Acetylating modification and the alkaline hydrolysis reaction

In order to isolate the novel mutant products, using the acetylation modification of the triterpene alcohol fraction and this method was performed according the previous literatures.⁴⁵ (Fig.2.2) The dry triterpene alcohol fraction was first dissolved in 2ml pyridine solvent, and then 1ml acetic anhydride was added into solution. The solution was stirred overnight at room temperature. The acetylation reaction was monitored by TLC analysis. After 16 hours, 5ml of water was added to terminate the reaction and three times extraction with 10ml CH Cl were carried out. The total organic phase was collected and

dried over with sodium sulfate, then evaporated in a rotary evaporator. The acetylation products were separated by using argentic column chromatography and analyzed by GC-MS.

Figure 2.2 The acetylation modification

3-impregnated silica gel chromatography

3 water, stirred and kept them away

3

eaction of the modified compound

ml methanol, and 0.5g

2 4

2.2.7 AgNO

8.6g AgNO and 25g silica gel dissolved in 50ml

from light in the oven under 110 for 16 hours. The gel was used to pack the column with℃ hexane, and then the acetylated products were fractioned by AgNO -impregnated silica gel chromatography using 2.5~3% diethyl ether in hexane.⁴⁶ Thus, each fractions were analyzed by GC-MS, and isolate the single product.

2.2.8 Deacetylation r

The dry acetylation triterpene fraction was dissolved in 10

potassium hydroxide (KOH) was added into the reaction. The reaction was performed with the closed system in the hood and stirred for 12-16 hours at room temperature. The deacetylation reaction was monitored by TLC analysis. After 16 hours, the reactant was dried by rotary evaporator and then 10 ml water was added to dissolve potassium hydroxide. The deacetylated products were thrice extracted with dichloromethane. The total organic phase was collected and dried over with anhydrous sodium sulfate (Na SO ), and then dried thoroughly in a rotary evaporator. The deacetylated products were separated by silica gel column chromatography using 19:1 hexane/ethyl acetate mixture. The

structure of finally novel product was characterized and identified by NMR spectroscopy (¹H, ¹³C, DEPT, COSY, HMQC, HMBC, and NOE).

2.2.9 GC-MS column chromatography condition

ackard model 5890 series II or Agilent

.2.10 Molecular modeling

udies were performed, using the DiscoveryStudio program with t

GC analyses were performed with a Hewlett-P

6890N chromatography equipped with a DB-5 column (30 m x 0.25 mm I.D., 0.25 μm film;

oven gradient at 50 °C for 2 min, and then 20 °C per min until 300°C, held at 300°C for 20 min, 300 °C injector; 250 °C interface; 1/40 split ratio using helium carrier gas at 13 psi column head pressure). GC/MS was performed on a Hewlett-Packard model 5890 II GC (J

&W DB-5MS column, 30 m x 0.25 mm I.D., 0.25 μm film; oven 280 °C, injector 270 °C, GC-MS transfer line: 280 °C) coupled to a TRIO-2000 micromass spectrometer.

2

Molecular-modeling st

he X-ray structure of lanosterol-complexed human OSC as the template. And then using GOLD program for ligand docking, finally using SYBYL program for minimum of the energy. The homologous model structures were obtained with optimum condition. All of the molecular modeling softwares are provided by National Center for High-Performance Computing (NCHC) institution.

Chapter 3 Results and Discussion

3.1 Functional analysis of ERG7

within S. cerevisiae

The saturated mutations of Ile705 within ERG7 from S. cerevisiae were constructed by using QuikChange Site-Directed Mutagenesis kit, and then using the restriction enzyme (Ava I) mapping check. The correct mutants were digested into two parts, one was approximately 6.9 kbp, and the other was 1.1 kbp compared to the wild-type plasmid pRS314OSC which was digested into one fragment of 8 kbp. Mutated plasmids were confirmed by DNA sequencing via the ABI PRISM 3100 DNA sequencer.

