), such as testicular cancer and acute lymphocytic leukemia. The "mammalian" lignans, enterolactone and enterodiol, apparently reduce incidence rates of breast and prostrate cancers in humans on high fiber diets (12) by modulating steroidal hormone synthesis (13); both lignans are presumed to be formed in the intestine via metabolism of ingested plant materials (14). Lastly, (-)-arctigenin and (-)-trachelogenin strongly inhibit in vitro replication of the human immunodeficiency virus (HIV-1) ( E ) , and like the mammalian lignans, both are presumed to be formed from secoisolariciresinol 1 and matairesinol 2 (14,16).
Lignans can be conveniently divided into several different * This work was supported by National Science Foundation Grant MCB9219586 and United States Department of Agriculture Grant 91371036638. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertzsement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. Tel.: 509-335-2682; Fax: 509-335-7643. structural subfamilies (1). Among the most common ( Fig. 1) are the dibenzylbutanes (e.g. secoisolariciresinol 11, the dibenzylbutyrolactones (e.g., matairesinol 2, arctigenin 3), the furofurans (e.g. pinoresinol4, medioresinol 5), the arylnaphthalenes ( e g . chinensin 6) and the aryltetrahydronaphthalenes (e.g. podophyllotoxin 7). In terms of biosynthesis, the enzymatic steps leading to formation of the CsC3 monomeric units (monolignols) have been firmly established and recently reviewed (17,18). Surprisingly, the subsequent enzymatic transformations involved in monomeric coupling and post-coupling modifications are only now being delineated (16, [19][20][21][22][23][24][25]. Thus, it has been established that insoluble enzyme preparations from Forsythia sp. catalyze the formation of (+)-pinoresin01 4 a from two achiral molecules of E-coniferyl alcohol 8 (22). (More recent studies targeted toward purification of this enzyme have revealed that O2 is required as a cofactor.)l Once formed, (+)-pinoresin01 4 a undergoes highly enantiospecific NADPHdependent reductions to first afford the benzylaryltetrahydrofuran lignan, (+)-lariciresinol9a (24,25) and then the dibenzylbutane lignan, (-)-secoisolariciresinol l b (23)(24)(25) Matairesinol2 has also been proposed to serve as a precursor of aryltetrahydronaphthalene lignans, such as podophyllotoxin 7 (26). Thus, the sequential reductive fission of (+)-pinoresin01 4 a to give (+)-lariciresinolga and (-)-secoisolariciresinol l b permits a rational and hitherto unexpected entry into the various lignan subgroups and, indeed, may represent key regulatory points in lignan pathway branching. Moreover, these highly unusual reductive steps raise intriguing questions regarding enzyme-catalyzed hydride transfer mechanisms. Using (+)-pinoresinol 4a as an example (Fig. 2), three possible hydride transfer scenarios can be envisaged, resulting in either the original C-H bond geometry of the benzylic C-7' carbon being retained, inverted, or undergoing "racemization" as shown. (Note that this is the case, regardless whether a pentacoordinate SN2 or a quinone methide enzyme-bound transition state is involved.) This report describes delineation of the stereochemical course of these unusual benzylic ether reductions, affording (+)-lariciresinol9a and (-1-secoisolariciresinol lb, respectively.