Electroporation

Amp Trp

Ergosterol supplement

MATa ERG7Δ::LEU2

hem1Δ::G418 ade2-101 his3Δ-200

Yeast TKW14C-2

pRS314

ERG7^mut PSY^mut Amp Trp

Analyze the product profiles

m/z =426

Assay for cyclase activity by complement viability

Figure 3.1 Strategies for the genetic selection method using TKW14C2 strains.

Following the mutations of the other 19 amino acids substituted at Ile705 position, these mutated plasmids were transformed into a yeast TKW14C2 strain (MATa or MATα ERG7Δ::LEU2 hem1Δ::G418 ade2-101 his3Δ-200 leu2-Δ1 lys2-801 trp1-Δ63 ura3-52) for the in vivo analysis. The yeast system, TKW14C2, is a CBY57-derived HEM1 ERG7 double-knockout mutant and is only viable when supplied with exogenous ergosterol or complemented with oxidosqualene cyclase activity derived from the ERG7^I705X mutants.

(Fig. 3.1) From the genetic selection among S. cerevisiae ERG7^I705X mutants, the I705X (X=G/A/V/L/D/F/N/Q/K/T/C/M/P/S) mutants complemented the ERG7 disruption strain, TKW14C2 in the absence of exogenous ergosterol, while the others (I705W/I705H/I705E/I705R/I705Y mutants) failed. (Table 3.1) These results suggested that the different side chain substitution of I705 might influence the enzymatic activity and abolish the cyclization process of oxidosqualene, and further contributed to the understanding of the function of I705.

ERG7^mut

Ergosterol supplement ( TKW14C-2 )

Table 3.1 The site-saturated mutants of S. cerevisiae ERG7^I705X and their genetic analysis

In order to analyze the function of I705 in the oxidosqualene cyclization/rearrangement cascade, the TKW14C2[pERG7^I705X] mutants were incubated in 2.5 L culture medium. Products were isolated by extracting the non-saponifable lipid (NSL), applying onto silica gel column separation, and analyzing the structures of products by using gas chromatography-mass spectrometry (GC-MS). The product profiles of the S.

cerevisiae ERG7^I705X mutants are listed in Table 3.2. There are eight triterpenoid products with a molecular mass of m/z = 426 including lanosterol,

(13αH)-isomalabarica-14E,17E,21-trien-3β-ol, (13αH)- isomalabarica-14Z,17E,21-trien-3β-ol, protosta-13(17),24-dien-3β-ol, (13αH)- isomalabarica-14(26),17,21-trien-3β-ol, 17α-protosta-20(21),24-dien-3β-ol and two novel products. The ERG7^I705L mutant has only one product, lanosterol, from wild-type ERG7.

Twelve mutations including ERG7^I705G, ERG7^I705A, ERG7^I705V, ERG7^I705D, ERG7^I705N, ERG7^I705Q, ERG7^I705K, ERG7^I705T, ERG7^I705C, ERG7^I705M, ERG7^I705S and ERG7^I705P, produce lanosterol, (13αH)-isomalabarica-14E,17E,21- trien-3β-ol, (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol, and protosta-13(17),24- dien-3β-ol.

Furthermore, the ERG7^I705G mutant also produced a small amount of the novel product.

Another mutation that produced lanosterol, (13αH)-isomalabarica-14(26),17,21-trien-3β-ol, 17α-protosta-20(21),24-dien-3β-ol and a large amount of novel product is ERG7^I705F. Finally, no products with m/z = 426 were observed in five mutations which were substituted with I705W, I705H, I705E, I705R and I705Y, respectively.

ERG7^I705X

Table 3.2 The product profiles of the S. cerevisiae ERG7^I705X mutants.