(+)-0,O'-Dibenzyl-7,7"dioxomatairesinols 1 3 a t l 3 b T o a solution of diastereomeric alcohols 12d12b (3 g, 5.3 mmol) in CHzClz (100 ml) was added pyridinium chlorochromate (1.25 g, 5.8 mmol), with the resulting suspension stirred for 18 h under N2 at room temperature. The suspension was diluted with diethyl ether (200 ml), filtered through silica gel (5 g) with the filtrate evaporated to dryness in vacuo (3.2 g). The whole was reconstituted in a minimum amount of EtOAc and applied to a silica gel column (5 x 20 cm) eluted with EtOAdhexanes (1:3). Fractions containing the desired product 13d13b were combined and evaporated to dryness in vacuo. Recrystallization from EtOAdhexanes afforded the diketones 13d13b (2. 77 mmol) in MeOH (5 ml) at 0 "C was added sodium borodeuteride (70 mg, 1.67 mmol, 98 atom % ,H) with the resulting solution stirred for 15 min at the same temperature. The reaction was quenched by adding acetone (0.5 ml), diluted with EtOAc (50 ml), washed with water (50 ml), and saturated NaCl (50 ml), then dried (Na2S0,), filtered, and evaporated to dryness in uacuo to give a foam (1.05 g). This was reconstituted in a minimum amount of EtOAc and applied to a silica gel column (2 x 30 cm) eluted with EtOAdhexanes (12). Fractions containing the desired products were combined and evaporated to dryness in uacuo to give the diastereomeric mixture of alcohols 14d14b (673 mg), which were converted as described (25,28)

Partial Purification of Pinoresinol 1 Lariciresinol Reductase
All steps were carried out a t 4 "C. F intermedia stems (20-25 cm long, 100 g) were cut into 1-cm sections, frozen (liquid N2) and pulverized (Waring Blender). The resulting powder was further homogenized in a Waring blender with potassium phosphate buffer (0.1 M, pH 7.0,300 ml) containing 10 m~ dithiothreitol. The homogenate was filtered through four layers of cheesecloth into a beaker containing PVPP slurry (6 g in 100 ml of potassium phosphate buffer, 0.1 M, pH 7.0) and stirred for 20 min. The filtrate was centrifuged (12,000 x g, 10 min) and the resulting supernatant fractionated with (NH,),SO,. Proteins precipitating between 40 and 60% saturation were recovered by centrifugation (12,000 x g, 15 min). The pellet was reconstituted in Tris-HC1 buffer (20 m, pH 8.0, 10 ml) containing 5 m~ dithiothreitol (buffer A) with aliquots (2.5 ml) applied to four prepacked PD-10 columns, (Pharmacia LKB Biotechnology Inc., Sephadex G-25 medium) equilibrated with Stereospecificity of Benzylic Ether Reductases 27029 buffer A. The eluted fraction (14.0 ml) was next applied to an Affi-Gel Blue Gel column (1 x 15 cm) equilibrated in buffer A (29, 30). ARer rinsing the column (buffer A, 20 ml), pinoresinolflariciresinol reductase was eluted with a NaCl gradient (0-5 M in 100 ml) in buffer A at a flow rate of 0.2 ml min". Proteins were detected at 280 nm and each fraction (1 ml) was assayed for pinoresinol and lariciresinol reductase activities (see below). Active fractions were combined and used as the enzyme preparation for stereospecificity studies. Protein contents were determined using the method of Bradford (31) using Bio-Rad dye reagent and bovine y-globulins as standard.
Enzyme Assays Pinoresinol Reductase-Pinoresinol reductase activity was assayed by monitoring the formation of [3H]lariciresinol 9 and secoisolariciresinol I . Each assay, conducted in quadruplicate, consisted of (a)-pinoresinols 4d4b ( . An aliquot (60 pl) from one assay was subjected to reversed-phase HPLC analysis with fractions collected every minute (t = 0-30 min) and analyzed by liquid scintillation counting. The remaining assays were divided into 6O-pl aliquots and individually subjected to reversed-phase HPLC, with (2)lariciresinols 9dSb and (a)-secoisolariciresinols l d l b (retention volume 5.9 and 6.7 ml, respectively) separately collected and evaporated to dryness in uacuo. Each lignan was reconstituted in MeOH (100 PI) and filtered (0.45 pm), with an aliquot (60 4) subjected to chiral column HPLC analysis with eluant analyzed by both UV and liquid scintillation counting. Controls were performed using either denatured enzyme (boiled 96 "C, 10 min) or in the absence of (2)-pinoresinols 4d4b.
Lariciresinol Reductase-Lariciresinol reductase activity was assayed by monitoring the formation of [3Hlsecoisolariciresinol 1. Assays were carried out exactly as described above, except that (a)-lariciresinols 9dSb (5 m~ in MeOH, 40 pl) were used as substrates and (a)secoisolariciresinols l d l b (40 pg) were added as radiochemical carriers.