3.1.2 The identification and characterization of novel product

In order to isolate the unknown product, 70 L of mutant yeast were incubated and the non-saponifable lipids (NSL) were extracted. One of the novel products was a major compound within the culture of the mutant for ERG7^I705F. (Fig. 3.2) The products mixture which with m/z = 426 were acetylated for increasing the polarity, then their m/z became 468. (Fig. 3.3) According to the principle that an argentic ion would bind with a different double bond of the compound, each single compound was separated by AgNO3-impregenated silica gel column chromatography with 2.5 - 3% diethyl ether in hexane as the mobile phase. By the end, through the deacetylation step, the isolated single compound (Fig. 3.2) could be identified by nuclear magnetic resonance (NMR).

Figure 3.2 The GC data of ERG7^I705F. After the novel product was isolated, the complex

Novel product

mixture was purified to became a sole product shown in picture below.

(13αH)-isomalabarica-14(26),17,21-trien-3β-ol 17α-protosta-20(21),24-dien-3β-ol

Lanosterol

I705F

Novel product

m/z = 426

m/z = 468

Figure 3.3 The mass spectra of novel products from the ERG7^I705F mutant.

The unknown product (Fig. 3.4) which contains a tetracyclic scaffold with a 17α side chain and a △^20/22 double bond, and its spectroscopic assignment was determined according to the following analysis of various NMR spectra (¹H, ¹³C NMR, DEPT, ¹H-¹H COSY, HMQC, HMBC, and NOE), and the spectra in depth are shown in Appendix section.

First, the compound showed ¹H NMR spectrum with three vinylic methyl singlets (δ 1.66, 1.6, and 1.52), and five methyl singlets (δ 0.95, 0.75, 0.9, 1.11, and 0.8), and two olefinic protons (δ 5.07, and 5.07). Second, the ¹³C NMR showed the presence of two tertiary-quaternary (δ = 131.85, 124.31, and 123.91, 137.7). These results indicated the presence of a tetracyclic nucleus with a terminal hydrocarbon side chain double bond.

Correlations of the HMQC, HMBC, and NOE spectra showed the following features: (1) the vinyl proton at δ 5.07 (δc 123.91, C-22) is coupled to carbons at 13.44 ppm (C-21), 27.77 ppm (C-23), and 51.29 ppm (C-17); (2) the vinyl proton at δ 5.07 (δc 131.85, C-24) is coupled to carbons at 18.18 ppm (C-27), 26.16 ppm (C-26), and 124.31 ppm (C-25); (3) the proton at δ 2.65 (δc 27.77, C-23) is coupled to carbons at 123.91 ppm (C-22), 124.31 ppm

(C-25), 131.85 ppm (C-24), and 137.70 ppm (C-20); (4) the proton at δ 1.59 (δc 44.94, C-13) is coupled to carbons at 16.93 ppm (C-30), 23.41 ppm (C-11), 25.78 ppm (C-12), 50.79 ppm (C-14), 137.70 ppm (C-20)and 51.29 ppm (C-17); (5) the proton at δ 0.90 (δc

23.2, C-19) is coupled to carbons at 33.87 ppm (C-1), 37.66 ppm (C-10), 46.72 ppm (C-9), and 48.72 ppm (C-5). These correlations established the bond connectivity between the tetracyclic nucleus skeleton and the exocyclic hydrocarbon side chain as well as the double bond positions. The distinct evidence of NOE spectra, which was observed among Me-19/Me-29, H-5/Me-18, and H-9/Me-19, confirmed the presence of chair-boat-chair conformation of the compound, and the tetracyclic structure contains the spatial NOE interaction between Me-30/H-17, and Me-30/H-9, confirmed the 17α side chain conformation.

Figure 3.4 The structure and the NOE correlation of the novel compound

0.8

Table 3.3 NMR assignments for 17α-protosta-20(22),24-dien-3β-ol for dilute CD2Cl2

solution

This is the second time to isolate the 17α tetracyclic triterpene alcohol derivative which has the chair-boat-chair skeleton simultaneously, while all of the other chair-boat-chair products are 17β-derivatives. The novel products, 17α-protosta-20(22),24-dien-3β-ol, and 17α-protosta-20(21),24-dien-3β-ol which was the first 17α-derivative isolated in our lab⁴⁷, are simultaneously produced by the ERG7^I705F mutant. These data showed that I705 is an important residue that contributes to the formation of 17α-products.