RESULTS AND DISCUSSION
The stereospecificity of the pinoresinolllariciresinol reductase-catalyzed hydride transfers was investigated from two perspectives, namely (i) the stereochemical consequence(s) of hydride addition during enzymatic product formation and (ii) the mode of hydride abstraction from the NADPH cofactor. In order to investigate these stereochemical questions, (+)-pi-noresinoY(+)-lariciresinol reductase was partially purified (50fold) by affinity chromatography (Affi-Gel Blue Gel eluted with a NaCl gradient; see "Experimental Procedures"), and used as the enzyme preparation for this study. It should be noted, however, that both pinoresinol and lariciresinol reductase activities eluted coincidentally during the NaCl gradient. Indeed, this was also observed to be the case even when the reductase was purified -1,000-fold via a combination of hydroxyapatite and affinity yellow chromatography3 With partially purified pinoresinolllariciresinol reductase in hand, attention was first directed toward defining the stereospecificity of hydride transfer. As can be seen in Fig. 3 A , the net stereochemical change during the reductive cleavage of the furan rings of 4a and 9a is destruction of the chiral centers at C-7/C-7'. But in order to address the stereospecificity of hydride transfer, it was necessary to unambiguously distinguish the C-7/C-7' pro-R and pro-S methylenic protons in the enzymatic products, (+)-lariciresinol 9a and (-)-secoisolariciresinol lb. This was achieved via a combination of 'H, ZD-COSY, and 2D-NOESY NMR spectroscopic experiments aided by molecular modeling. Using (2)-lariciresinols 9d9b as an example, the 'H and 2D-COSY NMR spectra revealed that the (2-7' methylenic protons were magnetically non-equivalent, with each individually observed as a pair of doublet of doublets at 62.55 ( J~c = 10.8 Hz, J,,, = 13.5 Hz) and 2.92 ( J~= = 5.2 Hz, Jgem = 13.5 Hz), respectively (Fig. 4A). Based on the magnitude of the vicinal coupling constants (5.2 and 10.8 Hz) between the (2-7' methylene protons and the adjacent C-8' proton, two possible conformers A and B (as shown by their Newman projections in Fig. 3 B ) bearing the requisite dihedral angles could be envisaged (32). But inspection of Dreiding molecular models suggests that only conformer A was favorable, since conformer B is apparently more sterically hindered via unfavorable interactions between the aromatic ring and the pendant C-9 hydroxymethylene group (Fig. 3B). That this was indeed the case was established by a 2D-NOESY experiment (500 ms mixing time), which reveals correlations between protons spatially located within 5 b; from each other (33). Thus the detection of a crosspeak between the C-2' proton (66.67) of the aromatic ring and the C-9'p proton (63.75) established that both protons were in close proximity to one another, as in conformer A (data not shown). By contrast, no cross-peak was observable between the C-2' aromatic proton at 66.67 and the C-9 hydroxymethylene protons at 6 3.78 and 3.92, indicating that conformer B was not favored. This result agreed with those obtained from a molecular modeling routine, using the program Macromodel with MM2 force field calculations for energy minimizations. This again revealed unfavorable steric interactions between the aromatic ring and the hydroxymethylene group (C-9) in conformer B (data not shown) which were not evident with conformer A.
Thus, based on the results from both 2D-NOESY experiments and energy minimization calculations, A was the preferred conformer (Fig. 3B). With the conformation so established, it was possible to assign the C-7' pro-R and C-7' pro-S protons on the basis of diaxial interactions with the C-7 proton via a 2D-NOESY experiment. Thus, a cross-peak between the C-7 proton a t 64.79 and a C-7' proton resonance a t 62.55, showed that both were in close proximity and that the latter corresponded to the C-7'pro-R proton. No cross-peaks were observed between the C-7 proton and the C-7' pro-S proton resonance at 62.92. Following a similar line of reasoning, the pair of doublet of doublets in the 'H NMR spectrum of (+secoisolariciresinols l d l b at 62.66 and 2.76 were assigned to the C-7pro-S and C-7pro-R protons, respectively. Having established a NMR spectroscopic method to distinguish the pro-S andpro-R protons at C-7' of (+)-lariciresinol9a and C-7E-7' of (-)-secoisolariciresinol lb, attention was directed to defining the stereochemistry of hydride transfer. This presented the new problem of differentiating the incoming hydride from that of the existing C-7 or C-7' proton present in the substrates 4a and 9a. One means to differentiate both protons was to employ selective deuterium labeling, either involving deuteride transfer from NADP'H or via selective deuteration at the C-7 and C-7' sites of pinoresinol 4 and lariciresinol 9; the second option was employed because of readily available synthetic methodology (25,28). Thus, p-ketolactones 13d13b were obtained from the known diastereomeric alcohols 12d12b following treatment with pyridinium chlorochromate; these were then reduced with sodium borodeuteride to give the deuterated diols 14d14b (Fig. 5). Subsequent LiAlH4 reduction afforded tetraols 15dl5b, which were treated with bromotrimethyl silane to give a -4:l mixture of (~)-O,O'-dibenzyl-[7,7'- 'Hz1pinoresinols 16d16b and (d-0,0'-dibenzyl-[7,7'-2H21epipinoresinols (25). Debenzylation via hydrogenation over Pd-C afforded the required (~)-[7,7'-2H21pinoresinols 4d4b. Subsequent comparison of the 'H NMR and mass spectra of deuterated 4d4b with its unlabeled analogue unequivocally established the positions of deuteration within the molecule. Thus, both 'H NMR spectra were essentially identical, except for the absence of a 2H doublet at 64.74 in the deuterated 4d4b sample; this resonance had previously been assigned to the C-7/C-7' protons in unlabeled (d-pinoresinols 4d4b (25,28). Analysis of the mass spectra of both deuterated and unlabeled In an analogous manner, (d-[7,7'-2H3]1ariciresinols 9d9b were prepared from (~)-[7,7'-2H21pinoresinols 9d9b via catalytic deuterolysis over 10% Pd-C. Comparison of the 'H NMR and mass spectra of both the deuterated and unlabeled lariciresinols 9d9b again unequivocally revealed the sites of deuteration. Thus, the 'H NMR spectra were essentially identical, except for the absence of resonances in the deuterated product a t 62.55, 2.92, and 4.79 corresponding to the C-7' pro-R, C-7' pro$, and C-7 protons, respectively. Analyses of the mass spectra of both provided additional support to the NMR assignments; the unlabeled (%)-lariciresinols 9d9b had a molecular ion at ( m / z ) 360, together with ion fragments at ( r n l z ) 236, With the required deuterated substrates in hand, the stereochemical basis of the enzyme-mediated hydride transfer was investigated. Thus, (~)-[7,7'-2H21pinoresinols 4d4b (0.42 mM) were incubated for 1 h at 30 "C with the partially purified pinoresinolllariciresinol reductase from F. intermedia in the presence of 1.6 mM NADPH. The resulting lariciresinol 9 so formed was purified using flash silica gel chromatography, and its 'H NMR and mass spectra, and chiral column HPLC profiles were recorded and compared to that of synthetic unlabeled (2)-lariciresinols 9d9b. Fig. 4, A and B, show the pertinent regions of the lH NMR spectra corresponding to the C-8, C-7' pro-R, C-8' and C-7' pro-S proton resonances for both the synthetic (Fig. 4A) and the enzymatically formed lariciresinol 9 (Fig. a). It can immediately be noted that in the enzymatic product, only the 1H doublet a t 62.51 ( J = 10.6 Hz) corresponding to the C-7'pro-R proton is observed, whereas the C-7' pr0-S proton signal at 62.92 is essentially absent (>99% reduction). Note also that the C-7'pro-R proton resonance at 62.51 is only a doublet ( J = 10.6 Hz) due to the absence of geminal coupling and that its chemical shift is moved upfield by 0.04 ppm; this shift is consistent with the reported shielding effects caused by replacing a proton with deuterium in a methylene or methyl group (34). Mass spectroscopic analyses of the enzymatic product further supported this finding; the enzymatically formed deuterated lariciresinol 9 had a molecular ion at ( m l z ) 362, corresponding to the presence of two deuterium atoms. That these deuterium atoms remained at C-7 and C-7' was established from the ion fragmentation pattern; thus, the enzymatic HPLC column analysis of the deuterated product revealed that only (+)-lariciresinol Sa was formed, in accordance with the known enantiospecificity of this transformation (25). Thus, it was concluded that the reductase-catalyzed hydride transfer was >99% stereospecific, where the incoming hydride exclusively took up the pro-R position with the existing carbondeuterium bond geometry at C-7' undergoing inversion to assume the pro-S position, c.e. the enzymatic product is (+)-[7,7'S-2H2]lariciresinol 9a.