3.1.3 Proposed cyclization/rearrangement pathways of TKW14C2 expressing ERG7^Ile705X

The various products derived from ERG7^I705X indicated that I705 is a crucial residue in the putative active site for catalytic function of oxidosqualene cyclase. Seven triterpenoid compounds include three tricyclic and four tetracyclic products. Three truncated tricyclic compounds including (13αH)-isomalabarica-14E,17E,21-trien-3β-ol, (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol and (13αH)-isomalabarica-14(26),17, 21-trien-3β-ol, suggest that after oxidosqualene was pre-folded into the chair-boat-chair conformation and then following cyclization step via cation-π interactions to a 6,6,5-tricyclic C-14 cation intermediate, the three (13αH)-isomalabarica-trien-3β-ols were derived from deprotonation at either the C-15 or C-26 of the 6,6,5-tricyclic C-14 cation.

Following the anti-Markovnikov expansion of the five-membered C-ring to form a 6,6,6-tricyclic C-13 cation, and then via D-ring closure to generate the protosteryl C-20 cation, the substrates through a series of methyl and hydride shifts, deprotonation at C-8 or C-9 yield lanosterol. The mutation of I705 may influence the F699 residue so that it affects the stability of the C-17 cation intermediate at or after the protosteryl cation formation, and the other parts of substrates generate the truncated triterpene, protosta-13(17),24-dien-3β-ol.⁴⁸

Different from the previous characterization, an amazing phenomenon appeared in the I705 mutation, there are two 17α-derivatives, 17α-protosta-20(22),24-dien-3β-ol and 17α-protosta-20(21),24-dien-3β-ol, observed in the ERG7^I705F mutant simultaneously. The speculated pathway is the unnatural D-ring closure, α-orientation of the large side-chain, and then it generates the 17α− protosteryl C-20 cation. This unnatural mechanism may arise from lack of correct amino acids in the putative active site of the cyclase, without rearrangement compared to the normal type, with deprotonation at C-21 or C-22 positions, yielding 17α-protosta-20(22),24-dien-3β-ol and 17α-protosta-20(21),24-dien-3β-ol. (Fig.

3.5)

Figure 3.5 Proposed cyclization/rearrangement pathway occurred in the ERG7^I705X site-saturated mutants

In a previous study, the near absence of 17α dammarenyl cyclases in higher plants reflects the rarity of the 17β to 17α evolutionary step.⁴⁴ Therefore, no matter what protosteryl cation or dammarenyl cation is present, the 17α configuration is unusual and rare in the cyclization process. Formation of 17α-derivatives is unnatural, and the proposed pathway via the 17α−protosteryl C-20 cation could not be defined in the mechanism of the wild-type cyclases. On the other hand, the 17α configuration intermediates did appear only in bacteria or rare plants species, indicating that the 17α configuration may exist in early evolutionary periods, which could rationally explain the rarity of the 17α−derivatives.

3.1.4 Analysis of the ERG7^Ile705X mutants with the ERG7 homology modeling

Molecular modeling studies were performed to investigate the roles of important residues in the cyclization/ rearrangement cascade. Because of the lack of a high-resolution crystal structure of S. cerevisiae ERG7, the homology models of S. cerevisiae ERG7 and its mutated ERG7 proteins were derived from the human OSC X-ray structure, as the template, and complexed with lanosterol or 6,6,5-tricyclic C-14 cation or 17α-protosteryl cation, together with product profiles, in order to investigate the relationship between enzyme structure and product specificity. The homology model of ERG7 showed that Ile705 is a hydrophobic residue different from neighboring aromatic residues, and I705 is spatially proximal to C-14 and C-17 positions of lanosterol (Fig. 3.6a). The hydrophobic property of I705 is considered to have some interaction with the substrate, and also affect the first-tired residues. According to the product profiles of ERG7^Ile705X, the models complexed with 6,6,5-tricyclic C-14 cation and 17α-protosteryl cation, take into account the variation of the residue at 705 position. Furthermore, the product profile of ERG7^I705X is similar to that of ERG7^F699X mutations⁴⁷; therefore we observe the variation of F699 for investigating the function of I705 in depth. (Table 3.4)