The possibility that lariciresinol9 reduction proceeded in an analogous manner was next investigated. Thus, (+)-[7,7'-2H3]lariciresinols 9d9b (0.11 m~) were incubated for 1 h at 30 "C with the partially purified reductases in the presence of 1.7 m~ NADPH. As before, the secoisolariciresinol 1 formed was purified, with its lH NMR and mass spectra and chiral HPLC profiles subsequently recorded and compared to authentic unlabeled (d-secoisolariciresinols l d l b . The 'H NMR spectroscopic findings are shown in Fig. 6, A and B, where only the pertinent regions of the 'H NMR spectra corresponding to the C-7 pro-S, C-7 pro-R, and C-9 proton resonances are shown. As can be seen, the deuterated enzymatic product 1 (Fig. 6B) displayed only a 1H doublet centered a t 62.70 ( J = 7.5 Hz), in contrast to the pair of doublets of doublets centered a t 62.66 and 2.76 corresponding to the C-7 pro-S and C-7 pro-R protons, respectively, in unlabeled (d-secoisolariciresinols l d l b (Fig.  6.4). Thus, given the small expected upfield proton resonance shift of -0.04-0.07 ppm due to deuterium substitution (341, it can again be proposed that hydride transfer is stereospecific (>99%) resulting in replacement of the C-7 pro-S proton by deuterium. The mass spectrum of the enzymatically synthesized (deuterated) secoisolariciresinol 1 confirmed the presence of three deuterium atoms, as evidenced by the molecular ion at ( r n l z ) 365. That these deuterium atoms remained at C-7 and C-7' positions was again established from the ion fragmentation patterns with ion fragments ( m / z ) at 139 and 138 corresponding to [ArC2H21+ and [ArCH2HI+, respectively, where Ar = 4-hydroxy-3-methoxyphenyl. Chiral HPLC analysis of the deuterated product revealed that only (-)-secoisolariciresinol l b synthesis had occurred, again in accordance with the known enantiospecificity of the conversion (24,25).
The enzymatically synthesized, deuterated, (-)-secoisolariciresinol l b formed was purified and subjected to 'H NMR and mass spectral analyses. As can be seen in Fig. 6C, a 2H doublet at 82.70 (J = 7.5Hz) was again evident in the 'H NMR spectrum. Since it had already been established that the hydride transferred during conversion of (+)-pinoresin01 4 a into (+I-lariciresinol9a becomes the C-7 pro-R proton, and that the 'H NMR spectrum o f (-)-deuterated l b shows only a single 2H doublet, it follows that its subsequent conversion into (-)-secoisolariciresinol lb has proceeded in an analogous manner, i.e. in both reduction steps, the incoming hydride takes up the pro-R position.
Lastly, it was also instructive to determine whether the 4 pro-R or 4 pro-S hydrogen on the nicotinamide ring of NADPH was abstracted during hydride transfer. [4R-3H] and [4S-3HlNADPH 10b and 10a were conveniently prepared by modification of the procedure by Moran et ai. (27). Each tritiated cofactor was individually incubated for 1 h at 30 "C with the partially purified reductases in the presence of ( a ) 0.5 mM (2)pinoresinols 4d4b and ( b ) 0.5 mM (+)-lariciresinols 9d9b, respectively. (Note that following incubation, unlabeled (*)-lariciresinols 9d9b (40 pg) and (2)-secoisolariciresinols l d l b (40 pg) were added as radiochemical carriers, with each lignan individually purified and resolved by chiral column HPLC.) The results obtained are summarized in Table I. With (*)-pinoresinols 4 d 4 b as substrates, radiolabeled lariciresinol9 was only formed (1.37 pkat mg" protein) when [4R-3HlNADPH lob was employed as cofactor. Moreover, chiral column HPLC analysis again revealed that only the (+)-enantiomer 9a was radiolabeled. The reduction of lariciresinol 9 to (-)-secoisolariciresinol l b proceeded in an analogous manner: radiolabeled secoisolariciresinol 1 was only synthesized (2.19 pkat mg-' protein) when [4R-3H]NADPH 10b was added as a cofactor; chiral In summary, the (+)-pinoresin01 and (+)-lariciresinol reductases function in a highly stereospecific manner. Both abstract the 4 pro-R hydrogen of NADPH, in such a manner that the incoming hydride adopts the pro-R position (>99%) at C-7fC-7' in the lignan product. These findings, therefore, rule out the possibility of a SN1 mechanism involving a planar transition state with random hydride delivery from either side of the molecule (Fig. 2). The next phases of our research will be to establish whether the enzyme-bound transition state is in either the furano or quinone methide form, and the regulatory roles of these enzymes in lignan biosynthesis.
State University NMR Spectroscopy Center) for obtaining the 2D-