ERG7^I705F and ERG7^I705G mutants both produce 17α-protosta-20(22),24-dien-3β-ol, which is the novel product in this study. According to the homology models, the distances between ERG7^I705F and C-14 and C-17 of lanosterol are closer than wild-type, and the distance between I705F and C-17 of 17α-protosteryl cation is also closer than ERG7^I705. (Fig. 3.6b) The aromatic Phe705 stabilized the 17α-protosteryl cation with π-cation interactions, therefore producing both 17α-protosta-20(22),24-dien-3β-ol and 17α-protosta-20(21),24-dien-3β-ol. On the other hand, substitution of Gly with a small aliphatic side chain possibly enlarged the cavity of the active site to cause the substrate to be more flexible, and then could not form lanosterol accurately. There are many truncated products discovered in the ERG7^I705G mutant, including a small amount of

17α-protosta-20(22),24-dien-3β-ol. Except for the 17α-product, the other truncated products are considered to form by variation of the F699 residue, where the flexible Gly possibly influences the F699 residue in this case. Formation of the 17α-protosteryl cation is rare and it was observed in F699X mutations before, so the 17α-protosteryl cation formation may be caused by the effect of F699.

Substitution of I705 into an aliphatic group such as Ala or Val produce truncated products, whereas Leu only produces lanosterol. The ratio of lanosterol decreases when the size of the side chain becomes smaller. Because Leu has a similar bulky size as Ile, the ERG7^I705L mutant only yielded lanosterol as was observed for the wild-type. The percentage of lanosterol for ERG7^I705A and ERG7^I705V are about 75 - 78%, and even the ERG7^I705G mutant only has about 35% of lanosterol shown in Table 3.2. This is the evidence that the steric bulk size at the 705 position is an important factor during catalytic cyclization. The change of the distance between I705A/V of lanosterol are both decreased, and the variation of the F699 residue within I705A/V mutants, complexed with 6,6,5-tricyclic C-14 cation, are closer than wild-type ERG7.

The obvious phenomenon within the acidic, amide and basic group such as I705D/

N/ Q/ K mutants represent smaller amounts of lanosterol compared to the aliphatic group, even without any other products yielded in the ERG7I705E/ H/ R mutations. The balance environments destroyed by substitution of hydrophobic residue into acidic, amide and basic group within the putative active site, leading to tendency of lanosterol formation reduce and generated some truncated products by the F699 effect. In addition, the mutated ERG7I705S/ T/ C/ M/ P proteins also yielded lanosterol and truncated products, similar to that observed for most ERG7^I705X mutations. Comparing the product profiles, we discovered the side chain polarity that could affect the proportion of lanosterol. Substitutions of I705 into Cys, Met and Pro with a nonpolar side chain have higher lanosterol amounts, while Ser and Thr residues with a polar side chain have a small amount of lanosterol. The analysis of the

lanosterol formation tendency show that the acidity and polarity of the residue at the 705 position influence the environment around the substrate within the active site of the oxidosqualene-lanosterol cyclase. But the distance between I705X (X=D, N, Q, K, E, H, R, S, T, C, M, P) and lanosterol, and the variation of the F699 residue in these mutants complexed with 6,6,5-tricyclic C-14 cation are irregular. The irregularly homological results perhaps are the results of a deviation arising from the computational calculation.

However, it also provided the information that the relative positions between the substrate and the cyclase were altered during the catalytic cyclization and the distance may become farther or nearer, when the cation intermediate formed.

Finally, there is no product observed in the ERG7^{I705Y/ W} mutants. Although Tyr and Trp are aromatic residues like Phe, the bulky size makes these two mutations difficult to complement yeast viability, causing the loss of cyclase activity. The steric bulk size may affect the F699 residue strongly, and the stabilization around F699 and the substrate destroyed, so that the catalytic processes were abolished. In conclusion, the key factors which influence the diverse product profiles and yeast viability of the I705X mutations are steric effect, acidity and side chain polarity. Variation at the I705 position certainly influences the substrate and the cyclase in significant ways.

a. b.

Figure 3.6 Homology models. (a) The homology model of wild-type ERG7 complexed with lanosterol, and the distance between I705 to C-14 and C-17 positions of lanosterol is 5.61 Å and 6.14 Å. (b) The homology model of ERG7^I705F mutant complexed with the

17α-protosteryl cation.

Amino acid substitution

Distance to C-17 of lanosterol (Å)

Distance to C-14 of lanosterol (Å)

Distance to C-17 of 17α-protosteryl cation

Table 3.4 The distance of I705 to C-14 and C-17 complexed with different ligands in the homology models, and observation of the variation of F699 within the ERG7^I705X mutants, using the homology model complexed with the 6,6,5-tricyclic C-14 cation.

3.1.5 Product analysis of the double mutant of ERG7I705F/F699X

The product profile of ERG7^I705X is similar to that of ERG7^F699X mutations investigated before in our lab, and F699 had been identified to be an important plasticity

residue with its product diversity⁴⁷. In order to prove that the hydrophobic residue at this position plays an important role that influences the first-tired residue in the putative enzymatic active site, some double mutants were constructed for study in depth. The various products of ERG7^I705X, 17α-protosta-20(22),24-dien-3β-ol is the only compound that ERG7^F699X mutations had not discovered before. The novel compound, 17α-protosta-20(22),24-dien-3β-ol, was only observed for the ERG7^I705F mutation, so that the combination of I705F/F699X double mutants were constructed and their products analysis will be discussed later.

In the experiment with ERG7F699C/I705F double mutants, as Table 3.4 describes, the single F699C mutation loses the activity of the oxidosqualene-lanosterol cyclase, causing the failed result in the absence of exogenous ergosterol, and the result of double mutants is the same as that observed for the single mutant. These data indicate that the F699 is the crucial residue while the I705 residue is critical as an “assistant＂ to affect the first-tired residue F699. Furthermore, in the single mutant F699M, the products yielded include lanosterol, protosta-13(17),24-dien-3β-ol, protosta-17(20),24-dien-3β-ol, (13αH)-isomalabarica-14E,17E,21-trien-3β-ol, (13αH) -isomalabarica-14Z,17E,21-trien-3β-ol, the chair-chair 6-6-5 tricyclic malabarica-14(15)E,17,21-trien-3β-ol and 17α-protosta-20(21),24-dien-3β-ol. Double mutants of I705F/F699M yield 17α-protosta-20(22),24-dien-3β-ol without 17α-protosta-20(21),24-dien-3β-ol, because F699 is crucial within the active site of cyclase, where the 17α-products might be formed by mutation of F699. With different orientations, the I705 affects the F699 residue and has a tendency to deprotonate the proton at C-22 simultaneously. Moreover, interesting data was discovered in the ERG7 I705F/F699T mutation shown in Table 3.5 that except for the formation of little amount of lanosterol, the single mutant of ERG7^F699T produced protosta-13(17),24-dien-3β-ol as the major compound. The generation of protosta-13(17),24-dien-3β-ol, 17α-protosta-20(21),24-dien-3β-ol and

17α-protosta-20(22),24-dien-3β-ol also appeared in the double mutation of I705F and F699T. This result showed that I705 is not only an important assistant residue to the first-tired residue F699, but it is also essential for contribution towards the forming of 17α-products. Furthermore, the results of homology models show the relative distance between F699M/I705F, F699T/I705F to the C-17 of lanosterol are shorter than the wild-type ERG7. However, the modeling data provide us with strong information that the I705F is influential at the C-17 position. (Fig. 3.7)

45

Table 3.5 The products analysis of double mutants between I705F and F699X

Table 3.5 The products analysis of double mutants between I705F and F699X